Advanced Therapy Medicinal Products: Clinical, Non-clinical, and Quality Considerations

Mastrobattista, Enrico; Doevendans, Erik; van Til, Niek P.; Kemp, Vera; de Vrij, Jeroen; Hoogendoorn, Karin

doi:10.1007/978-3-031-30023-3_14

Enrico Mastrobattista⁴,
Erik Doevendans⁴,
Niek P. van Til⁵,
Vera Kemp⁶,
Jeroen de Vrij⁷ &
…
Karin Hoogendoorn⁸

984 Accesses

Abstract

Cell therapy, tissue engineering, and gene therapy products, together called “advanced therapy medicinal products” (ATMPs), represent a heterogeneous group of innovative biopharmaceuticals. ATMPs are based on viable cells, tissue, or genetic material. In this chapter, after a brief introduction, first different classification systems for these products are discussed, illustrated with representative examples of products in clinical development or commercially available. Next, the challenges associated with successful pharmaceutical development, manufacturing, and testing of these products are covered. Finally, regulatory aspects are dealt with.

Access provided by Autonomous University of Puebla. Download chapter PDF

Advanced Therapies: Clinical, Non-clinical and Quality Considerations

Gene and Cell Therapy: How to Build a BioDrug

Regulatory Framework for Cell and Tissue Based Therapies in Europe

Keywords

Introduction

The most recent branch of the biotechnology revolution in medicine consists of gene and cell therapy medicinal products and tissue-engineered products. These are collectively called advanced therapies in the US and advanced therapy medicinal products (ATMPs) in the EU. The use of living cells, tissues, or organs in medical practice is not novel. The first successful kidney transplantation in a human took place in 1954, and human hematopoietic stem cell transplantation from a healthy donor to a cancer patient in 1959; the latter is now a routine clinical procedure for bone marrow regeneration. Cell therapies, including stem cell therapies, were not further explored until the early 1990s when the therapeutic relevance of mesenchymal stromal cells (MSCs) was considered for the regeneration of skeletal tissue and later for broader therapeutic use. Furthermore, the development of efficient gene transfer vectors to genetically modify cells ex vivo or directly in vivo in the 1980s and 1990s further boosted the development of therapies based on genetically corrected or augmented cells. Since the turn of the millennium, there has been a steady increase in the number of clinical studies, with a growing number of target indications. Particularly for the treatment of diseases and tissue/organ defects for which traditional therapies and medicinal products have not always provided positive benefit/risk outcomes, such as multiple sclerosis, Parkinson’s disease, cancer, and muscular dystrophy, advanced therapies hold high expectations. The inherent complexity of these products poses unique challenges compared to other therapeutics. The manufacture of gene transfer vectors or “living” materials (i.e., cells and tissues) comes with great challenges in terms of consistency and process and product characterization. Such challenges are analogous, in many ways, to those faced in the past when the first recombinant protein biopharmaceutical products were being developed and regulated. Bringing advanced therapies to market at an acceptable cost, benefit/risk ratio, and quality has proven extremely difficult for certain products.

This chapter discusses the current status and unique aspects of ATMPs. We explain the differences between traditional cell or tissue transplantation versus advanced therapies based on (genetically manipulated) somatic cells or tissue-engineered products. Then, we discuss in detail the various cell technologies and technologies for gene therapy and provide information on the manufacturing of advanced therapies. Finally, the regulatory aspects of ATMPs will be briefly highlighted.

ATMPs: Definitions, Classifications, and Modes of Action

Globally, different names and definitions for gene- and cell-based products are used in different jurisdictions. For example, in the EU, such a product is called an advanced therapy medicinal product (ATMP), whereas the term “advanced therapy” is used in the USA. Minor differences in the definition of (sub-)classes between the two jurisdictions exist, and importantly, in the US, human cells and tissues may also be regulated as devices (similarly to the combined ATMPs in Europe). Furthermore, because of the minor differences in definitions applied in the US and EU, hematopoietic stem cell (HSC) transplantation for the treatment of malignant blood disorders is an example of a product that would be classified as an advanced therapy in the US but would fall outside the scope of ATMPs in the EU (see section “Adult Stem Cells Used as Transplant Product”; Fig. 14.5).

Transplantation or Advanced Therapy?

Advanced therapies, when applied to humans, are considered biological medicinal products, meaning they are typically subject to either or both of the following regulatory regimes: public health legislation and pharmaceutical legislation. However, some clinical interventions for cell- and tissue-based advanced therapies are not considered “medicinal”; these products are subject to public health legislation only. These therapies are often called “cell and tissue transplant products” or “cell and tissue transplantations” and have to meet all of the following criteria (see also Fig. 14.1):

1.
A cell or tissue, which is not substantially manipulated. Table 14.1 provides guidance on the definition of substantial and nonsubstantial manipulations.
2.
Cells/tissues are used for the same essential function in the donor and recipients (sometimes called “homologous use”).
3.
It is not combined with a medical device or active implantable medical device.

A decision tree of the manipulation of cells and tissues by A T M P. If the cells can be manipulated ex vivo, then the A T M P options include ex vivo G T M P, s c t M P or somatic cell therapy medicinal product, tissue-engineered product, and combined A T M P. — **Fig. 14.1**

Table 14.1 Substantial and nonsubstantial manipulations of cells or tissues

Full size table

Cell and tissue transplant products require no clinical trials and no marketing authorization (MA) prior to commercial availability but public health legislations apply.

However, if cells or tissue are substantially manipulated, it fulfills the criteria for an advanced therapy medicinal product (ATMP) and, as such, will be regulated as a medicine, meaning the development must comply with medicines regulations.

ATMPs can be subdivided into somatic cell therapy medicinal products (SCTMP), consisting of somatic cells that have been expanded and/or manipulated ex vivo before being administered back into the patient; gene therapy medicinal products (GTMP), consisting of vectors for transfer of materials that can genetically modify cells ex vivo or in vivo; tissue-engineered products (TEP), involving cells or multiple cell types growing on a scaffold to form a 3D tissue culture; and cells or tissues that are being combined with a medical device (combined ATMP) (Fig. 14.1 and Table 14.2).

Table 14.2 EU-ATMP classification definitions according to the EU pharmaceutical legislation, adapted from Smith et al. (2015)

Full size table

Gene or Cell Therapy Medicinal Product?

There is considerable overlap between somatic cell therapy medicinal products (SCTMP) and gene therapy medicinal products (GTMP). If a cell is isolated from the body and substantially manipulated ex vivo before readministration, it is considered a cell therapy medicinal product. Suppose (part of) this substantial manipulation consists of genetic modification; the product of this manipulation is considered as an (ex vivo) gene therapy medicinal product (Fig. 14.1). For example, tabelecleucel (Ebvallo®) consists of allogeneic Epstein-Barr virus (EBV) specific T cells and is registered as a cell therapy medicinal product, whereas Zalmoxis® also consists of allogeneic T cells. Still, these were genetically modified with a retroviral vector to express the human low-affinity nerve growth factor receptor and the herpes simplex virus type 1 (HSV-1) thymidine kinase enzyme. This product was therefore registered as an ex vivo gene therapy medicinal product. Similarly, products based on ex vivo genetic modification of autologous T cells to express a chimeric antigen receptor (CAR-T cells) are categorized as ex vivo gene therapy medicinal products.

Classification

Besides the classification of ATMPs based on regulation as described above, ATMPs can be classified in many other ways, e.g., by:

1.
The therapeutic indication they aim to address, e.g., neurological, cardiovascular, or ophthalmological.
2.
Whether they comprise cells and/or tissues (see Fig. 14.2):
1. (a)
  derived from and administered to the same human individual (autologous = autogenic), hence the donor = the recipient (patient);
2. (b)
  derived from a human (healthy) donor, who is different from the patient (allogeneic);
3. (c)
  derived from an animal (xenogeneic; see Chap. 9), e.g., porcine islets to treat diabetes mellitus (DM).
3.
The potency of the cells, i.e., omnipotent, pluripotent, multipotent, oligopotent, and unipotent (see Table 14.3).
4.
The in vivo mode of action (i.e., pharmacological, immunological, metabolic, or regenerative; i.e., regenerate, repair, or replace a human tissue).
5.
Their underlying technology, as described in this chapter (Mount et al. 2015):
1. (a)
  somatic cell technologies;
2. (b)
  cell immortalization technologies;
3. (c)
  ex vivo gene modification of cells using viral vector technologies;
4. (d)
  in vivo gene modification of cells using viral vector technologies (see this chapter);
5. (e)
  genome editing technologies;
6. (f)
  cell plasticity technologies;
7. (g)
  three-dimensional technologies;
8. (h)
  combinations of the above technologies.
6.
The cell types, e.g., MSCs, dendritic cells (DCs), and T cells.

An illustration of the allogenic and xenogeneic transplants between a man and a woman and a man and a pig, respectively. Xenogeneic is labeled between different species. A cyclic arrow above the head of the man labeled autogenic, within the same individual. — **Fig. 14.2**

Table 14.3 Categorization of stem cells on their potency

Full size table

ATMPs: Possible Mode of Action(s)

The in vivo mode of action(s) (MoA(s)) of an advanced therapy depends on the type of cell/tissue, the ex vivo manipulations performed on the cells/tissue in the manufacturing facility, e.g., genetic modification, the route of administration and the in vivo environment of the cells/tissue:

1.
Pharmacological: cells/tissue release molecules such as cytokines and growth factors upon interaction with their/its environment. An example is the immunoregulatory effect of MSCs. E.g., darvadstrocel (Alofisel®) contains expanded adipose-derived MSCs, which, once activated, impair the proliferation of lymphocytes and reduce the release of pro-inflammatory cytokines at inflammation sites in patients with luminal Crohn’s disease. This immunoregulatory activity reduces inflammation and may allow the healing of the tissues around the fistula tract;
2.
Regenerative: ex vivo manipulated cells/tissue regenerate, repair, or replace a diseased or damaged human tissue. E.g., to replace damaged ß-cells of a patient suffering from DAM type I, whereby human (h)ESCs are ex vivo differentiated into pre-ß cells, loaded into a device, and administered subcutaneously.
3.
Immunological: cells of the immune system are ex vivo activated. E.g., cytotoxic T lymphocytes (CTL) or genetically modified cells (e.g., CAR-T cells) activate the patient’s own immune system upon administration, e.g., to treat cancer.

(Dis)similarities with Recombinant Therapeutic Proteins and Other Biologicals

Although ATMPs fall within the group of biologicals, there are substantial differences in the area of chemistry, manufacturing, and controls (CMC), nonclinical, clinical, regulatory, and costs/reimbursement compared to recombinant proteins, vaccines, and plasma-derived medicinal products. This is summarized in Table 14.4 and further discussed in this chapter.

Table 14.4 Example of differences between advanced therapies and biopharmaceuticals

Full size table

Part A: Technologies for Cell Therapy and Tissue Engineering

Although ATMPs can be classified by the regulatory regime to be applied (see above), the diversity of this new group of biologicals may be better illustrated by the underlying technology and their potential as therapeutics (Mount et al. 2015). Below, these technologies are briefly discussed with examples of products in clinical development or approved for commercial use.

Somatic Cell Technologies

This technology involves the use of adult stem cells, also known as somatic stem cells. The fundamental property of a stem cell is the capability to multiply, i.e., it has the self-renewal capacity, which is the ability to go through numerous cycles of cell division while maintaining the undifferentiated state and to give rise to a variety of differentiated cells. Stem cells can be characterized by their potency, which is the ability to differentiate into specific cell types. The more cell types it can generate, the higher the potency (Table 14.3). Different stem cells exist (Table 14.5) and can be isolated from embryos, blood cords, tissues, and organs, or they can be derived from differentiated somatic cells (mostly skin fibroblasts) by inducing pluripotency via forced expression of specific transcription factors, the so-called induced pluripotent stem cells (iPSC; see section “iPS Cell Technology”).

Table 14.5 Origin, characteristics, and uses of “stem” cells

Full size table

Adult stem cells are known to be present in many, if not all, individual organs in adults and are generally thought to be “multipotent”; that means that they have the ability to differentiate into all cell types within one particular lineage, i.e., they can give rise to the cells found in their organ of origin, but not in other organs (Fig. 14.3). In tissues, also in brain tissue in the subventricular zone and in the dentate gyrus (part of the hippocampus), they exist in an organized environment of supporting cells that define the architecture of the “stem cell niche” (Scadden 2006). A hallmark of adult stem cells is their ability to “self-renew” both in vivo and ex vivo and that they undergo asymmetric cell division.

An illustration of the niches of stem cells in parts of the human body. Brain has neural cells. Heart has cardiac muscle. Bone marrow has blood cells. A close-up of the bone marrow is provided that highlights the hematopoietic stem cell and stromal stem cell. — **Fig. 14.3**

This means that when they divide, they usually give rise to two different cells, one identical stem cell and the other a partly differentiated progenitor cell (Fig. 14.4). The common pattern in adult tissues is that the resulting progenitor cells are capable of expansion by symmetric division and can subsequently differentiate into the various cell types needed for repair or replenishment of the relevant tissue. Adult stem cells include chondrocytes, HSCs, MSCs, skin stem cells, and immune cells (see Table 14.5). Isolation of adult stem cells from organ-tissues is a challenge because only very small numbers of stem cells reside, and once removed from the body, these cells grow to senescence, a state in which cells stop dividing but do not die. Thus, obtaining large quantities of stem cells is difficult. Also, the separation of stem cells from other (unwanted) cell populations is far from trivial. For some products, master and working cell bank (MCB and WCB) strategies are applied. Still, genetic and phenotypic stability, i.e., certain markers present on the cell surface, must be closely monitored.

A flow diagram of the division of multipotent hematopoietic stem cells. The myeloid cell divides into G M C F C, E o C F C, B F U E, C F C mega, and T C F C, forming granulocytes, monocytes, eosinophils, erythrocytes, megakaryocytes, and T and B cells, respectively. — **Fig. 14.4**

Adult Stem Cells Used as Transplant Product

Adult stem cells have been used since the 1950s to treat cancers of blood cells as one of the components of bone marrow transplants (Santos 1983). This procedure involves whole-body irradiation to kill malignant cells in multiple myelomas and leukemia. The patient then receives a bone marrow transplant, not in itself a stem cell product, but the transplant contains a few HSCs which subsequently home to the bone marrow stem cell niches and begin to replenish the blood (Fig. 14.5). Rejection and graft-versus-host disease (GvHD) are still threatening complications of this form of therapy.

An illustration of the transplant of hematopoietic stem cells into a leukemia patient. In the patient, the stem cell transforms into multipotential stem cells, myeloid progenitor cells, and erythrocytes or red blood cells. — **Fig. 14.5**

Adult Stem Cells for Clinical Application: Immune Cells

Immune cell types currently investigated for their therapeutic value, mostly in the field of cancer, are DCs (see also Chap. 15), tumor-infiltrating lymphocytes (TILs), γδ T cells, regulatory T cells (Tregs), macrophages, and viral reconstitution T cells. Both autologous and allogeneic cells are used as cell sources. These immune cells have a highly specific mode of action and are in different stages of clinical development.

Adult Stem Cells for Clinical Application: MSCs

MSCs, sometimes called multipotent stromal cells or mesenchymal stem cells, have generated considerable interest in cell therapy applications (Bianco et al. 2008). However, the description of the cells, their source, and manufacturing processes are quite heterogeneous. MSCs can, e.g., be isolated from bone marrow, adipose tissue, corneal epithelial cells, and from a gelatinous substance within the umbilical cord (Wharton’s jelly) and umbilical cord blood. MSCs differentiate into various phenotypes, including chondrocytes, osteoblasts, and adipocytes. Due to their pleiotropic properties, e.g., growth factors and chemokines producing, anti-apoptotic, angiogenetic, anti-fibrotic, and neuroprotective, have been extensively tested in preclinical models. Hundreds of Phase I-III clinical trials have been performed and are ongoing globally in a wide variety of indications: cardiovascular diseases, GvHD, brain and neurological disorders, muscle, bone, and cartilage diseases, lung and bronchial diseases, wounds and tissue restorations, and immune system diseases (Galderisi et al. 2022), see Fig. 14.6.

A stacked bar chart has the following values. Recruiting status. (Cancers, 1), (Muscle disease, 45), (Covid 19, 31). Completed. (Eye disease, 10), (Muscle disease, 65), (Covid 19, 41). Active, not recruiting. (Digestive disease, 9), (Muscle disease, 71), (Covid 19, 51). Values are estimated. — **Fig. 14.6**

MSC can be administered locally, e.g., intralesionally, subcutaneously, or intravascularly. While local administration has been found effective in case of local injury, e.g., to treat bone and joint diseases, heart disease, for the repair of muscle and ligament damage, Crohn’s fistulas, and even for the repair of ischemic brain tissue, the systemic infusion is preferable in the case of systemic diseases such as GvHD (Kean et al. 2013). Therefore, both autologous and allogeneic cell sources have been studied. Examples of MSC products that have been approved globally are darvadstrocel (Alofisel®) in the EU for the treatment of Crohn’s fistulas, remestemcel-L (Prochymal®) in Canada and New Zealand, Holoclar and Alofisel in the United States and Temcell HS in Japan for the treatment of pediatric acute graft-versus-host disease (GvHD) (see also Table 14.6).

Table 14.6 Available gene and cell therapy medicinal products approved by the EMA and FDA (with the exception of blood cord-derived products; status December 20, 2022)

Full size table

Cell Immortalization Technologies

Another technology makes use of immortalized cell lines as starting material for the manufacture of cell-based products. An example of such a cell line is the neural stem cell line CTX0E03, derived from the human fetal cortical brain and genetically modified with a retroviral vector encoding the immortalizing gene, c-mycERTAM (Pollock et al. 2006; Stevanato et al. 2009). Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), this gene enables the large-scale production of the CTX cells using a two-tier cell banking system (MCB and WCB). Clinical testing of the CTX cell-based product is ongoing in a clinical phase II program for stroke. Although cell immortalization technologies have been in development for some time now, this is not a mainstream technology in the pharmaceutical world yet.

Cell Plasticity Technologies

The cell plasticity technology area takes advantage of discoveries that certain cells have the ability to evolve to cell types formerly considered outside their normal differentiation repertoire, i.e., hESCs and iPSCs. This technology has extensive clinical potential due to the high probability of an almost unlimited supply of cells (MCB and WCB approach) and also for the possibility to HLA-match the resulting cell-based product (partly) with the recipient patient. The application of pluripotent stem cells, such as ESCs and iPSCs, goes beyond the administration of cell-based medicinal products and is investigated as a source for tissue engineering and organogenesis (see section “Three-Dimensional Technologies”). In addition, autologous and allogeneic iPSCs are currently extensively used for disease modeling: i.e., patient-specific iPSC-derived cardiomyocytes, cultured in vitro, can be used to identify the genetic basis of cardiac disease, leading to the identification of pharmacogenetic biomarkers that support effective and personalized drug therapy and drug discovery including toxicity screening (Sayed et al. 2016).

Embryonic Stem Cells

During the earliest stages of mammalian development, soon after egg and sperm combine, the resulting diploid cells are said to be “totipotent,” i.e., they can give rise to both the embryo and placental tissue. At the blastocyst stage of embryogenesis (day 5 in humans), the “inner cell mass” or “embryoblast” is compacted and separated from the surrounding “trophoblast.” The latter combines with the maternal endometrium to form the placenta. The inner cell mass can be extracted and grown ex vivo as ESCs, which can give rise to all three germ cell types (mesoderm, endoderm, and ectoderm) and, therefore, potentially any cell type found in the adult (Fig. 14.7).

An illustration of the extraction of inner cell mass and grown ex vivo as E S C s. From the blastocyst, the cell mass is extracted as stem cells and cultured in an incubator. Stem cells are differentiated into pancreatic, hematopoietic cells, cardiomyocytes, neurons, and hepatocytes. — **Fig. 14.7**

Mouse ESCs were first isolated in 1981 (Evans and Kaufman 1981; Martin 1981), but it took until 1998 for a similar procedure to be described allowing human ESCs to be grown in culture (Thomson et al. 1998). ESCs can now be grown for many cell divisions, limited only by genetic damage that occurs by mutation after extensive culturing. The pluripotency of ESCs has been demonstrated in mice by injecting cells into a fertilized egg, resulting in the production of chimeric mice, i.e., mice made up of cells derived from both the donor and the injected ESCs, with this technology, transgenic mice for research purposes have been generated. HESCs are currently investigated by a set of cell surface markers (CD markers) and their capacity to differentiate. The criteria for this assessment include the expression of surface markers and transcription factors associated with an undifferentiated state. In addition, extended proliferative capacity, pluripotency, and a euploid karyotype are important characteristics of these cells. Recent advances in human pluripotent stem cell research revealed different subpopulations within stem cell cultures covering a wide spectrum of pluripotent states that hold distinct molecular and functional properties (Goodwin et al. 2020). Moreover, evidence suggests that the epigenetic status of the cells is also a relevant criterion for hESCs. Epigenetic alterations may accumulate when hESCs are cultured in vitro. Therefore, genetic stability over extended periods should be considered as a critical parameter, demonstrating that hESC characteristics do not change over time in terms of karyotype, expression of markers, expression of telomerase and their ability to differentiate into the three germlines (ecto-, meso- and endoderm) (Bar and Benvenisty 2019).

Maintenance and Differentiation of ESCs in Culture

HESCs are grown in the presence of high concentrations of basic fibroblast growth factor-2 (FGF2) and are unresponsive to leukemia inhibitory factor (LIF) (Levenstein et al. 2006). The technical challenge, now that hESCs can be maintained and expanded, is to develop robust methods to control and direct ESC differentiation, so that human cells of any desired phenotype can be obtained (Keller 2005; Murry and Keller 2008) with sufficient purity in terms of the absence of undesired cells, such as undifferentiated cells, or cells that are capable of de-differentiation into undifferentiated cells or into cells of a different lineage, either of which could cause tumor formation after implantation, both at the site of administration or elsewhere in the body after cell migration. This technology has not fully matured yet. Thus far, attention has focused on the differentiation of human ESCs toward products that could be of obvious use for clinical administration, e.g., midbrain dopaminergic neurons for Parkinson’s disease, cardiomyocytes for reinforcement of damaged heart tissue, and pancreatic pre-β-islet cells for implantation in Type I DM.

Since they were isolated for the first time, several first-in-human clinical studies with hESC have been initiated for various indications, including neural diseases (Parkinson’s disease, spinal injury), heart disease, cancer, and eye diseases (Table 14.7). In an open-label phase I/II study, hESC-derived retinal pigment epithelial cells (RPE) were given subretinally to patients with Stargardt’s disease or patients suffering from macular degeneration. Injected RPE cells showed no sign of hyperproliferation or tumor formation, albeit local immunosuppression was needed to prevent rejection. After 22 months, significant improvement in eyesight was reported for 19 out of 27 patients (Schwartz et al. 2015). Whereas researchers clearly have demonstrated their therapeutic potential, the use of hESC remains controversial. The opinions of scientists, regulators, and public are widely divided, from being very supportive to seeking a regulatory ban on hESC research for ethical/religious reasons. Besides these ethical barriers, some scientific barriers still need to be overcome. Progression towards clinical applications is hampered due to safety concerns, specifically immunogenicity and the unknown potential of undifferentiated escapees for teratomas.

Table 14.7 List of reported and registered hESC-based clinical trials. Adapted from Golchin et al. (2021)

Full size table

ESC Somatic Cell Nuclear Transfer (Therapeutic Cloning)

An alternative, particularly when an HLA-donor match cannot be found, is to produce ESCs for individual patients, by somatic cell nuclear transfer (SCNT) (Wilmut et al. 2002). This process, also known as “therapeutic cloning,” involves the implantation of a cell nucleus from the patient (i.e., genomic DNA extracted from a skin biopsy) into a human egg, which has undergone the removal of its own DNA. The environment in the enucleated egg can reprogram the DNA from the patient, removing epigenetic marks and restoring the DNA to an embryonic state. In addition, the development of an inner cell mass in the egg, after a period of incubation, allows extraction of ESCs that have the patient’s exact genotype. These cells could be used subsequently for the production of implants for cell therapy (Fig. 14.8).

An illustration of the process of somatic cell nuclear transfer. The nucleus from the patient cell inside an egg forms a blastocyst and the inner cell mass is extracted. The beta islet cells are injected into the pancreas. The stem cells divide into multipotential stem and then pancreatic cells. — **Fig. 14.8**

Somatic cell nuclear transfer (SCNT), moving nuclear DNA from a donor cell to an enucleated recipient cell to create an exact genetic match of the donor, is an inefficient process. Most eggs that have undergone SCNT cannot completely reprogram the donor DNA, and the surrogate pregnancy is usually unproductive. Moreover, even when the pregnancy comes to term, the cloned offspring are known to carry many epigenetic marks that may compromise normal development.

Given that defects are known to occur after SCNT, the subsequent derivation of cells for clinical uses might also be prone to failure due to defects in ESC differentiation. There is insufficient data available at this stage to judge whether this will be a limitation in practice. However, significant ethical concerns have limited the practice of SCNT. A human egg donor is required, and unless the process becomes more efficient, women who are prepared to donate eggs would need to provide several eggs to produce a single ESC line. There is concern that women could be exploited, particularly women from low economic backgrounds, and as a result, SCNT is not supported by government funding at present in most countries. A restricted number of ESC lines have been produced using spare eggs from in vitro fertilization programs, but SCNT remains a controversial topic and is subject to legal constraints that vary from country to country for mainly biomedical ethical reasons. An alternative source of cells for clinical application is umbilical cord blood stem cells, which are now being banked at childbirth (i.e., in a biobank), at least in private practice, and the first clinical trials have been initiated. Whether cord blood cells can be harnessed to produce all cell phenotypes is not clear at present (see also above “cord blood-derived MSCs”). However, many of the ethical issues surrounding SCNT and uncertainty of cord blood stem cell potency (in vivo activity), may become irrelevant if the promise of iPSCs can be fulfilled.

IPS Cell Technology

Initially, work on pluripotent stem cells (PSCs) was conducted using hESCs; however, the requirement to destroy early-stage embryos in the process of ESC derivation makes their use ethically controversial. In addition, practical considerations hinder their medical applications because any cells or tissues generated from hESCs, by definition, would be allotransplants into the recipient patient (see above).

However, since the discovery of Takahashi and Yamanaka in 2006 that differentiated somatic cells (in particular fibroblasts) can be reprogrammed to produce pluripotent cells by inducing the expression of four transcription factors (Sox2, Oct4, Klf4, and cMyc), the need for human embryo’s as a source of pluripotent stem cells has become obsolete (Takahashi and Yamanaka 2006; Takahashi et al. 2007). Over the last decades, induced pluripotent stem cells (iPSC) have exploded, and the technology is now in use in hundreds of stem cell biology laboratories around the world and has been tested for therapeutic applications in clinical trials (Figs. 14.9 and 14.10). The four genes initially identified can be partly substituted by alternatives, and several experiments have shown that integrated lentiviral constructs can be avoided to reduce safety concerns, by using nonviral plasmids (Jia et al. 2010), miRNA and mRNA (Yang et al. 2011; Liu and Verma 2015), protein transduction, and even by substituting some of the factors with small molecules (Yuan et al. 2011).

A cyclic chart of the production of i P S cells starts as follows. Collection of skin cells from humans with A M D, reprogram into E S like i P S cells, identify identical cells, correct mutation, differentiate into R P E, and injected into the body. — **Fig. 14.9**

A, B, and C are pie charts for clinical trials. D is a bar graph that plots the number of clinical trials versus countries. E is an illustration of the human body marking the frequency of clinical trials in different body organs. Visual Systems have 9. Blood and blood-forming organs have 6. — **Fig. 14.10**

Considerable effort has been directed at investigating how iPSCs differ from ESCs and whether reprogramming is complete enough to produce truly pluripotent cells. True pluripotency is difficult to demonstrate unequivocally in human iPSCs, so the development of methods to measure the extent of reprogramming will be important for practical applications. There are indications that iPSCs can have chromosomal defects and are not fully reprogrammed (Chin et al. 2010). Female human iPSCs appear to maintain the inactivated X chromosome that was present in the skin fibroblasts, although this has not been a problem with mouse iPSCs (Tchieu et al. 2010).

In recent years, progress has been made with improved culture techniques and differentiation protocols, which resulted in safer and clinically relevant iPS cells with lower tumorigenic risk. Various clinical trials with iPSC are being conducted worldwide with Japan and the US being the frontrunners (Fig. 14.10) (Kim et al. 2022). Similar to hESC, most clinical trials focus on retinal degenerative diseases, with Dr. Takahashi at the Riken Center for Developmental Biology in Japan being a pioneer. She led the first team to successfully transplant autologous iPSC-derived RPE cell sheets into a patient with age-related macular degeneration (AMD) in 2014 (Sayed et al. 2016) and is presently conducting studies on the transplantation of iPSC-derived corneal cells. Despite these individual successes, clinical development of iPSC therapies has been slow, possibly related to potential safety issues associated with these cells, including genomic instability and tumorigenicity (Nori et al. 2015). Further clinical research investigating the long-term safety of using iPSC in regenerative medicine is needed.

Transdifferentiation

Transdifferentiation is the process of converting cells from one lineage into another without going through a pluripotent cell stage, as is the case for reprogramming. Forced expression of genes has been used to convert fibroblasts directly into unrelated differentiated cells by skipping the iPSC stage, but also small chemical compounds can be used for this, although less efficient (Hybiak et al. 2020). Upregulation of apoptosis and cancer-related genes occurs in addition to chromatin remodeling during transdifferentiation. An important criterium of successful transdifferentiation is full morphological and molecular differentiation of both initial and final cells. Different cell types have been generated from fibroblasts, including neurons, hepatocytes, endothelial cells, and cardiomyocytes. The technique used is analogous to that used to derive iPSCs, except that genes associated with the desired somatic cell are expressed instead of pluripotency genes. The realization that cellular phenotypes can be transformed in this way has been met with astonishment and is certainly breakthrough technology. It opens the possibility of performing interconversion in vivo, although it does not allow for the expansion of cells in preparation for an implant. However, direct reprogramming of fibroblasts to neural stem cells, as reported in 2012 (Han et al. 2012; Thier et al. 2012), may be a shortcut to growing neurons. This approach may offer some advantages over the production of neurons by way of iPSCs.

Three-Dimensional Technologies

Another technology, tissue engineering, is combining somatic cell technologies or cell therapy technologies described above, with various types of biocompatible materials to solve structural challenges that are often surgical or immunological in nature. Three-dimensional (3D) technologies, including biomaterial scaffolds, can have many purposes, such as supporting cell viability, induction of cell differentiation, provision of a substrate for cell growth and support of tissue regeneration, provision of the shape, scale, and volume of a desired tissue, provision of growth factors, and encapsulation of cell-based products to protect the product from the host immune system to avoid rejection. This is schematically presented in Figs. 14.11 and 14.12 (Smith and Grande 2015). 3D technologies can be divided into four subtypes of technologies, as shown in Table 14.8. For further reading, see Pina et al. (2019), and Bajaj et al. (2014).

A Venn diagram consists of 3 overlapping circles, labeled scaffolds, cells, and bioactive molecules. The area where all 3 circles intersect is labeled tissue engineering. Within this overlapping region, each component of tissue engineering. Scaffolds act as drug delivery systems for improved repair. — **Fig. 14.11**

An illustration of the steps in matrix-assisted chondrocyte implantation. The chondrocytes are grown in vitro in a collagen membrane. The chondrocytes are implanted into the cartilage above the subchondrial bone. The cartilage is regenerated in vivo. — **Fig. 14.12**

Table 14.8 3D technologies and examples

Full size table

Combinations of the Above Technologies

A combination of the above technologies is currently in preclinical development in the cell therapy area, e.g., the self-formation of complex organ buds into organ-like structures, i.e., organoids (Takebe et al. 2015; Brassard and Lutolf 2019).

Part B: Technologies for Gene Therapy

Introduction

Gene therapy aims to treat or cure a disease by inserting, altering, or removing genetic material in affected cells in the human body. The concept of gene therapy arose during the 1960s and early 1970s and was a direct consequence of a series of discoveries made in the preceding years. It started with developing tools to select cells based on a functional trait that could be acquired via the transfer of exogenous DNA. For example, in the mid-1960s, it was demonstrated that cells from patients with Lesch-Nyhan syndrome, which lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HPRT), could be rescued upon uptake of exogenous DNA encoding HPRT, when grown in hypoxanthine-aminopterin-thymidine (HAT) medium which is selective for cells expression HPRT. Despite this proof-of-concept that exogenous DNA can be expressed in mammalian cells, the genetic transformation was inefficient. Methods to facilitate the delivery of exogenous DNA were developed, including the calcium phosphate chemical transfection method. Later, work on polyomaviruses, papovaviruses, and retroviruses provided the tools for more efficient transformation. With the parallel discovery of restriction enzymes in the early 1970s and pioneering work by Paul Berg, Stanley Cohen, and Herbert Boyer, recombinant DNA technology became a fact, enabling the manipulation of DNA, including viral genomes, to insert therapeutic genes that could be efficiently transduced (Cf. Chap. 1). Retroviral vectors were further optimized and became the workhorse for the transfer of therapeutic genes to correct disease phenotypes in vitro. By the mid-1980s, it was demonstrated that T cells from SCID patients lacking the enzyme adenosine deaminase (ADA) could be restored after retroviral vector-mediated delivery of genes encoding ADA. These findings formed the basis of the first human gene therapy clinical trial for ADA-SCID on September 14, 1990, at the National Institute of Health (NIH). The first patient included was Ashanti DeSilva, at the age of four. The medical team isolated the patient’s T lymphocytes through apheresis, exposed these cells ex vivo to a genetically engineered live nonvirulent retrovirus carrying the normal ADA gene, and transfused these genetically modified T cells back into the patient’s bloodstream. The treatment was successful in the sense that no adverse events were observed, and the low number of T cells that were transduced continued to express the recombinant transgene for over 12 years (Blaese et al. 1995; Muul et al. 2003), but treated patients still relied on enzyme replacement therapy. Now, 30 years later, over 3000 clinical trials with gene therapy were initiated, resulting in 18 gene therapies approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA).

In this section, we discuss the current state of gene therapy and the gene therapy medicinal products on the market, focusing on in vivo gene transfer. We will discuss how gene therapy can be applied and the various methods of gene transfer, including synthetic and viral vectors. Finally, we highlight the diseases currently subjected to gene therapy and touch upon the regulation of gene therapy products.

Gene Therapy: Definitions and Ways of Application

The definition of gene therapy continues to evolve to keep up with the ongoing technological advances. As a result, older definitions have become obsolete, and others are too broadly defined. Take, for example, the definition as applied by the European Medicines Agency: “gene therapy medicines contain genes that lead to a therapeutic, prophylactic or diagnostic effect. They work by inserting 'recombinant' genes into the body, usually to treat a variety of diseases, including genetic disorders, cancer or long-term diseases.” Strictly speaking, any modification applied to the genome that does not involve the insertion of recombinant DNA is not considered gene therapy. With the rise of CRISPR-Cas (clustered regularly interspaced short palindromic repeats) gene editing, base editing, and prime editing (see section “Designer Nucleases for Gene Editing”) that can introduce modifications in the human genome without the use of recombinant DNA, this definition falls short. Conversely, the FDA uses a much broader definition: “Human gene therapy seeks to modify or manipulate the expression of a gene or to alter the biological properties of living cells for therapeutic use.” FDA generally considers human gene therapy products to include all products that mediate their effects by transcription or translation of transferred genetic material, or by specifically altering host (human) genetic. According to this definition, any intervention that leads to altered gene expression would be considered a gene therapy, including those that do not directly change the genetic makeup of a cell such as gene silencing using RNA interference or the delivery of therapeutic mRNA. To bring some clarity and to draw a clear line as to where gene therapy ends and other nucleic acid therapies start (cf. Chap. 13), we propose the following definition: gene therapy is any intervention that leads to deliberate and long-lasting genomic alteration(s) or episomal expression of recombinant DNA with the aim to treat or cure a disease. This definition excludes interventions that affect gene expression at the level of transcription or translation, such as siRNA therapy, mRNA therapy, and antisense ON therapy, which are often transient and require frequently repeated dosing (see Fig. 14.13).

A flow diagram of the process in the intervention of therapeutic nucleic acids. The steps include transcription, R N A splicing, translation of D N A, pre-m R N A, m R N A, and protein, respectively. — **Fig. 14.13**

Gene therapy can be applied in three different ways: gene augmentation or addition, gene correction, and gene knockout.

Gene Augmentation

With gene augmentation or addition, an intact copy of a malfunctioning, disease-causing gene is inserted into the genome of patient cells (Fig. 14.14). This approach can only be used if the disease is caused by a “loss-of-function” gene mutation. These are mutations in a gene that causes the encoded protein to (partially) lose its function, which in turn causes disease. Loss-of-function mutations are typically recessive, meaning that both copies of the autosomal gene have defects. The addition of an intact copy of the gene under the control of a strong promoter thereby restores this function. Insertion of the therapeutic transgene into the genome can be random or targeted to a specific region within the genome. Both viral and synthetic vectors can be used to deliver such gene constructs into affected cells (see section “Delivery Systems”).

An illustration of a cell with a cell membrane, nucleus, and a new gene inside the nucleus. Cloud-shaped structures outside the nucleus are labeled protein creation is restored. — **Fig. 14.14**

Gene Knock Out

A gene mutation might sometimes lead to a mutant protein that acquires a new, disease-causing function. Such mutations are referred to as “gain-of-function” mutations and are often dominant. Take, for example, the hereditary form of transthyretin amyloidosis (ATTR), which occurs when mutations in the TTR gene for transthyretin lead to instability of the tetrameric protein and formation of aggregates and fibrils that damage cells, leading to clinical symptoms. Such a gain-of-function mutation can only be treated if the mutated protein production is halted. This can be done by gene knock out in which a targeted disruption of the mutated gene (but not the unaffected allele) is introduced. CRISPR-Cas is a technology often used for creating a targeted gene knock out (see Sect. “CRISPR-Cas9” below).

Gene Correction

Gene correction aims to alter the disease-causing gene mutation. These corrections can be small (point mutations) or require the insertion of large pieces of DNA in case the mutation involves partial deletion of a gene. Gene editing systems such as Zinc-finger nucleases, TALENs (Transcription activator-like effector nucleases), CRISPR-Cas, base editors, or prime editors (see section “Designer Nucleases for Gene Editing” and Cf. Chap. 9) have been developed for this purpose. As such corrections are often very precise and include the use of molecular scissors to cut open the genomic DNA at precise locations, gene correction is sometimes referred to as gene surgery.

Gene editing tools can be categorized based on those that introduce double-strand cuts, those that only cut one strand of the DNA or those that do not cut at all.

Somatic Versus Germline

Gene therapy can be applied to modify individual cells in the body. This is called somatic gene therapy and only affects the patient being treated. The corrected traits will not be inherited by potential offspring. Conversely, germline gene therapy aims to modify the germ cells (sperm cells and/or egg cells) to correct genetic mutations in the germline. If such modified germ cells are being used for reproduction, this will lead to offspring in which the gene correction is carried by all cells of the body, including the germline cells. Such corrections will therefore be passed down from generation to generation. Since gene therapy is a relatively new form of therapy, we do not yet know the long-term side effects of this, and as such, germline modifications would be unethical to perform at this stage. Many scientists, therefore, call for a global moratorium on germline gene editing (Lander et al. 2019).

Ex Vivo Versus In Vivo

Ex vivo gene therapy involves the genetic modification of cells outside of the body and their subsequent transplantation back into patients (Fig. 14.15). The advantage of this approach is that there is no patient exposure to the gene transfer vector, which can sometimes be harmful. Furthermore, the correctly modified target cells can be selected, expanded, and, if desired, differentiated before being transferred back to the patient to improve efficacy and safety. The limitation of such an ex vivo approach is that it can only be applied to cells easily isolated from the human body (e.g., hematopoietic stem cells).

An illustration of the processes of direct and cell-based deliveries. Direct delivery includes the therapeutic gene directly injected into the patient. Cell-based involves the in vitro differentiation of stem cells and their injection into the patient. — **Fig. 14.15**

In vivo, gene therapy uses gene transfer vectors locally (e.g., into the eye or muscle) or systemically (via the bloodstream) to reach distant organs or tissues in the patient. Both viral and synthetic vectors have been developed for this purpose, which will be detailed in the section below. The advantage of this approach is that it avoids cumbersome cell isolations and manipulations in the laboratory and can, in principle, be applied by a single injection. The downside is the relatively low gene transfer efficiencies and potential vector-related (immune)toxicities to which the patients are exposed.

Designer Nucleases for Gene Editing

Designer nucleases are engineered nucleases that can be targeted to unique sequences in the human genome to introduce a double-strand DNA cut (Merkert and Martin 2016). As a result of this genomic DNA damage, the endogenous DNA repair system is activated to repair these cuts and ligate the ends of the fragmented DNA. Several different DNA repair pathways can be employed by the cell, which include nonhomologous end-joining (NHEJ), microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR)(Fig. 14.16).

A 3-part illustration of the D N A double-strand break repair mechanisms. The mechanisms include nonhomologous end joining, microhomology-mediated end joining, and homology-directed repair. — **Fig. 14.16**

With NHEJ, the ends of the damaged DNA recruit a protein complex that polishes the DNA: it removes damaged or mismatched nucleotides and randomly fills in nucleotides before the ends are ligated back to each other. As a consequence, NHEJ often leads to small insertions or deletions (indels) at the cut site. If this cut is inside a gene, such indels often lead to a frameshift in the coding sequence, and loss of protein function. It can therefore be used to knock out genes with gain-of-function mutations. MMEJ uses microhomology regions that may be present after end resection of the DNA ends, leading to the base pairing of the single-strand DNA overlaps and removal of the remaining pieces of ssDNA. This leads to deletions at the cut site. Both NHEJ and MMEJ are active throughout the cell cycle and thus active in both dividing and nondividing cells. However, these pathways do not allow precise editing as they tend to lead to errors.

For precise editing, the homology-directed repair is preferred. HDR is mostly active in cells in the G2 and S phases and is meant to repair broken DNA strands prior to mitosis, making use of the homologous DNA from a sister chromatid to guide the repair. In a similar fashion, DNA double-strand break repair can be guided by providing an exogenous DNA template during repair. This HDR template can be single or double-stranded DNA with on the 5’ and 3’ ends sequences that are homologous to the sequences surrounding the double-strand cut. The length of such homology arms can vary from only 30 bases up to a few hundred or even thousand bases, depending on the overall size of the DNA to be inserted at the double-strand cut size. The exact working mechanism of HDR is still under debate, but it is generally believed that it involves 5′ to 3′ resection of the DNA ends at the double-strand cut site, after which the 3′ single-strand DNA overlap can invade the homologous DNA duplex of the HDR template to initiate the recombination repair process (Fig. 14.16). Compared to NHEJ and MMEJ, HDR is much less error-prone and often enables precise gene editing. Using single-stranded synthetic oligonucleotides of only 60 bases, specific point mutations can be introduced at a precise location within the genome (Shy et al. 2022). This technology offers great potential to correct debilitating diseases caused by a single point mutation in a single gene, such as sickle cell anemia, β-thalassemia, transthyretin amyloidosis (ATTR), or certain genetic eye diseases.

The first designer nucleases to be engineered were the zinc-finger nucleases (ZFNs), followed by transcription activator-like effector nucleases (TALEN), and more recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and its CRISPR associated Cas protein (CRISPR/Cas) (Fig. 14.17) (LaFountaine et al. 2015).

A 3-part illustration of the structures of nucleases. Zinc-finger nucleases. T A L effector nucleases. C R I S P R R N A-guided nucleases. The differences for each are listed in 3 columns. — **Fig. 14.17**

They consist of an engineered restriction endonuclease, often FokI, fused to 3-6 zinc-finger proteins, each recognizing a specific three-base pair sequence. FokI from the bacterium Flavobacterium okeanokoites, only cleaves DNA when it is bound to its recognition sequence GGATG and when it can form dimers. By directing FokI monomers to specific sequences on both strands of the DNA, FokI monomers can be positioned to form dimers and introduce a double-strand cut in the genomic DNA (Fig. 14.17). Although initially popular, the need for generating complex fusion proteins, which is often time-consuming, and the CRISPR-Cas technology's emergence has made this gene editing technique less popular.

TALEN

Similar to ZFNs, the transcription activator-like effector nucleases (TALEN) exploit customizable DNA-binding proteins fused to a FokI nuclease. The TALE DNA-binding domains were identified as secreted proteins from the Xanthomonas spp. bacteria and consist of highly conversed 33–35 amino acid repeats with two amino acid repeat-variable diresidues (RVD), which dictates individual nucleotide specificity. Assembling repeats into TALE arrays flanked by essential TALE-derived N and C-terminal domains fused to FokI repurposes the system for genome editing. As a result, TALENs are very precise with low off-target editing and are still popular in gene therapy applications. For example, several companies have used this technology for engineering chimeric antigen receptor (CAR) T cells for cancer immunotherapy.

CRISPR-Cas9

CRISPR, an acronym for Clustered Regularly Interspaced Short Palindromic Repeats, and its associated proteins (Cas) are part of the bacterial adaptive immune system to shield invading viruses and which has been repurposed for gene editing in human cells. The Cas9 gene editing system consists of a Cas9 endonuclease interacting with a trans-activating RNA (tracrRNA), which is directed to a target sequence by ~ 20 nt complementary sequences in the CRISPR RNA (crRNA) and flanked by the 3′ protospacer adjacent motif (PAM). The crRNA and tracrRNA can be joined by a tetraloop to form a single guide RNA (sgRNA) (Fig. 14.18). When a sgRNA:Cas9 ribonucleoprotein binds its target sequence in the presence of a flanking PAM sequence, the Cas9 protein will introduce a double-strand break (DSB). If a PAM sequence is lacking, the Cas9 will not cut. Cas9 proteins from different bacteria recognize different PAM sequences. The most frequently used are Cas9 from Streptococcus pyogenes (spCas9; PAM 5′-NGG-3′, where N is any nucleobase) and Cas9 from Staphylococcus aureus (saCas9; PAM 5’-NNGRRT-3’, where R is a purine), but many different natural and engineered variants of these Cas nucleases exist. For example, Cas12a, previously known as Cpf1 from Acidoaminococcus sp., does not require a tracrRNA but only a crRNA. After binding its target sequence and identifying its PAM (5’-YTN-3’, where Y is a pyrimidine) Cas12a introduces a sticky-end cut, with 4-5 nucleotides overhang. The advantage of CRISPR-Cas for gene editing over the use of ZFN or TALEN is the ease by which the system can be adapted to target a specific sequence in the genome. Whereas for TALEN and ZFN, it is required to engineer a new targeted nuclease for each target sequence, which can take weeks to months as it requires elaborate recombinant protein expression and purification, the target specificity of Cas9 and Cas12a is completely determined by the associated guide RNA, which can be easily synthesized. A downside of CRISPR-Cas gene editing system is its relatively high base mismatch tolerance, meaning that it can introduce DSB at places in the genome with near-identical 20-nt sequences compared to the intended target sequence (as long as a PAM sequence is present). It has been reported that off-target events can occur with as many as 3-5 base pair mismatches. This could introduce unintended mutations in the genome at potentially harmful places. For that reason, Cas9 proteins have been engineered to increase specificity. For example, spCas9-HF1 has very low levels of off-target effects, albeit at the cost of lower on-target cutting efficiency than wild-type spCas9 (Kleinstiver et al. 2016).

A 2-part illustration of the 2-part guide R N A and single guide R N A. — **Fig. 14.18**

Despite these improved Cas9 nucleases, introducing DSB still imposes a risk during therapeutic gene editing. Besides introducing unintended mutations at off-target sites, generating DSB in genomic DNA can also lead to large deletions and chromosomal translocations. As a result, Cas mutants have been generated that can still be targeted to specific sequences in the genome but are devoid of nuclease activity. These Cas mutants form the basis of an entirely new class of gene editing tools without DSB. The most frequently used will be discussed below.

Base Editors

DNA Base editors allow the introduction of point mutations in the DNA without generating DSB. They consist of two domains: a catalytically “dead” Cas9 enzyme (dCas9) or a nickase (nCas9), fused to a single-stranded DNA modifying enzyme for targeted nucleotide alteration (Fig. 14.19). Two classes of DNA base editors have been described: cytosine base editors (CBE) and adenine base editors (ABE). Cytosine deamination generates uracil, which base pairs as thymidine in DNA and thus converts C:G into A:T base pairs. Conversely, ABEs convert A:T into G:C base pairs. Collectively, all four transition mutations (C→T, T→C, A→G, and G→A) can be installed. Besides these two major base editors, new variants are being developed, including those that introduce a base transversion (C→G), expanding the scope of disease-causing point mutations that can be edited (Kurt et al. 2021). The distance at which the base editors operate within the protospacer (i.e., the target sequence to which the base editor is bound)

is called the editing window and is dependent on the type of BE being used. For CBE, the editing window spans positions 4-8 of the protospacer, and for ABE positions 4-7 (ABE7.10) or 8-10 (ABE6.3, ABE7.8, or ABE7.9).

Conversion of multiple nucleobases within the editing window is possible and may lead to undesired edits, called bystander edits. Adapted from Antoniou et al. (2021).

Prime Editors

While BEs can, in principle, correct the majority of pathogenic point mutations, they cannot perform all possible single-nucleotide conversions and also cannot mediate targeted insertions or deletions. The solution for this is prime editors (PEs) developed by Anzalone et al. (2019). A PE consists of a reverse transcriptase fused to a nCas9 nickase (Fig. 14.20). In combination with an engineered prime editing guide (pegRNA), this construct is directed to a target sequence in the genome, which nicks the noncoding DNA strand. Hybridization of the 3’ end of this DNA strand with the pegRNA initiates reverse transcription using the pegRNA extension as a template. This leads to a branched DNA intermediate with either a 3’ flap (containing the edited sequence) or 5’ flap (containing the original, unedited sequence) sticking out. Exonucleases remove these flaps, yielding in 50% of the cases a heteroduplex DNA composed of one edited and one unedited strand. The intrinsic mismatch DNA repair pathway can then repair this mismatch with two possible outcomes: the edited strand is copied to the complementary strand, or the unedited strand is restored.

An illustration of the five steps of prime editing. 1. Nicking of P A M strand. 2. Hybridization of prime-binding side to P A M strand. 3. Reverse transcription. 4. Hybridization of D N A strands and flap cleavage. 5. Ligation and mismatch repair. — **Fig. 14.20**

PE is a very flexible gene editing tool that can introduce base transitions, transversions, and deletions (up to 80 bp) or insertions (up to 60 bp). However, it is less suitable for inserting larger pieces of DNA. The downside of PEs is their low efficiency of editing, which might contribute to the edit's various outcomes and instability of the rather large pegRNA. Further research is needed to make this method of gene editing more efficient.

Delivery

For gene therapy to be effective, the transgene construct or gene editing tools must be delivered to diseased cells in the body. For inherited diseases, this would, in principle, be all somatic cells in the body, but this will technically not be feasible. Hence, delivery strategies focus on targeting cells in vivo or ex vivo that are mostly affected by the genetic disease. In the section below, we discuss the various gene delivery vectors that have been developed for gene therapy applications. This includes viral vectors, synthetic vectors, and vectors derived from extracellular vesicles.

Viral Vectors

Viruses have evolved to introduce their genetic material into the host cell efficiently. These properties can create a vehicle to deliver genes for expression into cells. This is called a viral vector. A viral vector differs from the native counterpart in that it cannot replicate and is less pathogenic: genes involved in viral replication and pathogenicity are removed to generate a safe vehicle with enough space to include transgene cassettes. The most commonly used viral vector systems for gene therapy purposes are adeno-associated viral (AAV) and retroviral vectors.

To produce functional viral vector particles, the genes required for the structural viral proteins need to be provided in trans with the transfer vector, i.e., a piece of DNA, often plasmid DNA containing the therapeutic gene construct. The genes encoding the viral proteins for replication and packaging of the transfer vector can be placed on plasmids and transfected into producer cells (Fig. 14.21). The transfer vector retains minimal sequences required for stability, replication, or integration. These elements include the terminal sequences called inverted terminal repeats (ITRs) for AAV vectors and long-terminal repeats (LTRs) for gammaretroviral vectors. Additional elements, such as cis-acting elements, e.g., a packaging signal (ψ), may be essential for the efficient incorporation of the transfer vector into the viral particle. Retroviral vectors, besides the packaging signal, may also require truncated leader regions for efficient packaging. Other virus-derived sequences may be included to enhance delivery to the nucleus, such as the central polypurine tract (cPPT) in lentiviral vectors (a subtype of retroviral vectors) (Zennou et al. 2000), or elements to enhance expression of the transgene, such as the commonly incorporated Woodchuck hepatitis posttranslational regulatory element (WPRE). This will be discussed in more detail below. Retroviruses, including gammaretroviral vectors, lentiviral vectors, more recently alpharetroviral vectors, and AAV, are the most extensively studied for ex vivo and in vivo use in monogenic diseases. Their characteristics are summarized in Table 14.9.

A 2-part schematic representation of a virus vector system for gene transfer. In A, terminal repeats are followed by viral vector construct and packaging construct. In B, from a packaging cell, recombinant viral vectors are formed. — **Fig. 14.21**

Table 14.9 Characteristics of viral vectors for gene transfer^a

Full size table

Retrovirus

Biology

Retroviruses are membrane-enveloped RNA viruses containing two copies of a positive single-stranded RNA genome (Fig. 14.22). The retroviral vector systems that are commonly used in clinical applications for monogenic diseases are derived from gammaretroviruses and lentiviruses. More recently, alpharetroviral vectors have also been developed. Retroviruses are ~80–145 nm in diameter and have a genome size of about 7–10 kb, composed of a group-specific antigen gene (gag), which codes for core and structural proteins of the virus; polymerase (pol) gene, which codes for reverse transcriptase, protease, and integrase; and envelope (env) gene encoding the retroviral envelope glycoproteins. The long-terminal repeats (LTRs) control the expression of viral genes, hence act as enhancer-promoter. The packaging signal (ψ) helps efficient incorporation of the viral positive-strand RNA into the virus particle before budding off the cell membrane (Verma 1990).

A is a cross-sectional illustration of the circular retrovirus with pol, e n v, and gag parts. B illustrates the retrovirus replication cycle. 1. Binding. 2. Endocytosis. 3. Reverse transcription. 4. Integration. 5. Transcription. 6. Translation. 7. Assembly. 8. Budding. 9. Maturation. — **Fig. 14.22**

After viral binding and introducing the viral RNA into the host cell, reverse transcriptase, which has both polymerase and RNase activity, converts the viral RNA to linear double-stranded DNA that integrates into the host genome with the help of the viral integrase. The integrated virus sequence, the provirus, will later undergo transcription and translation to produce viral genomic RNA encoding viral proteins. Virus particles then assemble in the cytoplasm and bud from the host cell to infect other cells.

Suitability of Retroviruses as Vectors for Gene Transfer

To generate replication-deficient retroviral vectors, the sequences encoding the virion proteins (gag, pol, and env) responsible for the viral replication and pathogenicity are removed if redundant or placed in a split packaging system to produce the vector particles. The space that is created by deleting viral genes can be used to insert a transgene cassette. The transgene can be controlled by the native LTRs, which have intrinsic enhancer/promoter activity, or by including exogenous enhancer-promoter sequences. For the production of retroviral particles, the vector containing plasmid is introduced into packaging cell lines, mostly HEK293 cells, to produce the retroviral vectors.

Retroviral vectors have several features for gene transfer applications that are important to consider (Table 14.9). They can accommodate transgene cassettes of 8 kb and integrate them into the host genome. Therefore, retroviral vectors can provide stable, long-term transgene expression in dividing cells with low immunogenic potential, particularly because these vectors are mostly used for ex vivo applications. However, there are several disadvantages to these vectors. Gammaretroviruses and alpharetroviruses cannot transduce nondividing cells, but lentiviral vectors can overcome this hurdle, by transporting the preintegration complex (PIC) through the nuclear pores by an active, energy-dependent process (Bukrinsky et al. 1992), as has been shown for transduction of neurons, hepatocytes and hematopoietic stem cells (HSCs). Current methods of viral vector production generate preparations in which the virus titer is sufficient (1 × 10⁵–1 × 10⁷ active viral vector particles/mL) but can generally only be used for a limited number of patients. Retroviruses are also inactivated by elements of the complement system and rapidly removed from the systemic circulation in response to cellular proteins incorporated in the viral envelope during the budding process. Therefore, there are limited clinical trials with retroviral vectors for direct in vivo gene therapy.

A limitation of retrovirus-based gene therapy is that gammaretrovirus tethers to the transcription start sites and promoters, inserting the genetic cargo semi-randomly into the host genome (Deichmann et al. 2007, 2011; Schwarzwaelder et al. 2007). This can lead to genotoxic events through multiple mechanisms. The vector integration could cause insertional mutagenesis, disrupt and alter the gene expression of a gene close to the integration site. In addition, gammaretroviral vectors have a bias to integrate into proto-oncogenes (Cattoglio et al. 2007), which could cause insertional oncogenesis. Indeed, this serious adverse event was observed in clinical trials, which pushed the field to create safer retroviral vector designs. For instance, modifications have been made to delete part of the U3 region in the 3’ LTR to create a self-inactivating (SIN) configuration. After reverse transcription, the 3’ U3 region is copied to the 5’ LTR creating two LTRs lacking enhancer/promoter activity. This modification enables the use of internal expression cassettes with physiological or cell type specific promoters reducing the risk of genotoxicity (insertional mutagenesis) and phenotoxicity (ectopic transgene expression) by restricting the expression level to certain cell types or tissues.

Gammaretrovirus

Biology

Gammaretroviruses, such as Moloney murine leukemia virus (MoMLV), were the first retroviral vectors used for clinical application. Gammaretroviruses were originally called oncoretroviruses, which caused tumors in mice. Cell lines derived from HEK293T are commonly used for the production of gammaretroviral vectors. These often generate producer cell lines using ecotropic or amphotropic MLV envelope protein for pseudotyping gammaretroviral vector particles. Pseudotyping means that different envelope glycoproteins are used instead of the wild-type glycoprotein. Amphotropic MLVs are subgroups that infect a broader range of cell types. The structural proteins and transfer vectors are commonly split over three expression plasmids. Vesicular stomatitis virus G (VSVg) glycoprotein can also be used to pseudotype gammaretroviral vectors, stabilizing particles and broadening their tropism. A drawback of using gammaretroviral vectors is that cell division is required for efficient transduction.

Clinical Use of Gammaretrovirus

Retroviral vectors are currently employed mostly for ex vivo gene therapy, such as hematopoietic stem cell (HSC) gene therapy or cellular immunotherapy applications. The gammaretrovirus MoMLV was the first viral vector used in the clinic for treating severe combined immunodeficiency ADA-SCID, a rare inherited disease in which the buildup of toxic deoxyadenosine due to lack of activity of the enzyme adenosine deaminase results in complete lack of T and B lymphocytes (Ferrua and Aiuti 2017). In addition, MoMLV-vector expressing recombinant ADA was used for ex vivo genetic modification of autologous peripheral blood lymphocytes (Ferrua and Aiuti 2017).

Other successful clinical trials employing gammaretroviral vectors were performed to treat a rare X-linked SCID (X-SCID) (Kohn and Kohn 2021). MoMLV-vectors expressing IL2RG cDNA (also known as common gamma chain) were used to transduce autologous HSCs isolated from X-SCID patients without an HLA-identical sibling donor for ex vivo transduction. Subsequently, the genetically modified HSCs were transfused back to patients without preceding cytoreductive chemotherapy to reconstitute lymphocyte and natural killer cell lineages. These initial gene therapy trials enrolled 20 patients (Kohn and Kohn 2021) and showed restoration of immune function in 18 out of 20 patients, but some patients still required immunoglobulin infusions. However, 6 out of 20 patients developed T-cell leukemias due to insertional oncogenesis (Fischer and Hacein-Bey-Abina 2020; Kohn and Kohn 2021). This initiated the construction of self-inactivating gammaretroviral vectors (Kohn and Kohn 2021), which include weaker physiological promoters, such as elongation factor 1 alpha short promoter lacking enhancer sequences. In another trial for Wiskott-Aldrich syndrome, nine out of ten patients developed T-cell leukemia using a similar vector design with transgene transcription driven by the viral LTR (Ferrua and Aiuti 2017). On the other hand, a similar gammaretroviral vector backbone has been used in ADA-SCID, with a much lower genotoxicity profile (Tucci et al. 2022). This emphasizes that vector design and possibly disease background are important factors that determine the outcome in clinical trials.

In addition to safety-enhanced features that have been incorporated into gammaretroviral vectors, other virus-derived vector systems with an enhanced safety profile, such as lentiviral vectors and more recently developed alpharetroviral vector systems, are being exploited.

Lentivirus

Biology

Lentiviruses are retroviruses that can replicate in both dividing and nondividing cells. The biology of lentiviruses resembles that of gammaretroviruses. Apart from the genes gag, pol, and env, lentivirus has six accessory genes, such as tat, rev, vpr, vpu, nef, and vif, which regulate the synthesis and processing of viral RNA and other replicative functions.

Human immunodeficiency virus (HIV) is the most commonly studied lentivirus and has served as the backbone for a viral vector system commonly used for ex vivo applications. Wild-type HIV infects human helper T cells and macrophages. Apart from the genes gag, pol, and env, the accessory genes that are required for HIV replication and pathogenesis in vivo are not required for viral particle production and can all be removed to create sufficient space to incorporate transgene cassettes. HEK293T cells are most frequently used as packaging cells for lentiviral vector production.

Lentiviruses as Vectors for Gene Transfer Vehicles

One of the advantages of using lentiviral vectors is that they can efficiently transduce nondividing cells or terminally differentiated cells such as neurons, macrophages, muscle, and liver cells for in vivo gene therapy, as well as HSCs and immune cells, such as T cells, NK (natural killer) cells and macrophages for ex vivo gene therapy. These cell types are more effectively transduced with lentiviral vectors than using gammaretrovirus-based gene therapy systems. Previous studies have shown that when injected into the rodent brain, liver, muscle, or pancreatic islet cells, lentivirus promoted a sustained gene expression for over 6 months (Miyoshi et al. 1997). Lentiviruses have a typical integration site pattern, which is different from gammaretroviral vectors. The lentiviral LTRs and proteins that bind the integration complex largely determine the integration pattern by tethering the preintegration complex into highly expressed genes (Biffi et al. 2011). These properties reduce the genotoxicity potential of lentiviral vectors compared to gammaretroviral vectors that integrate into transcription start sites (TSS) and have a preference for proto-oncogene integration (Cattoglio et al. 2007; Moiani et al. 2013). The creation of self-inactivating lentiviral vectors has even further reduced the potential of genotoxicity because physiological promoters can drive transgene expression instead of the viral enhancer/promoter (Tucci et al. 2022). The development of the third-generation lentiviral vectors by dividing the plasmid system over four plasmids separating the transfer sequences from gag/pol, the envelope glycoprotein, and the rev gene has reduced the risk of generating replication-competent lentivirus (RCL) significantly. The third-generation self-inactivating lentiviral vectors system is the most commonly used as opposed to the earlier generations. As an exogenous envelope glycoprotein, VSVg is also commonly used for pseudotyping to improve tropism and stability. In addition, rev is provided in trans to increase lentiviral vector titers by binding to the rev response element sequence in the transfer vector and accumulating unspliced viral RNA in the cytoplasm (Fritz and Green 1996). The introduction of point mutations into the HIV integrase or the LTRs creates lentiviral vectors that are integration defective. This application can provide long-term expression in nondividing tissues, such as skeletal muscle, eye, and liver (Gurumoorthy et al. 2022). The use of integration defective lentiviral vectors obviously reduces the risk for genotoxicity.

Clinical Use of Lentiviral Vectors

HIV-derived lentiviral vectors have been used in clinical trials for multiple indications, including inherited diseases. For example, to treat HIV infection, patient CD4 T cells were genetically modified ex vivo by the lentiviral vector VRX496 (MacGregor 2001). The VRX496 vector contained an antisense sequence targeted to the HIV env gene to interfere with HIV replication. Although no treatment-related severe adverse events were observed, no statistically significant anti-HIV effects were observed either (Manilla et al. 2005) (cf. the “Cell Therapy” part of this chapter). Lentiviral vectors have been mostly used in clinical trials for primary immune deficiencies (PIDs), such as X-SCID and Wiskott-Aldrich syndrome, and X-linked chronic granulomatous disease (CGD) (Tucci et al. 2022), other blood disorders (sickle cell disease/thalassemia) (Staal et al. 2019) and (neuro)metabolic disorders, such as metachromatic leukodystrophy (MLD), Hurler syndrome or Fabry disease (Chiesa and Bernardo 2022) using genetically modified HSCs. In PIDs and blood disorders, there is an inherent defect in the blood lineages and function development. In (neuro)metabolic disorders, the hematopoietic system may produce recombinant protein for cross-correction and deliver protein in the central nervous system (see also chapter below on monogenic diseases).

Alpharetroviral Vectors

Biology

Alpharetroviral vectors have been more recently developed in addition to gammaretroviral and lentiviral vectors. This system is based on the Rous sarcoma virus (RSV), which was originally isolated from chicken sarcoma cells. RSV contains the viral tyrosine kinase v-Src gene, which triggers uncontrolled growth in infected cells (Rubin 2011). The v-Src gene increases virulence of RSV. The RSV genome was used to design a split packaging system separating gag/pro and pol sequences from the transfer vector, with a transfer vector containing self-inactivating configuration and replacing the RSV envelope glycoprotein (Suerth et al. 2014). In this design, the packaging plasmids do not share any homology with the transfer vector, as opposed to the lentiviral vector systems, which do contain sequence overlap between transfer and packaging plasmids. In addition, the alpharetroviral vector leader region is gag-sequence free. The integration pattern of alpharetroviral vectors has a more random profile as opposed to gammaretroviral vectors and lentiviral vectors, and aberrant splicing has not been observed (Suerth et al. 2014), which may contribute to safety.

Retroviral Mechanisms of Genotoxicity

Retroviral vectors integrate into the genome and can therefore provide stable transgene expression. However, this could also lead to dysregulation of genes close to the integration site. Three main mechanisms contribute to gene dysregulation and potential genotoxicity. The most common is that the enhancer/promoter elements of the integrated vector could upregulate the expression of genes, potentially proto-oncogenes (Williams et al. 2022). Another mechanism of genotoxicity is related to splice donor and acceptor sites interfering with endogenous gene splicing, generating fusion or truncated transcripts. Finally, genotoxicity may be caused by read-through transcripts from retroviral vectors, and this may induce leukemia. Since the retroviral vector systems most commonly exploit the transcriptional termination signals in the 3’ LTR, which are generally poor terminators, transcript read-through may occur. Transcript read-though may be reduced by including elements that improve transcriptional termination (Suerth et al. 2014; David and Doherty 2017).

Genotoxicity risks also depend on cell source selection. For example, lentiviral vector integration sites in postmitotic tissues and dividing cells are different (Bartholomae et al. 2011), and such differences depend on gene expression profiles in the target cells (Biasco et al. 2011).

Adenovirus

Biology

Adenoviruses are nonenveloped (without an outer lipid bilayer), icosahedral, lytic DNA viruses composed of a nucleocapsid and a linear double-stranded genome (Fig. 14.11a). Adenoviruses are capable of infecting both dividing and nondividing cells. More than a hundred (sero)types of adenoviruses have been identified to date. They are grouped into seven subgroups or species (A-G) based on genome size, composition, hemagglutinating properties, and oncogenicity. The adenoviruses serotype 2 and 5 are the most extensively studied and the first to be used as vectors for gene therapy. The adenoviral genome is a linear, nonsegmented dsDNA, between 26 and 45 kb, composed of six early (E1a, E1b, E2a, E2b, E3, and E4) and five late (L1, L2, L3, L4, and L5) genes. The early genes encode proteins necessary for viral replication and prevention of cell death, while the late genes encode proteins for virus assembly, release, and cell death. The genome of adenoviruses is flanked by hairpin-like inverted terminal repeats (ITRs), functioning as self-priming structures that facilitate primase-independent DNA replication (Arrand and Roberts 1979; Shinagawa et al. 1980). A packaging signal sequence promotes viral genome packaging.

Adenovirus infection typically begins with the binding of the fiber knob on the surface of the viral capsid to the CAR and major histocompatibility complex (MHC) class I (Fig. 14.23). Several alternative entry receptors have been identified, including sialic acid, CD46, and Desmoglein-2 (Gaggar et al. 2003; Nilsson et al. 2011; Wang et al. 2011).

A is a cross-sectional illustration of the adenovirus with labeled parts. B is an electron micrograph image of the particles. C illustrates the replication cycle of the adenovirus particle in 8 steps. The steps are listed. — **Fig. 14.23**

After initial binding, the penton base interacts with integrins on the cell surface to initiate a series of cell signaling processes allowing internalization via receptor-mediated endocytosis (Nemerow and Stewart 1999; Medina-Kauwe 2003). As a result, adenovirus particles enter the nucleus via the nuclear envelope pore complex as early as 30 min after initial cellular contact (Wiethoff and Nemerow 2015). Viral DNA replication and particle assembly in the nucleus starts 8 h after infection and culminates in the release of 10⁴–10⁵ mature virus particles per cell 30–40 h postinfection by cell lysis (Majhen and Ambriovic-Ristov 2006).

Adenoviruses as Gene Therapy vectors

To construct an adenoviral vector for gene therapy, the E1 and E3 regions of the viral genome are often removed. This both prevents viral replication and creates space to accommodate transgene cassettes. Adenoviruses have a large genome capable of accommodating large transgene cassettes. The adenoviral genome is also easily manipulated to generate a vector with multiple deletions and inserts without affecting its transduction efficiency. Adenoviruses with both E1 and E3 inserts to simultaneously express two therapeutic genes have been reported (Panakanti and Mahato 2009). Moreover, adenoviruses with E1, E3, and E4 deletions and even “gutless” adenovirus (adenoviruses without viral coding regions) have been constructed to drive transgene expression (Armentano et al. 1995; Chen et al. 1997).

Other favorable characteristics of adenoviruses include that the biology of the virus is well understood, that recombinant virus can be generated with high titer and purity, that transgene expression from adenoviruses is rapid and robust, and that adenoviruses can infect a wide range of dividing and nondividing cells. Unfortunately, adenovirus genomes do not integrate into the host genome. While this minimizes the risk of insertional mutagenesis, gene expression is transient, making adenoviruses unsuitable for long-term correction of genetic defects.

The major drawback to the use of recombinant adenoviruses is the ability of the virus to elicit strong innate and adaptive immune responses and the existence of widespread preexisting neutralizing immunity in the population. Innate and adaptive immunity results in the killing of adenovirus-transduced cells and the production of antibodies to adenovirus, resulting in the clearance of the adenoviral vectors from the body (Dai et al. 1995). Preexisting neutralizing antibodies against one or more of the commonly used adenovirus (sero)types immunity in human populations, as a consequence of prior adenovirus infections, significantly reduce the efficacy of these vectors in both preclinical studies and clinical trials (Ertl 2005). To overcome this, researchers have started studying the use of human adenovirus types with low seroprevalence in the human population, such as Ad26. Importantly, these types seem to induce less potent immune responses than the most commonly studied Ad5 (Chen et al. 2010).

The immunogenicity of recombinant adenovirus raises serious safety concerns for its clinical applications. The massive immune responses caused by the administration of adenovirus could lead to multiple organ failures resulting in death. In 1999, a patient died 4 days after injection with an adenoviral vector. This was the first death of a participant in a clinical trial for gene therapy (Stolberg 1999). Another patient experienced a severe immune response syndrome characterized by multiple organ failure and sepsis and died soon after an adenoviral vector dose injection in 2003 (Raper et al. 2003). Preclinical studies also confirmed that the immune response generated by adenoviral vectors must be suppressed before a therapeutic effect can be expected. The transgene expression from adenovirus-transduced cells lasted for about 5–10 days, partially due to the clearance of the transduced cells by the host immune system (Lochmüller et al. 1996). Adenoviruses show an extended duration of expression when given to nude mice (mice with an “inhibited” immune system) or when an immunosuppressant is administered (Dai et al. 1995). Importantly, in some cases, the strong immunogenicity of adenovirus vectors benefits the therapy, e.g., in cancer or vaccines against pathogens. Less immunogenic adenovirus types such as Ad26 may not be the best choice in these cases. Alternatively, nonhuman primate-derived adenoviruses has been suggested as a source for vector development to avoid preexisting neutralizing immunity while maintaining a strong immune stimulation upon administration (Bots and Hoeben 2020).

A significant effort has been put forth to address the issue of the adenovirus-induced systemic immune response and potential regeneration of replication-competent adenovirus by engineering next-generation adenoviral vectors. First-generation adenoviral vectors were engineered by removing E1 and/or E3 to allow for transgene insertion of up to 4.5–6.5 kb. As the E1 region is vital for adenovirus replication, E1-expressing cell lines have been generated to produce these vectors, such as HEK293. E3 is dispensable for viral propagation in cultured cells.

Second-generation adenovirus vectors lack additional early gene regions (E2a/E2b/E4), enlarging the space for transgene cassettes to 10.5 kb. Like for first-generation adenoviral vectors, the gene deletions are compensated for by producer cell lines expressing the genes. However, titers of second-generation adenoviral vectors are typically lower than for first-generation vectors. Second-generation adenoviral vectors induce notably lower immunogenicity as less viral antigens are being produced, resulting in longer-lasting expression of the encoded transgenes. Nevertheless, the late genes that are still present in the adenovirus vector genome can still trigger undesired immune responses against the vector.

Third-generation adenoviral vectors are called “gutless,” “helper-dependent,” or “high-capacity” and carry none of the viral sequences except for the ITRs and the packaging signal. These vectors can accommodate ~36 kb of space for transgene cassettes. Production depends on additional helper viruses carrying loxP sites flanking the packaging signal and producer cells that express Cre recombinase. The viral proteins expressed by the genome of the helper viruses allow for replication and packaging. The Cre recombinase ensures the removal of the packaging signal from the helper virus genome to ensure that only the adenovirus genome can be packaged. Third-generation adenoviral vectors have even more reduced immunogenicity, longer-lasting transgene expression in the host cell, and a larger transgene capacity. The main challenge is to eliminate the helper virus from vector batches. Conditionally replicating adenoviral vectors carry tumor-specific gene promoters to make the viruses specifically replicate in tumors. Initially, these were generated by partial deletion of E1B, restricting genome replication to cells that lack p53 such as tumor cells. More recently, conditionally replicating adenoviral vectors have been generated by removing a specific stretch of 24 amino acids from the E1A protein. These so-called AdΔ24 or AdΔRb vectors cannot bind to the retinoblastoma (Rb) protein. Rb normally retains E2F, preventing the cells from entering the S-phase and thereby replicating the genome. Cancer cells often have an aberrated Rb pathway and thereby facilitate the S-phase and AdΔRb replication independent from the Rb-binding activities of E1A (Fig. 14.24). These vectors have been widely studied in oncolytic virotherapy, which is more elaborately discussed later in this chapter.

A 4-part illustration of the replication of wildtype adenovirus in a normal and tumor cell and delta R b binding oncocytic adenovirus in a normal and tumor cell. — **Fig. 14.24**

Another hurdle for the use of adenovirus vectors in the human body is the generation of replication-competent adenovirus (RCA). Although the early genes responsible for viral replication and pathogenicity are already removed in the vector construction process, RCA can still be generated by homologous recombination if there is some overlap between sequences in the virus genome and the packaging cell genome. Although adenoviruses typically cause mild respiratory illness, RCA in clinical products could be life-threatening for immune-compromised patients. Several groups have observed the production of RCA from HEK293 cells caused by sequence overlap (Louis et al. 1997). Some new packaging cell lines with less overlap have been reported to overcome such problems. Moreover, there are strict rules for the purity of vector batches, e.g., the FDA recommends a maximum level of 1 RCA in 3 × 10¹⁰ viral particles. RCA assays have been developed to screen vector preparations for the presence of RCA.

Clinical Use of Adenoviral Vectors

Over 500 clinical trials have been initiated using adenovirus vectors (Bulcha et al. 2021). Approximately 50% of all gene therapy clinical trials involving viral vectors use recombinant adenoviruses, making them the most widely applied vector for gene transfer. China was the first to approve a gene therapy, Gendicine, in 2003, for the treatment of head and neck cancer (Pearson et al. 2004). In this adenoviral vector, the E1 gene is replaced by the tumor-suppressor p53. Since its approval, it has been studied in various additional cancers. In 2005, another adenovirus-based gene therapy, Oncorine, was approved in China for the treatment of nasopharyngeal cancer in combination with chemotherapy (Liang 2018). In this vector, E1B is partially deleted. Oncorine is currently also studied in other cancer types. However, both Gendicine and Oncorine have not been approved for clinical use outside of China. A recent addition to the list of marketed adenovirus-based gene therapies is nadofaragene firadenovec (Adstiladrin™). It is a nonreplicating AdV vector expressing recombinant human interferon alfa-2b that is administered into the bladder to treat BCG-unresponsive nonmuscle-invasive bladder cancer. The results of a phase III clinical study showed that 55 out of 103 patients with bladder cancer (with or without a high-grade Ta or T1 tumor) had a complete response within 3 months of the first dose, and this response was maintained in 25 out of 55 patients at 12 months (Boorjian et al. 2021). The FDA approved it in December 2022. Additionally, the immunogenicity of adenovirus vectors has been used in vaccine development (Bulcha et al. 2021). In the last decade, human and chimpanzee Ad vectors have been developed and clinically tested for protection against Ebola, Influenza, or HIV. Especially the Ebola vaccines induced strong and specific cellular and humoral immunity that was long-lasting. Moreover, the global COVID-19 pandemic has prompted the development of additional adenovirus-based vaccines that deliver the SARS-CoV-2 spike protein. This led to the authorization of Vaxzevria (AstraZeneca, full market authorization) and Jcovden (Janssen, conditional market authorization) (see Chap. 15 Vaccines).

Adeno-Associated Virus (AAV)

Biology

The AAV genome is a 4.7 kb linear, single-stranded DNA molecule composed of two open reading frames (ORFs), rep, cap, and two inverted terminal repeats (ITRs) that define the start and end of the viral genome and packaging sequence. The rep genes encode proteins responsible for viral replication, while the cap genes encode structural capsid proteins. ITRs are required for genome replication, packaging, and integration.

The icosahedral AAV capsid is 25 nm in diameter. AAV is deficient in replication, and there are no packaging cells, which can express all the replication-related proteins of the AAV. Therefore, AAV requires coinfection with a helper virus, such as an adenovirus or a herpes simplex virus, to replicate (Fig. 14.25). Thirteen distinct AAV serotypes have been identified, and hundreds of AAV variants have been found in human and nonhuman tissues (Becker et al. 2022; Pupo et al. 2022). The biology of AAV serotype 2 (AAV2) has been the most extensively studied, and this serotype is most often used as a vector for gene transfer. Different serotypes are presumed to recognize different cell receptors and have distinct tissue and cell tropisms.

A flow diagram of the lifecycle of A A V genome. The A A V genome in A with the help of co-infection with A d or H S V under cell lysis. In B, the genome without the helper and site-specific integration undergoes rescue and lytic infection. — **Fig. 14.25**

Suitability of Adeno-Associated Viruses for Gene Transfer

Recombinant AAV vectors have rapidly gained popularity for gene therapy applications within the last decades, due to their lack of pathogenicity and ability to establish long-term gene expression (Table 14.6). The viral genome is simple, making it easy to manipulate. In addition, the virus is resistant to physical and chemical challenges during purification and long-term storage (Croyle et al. 2001; Wright et al. 2003). The ability of the virus to integrate into the human chromosome was an initial concern, but eventually, it turned out that AAV only integrates into a fixed human genomic location called AAVS1, and the integration frequency of recombinant AAV is quite low (Surosky et al. 1997).

The AAV vectors are produced by replacing the rep and cap genes with the transgene. Only one out of 100–1,000 viral particles is infectious. Apart from the production of AAV vectors being laborious, these vectors also have the drawback of limited packaging capacity (4.7 kb) for the transgene. Large genes are, therefore, not suitable for use in a standard AAV vector. To overcome the limited coding capacity, the ITRs of two AAV genomes can anneal to form a head-to-tail structure through trans-splicing between two genomes, almost doubling the capacity of the vector (Yan et al. 2000).

Since recombinant AAV vectors do not contain any viral ORFs, they induce only limited immune responses in humans. Intravenous administration of AAV vectors in mice causes the transient production of pro-inflammatory cytokines and limited infiltration of neutrophils, in contrast to an innate response lasting 24 h or longer induced by aggressive viruses (Zaiss et al. 2002). However, despite the limited innate immunity elicited by AAV vectors, the humoral immunity elicited by AAV is still common. Depending on the serotype, it is estimated that up to 80 % of individuals are positive for AAV antibodies in the human population. The associated neutralizing activity limits the usefulness of AAV in certain applications. To overcome this, capsids have been isolated from nonhuman sources such as nonhuman primates or other vertebrate species. Alternatively, the vector can be retargeted by inserting or removing specific sequences from the AAV that are known to bind to certain receptors, or by directed evolution.

Clinical Use of Adeno-Associated Virus Vectors

The first clinical use of recombinant AAV was to transfer the cDNA of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) to the respiratory epithelium for treating cystic fibrosis (Flotte et al. 1996). Since then, hundreds of clinical trials employing recombinant AAV vectors have been initiated worldwide. The interest in AAV for gene therapy has been boosted by the approval of Luxturna and Zolgensma, two AAV-based gene therapies, for retinal dystrophy and spinal muscular atrophy, respectively. The first approval of AAV-based gene therapy was granted by the European Commission to Glybera® (alipogene tiparvovec), which encodes the gene for lipoprotein lipase deficiency for the treatment of patients with familial lipoprotein lipase deficiency (LPLD, synonym: type I hyperlipidemia) (Büning 2013; Salmon et al. 2014). However, this therapy was withdrawn in 2017 due to limited use. Numerous clinical trials using AAV gene therapy have demonstrated its safety and efficacy in neurological, musculoskeletal, hematological, ophthalmological, and metabolic diseases (Kuzmin et al. 2021). Efforts are being made to avoid vector accumulation in the liver and potentially related toxicity and to retarget the vectors to other organs than the liver, eye, nervous system, and muscles (e.g., the heart).

Nonviral Vectors

The inherent problems with recombinant viruses such as limited packaging capacity of transgenes, high production costs, and immunogenicity, a.o. reflected in the generation of neutralizing antibodies, have called for the design of efficient, nonbiological delivery methods for human gene therapy. These nonviral methods can be categorized into physical methods of gene transfection and physicochemical methods that make use of synthetic biological molecules (e.g., lipids, polymers, peptides, or sugars) to encapsulate, complex, or conjugate genetic material (further detailed below).

Like viral vectors, synthetic, nonviral vectors can be used to deliver genetic material for transient, episomal expression, stable transgene integration into the genome, and gene editing.

Transient expression involves the delivery of either plasmid DNA (pDNA) or mRNA. Longevity of expression is dependent on many factors, including the speed at which a cell divides, the stability of the mRNA or pDNA, and the type of nonviral vector that was used. In general, expression fades within 1-2 weeks but can be longer in slowly dividing cells.

Stable expression with synthetic vectors can be achieved by introducing pDNA encoding integration competent genetic elements. These can be of viral origin (Chiang et al. 2020), such as retroviral constructs or derived from engineered transposable elements such as Sleeping Beauty, piggyBac, and Tol2 (Sandoval-Villegas et al. 2021). In both cases, the integrase (for retroviruses) or transposase (for transposable elements) need to be delivered in trans with the DNA inserted, often provided in the form of mRNA. Stable integration has been described for these systems after electroporation or nonviral delivery, but the efficiency drops with the size of the DNA to be integrated.

Synthetic vectors are also very suitable for delivering the necessary components for gene editing. Compared to viral vectors, synthetic vectors offer several advantages for this. First, for some viral vectors, especially the adeno-associated virus, the packaging capacity is limited, which can be a limitation when large or more than 1 gene construct needs to be delivered. Synthetic vectors do not have this intrinsic limitation, as the size of these systems can be readily adjusted to accommodate bulky cargo. Second, unlike viral vectors, synthetic vectors can accommodate a mixed cargo of DNA, RNA, and protein. For example, lipid nanoparticles have been used to co-deliver spCas9 ribonucleoprotein complexes and single-stranded HDR templates to cells in culture, with high editing efficiencies (Walther et al. 2022). Viral vectors would rely on co-expression of the Cas9 nuclease and the sgRNA, each with their own promoter, making it quite bulky, and from a safety point of view, this is not desired. Thirdly, synthetic vectors do not suffer from vector-induced antibody responses, allowing redosing with the same synthetic vector. Despite these advantages, their clinical utility still suffers from low transfection efficiencies, which stems from the nonspecific uptake of the vector by epithelial barriers and extracellular matrix and poor delivery into the therapeutic target cells (Fig. 14.26). New emerging delivery systems and vector-constructing technologies try to address these issues.

An illustration of the different barriers between a plasmid and translation. The barriers include blood vessels, extracellular matrix, cellular uptake, endosome, endosomal release, nuclear uptake, transcription, m R N A, and then translation. — **Fig. 14.26**

Nonviral delivery methods can be subdivided into physical delivery methods and synthetic and biomimetic vectors for gene transfer.

Physical Methods for Gene Transfer

Physical methods involve the transfer of naked nucleic acids (DNA or RNA) by direct disruption of (target) cell membranes.

The earliest techniques to deliver recombinant DNA to cellular targets include microinjection, particle bombardment, and electroporation (Table 14.10). Microinjection, the direct injection of DNA or RNA into the cytoplasm or nucleus of a single cell, is the simplest and most effective method for the physical delivery of genetic material to cells. This transfects 100 % of the treated cells and minimizes waste of plasmid DNA. However, it requires highly specialized equipment and skills. Moreover, microinjection is unsuitable for in vivo or in vitro gene transfer into tissues or organs composed of many cells. Particle bombardment, or gene gun treatment, starts with coating tungsten or gold particles with plasmid DNA. The coated particles are loaded into a gene gun barrel, accelerated with gas pressure, and shot into targeted cells or tissues in a petri dish. Particle bombardment can be used to introduce a variety of DNA vaccines into desirable cells in vitro. However, particle bombardment has a low penetration capacity, making it unsuitable for in vivo gene delivery apart from easily accessible tissue, e.g., the skin. Electroporation generates temporary pores in the plasma membrane to transfer plasmid DNA to the cells by an externally applied high-voltage electrical field. Electroporation increases the gene transfer efficiency by 100–1000 folds compared to naked DNA solutions and has met with great success in laboratory practices and clinical trials (Wells 2004). For example, GMP-compliant electroporation devices have been developed to transfect cells in a closed flow system, thereby preventing potential contamination during transfection. Such closed systems are ideally suited for cell-based therapies in which cells need to be genetically modified prior to infusion into the patient (see “Cell Therapy” section) (Li et al. 2013). Electroporation can also be applied directly in vivo and has demonstrated safety and efficacy in clinical trials to treat melanoma, prostate cancer, and HIV infection (Daud et al. 2008; Vasan et al. 2011). Other physical methods for gene transfer include sonoporation, photoporation, laser irradiation, magnetofection, and hydroporation (Raes et al. 2021). However, because most of the physical methods induce stress and disruption of cellular structure and function, physical methods are less widely studied compared with the use of viral and synthetic vectors (see below) and are generally restricted to in vitro gene transfer of cultured cells or embryonic stem cells (Table 14.10).

Table 14.10 Summary of nonbiological methods used for gene transfer

Full size table

Lipid-Based Vectors

Lipid-based vectors make use of cationic lipids to electrostatically bind negatively charged RNA/DNA to form complexes in the submicron scale (60-400 nm). Since the invention of lipofectamine in 1987, numerous cationic lipids have been synthesized and tested for gene delivery. Most of these cationic lipids are composed of three parts: (i) a hydrophobic lipid anchor group; (ii) a linker group, such as an ester, amide, or carbamate; and (iii) a positively charged head group (Fig. 14.27) (Mahato et al. 1997). 2,3-dioleyloxypropyl-1-trimethyl ammonium bromide (DOTMA) and 3-β[N(NV,NV-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) are two commonly used cationic lipids with different structures. Cationic lipids are usually mixed with other lipids to change the structural properties as well as toxicity profiles of the formed complexes. Typically, these consist of cholesterol, neutral helper lipids, and lipids with a flexible poly(ethylene glycol) (PEG) polymer attached to their polar headgroup to increase nanoparticle stability and prevent rapid clearance once injected into the circulation.

A features molecular structures of D O T M A, D C chol, and D O T A P molecules. The amine head group, linker group, and hydrophobic tails are marked. B features structures of ionization lipids and lipidoids. C features helper lipids and PEG lipids. D illustrated the structure of a liposome and lipid nanoparticle. — **Fig. 14.27**

Depending on the exact composition of the lipids and the ratio of lipids to nucleic acid cargo, the formed structures can either adopt a vesicular nature, containing lipid bilayers enclosing an aqueous core (liposomes), or form solid spherical structures in which the solid core consists of cationic lipid-complexed nucleic acids with some water, which are surrounded by a layer of neutral phospholipids and PEG lipids, the so-called lipid nanoparticles, LNPs.

Initially, permanently charged cationic lipids such as DOTMA were being used for in vivo transfection of pDNA, but direct exposure of cells to these highly cationic nanoparticles caused toxicity along with the activation of the innate immune system. This problem was partially solved by developing ionizable cationic lipids. In the LNP, the amino head groups are only charged at low pH (<6.5). In this way, nucleic acid encapsulation can be performed at low pH to enable optimal complexation, but once the LNPs are formed, and pH is neutralized, the LNPs hardly expose positive charges on their surface that could cause cytotoxicity. Screening of a vast library of such ionizable lipids and lipidoids (i.e., lipid-like materials) has led to the development of highly efficient LNPs for direct in vivo delivery of different therapeutic nucleic acids, including siRNA, mRNA, and pDNA (Sago et al. 2018) (cf. Chap. 5).

The mechanisms by which LNPs deliver their nucleic acid cargo is still a topic of investigation but involves endocytic uptake, followed by the exchange of lipids between the LNP and endosomal membranes, which in sporadic cases, leads to the endosomal escape of the nucleic acid cargo (Fig. 14.26). To enhance endosomal escape and transport to and through the nuclear membrane, additional functional elements may be attached: for endosomal escape (pH-sensitive fusogenic peptides), for transport in the cytoplasm, and nuclear membrane passage (a nuclear translocation peptide).

LNPs are being explored for gene editing. Hereditary Transthyretin Amyloidosis is a rare genetic disorder that causes the amyloid formation of the TTR protein due to a point mutation in the TTR gene. By encapsulating an mRNA encoding spCas9 together with a sgRNA targeting the mouse transthyretin (Ttr) gene in the liver, a single intravenous administration of the gene could be effectively knocked out, leading to levels of TTR protein in the serum that were reduced by 97% and which persisted for at least 12 months (Finn et al. 2018). Interim results of a phase I study in patients suffering from hereditary transthyretin amyloidosis showed serum TTR level reduction up to 87%, after a single intravenous dose, with only mild grade 1 adverse effect (Gillmore et al. 2021).

Peptide-Based Vectors

Just like cationic lipids, cationic peptides condense DNA in a similar manner and can be used as gene delivery carriers. Poly(l-lysine) (PLL), a polydisperse, synthetic repeat of the amino acid lysine, was one of the first cationic peptides to deliver genes. However, an increase in the length of PLL leads to increasing cytotoxicity. Besides, PLL shows limited transfection efficiency and needs the addition of endosomolytic agents such as fusogenic peptides to facilitate plasmid release into the cytoplasm. Due to these issues, many researchers have turned to the development of PLL-containing “active” peptides and have met with some success (McKenzie et al. 1999). Such peptides offer many advantages over PLL, such as lower toxicity, precise control of synthesis, and homogeneity of peptide length.

Another class of peptides that have been extensively explored for gene delivery are the cell-penetrating peptides (CPPs). CPPs are short, synthetic peptides that facilitate cellular uptake and endosomal escape of molecules ranging from small chemical compounds, and nucleic acids to entire nanoparticles. CPPs can be categorized as those with a high abundance of cationic amino acids (lysine and arginine) or a sequence with alternating hydrophobic and polar amino acids, creating an amphipathic alpha-helical structure (Cf. Chap. 2). The latter are known for their endosomolytic activity. When complexed with nucleic acids, the nanocomplexes are taken up by endocytosis, where the peptides – because of the low pH – destabilize the endosomal membrane to release the cargo into the cytosol. The stability of the nucleic acid/peptide complexes can be greatly enhanced by the inclusion of a lipid tail to the C- or N-terminus of the CPP.

Despite efficient transfection of a variety of cells in vitro, peptide-based vectors are not yet suitable for systemic in vivo administration because of rapid destabilization, nonspecific plasma protein binding, and uptake by the reticuloendothelial system (Männistö et al. 2002). Another unique challenge for peptide-based gene delivery systems is cytosolic proteasomes, which degrade unneeded or damaged proteins by proteolysis. Co-administration of proteasome inhibitors is the most effective strategy to address this issue.

Polymeric Vectors

Synthetic and naturally occurring cationic polymers constitute another category of nonviral vectors. The advantage of synthetic polymers is that their architecture can be fully tailored: they can be made from biodegradable materials, the molecular architecture can be adapted from linear to branched to star-like dendrimers, and the density of cationic charges can be varied. Several polymers that have been studied for gene delivery are polyethyleneimine (PEI), poly[(2-dimethylamino) ethyl methacrylate (pDMAEMA), polyamidoamine (PAMAM) and biodegradable poly(β-amino ester) polymers (PBAE). Natural polymers, such as chitosan, dextran, gelatin, pullulan, and synthetic analogs, were also explored. Polymeric systems can generally be tailored to specific needs but require PEGylation to prevent undesired aggregation and rapid clearance in vivo. Some polymers, such as PEI, work via the postulated proton-sponge effect: the proton buffering capacity of such polymers at acidic pH causes the buildup of osmotic pressure when they reside in acidifying endocytic compartments, leading to rare endosomal burst events that enable the endosomal escape of the nucleic acid cargo. So far, only a few polymeric vectors for gene therapy have reached clinical stage development. A PEG–PEI–cholesterol lipopolymer is under clinical investigation for immunotherapy of ovarian and colorectal cancers through forced expression of the cytokine interleukin-12 (IL-12) (NCT01489371) (Thaker et al. 2015).

In another study, local delivery of a CRISPR-Cas9 ribonucleoprotein complex with a polymeric vector based on disulfide-crosslinked acrylates into the eye resulted in robust gene editing in the retina of nonhuman primates. It could potentially be used for local treatment of genetic eye diseases (Chen et al. 2019).

Extracellular Vesicles

Extracellular vesicles (EVs) encompass different types of lipid vesicles that are naturally secreted by cells, including exosomes, microvesicles, and apoptotic bodies, and may serve as a new type of delivery system for gene therapies (Yáñez-Mó et al. 2015; Varderidou-Minasian and Lorenowicz 2020) (Figure 14.28). EVs are secreted by almost every cell type and play pivotal roles in both physiology and pathophysiology. EVs naturally transfer biological payload (DNA, RNA, proteins, lipids) from cell to cell. Also, EVs can overcome cytotoxicity and immunogenicity issues associated with most viral vectors and synthetic nanoparticles, as demonstrated in preclinical models (Corradetti et al. 2021). These characteristics have boosted the development of EV therapeutics, which concerns the (ex vivo) production of EVs that are loaded with therapeutic drugs, proteins, small RNAs, mRNAs, or DNA (Liu and Su 2019). For example, Cas9-gRNA ribonucleoprotein can be loaded in EVs and functionally delivered in recipient cells, which, due to the large size of Cas9, is less feasible with AAV viral vector systems. The first engineered EV therapeutics have found their way into clinical trials, and initial findings on safety and efficacy are promising (e.g., to deliver cytokine profiles locally to tumor cells). Many developments are currently ongoing to fully explore the potential of EVs as a new type of delivery system for gene therapeutics. These not only focus on the engineering aspects to optimize the loading (and unloading) of payload but also involve targeting studies, specificity studies and development of scalable, clinical-grade EV production technologies.

An illustration of the E V biogenesis and E V uptake between a healthy donor cell and a recipient cell. — **Fig. 14.28**

Delivery Systems for CRISPR-Based Gene Editing

CRISPR-Cas9-based genome editing holds great potential to provide an entirely new class of therapeutics. However, to achieve effective therapeutic efficacy, the delivery of CRISPR-Cas9 components to the target cells of the patients is still a major hurdle and a prime topic for research. Several studies suggest an efficient delivery of the ~4 kb Cas9 from Streptococcus pyogenes into mammalian cells using adenoviral and lentiviral vectors (Eyquem et al. 2017). Nonviral approaches, including cationic polymer-based vectors (Platt et al. 2014), cationic lipid-based vectors (Zuris et al. 2015), and conjugated vectors (Ramakrishna et al. 2014), are also studied as delivery vehicles. For instance, CRISPR-Cas9 was used to target frequently mutated oncogene KRAS alleles in cancer cells and in vivo tumors using lentivirus or AAV expressing Cas9 and single guide RNA (sgRNA) (Kim et al. 2018). Also, more than 97% reduction in serum transthyretin level was achieved in mice when the CRISPR-Cas9 system was delivered using lipid nanoparticles (Finn et al. 2018). Other ex vivo delivery methods such as electroporation and nucleofection, are also extensively applied for the delivery of CRISPR-Cas9 components.

Disease Targets for Gene Therapy

In 2021 3000+ active gene therapy clinical trials were registered worldwide (Fig. 14.29) (John Wiley and Sons LTD 2022). Approximately 65 % of these trials are for cancer treatment. Treatment of monogenetic, cardiovascular, and infectious diseases each takes ~6-12 %, whereas treatment of neurological diseases is close to 2% (Fig. 14.29). Currently, gene therapy trials are primarily performed in the United States (58.3 % of all trials), China (10.1 %), the United Kingdom (7.3 %), and Germany (3.5 %). The geographical distribution of gene therapy clinical trials is summarized in Fig. 14.29. General indications for gene therapy trials in the clinic are summarized in Table 14.11.

Three pie charts. 1. Indications addressed by gene therapy clinical trials. 2. Geographical distribution of gene therapy clinical trials. 3. Geographical distribution of gene therapy clinical trials. — **Fig. 14.29**

Table 14.11 Conditions for which human gene transfer trials have been approved

Full size table

Cancer Gene Therapy

Most of today’s gene therapy clinical trials are devoted to treating cancer. Gene therapy has the potential to target and destroy cancer cells in a way that is much more specific and tailored as compared to traditional cancer treatments with chemotherapy and radiation. This property has even become much more relevant in light of the current knowledge on the large intra- and inter-tumor heterogeneity of many cancer types, which advocates the use of therapeutics that attack cancer cells from multiple angles and in a directed fashion. The types of cancer gene therapies being explored vary widely and include oncolytic viruses, ex vivo immune cell modification (e.g., CAR and recombinant T cell receptor T cells), overexpression of pro-apoptotic genes (e.g., p53), introduction of suicide genes, suppression of oncogenes (e.g., using RNA interference), creating synergy toward other treatments (sensitization) and targeted interference with the cancer cell genome using CRISPR-Cas.

Correction of Genetic Abnormalities

In this approach, gene therapy corrects genetic abnormalities contributing to the malignant phenotype by replacing/reintroducing tumor-suppressor genes or downregulating certain oncogenic pathways. Understanding cancer at the molecular level is the starting point for gene correction in cancer therapy. The inactivation or activation of certain genes may contribute to tumor growth. Many cancer gene therapy clinical trials involve overexpression of tumor-suppressor genes such as p53, MDA-7, and ARF (Belete 2021). Mutations in the p53 gene are most commonly seen in a wide spectrum of tumors (Valente et al. 2018). Efficient delivery and expression of the wild-type p53 tumor-suppressor gene prevents the growth of human cancer cells in culture, causes regression of established human tumors in nude mice, or sensitizes existing tumors to the therapeutic effect of conventional chemotherapy and radiotherapy (Valente et al. 2018). The results from clinical trials indicated that the therapeutic effect of the adenoviral vector-based therapy Gendicine, the first gene therapy product, was promising in patients with head and neck squamous cancers (Xia et al. 2020). However, the results were only validated in China. Gendicine has also been used to treat various other cancers, which prolong overall survival when combined with other drugs. Although it does not show any adverse effects on the patients, vector-associated transient fever that lasts for only a few hours cannot be overcome in 50-60 % of the patients (Zhang et al. 2018). Efficient delivery of tumor-suppressor genes deep within tumor tissue is difficult, and restriction of gene expression in malignant tissue is challenging. Possibly this may be improved by the development of novel types of delivery systems. Gene silencing by this approach is also a limited success, especially when a prolonged effect is required. Tumor heterogeneity is another major bottleneck, and it will most likely require co-treatments or simultaneous “all-in-one-vector” delivery of multiple anti-cancer approaches at the same time. Despite these reservations, promising results have been obtained in clinical trials for different cancers, including prostate, lung, pancreatic, and brain tumors.

Immunotherapy

The past decade has witnessed some major breakthroughs in cancer treatment by applying immunotherapies. These consist of different approaches, all having in common that the patient’s immune system is being modified or boosted to create additional antitumor responses. One example is the class of the oncolytic viruses (e.g., Imlygic®, talimogene laherparepvec, based on herpex simplex virus-1), which not only kill the tumor cells (in a specific way) but also induce a strong antitumor immune response, as outlined in more detail below. Other more recent developments are the autologous or allogeneic immune cell therapies, most strikingly CAR-T cells (covered in detail elsewhere in this chapter). This concerns the modification of immune cells outside the body (ex vivo) to direct them against tumor targets and subsequent infusion into the patient. Driven by the successes of CAR-T cells for the treatment of different hematological malignancies, many efforts are ongoing to improve the efficacy, e.g., to become active in so-called “cold tumors,” that is, tumors with lower presence and influx of immune cells. Methods and technologies are improved to make this therapy more practical, easier, and cheaper. It is also being explored whether in vivo T cell therapy may be viable, which will require the use of vector systems that efficiently and safely deliver the transgenes into the immune cells inside the patient (Xin et al. 2022).

In recent years, a number of viral vectors have been developed that express therapeutic transgenes (Shaw and Suzuki 2019). Many of those represent oncolytic viruses (summarized below). Additionally, several finished and ongoing clinical trials have employed adenoviral vectors expressing tumor-associated antigens such as PSA or MAGEA3, or immunomodulatory molecules like IL-12 and interferon IFN-α/-β. As an example, nonreplicating adenoviral vectors encoding recombinant human interferon α-2b have been approved by the FDA for the treatment of BCG-unresponsive nonmuscle-invasive bladder cancer (Boorjian et al. 2021).

Oncolytic Viruses (Virotherapy)

Oncolytic virotherapy employs viruses that, either by nature or upon engineering, preferentially target tumor cells and induce antitumor immunity (Macedo et al. 2020). Moreover, it can be used to simultaneously deliver therapeutic genes into tumor cells. This represents a relatively novel anti-cancer approach that has gained interest in recent years. The first oncolytic virus that was approved was Rigvir, an ECHO-7 picornavirus. This virus was authorized in 2004 in Latvia for the treatment of melanoma patients. In 2005, China approved Oncorine, a genetically modified adenovirus (see “Adenovirus” section) for combination treatment with chemotherapy in nasopharyngeal carcinoma. More well-known is Imlygic (also called T-VEC), a modified herpes simplex virus, which was approved in the United States, the European Union, Australia, and Israel in 2015, for use in a subset of advanced melanoma patients (Kaufman et al. 2022). A number of other oncolytic viruses are currently under (pre)clinical investigation, including picorna-, adeno-, reo-, pox-, paramyxo-, rhabdo-, and retroviruses (Macedo et al. 2020). Their clinical antitumor effects as monotherapies, although long-lasting, seem moderate at best. Although combinations with other (chemo- and immuno-) therapies show promising results, alternative (nonviral) approaches with similar effects are often preferred. Many current research efforts are therefore focused on generating oncolytic viruses with enhanced potency. In doing so, viruses have, for example, been engineered with expanded tropisms, enhanced tumor selectivity, and/or increased immunostimulatory properties (Harrington et al. 2019). Additionally, efforts are made to evade circulating immunity in the human population by employing low-prevalent serotypes or nonhuman viruses (Uusi-Kerttula et al. 2015).

Monogenic Diseases

Great successes of gene therapy have been achieved in treating monogenic diseases (see Table 14.6 for a detailed list of approved gene therapy products). The approach has mainly been to apply gene augmentation or addition to restore a loss-of-function mutation, particularly successful for ex vivo applications. Severe combined immunodeficiency diseases (SCID) is a group of diseases in which ex vivo gene therapy has shown a lasting, clinically meaningful therapeutic benefit, e.g., gammaretroviral vector gene therapy for ADA-SCID (approved product Strimvelis™) and X-linked SCID. Gene-based therapy for other blood disorders, such as for β-thalassemia, has also been recently approved (betiglogene autotemcel/Zynteglo™). In inborn errors of metabolism, HSC gene therapy has been used to produce recombinant protein and shown beneficial effects in cerebral adrenoleukodystrophy (CALD, approved product elivaldogene autotemcel/SKYSONA™) and metachromatic leukodystrophy (MLD, approved product atidarsagene autotemce/Libmeldy™)(see Table 14.6) (Eichler et al. 2017; Fumagalli et al. 2022), and other metabolic disorders including Fabry, Gaucher, and mucopolysaccharidosis are underway.

In addition, in vivo gene therapy trials using AAV vectors for the treatment of monogenic diseases, such as spinal muscular atrophy (SMA), have been approved by regulatory agencies (onasemnogene abeparvovec – xioi/Zolgensma®). However, a rare but major risk of this type of in vivo AAV gene therapy is that it can cause acute serious liver injury or acute liver failure (Chand et al. 2021). It was also thought until recently that the safety profile of AAV vector integration is negligible, but recent studies indicate that long-term preclinical studies may be required to thoroughly understand these risks (Nguyen et al. 2021).

Other approaches have used CRISPR/Cas9 editing technology for blood disorders and inborn errors of metabolism. For instance, BCL11A, an erythroid-specific enhancer that represses γ-globin expression and fetal hemoglobin in erythroid cells, has been targeted in autologous CD34+ cells (Frangoul et al. 2021; Quintana-Bustamante et al. 2022). In another trial for transthyretin amyloidosis using lipid nanoparticles to deliver CRISPR/Cas9, misfolded transthyretin serum levels were lowered after a single dose and caused durable knockout (Gillmore et al. 2021).

Issues that have prevented gene transfer for monogenic diseases are (a) lack of suitable gene delivery technologies, (b) unfavorable interactions between the host and gene transfer vector, (c) complex biology and pathology of monogenetic diseases and target organs, and (d) lack of relevant measures, i.e., biomarkers, to assess the clinical efficacy and long-term efficacy of gene transfer. Challenges that remain in treating monogenic diseases are to induce gene expression sufficiently to correct or prevent further progression of clinical phenotypes without induction of host immune responses against the vector component or transgene product and minimize the risk of insertional mutagenesis for integrating vectors in target cells. Improvements in vector technology and advancements in the understanding of disease pathology will vastly improve methods for the correction of genetic diseases.

Cardiovascular Diseases

Cardiovascular diseases are the fourth largest group of diseases actively treated by gene therapy clinical trials (John Wiley and Sons LTD 2022). The current understanding of molecular mechanisms of cardiovascular diseases has uncovered many genes that could serve as potential targets for molecular therapies. For example, overexpression of genes involved in vasodilation such as endothelial nitric oxide synthase and heme oxygenase-1 or inhibition of molecules involved in vasoconstriction (angiotensin-converting enzyme, angiotensinogen) have reduced blood pressure in animal models of hypertension (Melo et al. 2005). Most clinical trials for cardiovascular diseases are designed for treating coronary and peripheral ischemia. Overexpression of pro-angiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) has been effective in myocardial and peripheral ischemia in preclinical studies (Shimamura et al. 2020). Despite the lack of significant benefit in several earlier clinical trials, VEGF gene therapy did show an excellent safety profile and improvement of symptoms in patients following adenovirus or plasmid intramyocardial administration in both pilot studies and long-term follow-ups (Stewart et al. 2006; Reilly et al. 2005). However, limited success was experienced in using gene therapy to treat cardiovascular diseases compared to other areas. The efficacy of gene therapy for cardiovascular disease will most likely be enhanced by strategies that incorporate multiple gene targets with cell-based approaches. Few of the gene therapy approaches for cardiovascular diseases are in phase II or III clinical trials. In 2019, a phase III clinical trial investigating the effect of intramuscular injection of pDNA encoding hepatocyte growth factor for the treatment of critical limb ischemia with ulcerations was successfully finalized, which led to the conditional approval of Collategene in Japan (Ylä-Herttuala 2019).

Infectious Diseases

Genetic vaccines based on DNA or mRNA, which are being discussed in Chap. 15 have shown to be very effective in the prevention of a number of infectious diseases, including COVID-19. For treating chronic infections, gene therapy approaches are being developed as well. Gene therapy for acquired immunodeficiency syndrome (AIDS) is the main application in this category. These interventions share the goal of inducing remission from HIV pathogenesis without the use of antiretroviral therapy (ART). The interest in gene therapy for an HIV cure was inspired by the elimination of the intact virus in Timothy Brown (also known as the Berlin patient) and Adam Casteljo (also known as the London patient), who both received stem-cell transplants from a CCR5-negative donor to treat their underlying malignancies (Gupta et al. 2019). This has spurred the research on genome editing approaches of CCR5 and co-receptor CXCR4 using CRISPR-Cas to confer resistance to CCR5-tropic HIV strains. Other strategies focus on boosting the immune system to reduce or eliminate the HIV pool. Many gene therapy trials for AIDS involve ex vivo transfer of genetic material to autologous T cells using self-inactivating or conditionally replicating viral vectors to improve the immune system of the patients (Manilla et al. 2005; Levine et al. 2006). Other trials employed overexpression of HIV inhibitors such as RevM10 to increase CD4⁺ T cell survival in HIV-infected individuals (Ranga et al. 1998; Morgan et al. 2005).

Besides HIV, gene therapy approaches are being developed against chronic infections with hepatitis B and hepatitis C virus, herpes simplex virus, malaria, and bacterial infections.

Neurological Diseases

Progress has been made in gene therapy for neurological diseases. For example, the approval of onasemnogene abeporvovec-xioi (Zolgensma®) for spinal muscular atrophy (SMA), which uses an AAV9 serotype with a human survival motor neuron 1 (SMN1) gene, is infused into the circulation but crosses the blood-brain barrier to transduce affected neurons.

Other more commonly investigated neurological diseases for gene therapy are Alzheimer’s disease (AD) and Parkinson’s disease. For Parkinson’s disease, both gene augmentation to restore loss of dopaminergic neurons and restoration of neurotrophic factors have been investigated in clinical trials using AAV vectors and lentiviral vectors (Serva et al. 2022).

AD can be divided into familial and sporadic, with the familial form having mutations in three major genes, amyloid precursor protein (APP), presenilin 1 (PSEN1), or presenilin 2 (PSEN2). Many genetic or environmental factors play a role in sporadic AD, which makes it difficult to apply therapies designed for monogenic disorders. Gene therapy trials have been performed using neurotrophic factors, such as nerve growth factor, to promote neuronal and synaptic repair, but these have been providing mixed results (Lennon et al. 2021).

In the neurogenerative disorder Huntington’s disease, many clinical gene therapy trials have been performed aiming at lowering the protein Huntingtin (Htt). Abnormal conformation of Htt results in the toxic gain-of-function. There are approaches using antisense oligonucleotides, but this can only transiently downregulate Htt. AAV5 vectors have been used to deliver miRNA to stably reduce Htt expression long-term (Byun et al. 2022).

Another group of neurological diseases encompasses leukodystrophies, which are a heterogeneous group of genetic disorders affecting the white matter of the central nervous system. Both AAV gene therapies as well as HSC gene therapy approaches have been tested, targeting different cell types in the brain and showing promising results in preclinical and clinical studies (von Jonquieres et al. 2021).

However, delivery of gene therapy technologies to the CNS requires careful selection of vector type and route of administration for optimal biodistribution to the affected cell types requiring correction or modulation of gene expression.

Nonclinical Animal Testing Considerations

A full pre-/nonclinical testing program during drug development, as presented in Chap. 8 for mAbs, may not always be feasible or necessary for advanced therapies due to the nature of these products that consist of a heterogeneous population of human cells or tissues (see also Table 14.2). Generally, the pre-/nonclinical testing package entails studies to provide data on the following:

(i)
safety (toxicity, including immunogenicity);
(ii)
tolerance (local, systemic);
(iii)
biodistribution;
(iv)
persistence (duration of exposure);
(v)
in vivo proliferation, maturation, and/or differentiation into an unwanted lineage of stem cells (ESCs, iPSCs);
(vi)
tumorigenicity;
(vii)
reproducibility;
(viii)
biological activity (potency) in vivo and/or in vitro; in vivo mechanism of action
(ix)
in vitro and in vivo efficacy studies to understand
(x)
which cells/cell-subpopulations and cell characteristics have therapeutic value;
(xi)
PK/PD to serve dose definition, e.g., number of (viable) cells;
(xii)
PK/PD to serve route of administration and schedule;
(xiii)
study duration to monitor for toxicity, and;
(xiv)
safety of the surgical procedure for local delivery of cells/tissues.

Nonclinical animal safety (toxicology) and efficacy (pharmacology) studies pose significant challenges when applied to advanced therapies, e.g., for the following reasons:

1.
Molecular incompatibility and immune rejection in xenogeneic human-animal combinations i.e., human tissues/cells tested in animal models. This is also true for genetically transduced cells, where the genetic modification leads to the expression of human protein(s), e.g., CAR-T cells.
2.
Cellular immunotherapy to treat cancer (e.g., TILs) relies on the interaction of the cellular product with the patient’s immune system for its effect. The in vivo immunological effect will very likely be different between species.
3.
Cells do not undergo ADME in a way conventional medicinal products often do.

Without nonclinical safety and pharmacology data, it may be difficult to predict the potential safety and efficacy of the cell therapy product in a first-in-human clinical study. Therefore, alternatives should be investigated that could yield evidence of safety and, evidence of efficacy or at least paucity of efficacy, including the use of models explained in Table 14.12.

Table 14.12 Examples of animal and other pre-clinical models applied for assessment of safety, efficacy, and product potency testing

Full size table

Relevant Animal Models

Mice are often the species of choice to study advanced therapies. They are relatively inexpensive, reproduce quickly, and can be easily manipulated genetically. However, the ability of mouse experiments to predict the effectiveness of advanced therapies remains controversial. The failure of many mouse models to mimic particular human diseases has compelled investigators to examine animal species that may be more predictive of humans. Larger animals, such as rabbits, dogs, pigs, goats, sheep, and nonhuman primates, are potentially better models than mice. They have a longer life span, which facilitates long-term (e.g., years) studies that are critical for some advanced therapy products with a lifelong effect. In addition, many physiological parameters, e.g., immune system properties that play an important role in the reaction of the host animal to advanced therapies, are much closer to humans than rodents. Large animals also have significant advantages regarding the number and types of cells or amounts of tissues that can be reproducibly isolated from a single donor animal and ex vivo manipulated in sufficient quantity for analysis and for various nonclinical applications.

In case animal safety data do not provide meaningful information based on which an extrapolation can be made to potential risks posed to humans, those studies may be (partially) waived by regulatory authorities. Study set-up and duration for evaluation of the toxicity and/or biodistribution have to be determined on a case-by-case basis and depend on, e.g.,

product half-life, which may vary between hours–days and months–years, the latter for cells that engraft in a specific niche in the human body;
potential alterations of cells over time upon administration;
dose regime of single or repeat dosing over a period of weeks–months–years;
chance for migration of the cells in the body to unwanted sites upon administration, e.g., local administration of an adult stem cell in the subretinal space of the eye may be safer than the systemic administration of an ESC/iPSC-derived product;
type and number of ex vivo cell manipulations performed during manufacture, i.e., in case cells are expanded for multiple passages close to the point where these cells senesce, and animal studies should be performed with cells beyond the cell passage used to manufacture the advanced therapy).

Generally, genotoxicity and specific safety pharmacology studies are not conducted for cell and tissue-based products unless there is a reason for concern, e.g., the use of a novel excipient or novel route of administration for an approved excipient. In addition, reproductive toxicity studies are only required when there is a potential risk for exposure to the reproductive organs. And finally, literature data may be used to support the (lack of) animal data. See Herberts et al. (2011) and Vestergaard et al. (2013) for further reading.

Clinical Testing Considerations

For investigating the safety and efficacy in humans, generally, the same principles apply to advanced therapies as to other medicinal products (see Table 14.4). However, considering the nature and complexity of the products and potential risks and benefits, there are some unique aspects to the clinical programs (Mount et al. 2015):

1.
Different set-up of trials compared to most conventional medicinal products:
1. (a)
  First-in-human trials are always in patients and never in healthy volunteers;
2. (b)
  A seamless development path rather than the traditional route of separate formal phase I (safety), phase II (hint of efficacy), and phase III (safety confirmation and efficacy) studies.
2.
Traditional PK (ADME)/PD studies may not be feasible.
1. (a)
  Dose (defined as the number of cells/mL; the number of cells/kg body weight) escalation studies may not be feasible as there may not be clear dose-response correlations. A low-, medium-, and high-dose is often selected based on literature data concerning the number of cells that have historically been administered to humans.
2. (b)
  Advanced therapies are frequently administered intravenously and rapidly cleared via the lungs, spleen, and liver (Leibacher and Henschler 2016). Other possible routes are intranodal (DCs to treat rheumatoid arthritis) or local administration via a surgical procedure, e.g., into the eye, brain, spinal cord, or knee.
3.
For safety evaluation, the following risks may need to be taken into account, depending on many factors, including the type of product, cell differentiation status upon administration, cell proliferation capacity, cell source being autologous, allogeneic or xenogeneic, the half-life of the cells in the body/lifelong persistence, site and method of administration/implantation, quality of the starting material (derived from a healthy donor or very sick patient), and disease environment(s) which cells may encounter in the patient’s body:
1. (a)
  Tumor formation (tumorigenicity), e.g., in case of ESC- and iPSC-derived products which are ex vivo expanded and differentiated;
2. (b)
  Potential adverse reactions at the site of administration, e.g., dimethyl sulfoxide (DMSO) related side effects upon i.v. administration;
3. (c)
  Cells, being subvisible particles, make it difficult to assess subvisible particles potentially present in the product. These foreign particles may damage the tissue upon administration, e.g., in the subretinal space of the eye;
4. (d)
  Inflammatory responses and infections (e.g., side effect of CAR-T cells);
5. (e)
  Implantation procedure for cells or 3-D tissue replacement therapies using a complex surgical procedure, e.g., to administer cells in the subretinal space of the eye, in the spinal cord, or in the brain; 3-D cultured trachea placed in the throat;
6. (f)
  Immuno-mediated side effects (CAR-T cells may cause cytokine release syndrome);
7. (g)
  Immunogenicity, which may depend on:
  - Relative immune privilege of the administration site (e.g., eye);
  - Allelic differences between product and patient cells (e.g., allogeneic dendritic cells);
  - Immune competence of the patient;
  - Need for repeat dosing (more doses may increase the chance of immune rejection of the advanced therapy);
  - Maturation status of the cells (e.g., ESCs).
Advanced therapies derived from an allogeneic cell source often require immune-suppressant medicines to be administered together with the cell-/tissue-based product. However, some allogeneic cell-/tissue-based products, such as MSCs, have shown relatively low immunogenicity profiles, in part due to the short half-life of the cells in the body. See more details below.
4.
Selecting the right patient population for the initial clinical program is challenging as there is a tension between choosing the patients most likely to benefit from an efficacious advanced therapy (e.g., early-stage cancer patients) and limiting the risk to which patients are exposed to the unlicensed therapy (late-stage cancer patients who may not benefit from the therapy at all due to their severe illness).
5.
Establishment of surrogate biomarkers for efficacy assessment may be needed to predict long-term clinical outcomes of cells that may persist in the body for years e.g., CAR-T cells which engraft in the peripheral blood and bone marrow and transduced CD34+ cells, which engraft in the bone marrow.
6.
Particularly for genetically modified cells, which may persist in the body for many years or lifelong, long-term (10–20 years) patient follow-up for safety, efficacy, and durability monitoring may be necessary.

Immunological Considerations in Advanced Therapy

The potential application of adult stem cell-based medicinal products derived from allogeneic sources and hESC-based therapies is limited by risks for graft-host rejection issues, as with all therapeutic strategies based on cell, tissue, and organ transplantation, unless the transplant is derived from an autologous source. A way to overcome this challenge is the use of a device to protect the allogeneic cellular product from the host immune system. An example of this strategy is Viacyte’s cell-based combination product, where the hESC-derived β-islet progenitors are contained in the Encaptra® cell delivery system, which is placed subcutaneously (see Table 14.13) and later Fig. 14.34). The additional advantage of this system is that cells cannot migrate in the body to unwanted sites, and the device can be taken out in case of, e.g., tumor formation. The disadvantages of such an immune-protective device are fibrosis and the lack of vascularization around the device, which is required for cell viability and insulin production. Certain human body sites have immune privilege, i.e., they tolerate the introduction of nonself-antigens without eliciting an inflammatory immune response. These sites include the eyes, the testicles, the fetus, and certain tumors. There is debate in the cell therapy world regarding the immunogenicity of allogeneic MSCs (Ankrum et al. 2014; Consentius et al. 2015). Clinical trials with standardized immune monitoring programs and a better understanding of the in vivo mode of action of allogeneic MSCs may provide answers.

Table 14.13 Example of clinical trials with pluripotent stem cells (hESCs and iPSC), adapted from Trounson and McDonald (2015) and Ilic et al. (2015)

Full size table

Administration of drugs to suppress the immune response is standard practice for patients undergoing transplantation, but with immunosuppression come side effects. The hope is that iPSC technology (see above) may overcome rejection problems for which several products are being tested in the clinic (Kim et al. 2022). Another approach is to bank a collection of ESC lines that allows the selection of a matched ABO and HLA haplotype or a close match (Lui et al. 2009). It has been estimated that with a bank of 70–100 ESC lines, a partially matched ESC line that is adequate for each recipient can be chosen. The downside of this approach is that at the time the cell lines are banked, it may not be clear yet for which diseases they will be used in the future, hence what the critical parameters are to characterize the banks, for example, purity of the cells, stability, potency, viral safety, see (Bravery 2015). Preparing cell banks, extensive testing and long-term storage under frozen conditions are very expensive undertakings.

Manufacturing and Testing Considerations

Manufacturing

Cell and tissue-based products are distinct from traditional biopharmaceuticals in that the modified cell/tissue itself is the active ingredient in the medicinal product rather than “simply” the means by which the cells produce an active ingredient (e.g., a recombinant protein; a viral vector). However, many of the production platforms, cell culture media, storage and transport bags, and product excipients and primary containers have been established for traditional cell-based recombinant protein manufacturing processes (cf. Chap. 4) and can be readily applied to these innovative products.

Since the vast majority of advanced therapies contain viable cells/tissue that can be easily destroyed through sterilization procedures and cannot be sterile filtered (≤0.2 μm filter pore size), as cells have a size of 10–30 μm on average and tissues are even bigger, the manufacturing of these products must take place under aseptic conditions. For nonsterile raw and starting materials and excipients, additional steps may need to be taken to ensure subsequent aseptic manufacturing, e.g., heat inactivation, gamma-irradiation or sterile filtration of the material. The facilities, equipment, raw materials, viral vectors, and cells/tissues used must be of suitable quality to allow for good manufacturing practice (GMP) production of the drug product for human application. At every stage of production, materials and the final product should be protected from microbial, viral, and other contamination. The manufacturing of advanced therapies typically requires many or all of the following “manipulation” steps, see Table 14.14.

Table 14.14 Typical advanced therapy manipulation steps and equipment used for each step

Full size table

Control of the Manufacturing Process

As with manufacturing process of biologics, process variables need to be chosen carefully and monitored to allow for adjustments to the process and to ensure a product of high quality is consistently produced. Process variables assessed are, e.g., medium perfusion or exchange rate, feeding regime, biomass, stirring speed, pH, dissolved oxygen (DO), and lactate production. Particularly in the case of open and manual culture steps, this is challenging because any handling of the cells/tissue may impact the quality of the viable material and could potentially contaminate the culture system. Examples of fully closed production systems enabling different manipulation steps in one system are the CliniMACs Prodigy® and the Octane Technology (see Figs. 14.30 and 14.31, respectively).

A close-up of the setup of an octane technology. It has multiple liquid bags hanging, a central processing station, numbered button-like and connected ports, and an output display device. — **Fig. 14.30**

A close-up view of circular closed containers organized in a grid pattern. On the left side is a cutaway view of one of the containers, revealing a securely sealed box. — **Fig. 14.31**

A fully closed processing system is the CliniMACS Prodigy. This single-use device performs all manufacturing steps (i.e., cell wash, enrichment, activation, genetic modification, expansion, final formulation, and sampling). This contrasts with other manufacturing approaches, which use separate machines for cell culture, cell washing, and other steps in the production chain.

The manufacturing process for advanced therapies parallels the processes for E. coli/mammalian production cells described in Chap. 4 for therapeutic proteins. But they differ considerably from those processes at a number of critical points. On top of that, the various types of cell therapy products vary widely from each other. Below are examples of manufacturing process flow charts for three different types of advanced therapy medicinal products:

1.
Off-the-shelf or nonoff-the-shelf MSC production process, as described below and presented in (Fig. 14.32);
2.
Non off-the-shelf CAR-T production process, as this procedure is a prime example of “personalized medicine” (see Chap. 9) the complexity is caught both in the text below and shown in Fig. 14.33;
3.
Off-the-shelf human ESC-derived prebeta cell production process, as described below and presented in Fig. 14.34.

A flow diagram of the manufacturing process of a cell-based product. 1. Adipose tissue biopsy. 2. Tissue digestion. 3. Cell separation. 4. Cell culture. 5. Cell wash. 6. Cell concentration. 7. Cell resuspension. 8. Filling of cell suspension. 9. Storage. 10. Shipment to clinic. 11. Administration. — **Fig. 14.32**

A flow diagram of the manufacturing process of C A R T cell. 1. Patient apheresis. 2. T-cell enrichment. 3. Activation. 4. T-cell transduction. 5. Cell expansion. 6. Cell harvest and concentration. 7. Cell resuspension. 7 b. Filling in infusion. 8. Storage. 9. Shipment. 10. Administration. — **Fig. 14.33**

A flow diagram of h E S C manufacturing. 1. Isolation. 2. Pre M C B. 3. M C B. 4. W C B. 5. Culture. 6. Anterior cells. 7. Foregut cells. 8. Posterior cells. 9. Pancreatic cells. 10. Harvest. 11. Cryopreservation. 12. Thew. 13. Recovery. 14. Harvest. 15. Formulation. 16. Upload. 17. Administration. — **Fig. 14.34**

A flow diagram illustrates the leukapheresis system. 1. Leukapheresis. 2. Leukocytes are washed out. 3. The leukocytes undergo counterflow centrifugal elutriation. 4. Monocytes, red blood cells, and platelets are removed from the product. — **Fig. 14.35**

Manufacturing of MSC Product

The manufacturing of an off-the-shelf (allogeneic) or nonoff-the-shelf (autologous or allogeneic) cell-based product, e.g., MSC-derived product, is a multi-step process with slight modifications for each specific product (see Fig. 14.32).

Step 1: Starting material procurement via bone marrow (BM) aspiration (1a) or adipose tissue biopsy (1b) from a healthy donor (allogeneic cell source) or patient (autologous cell source). Other sources of MSCs are not discussed here. The donor (healthy person or patient) is tested for specific human viruses before donating the starting material.
Step 2: Mononuclear cell separation from BM (2a) using separation techniques; adipose tissue digestion using enzymes, such as collagenase (2b).
Step 3: Mononuclear cell separation from digested adipose tissue.
Step 4: MSC expansion: MSCs are adherent cells and can, therefore, either be cultured in a culture flask (2D culture) or on micro-carriers in suspension culture (3D culture). Cells grow and multiply via mitosis and meiosis. By selecting the appropriate surface and culture medium, and culture conditions, unwanted cell populations do not adhere and are separated from the wanted cell populations.
Step 5: Cell detachment from the surface via trypsinization. Cells are washed to remove dead cells, unwanted cell populations, and trypsin. Steps 4 and 5 are repeated as many times as needed for the targeted dose or to freeze-down a cell bank (MCB/WCB strategy; which is an off-the-shelf product approach).
Step 6: Cell concentration.
Step 7: Resuspension of the cells in formulation buffer.
Step 8: Filling of the cell suspension in the primary container (vial or bag) and labeling of the primary container. This is considered the drug product (DP).
Step 9: For some products, the cells are immediately shipped by a qualified courier to the side of administration after step 8. In such cases, the hospital should be at a short distance, as the product cells are generally stable for hours to a couple of days at room temperature or at 2–8 °C (short-term storage; nonoff-the-shelf product). To allow for time between product manufacture plus quality control (QC) testing plus the release of the DP and administration, and to allow for easy shipment to distant hospitals, the product is stored and shipped frozen, often in the vapor phase of liquid nitrogen at < −120 °C (long-term storage).
Step 10: Shipment of the DP to the clinical site.
Step 11: Administration to the patient systemically (IV infusion) or locally with/without the use of a surgical procedure.

Manufacturing of CAR-T Product

The manufacturing of genetically modified T cells is a multi-step process with slight modifications for each specific product (Fig. 14.33):

Step 1: Harvest of blood cells by apheresis (whole blood collection) (Fig. 14.35)) or leukapheresis (collection of leukocytes) from the patient (autologous cell source). The so-called “starting material” is shipped either “fresh,” i.e., at room temperature or at 2–8 °C, or “frozen” (≤−80 °C) to the GMP manufacturing site. The patient is tested for specific human viruses prior to the donation of the starting material.
Step 2: From this starting material, lymphocytes can be enriched either by counter-flow centrifugal elutriation or by subset selection according to the cellular phenotype.
Step 3: The enriched lymphocyte population is placed in culture and stimulated with bead-based artificial antigen-presenting cells, e.g., magnetic beads, coupled with mAbs.
Step 4: The viral vector is added to transduce the genetic insert (CAR) into the T cells.
Step 5: The cell culture is expanded in a bioreactor for several days until sufficient numbers of CAR-T cells are obtained for dosing and QC testing. A magnet removes the beads from step 3 as they are considered a process impurity.
Step 6: The T cells are harvested, washed, and concentrated.
Step 7: The cells are resuspended in the final product formulation buffer (7a) and filled in the primary container (infusion bag or vial). This is the so-called “DP” (7b). Samples are taken for quality control testing.
Step 8: For some products, the cells are immediately shipped by a qualified courier to the side of administration after step 7. In such cases, the hospital should be at a short distance, as the product cells are generally stable for hours to a couple of days at room temperature or at 2–8 °C (short-term storage). To allow for time between product manufacture plus QC testing plus the release of the DP and administration, and to allow for easy shipment to distant hospitals, the product is stored and shipped frozen, often in the vapor phase of liquid nitrogen at <−120 °C (long-term storage).
Step 9: See step 10, manufacturing of MSC product
Step 10: At the site of administration, the product is either administered directly to the patient or first thawed and sometimes washed to remove certain excipients such as dimethyl sulfoxide (DMSO) and then administered, often via IV infusion.

The chain of the identity of the entire process, from leukapheresis to infusion and throughout all manufacturing steps and vice versa, i.e., from donor to recipient and from the recipient to donor, is controlled by a computer-based system to ensure the product’s identity and product traceability.

Manufacturing of hESC Product

The manufacturing of an hESC-derived combination product (cells in device) to treat DM type I is a multi-step process with expansion and complex differentiation steps, with slight modifications for each specific product (Fig. 14.34):

Step 1: Isolation of the starting material (hESCs) via the inner cell mass extraction. This procedure can only take place after informed consent from the parent(s) and testing of the mother’s blood for specific human viruses. In addition, this step does not occur at a manufacturing facility under GMP but at an accredited tissue establishment, which is often a hospital.
Step 2: Production of the pre-MCB by hESC culture initiation, cell expansion, cell wash, cell harvest, formulation of the cells in cryogenic medium, fill in a vial, and storage under cryogenic conditions in the vapor phase of liquid nitrogen.
Step 3: Production of the MCB from a pre-MCB. A pre-MCB vial is thawed, and cells are cultured and expanded as described under “step 2,” followed by release testing of the MCB.
Step 4: Production of a WCB from the MCB (see step 3) and release testing of the WCB.
Step 5: A WCB vial is thawed, and cells are expanded to obtain the required cell number for cell differentiation. Steps 2 through 5 take a couple of weeks.
Step 6: Differentiation of undifferentiated hESCs into anterior definitive endoderm cells by adding specific growth factors and other factors to the culture medium. This step takes about 2 days.
Step 7: Differentiation of anterior definitive endoderm cells into foregut endoderm cells by adding specific growth factors and other factors to the culture medium. This step takes about 3 days.
Step 8: Differentiation of foregut endoderm cells into posterior foregut cells by adding specific growth factors and other factors to the culture medium. This step takes about 3 days.
Step 9: Differentiation of posterior foregut cells into pancreatic endoderm cells by adding specific growth factors and other factors to the culture medium. This step takes about 4 days.
Step 10: Pancreatic endoderm cells are harvested, washed, resuspended in a cryo-preservation medium, and filled in cryovials. The cryovials are labeled. This is considered the “intermediate DP.”
Step 11: The intermediate DP is cryopreserved in the vapor phase of liquid nitrogen at < −120 °C (long-term storage) and extensively QC tested prior to the release of the intermediate DP.
Step 12: Intermediate DP cryovials are thawed. In case steps 2 through 11 take place at a GMP facility on long distance from the clinical site where the drug product will be administered to the patient, the cryopreserved intermediate DP is shipped frozen to a GMP facility, often the hospital pharmacy, for preparation of the final drug product.
Step 13: Intermediate DP cells are recovered from the freezing and thawing steps by placing them in culture for another 3–4 days.
Step 14: The recovered cells are harvested and washed to remove dead cells and culture medium.
Step 15: Cells are concentrated and formulated in a buffer.
Step 16: Cells are uploaded into the immune-protective device using a loading device. The pancreatic prebeta cells in the device are considered the DP. Limited QC release testing is performed on the DP.
Step 17: The device is administered to the patient via a surgical procedure.

Key Factors for a Successful Manufacturing Process

To consistently manufacture advanced therapies at a large-scale, automated manufacturing processes as well as the implementation of functionally closed systems are key success factors for the following reasons: (1) lower the risk of viral and bacterial contamination during manual and open-process steps; (2) decrease costs associated with manual handlings; (3) improve product consistency; (4) shorten production times. Other key factors for success are logistics around the manufacturing, supply chain of the product, and the cost of goods. Particularly animal and human-derived raw materials, for example, growth factors, fetal bovine serum (FBS), antibody-coupled beads, and viral vectors, are very expensive. Considering the high cost and increased risk of validating sterilization cycles of multiple-use bioreactors, these closed-processes for advanced therapies utilize single-use, disposable bioreactors, mimicking current recombinant protein platform approaches (see Chap. 4). Despite some progress made in this field, there remains a requirement for a better understanding of potential manufacturing platforms and how they can be best utilized for advanced therapies, taking the variety of cell and tissue types and clinical applications into account.

Viral Vector Production for Ex Vivo Gene Modification of Cells

Recombinant viral vectors, e.g., retroviruses like lentiviruses (cf. section on Viral Vectors in this chapter and Fig. 14.21), are produced by transfecting packaging cells, cultured with three to four plasmids that encode viral structural proteins, e.g., GAG, POL, Vesicular stomatitis virus (VSV)-G, and REV; the so-called packaging plasmids, and the plasmid encoding the therapeutic gene of interest, e.g., CAR, ADA-SCID; the so-called transfer plasmid. The transfer plasmid encoding the therapeutic gene contains the regulatory sequences that control its expression and a packaging sequence that enables its recognition. Within the packaging cell, e.g., the human embryonic kidney (HEK) 293 cell line, the RNA transcribed from the plasmid encoding the therapeutic gene is recognized by the viral proteins that assemble around it. The recombinant virus is then transported to the plasma membrane of the packaging cell that expresses viral envelope proteins (VSV-G). During budding, the virus acquires the lipid bilayer from the packaging cell surface and incorporates the envelope proteins. The viral vector particles are released from the cells cultured as adherent cells in culture flasks into the cell culture medium. The above-described steps are considered the upstream processing (USP) steps. The virus particles are subsequently harvested from the medium, formulated in a buffer, and filled in the primary container. These production steps are considered the downstream processing (DSP) steps (Morenweiser 2005). DSP steps applied for viral vector production are traditionally used in the biotechnology industry to manufacture recombinant proteins. These are membrane-based (filtration/clarification, concentration/diafiltration using tangential flow filtration, membrane-based chromatography) and chromatography-based (ion-exchange chromatography, affinity chromatography, and size exclusion chromatography) process steps. The combination of these different process steps is variable; in some cases, different purification principles are used for the same purpose. Furthermore, a benzonase/DNase treatment for the degradation of contaminating DNA from the packaging cells is either included in the USP or DSP part of the manufacturing process. Subsequently, the purified virus particles are formulated in a buffer, filled in the primary container, stored frozen, and tested until further use for transduction of the cells to make a genetically modified cell therapy product (Wright 2008). Figure 14.36 provides a schematic overview of the entire viral vector material manufacturing process used to produce a genetically modified cell therapy product. For the production of a viral vector product for in vivo gene therapy (see later in this chapter), the production process is identical.

A flow diagram of the lentiviral vector manufacturing process from day 1 to day 15. Transient transfection, crude vector harvest, Benzonase endonuclease treatment, and chromatography. Filtration and formulation is followed by aseptic processing on day 1. — **Fig. 14.36**

Excipients

Common excipients used in the formulation of advanced therapies are presented in Table 14.15. Most of these excipients overlap with those used in therapeutic protein products. However, KCl, MgCl₂, nucleosides, FBS, and DMSO are not found in therapeutic protein drug products.

Table 14.15 Examples of excipients used in the formulation of advanced therapy products

Full size table

Table 14.16 provides an overview of a few commercially available advanced therapies with their formulation and shelf-life

Table 14.16 Examples of approved advanced therapies, their formulation, and shelf-lives

Full size table

Add to legend under ^c: DMEM = Eagle’s minimal essential medium...and then the rest of the text

Primary Container

Generally, two types of containers are used for cell-based products: vials (small volume, low dose) and infusion bags (higher volume and dose), as shown in Fig. 14.37. However, tissue-based products often have a nonstandard container for storage and shipment.

On the left is a close-up of an infusion bag. On the right is a close-up of vials inside a box. — **Fig. 14.37**

Storage and Shipment

Stability of the starting material (cells or tissue and viral vector) and DP are an important element for the successful production, storage, shipment, and administration of advanced therapies. Starting materials and DPs either have a very short shelf-life of hours–days and are stored and transported at 2–8 °C or at room temperature or have a longer shelf-life (months–years) and are stored and shipped frozen (cryopreserved in the vapor phase of liquid nitrogen at <−120 °C or in a −80 °C or −150 °C freezer).

Manufacturing Model: Scale–Up Versus Scale-Out

Broadly speaking, there are two paradigms in advanced therapy manufacture: off-the-shelf (always allogeneic source of cells/tissue) and patient-specific (commonly autologous source of starting materials, but sometimes allogeneic) DPs. Off-the-shelf products represent a business model akin to current biopharmaceuticals, where one batch can be manufactured to treat multiple patients. This allows for increased economies of scale, which drives down the per-dose cost of the final product. This means that there is a wealth of engineering and process knowledge and technologies that can be leveraged to support the manufacture of off-the-shelf advanced therapies at an increasing scale.

However, scale-up is not just about making the reactor grow the cells bigger. Conventional scale-up bioprocesses typically use cells to produce therapeutic agents (e.g., mAbs), which can then be isolated and purified without the need to recover the cell. For the manufacture of advanced therapies, where the cells/tissue culture is the product of interest, retention of cell viability, phenotype, and function to assure quality, is of primary importance in order to preserve product safety and efficacy. As the number of cells increases during expansion, this can become increasingly challenging, as the greater cell numbers lead to an increased chance of inhomogeneity of culture and hence of cellular performance being altered. This means that the desired quality of the cells/tissue must be maintained through the entire manufacturing process, including the harvest and DSP, storage, shipment, and delivery to the patient. This will require the development of scalable harvesting, DSP, and formulation technologies to cope with the large batch size produced.

Patient-specific advanced therapies offer a new challenge for process scalability, where the manufacturing process must be scaled-out in order to produce one batch for each patient (Fig. 14.38). This introduces the concept of “personalized medicine” (see Chap. 9), where the cost of production per batch cannot be reduced by exploiting an increasing economy of scale by simply producing a larger batch. Reducing the cost of these patient-specific cell- and tissue-based products must therefore be achieved by advances in engineering and manufacturing technology, reducing the number of complex, labor-intensive, and open-process steps that are commonplace in the manufacture of these products at research labs. The developments of closed and automated processes as well as process simplification, are key factors for commercial success as this will allow multiple batches to be produced in parallel (scale-out), with reduced burden of oversight by highly-trained scientists. These new processes must be GMP-compliant and closed for sterility.

A 3-part illustration of rooms with 3 staff on the left, 20 rooms with 100 staff in the center, and 200 rooms and 850 staff on the right. — **Fig. 14.38**

Testing

As for any DP, cell- and tissue-based therapies are subject to detailed characterization. This involves the assessment of quality attributes, i.e., identity, purity and impurities, viability (Cadena-Herrera et al. 2015), bioactivity (potency; Bravery et al. 2013), safety, quantity, and general attributes, such as appearance, pH, morphology both of the cellular/tissue/vector starting material and the final DP, see Table 14.17. The latter includes QC testing to allow the release of the DP for administration. In addition, at different stages of production, in-process controls are performed to assess the quality and stability of the cells/tissue during manufacture. Finally, a subset of characterization tools is used to assess the stability of the starting material(s) and DP.

Table 14.17 Examples of techniques applied for the analysis of different quality attributes of cell- and tissue-based therapies

Full size table

However, for a lot of autologous and some allogeneic DPs that are not “off-the-shelf,” performing QC tests may be challenging due to the time constraints between manufacture and administration, i.e., the shelf-life of the drug product is hours–days. Moreover, for some autologous products, all the available cell/tissue material is needed for the dose. In such cases, product release may be justified by extensive process validation; in-process control testing and/or QC testing data becoming available after product administration. These approaches require a paradigm shift in the pharma world, where traditional products are only administered after extensive testing and batch release. Adequate QC of starting materials such as cells/ tissue biopsy and viral vectors is crucial as poor-quality starting material will affect the quality of the final product. Autologous or allogeneic cells/tissue can be very heterogeneous due to the inherent donor variability (age, sex, health status, medication), the variable number of cells other than the intended cells, and because the collected cells are not in a synchronized cell cycle. In addition, the origin of the cells, e.g., MSCs of bone marrow, adipose, and cord blood origin, may have a significant impact on the activity and phenotype of the cells after manufacture.

The challenge is that a lot of the techniques used for the characterization of this heterogeneous group of products are not sensitive methods; hence they are not able to pick-up subtle changes to the process and/or to the product.

For further reading oncell- and tissue-based product characterization, see BSI PAS 93:2011. For details on testing (lot release and additional characterization) of viral vectors for ex vivo and in vivo gene therapy products, see Gombold et al. (2006a, b). Table 14.18 provides an overview of the QC testing panel for an MSC-derived and a CAR-T product.

Table 14.18 Example of QC testing panel for an MSC-derived cell based product and a CAR-T ex vivo gene therapy product

Full size table

Flow Cytometry

One of the key technologies in advanced therapy manufacturing is flow cytometry. It can be operated in a QC test environment and in the production of advanced therapies products (see next section). As this technique is not used regularly to characterize therapeutic proteins, it is not discussed in Chap. 3. Therefore, we pay attention to it in this chapter.

Flow cytometry assays may be used to assess cell- and tissue-based product identity, active substance purity, cellular impurity, viability, and potency testing. It is a powerful technique that allows for a specific measurement of cellular components on the cell surface, e.g., CD73, CD90, and CD105, to characterize MSCs, and intracellular components. It is also amenable to the measurement of soluble analyte(s) such as cytokines, released by the cells in the extracellular environment, e.g., upon cell activation.

Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light (Fig. 14.39). The properties measured include a particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity. These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence. A flow cytometer is made up of three main systems: fluidics, optics, and electronics.

The fluidics system transports single particles (cells) in a stream to the laser beam for interrogation.
The optics system consists of a light source, mostly lasers, to illuminate the particles in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors. Light scattering or fluorescence emission from auto-fluorescence of the particle or from fluorophores, which are fluorescence labels, e.g., bound to specific antibodies, used to detect the expression of cellular molecules such as specific proteins or nucleic acids, provides information about the particle’s properties. (1) Light that is scattered in the forward direction after interacting with a particle, typically up to 20° offset from the axis of the laser, is collected by a photomultiplier tube or photodiode and is known as the forward scatter (FSC) channel. This FSC measurement can estimate a particle’s size, with larger particles refracting more light than smaller particles. (2) Light measured at a 90° angle to the excitation line is called side scatter (SSC). The SSC can provide information about the relative complexity, e.g., granularity and internal structures, of a cell or particle. However, as with forward scatter, this can depend on various factors. Both FSC and SSC are unique for every particle, and a combination of the two can be used to roughly differentiate cell types in a heterogeneous population such as blood or bone marrow aspirate. However, this scatter information and cell typing depend on the sample type and the quality of sample preparation, so fluorescent labeling is generally required to obtain more detailed information.
The electronics system converts the detected light signals into electronic signals that the computer can process.
In the flow cytometer, particles are carried to the laser intercept in a fluid stream. Any suspended particle or cell from 0.2 to 150 μm in size is suitable for analysis. Cells from solid tissue must be desegregated into single cells before analysis. The portion of the fluid stream where particles are located is called the sample core. When particles pass through the laser intercept, they scatter laser light. Any fluorescent molecule present on the particle fluoresces. The scattered and fluorescent light is collected by appropriately positioned lenses. A combination of beam splitters and filters steers the scattered and fluorescent light to the appropriate detectors. The detectors produce electronic signals proportional to the optical signals striking them. Readouts are collected on each particle or single event. The characteristics or parameters of each event are based on its light scattering and fluorescent properties. The data are collected and stored in the computer. This data can be analyzed to provide information about subpopulations of cells within the sample (see Fig. 14.40).

An illustration of the structure of a flow cytometer with labeled parts. Side scatter detector, 488 by 10 filter, fluorescence, light scatter by forward scatter detector, obstruction bar, and 488 n m laser. — **Fig. 14.39**

Three flow cytometry histograms plot percent positive cells versus C D 73, C D 90, and C D 105. The peaks in the graphs are single for the two markers tested. — **Fig. 14.40**

Fluorescence-Activated Cell Sorting (FACS)

Flow cytometry techniques can also be used to sort specific cell (sub) populations, e.g., to increase product yield and/or reduce the amount of unwanted cell populations, which are considered impurities. A FACS machine provides the ability to separate cells identified by flow cytometry. Droplet-based cell sorters first analyze the particles but also have hardware that can generate droplets and deflect or direct wanted particles into a collection tube. Cell dispersions are often purified based on surface markers such as CD34+ in HSCs or on their viability. Common uses of cell sorting include identifying and isolating cell populations or single cells followed by subsequent downstream applications where DNA, protein, or cellular function is investigated.

Improvements in Testing Strategies Needed

Developing robust, sensitive, rapid, and in-line analytical testing and characterization tools will be required as cell/tissue and viral vector processing platforms continue to evolve. Significant improvements are needed to establish next-generation analytics for (in-process) QC, stability, and additional characterization testing to assess the quality attributes of starting materials, intermediates, and advanced therapy products. Improvements are also to be made in the field of in-line and online testing of cell culture conditions, e.g., pH, morphology, and viability. Reducing the sampling frequency, technical complexity, amount of sample needed, and labor intensiveness of testing are especially critical for a nonoff-the-shelf autologous ex vivo gene therapy product. This contrasts with traditional biopharmaceuticals, where a single batch of QC tested products may treat hundreds or thousands of patients. Cell processing automation will also be enabled through the development of high throughput in-process and release assays providing results in a very short time frame (minutes–hours). Advanced cell/tissue characterization techniques based on nanofluidics, transcriptomics, and proteomics, and next-generation sequencing techniques may allow a better understanding of what happens to desired cell population(s)/tissue once they are processed and before patient administration (see Chap. 9 for more details on “-omics”), both in the cytosol as well as in the extracellular environment. Examples are changes in intracellular genetic profiles and patterns within the micro-RNA and exosome pools secreted into the culture medium by the cells.

Different advanced therapy technologies are currently at different translation stages and have their particular manufacturing and testing challenges, as summarized in Table 14.19.

Table 14.19 Development stage manufacturing and testing challenges for different advanced therapy technologies, adapted from Mount et al. (2015)

Full size table

Other Aspects of Advanced Therapies

Regulatory Bodies Involved in Regulating Advanced Therapies in Europe

In Europe, the responsibility for regulating transplant products according to the public health legislation lies with the national Competent Authority for tissues and cells in each member state. ATMPs, in contrast, are regulated by pharmaceutical legislation. Hence, marketing approval must be obtained before marketing an ATMP through the centralized procedure, as for any other biological medicinal product. The scientific evaluation of these products is led by a specialized committee within the EMA (the Committee for Advanced Therapies “CAT”). The CAT drafts an opinion for the Committee for Medicinal Products for Human Use (CHMP), which is responsible for providing a second scientific opinion. Based on a positive CHMP opinion, the approval of a marketing authorization application (MAA) is granted by the European Commission. Clinical trials involving ATMPs are regulated and authorized the same manner as other medicinal products, i.e., on a national level by the appropriate national competent authority (NCA).

Regulatory Bodies Involved in Regulating Advanced Therapies in the USA

The situation in the United States is simpler in that the FDA is responsible for both aspects of the legislation: the public health and pharmaceutical legislation. Within the FDA, the responsibility for the regulation of HCT/Ps and human gene therapy products lies with the Center for Biologics Evaluation and Research (CBER), both for clinical trials and marketing authorization. As of 2016, the CBER structure includes the Office of Blood Research and Review (OBRR), the Office of Vaccines Research and Review (OVRR), and the Office of Tissues and Advanced Therapies (OTAT), which was formerly known as the Office of Cellular, Tissues and Gene Therapies (OCTGT). To monitor activity, review data, and anticipate future needs, the FDA operates the Cellular, Tissue, and Gene Therapies Advisory Committee.

Regulatory Guidances

Links to the relevant regulatory bodies involved in advanced therapies in the EU and US, as well as applicable guidances, can be found in Table 14.20.

Table 14.20 Regulatory agencies and applicable guidances for advanced therapies in the US and EU

Full size table

Stem Cell Tourism

The general interest in advanced therapies worldwide has allowed unregulated practice, particularly of cell-based products, to develop in some countries, i.e., “stem cell tourism.” This is a major concern for many stakeholders in the field of ATMPs because treatments are being offered in the absence of a strong safety data package and any proven efficacy. In addition, there is suspicion that the products in use have been manufactured with insufficient attention to GMP, including quality control. Patients must be warned of the dangers of falling prey to unethical operations. An up-to-date source of information on private clinics and stem cell tourism is available at the website of the International Society for Stem Cell Research (www.isscr.org).

Concluding Remarks

Although progress has been made in the area of ATMPs, with about 80 products approved globally and 27 in the USA & EU for commercial use (see examples in Table 14.6) and many products in clinical development, this field was currently struggling with similar problems as the first recombinant proteins 20 years ago. Appropriate manufacturing platforms, supply chain models, healthcare systems, reimbursement models, and regulatory frameworks for these medicinal products need to be established by developers and other key stakeholders, while specific knowledge about quality (production and testing), safety, and efficacy of advanced therapies is steadily growing.

Self-Assessment Questions

Questions

1.
What is the difference between embryonic and adult stem cells?
2.
How is somatic cell nuclear transfer carried out, and what are the problems with this technique?
3.
What are iPSCs, and why are they important?
4.
What is the difference between in vivo gene therapy and ex vivo gene therapy?
5.
Which disease areas are predominantly investigated clinically with ATMPs?
6.
What problems could arise in the use of stem cell-derived products for clinical application?
7.
What was the disease target for the first gene therapy clinical trial? What vector was selected for gene transfer?
8.
Identify and describe five transcription regulatory elements discussed in the chapter.
9.
Several clinical trials involve gene transfer for treating malignant glioma. One approach involves the use of a recombinant retrovirus expressing the HSV-tk transgene. Another involves the use of a recombinant adenovirus expressing the p53 transgene.
1. (a)
  Which of the five current strategies to treat cancer by viral gene therapy does each of these trials employ? Describe the principle behind each strategy.
2. (b)
  List two advantages and two disadvantages associated with the vector used in each of these trials.
3. (c)
  Outline potential drawbacks to the use of each of these strategies for cancer therapy.
4. (d)
  What other approaches could have been selected to prevent the growth and spread of malignant tissue? Explain the principle behind each.
10.
What is the purpose of the packaging cell line during the production of recombinant viral vectors for gene transfer? What is the risk associated with using packaging cell lines for vector production?
11.
Provide two examples of how gene therapy is used to modulate the immune system to fight infection.
12.
Describe one clinical trial for retrovirus-based gene therapy and adenovirus-based gene therapy and identify the most significant adverse effects that have been reported for each trial.
13.
What can be incorporated into viral vector design to reduce genotoxic risk?
14.
How can preexisting immunity to viral vectors be circumvented?
15.
Name two advantages of adenovirus vectors as opposed to AAV vectors and vice versa.
16.
How can adenoviruses be engineered to selectively replicate in tumor cells?

Answers

1.
Embryonic stem cells are grown ex vivo after extraction of the inner cell mass from a blastocyst. Adult stem cells are found in vivo in many tissues, usually in the specialized environment of a stem cell niche that supports their asymmetric cell division.
2.
Somatic cell nuclear transfer (SCNT) involves the injection of a donor genome into an enucleated egg, such that the embryo develops as a clone of the donor genome. This allows the generation of embryonic stem cells using the donor genome and, in principle, allows implantation into the uterus of a recipient female leading to pregnancy. There are ethical problems concerning the supply of fertilized human eggs and technical problems caused by incomplete reprogramming of the donor nucleus.
3.
iPSCs are produced by transient expression of pluripotency genes in somatic cells, leading to reprogramming to form pluripotent cells resembling embryonic stem cells. The production of iPSCs allows pluripotent cells to be obtained from a patient without the need for SCNT. iPSCs can be used to derive differentiated cells for producing ATMPs for clinical application or disease modeling purposes.
4.
In vivo gene therapy refers to the direct introduction of genetic material into the human body, whereas ex vivo gene therapy refers to the use of cells, which are genetically modified outside the body (i.e., ex vivo) prior to administration of these genetically modified cells into the human body. In the latter case, the genetic material is introduced into the human body using cells as “delivery system”. See also “Gene Therapy” section.
5.
Various cancers, autoimmune disorders, such as DM type I and Crohn’s disease, neurological disorders, such as Parkinson’s disease and Alzheimer’s disease, myocardial infarction, and macular degeneration.
6.
One of the concerns with stem cell-derived ATMPs is the possibility that rare pluripotent or multipotent cells in the product could give rise to tumors after administration to humans, i.e., tumorigenicity risk. Thus, the quality control of medicinal products is of paramount importance. Often, in particular, in the treatment of neurological diseases, it is not clear whether a progenitor, precursor or fully mature cell should be administered. Careful preclinical work is required in each clinical indication to establish the most effective approach. Where the strategy is designed to replace a cell that is lost in a particular disease, the environment into which the cell-based medicinal product is placed may not be supportive of cell survival and integration/persistence. In general, one needs to pay attention to providing a protective environment for the medicinal product
7.
The first gene therapy clinical trial was initiated in 1990 for treating adenosine deaminase (ADA) deficiency. In this trial, patients with ADA deficiency were given peripheral blood lymphocytes treated with a retroviral vector expressing the ADA transgene.
8.
Promoter is a DNA sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase and transcriptional factors. Enhancer is a short DNA sequence that can bind transcription factors or activators to enhance transcription levels of a gene from a distance. Insulators are genetic boundary elements that block the enhancer-promoter interaction or rarely act as a barrier against condensed chromatin proteins. Finally, operators and silencers are usually short DNA sequences close to the promoter with binding affinity to a set of proteins named repressors and inducers.
9.
(a) Retrovirus trial

Gene-directed enzyme-prodrug therapy. Cells transduced by the virus express an enzyme capable of converting a prodrug (in this case, ganciclovir) to a cytotoxic metabolite. This conversion cannot occur in cells that do not express the transgene, limiting the cytotoxic effect to transduced cells and their neighbors through the bystander effect.

Adenovirus trial

Correction of genetic mutations that contribute to a malignant phenotype. Cells transduced by the virus express a gene such as p53 that is necessary for controlled cell division and development. This prevents the uncontrolled growth and division associated with malignant disease.

(b) Retrovirus

Advantages—(i) Retroviruses can infect dividing cells which are the therapeutic target in this trial. Despite this fact, the transduction efficiency of this vector in vivo has been low. (ii) Retroviruses can induce long-term gene expression, which should be sufficient to effectively remove malignant tissue.

Disadvantages—(i) Retroviruses have the potential for inducing insertional mutagenesis in normal, healthy cells. (ii) Transgene expression is sometimes limited by the host immune response to cellular components acquired by the virus during large-scale production.

Adenovirus

Advantages—(i) Adenoviruses can infect dividing cells, the therapeutic target in this trial. (ii) Adenoviruses can induce high levels of transgene expression in short periods of time. (iii) Adenoviruses do not have the risk of insertional mutagenesis. (iv) It is relatively easy to produce large amounts of recombinant adenovirus sufficient for clinical use.

Disadvantages—(i) Transgene expression is transient, making readministration necessary for continued effect. (ii) Adenoviral vectors can induce a potent immune response. This limits the success of gene transfer after a second dose of virus and is associated with severe toxicity at certain doses. (iii) Preexisting immunity to adenovirus serotype 5 is common in the general population. This may also limit gene transfer.

(c) Drawbacks to gene-directed enzyme-prodrug therapy.
1. (i)
  Efficacy relies on efficient transgene expression and drug bioavailability. (ii) The therapeutic effect may spread to healthy cells through the bystander effect.
Drawbacks to gene correction therapy.
1. (ii)
  Gene correction may stop tumor growth but not eliminate it. (ii) Expression is not limited to malignant tissue.
(d) Other approaches for cancer gene therapy
1. (i)
  Immunotherapy. A vector expressing pro-inflammatory cytokines, co-stimulatory molecules, or tumor-specific antigens is injected directly into the tumor mass. This facilitates the formation of an antitumor immune response that targets and destroys malignant cells.
2. (ii)
  Virotherapy. A replication-competent virus naturally targeting cancers is directly injected into the tumor mass. The virus can induce cell death during replication in malignant tissue by producing cytotoxic proteins and subsequent cell lysis.
10.
(i) The primary purpose of the packaging cell line is to provide genetic elements that support virus replication and assembly. These have been eliminated from the vector to prevent it from causing disease in the patient. (ii) The recombinant virus can incorporate elements for replication into its genome through homologous recombination during the production process. The potential for the generation of replication-competent virus (RCV) in this manner does exist for each vector but can vary due to specific features of a given packaging cell line.
11.
(i) Gene transfer into autologous immunocytes to increase the immune system of a patient. (ii) Overexpression of protein inhibitors that interfere with virus infection and replication. (iii) Overexpression of known antigenic epitopes of the pathogen by DNA vaccination to stimulate an immune response.
12.
(i) One trial employed aerosol administration of a recombinant adenovirus expressing cystic fibrosis transmembrane conductance regulator (CFTR) to treat cystic fibrosis (CF). Another trial employed a recombinant retrovirus expressing recombinant adenosine deaminase (ADA) to transduce autologous T lymphocytes isolated from patients for treating ADA deficiency-induced severe combined immunodeficiency (ADA-SCID). (ii) CF trial. Massive immune response to the recombinant viral vector.

ADA-SCID trial. Lymphoproliferative leukemia is caused by insertional mutagenesis.
13.
Self-inactivating configurations can be incorporated to enable the use of physiological and cell type specific promoters, which can reduce potential genotoxic risk. In addition, using viral vector systems with more random integration site profiles and with reduced ability to cause aberrant splicing.
14.
Preexisting immunity to viral vectors can be circumvented by using serotypes that have no or low prevalence in the human population, including virus variants of nonhuman origin.
15.
Adenovirus vectors can be used to achieve transient transgene expression, and it has a higher packaging capacity than AAV. On the other hand, AAV can be used to get potentially long-term transgene expression, and the risk for strong inducing (too) strong immune responses is lower.
16.
Initially, these were generated by partial deletion of E1B, restricting genome replication to cells that lack p53 such as tumor cells. More recently, they have been generated by removing a specific stretch of 24 amino acids from the E1A protein. These so-called AdΔ24 or AdΔRb vectors are unable to bind to the retinoblastoma (Rb) protein. Rb normally retains E2F, preventing the cells from entering the S-phase and thereby replicating the genome. Cancer cells often have an aberrated Rb pathway, facilitating the S-phase and AdΔRb24 replication independent from the Rb-binding activities of E1A.

References

Ankrum JA, Ong JF, Karp JM (2014) Mesenchymal stem cells: immune evasive, not immune privileged. Nat Biotechnol 32:252–260
Article CAS PubMed PubMed Central Google Scholar
Antoniou P, Miccio A, Brusson M (2021) Base and prime editing technologies for blood disorders. Front Genome Ed 3:618406
Google Scholar
Anzalone AV, Randolph PB, Davis JR et al (2019) Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. https://doi.org/10.1038/s41586-019-1711-4
Armentano D, Sookdeo CC, Hehir KM et al (1995) Characterization of an adenovirus gene transfer vector containing an E4 deletion. Hum Gene Ther 6:1343–1353
Article CAS PubMed Google Scholar
Arrand JR, Roberts RJ (1979) The nucleotide sequences at the termini of adenovirus-2 DNA. J Mol Biol 128:577–594
Article CAS PubMed Google Scholar
Bajaj P, Schweller RM, Khademhosseini A et al (2014) 3D biofabrication strategies for tissue engineering and regenerative medicine. Annu Rev Biomed Eng 16:247–276
Article CAS PubMed PubMed Central Google Scholar
Bar and Benvenisty. EMBO J. 2019. https://doi.org/10.15252/embj.2018101033
Bartholomae CC, Arens A, Balaggan KS et al (2011) Lentiviral vector integration profiles differ in rodent postmitotic tissues. Mol Ther 19:703–710
Article CAS PubMed PubMed Central Google Scholar
Becker J, Fakhiri J, Grimm D (2022) Fantastic AAV gene therapy vectors and how to find them-random diversification, rational design and machine learning. Pathogens 11:756
Article CAS PubMed PubMed Central Google Scholar
Belete TM (2021) The Current Status of Gene Therapy for the Treatment of Cancer. Biologics 15:67–77
PubMed PubMed Central Google Scholar
Bianco P, Robey PG, Simmons PJ (2008) Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2:313–319
Article CAS PubMed PubMed Central Google Scholar
Biasco L, Ambrosi A, Pellin D et al (2011) Integration profile of retroviral vector in gene therapy treated patients is cell-specific according to gene expression and chromatin conformation of target cell. EMBO Mol Med 3:89–101
Article CAS PubMed PubMed Central Google Scholar
Biffi A, Bartolomae CC, Cesana D et al (2011) Lentiviral vector common integration sites in preclinical models and a clinical trial reflect a benign integration bias and not oncogenic selection. Blood 117:5332–5339
Article CAS PubMed Google Scholar
Blaese RM, Culver KW, Miller AD et al (1995) T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years. Science 270:475–80
Google Scholar
Boorjian SA, Alemozaffar M, Konety BR et al (2021) Intravesical nadofaragene firadenovec gene therapy for BCG-unresponsive non-muscle-invasive bladder cancer: a single-arm, open-label, repeat-dose clinical trial. Lancet Oncol 22:107–117
Article CAS PubMed Google Scholar
Bots STF, Hoeben RC (2020) Non-human primate-derived adenoviruses for future use as oncolytic agents? Int J Mol Sci 21:4821
Article CAS PubMed PubMed Central Google Scholar
Brassard JA, Lutolf MP (2019) Engineering Stem Cell Self-organization to Build Better Organoids. Cell Stem Cell 24:860–876
Article CAS PubMed Google Scholar
Bravery CA (2015) Do human leukocyte antigen-typed cellular therapeutics based on induced pluripotent stem cells make commercial sense? Stem Cells Dev 24:1–10
Article PubMed Google Scholar
Bravery CA, Carmen J, Fong T et al (2013) Potency assay development for cellular therapy products: an ISCT review of the requirements and experiences in the industry. Cytotherapy 15:9–19
Article PubMed Google Scholar
Bukrinsky MI, Sharova N, Dempsey MP et al (1992) Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proc Natl Acad Sci U S A 89:6580–6584
Article CAS PubMed PubMed Central Google Scholar
Bulcha JT, Wang Y, Ma H et al (2021) Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther 6:53
Article CAS PubMed PubMed Central Google Scholar
Büning H (2013) Gene therapy enters the pharma market: the short story of a long journey. EMBO Mol Med 5:1–3
Article PubMed PubMed Central Google Scholar
Byun S, Lee M, Kim M (2022) Gene therapy for Huntington’s disease: The final strategy for a cure? J Mov Disord 15:15–20
Article PubMed Google Scholar
Cadena-Herrera D, Esparza-De Lara JE, Ramírez-Ibañez ND et al (2015) Validation of three viable-cell counting methods: Manual, semi-automated, and automated. Biotechnol Rep (Amst) 7:9–16
Article PubMed Google Scholar
Cattoglio C, Facchini G, Sartori D et al (2007) Hot spots of retroviral integration in human CD34 hematopoietic cells. Blood 110:1770–1778
Article CAS PubMed Google Scholar
Chand D, Mohr F, McMillan H et al (2021) Hepatotoxicity following administration of onasemnogene abeparvovec (AVXS-101) for the treatment of spinal muscular atrophy. J Hepatol 74:560–566
Article CAS PubMed Google Scholar
Chen G, Abdeen AA, Wang Y et al (2019) A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing. Nat Nanotechnol. https://doi.org/10.1038/s41565-019-0539-2
Chen H, Xiang ZQ, Li Y et al (2010) Adenovirus-based vaccines: comparison of vectors from three species of adenoviridae. J Virol 84:10522–10532
Article CAS PubMed PubMed Central Google Scholar
Chen HH, Mack LM, Kelly R et al (1997) Persistence in muscle of an adenoviral vector that lacks all viral genes. Proc Natl Acad Sci U S A 94:1645–1650
Article CAS PubMed PubMed Central Google Scholar
Chiang C-Y, Ligunas GD, Chin W-C, Ni C-W (2020) Efficient nonviral stable transgenesis mediated by retroviral integrase. Mol Ther Methods Clin Dev 17:1061–1070
Article CAS PubMed PubMed Central Google Scholar
Chiesa R, Bernardo ME (2022) Haematopoietic stem cell gene therapy in inborn errors of metabolism. Br J Haematol 198:227–243
Article CAS PubMed Google Scholar
Chin MH, Pellegrini M, Plath K, Lowry WE (2010) Molecular analyses of human induced pluripotent stem cells and embryonic stem cells. Cell Stem Cell 7:263–269
Article CAS PubMed PubMed Central Google Scholar
Consentius C, Reinke P, Volk H-D (2015) Immunogenicity of allogeneic mesenchymal stromal cells: what has been seen in vitro and in vivo? Regen Med 10:305–315
Article CAS PubMed Google Scholar
Corradetti B, Gonzalez D, Mendes Pinto I, Conlan RS (2021) Editorial: Exosomes as therapeutic systems. Front Cell Dev Biol 9:714743
Article PubMed PubMed Central Google Scholar
Croyle MA, Cheng X, Wilson JM (2001) Development of formulations that enhance the physical stability of viral vectors for human gene therapy. Gene Ther 8:1281–1291
Article CAS PubMed Google Scholar
Dai Y, Schwarz EM, Gu D et al (1995) Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc Natl Acad Sci U S A 92:1401–1405
Article CAS PubMed PubMed Central Google Scholar
Daud AI, DeConti RC, Andrews S et al (2008) Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol 26:5896–5903
Article CAS PubMed PubMed Central Google Scholar
David RM, Doherty AT (2017) Viral vectors: The road to reducing genotoxicity. Toxicol Sci 155:315–325
Article CAS PubMed Google Scholar
Deichmann A, Brugman MH, Bartholomae CC et al (2011) Insertion sites in engrafted cells cluster within a limited repertoire of genomic areas after gammaretroviral vector gene therapy. Mol Ther 19:2031–2039
Article CAS PubMed PubMed Central Google Scholar
Deichmann A, Hacein-Bey-Abina S, Schmidt M et al (2007) Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy. J Clin Invest 117:2225–2232
Article CAS PubMed PubMed Central Google Scholar
Dharan A, Bachmann N, Talley S et al (2020) Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus. Nat Microbiol 5:1088–1095
Article CAS PubMed PubMed Central Google Scholar
Edelstein ML, Abedi MR, Wixon J, Edelstein RM (2004) Gene therapy clinical trials worldwide 1989-2004-an overview. J Gene Med 6:597–602
Article PubMed Google Scholar
Eichler F, Duncan C, Musolino PL et al (2017) Hematopoietic stem-cell gene therapy for cerebral adrenoleukodystrophy. N Engl J Med 377:1630–1638
Article CAS PubMed PubMed Central Google Scholar
Ertl HCJ (2005) Challenges of immune responses in gene replacement therapy. IDrugs 8:736–738
PubMed Google Scholar
Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156
Article CAS PubMed Google Scholar
Eyquem J, Mansilla-Soto J, Giavridis T, et al (2017) Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543:113–117
Google Scholar
Ferrua F, Aiuti A (2017) Twenty-Five Years of Gene Therapy for ADA-SCID: From Bubble Babies to an Approved Drug. Hum Gene Ther 28:972–981
Article CAS PubMed Google Scholar
Finn JD, Smith AR, Patel MC et al (2018) A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing. Cell Rep 22:2227–2235
Article CAS PubMed Google Scholar
Fischer A, Hacein-Bey-Abina S (2020) Gene therapy for severe combined immunodeficiencies and beyond. J Exp Med 217. https://doi.org/10.1084/jem.20190607
Flotte T, Carter B, Conrad C et al (1996) A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease. Hum Gene Ther 7:1145–1159
Article CAS PubMed Google Scholar
Frangoul H, Altshuler D, Cappellini MD et al (2021) CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N Engl J Med 384:252–260
Article CAS PubMed Google Scholar
Fritz CC, Green MR (1996) HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs. Curr Biol 6:848–854
Article CAS PubMed Google Scholar
Fumagalli F, Calbi V, Natali Sora MG et al (2022) Lentiviral haematopoietic stem-cell gene therapy for early-onset metachromatic leukodystrophy: long-term results from a non-randomised, open-label, phase 1/2 trial and expanded access. Lancet 399:372–383
Article CAS PubMed PubMed Central Google Scholar
Gaggar A, Shayakhmetov DM, Lieber A (2003) CD46 is a cellular receptor for group B adenoviruses. Nat Med 9:1408–1412
Article CAS PubMed Google Scholar
Galderisi U, Peluso G, Di Bernardo G (2022) Clinical Trials Based on Mesenchymal Stromal Cells are Exponentially Increasing: Where are We in Recent Years? Stem Cell Rev Rep 18:23–36
Article PubMed Google Scholar
Gillmore JD, Gane E, Taubel J et al (2021) CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. N Engl J Med 385:493–502
Article CAS PubMed Google Scholar
Ginn SL, Amaya AK, Alexander IE et al (2018) Gene therapy clinical trials worldwide to 2017: An update. J Gene Med 20:e3015
Article PubMed Google Scholar
Golchin A, Chatziparasidou A, Ranjbarvan P et al (2021) Embryonic Stem Cells in Clinical Trials: Current Overview of Developments and Challenges. Adv Exp Med Biol 1312:19–37
Article CAS PubMed Google Scholar
Gombold J, Peden K, Gavin D et al (2006a) Lot Release and Characterization Testing of Live-Virus-Based Vaccines and Gene Therapy Products, Part 1 Factors Influencing Assay Choices. Bioprocess Int 4:46–54
CAS Google Scholar
Gombold J, Peden K, Gavin D et al (2006b) Lot release and characterization testing of live-virus based vaccines and gene therapy products part 2: Case Studies and Discussion. Bioprocess Int 4:56–65
CAS Google Scholar
Goodwin J, Laslett AL, Rugg-Gunn PJ (2020) The application of cell surface markers to demarcate distinct human pluripotent states. Exp Cell Res 387:111749
Article CAS PubMed PubMed Central Google Scholar
Gupta RK, Abdul-Jawad S, McCoy LE et al (2019) HIV-1 remission following CCR5Δ32/Δ32 haematopoietic stem-cell transplantation. Nature 568:244–248
Article CAS PubMed PubMed Central Google Scholar
Gurumoorthy N, Nordin F, Tye GJ et al (2022) Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through. Biomedicines 10. https://doi.org/10.3390/biomedicines10010107
Han DW, Tapia N, Hermann A et al (2012) Direct reprogramming of fibroblasts into neural stem cells by defined factors. Cell Stem Cell 10:465–472
Article CAS PubMed Google Scholar
Harrington K, Freeman DJ, Kelly B et al (2019) Optimizing oncolytic virotherapy in cancer treatment. Nat Rev Drug Discov 18:689–706
Article CAS PubMed Google Scholar
Herberts CA, Kwa MSG, Hermsen HPH (2011) Risk factors in the development of stem cell therapy. J Transl Med 9:29
Article PubMed PubMed Central Google Scholar
Hybiak J, Jankowska K, Machaj F et al (2020) Reprogramming and transdifferentiation - two key processes for regenerative medicine. Eur J Pharmacol 882:173202
Article CAS PubMed Google Scholar
Ilic D, Devito L, Miere C, Codognotto S (2015) Human embryonic and induced pluripotent stem cells in clinical trials. Br Med Bull 116:19–27
Google Scholar
Jia F, Wilson KD, Sun N et al (2010) A nonviral minicircle vector for deriving human iPS cells. Nat Methods 7:197–199
Article CAS PubMed PubMed Central Google Scholar
John Wiley and Sons LTD (2022) Gene Therapy Clinical Trials Worldwide. In: The Journal of Gene Medicine Clinical Trial Site. https://a873679.fmphost.com/fmi/webd/GTCT. Accessed 24 Dec 2022
Kaufman HL, Shalhout SZ, Iodice G (2022) Talimogene laherparepvec: Moving from first-in-class to best-in-class. Front Mol Biosci 9:834841
Article CAS PubMed PubMed Central Google Scholar
Kean TJ, Lin P, Caplan AI, Dennis JE (2013) MSCs: Delivery Routes and Engraftment, Cell-Targeting Strategies, and Immune Modulation. Stem Cells Int 2013:732742
Article PubMed PubMed Central Google Scholar
Keller G (2005) Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev 19:1129–1155
Article CAS PubMed Google Scholar
Kim JY, Nam Y, Rim YA, Ju JH (2022) Review of the Current Trends in Clinical Trials Involving Induced Pluripotent Stem Cells. Stem Cell Rev Rep 18:142–154
Article PubMed Google Scholar
Kim W, Lee S, Kim HS et al (2018) Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth. Genome Res 28:374–382
Article CAS PubMed PubMed Central Google Scholar
Kleinstiver BP, Pattanayak V, Prew MS et al (2016) High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490–495
Article CAS PubMed PubMed Central Google Scholar
Kohn LA, Kohn DB (2021) Gene Therapies for Primary Immune Deficiencies. Front Immunol 12:648951
Article CAS PubMed PubMed Central Google Scholar
Kurt IC, Zhou R, Iyer S et al (2021) CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat Biotechnol 39:41–46
Article CAS PubMed Google Scholar
Kuzmin DA, Shutova MV, Johnston NR et al (2021) The clinical landscape for AAV gene therapies. Nat Rev Drug Discov 20:173–174
Article CAS PubMed Google Scholar
LaFountaine JS, Fathe K, Smyth HDC (2015) Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9. Int J Pharm 494:180–194
Article CAS PubMed Google Scholar
Lander ES, Baylis F, Zhang F et al (2019) Adopt a moratorium on heritable genome editing. Nature Publishing Group UK. https://doi.org/10.1038/d41586-019-00726-5. Accessed 23 Dec 2022
Book Google Scholar
Leibacher J, Henschler R (2016) Biodistribution, migration and homing of systemically applied mesenchymal stem/stromal cells. Stem Cell Res Ther 7:7
Article PubMed PubMed Central Google Scholar
Lennon MJ, Rigney G, Raymont V, Sachdev P (2021) Genetic therapies for Alzheimer’s disease: A scoping review. J Alzheimers Dis 84:491–504
Article CAS PubMed Google Scholar
Levenstein ME, Ludwig TE, Xu R-H et al (2006) Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells 24:568–574
Article CAS PubMed Google Scholar
Levine BL, Humeau LM, Boyer J et al (2006) Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci U S A 103:17372–17377
Article CAS PubMed PubMed Central Google Scholar
Levine BL, Miskin J, Wonnacott K, Keir C (2017) Global Manufacturing of CAR T Cell Therapy. Mol Ther Methods Clin Dev 4:92–101
Google Scholar
Li L, Allen C, Shivakumar R, Peshwa MV (2013) Large volume flow electroporation of mRNA: clinical scale process. Methods Mol Biol 969:127–138
Article CAS PubMed Google Scholar
Liang M (2018) Oncorine, the world first oncolytic virus medicine and its update in China. Curr Cancer Drug Targets 18:171–176
Article CAS PubMed Google Scholar
Liu C, Su C (2019) Design strategies and application progress of therapeutic exosomes. Theranostics 9:1015–1028
Article CAS PubMed PubMed Central Google Scholar
Liu J, Verma PJ (2015) Synthetic mRNA Reprogramming of Human Fibroblast Cells. Methods Mol Biol 1330:17–28
Article CAS PubMed Google Scholar
Lochmüller H, Petrof BJ, Pari G et al (1996) Transient immunosuppression by FK506 permits a sustained high-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscles of adult dystrophic (mdx) mice. Gene Ther 3:706–716
PubMed Google Scholar
Louis N, Evelegh C, Graham FL (1997) Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line. Virology 233:423–429
Article CAS PubMed Google Scholar
Lui KO, Waldmann H, Fairchild PJ (2009) Embryonic stem cells: overcoming the immunological barriers to cell replacement therapy. Curr Stem Cell Res Ther 4:70–80
Article CAS PubMed Google Scholar
Macedo N, Miller DM, Haq R, Kaufman HL (2020) Clinical landscape of oncolytic virus research in 2020. J Immunother Cancer 8. https://doi.org/10.1136/jitc-2020-001486
MacGregor RR (2001) Clinical protocol. A phase 1 open-label clinical trial of the safety and tolerability of single escalating doses of autologous CD4 T cells transduced with VRX496 in HIV-positive subjects. Hum Gene Ther 12:2028–2029
CAS PubMed Google Scholar
Mahato RI, Rolland A, Tomlinson E (1997) Cationic lipid-based gene delivery systems: pharmaceutical perspectives. Pharm Res 14:853–859
Article CAS PubMed Google Scholar
Majhen D, Ambriovic-Ristov A (2006) Adenoviral vectors-how to use them in cancer gene therapy? Virus Res 119:121–133
Article CAS PubMed Google Scholar
Manilla P, Rebello T, Afable C et al (2005) Regulatory considerations for novel gene therapy products: a review of the process leading to the first clinical lentiviral vector. Hum Gene Ther 16:17–25
Article CAS PubMed Google Scholar
Manno CS, Pierce GF, Arruda VR, et al (2006) Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 12:342–347
Google Scholar
Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78:7634–7638
Article CAS PubMed PubMed Central Google Scholar
Männistö M, Vanderkerken S, Toncheva V, et al (2002) Structure-activity relationships of poly(L-lysines): effects of pegylation and molecular shape on physicochemical and biological properties in gene delivery. J Control Release 83:169–182
Google Scholar
McKenzie DL, Collard WT, Rice KG (1999) Comparative gene transfer efficiency of low molecular weight polylysine DNA-condensing peptides. J Pept Res 54:311–318
Article CAS PubMed Google Scholar
Medina-Kauwe LK (2003) Endocytosis of adenovirus and adenovirus capsid proteins. Adv Drug Deliv Rev 55:1485–1496
Article CAS PubMed Google Scholar
Melo LG, Pachori AS, Gnecchi M, Dzau VJ (2005) Genetic therapies for cardiovascular diseases. Trends Mol Med 11:240–250
Article CAS PubMed Google Scholar
Merkert S, Martin U (2016) Targeted genome engineering using designer nucleases: State of the art and practical guidance for application in human pluripotent stem cells. Stem Cell Res 16:377–386
Article CAS PubMed Google Scholar
Miyoshi H, Takahashi M, Gage FH, Verma IM (1997) Stable and efficient gene transfer into the retina using an HIVbased lentiviral vector. Proc Natl Acad Sci U S A 94:10319–10323
Google Scholar
Moiani A, Miccio A, Rizzi E et al (2013) Deletion of the LTR enhancer/promoter has no impact on the integration profile of MLV vectors in human hematopoietic progenitors. PLoS One 8:e55721
Article CAS PubMed PubMed Central Google Scholar
Morenweiser R (2005) Downstream processing of viral vectors and vaccines. Gene Ther 12(Suppl 1):S103–S110
Article CAS PubMed Google Scholar
Morgan RA, Walker R, Carter CS et al (2005) Preferential survival of CD4+ T lymphocytes engineered with anti-human immunodeficiency virus (HIV) genes in HIV-infected individuals. Hum Gene Ther 16:1065–1074
Article CAS PubMed Google Scholar
Mount NM, Ward SJ, Kefalas P, Hyllner J (2015) Cell-based therapy technology classifications and translational challenges. Philos Trans R Soc Lond B Biol Sci 370:20150017
Article PubMed PubMed Central Google Scholar
Muul LM, Tuschong LM, Soenen SL, et al (2003) Persistence and expression of the adenosine deaminase gene for 12 years and immune reaction to gene transfer components: long-term results of the first clinical gene therapy trial. Blood 101:2563–2569
Google Scholar
Murry CE, Keller G (2008) Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132:661–680
Article CAS PubMed Google Scholar
Nemerow GR, Stewart PL (1999) Role of alpha(v) integrins in adenovirus cell entry and gene delivery. Microbiol Mol Biol Rev 63:725–734
Article CAS PubMed PubMed Central Google Scholar
Nguyen GN, Everett JK, Kafle S et al (2021) A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells. Nat Biotechnol 39:47–55
Article CAS PubMed Google Scholar
Nilsson EC, Storm RJ, Bauer J et al (2011) The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis. Nat Med 17:105–109
Article CAS PubMed Google Scholar
Nori S, Okada Y, Nishimura S et al (2015) Long-term safety issues of iPSC-based cell therapy in a spinal cord injury model: oncogenic transformation with epithelial-mesenchymal transition. Stem Cell Reports 4:360–373
Article CAS PubMed PubMed Central Google Scholar
Panakanti R, Mahato RI (2009) Bipartite adenoviral vector encoding hHGF and hIL-1Ra for improved human islet transplantation. Pharm Res 26:587–596
Article CAS PubMed Google Scholar
Pearson S, Jia H, Kandachi K (2004) China approves first gene therapy. Nat Biotechnol 22:3–4
Article CAS PubMed PubMed Central Google Scholar
Pina S, Ribeiro VP, Marques CF et al (2019) Scaffolding Strategies for Tissue Engineering and Regenerative Medicine Applications. Materials 12. https://doi.org/10.3390/ma12111824
Platt RJ, Chen S, Zhou Y et al (2014) CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159:440–455
Article CAS PubMed PubMed Central Google Scholar
Pollock K, Stroemer P, Patel S et al (2006) A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke. Exp Neurol 199:143–155
Article PubMed Google Scholar
Pupo A, Fernández A, Low SH et al (2022) AAV vectors: The Rubik’s cube of human gene therapy. Mol Ther 30:3515–3541
Article CAS PubMed PubMed Central Google Scholar
Quintana-Bustamante O, Fañanas-Baquero S, Dessy-Rodriguez M et al (2022) Gene editing for inherited red blood cell diseases. Front Physiol 13:848261
Article PubMed PubMed Central Google Scholar
Raes L, De Smedt SC, Raemdonck K, Braeckmans K (2021) Non-viral transfection technologies for next-generation therapeutic T cell engineering. Biotechnol Adv 49:107760
Article CAS PubMed Google Scholar
Ramakrishna S, Kwaku Dad A-B, Beloor J et al (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 24:1020–1027
Article CAS PubMed PubMed Central Google Scholar
Ranga U, Woffendin C, Verma S et al (1998) Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals. Proc Natl Acad Sci U S A 95:1201–1206
Article CAS PubMed PubMed Central Google Scholar
Raper SE, Chirmule N, Lee FS et al (2003) Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 80:148–158
Article CAS PubMed Google Scholar
Reilly JP, Grise MA, Fortuin FD, et al (2005) Long-term (2-year) clinical events following transthoracic intramyocardial gene transfer of VEGF-2 in no-option patients. J Interv Cardiol 18:27–31
Google Scholar
Rubin H (2011) The early history of tumor virology: Rous, RIF, and RAV. Proc Natl Acad Sci U S A 108:14389–14396
Article CAS PubMed PubMed Central Google Scholar
Sago CD, Lokugamage MP, Paunovska K et al (2018) High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing. Proc Natl Acad Sci U S A 115:E9944–E9952
Article CAS PubMed PubMed Central Google Scholar
Salmon F, Grosios K, Petry H (2014) Safety profile of recombinant adeno-associated viral vectors: focus on alipogene tiparvovec (Glybera®). Expert Rev Clin Pharmacol 7:53–65
Article CAS PubMed Google Scholar
Sandoval-Villegas N, Nurieva W, Amberger M, Ivics Z (2021) Contemporary Transposon Tools: A Review and Guide through Mechanisms and Applications of Sleeping Beauty, piggyBac and Tol2 for Genome Engineering. Int J Mol Sci 22. https://doi.org/10.3390/ijms22105084
Santos GW (1983) History of bone marrow transplantation. Clin Haematol 12:611–639
Article CAS PubMed Google Scholar
Sayed N, Liu C, Wu JC (2016) Translation of Human-Induced Pluripotent Stem Cells: From Clinical Trial in a Dish to Precision Medicine. J Am Coll Cardiol 67:2161–2176
Article PubMed PubMed Central Google Scholar
Scadden DT (2006) The stem-cell niche as an entity of action. Nature 441:1075–1079
Article CAS PubMed Google Scholar
Schwartz SD, Regillo CD, Lam BL et al (2015) Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet 385:509–516
Article PubMed Google Scholar
Schwarzwaelder K, Howe SJ, Schmidt M et al (2007) Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo. J Clin Invest 117:2241–2249
Article CAS PubMed PubMed Central Google Scholar
Selyutina A, Persaud M, Lee K et al (2020) Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating. Cell Rep 32:108201
Article CAS PubMed PubMed Central Google Scholar
Serva SN, Bernstein J, Thompson JA et al (2022) An update on advanced therapies for Parkinson’s disease: From gene therapy to neuromodulation. Front Surg 9:863921
Article PubMed PubMed Central Google Scholar
Shaw AR, Suzuki M (2019) Immunology of Adenoviral vectors in cancer therapy. Mol Ther Methods Clin Dev 15:418–429
Article CAS PubMed PubMed Central Google Scholar
Shimamura M, Nakagami H, Sanada F, Morishita R (2020) Progress of Gene Therapy in Cardiovascular Disease. Hypertension 76:1038–1044
Article CAS PubMed Google Scholar
Shinagawa M, Padmanabhan RV, Padmanabhan R (1980) The nucleotide sequence of the right-hand terminal SmaI-K fragment of adenovirus type 2 DNA. Gene 9:99–114
Article CAS PubMed Google Scholar
Shy BR, Vykunta VS, Ha A et al (2022) High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat Biotechnol. https://doi.org/10.1038/s41587-022-01418-8
Singh S, Narang AS, Mahato RI (2011) Subcellular fate and off-target effects of siRNA, shRNA, and miRNA. Pharm Res 28:2996–3015
Article CAS PubMed Google Scholar
Smith BD, Grande DA (2015) The current state of scaffolds for musculoskeletal regenerative applications. Nat Rev Rheumatol 11:213–222
Article CAS PubMed Google Scholar
Staal FJT, Aiuti A, Cavazzana M (2019) Autologous Stem-Cell-Based Gene Therapy for Inherited Disorders: State of the Art and Perspectives. Front Pediatr 7:443
Article PubMed PubMed Central Google Scholar
Stewart DJ, Hilton JD, Arnold JMO, et al (2006) Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF(121) versus maximum medical treatment. Gene Ther 13:1503–1511
Google Scholar
Stevanato L, Corteling RL, Stroemer P et al (2009) c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain. BMC Neurosci 10:86
Article PubMed PubMed Central Google Scholar
Stolberg SG (1999) The biotech death of Jesse Gelsinger. N Y Times Mag 136–140:149–150
Google Scholar
Suerth JD, Labenski V, Schambach A (2014) Alpharetroviral vectors: from a cancer-causing agent to a useful tool for human gene therapy. Viruses 6:4811–4838
Article PubMed PubMed Central Google Scholar
Surosky RT, Urabe M, Godwin SG et al (1997) Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome. J Virol 71:7951–7959
Article CAS PubMed PubMed Central Google Scholar
Takahashi K, Tanabe K, Ohnuki M et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872
Article CAS PubMed Google Scholar
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676
Article CAS PubMed Google Scholar
Takebe T, Enomura M, Yoshizawa E et al (2015) Vascularized and Complex Organ Buds from Diverse Tissues via Mesenchymal Cell-Driven Condensation. Cell Stem Cell 16:556–565
Article CAS PubMed Google Scholar
Tchieu J, Kuoy E, Chin MH et al (2010) Female human iPSCs retain an inactive X chromosome. Cell Stem Cell 7:329–342
Article CAS PubMed PubMed Central Google Scholar
Thaker PH, Brady WE, Bradley WH et al (2015) Phase I study of intraperitoneal IL-12 plasmid formulated with PEG-PEI-cholesterol lipopolymer administered in combination with pegylated liposomal doxorubicin in recurrent or persistent epithelial ovarian, Fallopian tube, or primary peritoneal cancer patients: An NRG/GOG study. J Clin Orthod 33:5541–5541
Google Scholar
Thier M, Wörsdörfer P, Lakes YB et al (2012) Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell 10:473–479
Article CAS PubMed Google Scholar
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147
Article CAS PubMed Google Scholar
Trounson A, McDonald C (2015) Stem cell therapies in clinical trials: progress and challenges. Cell Stem Cell 17:11–22
Google Scholar
Tucci F, Galimberti S, Naldini L et al (2022) A systematic review and meta-analysis of gene therapy with hematopoietic stem and progenitor cells for monogenic disorders. Nat Commun 13:1315
Article CAS PubMed PubMed Central Google Scholar
Uusi-Kerttula H, Hulin-Curtis S, Davies J, Parker AL (2015) Oncolytic Adenovirus: Strategies and insights for vector design and immuno-oncolytic applications. Viruses 7:6009–6042
Article CAS PubMed PubMed Central Google Scholar
Valente JFA, Queiroz JA, Sousa F (2018) p53 as the Focus of Gene Therapy: Past, Present and Future. Curr Drug Targets 19:1801–1817
Article CAS PubMed Google Scholar
Varderidou-Minasian S, Lorenowicz MJ (2020) Mesenchymal stromal/stem cell-derived extracellular vesicles in tissue repair: challenges and opportunities. Theranostics 10:5979–5997
Article CAS PubMed PubMed Central Google Scholar
Vasan S, Hurley A, Schlesinger SJ et al (2011) In vivo electroporation enhances the immunogenicity of an HIV-1 DNA vaccine candidate in healthy volunteers. PLoS One 6:e19252
Article CAS PubMed PubMed Central Google Scholar
Vestergaard HT, D’Apote L, Schneider CK, Herberts C (2013) The evolution of nonclinical regulatory science: advanced therapy medicinal products as a paradigm. Mol Ther 21:1644–1648
Article PubMed Google Scholar
Verma IM (By Inder M. Verma on November 1 1990) Gene Therapy. Scientific American.
Google Scholar
von Jonquieres G, Rae CD, Housley GD (2021) Emerging concepts in vector development for glial gene therapy: Implications for leukodystrophies. Front Cell Neurosci 15:661857
Article Google Scholar
Walther J, Wilbie D, Tissingh VSJ et al (2022) Impact of Formulation Conditions on Lipid Nanoparticle Characteristics and Functional Delivery of CRISPR RNP for Gene Knock-Out and Correction. Pharmaceutics 14:213
Article CAS PubMed PubMed Central Google Scholar
Wang H, Li Z-Y, Liu Y et al (2011) Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14. Nat Med 17:96–104
Article PubMed Google Scholar
Weber W, Fussenegger M (2006) Pharmacologic transgene control systems for gene therapy. J Gene Med 8:535–556
Article CAS PubMed Google Scholar
Wells DJ (2004) Gene therapy progress and prospects: electroporation and other physical methods. Gene Ther 11:1363–1369
Article CAS PubMed Google Scholar
Wiethoff CM, Nemerow GR (2015) Adenovirus membrane penetration: Tickling the tail of a sleeping dragon. Virology 479–480:591–599
Article PubMed Google Scholar
Williams DA, Bledsoe JR, Duncan CN, et al (2022) Myelodysplastic syndromes after eli-cel gene therapy for cerebral adrenoleukodystrophy (CALD). In: MOLECULAR THERAPY. CELL PRESS 50 HAMPSHIRE ST, FLOOR 5, CAMBRIDGE, MA 02139 USA, pp 6–6
Google Scholar
Wilmut I, Beaujean N, de Sousa PA et al (2002) Somatic cell nuclear transfer. Nature 419:583–586
Article CAS PubMed Google Scholar
Wright JF (2008) Manufacturing and characterizing AAV-based vectors for use in clinical studies. Gene Ther 15:840–848
Article CAS PubMed Google Scholar
Wright JF, Qu G, Tang C, Sommer JM (2003) Recombinant adeno-associated virus: formulation challenges and strategies for a gene therapy vector. Curr Opin Drug Discov Devel 6:174–178
CAS PubMed Google Scholar
Xia Y, Li X, Sun W (2020) Applications of Recombinant Adenovirus-p53 Gene Therapy for Cancers in the Clinic in China. Curr Gene Ther 20:127–141
Article CAS PubMed Google Scholar
Xin T, Cheng L, Zhou C et al (2022) In-Vivo Induced CAR-T Cell for the Potential Breakthrough to Overcome the Barriers of Current CAR-T Cell Therapy. Front Oncol 12:809754
Article CAS PubMed PubMed Central Google Scholar
Yan Z, Zhang Y, Duan D, Engelhardt JF (2000) Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. Proc Natl Acad Sci U S A 97:6716–6721
Article CAS PubMed PubMed Central Google Scholar
Yáñez-Mó M, Siljander PR-M, Andreu Z et al (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4:27066
Article PubMed Google Scholar
Yang C-S, Li Z, Rana TM (2011) microRNAs modulate iPS cell generation. RNA 17:1451–1460
Article CAS PubMed PubMed Central Google Scholar
Ylä-Herttuala S (2019) Gene Therapy of Critical Limb Ischemia Enters Clinical Use. Mol. Ther. 27:2053
Article PubMed PubMed Central Google Scholar
Yuan X, Wan H, Zhao X et al (2011) Brief report: combined chemical treatment enables Oct4-induced reprogramming from mouse embryonic fibroblasts. Stem Cells 29:549–553
Article CAS PubMed Google Scholar
Zaiss A-K, Liu Q, Bowen GP et al (2002) Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors. J Virol 76:4580–4590
Article CAS PubMed PubMed Central Google Scholar
Zennou V, Petit C, Guetard D et al (2000) HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 101:173–185
Article CAS PubMed Google Scholar
Zhang W-W, Li L, Li D et al (2018) The first approved gene therapy product for cancer Ad-p53 (Gendicine): 12 years in the clinic. Hum Gene Ther 29:160–179
Article CAS PubMed Google Scholar
Zuris JA, Thompson DB, Shu Y et al (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33:73–80
Article CAS PubMed Google Scholar

Author information

Authors and Affiliations

Pharmaceutics Division, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, The Netherlands
Enrico Mastrobattista & Erik Doevendans
Department of Child Neurology, Amsterdam Leukodystrophy Center, Emma Children’s Hospital, Amsterdam University Medical Centers, VU University, and Neuroscience, Cellular & Molecular Mechanisms, Amsterdam, The Netherlands
Niek P. van Til
Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands
Vera Kemp
Department of Neurosurgery, Brain Tumor Center, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
Jeroen de Vrij
Hospital Pharmacy, Interdivisonal GMP Facility, Leiden University Medical Center, Leiden, The Netherlands
Karin Hoogendoorn

Authors

Enrico Mastrobattista
View author publications
You can also search for this author in PubMed Google Scholar
Erik Doevendans
View author publications
You can also search for this author in PubMed Google Scholar
Niek P. van Til
View author publications
You can also search for this author in PubMed Google Scholar
Vera Kemp
View author publications
You can also search for this author in PubMed Google Scholar
Jeroen de Vrij
View author publications
You can also search for this author in PubMed Google Scholar
Karin Hoogendoorn
View author publications
You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Enrico Mastrobattista .

Editor information

Editors and Affiliations

Utrecht University, Utrecht, The Netherlands
Daan J. A. Crommelin
Faculty of Pharmaceutical Sciences, The University of British Columbia (UBC), Vancouver, BC, Canada
Robert D. Sindelar
College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN, USA
Bernd Meibohm

Rights and permissions

Reprints and permissions

Copyright information

About this chapter

Cite this chapter

Mastrobattista, E., Doevendans, E., van Til, N.P., Kemp, V., de Vrij, J., Hoogendoorn, K. (2024). Advanced Therapy Medicinal Products: Clinical, Non-clinical, and Quality Considerations. In: Crommelin, D.J.A., Sindelar, R.D., Meibohm, B. (eds) Pharmaceutical Biotechnology. Springer, Cham. https://doi.org/10.1007/978-3-031-30023-3_14

Download citation

DOI: https://doi.org/10.1007/978-3-031-30023-3_14
Published: 30 January 2024
Publisher Name: Springer, Cham
Print ISBN: 978-3-031-30022-6
Online ISBN: 978-3-031-30023-3
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)

Publish with us

Policies and ethics

Advanced Therapy Medicinal Products: Clinical, Non-clinical, and Quality Considerations

Abstract

Similar content being viewed by others

Advanced Therapies: Clinical, Non-clinical and Quality Considerations

Gene and Cell Therapy: How to Build a BioDrug

Regulatory Framework for Cell and Tissue Based Therapies in Europe

Keywords

Introduction

ATMPs: Definitions, Classifications, and Modes of Action

Transplantation or Advanced Therapy?

Gene or Cell Therapy Medicinal Product?

Classification

ATMPs: Possible Mode of Action(s)

(Dis)similarities with Recombinant Therapeutic Proteins and Other Biologicals

Part A: Technologies for Cell Therapy and Tissue Engineering

Somatic Cell Technologies

Adult Stem Cells Used as Transplant Product

Adult Stem Cells for Clinical Application: Immune Cells

Adult Stem Cells for Clinical Application: MSCs

Cell Immortalization Technologies

Cell Plasticity Technologies

Embryonic Stem Cells

Maintenance and Differentiation of ESCs in Culture

ESC Somatic Cell Nuclear Transfer (Therapeutic Cloning)

IPS Cell Technology

Transdifferentiation

Three-Dimensional Technologies

Combinations of the Above Technologies

Part B: Technologies for Gene Therapy

Introduction

Gene Therapy: Definitions and Ways of Application

Gene Augmentation

Gene Knock Out

Gene Correction

Somatic Versus Germline

Ex Vivo Versus In Vivo

Designer Nucleases for Gene Editing

TALEN

CRISPR-Cas9

Base Editors

Prime Editors

Delivery

Viral Vectors

Retrovirus

Biology

Suitability of Retroviruses as Vectors for Gene Transfer

Gammaretrovirus

Biology

Clinical Use of Gammaretrovirus

Lentivirus

Biology

Lentiviruses as Vectors for Gene Transfer Vehicles

Clinical Use of Lentiviral Vectors

Alpharetroviral Vectors

Biology

Retroviral Mechanisms of Genotoxicity

Adenovirus

Biology

Adenoviruses as Gene Therapy vectors

Clinical Use of Adenoviral Vectors

Adeno-Associated Virus (AAV)

Biology

Suitability of Adeno-Associated Viruses for Gene Transfer

Clinical Use of Adeno-Associated Virus Vectors

Nonviral Vectors

Physical Methods for Gene Transfer

Lipid-Based Vectors

Peptide-Based Vectors

Polymeric Vectors

Extracellular Vesicles

Delivery Systems for CRISPR-Based Gene Editing

Disease Targets for Gene Therapy

Cancer Gene Therapy

Correction of Genetic Abnormalities

Immunotherapy

Oncolytic Viruses (Virotherapy)

Monogenic Diseases

Cardiovascular Diseases

Infectious Diseases

Neurological Diseases