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Construction and utilization of a new generation of bacteriophage-based particles, or TPA, for guided systemic delivery of nucleic acids to tumors

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Abstract

Successful delivery of nucleic acid therapeutics to diseased sites would present a pivotal advancement in cancer treatment. However, progress has been hindered by the lack of efficient tumor-selective vectors via clinical systemic routes, the blood–brain barrier for brain tumors and problems with repeated administrations. We present a new generation of M13 phage-based vectors termed transmorphic phage/adeno-associated virus (AAV) (TPA), wherein the phage genome has been excised to facilitate exclusive packaging of human AAV DNA by phage coat proteins. Here we provide a detailed protocol for the molecular cloning of DNA into the TPA construct, display of disease-specific ligands on the helper phage capsid for cell targeting and entry, and packaging of TPA DNA by helper phage coat proteins in a bacterial host. Furthermore, we provide methods for mammalian cell transduction and assessment of transgene expression in vitro as well as in vivo application of TPA particles in tumor-bearing mice. Unlike other similar methods, our protocol enables high-yield production and control of helper phage quantity in TPA preparations. Moreover, compared with existing M13 phage vectors, TPA particles can accommodate large size transgene inserts, despite being considerably more compact, providing superior gene delivery through enhanced diffusion across the extracellular matrix, improved cellular binding and entry and increased vector DNA accumulation in the nucleus. The protocol encompasses a timeline of 4–5 months, including construction and production of TPA particles with transgene and targeted ligand and in vitro/in vivo testing. This protocol can be conducted by researchers trained in basic molecular biology/bacteriology research techniques.

Key points

  • This protocol describes the construction and production of transmorphic phage/adeno-associated virus particles for the targeted delivery of nucleic acid payloads, in vitro and in vivo.

  • Transmorphic phage/adeno-associated virus particles ensure effective and safe delivery of nucleic acids (for example, luciferase, green fluorescent protein or cytokine genes) to solid tumors, thanks to their greater diffusion across the extracellular matrix and improved internalization and intracellular trafficking to the nucleus of target cells.

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Fig. 1: Schematic representation of the AAVP and TPA particles and the corresponding DNA constructs/genomes.
Fig. 2: Schematic overview of the protocol for the generation, production and functional analysis of TPA particles.
Fig. 3: Schematic overview of the protocol for the production of TPA particles using a bacterial superinfection production system.
Fig. 4: Lucia transgene expression in HEK293 cells.
Fig. 5: GFP expression in HEK293 cells.
Fig. 6: Biodistribution of TNF transgene expression after systemic TPA administration in tumor-bearing mice.
Fig. 7: Biodistribution and therapeutic evaluation of TPA-Il15 in subcutaneous tumor-bearing mice.

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Data availability

The data shown in the ‘Anticipated results’ section come from our previous publication26. The raw data are also available in the supplementary material section of the primary research publication26. Figures 6 and 7 are adapted with permission from ref. 26, EMBO.

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Acknowledgements

We thank the UK Medical Research Council for their support with a grant MR/T029226/1, Cancer Research UK for the C31277/A25887 award, Brain Research UK for the 202021-34 award and Children with Cancer UK for the 16-230 grant. We also thank P. Asavarut, S. Waramit, P. Vila-Gomez and T. Yata at Imperial College London. Source data are provided with this paper.

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Author notes

  1. These authors contributed equally: Lauren Gay, Keittisak Suwan.

Authors and Affiliations

  1. Cancer Phage Therapy, Department of Brain Sciences, Imperial College London, London, UK

    Lauren Gay, Keittisak Suwan & Amin Hajitou

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  1. Lauren Gay
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Contributions

L.G., K.S. and A.H. designed the methods and analyses, and performed experiments. A.H. founded, designed and supervised the whole study, and secured research funding. All authors wrote and edited the manuscript, and approved the protocol.

Corresponding author

Correspondence to Amin Hajitou.

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Competing interests

K.S. and A.H. are inventors on patent applications describing the vector constructs described here.

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Nature Protocols thanks Alfonso Jaramillo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key reference using this protocol

Asavarut, P. et al. EMBO Mol. Med. 14, e15418 (2022): https://doi.org/10.15252/emmm.202115418

Al-Bahrani, M. et al. FASEB J. 37, e23038 (2023): https://doi.org/10.1096/fj.202300045R

Chongchai, A. et al. FASEB J. 35, e21487 (2021): https://doi.org/10.1096/fj.202002539R

Supplementary information

Source data

Source Data Figs. 4, 6a and 7b,d–f

. Statistical source data for Figs. 4, 6a and 7b,d–f.

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Gay, L., Suwan, K. & Hajitou, A. Construction and utilization of a new generation of bacteriophage-based particles, or TPA, for guided systemic delivery of nucleic acids to tumors. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01040-9

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