Safety of Retroviral Vectors in Clinical Applications: Lessons from Retroviral Biology and Pathogenesis

Abstract

The ability of retroviruses to integrate a precise copy of the viral genome into host cell DNA (deoxyribonucleic acid) has been harnessed in the development of retroviral vectors for stable delivery of foreign genes to target cells. Over several decades, these tools have been utilised in human medicine in diverse applications from cell lineage tracing by gene marking to correction of single‐gene disorders and therapy of cancer. The unanticipated occurrence of leukaemias due to vector insertional mutagenesis in trials of gene therapy led to an urgent search for safer vectors and delivery systems. The advent of T‐cell therapy for cancer has led to renewed interest in retroviral vectors as stable and efficient delivery vehicles and is supported by observations suggesting that mature T cells are relatively resistant to oncogenic transformation by retroviruses. However, this evidence is circumstantial and the long‐term risks are poorly understood. Retroviral vector safety is considered here from the perspective of the biology and pathogenesis of their parental viruses. It is suggested that robust cell suicide strategies to remove transduced cells are likely to be important if retroviral vectors are to be adopted widely for delivery of T‐cell therapy for cancer and other diseases.

Key Concepts

  • Retroviral vectors are useful tools for delivery of foreign genes to host cells in vitro and in vivo.
  • The term retroviral vector is generally used for a vector based on a gamma‐retrovirus such as murine leukaemia virus.
  • The term lentiviral vector is generally used for a vector based on human immunodeficiency virus type 1.
  • Retroviral vectors have been tested in many clinical applications including cell lineage tracing, gene therapy and cancer therapy.
  • Retroviruses can cause disease due to insertional mutagenesis or transduction of host cell genes.
  • Retroviral and lentiviral integration are nonrandom processes that favour active regions of the genome, increasing the risks of insertional mutagenesis.
  • Examples of retroviral transduction of host genes include a T‐cell receptor β‐chain gene, providing a parallel with cells engineered for use in T‐cell therapy of cancer.

Keywords: retroviral vector; safety; insertional mutagenesis; gene marking; gene therapy; GDEPT; CAR T cell

Figure 1. Comparison of the natural retroviral life cycle with retroviral vector transduction. The integrated provirus is transcribed into mRNA (messenger ribonucleic acid) and proteins expressed by the host cell's machinery (a). Particles assemble at the cell membrane and bud from the surface, acquiring an outer membrane from the host cell. Virus‐specific enzymes are required to process the Gag‐pol polyprotein during and after virion assembly (protease), to copy the RNA genome to its double‐stranded DNA (deoxyribonucleic acid) proviral form (reverse transcriptase) and to integrate the provirus into host cell DNA (integrase). The essential information required for infectious particle production is encoded by the Gag, Pol and Env genes, which in later versions of retroviral packaging cell lines are produced from separate plasmids with nonretroviral promoters (b). This design minimises homology of the packaging constructs and with vector sequences and greatly reduces the likelihood of recombination to produce replication‐competent retroviruses. The vector is introduced to the packaging cell line by stable or transient transfection. Only the vector is engineered to carry the ψ packing signal (in red) and therefore expresses the only RNA that is efficiently incorporated in virions and transmitted to the target cell. These basic principles also apply to lentiviral vectors, although these agents require additional viral genes (e.g. Rev) to be supplied in trans for efficient viral gene expression and particle production, and the HIV (human immunodeficiency virus) Env gene is generally replaced by VSV‐G, a membrane surface glycoprotein that generates viral particles capable of entering many cell types.
Figure 2. Vector integration is inherently mutagenic and (a) can have direct effects on a gene at the site of integration by a variety of mechanisms such as promote insertion, truncation, displacement of 5′ regulatory sequences or 3′ miRNA regulatory sequences or antisense RNA. Also, due to chromatin looping, retroviral enhancers can activate gene expression at a considerable distance (up to 300 kb), whereas in some cases, the products encoded by the vector can play a direct oncogenic role. Proviral clones derived from Moloney murine leukaemia virus (MoMLV) formed the backbone of many retroviral vector systems. (b) The long terminal repeats of this virus carry an additional copy of the enhancer core with its characteristic array of binding sites for Myb, Runx and Ets proteins, a feature that confers significantly accelerated lymphoma onset in vivo (see text).
Figure 3. The preferential integration of retroviral families is influenced significantly by the interaction of the viral integrase (IN) protein with chromatin tether molecules. For HIV, the key interaction is with LEDGF/p75 (lens epithelium‐derived growth factor aka PSIP1), which directs interaction to actively transcribed genes. For MLV, several members of the BET (bromo‐ and extraterminal domain) family provide an analogous role, in this case targeting viral integration to active promoters and enhancer elements. These interactions with tether proteins appear to explain the bias of proviral integration towards genomic regions enriched for distinct histone modifications (HIV at H3K36me3 and MLV at H3K27Ac).
Figure 4. A naturally occurring case of T‐cell lymphoma in a domestic cat infected with feline leukaemia virus, a gamma‐retrovirus closely related to MLV, revealed apparent amplification of the T‐cell receptor β‐chain gene. Further molecular characterisation showed that this was due to the presence of multiple copies of a provirus in which the coding sequence of the Env gene was replaced by the coding sequence of an intact, functionally rearranged TCRβ. The same tumour harboured a second transduction of Myc, a frequent target for activation by insertional mutagenesis or transduction FeLV‐associated lymphomas (Fulton et al., ). Although the specificity of the v‐tcr is unknown, it appears highly likely that it activated TCR pathway signalling in the index tumour.
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Further Reading

Pedersen FS, Bahrami S and Duch M (2014) Retroviruses in human gene therapy. In: Encyclopedia of Life Sciences. Chichester: John Wiley & Sons Ltd. http://www.els.net.

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Zhang J, Kale V and Chen M (2015) Gene‐directed enzyme prodrug therapy. AAPS Journal 17: 102–110.

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Neil, James C(Nov 2017) Safety of Retroviral Vectors in Clinical Applications: Lessons from Retroviral Biology and Pathogenesis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0024847]