Retroviruses in Human Gene Therapy


A retrovirus‐based gene transfer system consists of two components: the transfer vector, which harbours a foreign gene linked to elements needed for retroviral replication, and the packaging cell, which supplies the necessary retroviral proteins for transfer of the vector through a single round of viral replication. A hallmark of retroviral replication is the stable maintenance of the transferred gene(s) during cell division. The first type of transfer vectors used in gene therapy trials were derived from mouse retroviruses and referred to as retroviral vectors. Lentiviral vectors are derived from human immunodeficiency virus type 1 and exhibit distinct features that provide advantages in some settings. Retroviral and lentiviral vectors are being used in clinical trials for several diseases, including monogenic diseases and cancer. The clinical protocols include gene transfer in vivo as well as ex vivo, the latter being followed by installation of the genetically modified cells into the body.

Key Concepts:

  • The retroviral replication machinery leads to stable maintenance of a transferred gene.

  • The stable integration of retroviral vectors into a chromosome of the target cell may have adverse effects on neighbouring genes.

  • Retroviral gene transfer does not require the expression of retroviral genes in the target cell.

  • The term retroviral vector is used for a vector based on murine leukaemia virus.

  • The term lentiviral vector is used for a vector based on human immunodeficiency virus type 1.

  • Retroviral vectors allow gene transfer only to dividing cells, whereas lentiviral vectors target dividing as well as nondividing cells.

  • In clinical protocols using retroviral or lentiviral vectors, gene transfer may take place ex vivo or in vivo.

  • The selectivity of retroviral vectors for dividing cells has been exploited to target tumour cells.

Keywords: vector; packaging cell; gene transfer; integration; lentivirus

Figure 1.

Gene transfer by MLV‐derived vectors. Generation of the two components of an MLV‐based gene transfer system. Top shows a diagram of the integrated DNA genome of MLV (∼9000 nucleotides). Among the cis‐acting control sequences are the packaging signal (ψ) for encapsidation of RNA into viral particles and the LTR regions that harbour control elements for transcription, reverse transcription and integration. The three open reading frames (gag, pol and env) encode all trans‐acting protein factors. The left column shows packaging constructs engineered to direct the production of viral proteins. Simple packaging constructs harbour all three open reading frames in one construct. In complex packaging constructs, the gag–pol and env reading frames are engineered into two separate constructs. Δψ, deleted packaging signal; p(A), heterologous polyadenylation signal; Prom, heterologous promoter. The right column shows various designs of MLV‐based transfer vectors. (a) Transfer vector derived by simple replacement of the coding regions of the viral proteins by the coding region of a foreign gene. (b) Transfer vector harbouring an internal heterologous promoter in addition to the viral LTR promoter. Owing to the scheme of duplication of LTR sequences during retroviral replication, a two‐promoter transfer vector may be designed such that transcriptional control sequences of the LTR are nonoperative in the target cell (self‐inactivating transfer vectors). (c) Transfer vector in which expression of two genes is directed by distinct promoters, the LTR promoter and an internal heterologous promoter, respectively. (d) Transfer vector with two genes in which gene product 1 is produced from unspliced RNA and gene product 2 from spliced messenger RNA directed by the splice donor (SD) and splice acceptor (SA) signals for viral env messenger RNA. (e) Transfer vector producing a single messenger RNA directing the translation of two open reading frames, translation of gene 2 being controlled by an internal ribosome entry site (IRES).

Figure 2.

Gene transfer by a two‐component retroviral vector system (see Figure ). A packaging cell line has been generated by stable insertion of packaging construct DNA by a nonviral method, such as transfection. Transfer vector DNA is introduced into the packaging cells. The transfer vector RNA is packaging competent, whereas the packaging construct RNAs are defective for incorporation into viral particles. Viral particles harbouring transfer vector RNA are used for transduction of target cells. Subsequently, target cells producing the desired transfer vector‐encoded RNA and protein may be identified and expanded.

Figure 3.

Activation of an oncogene by provirus insertion. In the native conformation the protooncogene is regulated by its native promotor and enhancer elements illustrated in the upper part of the figure. Insertion of retroviral vector enhancer elements from the integrated provirus may influence the transcriptional level of the oncogene and increase its level of transcription. Depicted is a case of enhancer activation of an oncogene where the provirus has been inserted in the opposite transcriptional direction. Curved twin lines represent chromatin; thin straight lines represent RNA transcripts; and thick arrow indicates enhancer activation of host promoter.

Figure 4.

Generation of RCVs by recombination between transfer vector and packaging constructs. Recombination takes place at the level of reverse transcription of two RNAs encapsidated into the same particle. All three examples cover a simple transfer vector as shown in Figure . In the case of a simple packaging construct with viral control sequences at both ends, only one crossover event is needed to generate a fully functional virus. In the simple packaging construct with a heterologous poly(A) signal, two crossover events are needed. In the complex packaging construct, three crossover events are needed. Not shown is the possible contribution of endogenous retroviral RNAs.

Figure 5.

HIV‐1‐based gene transfer systems. Top: Map of the HIV‐1 genome in the DNA form with indication of the LTR, ψ and Rev‐responsive element (RRE) regions and open reading frames encoding viral proteins. Left: Basic packaging construct impaired in the Vpu and Env. Heterologous envelopes, the amphotropic MLV (MLV (A) env) or the VSV G protein provided from separate expression constructs. All constructs harbour heterologous promoters (Prom) and polyadenylation signals (p(A)). Advanced packaging constructs express gag–pro–pol, lack all auxiliary HIV‐1 genes and retain the RRE. Separate expression constructs provide Rev and envelope function (in casuVSV G). Right: The simple transfer vector harbours the extended packaging signal (ψ) partly overlapping with gag, Gag production being impaired by mutation. The foreign gene is inserted after a heterologous promoter. In the self‐inactivating transfer vector the heterologous promoter (Prom) is independent of Tat in the packaging cell and the LTR translocated to the upstream position in the target cell impaired by mutation. The lines below the self‐inactivating transfer vector maps indicate predicted RNA species. SD, splice donor site; SA, splice acceptor site.

Figure 6.

Ex vivo and in vivo gene transfer. Transfer vector‐bearing virus particles are liberated into the supernatant of cultured packaging cells (upper left). The right part of the figure (blue arrows) illustrates the ex vivo scheme and the left part (red arrows) the in vivo scheme.



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Further Reading

Glauche I, Bystrykh L, Eaves C et al. (2013) Stem cell clonality – theoretical concepts, experimental techniques, and clinical challenges. Blood Cells, Molecules, and Diseases 50: 232–240.

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Naldini L (2011) Ex vivo gene transfer and correction for cell‐based therapies. Nature Reviews Genetics 12: 301–315.

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Pedersen, Finn Skou, Bahrami, Shervin, and Duch, Mogens(Apr 2014) Retroviruses in Human Gene Therapy. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001002.pub3]