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 constructs, which supply the necessary retroviral proteins for transfer of the vector from a producer cell 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 primarily ex vivo gene transfer followed by introduction 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 and lentiviral vectors differ in their integration patterns with respect to genes and gene regulatory regions.
  • To treat inherited monogenic disease affecting e.g. skin or blood, stem cells can be corrected ex vivo by using retroviral and lentiviral vectors.
  • Retroviral and lentiviral vectors are used in clinical protocols to generate modified T‐lymphocytes that target cancer cells.
  • New designs of lentiviral vectors are being developed as a toolbox for therapy by the CRISPR/Cas9 genome editing technology.

Keywords: vector; packaging constructs; gene transfer; transduction; integration; lentivirus

Figure 1. From virus to vector. Schematic representation of the integrated MLV genome (∼9000 basepairs) with key cis‐elements indicated in light green and genes encoding structural and enzymatic proteins indicated in blue. Above is shown a typical vector design carrying a gene‐of‐interest (GOI) with viral cis‐elements indicated (light green). Packaging constructs encoding Gag and Pol and Env (here 4070A envelope), respectively, are shown below. Among the cis‐acting control sequences are the LTR (long terminal repeat) that harbours control regions for transcription, reverse transcription, and integration and the packaging signal (ψ) for encapsidation of RNA into viral particles. Other cis elements include R (repeat region), att (integrase attachment site), PBS (primer binding site), PPT (polypurine tract). Expression of Gag/Pol and Env is driven by the cytomegalovirus promoter (CMV) and polyadenylation by the p(A) sequence.
Figure 2. Design of MLV‐based and lentiviral vector systems. Integrated genomes of MLV (A) and HIV (B) are shown at the top. Vif, vpu, vpr, tat, nef, and rev indicate genes encoding HIV accessory proteins; only Rev is required for vector production. RRE indicates Rev‐responsive element, whereas TAR is the trans‐activation response element. State‐of‐the‐art packaging constructs are shown for both MLV‐ and HIV‐based systems. MLV production is based on the expression of Gag/Pol and Env from two separate genetic entities. State‐of‐the‐art production of third‐generation lentiviral vectors features three packaging plasmids, one expressing gag‐pro‐pol, one encoding the Env protein (here the vesicular stomatitis virus glycoprotein; VSV‐G), and one encoding the Rev protein. Four different MLV vector designs are shown: (a) Transfer vector derived by simple replacement of the coding regions of the viral proteins by the coding region of a gene‐of‐interest (GOI). (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 or SIN transfer vectors; here indicated by the cross in the LTR region labelled ΔU3. (c) SIN transfer vector in which expression of two genes separated by a 2A peptide sequence (2A) is directed by a single promoter. (d) SIN transfer vector with two genes expressed in opposite directions from two promoters. GOI2 is expressed in the opposite orientation of vector RNA production and therefore has its own p(A) sequence, whereas GOI1 is expressed in the same orientation as the vector RNA with use of the p(A) present in the downstream LTR. Prom, promoter; Ψ, packaging signal. A single classical lentiviral vector type is shown carrying an internal promoter driving expression of the GOI flanked downstream by the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) for increased expression. Expression of the vector is driven by the heterologous CMV promoter and occurs independently of TAR. Below is shown the reverse‐transcribed vector with duplicated SIN LTRs.
Figure 3. Gene transfer by retroviral vector systems (a) Production and transfer of γ‐retroviral MLV‐based vectors. A packaging cell line has been generated by stable insertion of packaging construct DNA by a nonviral method, such as transfection. Transfer vector plasmid DNA is introduced into the packaging cells by DNA transfection (as shown) or alternatively by viral vector transduction. 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‐of‐interest (POI) may be identified and expanded. (b) Production of VSV‐G‐pseudotyped lentiviral vectors by co‐transfection of three packaging plasmid and a plasmid encoding transfer vector RNA. Vector‐containing virus particles are produced and can be transferred to target cells, which subsequently produces the POI.
Figure 4. Schematic representation of MLV and HIV vector integration profiles. (a) Differences in intragenic insertion profiles of MLV‐ and HIV‐derived vectors. MLV vectors are prone for insertion near the transcriptional start site (TSS) of genes, whereas lentiviral vectors are inserted preferentially in other regions of the genes and are not prone for integration near TSS. (b) Schematic representation of integration around the TSS of target genes demonstrating frequent insertion of MLV near TSS (but not within TSS) and minimal lentiviral vector insertion near the TSS. Source: Data from Cattoglio et al. .
Figure 5. Insertional activation or mutagenesis caused by genomic insertion of a retroviral vector. Different scenarios disturbing the normal function of an endogenous gene (indicated in dark green) driven by its natural promoter (yellow arrow labelled with ‘Prom’) are shown. (a) Activation of endogenous promoter by gene‐regulatory sequences in downstream LTR; (b) Activation by strong internal promoter driving expression of the transgene; (c) Reduced insertional gene activation by insulator elements incorporated in the vector design; (d) Disruption of endogenous gene by insertion inside the gene, potentially leading to production of truncated transcripts or affecting expression and/or splicing.
Figure 6. Examples of gene therapies utilizing gene transfer by MLV‐based vector. Top part illustrates MLV transfer of the IL2γR gene to hematopoietic stem cells for treatment of X‐linked severe combined immunodeficiency (SCID‐X1) by re‐infusion of corrected stem cells. The lower part shows a similar strategy for treatment of Junctional Epidermolysis Bullosa (JEB) by retroviral transfer of the LAMB3 gene to epidermal stem cells. In the latter case, ecotropic MLV vectors were utilized to transduce GP+envAm12 amphotropic packaging cells, allowing production of retroviral particles. Also, corrected cells were utilized for production of 3D grafts that were successfully transplanted onto the patient.
Figure 7. Delivery of CRISPR/Cas9 components and repair donor sequences by lentiviral gene transfer. Vectors shown at the top allow co‐delivery of sgRNA and Cas9 expression cassettes (left) and delivery of a donor sequence, which upon reverse transcription can serve as a template for homology‐directed repair. The two left panels illustrate the use of lentiviral vector transfer for Cas9/sgRNA delivery. Panel to the far left shows transduction with a conventional integration‐competent vector leading to genomic insertion of the Cas9 and sgRNA expression cassettes, whereas the middle panel illustrates transfer of a similar vector in the context of an integrase‐defective lentiviral vector (IDLV) carrying an inactive integrase protein. In this case, Cas9 and sgRNA are expressed from episomal DNA intermediates. The panel to the far right illustrates the use of IDLVs for delivery of vector RNA, which upon reverse transcription can serve as a donor for homologous recombination due to the presence of homology arms (HA, indicated as green boxes) in the sequence.


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Further Reading

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Pang Y, Hou X, Yang C, Liu Y and Jiang G (2018) Advances on chimeric antigen receptor‐modified T‐cell therapy for oncotherapy. Molecular Cancer 17: 91.

Skipper KA and Mikkelsen JG (2015) Delivering the goods for genome engineering and editing. Human Gene Therapy 26: 486–497.

Thrasher AJ and Williams DA (2017) Evolving gene therapy in primary immunodeficiency. Molecular Therapy 25: 1132–1141.

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Pedersen, Finn S, and Mikkelsen, Jacob G(Sep 2018) Retroviruses in Human Gene Therapy. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001002.pub4]