Using RT-prone recombination to promote re-building of complete retroviral vectors from two defective precursors: Low efficiency and sequence specificities

Bru, Thierry; Galetto, Román; Piver, Éric; Collin, Christine; Negroni, Matteo; Pagès, Jean‐Christophe

doi:10.1016/j.jviromet.2007.01.022

Cited by 2 publications

(3 citation statements)

References 48 publications

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“…To simplify the system during optimisation, we initially employed a smaller NIGW transgene cassette whilst screening the impact of modifications to vector architecture. The efficiency of NIGW provirus reconstitution in a lentiviral vector containing unmodified elements was comparable with previous reports in which IRES was used as a homologous region for recombination3132. Interestingly, mutation of core lentiviral cis and trans elements did not improve the efficiency of this technology.…”

Section: Discussionsupporting

confidence: 83%

“…Our heterozygous vector configuration produced significantly more GFP-positive cells, although the 0.1% return would require significant improvement for future gene therapy applications. In the NIGW model, recombination may have been facilitated by IRES, which produces a complex secondary structure in RNA molecules, potentially increasing the rate of pausing during reverse-transcription and promoting template-switching313536373839. Therefore, one potential avenue for improving dystrophin reconstitution could be to incorporate RNA secondary structure into the region of dystrophin homology to force recombination at the intended site.…”

Section: Discussionmentioning

confidence: 99%

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Lentiviral vectors can be used for full-length dystrophin gene therapy

Counsell

Asgarian

Meng

et al. 2017

Sci Rep

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Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a ‘template-switching’ lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

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Section: Discussionsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Lentiviral vectors can be used for full-length dystrophin gene therapy

Counsell

Asgarian

Meng

et al. 2017

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Our heterozygous vector configuration produced significantly more GFP-positive cells, although the 0.1% return would require significant improvement for future gene therapy applications. In the NIGW model, recombination may have been facilitated by IRES, which produces a complex secondary structure in RNA molecules, potentially increasing the rate of pausing during reverse-transcription and promoting template-switching 31, 35–39 . Therefore, one potential avenue for improving dystrophin reconstitution could be to incorporate RNA secondary structure into the region of dystrophin homology to force recombination at the intended site.…”

Section: Discussionmentioning

confidence: 99%