Abstract
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.
Highlights
Are preferred over AAV for ex vivo Duchenne Muscular Dystrophy (DMD) gene therapy as they enable stable transduction of a stem cell pool with a therapeutic cassette, whilst concurrently enhancing muscle stem cell functionality prior to transplantation
Titration of the stuffer constructs by green fluorescence protein (GFP) output showed that lentivirus functional titres reduce as the size of the payload increases (Fig. 1b)
Viral gene transfer is hindered by the packaging limits of clinically applicable viral vectors, which operate with reduced efficiency when delivering transgenes as large as dystrophin
Summary
Are preferred over AAV for ex vivo DMD gene therapy as they enable stable transduction of a stem cell pool with a therapeutic cassette, whilst concurrently enhancing muscle stem cell functionality prior to transplantation. We initially profiled the packaging capacity of standard lentiviral vectors and proof-of-concept for a method designed to circumvent restrictions on the length of transgenes that can be delivered to cells, before successfully demonstrating that lentiviruses can be used to deliver full-length dystrophin to DMD myoblasts as a proof-of-concept ex vivo gene therapy strategy. This approach could provide full, permanent dystrophin functionality, which has been unachievable with competing gene therapy technologies
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