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

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.

Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next‐generation human artificial chromosomes for Duchenne muscular dystrophy

“…“Permanent” exon skipping via adeno‐associated viral vector (AAV)‐mediated gene editing also appears to be a promising option (Long et al , ; Nelson et al , ; Tabebordbar et al , ; Young et al , ); however, immune reactions against AAVs (Boisgerault & Mingozzi, ) and the editing enzyme (which usually is of prokaryotic origin) plus off‐target effects might delay clinical translation. Although recent work provides encouraging in vitro evidence of lentiviral full‐length dystrophin transfer into human myoblasts (Counsell et al , ), HAC‐mediated gene therapy offers the chance to cover the entire spectrum of DMD mutations while preserving all the complex dystrophin regulatory elements (Muntoni et al , ). Additionally, safety profile and standard operating procedures for manufacturing both cell populations used in this study have been already developed in allogeneic and autologous clinical‐grade conditions (Perie et al , ; Cossu et al , ), thus paving the way for future clinical translation of genetically corrected cells.…”

Multiple Species Comparison of Cardiac Troponin T and Dystrophin: Unravelling the DNA behind Dilated Cardiomyopathy

“…The economic loss, food security threats and impact of loss and treatment costs make the condition important to both breeders and owners, the veterinary medicine community, healthcare insurance, pharmaceutical companies, and those involved in the food industry. Advances in genetic testing and more recently gene therapy proof of concept advances are now making mutation detection more accessible and treatment development more promising even in the large dystrophin gene [ 146 , 147 , 148 ]. In addition, finding causative mutations enables testing for at risk breeds and species prior to clinical detection and/or symptoms.…”

Gene therapies targeting the liver

“…3 Lentiviral vectors are single-stranded RNA viruses that can efficiently accommodate 10 kb of a transgene. 4 They integrate their genetic material into the host genome, allowing long-term expression of the therapeutic gene. Depending on design, they present a low risk of genotoxicity, as they integrate into intronic regions of actively transcribed genes.…”