527. Exploiting Retroviral Recombination for the Delivery of Full-Length Dystrophin cDNA

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin expression in patient muscle cells. Restoration of dystrophin expression could alleviate the symptoms of DMD, although the large size of full-length dystrophin cDNA (11.1kb) and the restricted packaging capacity of viral vectors limit the application of standard gene transfer methods.Retroviral vectors have a pseudo-diploid RNA genome which can co-package two distinct RNA genomes to form a heterozygous virion. Template-switching between co-packaged strands is a common event during HIV-1 reverse transcription, which can lead to recombination and the production of chimeric proviruses. Template-switching can be influenced by a number of cis and trans-acting factors such as structures in the viral packaging sequences and mutations of reverse transcriptase.In this work, the recombinogenic characteristics of HIV-1 based lentiviral vectors have been exploited for the purpose of reconstituting full-length dystrophin cDNA from overlapping 5’ and 3’ portions of the full-length coding sequence. The vector has been initially optimised using a small bicistronic cassette to evaluate the recombination efficiency in standard lentiviral vectors compared with those harbouring mutations in the HIV-1 packaging sequence and reverse transcriptase.Interestingly, the results suggested that a standard lentivirus vector could deliver a full-length, recombined provirus with greater efficiency than vectors harbouring mutations to cis and trans factors. A multiplicity of infection (MOI) of 40 heterozygous vector particles per target cell was sufficient to obtain a reconstituted full-length Neomycin-IRES-GFP provirus in 40% of transduced HeLa cells. When the viral RNA components were further modified to render functional provirus synthesis dependent on recombination (schematic displayed in figure 1), the vector was capable of reconstituting a full-length dystrophin-gfp fusion protein in HEK293t cells. Flow cytometry analysis showed that heterozygous vectors produced 0.1% GFP-positive cells following a MOI50 vector dose. Furthermore, genomic DNA extracted from these cells contained a full-length dystrophin provirus, which was detected by nested PCR and sequenced by clonal analysis.This work reveals that retroviral recombination offers a means to circumvent retroviral packaging limitations and may be of use in dystrophin gene therapy following further optimisation. View Large Image | Download PowerPoint Slide