Delivery of large transgene cassettes by foamy virus vector

Abstract

Viral vectors are effective tools in gene therapy, but their limited packaging capacity can be restrictive. Larger clinically-relevant vectors are needed. Foamy viruses have the largest genomes among mammalian retroviruses and their vectors have shown potential for gene therapy in preclinical studies. However, the effect of vector genome size on titre has not been determined. We inserted increasing lengths of the dystrophin open reading frame in a foamy virus vector and quantified packaged vector RNA and integrated DNA. For both measures, a semi-logarithmic reduction in titre was observed as genome size increased. Concentrated titres were reduced 100-fold to approximately 106 transducing units per ml when vector genomes harboured a 12 kb insert, approximately twice that reported for lentivirus vectors in a comparable study. This potential was applied by optimising foamy virus vectors carrying the full-length dystrophin open-reading frame for transduction of human muscle derived cells. Full-length dystrophin protein was expressed and transduced cells remained able to form myotubes in vitro. Foamy virus vectors are well-suited for stable delivery of large transgene cassettes and warrant further investigation for development as a therapy for Duchenne or Becker muscular dystrophy.

The impact of lentiviral vector genome size and producer cell genomic to gag-pol mRNA ratios on packaging efficiency and titre

“…Alternatively, this biological constraint of HIV-1 may be avoided by selecting an alternative integrating vector system, such as foamy viral vectors, which have been shown to tolerate large inserts with reduced impact on titer. 13 …”

“… 10 Similarly, Moloney murine leukemia virus was reported to lose titer as genome size increased due to a combination of packaging, entry, and reverse transcription defects, 11 whereas replication-competent vectors based on the spleen necrosis virus were shown to have a replication defect at sizes greater than 9.4 kb and unable to replicate at sizes above 10 kb, reportedly due to a packaging defect. 12 Foamy virus vectors lose functional titer with increasing genome size at half the rate of HIV-1-based vectors, 13 supporting a role of reverse transcription to the overall impact of genome size on vector titer, since the foamy virus reverse transcriptase is reported to have higher processivity than that of HIV-1. 14 …”

Restoring Dystrophin Expression in Duchenne Muscular Dystrophy: Current Status of Therapeutic Approaches

“…Additionally, there may be a length threshold in dystrophin [44,79], i.e., a protein that is small or truncated in length may not be protective. Further preclinical studies aim to deliver larger or full-length dystrophin via human artificial chromosome [80], lentiviral vectors [81], or foamy viral vector [82]. Moreover, the durability of the therapy is a cause for concern, but systemic administration of micro-dystrophin in the canine model resulted in significant and sustained levels of micro-dystrophin in skeletal muscles and reduced disease symptoms for at least 2 years [83].…”

Foamy Virus Integrase in Development of Viral Vector for Gene Therapy

“…FVs are the most promising vectors for large transgene delivery because they have the largest mammalian retroviral genome [117,125,126]. Sweeney et al investigated the ability of FVVs to incorporate the simian macaque FV envelope using physiological promoters to efficiently deliver large transgene cassettes by inserting increasing lengths of the dystrophin open reading frames in an FVV before quantifying the packaged vector RNA and integrated DNA [127,128]. Molecular assays showed that a 12-kb insert could be packaged, delivered, and integrated into a target human cell genome at a sufficient titer for ex vivo gene therapy.…”