Sustained Factor VIII Activity Following Single Dose of Non-Viral Integrating Gene Therapy (2022)

The current first-line treatment for Hemophilia A patients is factor replacement therapy and/or bi-specific antibodies; however, these treatments require continuous infusions over the course of the patient's life, and patients continue to have breakthrough bleeds, representing a high unmet need for a non-viral gene therapy. Unlike conventional AAV gene therapies, our proprietary piggyBac® DNA delivery platform facilitates stable genomic insertion of a therapeutic transgene as a means of developing lifelong cures of hereditary genetic deficiencies such as hemophilia A. The Super piggyBac (SPB) transposase enzyme functions by excising a DNA cargo flanked by SPB recognition ITRs and inserting into TTAA genomic sites. We have developed a non-viral, nanoparticle-based delivery system to enable piggyBac-based gene therapy. This system entails two liver-tropic nanoparticles: a lipid nanoparticle encapsulating the SPB transposase formulated as mRNA, and a second nanoparticle encapsulating a plasmid comprising the human FVIII gene, promoter, and piggyBac ITRs. This non-viral strategy provides certain advantages over AAV-based approaches: increased transgene cargo capacity beyond 4.7 kb, stable integration of the FVIII transgene into the genome, the potential for re-dosing, and potentially simpler manufacturing processes, relative to AAV-based vector production. Optimization of the human FVIII transposon was conducted, specifically to elucidate the role of promoter, transgene sequence, and UTR elements in FVIII antigen levels. A panel of transposons was prepared and FVIII levels assessed following single administration in vivo to wild type mice. The transposons with the highest FVIII performance were subsequently evaluated in long-term efficacy studies. Initial proof-of-concept studies demonstrated that LNPs separately optimized for SPB mRNA or FVIII transposon DNA delivery can be co-administered intravenously to mice. We observed that transposase mRNA delivered to the liver is converted to protein rapidly and expressed for several days. Transposon DNA delivered to mouse liver results in expression of functional human FVIII protein, though at sub-therapeutic levels at the doses evaluated here. Co-administration of the transposon and transposase nanoparticles resulted in sustained FVIII antigen levels in the anticipated therapeutic range (>50% of normal) in wild type animals. We observed a more dramatic response when the dual-nanoparticle system was evaluated in neonatal (post-natal day 1) wild type mice. Mice were co-administered the SPB mRNA LNP and the human FVIII transposon nanoparticle. Control mice were co-administered the same dose of a catalytically dead SPB transposase, which is unable to mediate genomic integration. FVIII protein reached therapeutic levels in plasma and were sustained over the full study duration (5 months), whereas control animals exhibited negligible (<2%) human FVIII levels. These findings illustrate the utility of the piggyBac DNA delivery system for treating genetic disease early in life, enabled by stable integration. Thus, piggyBac platform avoids the transient efficacy typical of conventional non-integrating gene therapy approaches. To further validate this long-term durability, a six-month efficacy study was conducted in a mouse model of hemophilia A (FVIII exon 16−/−/CD4−/−, C57BL/6). This model is deficient in FVIII as well as CD4, allowing measurement of FVIII activity levels afforded the absence of an immune response to human FVIII. We observed that a single treatment yielded 30 - 150% of human FVIII activity in a dose-responsive manner, which was generally sustained over the duration of the study. Mice treated with the same dose of human FVIII transposon and the catalytically dead transposase exhibited negligible (<2%) human FVIII activity at all timepoints evaluated. In conclusion, our results demonstrate the novelty of our piggyBac® platform and approach in providing a potential lifelong functional cure for hemophilia A. By providing stable transgene expression through genomic integration, this technology has the potential to greatly improve the lives of hemophilia A patients.