Achieving Long-Term Factor VIII Stability with Non-Viral Gene Therapy: A Breakthrough for Hemophilia A Treatment

The primary treatment for Hemophilia A involves factor replacement therapy or bi-specific antibodies, which necessitate ongoing infusions and still allow for bleeding incidents, indicating a significant need for a non-viral gene therapy. A new DNA delivery system, piggyBac, which is distinct from AAV gene therapies, offers stable genomic integration of therapeutic genes to potentially cure hereditary deficiencies like Hemophilia A. The Super piggyBac transposase enzyme operates by extracting a DNA segment surrounded by specific recognition sequences and inserting it into TTAA genomic sites.

A non-viral, nanoparticle-based gene therapy delivery system has been created using two liver-targeting nanoparticles: one containing the SPB transposase as mRNA in a lipid nanoparticle, and another with a plasmid containing the human FVIII gene, its promoter, and piggyBac ITRs. This strategy surpasses AAV methods by providing a higher transgene cargo capacity, stable genomic integration, the possibility of re-dosing, and potentially simpler manufacturing.

The optimization of the human FVIII transposon was achieved by examining the promoter, transgene sequence, and UTR elements' impact on FVIII antigen levels. A series of transposons were prepared, and their FVIII levels were assessed after a single in vivo administration to mice. The most effective transposons were then tested in long-term efficacy studies.

Initial studies showed that separately optimized lipid nanoparticles for SPB mRNA or FVIII transposon DNA can be administered intravenously to mice. The transposase mRNA was rapidly converted to protein in the liver and remained expressed for several days. The transposon DNA, when delivered to the mouse liver, resulted in the expression of functional human FVIII protein, albeit at levels below therapeutic requirements. However, the co-administration of both types of nanoparticles led to sustained FVIII antigen levels within the therapeutic range in wild type animals.

A more pronounced response was observed in neonatal mice administered with the SPB mRNA and human FVIII transposon nanoparticles. The FVIII protein reached therapeutic levels and were sustained throughout the entire study period (5 months), unlike control animals that showed minimal human FVIII levels. This highlights the piggyBac system's potential for early-life treatment of genetic diseases through stable integration, avoiding the temporary effects of traditional non-integrating gene therapies.

A six-month efficacy study in a hemophilia A mouse model (FVIII exon 16 knockout/CD4 knockout, C57BL/6) was conducted to validate the long-term effectiveness of the treatment. The model lacks both FVIII and CD4, which allows for the measurement of FVIII activity levels without an immune response to human FVIII. The single treatment resulted in a dose-responsive increase in human FVIII activity levels, which were generally sustained throughout the study. In contrast, mice treated with a catalytically dead transposase showed negligible human FVIII activity.

In summary, the piggyBac platform offers a novel approach with the potential to provide a lifelong cure for hemophilia A. By ensuring stable transgene expression through genomic integration, this technology could significantly enhance the quality of life for patients with hemophilia A.