Sustained FVIII Expression with a Tolerable, Titratable, Fully Non-Viral Gene Therapy for Hemophilia a
Authors: Schwerk Johannes , Barajas-Mora Mauricio , Wang Hua , Truong Brian , Bennett Michael , Cam Nicholas , Clancey James , Davis Alicia , Hajj Khal , Jacques Bonnie , Tamayo Johnnatan , Yen James , Rychak Joshua , Madison Blair 
Published: December 2024

Although the standard of care for patients with severe hemophilia A (HemA) has dramatically improved in recent decades, patients still face the risk of breakthrough bleeds, chronic joint damage, and pain. The clinical experience with most AAV-based gene therapies has not supported sustained circulating FVIII activity at functionally curative levels. Moreover, these products cannot be widely deployed due to pre-existing AAV immunity, frequently require sustained immunosuppression, and are not suitable for use in pediatric patients.
To overcome these challenges, we are developing a gene insertion platform coupled with a novel lipid nanoparticle (LNP)-based delivery system to power a durable and efficacious gene therapy for severe HemA. The piggyBac DNA insertion system is a transposon-based platform that enables controlled and stable integration of a therapeutic transgene into the patient's genome. The double stranded DNA (dsDNA) transposon containing the hFVIII expression cassette and an mRNA encoding the transposase enzyme that mediates integration of the FVIII transgene are co-encapsulated into a single LNP. Our data suggest that genomic integration of the therapeutic hFVIII transgene could enable lifelong durability following a single dose, while also opening the possibility of treating patients early in life. We present data demonstrating stable therapeutic hFVIII expression and restoration of clotting hemostasis in juvenile and adult hemophilic mice spanning for up to 13 months following a single treatment.
The non-viral delivery system reported here also has the potential for repeated administration, which could enable an individualized “titrate-to-efficacy” dosing strategy. We observed increasing levels of hFVIII expression following three repeated doses spaced by 3 weeks in adult hemophilic mice. To further ensure long-term efficacy and mitigate potential adaptive immunity against the SPB transposase after successive doses, the mRNA was engineered to incorporate a specific motif within the UTR intended to suppress expression in antigen-presenting cells. Repeat dosing (3X) of this construct in adult immunocompetent mice yielded negligible IFNg+ splenocytes, while controls showed approximately 300-fold above background.
Gene therapies are traditionally irreversible, which may present safety challenges in the event that a patient expresses supraphysiological FVIII as has been observed for some AAV therapeutics. To enable further control over FVIII levels, we incorporated an inducible “modulator switch” into the DNA transposon. In adult mice we demonstrate that hFVIII expression resulting from our integrating gene therapy can be reduced in a dose-responsive manner by intravenous administration of a small molecule. The modulator switch concept may enable more precise control over the dose-response on an individual patient basis or could theoretically be deployed to eliminate all transposed cells should an unwanted transformation event ever occur.
In addition, we also investigated methods for increasing potency to achieve greater DNA delivery into cells and thus higher overall FVIII levels. A novel class of amphipathic compounds, referred to here as “intracellular trafficking agents” (ICTAs's), were discovered that significantly potentiate the expression from LNP-delivered dsDNA. Incorporation of an ICTA into our LNP resulted in a ~10-fold increase in expression, achieving target hFVIII antigen levels in adult immunocompetent mice following a single dose at or below 1 mg/kg.
The use of dsDNA as a therapeutic payload introduces potential safety challenges arising from stimulation of the innate immune system and pro-inflammatory cytokine release. Thus, we are developing additional platform technologies to mitigate these challenges. We investigated novel solutions for preventing hepatotoxicity and unwanted immune stimulation by engineering a shielded LNP bearing a GalNAc targeting ligand that reduces uptake of the LNPs by macrophages. This strategy yields a dramatic (2-3 log) reduction of pro-inflammatory cytokines while maintaining robust hFVIII expression.
The non-viral platform components presented here-including novel ionizable lipids, inducible modulator switch, targeting ligands, and ICTAs-combined with the piggyBac DNA insertion system may enable a safer, tunable, and durable solution for FVIII restoration in HemA patients.