Mauricio Barajas-Mora

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.

Cutting-edge gene editing holds enormous promise for tackling devastating genetic diseases like hereditary angioedema (HAE). Here, we describe the efficient inactivation of the gene encoding pre-kallikrein, KLKB1, using our proprietary Cas-CLOVER™ high-fidelity nuclease with our non-viral, lipid nanoparticle (LNP) delivery system. Genetic inactivation of KLKB1 is an alternative clinical approach that provides durable relief to both Type I and II HAE. HAE is a rare genetic disease characterized by subcutaneous and submucosal edema, with swelling of the upper respiratory tract posing a life-threatening situation. Type I and II HAE are the most common types and are caused by mutations in the SERPING1 gene, which leads to compromised production or function of the C1 protease inhibitor. Strategies for treatment and prophylaxis include the restoration of C1 inhibitor function, or downstream antagonism of active plasma kallikrein. Safe and effective gene editing of KLKB1 could be a viable alternative for patients not adequately responding to the current standard of care. However, gene editing approaches must demonstrate an exquisitely high level of fidelity for optimal safety. To demonstrate such an approach with Cas-CLOVER, multiple guide RNAs (gRNA) targeting the human KLKB1 gene were screened in human hepatoma cell lines to identify gRNA pairs with optimal editing. Next, we evaluated KLKB1 protein reduction in primary human hepatocytes (PHH) that were incubated with LNPs encapsulating Cas-CLOVER mRNA along with each gRNA pair. Lead candidate gRNAs showed robust KLKB1 editing in a dose-responsive manner, achieving >65% editing and >85% reduction in KLKB1 protein secreted into culture medium at 0.5 ug/mL (EC90). To evaluate Cas-CLOVER off-target activity, oligo incorporation by iGUIDE was carried out by a licensed contract research organization. In this assay, double-stranded oligodeoxyribonucleotides (dsODNs) were co-electroporated with Cas-CLOVER mRNA, along with our lead KLKB1 gRNA pair, in the Huh7 cell line, and candidate off-target sites were identified by Illumina next-generation sequencing. Off-target activity was assessed by amplicon-seq at the eight top sites nominated by iGUIDE. In PHHs treated with 0.5 ug/mL of Cas-CLOVER LNPs, off-target editing was detected in 3/8 sites at very low levels (<0.25%). Remarkably, this low level of off-target editing remained unchanged when PHHs were treated with 10-fold higher concentrations of Cas-CLOVER LNP. For further evaluation of our platform, we sought to determine KLKB1 editing efficiency and fidelity in a mouse model of liver humanization. TK-Nog mice engrafted with PHHs were treated with a single intravenous injection of an LNP formulation co-encapsulating Cas-CLOVER mRNA and our lead KLKB1 gRNA pair. Amplicon-seq analysis demonstrated that 60% of KLKB1 alleles in the liver were edited. Importantly, no off-target editing was detected among the top eight sites identified by iGUIDE, including the three off-target sites validated in cultured PHHs. Next, we evaluated efficacy and tissue specificity of our platform in wild type mice. C57BL/6 male and female mice were dosed with LNP encapsulating Cas-CLOVER mRNA and mouse Klkb1-targeting gRNAs. A single intravenous LNP injection achieved high Klkb1 editing (>50% of haploid genomes) in the liver and >80% reduction in serum pre-kallikrein levels. No Klkb1 editing was detected in gonads. In summary, these results highlight the efficacy and specificity of our high-fidelity Cas-CLOVER gene editing platform that enables targeted and therapeutically relevant kallikrein reduction in a fully non-viral manner. These data provide a promising foundation for the development of a highly specific gene editing therapy for HAE.