Kevin Briseno

Base editors can correct disease-causing genetic variants. After a neonate had received a diagnosis of severe carbamoyl-phosphate synthetase 1 deficiency, a disease with an estimated 50% mortality in early infancy, we immediately began to develop a customized lipid nanoparticle-delivered base-editing therapy. After regulatory approval had been obtained for the therapy, the patient received two infusions at approximately 7 and 8 months of age. In the 7 weeks after the initial infusion, the patient was able to receive an increased amount of dietary protein and a reduced dose of a nitrogen-scavenger medication to half the starting dose, without unacceptable adverse events and despite viral illnesses. No serious adverse events occurred. Longer follow-up is warranted to assess safety and efficacy. (Funded by the National Institutes of Health and others.).

Current first-line treatments for Hemophilia A patients are clotting factor replacement or bi-specific antibodies. These treatments require continuous, lifelong infusions, yet many patients have breakthrough bleeds. There remains a high unmet need for safe, effective, and durable therapies. We have developed a liver-directed non-viral in vivo gene insertion approach using the piggyBac® DNA insertion system. Unlike conventional AAV-based gene therapy, our platform enables delivery of large transgenes, the ability to stably and efficiently integrate the therapeutic transgene into the genome, and the potential for re-dosing to titrate to target FVIII activity levels. A key challenge for all gene insertion systems relates to the need to safely and efficiently deliver the transgene DNA. In the current study we evaluated a novel hepatocyte-targeted non-viral platform able to co-deliver both DNA and mRNA with superior safety and specificity relative to the traditional liver-directed LNP concept. We first evaluated a two nanoparticle system using conventional 4-component liver-directed LNPs, with one lipid nanoparticle (LNP) encapsulating the mRNA for the super piggyBac (SPB) transposase (LNP-SPB), and a second LNP encapsulating a plasmid containing the hFVIII transposon DNA (LNP-hFVIII). We subsequently developed a novel co-encapsulated LNP formulation, comprising both SPB mRNA and transposon plasmid DNA (LNP-SPB-hFVIII). In juvenile WT mice we observed a 50% increase in hFVIII antigen expression compared to the dual nanoparticle approach. This was further validated in a severe hemophilia A mouse model (FVIII knock-out). Following a single dose of the co-encapsulated LNP-SPB-hFVIII to adult hemophilia A mice tolerized to human FVIII, we observed ~30% of normal hFVIII expression sustained over the duration of the 7 month study. To further support the concept of re-dosing, we treated immunocompetent adult hemophilia A mice tolerized to human FVIII with repeated administrations every 3 weeks. We observed a dose-proportionate increase in FVIII activity after each administration, reaching an average hFVIII activity of 96% of normal, following 3 repeated doses. Traditional LNPs use several lipid components that typically include an ionizable lipid, cholesterol, a polyethylene glycol (PEG) lipid, and a structural lipid, each with a unique function to effectively encapsulate and deliver the DNA and mRNA required for the piggyBac platform. Cellular transfection of these traditional LNPs has been demonstrated to occur by passive mechanisms such as micropinocytosis or Apolipoprotein E (ApoE)-mediated endocytosis by the low-density lipoprotein receptor (LDLR). However, this can also result in delivery to unwanted cell-types, particularly tissue-resident immune cells. To address this potential failure mode, we explored the addition of a N-Acetylgalactosamine (GalNac)-based targeting ligand to our co-encapsulated LNP formulation (LNP-SPB-hFVIII-G). In immunocompetent animals, targeted LNPs yielded a ~2-3-log reduction in pro-inflammatory serum cytokines (IL-6, IFNɣ) while maintaining high hFVIII expression and no elevation in transaminases (ALT, AST). In conclusion, our results demonstrate the capabilities of the piggyBac DNA insertion system and non-viral approach in providing stable FVIII transgene expression through genomic integration, along with the potential for redosing. Additionally, we have highlighted the tolerability profile of our current generation of liver-targeted non-viral delivery platform. Altogether, these data provide proof-of-principle toward developing an effective and durable therapy for Hemophilia A.

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.

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 &amp;gt;65% editing and &amp;gt;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 (&amp;lt;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 (&amp;gt;50% of haploid genomes) in the liver and &amp;gt;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.

Abstract Multiple myeloma (MM) is a clonal plasma cell malignancy characterized by bone marrow infiltration, monoclonal immunoglobulin production, and microenvironmental dysregulation that leads to systemic organ damage. The advent of B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell therapy has induced unprecedented responses and durability for patients with relapsed/refractory MM. These outcomes are rarely observed with prior salvage strategies, although relapse remains the predominant long-term challenge for most patients. The two currently approved BCMA CAR-T cell products use viral vectors to semi-randomly insert the CAR gene, which results in heterogeneous genomic composition and variability in efficacy, safety, and product consistency. To address these challenges, we integrated targeted CRISPR genome engineering with precise CAR transgene insertion at the T-cell receptor alpha constant ( TRAC ) locus, 1XX CAR signaling architecture to enhance potency and durability, and non-viral manufacturing with a single-stranded DNA repair template to improve efficiency and yield. This approach confers physiological CAR expression, reduces insertional mutagenesis, and improves persistence by mitigating tonic signaling and exhaustion. Our GMP manufacturing process consistently achieved high CAR integration (37.7–72.7%) and yields across all full-scale runs and met predefined release criteria for identity, purity, safety, and quality. In NSG mouse models of MM, the UCCT-BCMA-1 product exhibited exceptionally potent tumor control, CAR-T cell expansion 100–1000-fold greater than that of lentiviral constructs, and durable clearance of myeloma cells after multiple rechallenges. These findings establish a CRISPR-edited, fully non-viral manufacturing platform for next-generation 1XX-BCMA CAR-T therapies with enhanced persistence, safety, and efficacy. One Sentence Summary CRISPR-engineered, TRAC -targeted 1XX-BCMA CAR-T therapy with improved safety, potency, and persistence in relapsed and refractory multiple myeloma.