BACKGROUND AND AIMS:Alpha-1 antitrypsin (AAT), encoded by the SERPINA1 gene, is primarily synthesized in the liver and secreted into the blood. The SERPINA1 Z allele encodes Z-AAT (Glu342Lys), a variant that self-associates into polymers that are retained in hepatocytes and trigger inflammation and hepatic injury. Targeting and preventing Z-AAT polymerization allows a therapy to directly address the pathophysiology of AAT deficiency (AATD) associated liver disease. APPROACH AND RESULTS:A structure-based design approach was used to develop BMN 349, a small-molecule polymerization blocker capable of stabilizing Z-AAT to prevent new polymerization and promote hepatic clearance. BMN 349 bound Z-AAT ~150 times faster in vitro than wild-type M-AAT and promoted secretion and reduced polymer levels in a cell model of Z-AAT deficiency. Crystallography with M-AAT showed that BMN 349 partially displaces a region involved in polymerization, the reactive center loop proximal hinge. Oral dosing in female PiZ transgenic mice with 20-200 mg/kg/day BMN 349 for 30 days was associated with dose-dependent increases in total plasma Z-AAT and a concurrent decrease in circulating and liver Z-AAT polymers relative to vehicle-treated controls. Histopathologic and proteomic assessments of liver and plasma samples from these mice showed that doses ≥100 mg/kg/day of BMN 349 reduced liver inflammation and improved liver health biomarkers after 30 days. CONCLUSIONS:This study is the first to demonstrate a reduction in Z-AAT polymer burden without altering protein expression. Improvements in liver inflammation and function following BMN 349 treatment support further investigations of its therapeutic benefits for AATD liver disease.

Introduction Prior to commercial withdrawal, adeno-associated virus (AAV) gene therapy was approved for the treatment of adults with severe hemophilia A. Mechanisms underlying variability, durability, and liver transaminitis are largely uncharacterized. Objectives To evaluate the effects of prophylactic corticosteroid administration on AAV gene therapy outcomes in severe hemophilia A dogs. Methods Seven hemophilia A dogs received 6e13 vector genomes/kg AAV5-canine factor VIII (AAV5-cFVIII). Four dogs received oral prednisolone (1 mg/kg/day) starting 3 hours pre-infusion with dose-tapering over 6 weeks; three control dogs received no corticosteroids. Percutaneous liver biopsies were performed on detection of alanine transaminase (ALT) >2-fold the upper limit of normal (ULN). Results All dogs expressed therapeutic FVIII:C (8.7-56.1%), improved whole blood clot time, and decreased bleeding rates (pre=6.11 vs. post=1.42 bleeds/year, p=0.016) over 2 years post-AAV5-cFVIII. Corticosteroid-treated dogs demonstrated a modest increase in mean FVIII:C at two years by chromogenic substrate assay (CSA) (39.1%) compared with controls (29.5%), driven by one female with markedly elevated FVIII:C from day 54 onward. When analyzed by sex, all female dogs exhibited increased mean FVIII:C CSA at 2 years (49.3%) compared with males (9.1%), regardless of prophylactic corticosteroid use. Prophylactic corticosteroids did not impact transient post-treatment elevations of pro-inflammatory cytokines, anti-AAV antibody formation, or ALT levels. One corticosteroid-treated dog experienced ALT >4.3-fold ULN at 18 weeks without impacting long-term FVIII:C expression. A liver biopsy showed diffuse, minimal periportal lymphocyte and neutrophil infiltration, consistent with non-specific minimal hepatitis. Conclusions Prophylactic corticosteroids were not clearly associated with increased transgene expression in hemophilia A dogs.

The GM2 gangliosidoses, Tay-Sachs disease and Sandhoff disease, are devastating neurodegenerative disorders caused by β-hexosaminidase A (HexA) deficiency. In the Sandhoff disease mouse model, rescue potential was severely reduced when HexA was introduced after disease onset. Here, we assess the effect of recombinant HexA and HexD3, a newly engineered mimetic of HexA optimized for the treatment of Tay-Sachs disease and Sandhoff disease. Enzyme replacement therapy was administered by repeat intracerebroventricular injections in Sandhoff disease model mice with dosing beginning before and after signs of neurodegeneration. As previously observed, HexA effectively increased the lifespan of Sandhoff disease mice by 3.5-fold only when treatment was started before onset of neurodegeneration. In contrast, HexD3 halted motor decline and ameliorated late-stage disease severity even when dosing began late, after neurodegeneration onset. Additionally, HexD3 had advantages over HexA in enzyme stability, distribution potential, and homodimer activity. Overall, our data indicate that advanced therapeutics may widen the treatment window for neurodegenerative disorders.

Introduction Roctavian (AAV5-HLP-hFVIII-SQ) is an approved gene therapy in the EU for the treatment of severe hemophilia A. A single infusion provides therapeutic levels of FVIII expression in adult men. The precise mechanisms contributing to variability and durability in transgene expression are unknown. Multiple lines of evidence suggest low RNA production contributes to the decline of FVIII expression and low-response to AAV gene therapy. Previously, we have shown that decreased interaction of active histones with episomal genomes may mediate the decline in transgene expression. Additional studies suggested hepatocyte capacity to fold and secrete FVIII may contribute to variability. We hypothesize (1) modifying the chromatin interaction with AAV-episomes using epigenic regulators may increase accessibility of vector genomes and (2) the use of molecular chaperones may improve FVIII folding and secretion.

Adeno-associated virus (AAV) vectors are used to deliver therapeutic transgenes, but host immune responses may interfere with transduction and transgene expression. We evaluated prophylactic corticosteroid treatment on AAV5-mediated expression in liver tissue. Wild-type C57BL/6 mice received 6 × 1013 vg/kg AAV5-HLP-hA1AT, an AAV5 vector carrying a human α1-antitrypsin (hA1AT) gene with a hepatocyte-specific promoter. Mice received 4 weeks of daily 2 mg/kg prednisolone or water starting day -1 or 0 before vector dosing. Mice that received prophylactic corticosteroids had significantly higher serum hA1AT protein than mice that did not, starting at 6 weeks and persisting to the study end at 12 weeks, potentially through a decrease in the number of low responders. RNAseq and proteomic analyses investigating mechanisms mediating the improvement of transgene expression found that prophylactic corticosteroid treatment upregulated the AAV5 coreceptor platelet-derived growth factor receptor alpha (PDGFRα) on hepatocytes and downregulated its competitive ligand PDGFα, thus increasing the uptake of AAV5 vectors. Evidently, prophylactic corticosteroid treatment also suppressed acute immune responses to AAV. Together, these mechanisms resulted in increased uptake and preservation of the transgene, allowing more vector genomes to be available to assemble into stable, full-length structures mediating long-term transgene expression. Prophylactic corticosteroids represent a potential actionable strategy to improve AAV5-mediated transgene expression and decrease intersubject variability.

Introduction The investigation of AAV vectored gene therapies has increased exponentially over the past decade for the treatment of monogenic disorders. Many clinical programs have adopted the use of corticosteroids or other immune modulatory therapies to prevent or treat inflammatory responses and preserve transgene expression following gene therapy dose administration, however there is no clear consensus across sponsors on the optimal immunosuppressive regimen. In the current study, we evaluated the pharmacodynamic and immunomodulatory effects of seven different immunosuppressive agents administered prophylactically prior to gene therapy dose administration and continued for four weeks after.

Background: Hypertrophic cardiomyopathy (HCM) is a life-threatening inherited heart disease characterized by left ventricular hypertrophy and diastolic dysfunction. The most common cause of HCM is genetic variants in MYBPC3 , encoding cardiac myosin binding protein C (cMyBP-C), a sarcomeric protein with structural and regulatory roles. The majority of MYBPC3 gene variants are truncating leading to protein haploinsufficiency. Hypothesis: Transfer of a functional copy of MYBPC3 to heart muscle deficient in cMyBP-C will lead to sustained improvements in cardiac function. Aims: To determine whether BMN 293 (AAV-hMYBPC3) can restore cMyBP-C levels in the sarcomere and halt and/or reverse disease progression in non-clinical models of genetic HCM due to MYPBC3 deficiency. Methods: BMN 293 is an adeno-associated virus (AAV) vector that encodes wild-type human MYBPC3 under the control of a cardiomyocyte-selective promoter. We transduced human iPSC-derived cardiomyocytes and engineered heart tissues (EHTs) carrying a compound heterozygous truncating MYBPC3 mutation (MYBPC3 -/- ) with BMN 293 and assessed cMyBP-C levels and contractile parameters. We also systemically administered BMN 293 to MYBPC3 -/- mice and assessed cardiac distribution of human cMyBP-C by molecular and histological methods and determined its impact on left ventricular hypertrophy and function by echocardiography and other imaging techniques. Results: BMN 293 transduction of human iPSC MYBPC3 -/- cardiomyocytes and EHTs resulted in high levels of human MYBPC3 mRNA and cMyBP-C protein, correct incorporation of cMyBP-C into the sarcomere, and complete normalization of contractile kinetics. BMN 293 was well tolerated in MYBPC3 -/- mice and resulted in uniform restoration of cMyBP-C expression throughout the heart and significant correction of structural and functional cardiac abnormalities. Conclusions: A single IV infusion of BMN 293 to MYBPC3 -/- mice resulted in early and sustained reduction in left ventricular hypertrophy and durable improvements in diastolic function.

Recombinant adeno-associated virus (rAAV) vectors are often produced in HEK293 or Spodoptera frugiperda (Sf)-based cell lines. We compared expression profiles of "oversized" (∼5,000 bp) and "standard-sized" (4,600 bp) rAAV5-human α1-antitrypsin (rAAV5-hA1AT) vectors manufactured in HEK293 or Sf cells and investigated molecular mechanisms mediating expression decline. C57BL/6 mice received 6 × 1013 vg/kg of vector, and blood and liver samples were collected through week 57. For all vectors, peak expression (weeks 12-24) declined by 50% to week 57. For Sf- and HEK293-produced oversized vectors, serum hA1AT was initially comparable, but in weeks 12-57, Sf vectors provided significantly higher expression. For HEK293 oversized vectors, liver genomes decreased continuously through week 57 and significantly correlated with A1AT protein. In RNA-sequencing analysis, HEK293 vector-treated mice had significantly higher inflammatory responses in liver at 12 weeks compared with Sf vector- and vehicle-treated mice. Thus, HEK293 vector genome loss led to decreased transgene protein. For Sf-produced vectors, genomes did not decrease from peak expression. Instead, vector genome accessibility significantly decreased from peak to week 57 and correlated with transgene RNA. Vector DNA interactions with active histone marks (H3K27ac/H3K4me3) were significantly reduced from peak to week 57, suggesting that epigenetic regulation impacts transgene expression of Sf-produced vectors.

Recombinant adeno-associated virus (AAV) is an effective platform for therapeutic gene transfer; however, tissue-tropism differences between species are a challenge for successful translation of preclinical results to humans. We evaluated the use of in vitro primary hepatocyte cultures to predict in vivo liverdirected AAV expression in different species. We assessed whether in vitro AAV transduction assays in cultured primary hepatocytes from mice, nonhuman primates (NHPs), and humans could model in vivo liver-directed AAV expression of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), an experimental gene therapy for hemophilia A with a hepatocyte-selective promoter. Relative levels of DNA and RNA in hepatocytes grown in vitro correlated with in vivo liver transduction across species. Expression in NHP hepatocytes more closely reflected expression in human hepatocytes than in mouse hepatocytes. We used this hepatocyte culture model to assess transduction efficacy of a novel liver-directed AAV capsid across species and identified which of 3 different canine factor VIII vectors produced the most transgene expression. Results were confirmed in vivo. Further, we determined mechanisms mediating inhibition of AAV5-hFVIII-SQ expression by concomitant isotretinoin using primary human hepatocytes. These studies support using in vitro primary hepatocyte models to predict species translatability of liver-directed AAV gene therapy and improve mechanistic understanding of drug-drug interactions.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by N-sulfoglucosamine sulfohydrolase (SGSH) deficiency. SGSH removes the sulfate from N-sulfo-glucosamine residues on the nonreducing end of heparan sul-fate (HS-NRE) within lysosomes. Enzyme deficiency results in accumulation of partially degraded HS within lysosomes throughout the body, leading to a progressive severe neuro-logical disease. Enzyme replacement therapy has been pro-posed, but further evaluation of the treatment strategy is needed. Here, we used Chinese hamster ovary cells to produce a highly soluble and fully active recombinant human sulfami-dase (rhSGSH). We discovered that rhSGSH utilizes both the CI-MPR and LRP1 receptors for uptake into patient fibroblasts. A single intracerebroventricular (ICV) injection of rhSGSH in MPS IIIA mice resulted in a tissue half-life of 9 days and widespread distribution throughout the brain. Following a single ICV dose, both total HS and the MPS IIIA disease -specific HS-NRE were dramatically reduced, reaching a nadir 2 weeks post dose. The durability of effect for reduction of both substrate and protein markers of lysosomal dysfunction and a neuroimmune response lasted through the 56 days tested. Furthermore, seven weekly 148 lig doses ICV reduced those markers to near normal and produced a 99.5% reduction in HS-NRE levels. A pilot study utilizing every other week dosing in two animals supports further evaluation of less frequent dosing. Finally, our dose-response study also suggests lower doses may be efficacious. Our findings show that rhSGSH can normalize lysosomal HS storage and markers of a neuro-immune response when delivered ICV.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) gene transfer provided reduced bleeding for adult clinical trial participants with severe hemophilia A. However, pediatric outcomes are unknown. Using a mouse model of hemophilia A, we investigated the effect of vector dose and age at treatment on transgene production and persistence. We dosed AAV5hFVIII-SQ to neonatal and adult mice based on body weight or at a fixed dose and assessed human factor VIII-SQ variant (hFVIII-SQ) expression through 16 weeks. AAV5-hFVIII-SQ dosed per body weight in neonatal mice did not result in meaningful plasma hFVIII-SQ protein levels in adulthood. When treated with the same total vector genomes per mouse as adult mice, neonates maintained hFVIII-SQ expression into adulthood, although plasma levels were 3- to 4-fold lower versus mice dosed as adults. Mice <1 week old initially exhibited high hFVIII-SQ plasma levels and maintained meaningful levels into adulthood, despite a partial decline potentially due to age-related body mass and blood volume increases. Spatial transduction patterns differed between mice dosed as neonates versus adults. No features of hepatotoxicity or endoplasmic reticulum stress were observed with dosing at any age. These data suggest that young mice require the same total vector genomes as adult mice to sustain hFVIII-SQ plasma levels.

two large general population cohorts, and with MRIquantified hepatic inflammation.We hypothesize that Thr95Ile heterozygosity associates with a mild form of hepatic manganese accumulation leading to liver damage.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype 5 (AAV5)-based gene therapy vector containing a B-domain-deleted human coagulation factor VIII (hFVIII) gene controlled by a liver-selective promoter. AAV5-hFVIII-SQ is currently under clinical investigation as a treatment for severe hemophilia A. The full-length AAV5-hFVIII-SQ is >4.9 kb, which is over the optimal packaging limit of AAV5. Following administration, the vector must undergo a number of genome-processing, assembly, and repair steps to form full-length circularized episomes that mediate long-term FVIII expression in target tissues. To understand the processing kinetics of the oversized AAV5-hFVIII-SQ vector genome into circular episomes, we characterized the various molecular forms of the AAV5-hFVIII-SQ genome at multiple time points up to 6 months postdose in the liver of murine and non-human primate models. Full-length circular episomes were detected in liver tissue beginning 1 week postdosing. Over 6 months, quantities of circular episomes (in a predominantly head-to-tail configuration) increased, while DNA species lacking inverted terminal repeats were preferentially degraded. Levels of duplex, circular, full-length genomes significantly correlated with levels of hFVIII-SQ RNA transcripts in mice and non-human primates dosed with AAV5-hFVIII-SQ. Altogether, we show that formation of full-length circular episomes in the liver following AAV5-hFVIII-SQ transduction was associated with long-term FVIII expression.

Introduction AAV-mediated gene therapy vectors represent a complex drug design with multiple components that may impact immunogenicity. Clinical trials monitor immunogenicity directed toward both the vector delivery system and the expressed transgene product. Valoctocogene roxaparvovec is an AAV5-mediated gene therapy under investigation for the treatment of hemophilia A and encodes a codon-optimized B-domain deleted human FVIII protein (hFVIII-SQ) under control of a liver-selective promoter. This report describes clinical immunogenicity monitoring data from up to 1 year of follow up from GENEr8-1, a Phase 3, single-arm, open-label study in 134 male participants with severe hemophilia A.

Introduction Adeno-associated virus (AAV)-based gene therapy can initiate host-immune responses to the AAV vector components or the transgenic protein product which could reduce AAV-mediated expression. Data from preclinical studies suggest that corticosteroid treatment may limit these immune responses and improve gene therapy outcomes.