An improved, novel, systemically administered AAV gene therapy for treatment of CLN3 juvenile neuronal ceroid lipofuscinosis

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Abstracts / Molecular Genetics and Metabolism 126 (2019) S17–S156

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Seinäjoki Central Hospital, Seinäjoki, Finland, cHelsinki University Hospital, Helsinki, Finland, dTampere University Hospital, Tampere, Finland, eKainuu Central Hospital, Kajaani, Finland, fPori Central Hospital, Pori, Finland, gCentral Finland Central Hospital, Jyväskylä, Finland, hKuopio University Hospital, Kuopio, Finland, iVaasa Central Hospital, Vaasa, Finland, jSouth Karelia Central Hospital, Lappeenranta, Finland, kOulu University Hospital, Oulu, Finland Enzyme replacement therapy (ERT) has stabilized renal function but its effect on proteinuria is inconsistent. Also nephroprotective renin-angiotensin-aldosterone system (RAAS) inhibition is warranted. We calculated eGFR using CKD-EPI formula and albuminuria using urine albumin/creatinine ratio (ACR) once a year in 12 Fabry patients with ERT for 10 years (8 females, 4 males, age 53.6 (15.0) years) and in 24 patients with ERT for 5 years (11 females, 13 males, 53.3 (17.2) years). In the 10-year group 5 patients and in the 5-year group 12 patients used RAAS inhibition at the end of follow-up. General linear model for repeated measures and Pearson correlation were used. Two patients who got renal transplantation were excluded. EGFR decreased 5.0 (SD 15.3, median 5.0) ml/min/1.73 m2, slope -0.5/ year in the 10-year group and increased 0.7 (SD 9.2, median -2.2), slope +0.14/year in the 5-year group. No statistically significant change was seen in either group. ACR increased 2.5 (SD 6.7, median 0.07) mg/mmol, slope 0.25/year in the 10-year group and 1.3 (SD 4.6, median 2.2) mg/mmol, slope 0.26/year in the 5-year group. No statistically significant change was seen in either group. In the 10-year group eGFR decreased more than 15 ml/min/1.73 m² in 4 patients and in the 5-year group in one patient. ACR increased over 5 mg/mmol in 2 patients both in the 10-year and in the 5-year group. Neither age, age at the initiation of ERT, gender, use of agalsidase alfa versus beta nor use of RAAS inhibitor correlated to change in eGFR or ACR in the 10-year group. In the 5-year group, higher age correlated to higher change in eGFR (p=0.024). According to our results, ERT for five years regardless of RAAS inhibition prevented decline in kidney function. This finding was more consistent with younger age, suggesting earlier initiation of ERT.

doi:10.1016/j.ymgme.2018.12.200

185 Preliminary N-acetylcysteine results for LDN 6722 - Role of oxidative stress and inflammation in Gaucher disease type 1: Potential use of antioxidant anti-inflammatory medications Reena V. Karthaa, James Joersa, Marcia Terluka, Paul Tuitea, Usha Mishraa, Kyle Rudsera, Gulin Oza, Neal J. Weinrebb, Jeanine JarnesUtza,c, James C. Cloyda, aUniversity of Minnesota, Minneapolis, MN, United States, bLeonard School of Medicine of University of Miami, Miami, FL, United States, cFairview Health Systems, Minneapolis, MN, United States. Enzyme replacement and substrate reduction therapies have significantly improved outcomes in patients with Type 1 Gaucher disease (GD1), but do not completely return glucocerebroside levels to the normal range nor prevent continuing tissue injury. Preliminary biomarker analyses indicate a role for oxidative stress and inflammation in GD1 pathophysiology. Thus, additional treatment strategies that target oxidative stress and inflammation have the potential to ameliorate both systemic and brain symptoms and may alter

disease progression. N-acetylcysteine (NAC), a FDA-approved medication with antioxidant/anti-inflammatory activity, is also available as an inexpensive dietary supplement with a good safety profile. Our objective is to determine whether oxidative stress and inflammation in the blood and brains of individuals with GD1 can be altered with orally administered NAC. In this open-label study, we have currently enrolled 13 patients who received NAC at 3600mg/day dose. Blood sample collection for biomarker analysis and brain imaging using magnetic resonance spectroscopy (MRS) was done before and following 3 months of NAC. Total glutathione in red blood cells, which is a measure of oxidative stress, and plasma NAC concentrations were measured using a validated liquid chromatography/mass spectrometry method. Intracellular antioxidant enzyme activity and plasma markers for inflammation and lipid and protein modifications were measured using colorimetric and immunoassays. Interim analysis of data from 10 patients revealed that NAC at this dose did not significantly alter any of the neurochemical markers. However, validated MRS markers related to neuronal health such as N-acetyl aspartate (NAA) and glutamate showed trends toward improvement. Similar trends were also observed in blood antioxidant and inflammatory biomarkers. Pharmacokinetic analysis using serial samples collected 90-days after NAC regimen is underway to determine if a dose increase is warranted. If these favorable trends subsequently are shown to be significant, NAC could be a new adjunctive therapeutic option.

doi:10.1016/j.ymgme.2018.12.201

186 An improved, novel, systemically administered AAV gene therapy for treatment of CLN3 juvenile neuronal ceroid lipofuscinosis Scott Kernsa, M. Arnolda, A. Aldrichb, R. Falletb, Tammy Kielianb, Linas Padegimasa, Timothy J. MIllera, aAbeona Therapeutics, Cleveland, OH, United States, bDepartment of Pathology & Microbiology, University of Nebraska Medical Center, Omaha, NE, United States Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessively inherited mutation in the CLN3 gene. JNCL is a progressive neurodegenerative disorder in which the central nervous system (CNS) is greatly affected resulting in behavioral issues, vision loss, and other cognitive disabilities. We developed a novel AAV capsid with enhanced tropism for CNS tissue after systemic administration and optimized the CLN3 transgene cassette to improve biodistribution and expression in CNS and somatic tissues. AAV9 was used as a benchmark to assess the gene biodistribution in the Cln3Δex7/8 mice model. AAV9-CLN3 and AAV214-CLN3 were compared in mice that received 2.0 x 1013 vg/kg via intravenous administration. After 30 days the animals were sacrificed and tissues were harvested for biodistribution analysis. We evaluated capsid delivery in the primary regions of the CNS and the spinal cord (cervical and lumbar). In those tissues AAV214-CLN3 showed greater biodistribution efficacy when compared to AAV9-CLN3 (brain=160% of AAV9, Cervical SC=158% of AAV9 and lumbar SC=184% off AAV9). The sciatic nerve also demonstrated higher AAV214-CLN3 (744%) biodistribution compared to AAV9. The optimization of the CLN3 gene and expression cassette along with a novel AAV vector was able to show greater tropism into the CNS via systemic administration. Current studies assessing expression and dose response over a longer duration suggest AAV-214 can be used for effective systemic

Abstracts / Molecular Genetics and Metabolism 126 (2019) S17–S156

delivery and the ability to correct CLN3 in CNS tissues and peripheral tissues.

doi:10.1016/j.ymgme.2018.12.202

187 AAV gene therapy for the treatment of Fabry disease: A novel capsid with improved tropism to heart, kidney and CNS and improved GLA expression Brian M. Kevany, Scott Kerns, Linas Padegimas, Timothy J. Miller, Abeona Therapeutics, Cleveland, OH, United States Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency in alpha-galactosidase A (GLA) activity that results in the accumulation of the glycolipid products, globotriaosylceramide (Gb3) and lyso-Gb3 in the lysosome. Disease manifestations include frequent bouts of peripheral neurotrophic pain, angiokeratomas, reduced sweat production, corneal dystrophy, cardiomyopathy, renal insufficiency and cerebrovascular disease, which result in reduced lifespan in Fabry patients. While males are affected more severely due to the mutations in the GLA gene, female patients are also frequently symptomatic and are often misdiagnosed. Enzyme replacement therapy (ERT) is currently the only FDA-approved therapy to treat Fabry and requires bi-weekly injections of relatively large quantities of recombinant protein. While ERT reduces the accumulation of Gb3 in the heart, kidney and vasculature it fails to completely treat all symptoms of Fabry, primarily due to its inability to efficiently enter the CNS. Gene therapy strategies have been investigated and while many show great promise in correcting the glycolipid accumulation, most have failed to efficiently enter the CNS and also suffered from an immune response often seen during GLA replacement. Previous work has demonstrated that hepatic-specific expression can tolerize animals to the GLA protein and significantly reduce the humoral response but suffer from the same limitations as ERT. We have identified novel adeno-associated virus (AAV) capsids with improved tissue specificity and expression in the target tissues, namely CNS, heart, and kidney after intravenous administration. After identifying a lead AAV candidate, we have developed a novel, codon-optimized transgene and enhanced promoter to provide supraphysiological expression levels in target tissues as well as increased secretion for cross-correction to non-transduced cells. Proof of concept studies demonstrate significant improvements and enzymatic activity to facilitate IND-enabling studies.

doi:10.1016/j.ymgme.2018.12.203

188 A novel AAV capsid with improved CNS tropism for treating Pompe disease by intravenous administration Brian M. Kevany, Linas Padegimas, Timothy J. Miller, Abeona Therapeutics, Cleveland, OH, United States Pompe disease is a lysosomal storage disorder caused by a deficiency in acid alpha-glucosidase (GAA) activity that results in the accumulation of glycogen in the lysosome. The disease presents as a form of muscular dystrophy which primarily affects both smooth and

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striated musculature as well as the central nervous system (CNS), with early mortality. Enzyme replacement therapy (ERT) is currently the only FDA-approved therapy to treat Pompe and requires biweekly injections of relatively large quantities of recombinant protein. While ERT significantly reduces the mortality rate of infantile Pompe patients, who typically die by the age of two without therapy, it fails to completely ameliorate all symptoms of Pompe, primarily due to its inability to efficiently enter the CNS and resulting immune responses to the GAA protein. Gene therapy strategies have been investigated and while many show great promise in correcting the glycogen accumulation and other symptoms of Pompe, most have suffered from the severe immune response seen during GAA replacement. Previous work has demonstrated that hepatic-specific expression can tolerize animals to the GAA protein and significantly reduce the humoral response. We have identified a novel adeno-associated virus (AAV) capsid with improved tissue specificity and expression in the target tissues, namely heart, muscle and CNS after intravenous administration. We have developed a novel codon-optimized transgene and improved promoter for supraphysiological expression levels in target tissues as well as increased secretion for cross-correction to non-transduced cells. Proof of concept studies demonstrate significant protein expression and enzymatic activity en route to IND-enabling studies.

doi:10.1016/j.ymgme.2018.12.204

189 Non-clinical evaluation of a blood-brain barrier-penetrating enzyme for the treatment of mucopolysaccharidosis type I Sachiho Kida, Masafumi Kinoshita, Satowa Tanaka, Miho Okumura, Yuri Koshimura, Hideto Morimoto, JCR Pharmaceuticals Co., Ltd., Kobe, Japan Treatment of central nervous system (CNS) disorders in patients with lysosomal storage diseases by intravenous enzyme replacement therapy is still challenging because the blood-brain barrier (BBB) interferes the drug delivery to the brain. We have developed JR-141, a fusion protein consisting of anti-human transferrin receptor antibody (named J-Brain Cargo) and iduronate-2-sulfatase, which can cross the BBB utilizing receptor-mediated transcytosis of transferrin, and are currently conducting clinical trials for Mucopolysaccharidosis II (MPS II). Here we report a non-clinical evaluation of JR-171, another J-Brain Cargo-applied enzyme for the treatment of MPS I. MPS I is caused by deficiency of a lysosomal enzyme α-L-iduronidase (IDUA) and characterized by pathological accumulation of heparan sulfate (HS) and dermatan sulfate (DS) in cells throughout the body, resulting in a broad spectrum of somatic and CNS symptoms. We prepared JR-171, a fusion protein consisting of J-Brain Cargo and IDUA, and examined its biodistribution in cynomolgus monkeys. JR-171 was detected in the brain as well as in peripheral tissues of monkeys after intravenous administration, indicating its penetration of the BBB. We then evaluated the efficacy of repetitive intravenous administration of JR-171 in the reduction of accumulated HS and DS in peripheral tissues and the brain using human transferrin receptor knock-in mice lacking Idua gene as an animal model of MPS I. The intact IDUA decreased HS and DS concentrations in peripheral tissues but failed to reduce the substrates in the brain of the mice. In contrast, JR-171 markedly reduced HS and DS concentrations in the brain as well. These results demonstrate that the J-Brain Cargo-applied IDUA distributes to the