Efficacy, pharmacokinetics, and safety in the mouse and primate retina of dual AAV vectors for Usher syndrome type 1B.

Gene therapy of Usher syndrome type 1B (USH1B) due to mutations in the large Myosin VIIA (MYO7A) gene is limited by the packaging capacity of adeno-associated viral (AAV) vectors. To overcome this, we have previously developed dual AAV8 vectors which encode human MYO7A (dual AAV8.MYO7A). Here we show that subretinal administration of 1.37E+9 to 1.37E+10 genome copies of a good-manufacturing-practice-like lot of dual AAV8.MYO7A improves the retinal defects of a mouse model of USH1B. The same lot was used in non-human primates at doses 1.6× and 4.3× the highest dose proposed for the clinical trial which was based on mouse efficacy data. Long-lasting alterations in retinal function and morphology were observed following subretinal administration of dual AAV8.MYO7A at the high dose. These findings were modest and improved over time in the low-dose group, as also observed in other studies involving the use of AAV8 in non-human primates and humans. Biodistribution and shedding studies confirmed the presence of vector DNA mainly in the visual pathway. Accordingly, we detected human MYO7A mRNA expression predominantly in the retina. Overall, these studies pave the way for the clinical translation of subretinal administration of dual AAV vectors in USH1B subjects.

Overcoming the Challenges Imposed by Humoral Immunity to AAV Vectors to Achieve Safe and Efficient Gene Transfer in Seropositive Patients.

One of the major goals of in vivo gene transfer is to achieve long-term expression of therapeutic transgenes in terminally differentiated cells. The extensive clinical experience and the recent approval of Luxturna® (Spark Therapeutics, now Roche) and Zolgensma® (AveXis, now Novartis) place vectors derived from adeno-associated viruses (AAV) among the best options for gene transfer in multiple tissues. Despite these successes, limitations remain to the application of this therapeutic modality in a wider population. AAV was originally identified as a promising virus to derive gene therapy vectors because, despite infecting humans, it was not associated with any evident disease. Thee large proportion of AAV infections in the human population is now revealing as a limitation because after exposure to wild-type AAV, anti-AAV antibodies develops and may neutralize the vectors derived from the virus. Injection of AAV in humans is generally well-tolerated although the immune system can activate after the recognition of AAV vectors capsid and genome. The formation of high-titer neutralizing antibodies to AAV after the first injection precludes vector re-administration. Thus, both pre-existing and post-treatment humoral responses to AAV vectors greatly limit a wider application of this gene transfer modality. Different methods were suggested to overcome this limitation. The extensive preclinical data available and the large clinical experience in the control of AAV vectors immunogenicity are key to clinical translation and to demonstrate the safety and efficacy of these methods and ultimately bring a curative treatment to patients.

Liver-Directed Adeno-Associated Virus-Mediated Gene Therapy for Mucopolysaccharidosis Type VI.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is an inherited multisystem lysosomal disorder due to arylsulfatase B (ARSB) deficiency that leads to widespread accumulation of glycosaminoglycans (GAG), which are excreted in increased amounts in urine. MPS VI is characterized by progressive dysostosis multiplex, connective tissue and cardiac involvement, and hepatosplenomegaly. Enzyme replacement therapy (ERT) is available but requires life-long and costly intravenous infusions; moreover, it has limited efficacy on diseased skeleton and cardiac valves, compromised pulmonary function, and corneal opacities. METHODS: We enrolled nine patients with MPS VI 4 years of age or older in a phase 1/2 open-label gene therapy study. After ERT was interrupted, patients each received a single intravenous infusion of an adeno-associated viral vector serotype 8 expressing ARSB. Participants were sequentially enrolled in one of three dose cohorts: low (three patients), intermediate (two patients), or high (four patients). The primary outcome was safety; biochemical and clinical end points were secondary outcomes. RESULTS: The infusions occurred without severe adverse events attributable to the vector, meeting the prespecified end point. Participants in the low and intermediate dose cohorts displayed stable serum ARSB of approximately 20% of the mean healthy value but returned to ERT by 14 months after gene therapy because of increased urinary GAG. Participants in the high-dose cohort had sustained serum ARSB of 30% to 100% of the mean healthy value and a modest urinary GAG increase that did not reach a concentration at which ERT reintroduction was needed. In the high-dose group, there was no clinical deterioration for up to 2 years after gene therapy. CONCLUSIONS: Liver-directed gene therapy for participants with MPS VI did not have a dose-limiting side-effect and adverse event profile; high-dose treatment resulted in ARSB expression over at least 24 months with preliminary evidence of disease stabilization. (Funded by the Telethon Foundation ETS, the European Commission Seventh Framework Programme, and the Isaac Foundation; ClinicalTrials.gov number, NCT03173521; EudraCT number, 2016-002328-10.)

Enhancing the Therapeutic Potential of Sulfamidase for the Treatment of Mucopolysaccharidosis IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a lysosomal storage disorder (LSD) caused by inherited defect of sulfamidase, a lysosomal sulfatase. MPS-IIIA is one of the most common and severe forms of LSDs with CNS involvement. Presently there is no cure. Here we have developed a new gene delivery approach for the treatment of MPS-IIIA based on the use of a modified version of sulfamidase expression cassette. This cassette encodes both a chimeric sulfamidase containing an alternative signal peptide (sp) to improve enzyme secretion and sulfatase-modifying factor 1 (SUMF1) to increase sulfamidase post-translational activation rate. We demonstrate that improved secretion and increased activation of sulfamidase act synergistically to enhance enzyme biodistribution in wild-type (WT) pigs upon intrathecal adeno-associated virus serotype 9 (AAV9)-mediated gene delivery. Translating such gene delivery strategy to a mouse model of MPS-IIIA results in a rescue of brain pathology, including memory deficit, as well as improvement in somatic tissues. These data may pave the way for developing effective gene delivery replacement protocols for the treatment of MPS-IIIA patients.