Liver-Directed AAV Gene Therapy for Gaucher Disease

Abstract

Introduction: Gaucher disease (GD), one of the most common lysosomal storage disorders, is an autosomal recessive condition resulting from mutations in the GBA gene that codes for the b-glucocerebrosidase (GCase) enzyme. Over 90% of patients have type 1 GD, which is characterised by lipid engorged macrophages (known as Gaucher cells) in multiple organs, including spleen, liver and bone marrow, with no overt involvement of the central nervous system (CNS). The current standard of care for type 1 GD patients includes enzyme replacement therapy (ERT), which provides good overall therapeutic benefit. However, ERT is administered intravenously every other week, resulting in a high cumulative cost and a significant treatment burden. Furthermore, disease manifestations, such as pulmonary and skeletal disease, remain unresolved with ERT. Gene therapy is emerging as a very promising avenue of treatment for various monogenic disorders and has the potential to provide sustained levels of GCase enzyme expression after a single treatment. Here we have evaluated liver-directed gene therapy in vitro and in vivo for the treatment of GD. Methods: Adeno-associated virus (AAV) constructs were optimised to express full-length wild-type GCase protein (GBA AAV) and packaged in AAV8 capsids for in vivo mouse studies, or our novel AAVS3 capsid for in vitro studies in a human cell line. GCase activity was determined fluorometrically with 4-Methylumbelliferyl-β-D-glucopyranoside and activity was based on a 4-methylumbelliferone standard curve. Levels of GCase in plasma, and uptake in GD target organs were compared between our GBA AAV optimized construct and ERT treatment with velaglucerase alfa (VPRIV®) in C57BL/6 wild type mice. Doses used ranged from 2x109 to 2x1012 vg/kg for GBA AAV constructs and 60 U/kg for ERT. Results: Our initial proof of concept studies for liver-directed AAV gene therapy of GD used an AAV construct encoding the native full-length human GBA cDNA (RC-04-01). After a single intravenous injection into mice, RC-04-01 led to a dose-dependent expression of GCase in liver and robust levels of enzymatically active GCase in plasma. Based on these preliminary data, 37 GBA AAV constructs with optimisations to the coding sequence and changes to the promoter, signal peptide, and polyA sequences were designed and evaluated in vitro for GCase production. Among them, 6 constructs outperformed RC-04-01 and were further tested in mice. Construct RC-04-26 showed the highest GCase activity levels both in vivo and in vitro. At a dose of 2x1012 vg/kg, RC-04-26 showed plasma active GCase levels up to 9.4-fold higher than RC-04-01. Upon GBA AAV infusion, plasma GCase levels were steady and sustained for the duration of the entire study period (9 months). In addition, RC-04-26 infusion into mice resulted in robust uptake of GCase by cells in spleen, bone marrow and lung, demonstrating that liver-produced GCase is taken up by macrophages present in the GD target organs. Lastly, GCase bioavailability was evaluated after a single administration of RC-04-26 or ERT. With velaglucerase alfa, GCase was rapidly cleared from the bloodstream and tissues. However, RC-04-26 resulted in sustained and steady levels of GCase, with an overall bioavailability of GCase uptake over 170-fold higher than with ERT. Conclusions: Our current data support the hypothesis that a single administration of an optimised liver directed GBA AAV vector results in sustained elevation of GCase in the bloodstream and higher level of GCase bioavailability for uptake into macrophages than velaglucerase alfa. This observation supports further development of AAV gene therapy for Gaucher disease with the potential for enhanced therapeutic benefit from a one-off administration. Disclosures Miranda: Freeline Therapeutics: Employment, Equity Ownership. Canavese:Freeline Therapeutics: Employment, Equity Ownership. Chisari:Freeline Therapeutics: Employment, Equity Ownership. Pandya:Freeline: Employment, Equity Ownership. Cocita:Freeline Therapeutics: Employment, Equity Ownership. Portillo:Freeline: Employment, Equity Ownership. McIntosh:Freeline Therapeutics: Consultancy, Equity Ownership. Kia:Freeline Therapeutics: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Dane:Freeline: Employment, Equity Ownership. Jeyakumar:Freeline Therapeutics: Employment, Equity Ownership. Sheridan:Freeline Therapeutics: Employment, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Gene Therapy for Lysosomal Storage Disorders: Ongoing Studies and Clinical Development

“…This approach was successful in rescuing a mouse model of Pompe disease following systemic delivery of an AAV8 vector that expressed an engineered acid-α-glucosidase protein secreted by hepatocytes [ 168 ]. Freeline Therapeutics has recently used both AAV8 and novel capsid AAVS3 to deliver a GBA vector to cells and mice in a proof-of-concept study, supporting the development of a liver-directed therapy for Gaucher disease [ 169 ]. In addition, liver-specific promoters have been designed to further improve efficacy and safety, as demonstrated in pre-clinical studies in Fabry disease [ 170 , 171 ], Gaucher disease [ 172 ] and Pompe disease [ 173 , 174 ] mouse models.…”

The Liver and Lysosomal Storage Diseases: From Pathophysiology to Clinical Presentation, Diagnostics, and Treatment

“…Miranda et al reported promising results regarding liver-directed gene therapy in vitro and in vivo for the treatment of GD. Liver-directed GBA AAV vector administration resulted in a sustained elevation of glucocerebrosidase in the bloodstream and a higher level of its bioavailability for uptake into macrophages than with ERT (velaglucerase alfa) [ 74 ].…”

Translating CRISPR-Cas Therapeutics: Approaches and Challenges

“…167 Preclinical studies have shown efficacy in liverdirected gene transfer for lysosomal storage diseases (LSDs) such as Gaucher disease, Fabry disease, and glycogen storage disease type Ib and II. [169][170][171][172] LSDs are mostly monogenic and include more than 40 different metabolic diseases. The spectrum of disease severity also spans from disorders in the central nervous system (CNS) to systemic multi-organ pathology, as the enzymes are ubiquitously expressed.…”