Liver-directed AAV gene therapy for PHKG2-Glycogen Storage Disease type IX (GSD IX y2)

Abstract

Liver Glycogen Storage Disease type IX (GSD IX) is the most common hepatic GSD, accounting for approximately 25% of all GSD cases with an overall estimated prevalence of 1 in 100,000 patients. Liver GSD IX is caused by a deficiency in Phosphorylase Kinase (PhK), a key enzyme in the breakdown of glycogen to glucose. Liver PhK is a complex, hetero-tetrameric enzyme comprised of four subunits, with α2, β, and δ subunits regulating the activity of the catalytic γ2 domain. Biallelic pathogenic variants in the gene encoding the γ2 subunit (PHKG2) are associated with ~25% of Liver GSD IX cases (GSD IX γ2). While all patients with Liver GSD IX present with similar symptoms, over 95% of patients with GSD IX γ2 progress to liver fibrosis and cirrhosis, with increased risk for hepatocellular carcinoma, liver failure, and death. Despite the life-threatening severity of advanced liver disease, there is no definitive treatment for GSD IX γ2. The purpose of this work is to provide natural history and preclinical data for a minimally invasive, long-term therapy for GSD IX γ2.Aim 1 characterized the first mouse model of GSD IX γ2. Prior to this work, there was no known mouse model for GSD IX γ2, limiting the capacity to understand disease progression and evaluate novel therapeutics. Knockout (KO, Phkg2-/-) and wild-type (WT, Phkg2+/+) mice were characterized at 3 months of age. Characterization confirmed that the mouse recapitulates the liver-specific phenotype of GSD IX γ2 patients, with low PhK enzyme activity, elevated liver glycogen content, elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), increased urine glucose tetrasaccharide (Hex4), abnormal hepatocyte architecture, and early perisinusoidal fibrosis on pathology. Aim 2 evaluated GSD IX γ2 mouse model disease progression. KO and WT mice were characterized up to 24 months of age. Characterization showed that the mouse model recapitulates the disease progression seen in GSD IX γ2 patients. Histologic review of KO mice identified a progression of liver pathology from increased subcapsular pericellular fibrosis at 6 months, periportal fibrosis at 9 months, to bridging fibrosis at 12 months, and cirrhosis at 18 months, with a concomitant initial increase and then decrease in serum and urine biomarkers. Patient liver biopsies demonstrate bridging fibrosis. Longitudinal biomarker data show that ALT, AST, and urine Hex4 elevations decline with age. Additional exploratory analysis was done on glycogen metabolism, indicating that in the fasted state KO mice have compensatory upregulation of enzymes involved in glycogenolysis and downregulation of enzymes involved in glycogen synthesis. Aim 3 utilized the GSD IX γ2 mouse model to evaluate a novel, liver directed AAV gene therapy. Four cohorts of Phkg2-/- mice were injected at age 3 or 6 months respective to cohort with AAV9-LSP-mPhkg2 at a dose of 5e12 vg/kg. Mice were sacrificed 2 weeks, 3 months, or 12 months post injection. Efficacy was evaluated by the characterization parameters captured in the GSD IX γ2 mouse model natural history study. Short term treatment results confirmed efficacy across all evaluated metrics. Phkg2-/- mice injected at age 3 months sacrificed at age 3.5 months had decreased percent liver weight, increased PhK enzyme activity, decreased liver glycogen content, decreased serum ALT and AST, and decreased urine Hex4, all comparable to wild type controls. Subsequent studies confirmed that treatment efficacy persisted over 3 months. Phkg2-/- mice injected at age 3 months sacrificed at age 6 months had a restoration of phenotype across all metrics. Mice injected at older ages demonstrated a reduction in disease phenotype despite treatment at progressive stages of liver fibrosis, a common barrier to treatment efficacy. Phkg2-/- mice injected at age 6 months and sacrificed at age 9 months demonstrated a restoration of liver health across metrics, including a significant reduction in liver fibrosis based on blinded board certified hepatopathologist histologic review. Treated mice age 9 months had an average fibrosis score 1.5x lower than untreated controls. Finally, long term studies show that treatment efficacy can persist across the mouse life span. Mice injected at age 6 months and sacrificed at age 18 months demonstrated a restoration of disease phenotype across all metrics. Treated mice age 18 months had an average fibrosis score 2.5x lower than age 18 month untreated mice, and even 1.5x lower than untreated mice age 3.5 months. Results suggest that AAV gene therapy may serve as the first definitive treatment for patients with GSD IX γ2. Additional studies are needed to evaluate safety and identify the potential therapeutic dose-range for first-in-human clinical trials.