From Bench to Clinic: Developing Pre-Clinical Resources, Assays, and Therapeutics for GNE Myopathy

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Lek, Monkol

Abstract

GNE myopathy (GNE-M) is a rare, autosomal recessive disease, leading to progressive skeletal muscle atrophy and weakness in the limbs. Myopathy occurs due to pathogenic mutations in the GNE gene, resulting in reduced enzymatic activity of the GNE protein, a bifunctional enzyme involved in the sialic acid biosynthesis pathway. Over 160 pathogenic mutations in GNE have been reported, which can be found throughout the gene. No current treatment exists for GNE myopathy patients, leaving an unmet need for the development of a therapy. While much work has been done to try and elucidate the disease mechanism of GNE-M, the development of patient-derived resources and cellular disease models, particularly those that encompass a wide range of pathogenic variants, would be an invaluable tool to further GNE myopathy research. In the first part of this study, we aimed to create a biobank of GNE myopathy patient-derived resources, including whole genome sequencing of GNE-M affected and unaffected individuals, as well as the generation and RNA sequencing of patient-derived cell lines with a variety of known pathogenic mutations. To produce the cell biobank, primary fibroblasts from GNE myopathy patients and their unaffected family members were immortalized, then transdifferentiated into a myogenic lineage, enabling the study of a disease-relevant cell type. Additionally, a human myoblast line completely deficient of GNE was generated via CRISPR/Cas9 knockout. We then utilized these myogenic cell lines to develop several assays to implement in GNE myopathy research. GNE knockout myoblasts exhibited altered lectin binding activity compared to wildtype controls, indicating a loss of GNE activity changes cell surface sialylation patterns. Additionally, patient-derived transdifferentiated myotubes exhibit reduced sarcolemmal membrane repair capacity across several different pathogenic mutations. These results highlight the utility of a patient-derived biobank, particularly for a disease with a wide range of pathogenic variants, such as GNE-M. In the second part of this study we designed, optimized, and evaluated the efficacy of a gene replacement therapy for GNE myopathy. A library of AAV vectors was developed and evaluated in vitro for GNE expression due to: (1) the promoter used; (2) the inclusion of an intron; (3) the GNE isoform; and (4) the position and type of protein tag. Following selection of an optimal vector, two high expressing constructs were packaged into AAV9 particles, and a dose and biodistribution study was performed in C57BL/6 wildtype mice to determine the safety and efficiency of GNE expression across a variety of tissues. Lastly, the efficacy of gene replacement therapy was evaluated in a knock-in mouse model of GNE myopathy. In brief, both AAV9 and AAVmyo2 serotypes were utilized to treat 6-8 week-old mice with constructs that express the GNE coding sequence under control of muscle-specific promoters. At 12 weeks post-injection, several outcome measures were determined, including (1) the effects of gene replacement therapy on muscle hyposialylation, (2) the biodistribution of viral genomes depending on AAV serotype used, and (3) GNE protein expression across a variety of tissues. In vitro optimization demonstrated that the CK8e-SV40-hGNE1-V5 and MCK-SV40-hGNE1 constructs resulted in high GNE transcript and GNE protein expression in myoblasts. The AAV9 dose and biodistribution study in C57BL/6 mice indicated robust, dose-dependent transcript and GNE protein expression across a variety of muscle tissues at 8 weeks post-injection. An efficacy study in a GNE-M mouse model indicate a high dose (3e14 vg/kg) of AAVmyo2-MCK-SV40-hGNE1 led to strong exogenous GNE protein expression in skeletal muscle. Additionally, the use of the AAVmyo2 serotype resulted in significantly decreased liver targeting of the viral vector compared to AAV9, while exhibiting increased skeletal muscle targeting. Both phenotypes of the disease mouse model, muscle hyposialylation and proteinuria, were rescued in a subset of mice following AAV gene therapy treatment. This proof-of-concept study provides key insights into the optimal muscle-specific promoter, dose, and AAV serotype to be used within the context of GNE myopathy. This work has provided key pre-clinical research in the creation and evaluation of an AAV gene replacement therapy for GNE myopathy.

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