Project Abstract

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​Restoration of Cardiac and Neurological Functions in FA Mouse Models via rAAV-Based Gene Therapy with Endogenous Promoter-Controlled Frataxin Expression

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Friedreich’s ataxia (FA), the most common hereditary ataxia, affects about 1 in 50,000 in the US. This autosomal recessive disorder stems from GAA trinucleotide repeat expansions in the Frataxin (FXN) gene, reducing FXN levels in mitochondria. Symptoms typically emerge between ages 5-15, affecting mainly the heart and nervous system. FA progressively worsens, leading to reduced quality of life and early death, often from ventricular arrhythmia. Currently, only one approved treatment exists, but it fails to address the underlying defect of the disease. Gene therapy using recombinant adeno-associated viral vector (rAAV) with the human FXN gene offers hope. However, past studies noted toxicity from FXN overexpression in mice. Our team has engineered a novel construct (AAV9-DE7-hFXN), controlled by an endogenous promoter to regulate FXN levels, that has been tested in vitro and vivo in wild-type mice. Our team now aims to evaluate this construct in FA's cardiac and neurological models. This study will be divided into 2 aims: evaluation of AAV9-DE7-hFXN Construct in Cardiac (MCK-Cre) Conditional Mice and analysis of the construct in CNS (Pvalb-Cre) conditional mice. In both aims, a survival assessment and a Frataxin level analysis will be used and compared to wild-type mice. The cardiac evaluation will use echocardiography to investigate the effect of the construct and will assess parameters such as cardiac mass and volumes to determine functional and structural changes over time. For the CNS model, motor function will be analyzed using tests such as the rotarod performance test and a neuroscore exam to assess motor skills and coordination. The Pvalb-Cre FXN mouse line is engineered for FA research as they exhibit ataxia symptoms around 9-10 weeks and have an average lifespan of about 17 weeks, making them ideal for studying neuronal implications in FA. The MCK-Cre FXN mouse line is unique in that the absence of Frataxin is specifically directed to cardiac and skeletal muscles. Although they do not show early signs of ataxia, they have a reduced lifespan of about 8-12 weeks. This model is valuable for studying Frataxins' role in muscle tissues and exploring muscle-targeted therapies in Frataxin deficiency. The wild type mice come from the C56B6 background. These animals have no health concerns related to FA. This pilot study will evaluate dose response after systemic intraperitoneal (IP) dosing of AAV9-DE7-hFXN in Pvalb-Cre FXN mouse model neonate mice and MCK-Cre FXN mice compared to wild-type mice. An equal number of male and female animals will be randomized to each treatment group. Mice will receive a single IP injection of AAV9-DE7-hFXN at the selected dose of 5E13 vg/kg or of 1E14 vg/kg when the mouse is approximately 1-3 days old. Each animal will be monitored weekly for signs of ataxia onset (approximately 9-10 weeks at onset) with weekly neuroscore, body weight recording and clinical observation. The animals will be broken into two subgroups: groups A will be harvested 10 weeks postAAV injection, and groups B will be used as a survival study with the average lifespan of the line used as endpoint, Pvalb-Cre at week 17 and MCK-Cre at week 10. Multiple tissues from sub-group A will be collected for vector-derived FXN protein isolation and quantification using an Enzyme Linked Immunosorbent Assay (ELISA) specific for human FXN expression. Group B will be used to determine the timeline of progression until humane endpoint. The data obtained from this pilot grant, alongside findings from Dr. Corti's ongoing U01 project, will be instrumental in shaping robust grant applications targeted towards patient-centered foundations and the National Institutes of Health (NIH). This submission aims to garner support for advancing our gene therapy program to the crucial stage of Investigational New Drug (IND) submission.

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