Project 2 - McNally (NU)_Spencer (UCLA)

University Of Florida, Gainesville FL

Investigators

Abstract

Abstract Although there has been progress in human gene therapy for muscular dystrophy, multiple issues have arisen during the clinical trials that necessitate developing improved safety and efficacy. Human AAV clinical trials for Limb girdle muscular dystrophy (LGMD) have initiated for β-sarcoglycan- and FKRP-related LGMDs. Like Duchenne muscular dystrophy, for these LGMD subtypes there is a need to correct both skeletal and cardiac muscle since these forms of LGMD are also associated with cardiomyopathy. We developed the first genetically engineered mouse model of LGMD and conducted early proof-of-principle studies of viral gene replacement, demonstrating restoration of sarcoglycan components within the dystrophin complex. Gamma sarcoglycan is encoded by the SGCG gene and is an ideal small cargo for AAV therapy, since the promoter and entire coding region fit well within AAV carrying capacity, unlike micro-dystrophin which only accommodates 30% of the full-length dystrophin protein. LGMD due to gamma sarcoglycan mutations and DMD share a similar clinical timeline with skeletal muscle and cardiac muscle involvement. Therefore, investigating and comparing results between gene therapy for these two diseases should inform clinically relevant issues related to functional rescue for skeletal muscle and the heart. The human clinical trial experience with micro-dystrophin has acutely identified the need to better define immune responses to AAV vectors and to develop animal models that better predict human immune responses. Immune responses can arise to the capsid, viral genome, or expressed protein cargo, leading to liver toxicity and limiting transgene durability. Newer, myotropic vectors are expected to improve safety by having less liver targeting and the ability to deliver effective therapy at lower doses. Additionally, codon-optimized vectors need to consider the presence of unmethylated CpGs that can unintentionally trigger adverse Toll Like Receptor (TLR) activity which has been associated with liver toxicity. In Aim 1, we will test TLR stimulatory activity of AAV vectors in human THP1 cells and combine optimized coding sequences with myotropic AAVs to improve striated muscle transduction and reduce immune response by testing in mouse models and human engineered heart tissues and monitoring for adverse outcomes. In Aim 2, we will test efficacy of these AAVs in an LGMD preclinical model, determining minimal and maximally efficacious doses and correlating with functional rescue. In Aim 3 we will further optimize and apply a dual dosing strategy to create better models of human immune response to AAV for treating muscle disease.