Bicistronic Gene Therapies for the Muscular Dystrophies

Above, Bicistronic, or dual gene, AAV vectors bearing both FKRP gene replacement, to stop disease progression, and FST, to build new muscle mass and strength, will be compared to AAVs expressing either single gene alone. The dual gene approach can normalize ambulation, increase muscle mass and strength beyond wild type levels, and prevent muscle pathology in the FKPRP448L mouse model of LGMDR9. In addition, the dual gene AAV configuration can amplify expression of both therapeutic genes relative to AAVs expressing either single gene alone.

Principal Investigator

What Is the Goal of the Project?

Research Project 1 of our MDSRC Program is dedicated to completing remaining pre-clinical proof of concept and safety studies required to file a pre-IND application with the FDA for a first-in-class bicistronic AAV gene therapy for patients with Limb Girdle Muscular Dystrophy Type R9 (LGMDR9).

What Does This Project Entail?

A major lesson we can take from away from gene therapy clinical trials done thus far for muscular dystrophy (MD) is that introduction of a normal copy of a mutated disease gene, or gene replacement therapy, can be effective in stopping or slowing muscle disease. Such gene replacement therapies, however, do little to improve lost muscle strength in patients where disease is already present. So while gene replacement therapy is a clinically important step forward, this approach falls far short of the ultimate goal of gene therapy - to cure the disease. In Research Project 1, we seek to complete experiments that will allow for testing of a first-in-class bicistronic, or dual gene, AAV gene therapy that can both arrest ongoing muscle disease and reverse pre-existing disease.

If successful, this approach could be applied to almost two dozen other genetic forms of muscular dystrophy, providing a design platform for a new class of more effective gene therapy treatments.

Research Project 1 will complete the studies required to file a pre-IND application to treat Limb Girdle Muscular Dystrophy Type R9 (LGMDR) patients with a novel bicistronic, or dual gene, FKRP/FST AAV gene therapy. LGMDR9, formerly LGMD2i, is one of the most common forms of LGMD. LGMDR9, and the more severe Congenital Muscular Dystrophy 1C (MDC1C), are caused by recessive loss-of-function mutations in the fukutin-related protein (FKRP) gene. This therapy will provide a normal copy of the FKRP gene that is mutated in LGMDR9 as well as the follistatin (FST) gene, which can build new muscle mass and strength. There are currently no FDA-approved therapies any LGMD or CMD that impact ultimate disease outcome.

The Specific Aims, in brief, are:

• Specific Aim 1. Determine the tissues required for FST-depending muscle building therapy in FKRP/FST bicistronic vectors and optimize promoters. Previously, we have used a constitutive promoter to overexpress FST in all tissues. This approach yields both high local expression in muscle and high systemic expression in serum as a result of gene expression in liver. We will dissect the tissue-specific needs of FST to deliver its component of the curative bicistronic therapy, testing specific FST expression in muscle, liver, or both. Such experiments will determine the targeted tissue expression requirements for muscle building therapy in bicistronic FKRP/FST AAV vectors. We will also optimize the bicistronic configuration for translation to LGMDR9 patients.
• Specific Aim 2. Determine the superiority of optimized FKRP/FST bicistronic therapy in reversing disease relative to single FKRP or FST alone. In this grant proposal, we provide clear evidence that early treatment with FKRP/FST bicistronic AAV gene therapy can fully recover disease phenotypes in the FKRP_P448L mouse model of LGMDR9, while either single gene therapy alone cannot. Here, we will test later stages of disease to see if our optimized FKRP/FST bicistronic gene therapy can be equally effective at a more advanced disease stage and if increased doses are required to achieve normal muscle function under such circumstances.
• Specific Aim 3. Perform a GLP-like safety study of optimized FKRP/FST AAV gene therapy. Nationwide Children’s Hospital has core facilities that allow for a GLP-like toxicology to be performed on AAV gene therapy treatments, complete with QC audit of all data sets. Here, we will undertake such a study at 10x the optimal dose required for treatment of LGMDR9 mice. This study will provide essential safety data that will be presented to the FDA for consideration along with proof of concept data at a planned pre-IND meeting that will occur at the completion of this grant proposal.