Semi-rational design of AAV capsid for Collagen VI-related Congenital Muscular Dystrophies.

Collagen VI-related congenital muscular dystrophies (COL6-CMDs) are the second most common congenital muscular dystrophies (CMDs). Patients with COL6-CMDs experience congenital or early-onset progressive muscle weakness, joint contractures, loss of ambulation by early teen years, and respiratory insufficiency. It is estimated that COL6-CMDs occur in at least 0.9 per 100,000 individuals. COL6-CMDs are caused by deleterious variants in any of the three genes (COL6A1, COL6A2, and COL6A3) that encode Collagen VI (COLVI)’s three major a-chains and there are no disease modifying treatments or cure. COLVI is an extracellular matrix protein that plays multiple roles in skeletal muscles, such as providing biomechanical stability, counteracting apoptosis, and maintaining stemness. In skeletal muscles, COLVI is primarily synthesized by fibro-adipogenic progenitor cells (FAPs) a type of interstitial stromal cells. Inside FAPs, three COLVI a-chains, each encoded by a different gene, assemble in a 1:1:1 stoichiometric ratio to form the COLVI triple helical monomer. Then, the monomers dimerize, and the dimers form tetramers. The tetramers are then secreted into the extracellular space, where they assemble to form the COLVI microfibrils. The majority of COLVI monomers are composed of a1(VI), a2(VI), and a3(VI) chains that are encoded by COL6A1, COL6A2, and COL6A3, respectively (a1a2a3 monomer). Genetic therapy approaches for COL6-CMDs depend on consequences of deleterious genetic mutations and the modification of downstream pathological processes with approximately 50% dominant and 50% recessive mutations.These include a common aberrant splicing event that inserts COL6-CMD-causing COL6A1 pseudoexon that could be corrected through splice-modifying antisense oligonucleotides (ASO). We have developed an ASO showing compelling efficacy in vitro for one of the most common mutations, the de novo deep intronic c.930+189C > T mutation in COL6A1 gene. A significant barrier to translation stems from inadequate delivery to target cells that express COLVI, specifically muscle interstitial fibroblasts (MIFs) and FAPs. To improve ASO delivery we have developed a peptide that demonstrates improved internalisation in human MIFs and FAPs through targeting PDGFRa receptors that enhances target cell delivery, but does not negate the need for repeated ASO delivery for translation. An alternative method would be to use adeno-associated virus (AAV) to deliver a genetic therapy for COL6-CMDs. AAV delivered genetic therapies are potentially transformational treatments as seen in Spinal Muscular Atrophy. In this proposal we aim to accelerate the clinical translation of genetic therapies for COL6-CMDs by combining our peptide design to develop a peptide insertion AAV capsid variant that targets PDGFRa receptors expressed on FAPs as a delivery vehicle for COL6-CMDs genetic therapies. Our current unpublished in vivo studies demonstrate AAV8 shows highest transduction efficiency for COLVI expressing target cells in skeletal muscle. However, its broader tropism is not ideal for COL6- CMDs with associated liver tropism. To overcome this, we propose semi-rational design AAV capsid engineering approach to modify capsid receptor interactions to enhance target cell transduction and detarget hepatocytes. We will use the peptide we developed for ASO conjugation as reference, to design a peptide display library on parental serotype AAV8. We will generate a barcoded capsid library to screen for transduction selectivity in vivo and characterise leading variants by detailed biodistribution study. We will perform in vitro human fibroblasts transduction studies to ensure our capsid retains transduction properties across species. With this novel AAV variant we can deliver gene supplementation, U7 exon skipping or gene editing approaches for COL6-CMDs.