Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders

Abstract

Bioengineering of viral vectors for therapeutic gene delivery is a pivotal strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety. Here, we engineered myotropic adeno-associated viral (AAV) vectors via a semirational, combinatorial approach that merges AAV capsid and peptide library screens. We first identified shuffled AAVs with increased specificity in the murine skeletal muscle, diaphragm, and heart, concurrent with liver detargeting. Next, we boosted muscle specificity by displaying a myotropic peptide on the capsid surface. In a mouse model of X-linked myotubular myopathy, the best vectors—AAVMYO2 and AAVMYO3—prolonged survival, corrected growth, restored strength, and ameliorated muscle fiber size and centronucleation. In a mouse model of Duchenne muscular dystrophy, our lead capsid induced robust microdystrophin expression and improved muscle function. Our pipeline is compatible with complementary AAV genome bioengineering strategies, as demonstrated here with two promoters, and could benefit many clinical applications beyond muscle gene therapy.

Fast, multiplexable and highly efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9

“…We also tested systemic administration of additional capsid variants of AAVMYO, called AAVMYO2 and AAVMYO3, that were originally selected for their liver de-targeting qualities ( 8 ), which can be an advantage for clinical applications. AAVMYO2 and AAVMYO3 were less efficient than AAVMYO, but superior to AAV9, at transducing and therefore eliciting gene editing events in skeletal muscles (Fig.…”

Reprogramming Adeno-Associated Virus Tropism Via Displayed Peptides Tiling Receptor-Ligands

“…In our validation experiments, we used a standard promoter (CMV) to drive expression of the mCherry transgene. Alternatively, tissue-specific promoters could be used to increase the specificity and magnitude of transgene expression in the organ of interest ( 18, 89, 90 ). Furthermore, the hit capsids identified here could be further engineered for increased activity.…”

“…The copyright holder for this preprint this version posted September 26, 2022. ; https://doi.org/10.1101/2022.09. 26.509383 doi: bioRxiv preprint 3 tissues such as the brain (14,15) and muscle (16)(17)(18). This has predominantly been accomplished using strategies of iteratively screening random peptides inserted into the AAV capsid (19)(20)(21), capsid shuffling (22)(23)(24)(25)(26), randomly mutagenizing the capsid sequence as a whole (27)(28)(29)(30), or direct chemical engineering (31,32).…”

“…In this regard, groups in the field have progressively engineered AAV variants to specifically target 3 tissues such as the brain (14,15) and muscle (16)(17)(18). This has predominantly been accomplished using strategies of iteratively screening random peptides inserted into the AAV capsid (19)(20)(21), capsid shuffling (22)(23)(24)(25)(26), randomly mutagenizing the capsid sequence as a whole (27)(28)(29)(30), or direct chemical engineering (31,32).…”

Strategies for Bottlenecks of rAAV-Mediated Expression in Skeletal and Cardiac Muscle of Duchenne Muscular Dystrophy

“…Moreover, this advance led to the introduction of AAVMYO2 and AAVMYO3 two years later. In addition to retaining all the assets of AAVMYO, AAVMYO2 and AAVMYO3 exhibit a further substantial reduction of off-targeting after the peripheral intravenous delivery [ 62 , 63 ]. In 2021, Tabebordbar et al further optimized the RGD motif through directed evolution, specific MyoAAVs were selected and validated in mice and non-human primates for their ability to efficiently target the striated muscle transduction and reduce AAV retention in the liver [ 64 ].…”