Development of myotropic human bocavirus vectors for gene therapy - PROJECT SUMMARY Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene located on the X chromosome. AAV-based gene therapies for DMD are expected to provide functional compensation in both skeletal and cardiac muscles via systematic delivery of a micro-dystrophy minigene. However, the size limit of the rAAV vector poses a major barrier to the effort for improving expression specificity and therapeutic efficacy. Gene editing has the potential to permanently correct the mutations of exon deletions that lead to a premature truncated protein in the majority of DMD patients. However, the CRISPR-based approaches require multiple components working together to achieve a therapeutic effect in gene editing, and fitting all of these components within a single AAV vector is challenging. This challenge is particularly prominent in DMD gene therapy, due to the whole-body treatment via systematic delivery necessitating high vector doses and the inclusion of muscle-specific expression regulation elements. rAAV2/HBoV1, in which an rAAV2 genome is pseudopackaged by the capsid of a respiratory human bocavirus 1 (HBoV1), has been developed for cystic fibrosis gene therapy, due to its superior apical tropism to human airway epithelium and a large cargo size. The 5.9-kb package capacity of rAAV2/HBoV1 makes it suitable for DMD gene therapies through both gene replacement and gene editing approaches. However, in spite of the fact that rAAV2/HBoV1 can transduce the primary human myotube in in vitro cultures and mouse skeletal muscles in vivo, the transduction efficiency is lower than that of rAAV. To overcome this limitation, this proposal aims to develop myotropic rAAV2/HBoV1 vectors of improved tropisms to skeletal and cardiac muscles. Structural biology studies have revealed that AAV and HBoV share conserved features of the compositions and locations of their variable region (VR) loops on the capsid surface. Directed evolution of AAV capsid libraries in mouse muscle has identified several myotropic AAV (MyoAAV) that display a 7-mer peptide bearing an “RGD” motif at the VR VIII on the capsid surface. In preliminary studies, we demonstrated that the insertion of a 7-mer peptide at the VR IV or VIII of the HBoV1 capsid surface did not disrupt the production of mutants; and we also created an infectious hybrid parvovirus of AAV2 and HBoV1, iAAV2-HBoV1, to enable the production of high titer HBoV1 capsid libraries in the context of a replication competent rAAV2 genome. Thus, in this application, we propose to identify myotropic HBoV1 capsid (MyoBoV) variants from directed evolution of HBoV1 capsid libraries that display RGD containing the 7-mer peptide in HSA-Cre79 mice, which specifically express Cre recombinase (CRE) in striated muscles. For efficient variant screening, we will incorporate the CRE- based BoV Targeted Evolution (CREBTE) platform. We will characterize the transduction profile of the MyoBoV variants in various mouse tissues/organs, as well as their capability in transducing primary human myoblasts and differentiated myotubes. The proposed directed evolution platform is also of great utility to engineer a bocaviral capsid to target desired tissues/organs, and thus to expand the toolbox of parvovirus vectors.
