A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection

Abstract

To engineer gene vectors that target striated muscles after systemic delivery, we constructed a random library of adeno-associated virus (AAV) by shuffling the capsid genes of AAV serotypes 1 to 9, and screened for muscle-targeting capsids by direct in vivo panning after tail vein injection in mice. After 2 rounds of in vivo selection, a capsid gene named M41 was retrieved mainly based on its high frequency in the muscle and low frequency in the liver. Structural analyses revealed that the AAVM41 capsid is a recombinant of AAV1, 6, 7, and 8 with a mosaic capsid surface and a conserved capsid interior. AAVM41 was then subjected to a sideby-side comparison to AAV9, the most robust AAV for systemic heart and muscle gene delivery; to AAV6, a parental AAV with strong muscle tropism. After i.v. delivery of reporter genes, AAVM41 was found more efficient than AAV6 in the heart and muscle, and was similar to AAV9 in the heart but weaker in the muscle. In fact, the myocardium showed the highest gene expression among all tissues tested in mice and hamsters after systemic AAVM41 delivery. However, gene transfer in non-muscle tissues, mainly the liver, was dramatically reduced. AAVM41 was further tested in a genetic cardiomyopathy hamster model and achieved efficient long-term ␦-sarcoglycan gene expression and rescue of cardiac functions. Thus, direct in vivo panning of capsid libraries is a simple tool for the de-targeting and retargeting of viral vector tissue tropisms facilitated by acquisition of desirable sequences and properties.

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

“…Although some earlier studies reported higher vg in the liver than in the heart with AAV9 vectors, 39,47,48 another publication found a better transduction of the heart than the liver of neonatal-injected animals. 45 These variations in biodistribution between different studies might be explained by different experimental conditions such as vector dose, mouse strain, gender of the mice and time point of tissue harvesting.…”

“…Very recently, an alternative approach to select vectors for an improved cardiac gene transfer was established by in vivo selection of an AAV capsid library shuffled from AAV 1, 2, 3, 4, 6, 7, 8 and 9 cap genomes. 48 Similar to our random peptide display approach, the selected hybrid capsid provided highly preferential and efficient gene expression in the heart after systemic application. However, analysis of ratios of genome uptake and gene expression revealed that the targeting effect could be explained rather by the improved post-entry performance of the newly generated capsids than by an increased gene transfer of heart versus liver tissue.…”

microRNA122-regulated transgene expression increases specificity of cardiac gene transfer upon intravenous delivery of AAV9 vectors

“…This vector revealed a similar transgene expression efficiency in murine myocardium as AAV9 but significantly reduced gene transfer into the liver. 17 Transcriptional targeting with cardiac-specific promoters has been described as another valuable strategy to improve specificity of cardiac transgene delivery. We could recently show that expression of a transgene driven by a CMV-MLC1.5-hybrid promoter is suitable to increase specificity of cardiac transgene expression of AAV vectors.…”

“…3,10,[12][13][14][15] However, AAV9 vectors exhibit a broad tissue tropism and allow also transduction of the liver upon intravascular administration. 3,[12][13][14]16,17 Reduction of AAV-mediated transgene expression in the liver, however, may be a desirable aim to reduce unwanted side effects in cardiac gene therapy. Transcriptional control of gene expression is a promising approach to overcome this limitation.…”

Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues

“…Most of the studies have used positions 138 (VP2 N-terminal), 587 and 588 (HSPG binding domain) to insert peptides ranging from 5 to 272 amino acids (Loiler et al, 2003;Nicklin et al, 2001a;Perabo et al, 2006;Shi et al, 2001;White et al, 2007;White et al, 2004;Wu et al, 2000b;Yu et al, 2009). Capsid protein modifications have improved gene delivery to lung (Kwon & Schaffer, 2008), endothelial cells (Nicklin et al, 2001a), pancreatic islets (Loiler et al, 2003), vascular tissue (White et al, 2004), atherosclerotic lesions (White et al, 2007), muscle (Yu et al, 2009), myocardium (Yang et al, 2009), and cancer cells . Although the therapeutic effect of these vectors still remains to be seen, there is a long list of reports showing different advantages of peptide insertion within different positions of the viral capsid.…”