Adeno-associated virus serotype 9 efficiently targets ischemic skeletal muscle following systemic delivery

Abstract Targeting therapeutic gene expression to the skeletal muscle following intravenous (IV) administration is an attractive strategy for treating peripheral arterial disease (PAD), except that vector access to the ischemic limb could be a limiting factor. As adeno-associated virus serotype 9 (AAV-9) transduces skeletal muscle at high efficiency following systemic delivery, we employed AAV-9 vectors bearing luciferase or enhanced green fluorescent protein (eGFP) reporter genes to test the hypothesis that increased desialylation of cell-surface glycans secondary to hindlimb ischemia (HLI) might help offset the reduction in tissue perfusion that occurs in mouse models of PAD. The utility of the creatine kinase-based (CK6) promoter for restricting gene expression to the skeletal muscle was also examined by comparing it with the cytomegalovirus (CMV) promoter after systemic administration following surgically induced HLI. Despite reduced blood flow to the ischemic limbs, CK6 promoter-driven luciferase activities in the ischemic gastrocnemius (GA) muscles were ∼34-, ∼28- and ∼150-fold higher than in the fully perfused contralateral GA, heart and liver, respectively, 10 days after IV administration. Furthermore, luciferase activity from the CK6 promoter in the ischemic GA muscles was ∼twofold higher than with CMV, while in the liver CK6-driven activity was ∼42-fold lower than with CMV, demonstrating that the specificity of ischemic skeletal muscle transduction can be further improved with the muscle-specific promoters. Studies with Evans blue dye and fluorescently labeled lectins revealed that vascular permeability and desialylation of the cell-surface glycans were increased in the ischemic hindlimbs. Furthermore, AAV9/CK6/Luc vector genome copy numbers were ∼sixfold higher in the ischemic muscle compared with the non-ischemic muscle in the HLI model, whereas this trend was reversed when the same genome was packaged in the AAV-1 capsid (which binds sialylated, as opposed to desialylated glycans), further underscoring the importance of desialylation in the ischemic enhancement of transduction displayed by AAV-9. Taken together, these findings suggest two complementary mechanisms contributing to the preferential transduction of ischemic muscle by AAV-9: increased vascular permeability and desialylation. In conclusion, ischemic muscle is preferentially targeted following systemic administration of AAV-9 in a mouse model of HLI. Unmasking of the primary AAV-9 receptor as a result of ischemia may contribute importantly to this effect. Introduction Peripheral arterial disease (PAD) is mainly caused by atherosclerosis, which results in obstructions in arterial beds other than the coronary arteries, and the most common site is the lower extremity where occlusive disease leads to impaired perfusion. PAD affects about 3–10% of adults in the world and 15–20% in those >70 years.1, 2 Many patients are not candidates for surgical or catheter-based revascularization and while patients with PAD should be treated with medications that target atherosclerosis, medications like statins and angiotensin-converting enzyme inhibitors have yet to prove effective at increasing blood flow to ischemic limbs. Gene therapy protocols for PAD using genes that, for example, encode angiogenic growth factors to augment collateral blood flow to the ischemic tissues have been pursued for more than a decade.3 Results from clinical trials of gene therapy for PAD, which to date have primarily used plasmid and adenovirus-based vectors delivered intra-muscularly, or sometimes intravascularly, have been almost uniformly disappointing.4, 5 Among the likely reasons for previous failures in human studies are the use of vectors that have short durations of expression and are inefficient at gene delivery when they are present in the target tissue, but perhaps no gap is greater than the fact that most of the ischemic muscle mass in a patient with PAD never receives gene therapy using the intra-muscular injection methods employed in most clinical trials. An ideal vector for skeletal muscle gene transfer would provide sustained gene expression and could be administered with minimally invasive procedures without inducing significant vector-related inflammatory responses in the host. Over the past decade, adeno-associated viral (AAV) vectors have emerged as arguably the single most promising gene delivery system for human gene therapy. Recombinant AAV vectors transduce a wide variety of tissues in vivo and provide for long-term gene expression without provoking significant cell-mediated immune responses.6, 7, 8 To date, >100 AAV serotypes have been reported.9 A comparison of the more recently discovered serotypes showed that AAV-9 transduction to heart, lung and tibialis anterior (TA) muscle after intravenous (IV) injection is superior to all other serotypes and is age independent, whereas transduction to liver and kidney is age dependent.10 The natural tissue tropism of the various AAV serotypes can be exploited to favor gene delivery to one organ over another. This tropism is based on the viral capsids recognizing specific viral receptors expressed on specific cell types, thus allowing a degree of cell-specific targeting within a given organ.11 Cell-specific expression may be further aided by the use of tissue-specific promoters conferring gene expression restricted to a specific cell type.11 This is desirable for gene therapy applications targeting organ-specific diseases, as this will help avoid any possible harmful side effects due to gene expression in off-target organs. Recently, several muscle-specific promoter constructs based on the muscle creatine kinase (MCK) regulatory region were shown to provide striated muscle-restricted gene expression.12, 13, 14, 15 Of the several regulatory cassettes based on the MCK regulatory element, the CK6 promoter has been shown to provide skeletal muscle-restricted gene expression with reduced expression in cardiac muscle.12, 13 This is particularly desirable in the context of using AAV-9 for PAD gene therapy via systemic administration as AAV-9 has a known preference for cardiac over skeletal muscle. However, the use of skeletal muscle-specific promoters in combination with the more recent AAV serotypes in the context of PAD is largely unexplored and indeed the entire approach could, in theory, be limited by the fact that blood flow to the ischemic limb is reduced thus creating a barrier to intravascular gene delivery. Recently, cell-surface N-linked glycans with terminal galactosyl residues were shown to serve as the primary receptor for AAV-9.16 Desialylation of these galactosylated glycans was shown to markedly increase cell-surface binding and transduction by AAV-9 and significantly decrease that by other AAV serotypes, like AAV-1.16 We hypothesized that ischemia induces desialylation of the cell-surface glycans, resulting in increased availability of AAV-9 recep (...truncated)