Intracellular trafficking of adeno-associated virus (AAV) vectors: challenges and future directions.

In the last two decades, recombinant adeno-associated virus has emerged as the most popular gene therapy vector. Recently AAV gene therapy has been approved by the FDA for the treatment of two rare genetic disorders, namely the early childhood blindness disease Leber congenital amaurosis and spinal muscular atrophy (SMA). As is the case for the treatment of SMA, if the AAV vector must be administered systemically, very high vector doses are often required for therapeutic efficacy. But higher vector doses inevitably increase the risk of adverse events. The tragic death of three children in a clinical trial to treat X-linked myotubular myopathy with an AAV vector has thrown this limitation into sharp relief. Regardless of the precise cause(s) that led to the death of the two children, it is critical that we develop better AAV vectors to achieve therapeutic levels of expression with lower vector doses. To transduce successfully a target cell, AAV has to overcome both systemic as well as cellular roadblocks. In this review, we discuss some of the most prominent cellular roadblocks that AAV must get past to deliver successfully its therapeutic payload. We also highlight recent advancements in our knowledge of AAV biology that can potentially be harnessed to improve AAV vector performance and thereby make AAV gene therapy safer.

A Calsequestrin Cis-Regulatory Motif Coupled to a Cardiac Troponin T Promoter Improves Cardiac Adeno-Associated Virus Serotype 9 Transduction Specificity.

Adeno-associated virus serotype 9 (AAV9) is an efficient vector for gene transfer to the myocardium. However, the use of ubiquitous promoters, such as the cytomegalovirus (CMV) promoter, can result in expression of the transgene in organs other than the heart. This study tested if the efficiency and specificity of cardiac transcription from a chicken cardiac troponin T (TnT) promoter could be further increased by incorporating a cardiomyocyte-specific transcriptional cis-regulatory motif from human calsequestrin 2 (CS-CRM4) into the expression cassette (Enh.TnT). The efficiency of luciferase expression from the TnT and Enh.TnT constructs was compared to expression of luciferase under the control of the CMV promoter in both adult and neonatal mice. Overall, expression levels of luciferase in the heart were similar in mice injected with AAV9.TnT.Luc, AAV9.Enh.TnT.Luc and AAV9.CMV.Luc. In contrast, expression levels of luciferase activity in nontarget organs, including the liver and muscle, was lower in mice injected with the AAV9.TnT.Luc compared to AAV9.CMV.Luc and was negligible with AAV9.Enh.TnT. In neonates, in organs other than the heart, luciferase expression levels were too low to be quantified for all constructs. Taken together, the data show that the AAV9 Enh.TnT constructs drives high levels of expression of the transgene in the myocardium, with insignificant expression in other organs. These properties reduce the risks associated with the AAV9-mediated expression of the therapeutic protein of interest in nontarget organs. The excellent cardiac specificity should allow for the use of higher vector doses than are currently used, which might be essential to achieve the levels of transgene expression necessary for therapeutic benefits. Taken together, the findings suggest that the Enh.TnT transcription unit is a potentially attractive tool for clinical cardiac gene therapy in adults.

Protein S Protects against Podocyte Injury in Diabetic Nephropathy.

Background Diabetic nephropathy (DN) is a leading cause of ESRD in the United States, but the molecular mechanisms mediating the early stages of DN are unclear.Methods To assess global changes that occur in early diabetic kidneys and to identify proteins potentially involved in pathogenic pathways in DN progression, we performed proteomic analysis of diabetic and nondiabetic rat glomeruli. Protein S (PS) among the highly upregulated proteins in the diabetic glomeruli. PS exerts multiple biologic effects through the Tyro3, Axl, and Mer (TAM) receptors. Because increased activation of Axl by the PS homolog Gas6 has been implicated in DN progression, we further examined the role of PS in DN.Results In human kidneys, glomerular PS expression was elevated in early DN but suppressed in advanced DN. However, plasma PS concentrations did not differ between patients with DN and healthy controls. A prominent increase of PS expression also colocalized with the expression of podocyte markers in early diabetic kidneys. In cultured podocytes, high-glucose treatment elevated PS expression, and PS knockdown further enhanced the high-glucose-induced apoptosis. Conversely, PS overexpression in cultured podocytes dampened the high-glucose- and TNF-α-induced expression of proinflammatory mediators. Tyro3 receptor was upregulated in response to high glucose and mediated the anti-inflammatory response of PS. Podocyte-specific PS loss resulted in accelerated DN in streptozotocin-induced diabetic mice, whereas the transient induction of PS expression in glomerular cells in vivo attenuated albuminuria and podocyte loss in diabetic OVE26 mice.Conclusions Our results support a protective role of PS against glomerular injury in DN progression.

Cardiac gene therapy with adeno-associated virus-based vectors.

PURPOSE OF REVIEW: Cardiac gene therapy with adeno-associated virus (AAV)-based vectors is emerging as an entirely new platform to treat, or even cure, so far intractable cardiac disorders. This review describes our current knowledge of cardiac AAV gene therapy with a particular focus on the biggest obstacle for the successful translation of cardiac AAV gene therapy into the clinic, namely the efficient delivery of the therapeutic gene to the myocardium. RECENT FINDINGS: We summarize the significant recent progress that has been made in treating heart failure in preclinically relevant animal models with AAV gene therapy and the recent results of clinical trials with cardiac AAV gene therapy for the treatment of heart failure. We also discuss the benefits and shortcomings of the currently available delivery methods of AAV to the heart. Finally, we describe the current state of identifying novel AAV variants that have enhanced tropism for human cardiomyocytes and that show increased resistance to preexisting neutralizing antibodies. SUMMARY: Here, we describe the successes and challenges in cardiac AAV gene therapy, a treatment modality that has the potential to transform current treatment approaches for cardiac diseases.