Peripheral artery disease (PAD) is a vascular disorder caused by occlusive atherosclerosis, which commonly impairs blood flow to the lower extremities. The prevalence of PAD is increasing globally with >200 million people affected. PAD remains a growing global health problem as the population continues to age and diabetes incidence grows. Many patients with PAD, most notably those with critical limb ischemia, fail attempts at surgical and percutaneous intervention to improve blood flow and are at risk of amputation. Gene therapy provides an opportunity to change the clinical course of PAD in these patients via strategies that increase vascular supply through angiogenesis and arteriogenesis improving muscle perfusion and function in ischemic legs. This article discusses gene therapy approaches in the context of PAD, both intermittent claudication and critical limb ischemia, and the promise of adeno-associated virus–based strategies delivering not just VEGFs (vascular endothelial growth factors) but a range of other mediators as potential new therapeutics. We also highlight challenges and failures in the clinical translation of gene therapy for PAD and how at least some of these obstacles may be overcome using adeno-associated virus.

Adeno-associated virus (AAV) is currently the most popular gene delivery vector for in vivo gene therapy. However, variability in titration methods between different laboratories affects the reproducibility of experiments and evaluation of safety and efficacy in clinical trials. We describe an optimized protocol for AAV titration, including quantitative PCR (qPCR) standard preparation and quantitation and treatment of AAV samples before qPCR and droplet digital PCR (ddPCR) titration. During the protocol development, we observed that quantitation of the qPCR standard was dependent on its conformation and that A260-based quantitation overestimated the plasmid copy numbers, introducing significant error. Linearized, free inverted terminal repeat (free-ITR), and supercoiled standards were compared with enhanced green fluorescent protein (EGFP), SV40p(A), and AAV2-ITR qPCR assays and we found that using the AAV2-ITR assay together with either linearized or supercoiled standard led to overestimation of the titers, while EGFP and SV40p(A) assays were more accurate with the linearized standard. Finally, we compared extraction of AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 genomes by heat denaturation, proteinase K treatment, and kit extraction. Kit extraction, which contained proteinase K treatment in denaturing buffer before spin-column purification, significantly increased the titers acquired for all the serotypes in both qPCR and ddPCR. These improvements resulted in an accurate quantitation of the ATCC reference standard and in a robust and reliable protocol for AAV titration.

The 9p21.3 genomic locus is a hot spot for disease-associated single-nucleotide polymorphisms (SNPs), and its strongest associations are with coronary artery disease (CAD). The disease-associated SNPs are located within the sequence of a long noncoding RNA ANRIL, which potentially contributes to atherogenesis by regulating vascular cell stress and proliferation, but also affects pancreatic β-cell proliferation. Altered expression of a neighboring gene, CDKN2B, has been also recognized to correlate with obesity and hepatic steatosis in people carrying the risk SNPs. In the present study, we investigated the impact of 9p21.3 on obesity accompanied by hyperlipidemia in mice carrying a deletion of the murine ortholog for the 9p21.3 (Chr4^Δ70/Δ70) risk locus in hyperlipidemic Ldlr^−/−ApoB^100/100 background. The Chr4^Δ70/Δ70 mice showed decreased mRNA expression of insulin receptors in white adipose tissue already at a young age, which developed into insulin resistance and obesity by aging. In addition, the Sirt1-Ppargc1a-Ucp2 pathway was downregulated together with the expression of Cdkn2b, specifically in the white adipose tissue in Chr4^Δ70/Δ70 mice. These results suggest that the 9p21.3 locus, ANRIL lncRNA, and their murine orthologues may regulate the key energy metabolism pathways in a white adipose tissue-specific manner in the presence of hypercholesterolemia, thus contributing to the pathogenesis of metabolic syndrome.

Despite the advancements and release of new therapeutics in the past few years, cardiovascular diseases (CVDs) have remained the number one cause of death worldwide. Genetic variation of a 9p21.3 genomic locus has been identified as the most significant and robust genetic CVD risk marker on the population level, with the strongest association with coronary artery disease (CAD) and other diseases, including diabetes and cancer. Several mechanisms of 9p21.3 in CVDs have been proposed, but their effects on CVDs have remained elusive. Moreover, most of the single nucleotide polymorphisms (SNPs) associated with CAD are located on a sequence of a long noncoding RNA (lncRNA) called ANRIL. ANRIL has several linear and circular splicing isoforms, which seem to have different effects and implications for CVDs. The mechanisms of the 9p21.3 locus and the interplay of its coding and noncoding transcripts in different diseases require further research. Circular RNAs have generally raised interest due to their beneficial features as biomarkers and therapeutic molecules. Here, we review the literature of 9p21.3 from its identification in 2007 and draw the current knowledge on its function, implications in CVDs, and therapeutic potential.