Deletion of the Murine Ortholog of the Human 9p21.3 Locus Leads to Insulin Resistance and Obesity in Hypercholesterolemic Mice.

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 ß-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 (Chr4Δ70/Δ70) risk locus in hyperlipidemic Ldlr-/-ApoB100/100 background. The Chr4Δ70/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 Chr4Δ70/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.

Gene Therapeutic Strategies for Peripheral Artery Disease and New Opportunities Provided by Adeno-Associated Virus Vectors.

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.

Deletion of the murine ortholog of human 9p21.3 locus promotes atherosclerosis by increasing macrophage proinflammatory activity.

Background: Several genome-wide association studies have reported a risk locus for coronary artery disease (CAD) in the 9p21. 3 chromosomal region. This region encodes a lncRNA in the INK4 locus (ANRIL) and its genetic variance has a strong association with CAD, but its mechanisms in atherogenesis remain unclear. Objectives: This study aimed to investigate the role of the murine ortholog of human 9p21.3 locus in atherogenesis in hypercholesterolemic mice. Methods: Murine 9p21.3 ortholog knockout mice (Chr4Δ70kb/Δ70kb ) were crossbred with Ldlr -/- ApoB 100/100 mice, and atherosclerotic plaque size and morphology were analyzed on a standard or a high-fat diet (HFD). The hematopoietic cell-specific effect of Chr4Δ70kb/Δ70kb on atherosclerotic plaque development was studied via bone marrow (BM) transplantation, where Chr4Δ70kb/Δ70kb or wild-type BM was transplanted into Ldlr -/- ApoB 100/100 mice. The role of Chr4Δ70kb/Δ70kb in macrophage M1/M2 polarization was studied. In addition, single-cell sequencing data from human and mouse atheroma were analyzed to show the expression profiles of ANRIL and its murine equivalent, Ak148321, in the plaques. Results: Both systemic and hematopoietic Chr4Δ70kb/Δ70kb increased atherosclerosis in Ldlr -/- ApoB 100/100 mice after 12 weeks of HFD. The systemic Chr4Δ70kb/Δ70kb also elevated the number of circulating leukocytes. Chr4Δ70kb/Δ70kb BMDMs showed enhanced M1 polarization in vitro. Single-cell sequencing data from human and mouse atheroma revealed that ANRIL and Ak148321 were mainly expressed in the immune cells. Conclusion: These data demonstrate that both systemic and BM-specific deletion of the murine 9p21.3 risk locus ortholog promotes atherosclerosis and regulates macrophage pro-inflammatory activity, suggesting the inflammation-driven mechanisms of the risk locus on atherogenesis.

Optimized Protocol for Accurate Titration of Adeno-Associated Virus Vectors.

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.