Critical limb-threatening ischaemia and microvascular transformation: clinical implications.

BACKGROUND AND AIMS: Clinical management of critical limb-threatening ischaemia (CLTI) is focused on prevention and treatment of atherosclerotic arterial occlusions. The role of microvascular pathology in disease progression is still largely unspecified and more importantly not utilized for treatment. The aim of this explorative study was to characterize the role of the microvasculature in CLTI pathology. METHODS: Clinical high-resolution imaging of CLTI patients (n = 50) and muscle samples from amputated CLTI limbs (n = 40) were used to describe microvascular pathology of CLTI at the level of resting muscle blood flow and microvascular structure, respectively. Furthermore, a chronic, low arterial driving pressure-simulating ischaemia model in rabbits (n = 24) was used together with adenoviral vascular endothelial growth factor A gene transfers to study the effect of microvascular alterations on muscle outcome. RESULTS: Resting microvascular blood flow was not depleted but displayed decreased capillary transit time (P < .01) in CLTI muscles. Critical limb-threatening ischaemia muscle microvasculature also exhibited capillary enlargement (P < .001) and further arterialization along worsening of myofibre atrophy and detaching of capillaries from myofibres. Furthermore, CLTI-like capillary transformation was shown to worsen calf muscle force production (P < .05) and tissue outcome (P < .01) under chronic ischaemia in rabbits and in healthy, normal rabbit muscle. CONCLUSIONS: These findings depict a progressive, hypoxia-driven transformation of the microvasculature in CLTI muscles, which pathologically alters blood flow dynamics and aggravates tissue damage under low arterial driving pressure. Hypoxia-driven capillary enlargement can be highly important for CLTI outcomes and should therefore be considered in further development of diagnostics and treatment of CLTI.

Hypoxic regulation of hypoxia inducible factor 1 alpha via antisense transcription.

Impaired oxygen homeostasis is a frequently encountered pathophysiological factor in multiple complex diseases, including cardiovascular disease and cancer. While the canonical hypoxia response pathway is well characterized, less is known about the role of noncoding RNAs in this process. Here, we investigated the nascent and steady-state noncoding transcriptional responses in endothelial cells and their potential roles in regulating the hypoxic response. Notably, we identify a novel antisense long noncoding RNA that convergently overlaps the majority of the hypoxia inducible factor 1 alpha (HIF1A) locus, which is expressed across several cell types and elevated in atherosclerotic lesions. The antisense (HIF1A-AS) is produced as a stable, unspliced, and polyadenylated nuclear retained transcript. HIF1A-AS is highly induced in hypoxia by both HIF1A and HIF2A and exhibits anticorrelation with the coding HIF1A transcript and protein expression. We further characterized this functional relationship by CRISPR-mediated bimodal perturbation of the HIF1A-AS promoter. We provide evidence that HIF1A-AS represses the expression of HIF1a in cis by repressing transcriptional elongation and deposition of H3K4me3, and that this mechanism is dependent on the act of antisense transcription itself. Overall, our results indicate a critical regulatory role of antisense mediated transcription in regulation of HIF1A expression and cellular response to hypoxia.

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.

Establishment and characterization of cytochrome P450 1A1 CRISPR/Cas9 Knockout Bovine Foetal Hepatocyte Cell Line (BFH12).

The cytochrome P450 1A (CYP1A) subfamily of xenobiotic metabolizing enzymes (XMEs) consists of two different isoforms, namely CYP1A1 and CYP1A2, which are highly conserved among species. These two isoenzymes are involved in the biotransformation of many endogenous compounds as well as in the bioactivation of several xenobiotics into carcinogenic derivatives, thereby increasing the risk of tumour development. Cattle (Bos taurus) are one of the most important food-producing animal species, being a significant source of nutrition worldwide. Despite daily exposure to xenobiotics, data on the contribution of CYP1A to bovine hepatic metabolism are still scarce. The CRISPR/Cas9-mediated knockout (KO) is a useful method for generating in vivo and in vitro models for studying xenobiotic biotransformations. In this study, we applied the ribonucleoprotein (RNP)-complex approach to successfully obtain the KO of CYP1A1 in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP1A1 excision was confirmed at the DNA, mRNA and protein level. Therefore, RNA-seq analysis revealed significant transcriptomic changes associated with cell cycle regulation, proliferation, and detoxification processes as well as on iron, lipid and mitochondrial homeostasis. Altogether, this study successfully generates a new bovine CYP1A1 KO in vitro model, representing a valuable resource for xenobiotic metabolism studies in this important farm animal species.

Characterization of a functional endothelial super-enhancer that regulates ADAMTS18 and angiogenesis.

Super-enhancers are clusters of enhancers associated with cell lineage. They can be powerful gene-regulators and may be useful in cell-type specific viral-vector development. Here, we have screened for endothelial super-enhancers and identified an enhancer from within a cluster that conferred 5-70-fold increase in transgene expression. Importantly, CRISPR/Cas9 deletion of enhancers demonstrated regulation of ADAMTS18, corresponding to evidence of chromatin contacts between these genomic regions. Cell division-related pathways were primarily affected by the enhancer deletions, which correlated with significant reduction in cell proliferation. Furthermore, we observed changes in angiogenesis-related genes consistent with the endothelial specificity of this SE. Indeed, deletion of the enhancers affected tube formation, resulting in reduced or shortened sprouts. The super-enhancer angiogenic role is at least partly due to its regulation of ADAMTS18, as siRNA knockdown of ADAMTS18 resulted in significantly shortened endothelial sprouts. Hence, functional characterization of a novel endothelial super-enhancer has revealed substantial downstream effects from single enhancer deletions and led to the discovery of the cis-target gene ADAMTS18 and its role in endothelial function.

Susceptibility to Cardiac Arrhythmias and Sympathetic Nerve Growth in VEGF-B Overexpressing Myocardium.

VEGF-B gene therapy is a promising proangiogenic treatment for ischemic heart disease, but, unexpectedly, we found that high doses of VEGF-B promote ventricular arrhythmias (VAs). VEGF-B knockout, alpha myosin heavy-chain promoter (αMHC)-VEGF-B transgenic mice, and pigs transduced intramyocardially with adenoviral (Ad)VEGF- B186 were studied. Immunostaining showed a 2-fold increase in the number of nerves per field (76 vs. 39 in controls, p < 0.001) and an abnormal nerve distribution in the hypertrophic hearts of 11- to 20-month-old αMHC-VEGF-B mice. AdVEGF-B186 gene transfer (GT) led to local sprouting of nerve endings in pig myocardium (141 vs. 78 nerves per field in controls, p < 0.05). During dobutamine stress, 60% of the αMHC-VEGF-B hypertrophic mice had arrhythmias as compared to 7% in controls, and 20% of the AdVEGF-B186-transduced pigs and 100% of the combination of AdVEGF-B186- and AdsVEGFR-1-transduced pigs displayed VAs and even ventricular fibrillation. AdVEGF-B186 GT significantly increased the risk of sudden cardiac death in pigs when compared to any other GT with different VEGFs (hazard ratio, 500.5; 95% confidence interval [CI] 46.4-5,396.7; p < 0.0001). In gene expression analysis, VEGF-B induced the upregulation of Nr4a2, ATF6, and MANF in cardiomyocytes, molecules previously linked to nerve growth and differentiation. Thus, high AdVEGF-B186 overexpression induced nerve growth in the adult heart via a VEGFR-1 signaling-independent mechanism, leading to an increased risk of VA and sudden cardiac death.

Differential but Complementary HIF1α and HIF2α Transcriptional Regulation.

Effective vascular regeneration could provide therapeutic benefit for multiple pathologies, especially in chronic peripheral artery disease (PAD) and myocardial ischemia. The hypoxia inducible factors (HIFs) mediate the cellular transcriptional response to hypoxia and regulate multiple processes that are required for angiogenesis to ultimately restore perfusion and oxygen supply. In endothelial cells, both HIF1α and HIF2α are known to contribute to this role; however, the extent and individual roles of each of these HIFα remain unclear. To characterize the individual roles of HIFα, we sequenced the transcriptional outputs of stabilized forms of HIF1α and HIF2α, where they regulated 701 and 1,454 genes, respectively. HIF1α transcription primarily regulated metabolic reprogramming, whereas HIF2α exerted a larger role in regulating angiogenic extracellular signaling, guidance cues, and extracellular matrix remodeling factors. Furthermore, HIF2α almost exclusively regulated a large and diverse subset of transcription factors and coregulators that contribute to its diverse roles in hypoxia. Further understanding of how HIFs regulate cellular processes in hypoxia and angiogenesis could offer new avenues to modulate physiological angiogenesis to enhance revascularisation in ischemic conditions and other pathologies.

p12 tethers the murine leukemia virus pre-integration complex to mitotic chromosomes.

The p12 protein of the murine leukemia virus (MLV) is a constituent of the pre-integration complex (PIC) but its function in this complex remains unknown. We developed an imaging system to monitor MLV PIC trafficking in live cells. This allowed the visualization of PIC docking to mitotic chromosomes and its release upon exit from mitosis. Docking occurred concomitantly with nuclear envelope breakdown and was impaired for PICs of viruses with lethal p12 mutations. Insertion of a heterologous chromatin binding module into p12 of one of these mutants restored PICs attachment to the chromosomes and partially rescued virus replication. Capsid dissociated from wild type PICs in mitotic cells but remained associated with PICs harboring tethering-negative p12 mutants. Altogether, these results explain, in part, MLV restriction to dividing cells and reveal a role for p12 as a factor that tethers MLV PIC to mitotic chromosomes.

Cell-Specific Targeting of the Endothelium in the Cardiorenal Syndrome.

BACKGROUND: The vascular endothelium serves as a semi-selective permeable barrier as a conduit for transport of fluid, solutes, and various cell populations between the vessel lumen and tissues. The endothelium thus has a dynamic role in the regulation of coagulation, immune system, lipid and electrolyte transport, as well as neurohumoral influences on vascular tone and end-organ injury to tissues such as the heart and kidney. SUMMARY: Within this framework, pharmacologic strategies for heart and kidney diseases including blood pressure, glycemic control, and lipid reduction provide significant risk reduction, yet certain populations are at risk for substantial residual risk for disease progression and treatment resistance and often have unwanted off-target effects leaving the need for adjunct, alternative targeted therapies. Recent advances in techniques in sequencing and spatial transcriptomics have paved the way for the development of new therapies for targeting heart and kidney disease that include various gene, cell, and nano-based therapies. Cell-specific endothelium-specific targeting of viral vectors will enable their use for the treatment of heart and kidney diseases with gene therapy that can avoid unwanted off-target effects, improve treatment resistance, and reduce residual risk for disease progression. KEY MESSAGES: The vascular endothelium is an important therapeutic target for chronic kidney and cardiovascular diseases. Developing endothelial-specific gene therapies can benefit patients who develop resistance to current treatments.

Caridac vein retroinjections provide an efficient approach for global left ventricular gene transfer with adenovirus and adeno-associated virus.

Heart failure (HF) is a major burden worldwide, and new therapies are urgently needed. Gene therapy is a promising new approach to treat myocardial diseases. However, current cardiac gene delivery methods for producing global myocardial effects have been inefficient. The aim of this study was to develop an endovascular, reproducible, and clinically applicable gene transfer method for global left ventricular (LV) transduction. Domestic pigs (n = 52) were used for the experiments. Global LV myocardium coverage was achieved by three retrograde injections into the three main LV vein branches. The distribution outcome was significantly improved by simultaneous transient occlusions of the corresponding coronary arteries and the main anastomotic veins of the retroinjected veins. The achieved cardiac distribution was visualized first by administering Indian Ink solution. Secondly, AdLacZ (2 × 1012vp) and AAV2-GFP (2 × 1013vg) gene transfers were performed to study gene transduction efficacy of the method. By retrograde injections with simultaneous coronary arterial occlusions, both adenovirus (Ad) and adeno-associated virus (AAV) vectors were shown to deliver an efficient transduction of the LV. We conclude that retrograde injections into the three main LV veins is a potential new approach for a global LV gene transfer.

Proteomics on human cerebral cavernous malformations reveals novel biomarkers in neurovascular dysfunction for the disease pathology.

BACKGROUND: Cerebral cavernous malformation (CCM) is a disease associated with an elevated risk of focal neurological deficits, seizures, and hemorrhagic stroke. The disease has an inflammatory profile and improved knowledge of CCM pathology mechanisms and exploration of candidate biomarkers will enable new non-invasive treatments. METHODS: We analyzed protein signatures in human CCM tissue samples by using a highly specific and sensitive multiplexing technique, proximity extension assay. FINDINGS: Data analysis revealed CCM specific proteins involved in endothelial dysfunction/inflammation/activation, leukocyte infiltration/chemotaxis, hemostasis, extracellular matrix dysfunction, astrocyte and microglial cell activation. Biomarker expression profiles matched bleeding status, especially with higher levels of inflammatory markers and activated astrocytes in ruptured than non-ruptured samples, some of these biomarkers are secreted into blood or urine. Furthermore, analysis was also done in a spatially resolving manner by separating the lesion area from the surrounding brain tissue. Our spatial studies revealed that although appearing histologically normal, the CCM border areas were pathological when compared to control brain tissues. Moreover, the functional relevance of CD93, ICAM-1 and MMP9, markers related to endothelial cell activation and extracellular matrix was validated by a murine pre-clinical CCM model. INTERPRETATION: Here we present a novel strategy for proteomics analysis on human CCMs, offering a possibility for high-throughput protein screening acquiring data on the local environment in the brain. Our data presented here describe CCM relevant brain proteins and specifically those which are secreted can serve the need of circulating CCM biomarkers to predict cavernoma's risk of bleeding.

Generation and characterization of cytochrome P450 3A74 CRISPR/Cas9 knockout bovine foetal hepatocyte cell line (BFH12).

In human, the cytochrome P450 3A (CYP3A) subfamily of drug-metabolizing enzymes (DMEs) is responsible for a significant number of phase I reactions, with the CYP3A4 isoform superintending the hepatic and intestinal metabolism of diverse endobiotic and xenobiotic compounds. The CYP3A4-dependent bioactivation of chemicals may result in hepatotoxicity and trigger carcinogenesis. In cattle, four CYP3A genes (CYP3A74, CYP3A76, CYP3A28 and CYP3A24) have been identified. Despite cattle being daily exposed to xenobiotics (e.g., mycotoxins, food additives, drugs and pesticides), the existing knowledge about the contribution of CYP3A in bovine hepatic metabolism is still incomplete. Nowadays, CRISPR/Cas9 mediated knockout (KO) is a valuable method to generate in vivo and in vitro models for studying the metabolism of xenobiotics. In the present study, we successfully performed CRISPR/Cas9-mediated KO of bovine CYP3A74, human CYP3A4-like, in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP3A74 ablation was confirmed at the DNA, mRNA, and protein level. The subsequent characterization of the CYP3A74 KO clone highlighted significant transcriptomic changes (RNA-sequencing) associated with the regulation of cell cycle and proliferation, immune and inflammatory response, as well as metabolic processes. Overall, this study successfully developed a new CYP3A74 KO in vitro model by using CRISPR/Cas9 technology, which represents a novel resource for xenobiotic metabolism studies in cattle. Furthermore, the transcriptomic analysis suggests a key role of CYP3A74 in bovine hepatocyte cell cycle regulation and metabolic homeostasis.

Synonymous site conservation in the HIV-1 genome.

BACKGROUND: Synonymous or silent mutations are usually thought to evolve neutrally. However, accumulating recent evidence has demonstrated that silent mutations may destabilize RNA structures or disrupt cis regulatory motifs superimposed on coding sequences. Such observations suggest the existence of stretches of codon sites that are evolutionary conserved at both DNA-RNA and protein levels. Such stretches may point to functionally important regions within protein coding sequences not necessarily reflecting functional constraints on the amino-acid sequence. The HIV-1 genome is highly compact, and often harbors overlapping functional elements at the protein, RNA, and DNA levels. This superimposition of functions leads to complex selective forces acting on all levels of the genome and proteome. Considering the constraints on HIV-1 to maintain such a highly compact genome, we hypothesized that stretches of synonymous conservation would be common within its genome. RESULTS: We used a combined computational-experimental approach to detect and characterize regions exhibiting strong purifying selection against synonymous substitutions along the HIV-1 genome. Our methodology is based on advanced probabilistic evolutionary models that explicitly account for synonymous rate variation among sites and rate dependencies among adjacent sites. These models are combined with a randomization procedure to automatically identify the most statistically significant regions of conserved synonymous sites along the genome. Using this procedure we identified 21 conserved regions. Twelve of these are mapped to regions within overlapping genes, seven correlate with known functional elements, while the functions of the remaining four are yet unknown. Among these four regions, we chose the one that deviates most from synonymous rate homogeneity for in-depth computational and experimental characterization. In our assays aiming to quantify viral fitness in both early and late stages of the replication cycle, no differences were observed between the mutated and the wild type virus following the introduction of synonymous mutations. CONCLUSIONS: The contradiction between the inferred purifying selective forces and the lack of effect of these mutations on viral replication may be explained by the fact that the phenotype was measured in single-cycle infection assays in cell culture. Such a system does not account for the complexity of HIV-1 infections in vivo, which involves multiple infection cycles and interaction with the host immune system.

Comparison of Automated and Traditional Western Blotting Methods.

Traditional Western blotting is one of the most used analytical techniques in biological research. However, it can be time-consuming and suffer from a lack of reproducibility. Consequently, devices with different degrees of automation have been developed. These include semi-automated techniques and fully automated devices that replicate all stages downstream of the sample preparation, including sample size separation, immunoblotting, imaging, and analysis. We directly compared traditional Western blotting with two different automated systems, iBind™ Flex, which is a semi-automated system designed to perform the immunoblotting, and JESS Simple Western™, a fully automated and capillary-based system performing all steps downstream of sample preparation and loading, including imaging and image analysis. We found that a fully automated system can save time and importantly offer valuable sensitivity. This is particularly beneficial for limited sample amounts. The downside of automation is the cost of devices and reagents. Nevertheless, automation can be a good option to increase output and facilitate sensitive protein analyses.

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.

ErbB signaling is a potential therapeutic target for vascular lesions with fibrous component.

Background: Sporadic venous malformation (VM) and angiomatosis of soft tissue (AST) are benign, congenital vascular anomalies affecting venous vasculature. Depending on the size and location of the lesion, symptoms vary from motility disturbances to pain and disfigurement. Due to the high recurrence of the lesions, more effective therapies are needed. Methods: As targeting stromal cells has been an emerging concept in anti-angiogenic therapies, here, by using VM/AST patient samples, RNA-sequencing, cell culture techniques, and a xenograft mouse model, we investigated the crosstalk of endothelial cells (EC) and fibroblasts and its effect on vascular lesion growth. Results: We report, for the first time, the expression and secretion of transforming growth factor A (TGFA) in ECs or intervascular stromal cells in AST and VM lesions. TGFA induced secretion of vascular endothelial growth factor (VEGF-A) in paracrine fashion, and regulated EC proliferation. Oncogenic PIK3CA variant in p.H1047R, a common somatic mutation found in these lesions, increased TGFA expression, enrichment of hallmark hypoxia, and in a mouse xenograft model, lesion size, and vascularization. Treatment with afatinib, a pan-ErbB tyrosine-kinase inhibitor, decreased vascularization and lesion size in a mouse xenograft model with ECs expressing oncogenic PIK3CA p.H1047R variant and fibroblasts. Conclusions: Based on the data, we suggest that targeting of both intervascular stromal cells and ECs is a potential treatment strategy for vascular lesions having a fibrous component. Funding: Academy of Finland, Ella and Georg Ehnrooth foundation, the ERC grants, Sigrid Jusélius Foundation, Finnish Foundation for Cardiovascular Research, Jane and Aatos Erkko Foundation, GeneCellNano Flagship program, and Department of Musculoskeletal and Plastic Surgery, Helsinki University Hospital.

The Gag cleavage product, p12, is a functional constituent of the murine leukemia virus pre-integration complex.

The p12 protein is a cleavage product of the Gag precursor of the murine leukemia virus (MLV). Specific mutations in p12 have been described that affect early stages of infection, rendering the virus replication-defective. Such mutants showed normal generation of genomic DNA but no formation of circular forms, which are markers of nuclear entry by the viral DNA. This suggested that p12 may function in early stages of infection but the precise mechanism of p12 action is not known. To address the function and follow the intracellular localization of the wt p12 protein, we generated tagged p12 proteins in the context of a replication-competent virus, which allowed for the detection of p12 at early stages of infection by immunofluorescence. p12 was found to be distributed to discrete puncta, indicative of macromolecular complexes. These complexes were localized to the cytoplasm early after infection, and thereafter accumulated adjacent to mitotic chromosomes. This chromosomal accumulation was impaired for p12 proteins with a mutation that rendered the virus integration-defective. Immunofluorescence demonstrated that intracellular p12 complexes co-localized with capsid, a known constituent of the MLV pre-integration complex (PIC), and immunofluorescence combined with fluorescent in situ hybridization (FISH) revealed co-localization of the p12 proteins with the incoming reverse transcribed viral DNA. Interactions of p12 with the capsid and with the viral DNA were also demonstrated by co-immunoprecipitation. These results imply that p12 proteins are components of the MLV PIC. Furthermore, a large excess of wt PICs did not rescue the defect in integration of PICs derived from mutant p12 particles, demonstrating that p12 exerts its function as part of this complex. Altogether, these results imply that p12 proteins are constituent of the MLV PIC and function in directing the PIC from the cytoplasm towards integration.

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.

Efficacy and Safety of Clinical-Grade Human Vascular Endothelial Growth Factor-DΔNΔC Gene Therapy Containing Residual Replication-Competent Adenoviruses.

Biological bypass through induced angiogenesis by vascular endothelial growth factor D (VEGF-D) gene therapy (GT) is a new concept for the treatment of cardiac ischemia. Serotype 5 adenoviruses are used in the clinical trials for transferring the VEGF-D cDNA into the ischemic myocardium. However, the presence of replication-competent vectors in the adenovirus products is a widely recognized problem that may pose a potential safety risk to the treated patients. We compared three different VEGF-D GT production lots containing different levels of replication-competent adenoviruses (RCA) tested in 3 × 1010 viral particles (vp): <10 RCA (VEGF-D L-RCA1), 10-100 RCA (VEGF-D H-RCA2), and 100-200 RCA (VEGF-D H-RCA3), as measured by a novel droplet digital polymerase chain reaction (PCR) RCA assay in a preclinical rabbit model (n = 21). ß-galactosidase encoding nonclinical-grade preparation was used as a nonangiogenic control. Each preparation was injected into the right semimembranosus muscle using dose of 1 × 1011 vp. Efficacy of the products was tested by the combination of contrast pulse sequencing ultrasound and modified Miles assay as well as quantifying the total cross-sectional area of capillaries. Safety, immunogenicity, toxicity, biodistribution, and shedding were assessed by general histology, serial measurements of C-reactive protein, white blood cell count and body temperature as well as using quantitative real-time PCR with primers targeted to the VEGF-D and replication-permitting E1 sequences. We found no significant differences in the efficacy or safety between the study groups. Most importantly, no detectable presence of RCA-specific E1 sequence was found in any samples tested, indicating that no detectable vector replication took place in vivo. We conclude that relatively low levels of RCA in adenoviral GT products may not be as important major safety issue as previously anticipated.

Genomic Landscapes of Noncoding RNAs Regulating VEGFA and VEGFC Expression in Endothelial Cells.

Vascular endothelial growth factors (VEGFs) are best known as key regulators of angiogenesis and lymphangiogenesis. Although VEGFs have been promising therapeutic targets for various cardiovascular diseases, their regulatory landscape in endothelial cells remains elusive. Several studies have highlighted the involvement of noncoding RNAs (ncRNAs) in the modulation of VEGF expression. In this study, we investigated the role of two classes of ncRNAs, long ncRNAs (lncRNAs) and enhancer RNAs (eRNAs), in the transcriptional regulation of VEGFA and VEGFC. By integrating genome-wide global run-on sequencing (GRO-Seq) and chromosome conformation capture (Hi-C) data, we identified putative lncRNAs and eRNAs associated with VEGFA and VEGFC genes in endothelial cells. A subset of the identified putative enhancers demonstrated regulatory activity in a reporter assay. Importantly, we demonstrate that deletion of enhancers and lncRNAs by CRISPR/Cas9 promoted significant changes in VEGFA and VEGFC expression. Transcriptome sequencing (RNA-Seq) data from lncRNA deletions showed downstream factors implicated in VEGFA- and VEGFC-linked pathways, such as angiogenesis and lymphangiogenesis, suggesting functional roles for these lncRNAs. Our study uncovers novel lncRNAs and eRNAs regulating VEGFA and VEGFC that can be targeted to modulate the expression of these important molecules in endothelial cells.