Adeno-associated Virus Vectors Research Articles

Targeting resident astrocytes attenuates neuropathic pain after spinal cord injury.

Astrocytes derive from different lineages and play a critical role in neuropathic pain after spinal cord injury (SCI). Whether selectively eliminating these main origins of astrocytes in lumbar enlargement could attenuate SCI-induced neuropathic pain remains unclear. Through transgenic mice injected with an adeno-associated virus vector and diphtheria toxin, astrocytes in lumbar enlargement were lineage traced, targeted, and selectively eliminated. Pain-related behaviors were measured with an electronic von Frey apparatus and a cold/hot plate after SCI. RNA sequencing, bioinformatics analysis, molecular experiment, and immunohistochemistry were used to explore the potential mechanisms after astrocyte elimination. Lineage tracing revealed that the resident astrocytes but not ependymal cells were the main origins of astrocytes-induced neuropathic pain. SCI-induced mice to obtain significant pain symptoms and astrocyte activation in lumbar enlargement. Selective resident astrocyte elimination in lumbar enlargement could attenuate neuropathic pain and activate microglia. Interestingly, the type I interferons (IFNs) signal was significantly activated after astrocytes elimination, and the most activated Gene Ontology terms and pathways were associated with the type I IFNs signal which was mainly activated in microglia and further verified in vitro and in vivo. Furthermore, different concentrations of interferon and Stimulator of interferon genes (STING) agonist could activate the type I IFNs signal in microglia. These results elucidate that selectively eliminating resident astrocytes attenuated neuropathic pain associated with type I IFNs signal activation in microglia. Targeting type I IFNs signals is proven to be an effective strategy for neuropathic pain treatment after SCI.

Targeting resident astrocytes attenuates neuropathic pain after spinal cord injury

Astrocytes derive from different lineages and play a critical role in neuropathic pain after spinal cord injury (SCI). Whether selectively eliminating these main origins of astrocytes in lumbar enlargement could attenuate SCI-induced neuropathic pain remains unclear. Through transgenic mice injected with an adeno-associated virus vector and diphtheria toxin, astrocytes in lumbar enlargement were lineage traced, targeted, and selectively eliminated. Pain-related behaviors were measured with an electronic von Frey apparatus and a cold/hot plate after SCI. RNA sequencing, bioinformatics analysis, molecular experiment, and immunohistochemistry were used to explore the potential mechanisms after astrocyte elimination. Lineage tracing revealed that the resident astrocytes but not ependymal cells were the main origins of astrocytes-induced neuropathic pain. SCI-induced mice to obtain significant pain symptoms and astrocyte activation in lumbar enlargement. Selective resident astrocyte elimination in lumbar enlargement could attenuate neuropathic pain and activate microglia. Interestingly, the type I interferons (IFNs) signal was significantly activated after astrocytes elimination, and the most activated Gene Ontology terms and pathways were associated with the type I IFNs signal which was mainly activated in microglia and further verified in vitro and in vivo. Furthermore, different concentrations of interferon and Stimulator of interferon genes (STING) agonist could activate the type I IFNs signal in microglia. These results elucidate that selectively eliminating resident astrocytes attenuated neuropathic pain associated with type I IFNs signal activation in microglia. Targeting type I IFNs signals is proven to be an effective strategy for neuropathic pain treatment after SCI.

Adeno-Associated Virus-Mediated Dickkopf-1 Gene Transduction Reduces Silica-Induced Oxidative Stress and Silicosis in Mouse Lung.

Aims: Silicosis is a lung disease caused by inhalation of silica particles. Both silica-induced oxidative stress and aberrant activation of the Wnt/β-catenin signaling pathway are potential targets in the treatment of pulmonary fibrosis. Dickkopf-1 (Dkk1), an inhibitor of the Wnt/β-catenin signaling pathway, plays regulatory roles in cell fate determination and immune responses. Our previous study demonstrated that adenoviral vector-mediated Dkk1 gene transfer alleviated the silica-induced mouse silicosis. However, the mechanism of therapeutic action of Dkk1 in silicosis is yet completely understood; together with the drawbacks of adenoviral vectors in gene therapy, we investigated the therapeutic effect and mechanisms of Dkk1 by employing an adeno-associated virus (AAV) vector in a silicosis mouse model. Results: The AAV vector could efficiently transduce the Dkk1 gene in silicotic lung during both the early and the late phases of disease, resulting in an alleviation of silicotic lesions, improvement of pulmonary compliance, and radiological findings. Mechanistic studies further demonstrated that the transduction of Dkk1 inhibited the silica-activated Wnt/β-catenin signaling and reduced the silica-induced reactive oxygen species-producing enzyme NADPH oxidase 4, oxidative stress regulator nuclear factor erythroid 2-related factor 2, and signaling molecules binding immunoglobulin protein and C/EBP homologous protein. In addition, shRNA-mediated downregulation of Dkk1 exacerbated the progression of silicosis in mice, whereas the treatment of ROS scavenger n-acetylcysteine showed a comparable mitigation of silicosis that was seen in the AAV-Dkk1 treatment. Innovation and Conclusion: This study provides an insight into the mechanism by which Dkk1 inhibits the silica-induced Wnt signaling and oxidative stress to mitigate the pathogenesis of lung silicosis and evidence of the potential of AAV-mediated Dkk1 gene transfer as an alternative approach in silicosis treatment. Antioxid. Redox Signal. 00, 000-000.

Adeno-Associated Viral Vectors in the Treatment of Epilepsy

Epilepsy is a brain disorder characterized by a persistent predisposition to epileptic seizures. With various etiologies of epilepsy, a significant proportion of patients develop pharmacoresistance to antiepileptic drugs, which necessitates the search for new therapeutic methods, in particular, using gene therapy. This review discusses the use of adeno-associated viral (AAV) vectors in gene therapy for epilepsy, emphasizing their advantages, such as high efficiency of neuronal tissue transduction and low immunogenicity/cytotoxicity. AAV vectors provide the possibility of personalized therapy due to the diversity of serotypes and genomic constructs, which allows for increasing the specificity and effectiveness of treatment. Promising orientations include the modulation of the expression of neuropeptides, ion channels, transcription, and neurotrophic factors, as well as the use of antisense oligonucleotides to regulate seizure activity, which can reduce the severity of epileptic disorders. This review summarizes the current advances in the use of AAV vectors for the treatment of epilepsy of various etiologies, demonstrating the significant potential of AAV vectors for the development of personalized and more effective approaches to reducing seizure activity and improving patient prognosis.

Cure of Congenital Purpura Fulminans via Expression of Engineered Protein C Through Neonatal Genome Editing in Mice.

PC (protein C) is a plasma anticoagulant encoded by PROC; mutation in both PROC alleles results in neonatal purpura fulminans-a fatal systemic thrombotic disorder. In the present study, we aimed to develop a genome editing treatment to cure congenital PC deficiency. We generated an engineered APC (activated PC) to insert a furin-cleaving peptide sequence between light and heavy chains. The engineered PC was expressed in the liver of mice using an adeno-associated virus vector or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated genome editing using an adeno-associated virus vector in vivo. The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of PS (protein S) in vitro. The adeno-associated virus vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation FV (factor V) in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6J mice. The insertion of EGFP sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using adeno-associated virus vector resulted in the expression of EGFP in 7% of liver cells, mainly via homology-directed repair, in mice. Finally, we succeeded in improving the survival of PC-deficient mice by expressing the engineered PC via neonatal genome editing in vivo. These results suggest that the expression of engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency.

A single dose recombinant AAV based CHIKV vaccine elicits robust and durable protective antibody responses in mice.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that is responsible for Chikungunya fever, which is characterized by fever, rash, and debilitating polyarthralgia. Since its re-emergence in 2004, CHIKV has continued to spread to new regions and become a severe health threat to global public. Development of safe and single dose vaccines that provide durable protection is desirable to control the spread of virus. The recombinant adeno-associated virus (rAAV) vectors represent promising vaccine platform to provide prolonged protection with a single-dose immunization. In this study, we developed a rAAV capsid serotype 1 vector based CHIKV vaccine and evaluated its protection effect against CHIKV challenge. The recombinant AAV1 encoding the full-length structural proteins of CHIKV (named as rAAV1-CHIKV-SP) was generated in vitro by transfecting the plasmids of AAV helper-free system into HEK-293T cells. The safety and immunogenicity of rAAV1-CHIKV-SP were tested in 4-week-old C57BL/6 mice. The antibody responses of the mice receiving prime-boost or single-dose immunization of the vaccine were determined by ELISA and plaque reduction neutralizing test. The immunized mice were challenged with CHIKV to evaluate the protection effect of the vaccine. The rAAV1-CHIKV-SP showed remarkable safety and immunogenicity in C57BL/6 mice. A single dose intramuscular injection of rAAV1-CHIKV-SP elicited high level and long-lasting antibody responses, and conferred complete protection against a heterologous CHIKV strain challenge. These results suggest rAAV1-CHIKV-SP represents a promising vaccine candidate against different CHIKV clades with a simplified immunization strategy.

Comprehensive Analysis Of Off-Target And On-Target Effects Resulting From Liver-Directed Crispr-Cas9–Mediated Gene Targeting With Adeno-Associated Viral Vectors

Comprehensive Analysis Of Off-Target And On-Target Effects Resulting From Liver-Directed Crispr-Cas9–Mediated Gene Targeting With Adeno-Associated Viral Vectors

Development of an optimized SEC method for characterization of genome DNA leakage from adeno-associated virus products.

Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes due to their superior safety, relatively low immunogenicity, and ability to target diverse tissues. AAV gene therapy products are typically formulated as frozen liquid and stored below - 60 °C, and therefore are subjected to multiple freeze/thaw cycles during manufacturing and administration. Recent studies have shown that genome DNA leakage could be induced by freeze/thaw stress. DNA leakage from AAV capsids has been reported to potentially impact product stability, induce immune responses, and compromise product efficacy. Thus, further characterization to improve the understanding of genome DNA leakage is necessary for mitigating the risks associated with genome DNA leakage during AAV product development. In this work, we developed an optimized size-exclusion chromatography (SEC) method for quantifying the leakage of genome DNA across multiple different AAV serotypes and demonstrated satisfactory assay performance in sensitivity, precision, and linearity. Furthermore, we showed that this method could also be applied to quantifying additional quality attributes of AAV, including the percentage of full capsids and quantification of AAV dimers. By using this optimized SEC method, we demonstrated that significantly increased free DNA was observed with increasing freeze/thaw cycles or at a temperature approaching the onset temperature for genome DNA ejection, which was effectively mitigated by the addition of 1.5% w/v sucrose in the AAV formulation. Thus, this optimized SEC method can serve as an invaluable tool for AAV formulation, product, and process development in ensuring the quality and stability of AAV gene therapy products.

Chaperone-mediated autophagy (CMA) confers neuroprotection of HBO preconditioning against stroke

Previous attempts to identify neuroprotective targets for acute ischemic stroke by studying ischemic cascades and devising ways to suppress these pathways have failed in translational research. We hypothesized that studying the molecular determinants of endogenous neuroprotection, namely, the tolerance against ischemic stroke conferred by hyperbaric oxygen (HBO) preconditioning, via a well-established paradigm would reveal new neuroprotective targets. By a combination of proteomics, KEGG pathway analysis, lysosome fraction and western blot analysis, we found that chaperone-mediated autophagy (CMA) was activated by HBO preconditioning. In addition, LAMP2A is uniquely decreased in cortical neurons in the early stage of stroke. Suppression of CMA with recombinant adeno-associated viral vector (rAAV)-mediated delivery of short hairpin RNAs (shRNAs) targeting the LAMP2A transcript increased the neuronal susceptibility of apoptosis and abolished the neuroprotection induced by HBO preconditioning. Administration of the clinically utilized FDA-approved drug mycophenolate mofetil induced long-term neuroprotection post-stroke in a CMA-dependent manner. In summary, HBO preconditioning confers neuroprotection against ischemia by inducing CMA, which is a promising translational treatment target for stroke.

Challenges in Cardiomyopathy Gene Therapy Clinical Trial Design.

Gene therapy has emerged as a possible treatment for progressive, debilitating Mendelian cardiomyopathies with limited therapeutic options. This paper arises from discussions at the 2023 Cardiovascular Clinical Trialists Forum and highlights several challenges relevant to gene therapy clinical trials, including low prevalence and high phenotypic heterogeneity of Mendelian cardiomyopathies, outcome selection complexities and resulting regulatory uncertainty, and immune responses to the adeno-associated viral vectors that are being used in ongoing studies. Avenues to address these challenges such as natural history studies, external controls, novel regulatory pathways, and immunosuppression are discussed. Relevant cases of recent therapy approvals are highlighted. Ultimately, this work aims to broadly frame discussions on and provide potential future avenues for clinical trial design for rare cardiomyopathy gene therapies.

Investigation in the cannabigerol derivative VCE-003.2 as a disease-modifying agent in a mouse model of experimental synucleinopathy

BackgroundThe cannabigerol derivative VCE-003.2, which has activity at the peroxisome proliferator-activated receptor-γ has afforded neuroprotection in experimental models of Parkinson’s disease (PD) based on mitochondrial dysfunction (6-hydroxydopamine-lesioned mice) and neuroinflammation (LPS-lesioned mice). Now, we aim to explore VCE-003.2 neuroprotective properties in a PD model that also involves protein dysregulation, other key event in PD pathogenesis.MethodsTo this end, an adeno-associated viral vector serotype 9 coding for a mutated form of the α-synuclein gene (AAV9-SynA53T) was unilaterally delivered in the substantia nigra pars compacta (SNpc) of mice. This model leads to motor impairment and progressive loss of tyrosine hydroxylase-labelled neurons in the SNpc.ResultsOral administration of VCE-003.2 at 20 mg/kg for 14 days improved the performance of mice injected with AAV9-SynA53T in various motor tests, correlating with the preservation of tyrosine hydroxylase-labelled neurons in the SNpc. VCE-003.2 also reduced reactive microgliosis and astrogliosis in the SNpc. Furthermore, we conducted a transcriptomic analysis in the striatum of mice injected with AAV9-SynA53T and treated with either VCE-003.2 or vehicle, as well as control animals. This analysis aimed to identify gene families specifically altered by the pathology and/or VCE-003.2 treatment. Our data revealed pathology-induced changes in genes related to mitochondrial function, lysosomal cell pathways, immune responses, and lipid metabolism. In contrast, VCE-003.2 treatment predominantly affected the immune response through interferon signaling.ConclusionOur study broadens the neuroprotective potential of VCE-003.2, previously described against mitochondrial dysfunction, oxidative stress, glial reactivity and neuroinflammation in PD. We now demonstrate its efficacy against another key pathogenic event in PD as α-synuclein dysregulation. Furthermore, our investigation sheds light on the molecular mechanisms underlying VCE-003.2 revealing its role in regulating interferon signaling. These findings, together with a favorable ADMET profile, enhance the preclinical interest of VCE-003.2 towards its future clinical development in PD.

Comparative Analysis of Six Adeno-Associated Viral Vector Serotypes in Mouse Inferior Colliculus and Cerebellum.

Adeno-associated viral vector (AAV) serotypes vary in how effectively they express genes across different cell types and brain regions. Here we report a systematic comparison of the AAV serotypes 1, 2, 5, 8, 9, and the directed evolution derived AAVrg, in the inferior colliculus (IC) and cerebellum. The AAVs were identical apart from their different serotypes, each having a synapsin promotor and expressing GFP (AAV-hSyn-GFP). Identical titers and volumes were injected into the IC and cerebellum of adult male and female mice, and brains were sectioned and imaged 2 weeks later. Transduction efficacy, anterograde labeling of axonal projections, and retrograde labeling of somata were characterized and compared across serotypes. Cell-type tropism was assessed by analyzing the morphology of the GFP-labeled neurons in the cerebellar cortex. In both the cerebellum and IC, AAV1 expressed GFP in more cells, labeled a larger volume, and produced significantly brighter labeling than all other serotypes, indicating superior transgene expression. AAV1 labeled more Purkinje cells, unipolar brush cells, and molecular layer interneurons than the other serotypes, while AAV2 labeled a greater number of granule cells. These results provide guidelines for the use of AAVs as gene delivery tools in these regions.

Conditional over-expression of heme-oxygenase 1 in myelomonocytic cells reduces AngII-induced hypertension and vascular inflammation in mice

Abstract Background Systemic arterial Hypertension is one of the most important risk factor responsible for morbidity and mortality world-wide. Angiotensin II (Ang II), a potent vasoconstrictor and key player in the renin-angiotensin-aldosterone system (RAAS) is responsible for vascular dysfunction, inflammation, and tissue damage. Heme oxygenase-1 (HO-1) overexpression may confer antioxidant and anti-inflammatory properties in Ang II-induced hypertension through its action on heme catabolism, generating carbon monoxide (CO), ferritin and biliverdin/bilirubin by the action of biliverdin reductase A (BLVRA). Methods and results Male 9–12-weeks-old HO-1indxLySMCre/wt and LySMCre/wt mice were infused with AngII (1mgAngII/Day/Kg) for 7 days to induce hypertension and compared to sham treatment. Blood pressure monitoring by tail-cuff method, and endothelium-dependent vasodilator studies via organ chamber system using acetylcholine (ACh) in isolated aortic segments (~4 mm), revealed that overexpression of HO-1 in myeloid cells resulted in decreased systolic blood pressure and improved endothelial function in AngII-infused HO-1indxLySMCre/wt mice compared to controls. Concurrently, increased BLVRA expression in liver tissue and elevated plasma bilirubin levels were detected by transcriptome analysis and high-performance liquid chromatography, respectively. These results were supported by a reduction in the expression of inducible cytokines and cell adhesion molecules (CAMs) in the aorta, assessed using qPCR. Bone marrow transplant experiments demonstrated that bone marrow from LysMcre mice in HO-1indxLySMCre/wt mice abrogated the protective effect of HO-1, resulting in endothelial damage and increased blood pressure, potentially due to decreased liver BLVRA expression and the accumulation of bone marrow-derived cells in the vessel wall in response to AngII. Additionally, adeno-associated viral vector (AAV) transfection targeting specifically BLVRA activity in liver lead to an increase in blood pressure values and worse endothelium relaxation, in parallel with higher oxidative stress and the blood neutrophils concentration, as assessed in whole blood by using oxidative burst method and blood count system respectively. Conclusion The overexpression of HO-1 in myeloid cells confers anti-inflammatory protection in AngII-induced arterial hypertension in mice. Myeloid cells bone marrow-derived overexpressing HO-1 regulate the differentiation and infiltration of pro-inflammatory immune cells in the vasculature. BLVRA expression and bilirubin levels downstream of HO-1 play a critical role in protecting against vascular damage by reducing leukocyte infiltration.

Phase I gene therapy clinical trial design of RP-A601 in adult patients with PKP2-arrhythmogenic cardiomyopathy (PKP2-ACM)

Abstract Background Arrhythmogenic cardiomyopathy (ACM) is a rare, progressive, autosomal dominant disorder characterized by increased risk of ventricular arrhythmias and sudden cardiac death. Up to 45% of all ACM cases are caused by pathogenic variants in the PKP2 gene, which encodes for the desmosomal protein Plakophilin-2. This genetic cardiomyopathy is referred to as PKP2-ACM. AAVrh.74-PKP2a is a recombinant adeno-associated viral (AAV) vector containing the coding sequence of human PKP2 isoform A (PKP2a) and delivers the functional PKP2 gene to cardiomyocytes. Preclinical testing conducted in a clinically relevant mouse model of PKP2-ACM (with survival ≤50 days post disease onset) demonstrated that administration of a single AAVrh.74-PKP2a dose after disease onset extended survival through 5 months with improved right ventricular (RV) function, and decreased cardiac dilation, myocardial fibrosis, and arrhythmias. Additional studies in rodents and nonhuman primates demonstrated AAVrh.74-PKP2a safety. These preclinical findings support clinical initiation. Purpose We describe the design of a phase I trial to assess safety and preliminary efficacy of AAVrh.74-PKP2a (RP-A601) in high-risk adult patients with PKP2-ACM. Methods This study (NCT05885412) is a multi-center, open-label, dose escalation trial evaluating the safety and preliminary efficacy of a single intravenous infusion of RP-A601 in adult patients (age ≥18 yrs) with clinical and genetic PKP2-ACM diagnosis and an implanted ICD. Patients with severe right ventricular dysfunction, LV ejection fraction ≤50% (echocardiogram or cardiac MRI) and/or NYHA Class IV heart failure are excluded. Preliminary efficacy will be assessed at 12 months after RP-A601 infusion and includes evaluation of change in PKP2 myocardial tissue expression and clinical markers of arrhythmia burden. Conclusions PKP2-ACM is a devastating disease associated with high morbidity and mortality. This Phase I trial will evaluate the safety and preliminary efficacy of RP-A601 and was initiated based on robust preclinical efficacy and safety data.

GenePHIT phase 2 study design: a double blind, placebo-controlled trial to assess efficacy, safety, and tolerability of AB-1002 gene therapy in adults with heart failure

Abstract Introduction Heart failure (HF) is associated with substantial morbidity and mortality, highlighting a critical need for novel therapies. AB-1002, an engineered adeno-associated virus (AAV) vector gene therapy candidate, delivers a constitutively active form of protein phosphatase inhibitor (I-1c) to restore cardiomyocyte intracellular calcium homeostasis. Preliminary results from an ongoing Phase (Ph) 1 trial (NCT04179643) of AB-1002 showed favourable safety and tolerability. Here we describe the design of AB-1002 Ph 2 trial (GenePHIT; NCT05598333) to evaluate efficacy, safety, and tolerability. Methods This phase 2 adaptive, double-blind, placebo-controlled, randomised, multi-centre trial (currently United States; European sites planned) includes a 52-week observation and 4-year follow-up period. Eligible patients are ≥18 years of age with non-ischaemic cardiomyopathy and New York Health Association (NYHA) Class III heart failure symptoms (Figure). Patients will be randomised 1:1:1 (n=30–50/treatment arm), including a single dose of AB-1002 administered via antegrade intracoronary infusion at 1 of 2 different doses, or placebo, all in addition to standard of care. The primary endpoint will evaluate efficacy at 52 weeks based on the composite endpoint by modified win ratio, which includes cardiovascular-related death, change in NYHA classification, and for the responders, improvements in left ventricular ejection fraction (LVEF) (≥5%), peak oxygen uptake (pVO2) ≥1.5 mL/kg/min, and 6-min walk test (6MWT) >30 metres. Key secondary efficacy endpoints include functional status and hospitalisations at 24 and 52 weeks, by assessing changes over time in NYHA classification, LVEF, pVO2, 6MWT, biomarkers (N-terminal pro-hormone b-type natriuretic peptide and troponin), quality of life questionnaires (Minnesota Living with Heart Failure, Kansas City Cardiomyopathy), and physiological assessments. Key safety endpoints will be assessed through 52 weeks and include treatment-emergent adverse events (AEs) and serious AEs (SAEs). Exploratory endpoints will evaluate AAV2i8 antibody titres, T-cell response to AAV2i8 capsid and I-1c, and optional right ventricular biopsy testing. Long-term follow-up will assess treatment-emergent AEs and SAEs, and number of HF hospitalisations. Data safety monitoring board assessment will occur every 3 months. Primary efficacy endpoint will compare both active dose groups combined versus placebo using the joint rank test (1). Key secondary endpoints will be analysed using Analysis of Covariance with baseline as a continuous covariate. Multiple imputations will be used to address missing data. Interim efficacy analysis will be performed using a Bayesian adaptive sample size algorithm. Conclusions GenePHIT is currently recruiting and will evaluate the efficacy, safety, and tolerability of intracoronary infusion of AB-1002, an innovative gene therapy targeting ventricular dysfunction in HF.

Suppression of Fibulin 5 Attenuated Pulmonary Arterial Hypertension Associated with Congenital Heart Disease via Mitigating Pulmonary Arterial Smooth Muscle Cell Dysfunction

Abstract Backgrounds: The specific molecular pathogenesis of pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH) and the reversal still remain elusive. Fibulin 5 was involved in the regulation of cardiovascular system while little is known about the expression and functions of fibulin 5 in the development of CHD-PAH. This study aimed to investigate the role of fibulin 5 in the pathogenesis of the disease. Methods Lung samples were obtained both from patients with CHD-PAH and aortocaval shunt-associated PAH rat models. TGF-β1was used to induce human pulmonary artery smooth muscle cells (hPASMCs) dysfunction and lentiviruses were responsible for alteration of fibulin 5 expression. In an effort to explore the specific role of fibulin 5, an inhibitor of phosphatidylinositol-3-kinases (PI3K) /protein-serine-threonine kinase (AKT) which was defined as the TGF-β1-related downstream signaling was applied for further exploration. Results Fibulin 5 was pronouncedly elevated in the PASMCs of the remodeled pulmonary arterioles from patients with CHD-PAH and shunt-associated PAH rats. We found that over-expression of fibulin 5 could promote hPASMCs dysfunction induced by TGF-β1 and it could be reversed by the PI3K/AKT inhibitor LY294002 suggesting the essential role of TGF-β1/PI3K/AKT signaling activation in phenotypic switch, proliferation and migration of PASMCs. Moreover, an adeno-associated virus vector encoding fibulin 5 by intratracheally instillation with rats improve the hemodynamic parameters in shunt-associated PAH rats and its related pulmonary artery remodeling. Conclusion Over-expression of fibulin 5 could significantly promote hPASMCs dysfunction and pulmonary artery remodeling via reinforcing TGF-β1/PI3K/AKT signaling activation, which highlighted the potential valuable pharmacological target for the treatment of CHD-PAH.Fibulin 5 was over-expressed in PAHInhibition of fibulin 5 attenuated PAH

Targeting an altered sphingolipid profile with consecutive lipotoxicity as therapeutic approach to calcific aortic valve disease

Abstract Background Aortic stenosis due to calcific aortic valve disease (CAVD) is the most common valvular heart disease. In symptomatic patients, without repair the prognosis is poor. Treatment to date is based on surgery or catheter-based interventions. Pharmacological therapy options to prevent the progression of valve calcification are not available. In vascular smooth muscle cells, cellular accumulation of sphingolipids is associated with a proinflammatory response in the sense of lipotoxicity. Chronic inflammatory processes are indeed also essential for aortic valve calcification. Purpose With the subsequent goal of developing innovative treatment approaches for CAVD, we therefore aim to elucidate the unclear role of sphingolipids in the development of CAVD. Methods Human aortic valve and plasma samples of CAVD patients and controls (n=26) were analysed by liquid chromatography–mass spectrometry for lipidomics and proteomics. Human aortic valve interstitial cells (hVICs) were stimulated with ceramides. Calcification was detected by alizarin red staining. mRNA expression of bone-related proteins was determined by qPCR. NF-kB pathway was assessed by proteome profiler. Myriocin and GW4869 were applied to inhibit ceramide biosynthesis. PDTC and MCC950 were used to inhibit NF-kB Pathway and NLRP3 activation. ApoE-/- mice received a gain-of-function PCSK9 adeno-associated virus vector and fed high cholesterol diet for 14 weeks. Myriocin was applied orally over the treatment period to inhibit ceramide de novo synthesis in vivo. Murine echocardiography was used to measure transvalvular velocity and left ventricular function. Calcification degree of murine aortic valve was determined by histological staining. Results A significantly altered sphingolipid profile was observed in aortic valve tissue of CAVD patients (Fig. 1). Ceramide species e.g. Cer (17:1;2O/16:0) were increased. Verifying the biological relevance of these findings in vitro, C2-Cer (d18:1/2:0) enhanced hVIC calcification (Fig. 2). Accordingly, ALP activity and mRNA expression of osteogenic genes RUNX2, ALPL and MSX2 were increased. Furthermore, C2-Cer (d18:1/2:0) enhanced NF-kB p65 phosphorylation and NLRP3 activation, while treatment with both PDTC and MCC950 protected against C2-Cer (d18:1/2:0) induced calcification. Supporting the role of sphingolipid biosynthesis in hVIC calcification, Myriocin and GW4869 both reduced ox-LDL-induced VIC calcification which was reversed by C2-Cer(d18:1/2:0). Finally, Myriocin reduced the degree of calcification and transvalvular jet velocity of aortic valve in the PCSK9-AAV ApoE-/- mice model. Conclusion CAVD is accompanied by an accumulation of ceramides causing lipotoxicity in human aortic valve tissue. Pharmacological modulation of sphingolipid metabolism is an effective approach to prevent the development and progression of aortic valve calcification in vivo and should be considered as a future therapeutic strategy in CAVD patients.Volcano Plot of CAVD tissue lipidomicsC2 Ceramide enhances hVIC calcification

Recent Advances in Designing Adeno-Associated Virus-Based Vaccines Against Viral Infections

Over 80% of the world’s deadliest pandemics are caused by viral infections, and vaccination remains the most effective way to prevent these infections from spreading. Since the discovery of the first vaccine over two centuries ago, several vaccine design technologies have been developed. Next-generation vaccines, based on mRNA and viral vector technologies, have recently emerged as alternatives to traditional vaccines. Adenoviral vector-based vaccines against coronavirus disease 2019 have demonstrated a more sustained antibody response as compared to mRNA vaccines. However, this has not been without complications, with a few cases of severe adverse events identified in vaccinated individuals, and the underlying mechanism is the subject of intense investigation. Adeno-associated viral vectors induce a weaker cellular immune response compared to adenoviral vectors, and it is mainly for this reason that there has been a diminished interest in exploring them as a vaccine platform until recently. This review will discuss recent developments and the potential of adeno-associated viral vectors as anti-viral vaccines.

Multimass Analysis of Adeno-Associated Virus Vectors by Orbitrap-Based Charge Detection Mass Spectrometry.

Adeno-associated virus (AAV) vectors have attracted significant attention as the main platform for gene therapy. To ensure the safety and efficacy of AAV vectors when used as gene therapy drugs, it is essential to assess their critical quality attributes (CQAs). These CQAs include the genome packaging status, the size of the genome encapsidated within the AAV capsid, and the stoichiometry of viral proteins (VPs) that constitute the AAV capsids. Analytical methods have been established for evaluating CQAs, such as analytical ultracentrifugation, capillary gel electrophoresis with laser-induced fluorescence detection, and capillary gel electrophoresis using sodium dodecyl sulfate with UV detection. Here, we present a multimass analysis of AAV vectors using orbitrap-based charge detection mass spectrometry (CDMS), a single-ion mass spectrometry. Orbitrap-based CDMS facilitates the quantitative evaluation of the genome packaging status based on the mass distribution of empty and full particles. Additionally, we established a novel method to analyze the encapsidated genome directly without pretreatment, such as protein digestion or heat treatment, and to estimate the stoichiometric variation of VP for the capsid based on the mass distribution constituted by the single peak corresponding to AAV particles. Orbitrap-based CDMS is a distinctive method that allows multiple mass characterizations of AAV vectors with a small sample volume of 20 μL for 1013 cp/mL in a short time (30 min), and it holds the potential to become a new standard method in the assessment of CQAs for AAV vectors.

Comparative analysis of six adeno-associated viral vector serotypes in mouse inferior colliculus and cerebellum.

AAVs have become ubiquitous gene expression tools in neuroscience research and are becoming more common in clinical settings. Naturally occurring and engineered serotypes have varying abilities to infect neurons and cause them to produce proteins of interest. The efficacy of AAV transduction in specific cell types depends on many factors and remains difficult to predict, so an empirical approach is often required to determine the best performing serotype in each population of cells. In the present study we show that AAV1 produces the highest expression in these two regions, labels the most axonal projections, and labels Purkinje cells and unipolar brush cells better than the other serotypes tested, while AAV2 labels granule cells most effectively.