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Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy. Adeno-associated virus is the basis of many gene therapies and gene transfer vectors. Here the authors report a pipeline to enable side-by-side comparison of pre-selected capsids in a high throughput manner.

Pathological remodeling of the extracellular matrix is a hallmark of cardiovascular disease. Abnormal fibrosis causes cardiac dysfunction by reducing ejection fraction and impairing electrical conductance, leading to arrhythmias. Hence, accurate quantification of fibrosis deposition in histological sections is of extreme importance for preclinical and clinical studies. Current automatic tools do not perform well under variant conditions. Moreover, users do not have the option to evaluate data from staining methods of their choice according to their purpose. To overcome these challenges, we underline a novel machine learning-based tool (FibroSoft) and we show its feasibility in a model of cardiac hypertrophy and heart failure in mice. Our results demonstrate that FibroSoft can identify fibrosis in diseased myocardium and the obtained results are user-independent. In addition, the results acquired using our software strongly correlate to those obtained by Western blot analysis of collagen 1 expression. Additionally, we could show that this method can be used for Masson’s Trichrome and Picosirius Red stained histological images. The evaluation of our method also indicates that it can be used for any particular histology segmentation and quantification. In conclusion, our approach provides a powerful example of the feasibility of machine learning strategies to enable automatic analysis of histological images.

Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach.

Marfan syndrome (MFS) is a connective tissue disorder caused by FBN1 mutations, characterised by aortic aneurysms leading to life-threatening dissections. Asprosin is the C-terminal propeptide of fibrillin-1 that was shown to function as a glucogenic hormone with critical implications in vascular pathology. However, its role in MFS remains unknown. Here, we investigate the effect of asprosin overexpression in vascular smooth muscle cells (VSMCs) and in the Marfan mouse model mgR/mgR via endothelial-targeted transduction by adeno-associated viruses (AAVs). In both human and murine VSMCs, asprosin overexpression did not alter VCAM1 or MMP9 expression, while TNF-α-stimulated asprosin-overexpressing MOVAS cells exhibited reduced IL6 levels. In mgR/mgR mice, asprosin overexpression did not affect aortic diameter or elastin integrity. Molecular analyses revealed no significant change in inflammatory or epithelial-to-mesenchymal transition (EMT) markers at the mRNA or protein level, with VCAM1 and MMP9 remaining unchanged. Together, these findings indicate that asprosin overexpression does not ameliorate the aneurysmal phenotype in MFS.

Background Parkinson’s disease is a complex, multifactorial neurodegenerative disorder characterized by aggregation of α-Synuclein into Lewy bodies, with phosphorylation at serine 129 (pSer129), serving as a key regulatory site and pathological hallmark. However, the exact mechanisms by which environmental triggers lead to this disease phenotype remain poorly understood. In this study, we investigate the impact of representative infectious and pesticide exposures on pSer129 α-Synuclein, with a particular focus on the role of cellular kinases in mediating this process. Methods Neuronal cells were exposed to two distinct environmental stressors: the pesticide rotenone and the well-characterized gastric bacterium Helicobacter pylori (H. pylori) . Phosphorylation of Ser129 α-Synuclein and mitochondrial damage were assessed by immunofluorescence staining or Western blotting. To investigate the involvement of c-Abl, cells were treated with mechanistically distinct c-Abl inhibitors and siRNA. Levels of pSer129 α-Synuclein were quantified by Western blotting, while the activities of the upstream serine/threonine kinase were predicted by kinase profiling and validated by Western blotting. Additionally, transcriptome analyses of treated cells were performed and ingenuity pathway analysis and DESeq2 were applied to identify neurodegenerative pathways affected by the infection/treatment. Results The functional analysis of our RNA-sequencing data revealed that both H. pylori and rotenone induce neuroinflammatory and cellular stress response pathways. Although they likely activate c-Abl through distinct upstream mediators, both triggers ultimately promote α-synuclein phosphorylation. Treatment with the c-Abl inhibitors, Ponatinib and Asciminib, effectively prevented the accumulation of pSer129 α-synuclein and reversed the associated gene expression changes induced by H. pylori or rotenone. Additionally, GSK3β has been identified as a contributor to Ser129 phosphorylation occurring downstream of activated c-Abl signaling. Notably, the vacuolating cytotoxin (VacA) produced by H. pylori appears to play a critical role in c-Abl–mediated phosphorylation of α-synuclein at Ser129. Conclusions These findings highlight the pivotal role of c-Abl in α-Synucleinopathies and provide insights into shared mechanisms between infection and pesticide exposure, offering potential therapeutic targets for Parkinson’s disease and related pathologies involving α-Synuclein modification.

Marfan syndrome is characterized by high expression of matrix metalloproteinases (MMPs) in aortic smooth muscle cells (AoSMCs) associated with medial elastolysis and aortic root aneurysm. We aimed to reduce aortic elastolysis through decrease of MMP expression with decoy oligodeoxynucleotides (dODNs) neutralizing the transcription factor activating factor-1 (AP-1). AP-1 abundance in nuclear extracts as well as MMP-2 and MMP-9 expression were significantly increased in isolated mAoSMC of mgR/mgR Marfan mice compared to wild-type cells. Exposure to AP-1 neutralizing dODNs resulted in a significant reduction of basal and interleukin-1β-stimulated MMP expression and activity in mAoSMCs. Moreover, increased migration and formation of superoxide radical anions was substantially decreased in mAoSMCs by AP-1 dODN treatment. Aortic grafts from donor Marfan mice were treated with AP-1- dODN ex vivo and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Pretreatment of aortic grafts with AP-1 dODN led to reduced elastolysis, macrophage infiltration, and MMP activity. Permeability of the endothelial monolayer was increased for dODN in mgR/mgR aortae with observed loss of tight junction proteins ZO-1 and occludin, enabling dODN to reach the tunica media. Targeting AP-1 activity offers a new potential strategy to treat the vascular phenotype associated with Marfan syndrome.

Transplant vasculopathy (TV), characterized by obstructive lesions in affected vessels, represents one of the long-term complications of cardiac transplantation. Activation of the transcription factor activator protein-1 (AP-1) is implicated in smooth muscle cell (SMC) phenotypic switch from contractile to synthetic function, increasing the migration and proliferation rate of these cells. We hypothesize that adeno-associated virus (AAV)-mediated delivery of an RNA hairpin AP-1 decoy oligonucleotide (dON) might effectively ameliorate TV severity in a mouse aortic allograft model. Aortic allografts from DBA/2 mice ex vivo transduced with modified AAV9-SLR carrying a targeting peptide within the capsid surface were transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg BW) was administered daily. AP-1 dONs were intracellularly expressed in the graft tissue as small hairpin RNA proved by fluorescent in situ hybridization. Explantation after 30 days and histomorphometric evaluation revealed that AP-1 dON treatment significantly reduced intima-to-media ratio by 41.5% (p < 0.05) in the grafts. In addition, expression of adhesion molecules, cytokines, as well as numbers of proliferative SMCs, matrix metalloproteinase-9-positive cells, and inflammatory cell infiltration were significantly decreased in treated aortic grafts. Our findings demonstrate the feasibility, efficacy, and specificity of the anti-AP-1 RNA dON approach for the treatment of allograft vasculopathy in an animal model. Moreover, the AAV-based approach in general provides the possibility to achieve a prolonged delivery of nucleic-acids-based therapeutics in to the blood vessel wall.

Targeted gene delivery to vascular smooth muscle cells (VSMCs) could prevent or improve a variety of diseases affecting the vasculature and particularly the aorta. Thus, we aimed to develop a delivery vector that efficiently targets VSMCs. We selected engineered adeno-associated virus (AAV) capsids from a random AAV capsid library and tested the top enriched motifs in parallel screening through individual barcoding. This approach allowed us to distinguish capsids that only transduce cells based on genomic DNA (gDNA) from those also mediating transgene expression based on transcribed cDNA reads. After three rounds of selection on primary murine VSMCs (mVSMCs), we identified a novel targeting motif (RFTEKPA) that significantly improved transduction and gene expression efficiency over AAV9-wild type (WT) and increased expression in mVSMCs by 70% compared to the previously identified SLRSPPS peptide. Further analysis showed that the novel motif also improved expression in human aortic smooth muscle cells (HAoSMCs) and human aortic tissue ex vivo up to threefold compared to SLRSPPS and approximately 70-fold to AAV9-WT. This high cross-species transduction efficiency makes the novel capsid motif a potential candidate for future clinical application in vascular diseases.

Parkinson's disease is a complex, multifactorial neurodegenerative disorder characterized by aggregation of [alpha]-Synuclein into Lewy bodies, with phosphorylation at serine 129 (pSer129), serving as a key regulatory site and pathological hallmark. However, the exact mechanisms by which environmental triggers lead to this disease phenotype remain poorly understood. In this study, we investigate the impact of representative infectious and pesticide exposures on pSer129 [alpha]-Synuclein, with a particular focus on the role of cellular kinases in mediating this process. Neuronal cells were exposed to two distinct environmental stressors: the pesticide rotenone and the well-characterized gastric bacterium Helicobacter pylori (H. pylori). Phosphorylation of Ser129 [alpha]-Synuclein and mitochondrial damage were assessed by immunofluorescence staining or Western blotting. To investigate the involvement of c-Abl, cells were treated with mechanistically distinct c-Abl inhibitors and siRNA. Levels of pSer129 [alpha]-Synuclein were quantified by Western blotting, while the activities of the upstream serine/threonine kinase were predicted by kinase profiling and validated by Western blotting. Additionally, transcriptome analyses of treated cells were performed and ingenuity pathway analysis and DESeq2 were applied to identify neurodegenerative pathways affected by the infection/treatment. The functional analysis of our RNA-sequencing data revealed that both H. pylori and rotenone induce neuroinflammatory and cellular stress response pathways. Although they likely activate c-Abl through distinct upstream mediators, both triggers ultimately promote [alpha]-synuclein phosphorylation. Treatment with the c-Abl inhibitors, Ponatinib and Asciminib, effectively prevented the accumulation of pSer129 [alpha]-synuclein and reversed the associated gene expression changes induced by H. pylori or rotenone. Additionally, GSK3[beta] has been identified as a contributor to Ser129 phosphorylation occurring downstream of activated c-Abl signaling. Notably, the vacuolating cytotoxin (VacA) produced by H. pylori appears to play a critical role in c-Abl-mediated phosphorylation of [alpha]-synuclein at Ser129. These findings highlight the pivotal role of c-Abl in [alpha]-Synucleinopathies and provide insights into shared mechanisms between infection and pesticide exposure, offering potential therapeutic targets for Parkinson's disease and related pathologies involving [alpha]-Synuclein modification.

Marfan syndrome (MFS) is one of the most common inherited disorders of connective tissue caused by mutations of the fibrillin-1 gene (FBN1). Vascular abnormalities, such as the enlargement of the aorta with the risk of life-threatening rupture are frequently observed. However, current treatment is limited and therapeutic options focus solely on symptomatic therapy. Gene therapy focuses on genetically modifying cells to produce a therapeutic effect and may be a promising treatment option for MFS. Here, we first provide an overview of the historical background and characterization of MFS. Subsequently, we summarise current gene therapy options and possible translational concepts for this inherited disorder that affects connective tissue.