Adeno-associated virus

Genome Editing is widely used in biomedical research and medicine. Zinc finger nucleases (ZFNs) are smaller in size than transcription activator-like effector (TALE) nucleases (TALENs) and CRISPR-Cas9. Therefore, ZFN-encoding DNAs can be easily packaged into a viral vector with limited cargo space, such as adeno-associated virus (AAV) vectors, for in vivo and clinical applications. ZFNs have great potential for translational research and clinical use. However, constructing functional ZFNs and improving their genome editing efficiency is extremely difficult...

Friedreich's ataxia is a degenerative and progressive multisystem disorder caused by mutations in the highly conserved frataxin (FXN) gene that results in FXN protein deficiency and mitochondrial dysfunction. While gene therapy approaches are promising, consistent induction of therapeutic FXN protein expression that is sub-toxic has proven challenging, and numerous therapeutic approaches are being tested in animal models. FXN (hFXN in humans, mFXN in mice) is proteolytically modified in mitochondria to produce mature FXN...

Microglial polarization and associated inflammatory activity are the key mediators of depression pathogenesis. The natural Smilax glabra rhizomilax derivative engeletin has been reported to exhibit robust anti-inflammatory activity, but no studies to date have examined the mechanisms through which it can treat depressive symptoms. We showed that treatment for 21 days with engeletin significantly alleviated depressive-like behaviours in chronic stress social defeat stress (CSDS) model mice. T1-weighted imaging (T1WI), T2-weighted imaging (T2WI) imaging revealed no significant differences between groups, but the bilateral prefrontal cortex of CSDS mice exhibited significant increases in apparent diffusion coefficient and T2 values relative to normal control mice, with a corresponding reduction in fractional anisotropy, while engeletin reversed all of these changes...

BACKGROUND: MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), original found in synthetic heroin, causes Parkinson's disease (PD) in human through its metabolite MPP+ by inhibiting complex I of mitochondrial respiratory chain in dopaminergic neurons. This study explored whether yeast internal NADH-quinone oxidoreductase (NDI1) has therapeutic effects in MPTP- induced PD models by functionally compensating for the impaired complex I. MPP+ -treated SH-SY5Y cells and MPTP-treated mice were used as the PD cell culture and mouse models respectively...

Hand dysfunction is a common observation after arteriovenous fistula (AVF) creation for hemodialysis access and has a variable clinical phenotype; however, the underlying mechanism responsible is unclear. Grip strength changes are a common metric used to assess AVF-associated hand disability but has previously been found to poorly correlate with the hemodynamic perturbations post-AVF placement implicating other tissue-level factors as drivers of hand outcomes. In this study, we sought to test if expression of a mitochondrial targeted catalase (mCAT) in skeletal muscle could reduce AVF-related limb dysfunction in mice with chronic kidney disease (CKD)...

Mitochondria are important for the activation of endothelial cells and the process of angiogenesis. NDUFS8 (NADH:ubiquinone oxidoreductase core subunit S8) is a protein that plays a critical role in the function of mitochondrial Complex I. We aimed to investigate the potential involvement of NDUFS8 in angiogenesis. In human umbilical vein endothelial cells (HUVECs) and other endothelial cell types, we employed viral shRNA to silence NDUFS8 or employed the CRISPR/Cas9 method to knockout (KO) it, resulting in impaired mitochondrial functions in the endothelial cells, causing reduction in mitochondrial oxygen consumption and Complex I activity, decreased ATP production, mitochondrial depolarization, increased oxidative stress and reactive oxygen species (ROS) production, and enhanced lipid oxidation...

The US Food and Drug Administration (FDA)'s authorization of etranacogene dezaparvovec (Hemgenix) is a significant milestone, constituting not only the first FDA approval of a gene therapy for hemophilia but also the first approval of a liver-targeted adeno-associated virus vector gene therapy. This review summarizes the nonclinical studies and clinical development that supported regulatory clearance. Similar to other gene therapies for single gene disorders, both the short-term safety and the phenotypic improvement were unequivocal, justifying the modest-sized safety and efficacy database, which included 57 participants across the phase 2b (3 participants) and phase 3 (54 participants) studies...

CRISPR-Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering. The concatemeric knockins occurred regardless of locus, vector concentration, cell line or cell type, including human pluripotent and hematopoietic stem cells...

The neurotoxic α-synuclein (α-syn) oligomers play an important role in the occurrence and development of Parkinson's disease (PD), but the factors affecting α-syn generation and neurotoxicity remain unclear. We here first found that thrombomodulin (TM) significantly decreased in the plasma of PD patients and brains of A53T α-syn mice, and the increased TM in primary neurons reduced α-syn generation by inhibiting transcription factor p-c-jun production through Erk1/2 signaling pathway...

OBJECTIVES: Glucocorticoid-induced Tumor-Necrosis-Factor-Receptor Family-related Protein (GITR), with its ligand (GITRL), plays an important role in CD4+ T-cell-mediated autoimmunity. This study aimed to investigate the underlying mechanisms of GITRL in primary Sjögren's Syndrome (pSS). METHODS: pSS patients and healthy controls were recruited. Serum GITRL and Th17-related cytokines were determinated. RNA-Sequencing was performed to decipher key signal pathways...

INTRODUCTION: Combined allergic rhinitis and asthma syndrome (CARAS) is a concurrent allergic symptom of diseases of allergic rhinitis and asthma. However, the mechanism of CARAS remains unclear. The study aimed to investigate the impact of microRNA-21 (miR-21) on CARAS via targeting poly (ADP-ribose) polymerase-1 (PARP-1) and phosphoinositide 3-kinase (PI3K)/AKT pathways. METHODS: The levels of miR-21-5p and PARP-1 in CARAS patients were detected by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA)...

Hemojuvelin (HJV) is a GPI-anchored protein of the repulsive guidance molecule (RGM) family acting as a bone morphogenetic protein (BMP) co-receptor to induce the hepatic iron regulatory protein hepcidin. Hepcidin causes ubiquitination and degradation of the sole known iron exporter ferroportin thereby limiting iron availability. The detailed signaling mechanism of HJV in vivo has yet to be investigated. In the current manuscript, we used an established model of adeno-associated virus (AAV) mediated liver-specific overexpression of HJV in murine models of hepatocyte-specific deficiency of the BMP type I receptors Alk2 or Alk3...

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes an asymptomatic latent infection of sensory neurons of dorsal root ganglia (DRG). Chemical and physical stress cause intermittent virus reactivation from latently infected DRG and recurrent virus shedding in the genital mucosal epithelium causing genital herpes in symptomatic patients. While T cells appear to play a role in controlling virus reactivation from DRG and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T cells into DRG and the vaginal mucosa (VM) remain to be fully elucidated...

BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition...

Adeno-associated virus has been remarkably successful in the clinic, but its broad tropism is a practical limitation of precision gene therapy. A promising path to engineer AAV tropism is the addition of binding domains to the AAV capsid that recognize cell surface markers present on a targeted cell type. We have recently identified two previously unexplored capsid regions near the 2-fold valley and 5-fold pore of the AAV capsid that are amenable to insertion of larger protein domains including nanobodies. Here, we demonstrate that these hotspots facilitate AAV tropism switching through simple nanobody replacement without extensive optimization in both VP1 and VP2...

Genetic variations in the glucocorticoid receptor (GR) gene NR3C1 can impact metabolism. The single nucleotide polymorphism (SNP) rs6190 (p.R23K) has been associated in humans with enhanced metabolic health, but the SNP mechanism of action remains completely unknown. We generated a transgenic knock-in mice genocopying this polymorphism to elucidate how the mutant GR impacts metabolism. Compared to non-mutant littermates, mutant mice showed increased muscle insulin sensitivity and strength on regular chow and high-fat diet, blunting the diet-induced adverse effects on weight gain and exercise intolerance...

Targeting cancer cell mitochondria holds great therapeutic promise, yet current strategies to specifically and effectively destroy cancer mitochondria in vivo are limited. Here, we introduce mLumiOpto, an innovative mitochondrial-targeted luminoptogenetics gene therapy designed to directly disrupt the inner mitochondrial membrane (IMM) potential and induce cancer cell death. We synthesize a blue light-gated channelrhodopsin (CoChR) in the IMM and co-express a blue bioluminescence-emitting Nanoluciferase (NLuc) in the cytosol of the same cells...

Dystrophic cardiomyopathy is a significant feature of Duchenne muscular dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca2+ ) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and mothers against decapentaplegic homolog-7 (Smad7) proteins. In this study, we created a gene transfer using an miR-25 tough decoy (TuD) RNA inhibitor delivered via recombinant adeno-associated virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice mdx/utrn ( +/- ), a validated transgenic murine model of DMD...

Transient receptor potential melastatin 7 (TRPM7) is a cation channel that plays a role in the progression of rheumatoid arthritis (RA), yet its involvement in synovial hyperplasia and inflammation has not been determined. We previously reported that TRPM7 affects the destruction of articular cartilage in RA. Herein, we further confirmed the involvement of TRPM7 in fibroblast-like synoviocyte (FLS) proliferation, metastasis and inflammation. We observed increased TRPM7 expression in FLSs derived from human RA patients...

Recombinant adeno-associated virus (rAAV) vectors appear, more than ever, to be efficient viral vectors for in vivo gene transfer as illustrated by the approvals of 7 drugs across Europe and the USA. Nevertheless, pre-existing immunity to AAV capsid in humans remains one of the major limits for a successful clinical translation. Whereas pre-existing humoral response to AAV capsid is well documented, the prevalence of pre-existing capsid-specific T cell responses still needs to be studied and characterized. Here, we investigated the prevalence of AAV-specific circulating T cells towards AAV2, 4, 5, 8, 9 and rh10 in a large cohort of healthy donors using the standard IFNγ ELISpot assay...