Adeno-associated Virus Serotype Research Articles

Progranulin AAV gene therapy for frontotemporal dementia: translational studies and phase 1/2 trial interim results.

GRN mutations cause progranulin haploinsufficiency, which eventually leads to frontotemporal dementia (FTD-GRN). PR006 is an investigational gene therapy delivering the granulin gene (GRN) using an adeno-associated virus serotype 9 (AAV9) vector. In non-clinical studies, PR006 transduced neurons derived from induced pluripotent stem cells of patients with FTD-GRN, resulted in progranulin expression and improvement of lipofuscin, lysosomal and neuroinflammation pathologies in Grn-knockout mice, and was well tolerated except for minimal, asymptomatic dorsal root ganglionopathy in non-human primates. We initiated a first-in-human phase 1/2 open-label trial. Here we report results of a pre-specified interim analysis triggered with the last treated patient of the low-dose cohort (n = 6) reaching the 12-month follow-up timepoint. We also include preliminary data from the mid-dose cohort (n = 7). Primary endpoints were safety, immunogenicity and change in progranulin levels in cerebrospinal fluid (CSF) and blood. Secondary endpoints were Clinical Dementia Rating (CDR) plus National Alzheimer's Disease Coordinating Center (NACC) Frontotemporal Lobar Degeneration (FTLD) rating scale and levels of neurofilament light chain (NfL). One-time administration of PR006 into the cisterna magna was generally safe and well tolerated. All patients developed treatment-emergent anti-AAV9 antibodies in the CSF, but none developed anti-progranulin antibodies. CSF pleocytosis was the most common PR006-related adverse event. Twelve serious adverse events occurred, mostly unrelated to PR006. Deep vein thrombosis developed in three patients. There was one death (unrelated) occurring 18 months after treatment. CSF progranulin increased after PR006 treatment in all patients; blood progranulin increased in most patients but only transiently. NfL levels transiently increased after PR006 treatment, likely reflecting dorsal root ganglia toxicity. Progression rates, based on the CDR scale, were within the broad ranges reported for patients with FTD. These data provide preliminary insights into the safety and bioactivity of PR006. Longer follow-up and additional studies are needed to confirm the safety and potential efficacy of PR006. ClinicalTrials.gov identifier: NCT04408625 .

Direct reprogramming of fibroblasts into functional hepatocytes via CRISPRa activation of endogenous Gata4 and Foxa3.

The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments. Clustered regularly interspaced short palindromic repeats (CRISPR) activator (CRISPRa) is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously, providing a new strategy for cell fate determination. The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis. The differentiation of mouse embryonic fibroblasts (MEFs) into functional induced hepatocyte-like cells (iHeps) was achieved by utilizing the CRISPRa synergistic activation mediator (SAM) system, which drove the combined expression of three endogenous transcription factors-Gata4, Foxa3, and Hnf1a-or alternatively, the expression of two transcription factors, Gata4 and Foxa3. In vivo, we injected adeno-associated virus serotype 6 (AAV6) carrying the CRISPRa SAM system into liver fibrotic Col1a1-CreER; Cas9fl/fl mice, effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts. The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction (RT-qPCR) and RNA-seq, and the morphology and characteristics of the induced hepatocytes were observed through microscopy. The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining, while the improvement of liver fibrosis is evaluated through Sirius red staining, alpha-smooth muscle actin (α-SMA) immunofluorescence staining, and blood alanine aminotransferase (ALT) examination. Activation of only two factors, Gata4 and Foxa3, via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps. These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes, such as drug metabolism and glycogen storage. Additionally, AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis. After 8 weeks of induction, the reprogrammed hepatocytes comprised 0.87% of the total hepatocyte population in the mice, significantly reducing liver fibrosis. CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.

Characterizing Glycosylation of Adeno-Associated Virus Serotype 9 Capsid Proteins Generated from HEK293 Cells through Glycopeptide Mapping and Released Glycan Analysis

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography–tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development.

Advances in Bone-Targeting Drug Delivery: Emerging Strategies Using Adeno-Associated Virus.

The development of bone-targeting drug delivery systems holds immense promise for improving the treatment of skeletal diseases. By precisely delivering therapeutic agents to the affected areas of bone, these strategies can enhance drug efficacy, minimize off-target effects, and promote patient adherence, ultimately leading to improved treatment outcomes and an enhanced quality of life for patients. This review aims to provide an overview of the current state of affinity-based bone-targeting agents and recent breakthroughs in innovative bone-targeting adeno-associated virus (AAV) strategies to treat skeletal diseases in mice. In particular, this review will delve into advanced AAV engineering, including AAV serotype selection for bone targeting and capsid modifications for bone-specific tropism. Additionally, we will highlight recent advancements in AAV-mediated gene therapy for skeletal diseases and discuss challenges and future directions of this promising therapeutic approach.

Viral Delivery of Compact CRISPR-Cas12f for Gene Editing Applications.

Treating human genetic conditions invivo requires efficient delivery of the CRISPR gene editing machinery to the affected cells and organs. The gene editing field has seen clinical advances with ex vivo therapies and with invivo delivery to the liver using lipid nanoparticle technology. Adeno-associated virus (AAV) serotypes have been discovered and engineered to deliver genetic material to nearly every organ in the body. However, the large size of most CRISPR-Cas systems limits packaging into the viral genome and reduces drug development flexibility and manufacturing efficiency. Here, we demonstrate efficient CRISPR gene editing using a miniature CRISPR-Cas12f system with expanded genome targeting packaged into AAV particles. We identified efficient guides for four therapeutic gene targets and encoded the guides and the Cas12f nuclease into a single AAV. We then demonstrate editing in multiple cell lines, patient fibroblasts, and primary hepatocytes. We then screened the cells for off-target editing, demonstrating the safety of the therapeutics. These results represent an important step in applying CRISPR editing to diverse genetic sequences and organs in the body.

14-3-3ε/YWHAE regulates the transcriptional expression of cardiac sodium channel NaV1.5

BackgroundCardiac sodium channel NaV1.5 encoded by SCN5A is associated with arrhythmia disorders. However, the molecular mechanism underlying NaV1.5 expression remains to be fully elucidated. Previous studies have reported that 14-3-3 family acts as an adaptor involved in regulating kinetic characteristics of cardiac ion channels. ObjectiveThe purpose of this study was to establish 14-3-3ε/YWHAE, a member of 14-3-3 family, as a crucial regulator of NaV1.5 and explore the potential role of 14-3-3ε in the heart. MethodsWestern blotting, patch-clamping, real-time RT-PCR, RNA immunoprecipitation, electrocardiogram recording, echocardiography and histological analysis were performed. ResultsYWHAE overexpression significantly reduced the expression level of SCN5A mRNA and sodium current density, whereas YWHAE knockdown significantly increased SCN5A mRNA expression and sodium current density in HEK293/NaV1.5 and H9c2 cells. Similar results were observed in mice injected with adeno-associated virus serotype 9 (AAV9)-mediated YWHAE knockdown. The effect of 14-3-3ε on NaV1.5 expression was abrogated by knockdown of TBX5, a transcription factor of NaV1.5. An interaction between 14-3-3ε protein and TBX5 mRNA was identified, and YWHAE overexpression significantly decreased TBX5 mRNA stability without affecting SCN5A mRNA stability. Additionally, mice subjected to AAV9-mediated YWHAE knockdown exhibited shorter R-R intervals and higher prevalence of premature ventricular contractions. ConclusionOur data unveil a novel regulatory mechanism of NaV1.5 by 14-3-3ε, and highlight the significance of 14-3-3ε in transcriptional regulation of NaV1.5 expression and cardiac arrhythmias.

Prenatal AAV9-GFP administration in fetal lambs results in transduction of female germ cells and maternal exposure to virus

Prenatal somatic cell gene therapy (PSCGT) could potentially treat severe, early-onset genetic disorders such as spinal muscular atrophy (SMA) or muscular dystrophy. Given the FDA-approval of adeno-associated virus serotype 9 (AAV9) vectors in infants with SMA, we tested the safety and biodistribution of AAV9-GFP (clinical-grade and dose) in fetal lambs to understand safety and efficacy after umbilical vein (UV) or intracranial (IC) injection on embryonic day 75 (E75). UV-injection led to widespread biodistribution of vector genomes in all examined lamb tissues and in maternal uteruses at harvest (E96 or E140; term=E150). There was robust GFP expression in brain, spinal cord, dorsal root ganglia (DRGs), without DRG toxicity and excellent transduction of diaphragm and quadriceps muscles. However, we found evidence of systemic toxicity (fetal growth restriction) and maternal exposure to the viral vector (transient elevation of total bilirubin and a trend towards elevation in anti-AAV9 antibodies). There were no antibodies against GFP in ewes or lambs. Analysis of fetal gonads demonstrated GFP expression in female (but not male) germ cells, with low levels of integration-specific reads, without integration in select protooncogenes. These results suggest potential therapeutic benefit of AAV9 PSCGT for neuromuscular disorders but warrant caution for exposure of female germ cells.

Noninvasive Focused Ultrasound-Mediated Delivery of rAAV9-EGFP Vectors for Neuronal Targeting in Rats

Objective: To evaluate the synergistic potential of Focused Ultrasound (FUS) in conjunction with microbubbles (MB) and recombinant adeno-associated virus serotype 9 (rAAV9) vectors for targeted gene delivery to neuronal cells in rats, optimizing gene expression conditions and assessing any adverse effects.Methods: The parameters for permeability enhancement of the rat's blood-brain barrier (BBB) were established using FUS+MB, with MRI scans and Evans Blue (EB) dye assisting in the evaluation. Rats underwent FUS-mediated transfection using rAAV9-Syn-EGFP vectors produced via a triple-transfection in HEK293T cells. Following this, the uptake and expression of GFP in targeted brain regions were evaluated using confocal fluorescence microscopy at various time intervals. Inflammatory responses post-FUS treatment were tracked by observing levels of GFAP, a marker for astrocytic activation, and TNF-α, a pro-inflammatory cytokine. Motor behavior effects post-intervention were gauged using the Rotarod test across multiple groups over a span of four weeks.Results: FUS+MB affected BBB permeability, with optimal results at 4 W for 200 seconds showing 85% permeability and evident Gd-DTPA leakage. Settings beyond these resulted in tissue damage. Control groups exhibited a basal GFP expression of 2% ± 0.5%, whereas FUS+MB with rAAV-EGFP injections substantially increased GFP expression to about 67% ± 6% in targeted neurons. This GFP expression peaked at three weeks post-treatment and remained evident six months later. Following FUS treatment, both GFAP and TNF-α levels underwent fluctuations before eventually nearing their baseline values. The Rotarod test revealed no significant behavioral differences post-treatments among the groups.Conclusions: Combining FUS+MB with rAAV offers an innovative approach to enhance therapeutic delivery to the central nervous system (CNS) by transiently adjusting BBB permeability.

YBX1 inhibits mitochondrial-mediated apoptosis in ischemic heart through the PI3K/AKT signaling pathway

Abstract Background Myocardial infarction (MI) is associated with higher morbidity and mortality in the world, especially in cold weather. YBX1 is an RNA-binding protein that is required for pathological growth of cardiomyocyte by regulating cell growth and protein synthesis. But YBX1, as an individual RNA-binding protein, regulates cardiomyocytes through signaling cascades during myocardial infarction remain largely unexplored. Methods In vivo, the mouse MI model was induced by ligating the left anterior descending coronary artery (LAD), and randomly divided into sham operation group, MI group, MI+ YBX1 knockdown/overexpression group and MI+ negative control (NC) group. The protective effect of YBX1 was verified by echocardiography and triphenyltetrazolium chloride staining. In vitro, mitochondrial-dependent apoptosis was investigated by using CCK8, TUNEL staining, reactive oxygen species (ROS) staining and JC-1 staining in hypoxic neonatal mouse cardiomyocytes (NMCMs). Results YBX1 expression of cardiomyocytes was downregulated in a mouse model and a cellular model on the ischemic condition. Compared to mice induced by MI, YBX1 overexpression mediated by adeno-associated virus serotype 9 (AAV9) vector reduced the infarcted size and improved cardiac function. Knockdown of endogenous YBX1 by shRNA partially aggravated ischemia-induced cardiac dysfunction. In hypoxic cardiomyocytes, YBX1 overexpression decreased lactic dehydrogenase (LDH) release, increased cell viability, and inhibited apoptosis by affecting the expression of apoptosis related proteins, while knockdown of endogenous YBX1 by siRNA had the opposite effect. Overexpression of YBX1 restored mitochondrial dysfunction in hypoxic NMCMs by increasing mitochondrial membrane potential and ATP content and decreasing ROS. In hypoxic NMCMs, YBX1 overexpression increased the expression of phosphorylated phosphatidylinositol 3 kinase (PI3K)/AKT, and the antiapoptosis effect of YBX1 was eliminated t by LY294002, PI3K/AKT inhibitor. Conclusion YBX1 protected the heart from ischemic damage by inhibiting the mitochondrial-dependent apoptosis through PI3K/AKT pathway. It is anticipated that YBX1 may serve as a novel therapeutic target for MI.

Spatiotemporal ATF3 Expression Determines VSMC Fate in Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy, likely due to the limited understanding of the mechanisms underlying AAA development and progression. Activating transcription factor (ATF) 3 has been increasingly recognized as a key regulator of cardiovascular diseases. However, the role of ATF3 (activating transcription factor 3) in AAA development and progression remains elusive. Genome-wide RNA sequencing analysis was performed on the aorta isolated from saline or Ang II (angiotensin II)-induced AAA mice, and ATF3 was identified as the potential key gene for AAA development. To examine the role of ATF3 in AAA development, vascular smooth muscle cell-specific ATF3 knockdown or overexpressed mice by recombinant adenoassociated virus serotype 9 vectors carrying ATF3, or shRNA-ATF3 with SM22α (smooth muscle protein 22-α) promoter were used in Ang II (angiotensin II)-induced AAA mice. In human and murine vascular smooth muscle cells, gain or loss of function experiments were performed to investigate the role of ATF3 in vascular smooth muscle cell proliferation and apoptosis. In both Ang II-induced AAA mice and patients with AAA, the expression of ATF3 was reduced in aneurysm tissues but increased in aortic lesion tissues. The deficiency of ATF3 in vascular smooth muscle cell promoted AAA formation in Ang II-induced AAA mice. PDGFRB (platelet-derived growth factor receptor β) was identified as the target of ATF3, which mediated vascular smooth muscle cell proliferation in response to TNF-alpha (tumor necrosis factor-α) at the early stage of AAA. ATF3 suppressed the mitochondria-dependent apoptosis at the advanced stage by upregulating its direct target BCL2. Our chromatin immunoprecipitation results also demonstrated that the recruitment of NFκB1 and P300/BAF/H3K27ac complex to the ATF3 promoter induces ATF3 transcription via enhancer activation. NFKB1 inhibitor (andrographolide) inhibits the expression of ATF3 by blocking the recruiters NFKB1 and ATF3-enhancer to the ATF3-promoter region, ultimately leading to AAA development. Our results demonstrate a previously unrecognized role of ATF3 in AAA development and progression, and ATF3 may serve as a novel therapeutic and prognostic marker for AAA.

Hepatocyte-Specific Fads1 Overexpression Attenuates Western Diet-Induced Metabolic Phenotypes in a Rat Model.

Fatty acid desaturase 1 (FADS1) is a rate-limiting enzyme in long-chain polyunsaturated fatty acid (LCPUFA) synthesis. Reduced activity of FADS1 was observed in metabolic dysfunction-associated steatotic liver disease (MASLD). The aim of this study was to determine whether adeno-associated virus serotype 8 (AAV8) mediated hepatocyte-specific overexpression of Fads1 (AAV8-Fads1) attenuates western diet-induced metabolic phenotypes in a rat model. Male weanling Sprague-Dawley rats were fed with a chow diet, or low-fat high-fructose (LFHFr) or high-fat high-fructose diet (HFHFr) ad libitum for 8 weeks. Metabolic phenotypes were evaluated at the endpoint. AAV8-Fads1 injection restored hepatic FADS1 protein levels in both LFHFr and HFHFr-fed rats. While AAV8-Fads1 injection led to improved glucose tolerance and insulin signaling in LFHFr-fed rats, it significantly reduced plasma triglyceride (by ~50%) and hepatic cholesterol levels (by ~25%) in HFHFr-fed rats. Hepatic lipidomics analysis showed that FADS1 activity was rescued by AAV8-FADS1 in HFHFr-fed rats, as shown by the restored arachidonic acid (AA)/dihomo-γ-linolenic acid (DGLA) ratio, and that was associated with reduced monounsaturated fatty acid (MUFA). Our data suggest that the beneficial role of AAV8-Fads1 is likely mediated by the inhibition of fatty acid re-esterification. FADS1 is a promising therapeutic target for MASLD in a diet-dependent manner.

BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts

Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the aging phenotype. Our data indicate that age-associated overexpression of BIN1 occurs alongside dysregulated endosomal recycling and disrupted trafficking of cardiac CaV1.2 and type 2 ryanodine receptors. These deficiencies affect channel function at rest and their upregulation during acute stress. In vivo echocardiography reveals reduced systolic function in old mice. BIN1 knockdown using an adeno-associated virus serotype 9 packaged shRNA-mBIN1 restores the nanoscale distribution and clustering plasticity of ryanodine receptors and recovers Ca2+ transient amplitudes and cardiac systolic function toward youthful levels. Enhanced systolic function correlates with increased phosphorylation of the myofilament protein cardiac myosin binding protein-C. These results reveal BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium.

Imaging of adeno-associated viral capsids for purposes of gene editing using CEST NMR/MRI.

Gene therapy using adeno-associated virus (AAV) vector-mediated gene delivery has undergone substantial growth in recent years with promising results in both preclinical and clinical studies, as well as emerging regulatory approval. However, the inability to quantify the efficacy of gene therapy from cellular delivery of gene-editing technology to specific functional outcomes is an obstacle for efficient development of gene therapy treatments. Building on prior works that used the CEST reporter gene lysine rich protein, we hypothesized that AAV viral capsids may generate endogenous CEST contrast from an abundance of surface lysine residues. NMR experiments were performed on isolated solutions of AAV serotypes 1-9 on a Bruker 800-MHz vertical scanner. In vitro experiments were performed for testing of CEST-NMR contrast of AAV2 capsids under varying pH, density, biological transduction stage, and across multiple serotypes and mixed biological media. Reverse transcriptase-polymerase chain reaction was used to quantify virus concentration. Subsequent experiments at 7 T optimized CEST saturation schemes for AAV contrast detection and detected AAV2 particles encapsulated in a biocompatible hydrogel administered in the hind limb of mice. CEST-NMR experiments revealed CEST contrast up to 52% for AAV2 viral capsids between 0.6 and 0.8 ppm. CEST contrast generated by AAV2 demonstrated high levels of CEST contrast across a variety of chemical environments, concentrations, and saturation schemes. AAV2 CEST contrast displayed significant positive correlations with capsid density (R2 > 0.99, p < 0.001), pH (R2 = 0.97, p = 0.01), and viral titer per cell count (R2 = 0.92, p < 0.001). Transition to a preclinical field strength yielded up to 11.8% CEST contrast following optimization of saturation parameters. In vivo detection revealed statistically significant molecular contrast between viral and empty hydrogels using both mean values (4.67 ± 0.75% AAV2 vs. 3.47 ± 0.87% empty hydrogel, p = 0.02) and quantile analysis. AAV2 viral capsids exhibit strong capacity as an endogenous CEST contrast agent and can potentially be used for monitoring and evaluation of AAV vector-mediated gene therapy protocols.

Isthmin-1 alleviates cardiac ischemia/reperfusion injury through cGMP-PKG signaling pathway.

Ischemia/reperfusion (I/R) injury is an important complication of reperfusion therapy for acute myocardial infarction, extremely compromising the cardiac benefits of revascularization, however, specific and efficient treatment for cardiac I/R injury is still lacking. Isthmin-1 (ISM1) is a novel adipokine, and plays indispensable roles in regulating glycolipid metabolism and cell survival. The present study aims to investigate the potential role and molecular mechanism of ISM1 in cardiac I/R injury using gain- and loss-of-function approaches. Cardiac-specific ISM1 overexpression and silence were achieved using an adeno-associated virus serotype 9 system, and then these mice were subjected to I/R surgery, followed by biochemical test, echocardiography and histopathologic examinations, etc. Meanwhile, neonatal rat cardiomyocytes (NRCMs) with ISM1 silence or overexpression also received simulated I/R (sI/R) injury to further verify its role in vitro. The potential downstream pathways and molecular targets of ISM1 were screened by RNA-sequencing. We also treated injured mice and NRCMs with recombinant ISM1 (rISM1) to explore whether supplementation with ISM1 was sufficient to protect against I/R injury. Furthermore, acute myocardial infarction patients with percutaneous coronary intervention (PCI) and paired healthy controls were included to reveal the clinical relevance of circulating ISM1. Cardiac-specific ISM1 silencing aggravated while ISM1 overexpression alleviated I/R-induced acute cardiac injury and cardiac remodeling and dysfunction. Mechanistically, ISM1 targeted αvβ5 integrin to facilitate the nuclear accumulation of nuclear transcription factor Y subunit alpha, transcriptionally increased soluble guanylyl cyclase beta subunit expression, and eventually enhanced cGMP generation. Besides, we confirmed that treatment with rISM1 before or after reperfusion could confer cardioprotective effects in mice. Clinically, lower ISM1 levels post-PCI was associated with worse outcome in patients. ISM1 can protect against cardiac I/R injury through cGMP-PKG signaling pathway, and it is a promising therapeutic and predictive target of cardiac I/R injury.

TRPC4 aggravates hypoxic pulmonary hypertension by promoting pulmonary endothelial cell apoptosis

Pulmonary hypertension (PH) is a devastating disease that lacks effective treatment options and is characterized by severe pulmonary vascular remodeling. Pulmonary arterial endothelial cell (PAEC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension. Canonical transient receptor potential (TRPC) channels, a family of Ca2+-permeable channels, play an important role in various diseases. However, the effect and mechanism of TRPCs on PH development have not been fully elucidated. Among the TRPC family members, TRPC4 expression was markedly upregulated in PAECs from hypoxia combined with SU5416 (HySu)-induced PH mice and monocrotaline (MCT)-treated PH rats, as well as in hypoxia-exposed PAECs, suggesting that TRPC4 in PAECs may participate in the occurrence and development of PH. In this study, we aimed to investigate whether TRPC4 in PAECs has an aggravating effect on PH and elucidate the molecular mechanisms. We observed that hypoxia treatment promoted PAEC apoptosis through a caspase-12/endoplasmic reticulum stress (ERS)-dependent pathway. Knockdown of TRPC4 attenuated hypoxia-induced apoptosis and caspase-3/caspase-12 activity in PAECs. Accordingly, adeno-associated virus (AAV) serotype 6-mediated pulmonary endothelial TRPC4 silencing (AAV6-Tie-shRNA-TRPC4) or TRPC4 antagonist suppressed PH progression as evidenced by reduced right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, PAEC apoptosis and reactive oxygen species (ROS) production. Mechanistically, unbiased RNA sequencing (RNA-seq) suggested that TRPC4 deficiency suppressed the expression of the proapoptotic protein sushi domain containing 2 (Susd2) in hypoxia-exposed mouse PAECs. Moreover, TRPC4 activated hypoxia-induced PAEC apoptosis by promoting Susd2 expression. Therefore, inhibiting TRPC4 ameliorated PAEC apoptosis and hypoxic PH in animals by repressing Susd2 signaling, which may serve as a therapeutic target for the management of PH.

Higher-Order Structure of Adeno-Associated Virus Serotype 8 by Hydrogen/Deuterium Exchange Mass Spectrometry.

The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids. We obtained 486 peptides representing 85% sequence coverage. Surprisingly, the VP1u region showed rapid deuterium uptake even though this region contains the phospholipase A2 domain composed primarily of α-helices. The comparison of deuterium uptake between full and empty capsids showed significant protection from hydrogen/deuterium exchange in the full capsid at the channel structure of the 5-fold symmetry axis. This corresponds to cryo-electron microscopy studies in which the extended densities were observed only in the full capsid. In addition, deuterium uptake was reduced in the VP1u region of the full capsid, suggesting the folding and/or interaction of this region with the encapsidated genome. This study demonstrated HDX-MS as a powerful method for probing the structure of the entire rAAV capsid.

Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors.

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. Although these highly abundant errors were found in more than half of the edited cells, they could not be readily detected by common analytical methods. We describe strategies to detect and thoroughly characterize the concatemeric viral vector insertions, and we highlight analytical pitfalls that mask their prevalence. We then describe strategies to prevent the concatemeric inserts by cutting the vector genome after transduction. This approach is compatible with established gene editing pipelines, enabling robust genetic knockins that are safer, more reliable and more reproducible.

Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation.

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. Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

Isolation of Adeno-associated Viral Vectors Through a Single-step and Semi-automated Heparin Affinity Chromatography Protocol.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs. This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

LRP1 facilitates hepatic glycogenesis by improving the insulin signaling pathway in HFD-fed mice.

LDL receptor-related protein-1 (LRP1) is a cell-surface receptor that functions in diverse physiological pathways. We previously demonstrated that hepatocyte-specific LRP1 deficiency (hLRP1KO) promotes diet-induced insulin resistance and increases hepatic gluconeogenesis in mice. However, it remains unclear whether LRP1 regulates hepatic glycogenesis. Insulin signaling, glycogenic gene expression, and glycogen content were assessed in mice and HepG2 cells. The pcDNA 3.1 plasmid and adeno-associated virus serotype 8 vector (AAV8) were used to overexpress the truncated β-chain (β∆) of LRP1 both invitro and invivo. On a normal chow diet, hLRP1KO mice exhibited impaired insulin signaling and decreased glycogen content. Moreover, LRP1 expression in HepG2 cells was significantly repressed by palmitate in a dose- and time-dependent manner. Both LRP1 knockdown and palmitate treatment led to reduced phosphorylation of Akt and GSK3β, increased levels of phosphorylated glycogen synthase (GYS), and diminished glycogen synthesis in insulin-stimulated HepG2 cells, which was restored by exogenous expression of the β∆-chain. By contrast, AAV8-mediated hepatic β∆-chain overexpression significantly improved the insulin signaling pathway, thus activating glycogenesis and enhancing glycogen storage in the livers of high-fat diet (HFD)-fed mice. Our data revealed that LRP1, especially its β-chain, facilitates hepatic glycogenesis by improving the insulin signaling pathway, suggesting a new therapeutic strategy for hepatic insulin resistance-related diseases.