Adeno-associated Virus Serotype Research Articles

Serotype-agnostic affinity purification of adeno-associated virus (AAV) via peptide-functionalized chromatographic resins

Adeno-associated viruses (AAVs) have emerged as a prominent family of vectors for gene delivery, providing therapeutic options to diseases once deemed incurable. At the same time, they necessitate efficient and affordable purification methods that can be platformed to serve all AAV serotypes. Current chromatographic tools, while affording high product purity, fail to bind certain serotypes, provide limited yield and lifetime, and impose harsh elution conditions that can compromise the vector's activity and safety. Addressing these challenges, this work demonstrates the application of new peptide ligands as the first serotype-agnostic technology for AAV purification by affinity chromatography. Our study reveals a pH-dependent affinity interaction: AAV2, AAV3, AAV6, AAV9, and AAVrh.10 are effectively captured at neutral pH, while binding AAV1, AAV5, AAV7, and AAV8 is stronger in a slightly acidic environment. The elution of bound AAVs was achieved using magnesium chloride at neutral pH for all serotypes, consistently affording capsid yields above 50% and genome yields above 80%, together with a >100-fold reduction in host cell proteins and nucleic acids. In particular, peptide ligand A10 exhibited remarkable binding capacity (> 1014 vp per mL of resin) and purification performance for all AAV serotypes, demonstrating broad applicability for gene therapy manufacturing. Finally, this work introduces novel alkaline-stable variants of A10 and demonstrates their use as the first affinity ligands capable of performing multiple cycles of AAV2, AAV8, and AAV9 purification with intermediate caustic cleaning without loss of capacity or product quality. Collectively, these results demonstrate the promise of this technology to further the impact and affordability of gene therapy.

KIAA1199/CEMIP knockdown attenuates cardiac remodeling post myocardial infarction by activating TSP4 pathway in mice

BackgroundExcessive activation of cardiac fibroblasts (CFs) significantly contributes to adverse cardiac remodeling post-myocardial infarction (MI). CEMIP, initially recognized as an enzyme involved in hyaluronic acid (HA) degradation, has also been implicated in the activation of pulmonary fibroblasts. Nevertheless, the role and mechanism of CEMIP in adverse cardiac remodeling following MI remain largely unexplored. Materials and methodsRNA sequencing (RNA-seq) was performed on cardiac tissue harvested from the infarct/peri-infarct region of mice 28 days post-MI. RNA-seq was conducted on primary cardiac fibroblasts (CFs) transfected with adenovirus overexpressing CEMIP. Adeno-associated virus serotype 9 (AAV9) was engineered for in vivo CEMIP knockdown to elucidate its impact on cardiac remodeling. Immunoprecipitation coupled with mass spectrometry (IP-MS) and co-immunoprecipitation (co-IP) were employed to elucidate the mechanism by which CEMIP affected cardiac remodeling. Key findingsRNA-seq of fibrotic heart tissue at day 28 post-MI revealed a significant upregulation of CEMIP. In vitro, CEMIP facilitated the activation of cardiac fibroblasts. In vivo, knockdown of CEMIP markedly reduced cardiac fibrosis and improved cardiac function post-MI. IP-MS and co-immunoprecipitation (co-IP) confirmed that CEMIP interacted with TSP4 through the G8 domain. Further experiments confirmed that CEMIP promoted TSP4 degradation in lysosomes in an ACTN4-dependent manner, thereby activating the FAK signaling pathway. SignificanceOur findings suggest that CEMIP significantly contributes to cardiac remodeling post-MI, which might be a novel approach for treating cardiac fibrosis following MI.

NLRP3 inflammasome inhibition decreases Schistosomiasis japonica-induced granulomatous inflammation and fibrosis in BALB/c mice.

To research the role of the NLRP3 inflammasome in Schistosoma japonicum-induced granuloma formation and liver fibrosis. In in vivo tests, BALB/c mice were used. shNLRP3 plasmid based on adeno-associated virus serotype 8 (AAV8-shNLRP3) was injected to block NLRP3 inflammasome via tail vein. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected to assess liver injury. H&E staining was used for routine histopathological assessment; Masson's trichrome staining was used to detect fibrous tissues and collagen fibers. Hepatic expression of NLRP3, procaspase-1, bioactive caspase-1, collagen-1, tissue inhibitor of metalloproteinases-1 (TIMP-1), and α-smooth muscle actin (α-SMA) were detected by western blot. Serum levels of IL-1β were detected by enzyme-linked immunosorbent assay (ELISA). The inflammatory cell infiltration and hepatic expression of IL-1β around the granuloma were detected by immunohistochemistry staining. Treatment of S. japonicum infected mice with AAV8-shNLRP3 significantly reduced the hepatic levels of bioactive caspase-1 and IL-1β, as well as circulating IL-1β concentrations, while reducing the amounts of myeloperoxidase (MPO) and F4/80 positive cells around the granuloma. Moreover, collagen deposition, TIMP-1, and α-SMA, which are markers of hepatic stellate cell (HSC) activation, were reduced around the liver granuloma. These findings highlight a therapeutic potential of AAV8-shNLRP3 in schistosomiasis cirrhosis.

Exon 1-targeting miRNA reduces the pathogenic exon 1 HTT protein in Huntington disease models.

Huntington disease (HD) is a fatal neurodegenerative disease caused by a trinucleotide repeat expansion in exon 1 of the huntingtin gene (HTT) resulting in toxic gain-of-function and cell death. Despite its monogenic cause, the pathogenesis of HD is highly complex and increasing evidence indicates that, in addition to the full-length (FL) mutant HTT protein, the expanded exon 1 HTT (HTTexon1) protein that is translated from the HTT1a transcript generated by aberrant splicing is prone to aggregate and may contribute to HD pathology. This finding suggests that reducing the expression of HTT1a may achieve a greater therapeutic benefit than targeting only FL mutant HTT. Conversely, strategies that exclusively target FL HTT may not fully prevent the pathogenesis of HD. We have developed an engineered microRNA targeting the HTT exon 1 sequence (miHTT), delivered via adeno-associated virus serotype 5 (AAV5). The target sequence of miHTT is present in both FL HTT and HTT1a transcripts. Preclinical studies with AAV5-miHTT have demonstrated efficacy in several rodent and large animal models by reducing FL HTT mRNA and protein and rescuing HD-like phenotypes, and have been the rationale for phase I/II clinical studies now ongoing in the US and Europe. In the present study, we evaluated the ability of AAV5-miHTT to reduce the levels of aberrantly spliced HTT1a mRNA and the HTTexon1 protein in the brain of two mouse models of HD (heterozygous zQ175 knock-in mice and humanized Hu128/21 mice). Polyadenylated HTT1a mRNA and HTTexon1 protein were detected in the striatum and cortex of heterozygous zQ175 knock-in mice, but not in wild-type, littermate control mice. Intrastriatal administration of AAV5-miHTT resulted in dose-dependent expression of mature miHTT microRNA in cortical brain regions, accompanied by significant lowering of both FL HTT and HTT1a mRNA expression at two months post-injection. Mutant HTT and HTTexon1 protein levels were also significantly reduced in the striatum and cortex of heterozygous zQ175 knock-in at 2 months after AAV5-miHTT treatment and in humanized Hu128/21 mice 7 months post-treatment. The effects were confirmed in primary Hu128/21 neuronal cultures. These results demonstrate that AAV5-miHTT gene therapy is an effective approach to lower both FL HTT and the pathogenic HTTexon1 levels, which could potentially have an additive therapeutic benefit compared to other HTT-targeting modalities.

Adeno-Associated Virus (AAV)-Delivered Exosomal TAT and BiTE Molecule CD4-αCD3 Facilitate the Elimination of CD4 T Cells Harboring Latent HIV-1.

Combinatorial antiretroviral therapy (cART) has transformed HIV infection from a death sentence to a controllable chronic disease, but cannot eliminate the virus. Latent HIV-1 reservoirs are the major obstacles to cure HIV-1 infection. Previously, we engineered exosomal Tat (Exo-Tat) to reactivate latent HIV-1 from the reservoir of resting CD4+ T cells. Here, we present an HIV-1 eradication platform, which uses our previously described Exo-Tat to activate latent virus from resting CD4+ T cells guided by the specific binding domain of CD4 in interleukin 16 (IL16), attached to the N-terminus of exosome surface protein lysosome-associated membrane protein 2 variant B (Lamp2B). Cells with HIV-1 surface protein gp120 expressed on the cell membranes are then targeted for immune cytolysis by a BiTE molecule CD4-αCD3, which colocalizes the gp120 surface protein of HIV-1 and the CD3 of cytotoxic T lymphocytes. Using primary blood cells obtained from antiretroviral treated individuals, we find that this combined approach led to a significant reduction in replication-competent HIV-1 in infected CD4+ T cells in a clonal in vitro cell system. Furthermore, adeno-associated virus serotype DJ (AAV-DJ) was used to deliver Exo-Tat, IL16lamp2b and CD4-αCD3 genes by constructing them in one AAV-DJ vector (the plasmid was named pEliminator). The coculture of T cells from HIV-1 patients with Huh-7 cells infected with AAV-Eliminator viruses led to the clearance of HIV-1 reservoir cells in the in vitro experiment, which could have implications for reducing the viral reservoir in vivo, indicating that Eliminator AAV viruses have the potential to be developed into therapeutic biologics to cure HIV-1 infection.

Adeno-associated virus-based gene therapy treats inflammatory kidney disease in mice.

Adeno-associated virus (AAV) is a promising in vivo gene delivery platform showing advantages in delivering therapeutic molecules to difficult or undruggable cells. However, natural AAV serotypes have insufficient transduction specificity and efficiency in kidney cells. Here, we developed an evolution-directed selection protocol for renal glomeruli and identified what we believe to be a new vector termed AAV2-GEC that specifically and efficiently targets the glomerular endothelial cells (GEC) after systemic administration and maintains robust GEC tropism in healthy and diseased rodents. AAV2-GEC-mediated delivery of IdeS, a bacterial antibody-cleaving proteinase, provided sustained clearance of kidney-bound antibodies and successfully treated antiglomerular basement membrane glomerulonephritis in mice. Taken together, this study showcases the potential of AAV as a gene delivery platform for challenging cell types. The development of AAV2-GEC and its successful application in the treatment of antibody-mediated kidney disease represents a significant step forward and opens up promising avenues for kidney medicine.

Gene therapy in Aβ-induced cell and mouse models of Alzheimer’s disease through compensating defective mitochondrial complex I function

BackgroundAlzheimer’s disease (AD) is the most common neurogenerative disorder without effective treatments. Defects in mitochondrial complex I are thought to contribute to AD pathogenesis. The aim of this study is to explore whether a novel gene therapy transducing yeast complex I gene NDI1 can be used to treat AD with severely reduced complex I function in cell and animal models.MethodsThe differentiated human neural cells were induced by Aβ1–42 to establish the AD cell model, and adeno-associated virus serotype 9 (AAV9) was used to transduce yeast NDI1 into the cell model. Aβ1–42 was injected into the hippocampus area of the brain to establish the AD mouse model. AAV9-NDI1 was injected stereotaxically into the hippocampus area to test the therapeutic effect.ResultsThe expressed yeast complex I had an ameliorating effect on the defective function of human complex I and cellular pathological characteristics in the AD cell model. Furthermore, AAV9-NDI1 gene therapy in the hippocampus had a therapeutic effect on various aspects of mitochondrial function, histopathological characteristics and neurological defects in the AD mouse model. In addition, AAV9-NDI1 injection into the hippocampus of normal mice did not cause any adverse effect.ConclusionsCompensating mitochondrial complex I function with yeast NDI1 is effective for gene therapy in Aβ-induced AD cell and mouse models. The results of this study offer a novel strategy and approach for treating AD types characterized by complex I abnormalities.

Arming AAV9 with a Single-Chain Fragment Variable Antibody Against PD-1 for Systemic Glioblastoma Therapy.

Glioblastoma (GBM) is a highly aggressive brain cancer with a low survival rate, prompting the exploration of novel therapeutic strategies. Immune checkpoint inhibitors have shown promise in cancer treatment but are associated with immune-related toxicities and brain penetration. Here, we present a targeted approach using an adeno-associated virus serotype 9 (AAV9) to systemically deliver a single-chain fragment variable antibody against PD-1 (scFv-PD-1) into the tumor microenvironment (TME). Single-cell RNA sequencing analysis revealed robust PD-1 expression in GBM TME, predominantly on T cells. AAV9-scFv-PD-1 expressed and secreted scFv-PD-1, which effectively binds to PD-1. Systemic administration of AAV9-scFv-PD-1 in an immunocompetent GBM mouse model resulted in a robust cytolytic T-cell activation at the tumor site, marked by accumulation of IFN-γ and Granzyme B, leading to a significant reduction in tumor growth. Importantly, AAV9-scFv-PD-1 treatment conferred a survival benefit, highlighting its therapeutic potential. This study demonstrates the feasibility of systemically delivered AAV9-mediated local expression of scFv-PD-1 for targeted immunotherapy in GBM and warrants further investigation for clinical translation.

Targeting OGF/OGFR signal to mitigate doxorubicin-induced cardiotoxicity

Enkephalins are reportedly correlated with heart function. However, their regulation in the heart remains unexplored. This study revealed a substantial increase in circulating levels of opioid growth factor (OGF) (also known as methionine enkephalin) and myocardial expression levels of both OGF and its receptor (OGFR) in subjects treated with doxorubicin (Dox). Silencing OGFR through gene knockout or using adeno-associated virus serotype 9 carrying small hairpin RNA effectively alleviated Dox-induced cardiotoxicity (DIC) in mice. Conversely, OGF supplementation exacerbated DIC manifestations, which could be abolished by administration of the OGFR antagonist naltrexone (NTX). Mechanistically, the previously characterized OGF/OGFR/P21 axis was identified to facilitate DIC-related cardiomyocyte apoptosis. Additionally, OGFR was observed to dissociate STAT1 from the promoters of ferritin genes (FTH and FTL), thereby repressing their transcription and exacerbating DIC-related cardiomyocyte ferroptosis. To circumvent the compromised therapeutic effects of Dox on tumors owing to OGFR blockade, SiO2-based modifiable lipid nanoparticles were developed for heart-targeted delivery of NTX. The pretreatment of tumor-bearing mice with the assembled NTX nanodrug successfully provided cardioprotection against Dox toxicity without affecting Dox therapy in tumors. Taken together, this study provides a novel understanding of Dox cardiotoxicity and sheds light on the development of cardioprotectants for patients with tumors receiving Dox treatment.

KCNQ1 suppression-replacement gene therapy in transgenic rabbits with type 1 long QT syndrome.

Type 1 long QT syndrome (LQT1) is caused by pathogenic variants in the KCNQ1-encoded Kv7.1 potassium channels, which pathologically prolong ventricular action potential duration (APD). Herein, the pathologic phenotype in transgenic LQT1 rabbits is rescued using a novel KCNQ1 suppression-replacement (SupRep) gene therapy. KCNQ1-SupRep gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement), packaged into adeno-associated virus serotype 9, and delivered in vivo via an intra-aortic root injection (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms were assessed in adult LQT1 and wild-type (WT) rabbits and patch-clamp experiments were performed on isolated ventricular cardiomyocytes. KCNQ1-SupRep treatment of LQT1 rabbits resulted in significant shortening of the pathologically prolonged QT index (QTi) towards WT levels. Ventricular cardiomyocytes isolated from treated LQT1 rabbits demonstrated pronounced shortening of APD compared to LQT1 controls, leading to levels similar to WT (LQT1-UT vs. LQT1-SupRep, P < .0001, LQT1-SupRep vs. WT, P = ns). Under β-adrenergic stimulation with isoproterenol, SupRep-treated rabbits demonstrated a WT-like physiological QTi and APD90 behaviour. This study provides the first animal-model, proof-of-concept gene therapy for correction of LQT1. In LQT1 rabbits, treatment with KCNQ1-SupRep gene therapy normalized the clinical QTi and cellular APD90 to near WT levels both at baseline and after isoproterenol. If similar QT/APD correction can be achieved with intravenous administration of KCNQ1-SupRep gene therapy in LQT1 rabbits, these encouraging data should compel continued development of this gene therapy for patients with LQT1.

Abstract We118: Phosphoglycerate Dehydrogenase Gene Therapy for Dilated Cardiomyopathy

Introduction: Dilated cardiomyopathy (DCM) is a multifaceted cardiac disorder affecting approximately 1 in 250 to 2000 individuals and stands as a predominant cause of heart failure. Despite advancements in identifying genetic mutations associated with DCM, the precise mechanisms driving its pathogenesis are unknown, translating to the lack of disease-modifying interventions. Hypothesis: Modulating serine biosynthesis and one-carbon metabolism in cardiomyocytes by forced expression of Phosphoglycerate Dehydrogenase ( PHGDH) will ameliorate cardiac dysfunction in DCM and halt the disease progression. Methods: We developed a gene therapy vector expressing the PHGDH gene, controlled by a chicken cardiac-specific promoter (cardiac troponin T, cTnT) (AAV9.cTnT.hPHGDH). We delivered this construct via an AAV-based gene transfer approach using the cardiotropic adeno-associated virus serotype-9 in the TM54 DCM mouse – a well-established transgenic mouse that expresses a mutant tropomyosin cDNA (Tpm1 p. E54K; Tm54) under the control of the cardiac αMHC promoter. At 6 weeks old, when the animals exhibit DCM, they were treated with AAV9.cTnT.hPHGDH or control (AAV9.cTnT.GFP). Serial echocardiography was employed to assess heart function for 10 weeks. Morphometric data including heart weight, body weight, and tibia length were recorded. Subsequently, WGA and picrosirius red staining were performed to evaluate cardiomyocyte size and fibrosis, respectively. Finally, metabolic changes were assessed by untargeted metabolomics. Results: AAV9.cTnT.hPHGDH treatment of DCM TM54 mice improved the systolic function and halted the progression of DCM compared to the control mice. Furthermore, PHGDH treatment prevented the development of interstitial fibrosis and cardiomyocyte hypertrophy. Finally, these functional and structural improvements correlate with metabolic rewiring of the glucose metabolism in the DCM heart. Conclusion: These findings suggest that metabolic rewiring of the glucose metabolism by enhancing serine biosynthesis and one-carbon metabolism in the DCM heart may be cardioprotective. Thus, PHGDH gene therapy may be a novel therapeutic approach for DCM and heart failure.

Efficient AAV9 Purification Using a Single-Step AAV9 Magnetic Affinity Beads Isolation.

Adeno-associated viruses (AAVs) have emerged as promising tools for gene therapy due to their safety and efficacy in delivering therapeutic genes or gene editing sequences to various tissues and organs. AAV serotype 9 (AAV9), among AAV serotypes, stands out for its ability to efficiently target multiple tissues, thus holding significant potential for clinical applications. However, existing methods for purifying AAVs are cumbersome, expensive, and often yield inconsistent results. In this study, we explore a novel purification strategy utilizing Dynabeads™ CaptureSelect™ magnetic beads. The AAV9 magnetic beads capture AAV9 with high specificity and recovery between 70 and 90%, whereas the AAVX magnetic beads did not bind to the AAV9. Through continuous interaction with AAVs in solution, these beads offer enhanced clearance of genomic DNA and plasmids even in the absence of endonuclease. The beads could be regenerated at least eight times, and the used beads could be stored for up to six months and reused without a significant reduction in recovery. The potency of the AAV9-purified vectors in vivo was comparable to that of iodixanol purified vectors.

Vaccine Therapy for Heart Failure Targeting the Inflammatory Cytokine Igfbp7.

The heart comprises many types of cells such as cardiomyocytes, endothelial cells (ECs), fibroblasts, smooth muscle cells, pericytes, and blood cells. Every cell type responds to various stressors (eg, hemodynamic overload and ischemia) and changes its properties and interrelationships among cells. To date, heart failure research has focused mainly on cardiomyocytes; however, other types of cells and their cell-to-cell interactions might also be important in the pathogenesis of heart failure. Pressure overload was imposed on mice by transverse aortic constriction and the vascular structure of the heart was examined using a tissue transparency technique. Functional and molecular analyses including single-cell RNA sequencing were performed on the hearts of wild-type mice and EC-specific gene knockout mice. Metabolites in heart tissue were measured by capillary electrophoresis-time of flight-mass spectrometry system. The vaccine was prepared by conjugating the synthesized epitope peptides with keyhole limpet hemocyanin and administered to mice with aluminum hydroxide as an adjuvant. Tissue samples from heart failure patients were used for single-nucleus RNA sequencing to examine gene expression in ECs and perform pathway analysis in cardiomyocytes. Pressure overload induced the development of intricately entwined blood vessels in murine hearts, leading to the accumulation of replication stress and DNA damage in cardiac ECs. Inhibition of cell proliferation by a cyclin-dependent kinase inhibitor reduced DNA damage in ECs and ameliorated transverse aortic constriction-induced cardiac dysfunction. Single-cell RNA sequencing analysis revealed upregulation of Igfbp7 (insulin-like growth factor-binding protein 7) expression in the senescent ECs and downregulation of insulin signaling and oxidative phosphorylation in cardiomyocytes of murine and human failing hearts. Overexpression of Igfbp7 in the murine heart using AAV9 (adeno-associated virus serotype 9) exacerbated cardiac dysfunction, while EC-specific deletion of Igfbp7 and the vaccine targeting Igfbp7 ameliorated cardiac dysfunction with increased oxidative phosphorylation in cardiomyocytes under pressure overload. Igfbp7 produced by senescent ECs causes cardiac dysfunction and vaccine therapy targeting Igfbp7 may be useful to prevent the development of heart failure.

Induction of hepatic fibrosis in mice with schistosomiasis by extracellular microRNA-30 derived from Schistosoma japonicum eggs.

Schistosomiasis is a zoonotic parasitic disorder induced by the infestation of schistosomes, a genus of trematodes. MicroRNAs (miRNAs) in egg-derived exosomes are crucial for modulating the host's immune responses and orchestrating the pathophysiological mechanisms. Although the exosomes secreted by S. japonicum contain abundant miRNAs, the specific roles of these miRNAs in the pathogenesis of schistosomiasis-induced hepatic fibrosis are yet to be comprehensively elucidated. The egg exosomes of S. japonicum secrete miRNA-30, a novel miRNA. In vitro, the effect of miRNA-30 was evaluated by transfecting HSCs with miRNA mimics. The target gene biosignature for miRNA-30 was predicted using the miRDB software. The effect of miRNA-30 in hepatic fibrosis was evaluated by either elevating its expression in healthy mice or by inhibiting its activity in infected mice by administration of recombinant adeno-associated virus serotype eight vectors expressing miRNA-30 or miRNA sponges. This novel miRNA can activate hepatic stellate cells (HSCs), the prinary effector cells of hepatic fibrosis, in vitro, i.e., it significantly increases the fibrogenic factors Col1(α1), Col3(α1), and α-SMA at both mRNA and protein levels. In addition, miRNA-30 may activate HSCs by targeting the host RORA gene. In addition, in vivo experiments were conducted by administering a recombinant adeno-associated viral vector to modulate the expression levels of miRNA-30. The overexpression of miRNA-30 in healthy mice significantly elevated the expression of Col1(α1), Col3(α1), and α-SMA at both the transcriptomic and proteomic scales. This overexpression was coupled with a pronounced augmentation in the hepatic hydroxyproline content. Conversely, the in vivo silencing of miRNA-30 in infected mice induced a considerable reduction in the size of hepatic granulomas and areas of collagen deposition. Hence, in vivo, modulation of miRNA-30 expression may play a pivotal role in ameliorating the severity of hepatic fibrosis in mice afflicted with S. japonica. The study results suggest that miRNA-30 may augment schistosomiasis-induced hepatic fibrosis through a probable interaction with the host RORA. Our study may improve the current theoretical framework regarding cross-species regulation by miRNAs of hepatic fibrosis in schistosomiasis.

The Expression and Function of the Small Nonstructural Proteins of Adeno-Associated Viruses (AAVs).

Adeno-associated viruses (AAVs) are small, non-enveloped viruses that package a single-stranded (ss)DNA genome of 4.7 kilobases (kb) within their T = 1 icosahedral capsid. AAVs are replication-deficient viruses that require a helper virus to complete their life cycle. Recombinant (r)AAVs have been utilized as gene delivery vectors for decades in gene therapy applications. So far, six rAAV-based gene medicines have been approved by the US FDA. The 4.7 kb ssDNA genome of AAV encodes nine proteins, including three viral structural/capsid proteins, VP1, VP2, and VP3; four large nonstructural proteins (replication-related proteins), Rep78/68 and Rep52/40; and two small nonstructural proteins. The two nonstructured proteins are viral accessory proteins, namely the assembly associated protein (AAP) and membrane-associated accessory protein (MAAP). Although the accessory proteins are conserved within AAV serotypes, their functions are largely obscure. In this review, we focus on the expression strategy and functional properties of the small nonstructural proteins of AAVs.

Restoration of peripheral neuropathy in Fabry mice via intrathecal administration of an adeno-associated virus vector encoding mGLA cDNA

Anderson-Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a pathological variant of the α-galactosidase A (GLA) gene that results in deficient GLA activity. GLA deficiency leads to the accumulation of globotriaosylceramide (Gb3) and lyso-Gb3 in many tissues. A certain number of FD patients have burning pain or acroparesthesia in the feet and hands since childhood. Enzyme replacement therapy (ERT) is available for FD patients. However, ERT does not dramatically improve these FD-related peripheral neuropathic pain. We generated an adeno-associated virus serotype PHP.eB (AAV-PHP.eB) vector encoding mouse GLA cDNA, which was administered to FD mice intrathecally (it) or intravenously (iv). In the it-administered AAV (it-AAV) FD mice, the GLA enzyme activity in the lumbar dorsal root ganglion (DRG) was significantly greater than that in the untreated (NT) FD mice, and the level of activity was similar to that in wild-type (WT) B6 mice. However, in iv-administered AAV (iv-AAV) FD mice, GLA activity in the DRG did not increase compared to that in NT FD mice. Gb3 storage in the DRG of it-AAV FD mice was reduced compared to that in the DRG of NT FD mice. However, compared with NT FD mice, iv-AAV FD mice did not exhibit a significant reduction in the expression of the Gb3 substrate. Compared with WT mice, FD mice were thermally hyposensitive at 52 °C according to the hot plate test. The it-AAV FD mice showed significant recovery from thermal hyposensitivity. However, the iv-AAV FD mice did not exhibit significant improvement in thermal hyposensitivity. These results suggest that the intrathecal delivery of AAV-PHP.eB-mGLA may be a valuable tool for the treatment of FD-related peripheral neuropathic pain.

RNA Helicase DDX5 Maintains Cardiac Function by Regulating CamkIIδ Alternative Splicing.

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. RNA-binding proteins are identified as regulators of cardiac disease; DDX5 (dead-box helicase 5) is a master regulator of many RNA processes, although its function in heart physiology remains unclear. We assessed DDX5 expression in human failing hearts and a mouse HF model. To study the function of DDX5 in heart, we engineered cardiomyocyte-specific Ddx5 knockout mice. We overexpressed DDX5 in cardiomyocytes using adeno-associated virus serotype 9 and performed transverse aortic constriction to establish the murine HF model. The mechanisms underlined were subsequently investigated using immunoprecipitation-mass spectrometry, RNA-sequencing, alternative splicing analysis, and RNA immunoprecipitation sequencing. We screened transcriptome databases of murine HF and human dilated cardiomyopathy samples and found that DDX5 was significantly downregulated in both. Cardiomyocyte-specific deletion of Ddx5 resulted in HF with reduced cardiac function, an enlarged heart chamber, and increased fibrosis in mice. DDX5 overexpression improved cardiac function and protected against adverse cardiac remodeling in mice with transverse aortic constriction-induced HF. Furthermore, proteomics revealed that DDX5 is involved in RNA splicing in cardiomyocytes. We found that DDX5 regulated the aberrant splicing of Ca2+/calmodulin-dependent protein kinase IIδ (CamkIIδ), thus preventing the production of CaMKIIδA, which phosphorylates L-type calcium channel by serine residues of Cacna1c, leading to impaired Ca2+ homeostasis. In line with this, we found increased intracellular Ca2+ transients and increased sarcoplasmic reticulum Ca2+ content in DDX5-depleted cardiomyocytes. Using adeno-associated virus serotype 9 knockdown of CaMKIIδA partially rescued the cardiac dysfunction and HF in Ddx5 knockout mice. These findings reveal a role for DDX5 in maintaining calcium homeostasis and cardiac function by regulating alternative splicing in cardiomyocytes, identifying the DDX5 as a potential target for therapeutic intervention in HF.

Effect of optogenetic excitation of non-orexinergic neurons in the hypothalamic perifornical area on motor behaviors and cardiovascular parameters in rats

Central command, a motor volition originating in the rostral part of the brain, plays a pivotal role in the precise regulation of autonomic nervous and cardiovascular systems. Central neuronal substrates responsible for transmitting central command signals remain incompletely understood. This study aimed to investigate the effect of optogenetic excitation of non-orexinergic (NOrx) neurons in the hypothalamic perifornical area (PeFA), where orexinergic neurons are densely distributed, on motor behaviors and cardiovascular parameters in rats. An adeno-associated viral serotype 2 vector carrying the human synapsin promoter encoding channelrhodopsin 2 (ChR2) fused to EYFP was injected into the PeFA of Sprague-Dawley rats, resulting in selective expression of ChR2-EYFP in NOrx PeFA neurons. In conscious rats, optogenetic excitation of NOrx PeFA neurons rapidly elicited walking or biting behavior, simultaneously causing pressor and tachycardiac responses regardless of the observed behavioral patterns. Under anesthesia, this excitation rapidly increased renal sympathetic nerve activity, immediately followed by sympathoinhibition. These findings suggest that NOrx PeFA neurons transmit central command signals, concurrently regulating somatomotor and autonomic nervous systems for locomotor exercise or biting behavior.

Membrane chromatography for AAV full capsid enrichment: Process development to scale up

The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing “empty” capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model.

Cardiomyocyte LGR6 alleviates ferroptosis in diabetic cardiomyopathy via regulating mitochondrial biogenesis

AimsThe majority of people with diabetes are susceptible to cardiac dysfunction and heart failure, and conventional drug therapy cannot correct the progression of diabetic cardiomyopathy. We assessed the potential role and therapeutic value of LGR6 (G protein-coupled receptor containing leucine-rich repeats 6) in diabetic cardiomyopathy. Methods and resultsType 2 diabetes models were established using high-fat diet/streptozotocin-induced diabetes in mice. LGR6 knockout mice were generated. Recombinant adeno-associated virus serotype 9 carrying LGR6 under the cardiac troponin T promoter was injected into diabetic mice. Cardiomyocytes incubated with high glucose (HG) were used to imitate diabetic cardiomyopathy in vitro. The molecular mechanism was explored through RNA sequencing and a chromatin immunoprecipitation assay. We found that LGR6 expression was upregulated in diabetic hearts and HL1 cardiomyocytes treated with HG. The LGR6 knockout aggravated, but cardiomyocyte-specific LGR6 overexpression ameliorated, cardiac dysfunction and remodeling in diabetic mice. Mechanistically, in vivo and in vitro experiments revealed that LGR6 deletion aggravated, whereas LGR6 overexpression alleviated, ferroptosis and disrupted mitochondrial biogenesis by regulating STAT3/Pgc1a signaling. STAT3 inhibition and Pgc1a activation abrogated LGR6 knockout-induced mitochondrial dysfunction and ferroptosis in diabetic mice. In addition, LGR6 activation by recombinant RSPO3 treatment ameliorated cardiac dysfunction, ferroptosis and mitochondrial dysfunction in diabetic mice. ConclusionsWe identified a previously undescribed signaling pathway of the LGR6-STAT3-Pgc1a axis that plays a critical role in ferroptosis and mitochondrial disorders during diabetic cardiomyopathy and provides an option for treatment of diabetic hearts.