Sirt6 protects retinal ganglion cells and optic nerve from degeneration during aging and glaucoma.

Glaucoma is characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, and its risk increases with aging. Yet comprehensive insights into the complex mechanisms are largely unknown. Here, we found that anti-aging molecule Sirt6 was highly expressed in RGCs. Deleting Sirt6 globally or specifically in RGCs led to progressive RGC loss and optic nerve degeneration during aging, despite normal intraocular pressure (IOP), resembling a phenotype of normal-tension glaucoma. These detrimental effects were potentially mediated by accelerated RGC senescence through Caveolin-1 upregulation and by the induction of mitochondrial dysfunction. In mouse models of high-tension glaucoma, Sirt6 level was decreased after IOP elevation. Genetic overexpression of Sirt6 globally or specifically in RGCs significantly attenuated high tension-induced degeneration of RGCs and their axons, whereas partial or RGC-specific Sirt6 deletion accelerated RGC loss. Importantly, therapeutically targeting Sirt6 with pharmacological activator or AAV2-mediated gene delivery ameliorated high IOP-induced RGC degeneration. Together, our studies reveal a critical role of Sirt6 in preventing RGC and optic nerve degeneration during aging and glaucoma, setting the stage for further exploration of Sirt6 activation as a potential therapy for glaucoma.

Evolving membrane-associated accessory protein variants for improved adeno-associated virus production.

Manufacturing sufficient adeno-associated virus (AAV) to meet current and projected clinical needs is a significant hurdle to the growing gene therapy industry. The recently discovered membrane-associated accessory protein (MAAP) is encoded by an alternative open reading frame in the AAV cap gene that is found in all presently reported natural serotypes. Recent evidence has emerged supporting a functional role of MAAP in AAV egress, although the underlying mechanisms of MAAP function remain unknown. Here, we show that inactivation of MAAP from AAV2 by a single point mutation that is silent in the VP1 open reading frame (ORF) (AAV2-ΔMAAP) decreased exosome-associated and secreted vector genome production. We hypothesized that novel MAAP variants could be evolved to increase AAV production and thus subjected a library encoding over 1 × 106 MAAP protein variants to five rounds of packaging selection into the AAV2-ΔMAAP capsid. Between each successive packaging round, we observed a progressive increase in both overall titer and ratio of secreted vector genomes conferred by the bulk-selected MAAP library population. Next-generation sequencing uncovered enriched mutational features, and a resulting selected MAAP variant containing missense mutations and a frameshifted C-terminal domain increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids.

Neuroglobin overexpression in cerebellar neurons of Harlequin mice improves mitochondrial homeostasis and reduces ataxic behavior.

Neuroglobin, a member of the globin superfamily, is abundant in the brain, retina, and cerebellum of mammals and localizes to mitochondria. The protein exhibits neuroprotective capacities by participating in electron transfer, oxygen supply, and protecting against oxidative stress. Our objective was to determine whether neuroglobin overexpression can be used to treat neurological disorders. We chose Harlequin mice, which harbor a retroviral insertion in the first intron of the apoptosis-inducing factor gene resulting in the depletion of the corresponding protein essential for mitochondrial biogenesis. Consequently, Harlequin mice display degeneration of the cerebellum and suffer from progressive blindness and ataxia. Cerebellar ataxia begins in Harlequin mice at the age of 4 months and is characterized by neuronal cell disappearance, bioenergetics failure, and motor and cognitive impairments, which aggravated with aging. Mice aged 2 months received adeno-associated viral vectors harboring the coding sequence of neuroglobin or apoptosis-inducing factor in both cerebellar hemispheres. Six months later, Harlequin mice exhibited substantial improvements in motor and cognitive skills; probably linked to the preservation of respiratory chain function, Purkinje cell numbers and connectivity. Thus, without sharing functional properties with apoptosis-inducing factor, neuroglobin was efficient in reducing ataxia in Harlequin mice.

Production of VP3-only virus-like particles of Adeno-associated virus 2 in E. coli cells.

Adeno-associated Virus (AAV) is a promising vector for gene therapy. However, few studies have focused on producing virus-like particles (VLPs) of AAV in cells, especially in E. coli. In this study, we describe a method to produce empty VP3-only VLPs of AAV2 in E. coli by co-expressing VP3 and assembly-activating protein (AAP) of AAV2. Although the yields of VLPs produced with our method were low, the VLPs were able to self-assemble in E. coli without the need of in vitro capsid assembly. The produced VLPs were characterized by immunological detection and transmission electron microscopy (TEM). In conclusion, this study demonstrated that capsid assembly of AAV2 is possible in E. coli, and E. coli may be a candidate system for production of VLPs of AAV.

Inhibition of complement C3 signaling ameliorates locomotor and visual dysfunction in autoimmune inflammatory diseases.

Neuromyelitis optica (NMO) is an autoimmune inflammatory disease of the central nervous system (CNS) characterized by transverse myelitis and optic neuritis. The pathogenic serum IgG antibody against the aquaporin-4 (AQP4) on astrocytes triggers the activation of the complement cascade, causing astrocyte injury, followed by oligodendrocyte injury, demyelination, and neuronal loss. Complement C3 is positioned as a central player that relays upstream initiation signals to activate downstream effectors, potentially stimulating and amplifying host immune and inflammatory responses. However, whether targeting the inhibition of C3 signaling could ameliorate tissue injury, locomotor defects, and visual impairments in NMO remains to be investigated. In this study, using the targeted C3 inhibitor CR2-Crry led to a significant decrease in complement deposition and demyelination in both slice cultures and focal intracerebral injection models. Moreover, the treatment downregulated the expression of inflammatory cytokines and improved motor dysfunction in a systemic NMO mouse model. Similarly, employing serotype 2/9 adeno-associated virus (AAV2/9) to induce permanent expression of CR2-Crry resulted in a reduction in visual dysfunction by attenuating NMO-like lesions. Our findings reveal the therapeutic value of inhibiting the complement C3 signaling pathway in NMO.

Association between adeno-associated virus 2 and severe acute hepatitis of unknown etiology in Japanese children.

INTRODUCTION: Outbreaks of acute hepatitis of unknown etiology (AHUE) in children were reported in Western countries in 2022. Previous studies found that adeno-associated virus 2 (AAV2) and its helper viruses, such as human adenovirus (HAdV) and human herpesvirus-6 (HHV-6), are frequently detected in patients with AHUE. However, the existence of hepatitis associated with AAV2 prior to AHUE outbreaks in 2022 had not yet been investigated. We aimed to investigate the association between AAV2 and pediatric acute hepatitis in Japanese children, as well as the incidence of AAV2-related hepatitis prior to 2022. METHODS: Preserved blood samples obtained from 49 pediatric patients with acute hepatitis between 2017 and 2023 were retrospectively analyzed. Blood samples from 50 children with acute illnesses and 50 children with chronic conditions were used as controls. Viral DNA loads were quantitated using real-time PCR. RESULTS: AAV2 DNA was detected in 12 % (6/49) of acute hepatitis cases but in only one acute illness and none of the chronic-condition control cases. The concentration of AAV2 DNA in the six acute hepatitis cases was higher than that in the acute-illness control case. Co-infection with one or more helper viruses, including HAdV, HHV-6, cytomegalovirus, and Epstein-Barr virus, was observed in five AAV2-positive cases. CONCLUSIONS: Our results indicated the sporadic occurrence of pediatric severe hepatitis associated with AAV2 infection in Japan prior to the AHUE outbreaks in 2022. Our findings suggest that co-infection with AAV2 and helper viruses plays a role in developing severe hepatitis.

Single Nuclei Analyses Reveal Transcriptional Profiles and Marker Genes for Diverse Supraspinal Populations.

The mammalian brain contains numerous neurons distributed across forebrain, midbrain, and hindbrain that project axons to the lower spinal cord and work in concert to control movement and achieve homeostasis. Extensive work has mapped the anatomic location of supraspinal cell types and continues to establish specific physiological functions. The patterns of gene expression that typify and distinguish these disparate populations, however, are mostly unknown. Here, using adult mice of mixed sex, we combined retrograde labeling of supraspinal cell nuclei with fluorescence-activated nuclei sorting and single-nuclei RNA sequencing analyses to transcriptionally profile neurons that project axons from the brain to lumbar spinal cord. We identified 14 transcriptionally distinct cell types and used a combination of established and newly identified marker genes to assign an anatomic location to each. To validate the putative marker genes, we visualized selected transcripts and confirmed selective expression within lumbar-projecting neurons in discrete supraspinal regions. Finally, we illustrate the potential utility of these data by examining the expression of transcription factors that distinguish different supraspinal cell types and by surveying the expression of receptors for growth and guidance cues that may be present in the spinal cord. Collectively, these data establish transcriptional differences between anatomically defined supraspinal populations, identify a new set of marker genes of use in future experiments, and provide insight into potential differences in cellular and physiological activity across the supraspinal connectome.SIGNIFICANCE STATEMENT The brain communicates with the body through a wide variety of neuronal populations with distinct functions and differential sensitivity to damage and disease. We have used single-nuclei RNA sequencing technology to distinguish patterns of gene expression within a diverse set of neurons that project axons from the mouse brain to the lumbar spinal cord. The results reveal transcriptional differences between populations previously defined on the basis of anatomy, provide new marker genes to facilitate rapid identification of cell type in future work, and suggest distinct responsiveness of different supraspinal populations to external growth and guidance cues.

A novel strategy for delivering Niemann-Pick type C2 proteins across the blood-brain barrier using the brain endothelial-specific AAV-BR1 virus.

Treating central nervous system (CNS) diseases is complicated by the incapability of numerous therapeutics to cross the blood-brain barrier (BBB), mainly composed of brain endothelial cells (BECs). Genetically modifying BECs into protein factories that supply the CNS with recombinant proteins is a promising approach to overcome this hindrance, especially in genetic diseases, like Niemann Pick disease type C2 (NPC2), where both CNS and peripheral cells are affected. Here, we investigated the potential of the BEC-specific adeno-associated viral vector (AAV-BR1) encoding NPC2 for expression and secretion from primary BECs cultured in an in vitro BBB model with mixed glial cells, and in healthy BALB/c mice. Transduced primary BECs had significantly increased NPC2 gene expression and secreted NPC2 after viral transduction, which significantly reversed cholesterol deposition in NPC2 deficient fibroblasts. Mice receiving an intravenous injection with AAV-BR1-NCP2-eGFP were sacrificed 8 weeks later and examined for its biodistribution and transgene expression of eGFP and NPC2. AAV-BR1-NPC2-eGFP was distributed mainly to the brain and lightly to the heart and lung, but did not label other organs including the liver. eGFP expression was primarily found in BECs throughout the brain but occasionally also in neurons suggesting transport of the vector across the BBB, a phenomenon also confirmed in vitro. NPC2 gene expression was up-regulated in the brain, and recombinant NPC2 protein expression was observed in both transduced brain capillaries and neurons. Our findings show that AAV-BR1 transduction of BECs is possible and that it may denote a promising strategy for future treatment of NPC2.

Adeno-Associated Virus 2 and Human Adenovirus F41 in Wastewater during Outbreak of Severe Acute Hepatitis in Children, Ireland.

During April-July 2022, outbreaks of severe acute hepatitis of unknown etiology (SAHUE) were reported in 35 countries. Five percent of cases required liver transplantation, and 22 patients died. Viral metagenomic studies of clinical samples from SAHUE cases showed a correlation with human adenovirus F type 41 (HAdV-F41) and adeno-associated virus type 2 (AAV2). To explore the association between those DNA viruses and SAHUE in children in Ireland, we quantified HAdV-F41 and AAV2 in samples collected from a wastewater treatment plant serving 40% of Ireland's population. We noted a high correlation between HAdV-F41 and AAV2 circulation in the community and SAHUE clinical cases. Next-generation sequencing of the adenovirus hexon in wastewater demonstrated HAdV-F41 was the predominant HAdV type circulating. Our environmental analysis showed increased HAdV-F41 and AAV2 prevalence in the community during the SAHUE outbreak. Our findings highlight how wastewater sampling could aid in surveillance for respiratory adenovirus species.

Potential involvement of miR-183/96/182 cluster-gene target interactions in transdifferentiation of human retinal pigment epithelial cells into retinal neurons.

miR-183/96/182 cluster plays a critical role in the developing retina by regulating many target genes involved in signaling pathways. This study aimed to survey the miR-183/96/182 cluster-target interactions that, potentially contribute to human retinal pigmented epithelial (hRPE) cell differentiation into photoreceptors. Target genes of the miR-183/96/182 cluster were obtained from miRNA-target databases and applied to construct miRNA-target networks. Gene ontology and KEGG pathway analysis was performed. miR-183/96/182 cluster sequence was cloned into an eGFP-intron splicing cassette in an AAV2 vector and overexpressed in hRPE cells. The expression level of target genes including HES1, PAX6, SOX2, CCNJ, and RORΒ was evaluated using qPCR. Our results showed that miR-183, miR-96, and miR-182 share 136 target genes that are involved in cell proliferation pathways such as PI3K/AKT and MAPK pathway. qPCR data indicated a 22-, 7-, and 4-fold overexpression of miR-183, miR-96, and miR-182, respectively, in infected hRPE cells. Consequently, the downregulation of several key targets such as PAX6, CCND2, CDK5R1, and CCNJ and upregulation of a few retina-specific neural markers such as Rhodopsin, red opsin, and CRX was detected. Our findings suggest that the miR-183/96/182 cluster may induce hRPE transdifferentiation by targeting key genes that involve in the cell cycle and proliferation pathways.

Postpandemic rebound of adeno-associated virus type 2 (AAV2) infections temporally associated with an outbreak of unexplained severe acute hepatitis in children in the United Kingdom.

Over 1000 cases of unexplained severe acute hepatitis in children have been reported to date worldwide. An association with adeno-associated virus type 2 (AAV2) infection, a human parvovirus, prompted us to investigate the epidemiology of AAV in the United Kingdom. Three hundred pediatric respiratory samples collected before (April 03, 2009-April 03, 2013) and during (April 03, 2022) the COVID-19 pandemic were obtained. Wastewater samples were collected from 50 locations in London (August 2021-March 2022). Samples were tested for AAV using real-time polymerase chain reaction followed by sequencing. Selected adenovirus (AdV)-positive samples were also sequenced. The detection frequency of AAV2 was a sevenfold higher in 2022 samples compared with 2009-2013 samples (10% vs. 1.4%) and highest in AdV-positive samples compared with negatives (10/37, 27% vs. 5/94, 5.3%, respectively). AAV2-positive samples displayed high genetic diversity. AAV2 sequences were either very low or absent in wastewater collected in 2021 but increased in January 2022 and peaked in March 2022. AAV2 was detected in children in association with AdV of species C, with a highest frequency in 2022. Our findings are consistent with the expansion of the population of children unexposed to AAV2, leading to greater spread of the virus once distancing restrictions were lifted.

Adenovirus F41 infection and liver cytolysis in adult hematopoietic stem cell transplant recipients.

Human adenoviruses (HAdVs) of the F species are commonly responsible for acute gastroenteritis. A few cases of systemic infections have been described in adults or children who have received a hematopoietic stem cell transplant (HSCT), but with no report of liver cytolysis. Since January 2022, several countries have reported an increase in cases of acute hepatitis of unknown cause in children. Adenovirus species F type 41 (HAdV-F41) infection was predominantly identified. The objective of this study is to describe HAdV-F41 infections diagnosed since January 2022 in adult HSCT recipients in two French hospitals. All four patients had diarrhea and liver cytolysis at the time of diagnosis of infection. HAdV viremia was observed in three patients (#1, #3, and #4), but no disseminated disease was reported. HAdV whole genome sequencing and metagenomics characterization were performed on stool and blood samples. The complete HAdV-F41 genome sequence was obtained for three patients and phylogenetic analysis showed that the strains consisted of similar lineage (2b). We did not identify any new HAdV-F41 strains. Metagenomics analysis found adeno-associated virus 2 and torque-teno virus infection in patient #1 and Epstein-Barr virus in patient #4. This is the first case series reporting liver cytolysis during HAdV-F41 infection in adult HSCT patients.

AAV capsid engineering identified two novel variants with improved in vivo tropism for cardiomyocytes.

AAV vectors are promising delivery tools for human gene therapy. However, broad tissue tropism and pre-existing immunity against natural serotypes limit their clinical use. We identified two AAV capsid variants, AAV2-THGTPAD and AAV2-NLPGSGD, by in vivo AAV2 peptide display library screening in a murine model of pressure overload-induced cardiac hypertrophy. Both variants showed significantly improved efficacy in in vivo cardiomyocyte transduction compared with the parental serotype AAV2 as indicated by a higher number of AAV vector episomes in the nucleus and significant improved transduction efficiency. Both variants also outcompeted the reference serotype AAV9 regarding cardiomyocyte tropism, reaching comparable cardiac transduction efficiencies accompanied with liver de-targeting and decreased transduction efficiency of non-cardiac cells. Capsid modification influenced immunogenicity as sera of mice treated with AAV2-THGTPAD and AAV2-NLPGSGD demonstrated a poor neutralization capacity for the parental serotype and the novel variants. In a therapeutic setting, using the long non-coding RNA H19 in low vector dose conditions, novel AAV variants mediated superior anti-hypertrophic effects and revealed a further improved target-to-noise ratio, i.e., cardiomyocyte tropism. In conclusion, AAV2-THGTPAD and AAV2-NLPGSGD are promising novel tools for cardiac-directed gene therapy outperforming AAV9 regarding the specificity and therapeutic efficiency of in vivo cardiomyocyte transduction.

Non-invasive administration of AAV to target lung parenchymal cells and develop SARS-CoV-2-susceptible mice.

Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice, and this can be used to study diverse pulmonary diseases.

Local magnetic delivery of adeno-associated virus AAV2(quad Y-F)-mediated BDNF gene therapy restores hearing after noise injury.

Moderate noise exposure may cause acute loss of cochlear synapses without affecting the cochlear hair cells and hearing threshold; thus, it remains "hidden" to standard clinical tests. This cochlear synaptopathy is one of the main pathologies of noise-induced hearing loss (NIHL). There is no effective treatment for NIHL, mainly because of the lack of a proper drug-delivery technique. We hypothesized that local magnetic delivery of gene therapy into the inner ear could be beneficial for NIHL. In this study, we used superparamagnetic iron oxide nanoparticles (SPIONs) and a recombinant adeno-associated virus (AAV) vector (AAV2(quad Y-F)) to deliver brain-derived neurotrophic factor (BDNF) gene therapy into the rat inner ear via minimally invasive magnetic targeting. We found that the magnetic targeting effectively accumulates and distributes the SPION-tagged AAV2(quad Y-F)-BDNF vector into the inner ear. We also found that AAV2(quad Y-F) efficiently transfects cochlear hair cells and enhances BDNF gene expression. Enhanced BDNF gene expression substantially recovers noise-induced BDNF gene downregulation, auditory brainstem response (ABR) wave I amplitude reduction, and synapse loss. These results suggest that magnetic targeting of AAV2(quad Y-F)-mediated BDNF gene therapy could reverse cochlear synaptopathy after NIHL.

A link between severe hepatitis in children and adenovirus 41 and adeno-associated virus 2 infections.

Over the past few months there have been reports of severe acute hepatitis in several hundred, otherwise healthy, immunocompetent young children. Several deaths have been recorded and a relatively large proportion of the patients have needed liver transplants. Most of the cases, so far, have been seen in the UK and in North America, but it has also been reported in many other European countries, the Middle East and Asia. Most common viruses have been ruled out as a causative agent; hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) were not detected, nor were Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human immunodeficiency virus (HIV) in many cases. A small proportion of the children had been infected with SARS-CoV-2 but these seem to be in a minority; similarly, almost none of the children had been vaccinated against COVID-19. Significantly, many of the patients were infected with adenovirus 41 (HAdV-F41). Previously, HAdV-41 had not been linked to hepatitis and is usually considered to cause gastroenteritis in both immunocompetent and immunocompromised patients. In two most recent studies, adeno-associated virus 2 (AAV2) was detected in almost all patients, together with species C and F HAdVs and human herpesvirus 6B (HHV6B). Here, I discuss the possibility that a change in tropism of HAdV-41 and changes in AAV2 may be responsible for their links to acute hepatitis.

Targeted deletion of PAC1 receptors in retinal neurons enhances neuron loss and axonopathy in a model of multiple sclerosis and optic neuritis.

Chronic inflammation drives synaptic loss in multiple sclerosis (MS) and is also commonly observed in other neurodegenerative diseases. Clinically approved treatments for MS provide symptomatic relief but fail to halt neurodegeneration and neurological decline. Studies in animal disease models have demonstrated that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1) exhibits anti-inflammatory, neuroprotective and regenerative properties. Anti-inflammatory actions appear to be mediated primarily by two receptors, VPAC1 and VPAC2, which also bind vasoactive intestinal peptide (VIP). Pharmacological experiments indicate that another receptor, PAC1 (ADCYAP1R1), which is highly selective for PACAP, provides protection to neurons, although genetic evidence and other mechanistic information is lacking. To determine if PAC1 receptors protect neurons in a cell-autonomous manner, we used adeno-associated virus (AAV2) to deliver Cre recombinase to the retina of mice harboring floxed PAC1 alleles. Mice were then subjected to chronic experimental autoimmune encephalomyelitis (EAE), a disease model that recapitulates major clinical and pathological features of MS and associated optic neuritis. Unexpectedly, deletion of PAC1 in naïve mice resulted in a deficit of retinal ganglionic neurons (RGNs) and their dendrites, suggesting a homeostatic role of PAC1. Moreover, deletion of PAC1 resulted in increased EAE-induced loss of a subpopulation of RGNs purported to be vulnerable in animal models of glaucoma. Increased axonal pathology and increased secondary presence of microglia/macrophages was also prominently seen in the optic nerve. These findings demonstrate that neuronal PAC1 receptors play a homeostatic role in protecting RGNs and directly protects neurons and their axons against neuroinflammatory challenge. SIGNIFICANCE STATEMENT: Chronic inflammation is a major component of neurodegenerative diseases and plays a central role in multiple sclerosis (MS). Current treatments for MS do not prevent neurodegeneration and/or neurological decline. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to have anti-inflammatory, neuroprotective and regenerative properties but the cell type- and receptor-specific mechanisms are not clear. To test whether the protective effects of PACAP are direct on the PAC1 receptor subtype on neurons, we delete PAC1 receptors from neurons and investigate neuropathologigical changes in an animal model of MS. The findings demonstrate that PAC1 receptors on neurons play a homeostatic role in maintaining neuron health and can directly protect neurons and their axons during neuroinflammatory disease.

Selection of a novel AAV2/TNFAIP3 vector for local suppression of islet xenograft inflammation.

BACKGROUND: Neonatal porcine islets (NPIs) can restore glucose control in mice, pigs, and non-human primates, representing a potential abundant alternative islet supply for clinical beta cell replacement therapy. However, NPIs are vulnerable to inflammatory insults that could be overcome with genetic modifications. Here, we demonstrate in a series of proof-of-concept experiments the potential of the cytoplasmic ubiquitin-editing protein A20, encoded by the TNFAIP3 gene, as an NPI cytoprotective gene. METHODS: We forced A20 expression in NPI grafts using a recombinant adenovirus 5 (Ad5) vector and looked for impact on TNF-stimulated NF-κB activation and NPI graft function. As adeno-associated vectors (AAV) are clinically preferred vectors but exhibit poor transduction efficacy in NPIs, we next screened a series of AAV serotypes under different transduction protocols for their ability achieve high transduction efficiency and suppress NPI inflammation without impacting NPI maturation. RESULTS: Forcing the expression of A20 in NPI with Ad5 vector blocked NF-κB activation by inhibiting IκBα phosphorylation and degradation, and reduced the induction of pro-inflammatory genes Cxcl10 and Icam1. A20-expressing NPIs also exhibited superior functional capacity when transplanted into diabetic immunodeficient recipient mice, evidenced by a more rapid return to euglycemia and improved GTT compared to unmodified NPI grafts. We found AAV2 combined with a 14-day culture period maximized NPI transduction efficiency (>70% transduction rate), and suppressed NF-κB-dependent gene expression without adverse impact upon NPI maturation. CONCLUSION: We report a new protocol that allows for high-efficiency genetic modification of NPIs, which can be utilized to introduce candidate genes without the need for germline engineering. This approach would be suitable for preclinical and clinical testing of beneficial molecules. We also report for the first time that A20 is cytoprotective for NPI, such that A20 gene therapy could aid the clinical development of NPIs for beta cell replacement.

PSCs Reveal PUFA-Provoked Mitochondrial Stress as a Central Node Potentiating RPE Degeneration in Bietti's Crystalline Dystrophy.

Bietti's crystalline dystrophy (BCD) is an incurable retinal disorder caused by the polypeptide 2 of cytochrome P450 family 4 subfamily V (CYP4V2) mutations. Patients with BCD present degeneration of retinal pigmented epithelial (RPE) cells and consequent blindness. The lack of appropriate disease models and patients' RPE cells limits our understanding of the pathological mechanism of RPE degeneration. In this study, using CYP4V2 mutant pluripotent stem cells as disease models, we demonstrated that RPE cells with CYP4V2 mutations presented a disrupted fatty acid homeostasis, which were characterized with excessive accumulation of poly-unsaturated fatty acid (PUFA), including arachidonic acid (AA) and eicosapentaenoic acid (EPA). The PUFA overload increased mitochondrial reactive oxygen species, impaired mitochondrial respiratory functions, and triggered mitochondrial stress-activated p53-independent apoptosis in CYP4V2 mutant RPE cells. Restoration of the mutant CYP4V2 using adeno-associated virus 2 (AAV2) can effectively reduce PUFA deposition, alleviate mitochondria oxidative stresses, and rescue RPE cell death in BCD RPE cells. Taken together, our results highlight a role of PUFA-induced mitochondrial damage as a central node to potentiate RPE degeneration in BCD patients. AAV2-mediated gene therapy may represent a feasible strategy for the treatment of BCD.

Structure comparison of the chimeric AAV2.7m8 vector with parental AAV2.

The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration. Directed evolution and in vivo screening in the mouse retina isolated this vector. In the present study, we sought to identify the structural differences between a recombinant AAV2.7m8 (rAAV2.7m8) vector packaging a GFP genome and its parental serotype, AAV2, by cryo-electron microscopy (cryo-EM) and image reconstruction. The structures of rAAV2.7m8 and AAV2 were determined to 2.91 and 3.02 Å resolution, respectively. The rAAV2.7m8 amino acid side-chains for residues 219-745 (the last C-terminal residue) were interpretable in the density map with the exception of the 10 inserted amino acids. While observable in a low sigma threshold density, side-chains were only resolved at the base of the insertion, likely due to flexibility at the top of the loop. A comparison to parental AAV2 (ordered from residues 217-735) showed the structures to be similar, except at some side-chains that had different orientations and, in VR-VIII containing the 10 amino acid insertion. VR-VIII is part of an AAV2 antigenic epitope, and the difference is consistent with rAAV2.7m8's escape from a known AAV2 monoclonal antibody, C37-B. The observations provide valuable insight into the configuration of inserted surface peptides on the AAV capsid and structural differences to be leveraged for future AAV vector rational design, especially for retargeted tropism and antibody escape.