Adeno-associated Virus Vectors Research Articles

Immunogenicity risk assessment of empty capsids present in adeno-associated viral vectors using predictive innate immune responses

Immunogenicity of gene therapy and the impacts on safety and efficacy are of increasing interest in the pharmaceutical industry. Unique structural aspects of gene therapy delivery vectors, such as adeno-associated viral (AAV) vectors, are expected to activate the innate immune system. The risk of innate immune activation is critical to understand due to the potential impacts on safety and on subsequent adaptive immune responses. In this study, we investigated the responses of key innate immune players—dendritic cells, natural killer (NK) cells, and the complement system—to AAV8 capsids. Immunogenicity risk was also predicted in the presence empty AAV capsids for AAV gene therapy. Compared to genome-containing "full" AAV8 capsids, empty AAV8 capsids more strongly induced proinflammatory cytokine production and migration by human and mouse dendritic cells, but the “full” capsid increased expression of co-stimulatory markers. Furthermore, in an NK cell degranulation assay, we found mixtures of empty and full AAV8 capsids to activate expression of TNF-α, IFN-γ, and CD107a more strongly in multiple NK cell populations compared to either capsid type alone. Serum complement C3a was also induced more strongly in the presence of mixed empty and full AAV8 capsid formulations. Risk for innate immune activation suggests the importance to determine acceptable limits of empty capsids. Immunogenicity risk assessment of novel biological modalities will benefit from the aforementioned in vitro innate immune activation assays providing valuable mechanistic information.

Leukocyte cell-derived chemotaxin 2 (LECT2) regulates liver ischemia–reperfusion injury

Background and aimHepatic ischemia–reperfusion injury (IRI) is a significant challenge in liver transplantation, trauma, hypovolemic shock, and hepatectomy, with limited effective interventions available. This study aimed to investigate the role of leukocyte cell-derived chemotaxin 2 (LECT2) in hepatic IRI and assess the therapeutic potential of Lect2-short hairpin RNA (shRNA) delivered through adeno-associated virus (AAV) vectors. Materials and methodsThis study analyzed human liver and serum samples from five patients undergoing the Pringle maneuver. Lect2-knockout and C57BL/6J mice were used. Hepatic IRI was induced by clamping the hepatic pedicle. Treatments included recombinant human LECT2 (rLECT2) and AAV-Lect2-shRNA. LECT2 expression levels and serum biomarkers including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and blood urea nitrogen (BUN) were measured. Histological analysis of liver necrosis and quantitative reverse-transcription polymerase chain reaction were performed. ResultsSerum and liver LECT2 levels were elevated during hepatic IRI. Serum LECT2 protein and mRNA levels increased post reperfusion. Lect2-knockout mice had reduced weight loss; hepatic necrosis; and serum ALT, AST, creatinine, and BUN levels. rLECT2 treatment exacerbated weight loss, hepatic necrosis, and serum biomarkers (ALT, AST, creatinine, and BUN). AAV-Lect2-shRNA treatment significantly reduced weight loss, hepatic necrosis, and serum biomarkers (ALT, AST, creatinine, and BUN), indicating therapeutic potential. ConclusionsElevated LECT2 levels during hepatic IRI increased liver damage. Genetic knockout or shRNA-mediated knockdown of Lect2 reduced liver damage, indicating its therapeutic potential. AAV-mediated Lect2-shRNA delivery mitigated hepatic IRI, offering a potential new treatment strategy to enhance clinical outcomes for patients undergoing liver-related surgeries or trauma.

AAV-mouse DNase I sustains long-term DNase I expression invivo and suppresses breast cancer metastasis.

Neutrophil extracellular traps (NETs) have been implicated in the pathology of various inflammatory conditions. In cancer, NETs have been demonstrated to induce systemic inflammation, impair peripheral vessel and organ function and promote metastasis. Here we show that the plasma level of NETs is significantly higher in patients with metastatic breast cancer compared to those with local disease, or those that were considered cured at a 5-year follow-up, confirming NETs as interesting therapeutic targets in metastatic breast cancer. Administration of DNase I is one strategy to eliminate NETs but long-term treatment requires repeated injections and species-specific versions of the enzyme. To enhance administration and therapeutic efficacy, we have developed an adeno-associated virus (AAV) vector system for delivery of murine DNase I and addressed its potential to counteract cancer-associated pathology in the murine MMTV-PyMT model for metastatic mammary carcinoma. The AAV vector is comprised of capsid KP1 and an expression cassette encoding hyperactive murine DNase I (AAV-mDNase I) under the control of a liver-specific promotor. This AAV-mDNase I vector could support elevated expression and serum activity of murine DNase I over at least 8 months. Neutrophil Gelatinase-Associated Lipocalin (NGAL), a biomarker for kidney hypoperfusion that is upregulated in urine from MMTV-PyMT mice, was suppressed in mice receiving AAV-mDNase I compared to an AAV-null control group. Furthermore, the proportion of mice that developed lung metastasis was reduced in the AAV-mDNase I group. Altogether, our data indicate that AAV-mDNase I has the potential to reduce cancer-associated impairment of renal function and development of metastasis. We conclude that AAV-mDNase I could represent a promising therapeutic strategy in metastatic breast cancer.

Multistep allelic conversion in mouse pre-implantation embryos by AAV vectors

Site-specific recombinases (SSRs) are critical for achieving precise spatiotemporal control of engineered alleles. These enzymes play a key role in facilitating the deletion or inversion of loci flanked by recombination sites, resulting in the activation or repression of endogenous genes, selection markers or reporter elements. However, multiple recombination in complex alleles can be laborious. To address this, a new and efficient method using AAV vectors has been developed to simplify the conversion of systems based on Cre, FLP, Dre and Vika recombinases. In this study, we present an effective method for ex vivo allele conversion using Cre, FLP (flippase), Dre, and Vika recombinases, employing adeno-associated viruses (AAV) as delivery vectors. AAVs enable efficient allele conversion with minimal toxicity in a reporter mouse line. Moreover, AAVs facilitate sequential allele conversion, essential for fully converting alleles with multiple recombination sites, typically found in conditional knockout mouse models. While simple allele conversions show a 100% efficiency rate, complex multiple conversions consistently achieve an 80% conversion rate. Overall, this strategy markedly reduces the need for animals and significantly speeds up the process of allele conversion, representing a significant improvement in genome engineering techniques.

Current Treatment Process and Challenges for Spinal Muscular Atrophy (SMA)

Spinal muscular atrophy (SMA) presents as a pediatric neuromuscular disorder resulting from mutations in the survival motor neuron 1(SMN1) gene, with an incidence of approximately 10-13 per 100,000 live births. This condition manifests primarily through muscle weakness and atrophy, particularly affecting the proximal limb muscles. A homozygous deletion of exon 7 in the SMN1 gene is the most common genetic mutation in SMA and serves as a sensitive diagnostic marker. This article comprehensively explores various aspects of SMA, including its types, diagnostic methods, and management strategies. Pulmonary care, gastrointestinal (GI) management, nutritional supplementation, orthopedic spinal care, and physiotherapy play crucial roles in delaying disease progression. Current pharmacological treatments for SMA are diverse and evolving. Notable among them is Zolgensma (formerly AVXS-101 or Onasemnogene abeparvovec), a gene replacement therapy utilizing an adeno-associated virus vector to boost functional SMN protein levels. Sodium vanadate, a phosphatase inhibitor, and risdiplam (RO703406) are agents that modulate SMN2 splicing to augment the production of functional SMN protein. Riluzole acts as a neuroprotective agent by preventing SMN deficiency, while reldesemtive (CK-2127107) is under development as a muscle-enhancing therapy to ameliorate skeletal muscle function during fatigue. While pharmacological therapies for SMA are advancing, a holistic approach involving comprehensive multidisciplinary care, encompassing pulmonary support, proper nutritional supplementation, and adept orthopedic management, significantly contributes to the effective management of SMA.

Direct dorsal root ganglia (DRG) injection in mice for analysis of adeno-associated viral (AAV) gene transfer to peripheral somatosensory neurons

BackgroundDelivering optogenetic genes to the peripheral sensory nervous system provides an efficient approach to study and treat neurological disorders and offers the potential to reintroduce sensory feedback to prostheses users and those who have incurred other neuropathies. Adeno-associated viral (AAV) vectors are a common method of gene delivery due to efficiency of gene transfer and minimal toxicity. AAVs are capable of being designed to target specific tissues, with transduction efficacy determined through the combination of serotype and genetic promoter selection, as well as location of vector administration. The dorsal root ganglia (DRGs) are collections of cell bodies of sensory neurons which project from the periphery to the central nervous system (CNS). The anatomical make-up of DRGs make them an ideal injection location to target the somatosensory neurons in the peripheral nervous system (PNS). Comparison to existing methodsPrevious studies have detailed methods of direct DRG injection in rats and dorsal horn injection in mice, however, due to the size and anatomical differences between rats and strains of mice, there is only one other published method for AAV injection into murine DRGs for transduction of peripheral sensory neurons using a different methodology. New Method/ResultsHere, we detail the necessary materials and methods required to inject AAVs into the L3 and L4 DRGs of mice, as well as how to harvest the sciatic nerve and L3/L4 DRGs for analysis. This methodology results in optogenetic expression in both the L3/L4 DRGs and sciatic nerve and can be adapted to inject any DRG.

Targeting Neurological Aspects of Mucopolysaccharidosis Type II: Enzyme Replacement Therapy and Beyond.

Mucopolysaccharidosis type II (MPS II) is a rare, pediatric, neurometabolic disorder due to the lack of activity of the lysosomal hydrolase iduronate 2-sulfatase (IDS), normally degrading heparansulfate and dermatansulfate within cell lysosomes. The deficit of activity is caused by mutations affecting the IDS gene, leading to the pathological accumulation of both glycosaminoglycans in the lysosomal compartment and in the extracellular matrix of most body districts. Although a continuum of clinical phenotypes is described, two main forms are commonly recognized-attenuated and severe-the latter being characterized by an earlier and faster clinical progression and by a progressive impairment of central nervous system (CNS) functions. However, attenuated forms havealso been recently described as presenting some neurological involvement, although less deep, such as deficits of attention and hearing loss. The main treatment for the disease is represented by enzyme replacement therapy (ERT), applied in several countries since 2006, which, albeit showing partial efficacy on some peripheral organs, exhibited a very poor efficacy on bones and heart, and a total inefficacy on CNS impairment, due to the inability of the recombinant enzyme to cross the blood-brain barrier (BBB). Together with ERT, whose design enhancements, performed in the last few years, allowed a possible brain penetration of the drug through the BBB, other therapeutic approaches aimed at targeting CNS involvement in MPS II were proposed and evaluated in the last decades, such as intrathecal ERT, intracerebroventricular ERT, ex vivo gene therapy, or adeno-associated viral vector (AAV) gene therapy. The aim of this review is to summarize the main clinical aspects of MPS II in addition to current therapeutic options, with particular emphasis on the neurological ones and on the main CNS-targeted therapeutic approaches explored through the years.

In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 is an autosomal recessive disorder caused by mutations (pathogenic variants) in fumarylacetoacetate hydrolase, an enzyme involved in tyrosine degradation. Its loss results in the accumulation of toxic metabolites that mainly affect the liver and kidneys and can lead to severe liver disease and liver cancer. Tyrosinemia type 1 has a global prevalence of approximately 1 in 100,000 births but can reach up to 1 in 1,500 births in some regions of Québec, Canada. Mutating functionally related "modifier' genes (i.e. genes that, when mutated, affect the phenotypic impacts of mutations in other genes) is an emerging strategy for treating human genetic diseases. In vivo somatic genome editing in animal models of these diseases is a powerful means to identify modifier genes and fuel treatment development. In this study, we demonstrate that mutating additional enzymes in the tyrosine catabolic pathway through liver-specific genome editing can relieve or worsen the phenotypic severity of a murine model of tyrosinemia type 1. Neonatal gene delivery using recombinant adeno-associated viral vectors expressing Staphylococcus aureus Cas9 under the control of a liver-specific promoter led to efficient gene disruption and metabolic rewiring of the pathway, with systemic effects that were distinct from the phenotypes observed in whole-body knockout models. Our work illustrates the value of using in vivo genome editing in model organisms to study the direct effects of combining pathological mutations with modifier gene mutations in isogenic settings.

AAV-Mediated Expression of miR-17 Enhances Neurite and Axon Regeneration In Vitro.

Neurodegenerative disorders, including traumatic injuries to the central nervous system (CNS) and neurodegenerative diseases, are characterized by early axonal damage, which does not regenerate in the adult mammalian CNS, leading to permanent neurological deficits. One of the primary causes of the loss of regenerative ability is thought to be a developmental decline in neurons' intrinsic capability for axon growth. Different molecules are involved in the developmental loss of the ability for axon regeneration, including many transcription factors. However, the function of microRNAs (miRNAs), which are also modulators of gene expression, in axon re-growth is still unclear. Among the various miRNAs recently identified with roles in the CNS, miR-17, which is highly expressed during early development, emerges as a promising target to promote axon regeneration. Here, we used adeno-associated viral (AAV) vectors to overexpress miR-17 (AAV.miR-17) in primary cortical neurons and evaluate its effects on neurite and axon regeneration in vitro. Although AAV.miR-17 had no significant effect on neurite outgrowth and arborization, it significantly enhances neurite regeneration after scratch lesion and axon regeneration after axotomy of neurons cultured in microfluidic chambers. Target prediction and functional annotation analyses suggest that miR-17 regulates gene expression associated with autophagy and cell metabolism. Our findings suggest that miR-17 promotes regenerative response and thus could mitigate neurodegenerative effects.

Qingshen Granules alleviates renal fibrosis in mice by regulating exosomes, miR-330-3p, and CREBBP expression

To explore the effects of Qingshen Granules (QSG) on adenine-induced renal fibrosis in mice and in uric acid (UA)-stimulated NRK-49F cells and its mechanism for regulating exosomes, miR-330-3p and CREBBP. A mouse model of adenine-induced renal fibrosis were treated daily with QSG at 8.0 g·kg-1·d-1 via gavage for 12 weeks. An adenoassociated virus vector was injected into the tail vein, and renal tissues of the mice were collected for analyzing exosomal marker proteins CD9, Hsp70, and TSG101 and expressions of Col-III, α-SMA, FN, and E-cad using Western blotting and immunofluorescence and for observing pathological changes using HE and Masson staining. In the cell experiment, NRK-49F cells were stimulated with uric acid (400 μmol/L) followed by treatment with QSG-medicated serum from SD rats, and the changes in expressions of the exosomal markers and Col-III, α-SMA, FN, and E-cad were analyzed. Dual luciferase reporter assay was employed to examine the targeting relationship between miR-330-3p and CREBBP, whose expressions were detected by RT-qPCR and Western blotting in treated NRK-49F cells. The mouse models of adenine-induced renal fibrosis showed significantly increased levels of CD9, Hsp70, and TSG101, which were decreased by treatment with QSG. The expressions of Col-III, α-SMA, and FN increased and Ecad decreased in the mouse models but these changes were reversed by QSG treatment. QSG treatment obviously alleviated renal fibrosis in the mouse models. Intravenous injection of adeno-associated viral vector obviously inhibited miR-330-3p, increased CREBBP levels, and reduced fibrosis in the mouse models. Dual luciferase assay confirmed CREBBP as a target of miR-330-3p, which was consistent with the results of the cell experiments. QSG inhibits renal fibrosis in mice by regulating the exosomes, reducing miR-330-3p levels, and increasing CREBBP expression.

Predictive power of deleterious single amino acid changes to infer on AAV2 and AAV2-13 capsids fitness

Adeno-associated virus (AAV) is the most widely used vector for invivo gene transfer. A major limitation of capsid engineering is the incomplete understanding of the consequences of multiple amino acid variations on AAV capsid stability resulting in high frequency of non-viable capsids. In this context, the study of natural AAV variants can provide valuable insights into capsid regions that exhibit greater tolerance to mutations. Here, the characterization of AAV2 variants and the analysis of two public capsid libraries highlighted common features associated with deleterious mutations, suggesting that the impact of mutations on capsid viability is strictly dependent on their 3D location within the capsid structure. We developed a novel prediction method to infer the fitness of AAV2 variants containing multiple amino acid variations with 98% sensitivity, 98% accuracy, and 95% specificity. This novel approach might streamline the development of AAV vector libraries enriched in viable capsids, thus accelerating the identification of therapeutic candidates among engineered capsids.

A novel approach to quantitate biodistribution and transduction of adeno-associated virus gene therapy using radiolabeled AAV vectors in mice

An understanding of recombinant adeno-associated viruses (AAVs) biodistribution profiles is an important element of a preclinical development program. Here, we have developed a radiolabeling strategy utilizing the co-delivery of 125I (non-residualizing) and 111In (residualizing) radionuclides conjugated AAVs to provide a detailed distribution quantification at tissue level delineating between the cellular internalized AAV (degraded, 111In-125I) and AAV remaining in the extracellular matrix (intact, 125I). This labeling method has been successfully applied to AAV9 and AAV-PHP.eB as tool molecules without altering the physical properties and biological activities of the AAVs. Upon labeling with either of the radioactive probes, these molecules were systemically injected into C57BL/6 mice. The biodistribution results indicate AAVs, with a fast distribution profile, were mainly located in the extracellular matrix of highly perfused organs such as liver and spleen at early time points, leading to a difference between capsid quantification and vector genome quantification. The results suggest that the 125I-AAV/111In-AAV co-delivery approach offers a robust and efficient analytical strategy to investigate the detailed tissue distribution of AAV vectors, including both vector genome and protein capsids. This novel method has the potential to be applied to capsid optimization, selection, and lead candidate development.

AAV-NDI1 Therapy Provides Significant Benefit to Murine and Cellular Models of Glaucoma.

Glaucoma, a leading cause of blindness, is a multifactorial condition that leads to progressive loss of retinal ganglion cells (RGCs) and vision. Therapeutic interventions based on reducing ocular hypertension are not always successful. Emerging features of glaucoma include mitochondrial dysfunction and oxidative stress. In the current study, NDI1-based gene therapy, which improves mitochondrial function and reduces reactive oxygen species, was delivered intraocularly via an adeno-associated viral vector (AAV). This AAV-NDI1 therapy protected RGCs from cell death in treated (1552.4 ± 994.0 RGCs/mm2) versus control eyes (1184.4 ± 978.4 RGCs/mm2, p < 0.05) in aged DBA/2J mice, a murine model of glaucoma. The photonegative responses (PhNRs) of RGCs were also improved in treated (6.4 ± 3.3 µV) versus control eyes (5.0 ± 3.1 µV, p < 0.05) in these mice. AAV-NDI1 also provided benefits in glaucomatous human lamina cribrosa (LC) cells by significantly increasing basal and maximal oxygen consumption rates and ATP production in these cells. Similarly, NDI1 therapy significantly protected H2O2-insulted primary porcine LC cells from oxidative stress. This study highlights the potential utility of NDI1 therapies and the benefits of improving mitochondrial function in the treatment of glaucoma.

Emerging Therapeutic Approaches and Genetic Insights in Stargardt Disease: A Comprehensive Review.

Stargardt disease, one of the most common forms of inherited retinal diseases, affects individuals worldwide. The primary cause is mutations in the ABCA4 gene, leading to the accumulation of toxic byproducts in the retinal pigment epithelium (RPE) and subsequent photoreceptor cell degeneration. Over the past few years, research on Stargardt disease has advanced significantly, focusing on clinical and molecular genetics. Recent studies have explored various innovative therapeutic approaches, including gene therapy, stem cell therapy, and pharmacological interventions. Gene therapy has shown promise, particularly with adeno-associated viral (AAV) vectors capable of delivering the ABCA4 gene to retinal cells. However, challenges remain due to the gene's large size. Stem cell therapy aims to replace degenerated RPE and photoreceptor cells, with several clinical trials demonstrating safety and preliminary efficacy. Pharmacological approaches focus on reducing toxic byproduct accumulation and modulating the visual cycle. Precision medicine, targeting specific genetic mutations and pathways, is becoming increasingly important. Novel techniques such as clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 offer potential for directly correcting genetic defects. This review aims to synthesize recent advancements in understanding and treating Stargardt disease. By highlighting breakthroughs in genetic therapies, stem cell treatments, and novel pharmacological strategies, it provides a comprehensive overview of emerging therapeutic options.

Intra-Articular Delivery of an AAV-Anti-TNF-α Vector Alleviates the Progress of Arthritis in a RA Mouse Model.

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease marked by joint destruction and functional impairment. Tumor necrosis factor (TNF) plays a critical role in RA pathogenesis. Although TNF-targeting drugs are clinically effective, their need for frequent and long-term administration often results in poor patient adherence and suboptimal outcomes. This study developed a gene therapy approach using engineered adeno-associated virus (AAV) vectors to deliver an anti-TNF agent directly into the joint cavity of RA animal models. Animals receiving this therapy demonstrated sustained improvement in clinical scores, inflammatory markers, and joint tissue health. Immunofluorescence staining revealed that AAV vectors could transduce various cell types, including T cells, type A synoviocytes, and dendritic cells. Our results indicate that a single administration of this gene therapy provided long-term efficacy. This suggests that AAV-mediated anti-TNF gene therapy can offer prolonged relief from clinical symptoms and reduce inflammatory damage in a mouse model of RA. This innovative approach presents a promising new therapy with significant clinical prospects to treat patients with RA.

Advances in Pompe Disease Treatment: From Enzyme Replacement to Gene Therapy.

Pompe disease is a neuromuscular disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA), hydrolyzing glycogen to glucose. Pathological glycogen storage, the hallmark of the disease, disrupts the metabolism and function of various cell types, especially muscle cells, leading to cardiac, motor, and respiratory dysfunctions. The spectrum of Pompe disease manifestations spans two main forms: classical infantile-onset (IOPD) and late-onset (LOPD). IOPD, caused by almost complete GAA deficiency, presents at birth and leads to premature death by the age of 2 years without treatment. LOPD, less severe due to partial GAA activity, appears in childhood, adolescence, or adulthood with muscle weakness and respiratory problems. Since 2006, enzyme replacement therapy (ERT) has been approved for Pompe disease, offering clinical benefits but not a cure. However, advances in early diagnosis through newborn screening, recognizing disease manifestations, and developing improved treatments are set to enhance Pompe disease care. This article reviews recent progress in ERT and ongoing translational research, including the approval of second-generation ERTs, a clinical trial of in utero ERT, and preclinical development of gene and substrate reduction therapies. Notably, gene therapy using intravenous delivery of adeno-associated virus (AAV) vectors is in phase I/II clinical trials for both LOPD and IOPD. Promising data from LOPD trials indicate that most participants met the criteria to discontinue ERT several months after gene therapy. The advantages and challenges of this approach are discussed. Overall, significant progress is being made towards curative therapies for Pompe disease. While several challenges remain, emerging data are promising and suggest the potential for a once-in-a-lifetime treatment.

Abstract Tu046: Mechanisms of Epicardium-Directed Cardiac Repair

Background: In the adult heart, the epicardium becomes activated by myocardial infarction (MI), contributing to cardiac remodeling primarily through the secretion of paracrine factors. However, there is a limited understanding of the cellular and molecular mechanisms that govern epicardial function and promote pro-reparative signaling pathways. Thus, enhancing or restoring the pro-regenerative characteristics of the epicardium may benefit cardiac repair after MI. We hypothesize that epicardial-derived cells preserve cardiac function during ischemic injury through the limited secretion of pro-reparative paracrine factors, such as slit guidance ligand 2 (Slit2), which enhances angiogenesis and vascular stability. Methods: We used the Wt1 (Wilms Tumor 1) CreERT2 ; R26 tdTomato mouse line for tamoxifen-inducible epicardial-specific lineage tracing and Pdgfra nGFP to label cardiac fibroblasts ( Wt1 CreERT2 ; R26 tdT ; Pdgfra nGFP ). Tagged epicardial-derived cells (tdTomato + ) from sham and 7-day post-MI mice were isolated by fluorescence-activated cell sorting and sequenced by single-cell RNA sequencing. To delineate Slit2-directed cell and molecular mechanisms, we performed in vitro studies and utilized adeno-associated viral vectors for gene therapy in a mouse model of MI. Results: We identified 9 transcriptionally distinct epicardial subpopulations contributing to paracrine signaling induced by MI. We found enriched expression of various chemokines in epicardial-derived stromal cells, while genes associated with Wnt signaling and epithelial-to-mesenchymal transition (EMT) were highly expressed in Wt1 -expressing clusters. Isolated cardiac fibroblasts treated with an adenoviral vector expressing Slit2 exhibited no differences in myofibroblast formation or collagen production following treatment with activation stimuli (TGFβ1/Ang II) but showed significant increases in the expression of pro-angiogenic gene programs. Cardiac-targeted AAV9-Slit2 overexpression in mice subjected to MI led to a striking increase in cardiac ejection fraction, expression of angiogenic-modulating genes, vessel density, and endothelial cell proliferation but did not alter fibrotic deposition. Conclusions: Our study reveals the dynamic role of epicardial cells during the post-MI cardiac repair process and delineates a Slit2-Robo dependent mechanism governing cardiac fibroblast and endothelial cell crosstalk to modulate angiogenesis.

Retinal Penetrating Adeno-Associated Virus.

The most common method of delivery of genes to the outer retina uses recombinant adeno-associated virus (AAV) injected into the subretinal space using a surgical procedure. In contrast, most drugs are delivered to the retina using an intravitreal approach in an office setting. The objective of the current study was to develop AAV vectors that can reach the outer retina via intravitreal injection. Recently, we described a molecular chaperone (Nuc1) that enhanced the penetration of small and large molecules, including AAV, into the retina. The Nuc1 amino acid sequence or a truncated version of Nuc1 (IKV) was genetically incorporated into an exposed loop of AAV2/9 VP1 protein. These novel recombinant AAV vectors expressing green fluorescent protein (GFP) or nuclear factor erythroid 2 p45-related factor 2 (Nrf2) were injected into the vitreous of C57Bl/6J or Nrf2 knockout mice, respectively. The amount of GFP expression or oxidative stress as measured by 8-Hydroxy-2'-deoxyguanosine staining in C57Bl/6J or Nrf2 knockout mice, respectively, was quantified. Incorporation of Nuc1 into AAV2/9 did not lead to significant expression of GFP in the murine retina. However, incorporation of IKV into AAV2/9 led to robust expression of GFP in photoreceptors and retinal pigment epithelium (RPE) via the intravitreal and subretinal routes of delivery. Furthermore, expression of Nrf2 using an IKV vector led to a reduction in oxidative stress in the retina of C57Bl/6J and Nrf2 knockout mice. We have developed a novel AAV vector that enables delivery of transgenes to the outer retina of mice, including photoreceptors and RPE following intravitreal injection.

Brain-Derived Neurotrophic Factor (BDNF) in the Frontal Cortex Enhances Social Interest in the BTBR Mouse Model of Autism.

A large body of evidence implies the involvement of brain-derived neurotrophic factor (BDNF) in the pathogenesis of autism spectrum disorders (ASDs). A deficiency of BDNF in the hippocampus and frontal cortex of BTBR mice (a model of autism) has been noted in a number of studies. Earlier, we showed that induction of BDNF overexpression in the hippocampus of BTBR mice reduced anxiety and severity of stereotyped behavior, but did not affect social interest. Here, we induced BDNF overexpression in the frontal cortex neurons of BTBR mice using an adeno-associated viral vector, which resulted in a significant increase in the social interest in the three-chamber social test. At the same time, the stereotypy, exploratory behavior, anxiety-like behavior, and novel object recognition were not affected. Therefore, we have shown for the first time that the presence of BDNF in the frontal cortex is critical for the expression of social interest in BTBR mice, since compensation for its deficiency in this structure eliminated the autism-like deficiencies in the social behavior characteristic for these animals.

Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system.

Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored. We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC). At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spfash mouse following elimination of residual endogenous murine OTC. The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.