Administration Of Adeno-associated Virus Vector Research Articles

Induction of tauopathy in a mouse model of amyloidosis using intravenous administration of adeno-associated virus vectors expressing human P301L tau.

Alzheimer's disease (AD) is a progressive neurodegenerative disease in which extracellular aggregates of the amyloid beta (Aβ) peptide precede widespread intracellular inclusions of the microtubule-associated protein tau. The autosomal dominant form of AD requires mutations that increase production or aggregation of the Aβ peptide. This has led to the hypothesis that amyloid deposition initiates downstream responses that lead to the hyperphosphorylation and aggregation of tau. Here we use a novel approach, somatic gene transfer via intravenous adeno-associated virus (AAV), to further explore the effects of pre-existing amyloid deposits on tauopathy. APP+PS1 mice, which develop amyloid deposits at 3 to 6 months of age, and non-transgenic littermates were injected at 8 months of age intravenously with AAV-PHP.eB encoding P301L human tau. Tissue was collected at 13 months and tauopathy was assessed. Total human tau expression was observed to be relatively uniform throughout the brain, reflecting the vascular route of AAV administration. Phospho-tau deposition was not equal across brain regions and significantly increased in APP+PS1 mice compared to non-transgenic controls. Interestingly, the rank order of phospho-tau deposition of affected brain regions in both genotypes paralleled the rank order of amyloid plaque deposits in APP+PS1 mice. We also observed significantly increased MAPT RNA expression in APP+PS1 mice compared to non-transgenic despite equal AAV transduction efficiency between groups. This model has advantages over prior approaches with widespread uniform human tau expression throughout the brain and the ability to specify the stage of amyloidosis when the tau pathology is initiated. These data add further support to the amyloid cascade hypothesis and suggest RNA metabolism as a potential mechanism for amyloid-induced tauopathy.

Long-Term Persistence of Antibodies to Adeno-Associated Viral Vectors Following Gene Therapy with scAAV8-LP1-Fixco

Background Gene therapy for hemophilia B (HB) consisting of a one-time, systemic, administration of adeno-associated viral (AAV) vectors encoding a functional Factor-IX gene is now approved and represents an important and long-awaited milestone. A rise in anti-AAV antibody titers follows systemic administration of AAV vectors. This does not have direct clinical consequences; however, persistence of neutralising anti-AAV antibodies (NAB) at high titers will preclude subsequent successful gene transfer with vector of the same serotype. This would be problematic, in the event that transgene expression should fall below therapeutic levels and repeat dosing might be needed. Furthermore, high titer NAB response to one serotype can cross-react with other serotypes thereby limiting the option of using alternative serotype AAV vectors. We report a longitudinal analysis of anti-AAV antibody response over a 10-year period in severe HB participants following systemic administration of scAAV2/8-LP1-hFIXco from the St Jude-UCL Phase I/II, gene therapy trial (AGT4HB ClinicalTrials.gov:NCT00979238). Methods Anti-AAV antibody levels were evaluated in the 10 trial participants following a bolus intravenous infusion of scAAV2/8-LP1-hFIXco at a dose of 2x10 11vg/kg (N=2), 6x10 11vg/kg (N=2), or 2x10 12vg/kg (N=6) between 2010 and 2012. Total anti-AAV antibodies (TAB) were measured by ELISA, and levels quantitated using a pooled IgG reference. NAB were quantitated by an in-vitro transduction inhibition assay (TIA) in which plasma samples were preincubated with AAV CMV NanoLuc (MOI: AAV3 1, AAV5 100 and 8 100) prior to evaluation of transduction of HEK 293T cells. Results were normalised to virus control and the 50% inhibition dilution (IC50) was calculated. For comparison, we assessed anti-AAV antibody titers in 38 normal human plasma samples using the same methodology. Results At the time of data cut-off (31 st Dec 2022) the median follow-up for the cohort was 10.7 years (range: 4-12 years) with follow-up in two participants (one mid dose and the other high dose) being limited to 4 years. A dose dependent AAV8 TAB response was observed with low dose participants having a mean of 84±101mg/ml at 1-year, mid dose 507 mg/ml and high dose 1650±603 mg/ml. This represented a >1400-fold increase from baseline, pre-AAV administration values. These TAB levels were significantly higher than levels following natural infection in 38 seropositive individuals not exposed to recombinant vector (normal range 0.31-34.02mg/ml). In the high dose cohort, TAB titer against AAV3 and AAV5 at 1 year after gene transfer were 145±167 mg/ml (natural infection reference range 0.31-29.61mg/ml) and 96±55 mg/ml (normal range 0.31-36.13mg/ml) respectively, consistent with cross-reactivity between AAV serotypes. There was a decline in TAB levels against all serotypes prior to reaching a plateau. At year 5 the high dose cohort was 928.9 ±556.9mg/ml for AAV8, 90.4±95.6 mg/ml for AAV3 and, 72.8 ±49.1 mg/ml for AAV5. AAV8 NAB titers had a similar kinetic with IC50 at 1 year 7261.5 ±9812 (low-dose), 15000 (mid-dose) and, 40204± 37823 (high dose). For comparison, AAV8 IC50 were significantly higher than levels following natural infection with wild-type AAV in seropositive individuals (range 5-747) (Mann Whitney p<0.0001). NABs levels declined to an IC50 level of 12907±8246 at 5 years for the high dose cohort, which was still 3000-fold higher than their baseline value (range 5-70.1) (Mann Whitney p<0.0001), and above the threshold for efficient gene transfer. The development of cross-reactive NAB levels was relatively modest and within the range observed in natural seropositive individuals. For the high dose cohort NAB to AAV3 at year 1 was 6536± 1869 (normal range 5- 13502), and for AAV5 at year 1 852± 596 (normal range 5-2143). Conclusion Following AAV gene therapy there is a robust dose dependent rise in anti-AAV 8 antibody levels that decline over time, but at 10 years are still significantly above the threshold for re-administration of vector of the same serotype. There was clear evidence of cross reactivity against AAV3 and 5. AAV5 NAB titres were lower than other serotypes declining to levels below a threshold required for efficient gene transfer in the low and mid dose cohort within 10 years. This suggests that, should FIX levels fall below therapeutic levels in this cohort, successful re-administration of an alternative vector may be possible.

Prevention of severe lung immunopathology associated with influenza infection through adeno-associated virus vector administration

BackgroundInfluenza A viruses (IAVs) have long posed a threat to humans, occasionally causing significant morbidity and mortality. The initial immune response is triggered by infected epithelial cells, alveolar macrophages and dendritic cells. However, an exaggerated innate immune response can result in severe lung injury and even host mortality. One notable pathology observed in hosts succumbing to severe influenza is the excessive influx of neutrophils and monocytes into the lung. In this study, we investigated a strategy for controlling lung immunopathology following severe influenza infection.ResultsTo evaluate the impact of innate immunity on influenza-associated lung injury, we employed CB17.SCID and NOD.SCID mice. NOD.SCID mice exhibited slower weight loss and longer survival than CB17.SCID mice following influenza infection. Lung inflammation was reduced in NOD.SCID mice compared to CB17.SCID mice. Bulk RNA sequencing analysis of lung tissue showed significant downregulation of 827 genes, and differentially expressed gene analysis indicated that the cytokine-cytokine receptor interaction pathway was predominantly downregulated in NOD.SCID mice. Interestingly, the expression of the Cxcl14 gene was higher in the lungs of influenza-infected NOD.SCID mice than in CB17.SCID mice. Therefore, we induced overexpression of the Cxcl14 gene in the lung using the adeno-associated virus 9 (AAV9)-vector system for target gene delivery. However, when we administered the AAV9 vector carrying the Cxcl14 gene or a control AAV9 vector to BALB/c mice from both groups, the morbidity and mortality rates remained similar. Both groups exhibited lower morbidity and mortality than the naive group that did not receive the AAV9 vector prior to IAV infection, suggesting that the pre-administration of the AAV9 vector conferred protection against lethal influenza infection, irrespective of Cxcl14 overexpression. Furthermore, we found that pre-inoculation of BALB/c mice with AAV9 attenuated the infiltration of trans-macrophages, neutrophils and monocytes in the lungs following IAV infection. Although there was no difference in lung viral titers between the naive group and the AAV9 pre-inoculated group, pre-inoculation with AAV9 conferred lung injury protection against lethal influenza infection in mice.ConclusionsOur study demonstrated that pre-inoculation with AAV9 prior to IAV infection protected mouse lungs from immunopathology by reducing the recruitment of inflammatory cells.

Positron Emission Tomography Quantitative Assessment of Off-Target Whole-Body Biodistribution of I-124-Labeled Adeno-Associated Virus Capsids Administered to Cerebral Spinal Fluid.

Based on studies in experimental animals demonstrating that administration of adeno-associated virus (AAV) vectors to the cerebrospinal fluid (CSF) is an effective route to transfer genes to the nervous system, there are increasing number of clinical trials using the CSF route to treat nervous system disorders. With the knowledge that the CSF turns over four to five times daily, and evidence in experimental animals that at least some of CSF administered AAV vectors are distributed to systemic organs, we asked: with AAV administration to the CSF, what fraction of the total dose remains in the nervous system and what fraction goes off target and is delivered systemically? To quantify the biodistribution of AAV capsids immediately after administration, we covalently labeled AAV capsids with iodine 124 (I-124), a cyclotron generated positron emitter, enabling quantitative positron emission tomography scanning of capsid distribution for up to 96 h after AAV vector administration. We assessed the biodistribution to nonhuman primates of I-124-labeled capsids from different AAV clades, including 9 (clade F), rh.10 (E), PHP.eB (F), hu68 (F), and rh91(A). The analysis demonstrated that 60-90% of AAV vectors administered to the CSF through either the intracisternal or intrathecal (lumbar) routes distributed systemically to major organs. These observations have potentially significant clinical implications regarding accuracy of AAV vector dosing to the nervous system, evoking systemic immunity at levels similar to that with systemic administration, and potential toxicity of genes designed to treat nervous system disorders being expressed in non-nervous system organs. Based on these data, individuals in clinical trials using AAV vectors administered to the CSF should be monitored for systemic as well as nervous system adverse events and CNS dosing considerations should account for a significant AAV systemic distribution.

826-P: Treatment of MODY3 Disease by AAV-Hnf1a-Mediated Gene Therapy

Maturity-onset diabetes of the young (MODY) are a group of monogenic diabetes characterized by onset of hyperglycemia at an early adult age, normally before 25 years. MODY3, the most common type of MODY, is caused by mutations in the gene encoding for the transcription factor hepatocyte nuclear factor 1A (HNF1A). MODY3 patients are usually treated with sulfonylureas, but they tend to become unresponsive to these drugs and adverse effects associated to chronic administration of sulfonylureas have been reported. Presently, there are no current treatments addressing the root cause of MODY3. Here, we have developed a new gene therapy approach to treat this disease. To this end, using the CRISPR/Cas9 technology, we first generated a novel MODY3 mouse model that showed pancreatic down-regulation of HNF1A protein production. These mice developed β-cell dysfunction and hyperglycemia, recapitulating the main alterations observed in human patients. Treatment of MODY3 mice by intra-pancreatic duct administration of adeno-associated viral vectors (AAVs) encoding Hnf1α, under control of β-cell-specific promoter, resulted in HNF1A expression in β-cells, which upregulated the expression of HNF1A main target genes, such as GLUT2 and L-PK. AAV-Hnf1α-mediated gene therapy counteracted hyperglycemia, increased insulinemia, improved glucose tolerance and enhanced insulin secretion by pancreatic β-cells, thus reverting the MODY3 disease. This study represents the first demonstration for the genetic counteraction of a monogenic diabetes and paves the way for the clinical translation of AAV-Hnf1α-based gene therapy to treat MODY 3 patients in the future. Moreover, these results may also contribute to the development of new gene therapy strategies for other monogenic forms of diabetes. Disclosure E.Casana lorente: None. X.Leon: None. S.Franckhauser: None. F.Bosch: None. V.Jimenez: None. M.Garcia: None. A.Casellas: None. M.Morró: None. A.Pujol: None. T.Ferré: None. V.Sacristán: None. C.Jambrina: None. Funding Fundació La Marató de TV3 (201603-31-32-33); Ministerio de Economía, Industria y Competitividad (SAF2017-86266R); Agencia Estatal de Investigación; Fondo Europeo de Desarrollo Regional; Programa Estatal de I+D+i Orientada a los Retos de la Sociedad; MCIN/AEI/10.13039/501100011033 (PID2020-113864RB-I00); Generalitat de Catalunya, Spain 2017 SGR 1508 (to F.B.)

Successful liver transduction by re-administration of different adeno-associated virus vector serotypes in mice.

Intravenous administration of adeno-associated virus (AAV) vectors is a promising gene therapy approach for monogenic diseases. However, re-administration of the same AAV serotype is impossible because of the induction of anti-AAV neutralizing antibodies (NAbs). Here, we examined the feasibility of re-administrating AAV vector serotypes different from the initial AAV vector serotype. Liver-targeting AAV3B, AAV5, and AAV8 vectors were intravenously injected in C57BL/6 mice, and the emergence of NAbs and the transduction efficacy following re-administration were evaluated. For all serotypes, re-administration of the same serotype was not possible. Although the highest neutralizing activity of NAb was induced by AAV5, anti-AAV5 NAbs did not react with other serotypes, resulting in successful re-administration with the other serotypes. AAV5 re-administration was also successful in all mice treated with AAV3B and AAV8. Effective secondary administration of AAV3B and AAV8 was observed in most mice initially administrated AAV8 and AAV3B, respectively. However, few mice developed NAbs cross-reacting with the other serotypes, especially those with close sequence homology. In summary, AAV vector administration induced NAbs relatively specific to the administrated serotype. Secondary administration of AAVs targeting liver transduction could be successfully achieved by switching AAV serotypes in mice.

Efficacy, pharmacokinetics, and safety in the mouse and primate retina of dual AAV vectors for Usher syndrome type 1B

Gene therapy of Usher syndrome type 1B (USH1B) due to mutations in the large Myosin VIIA (MYO7A) gene is limited by the packaging capacity of adeno-associated viral (AAV) vectors. To overcome this, we have previously developed dual AAV8 vectors which encode human MYO7A (dual AAV8.MYO7A). Here we show that subretinal administration of 1.37E+9 to 1.37E+10 genome copies of a good-manufacturing-practice-like lot of dual AAV8.MYO7A improves the retinal defects of a mouse model of USH1B. The same lot was used in non-humanprimates at doses 1.6× and 4.3× the highest dose proposedfor the clinical trial which was based on mouse efficacy data. Long-lasting alterations in retinal function and morphology were observed following subretinal administration of dual AAV8.MYO7A at the high dose. These findings were modest and improved over time in the low-dose group, as also observed in other studies involving the use of AAV8 in non-human primates and humans. Biodistribution andshedding studies confirmed the presence of vector DNA mainly in the visual pathway. Accordingly, we detected human MYO7A mRNA expression predominantly in the retina. Overall, these studies pave the way for the clinical translation of subretinal administration of dual AAV vectors in USH1B subjects.

Local and systemic administration of AAV vectors with alphaherpesvirus latency-associated promoter 2 drives potent transgene expression in mouse liver, kidney, and skeletal muscle

Adeno-associated virus (AAV) has great potential as a source of treatments for conditions that might respond to potent and ubiquitous transgene expression. However, among its drawbacks, the genetic “payload” of AAV vectors is limited to <4.9 kb and some commonly used gene promoters are sizeable and susceptible to transcriptional silencing. We recently described a short (404 bp), potent, and persistent promoter obtained from the genome of pseudorabies virus (PrV) called alphaherpesvirus latency-associated promoter 2 (LAP2). Here, we evaluated the biodistribution and potency of transgene expression in mouse peripheral tissues in response to local and systemic administration of AAV8-LAP2 and AAV9-LAP2. We found that administration of these vectors resulted in levels of transgene expression that were similar to the larger EF1α promoter. LAP2 drives potent transgene expression in mouse liver and kidney when administered systemically and in skeletal muscle in response to intramuscular delivery. Notably, in skeletal muscle, administration of vectors with LAP2 and EF1α promoters resulted in preferential transduction of myofibers type 2. A direct side-by-side comparison between LAP2 and the EF1α promoter revealed that, despite its smaller size, LAP2 was equally potent to the EF1α promoter and resulted in widespread gene expression after IV and IM administration of AAV8 or AAV9 vectors. Collectively, these findings suggest that constructs that include LAP2 may have the capacity to deliver large therapeutically effective payloads in support of future gene therapy protocols.

Key Role of Kupffer Cells in IL-1 Dependent Activation of CD8+ T Cell Responses to AAV Transgene Product in Liver

Introduction: Adeno-associated virus (AAV) based viral vectors have emerged as preferred vectors for in vivo gene therapy and are being evaluated in multiple phase I/II and phase III clinical studies. However, immune responses to both the AAV capsid and its encoded transgene still remain a major hurdle. Previously, we demonstrated in a murine model that cellular responses to AAV encoded transgene in the liver are dependent on IL-1α/β mediated activation of IL-1R1-MyD88 signaling. Using flow cytometry and tissue staining we provided evidence that plasmacytoid dendritic cells (pDCs) and Kupffer cells are central players in initiation of IL-1 signaling. Further, we showed that cross-presenting XCR1+ dendritic cells (DCs) are critical for antigen presentation to transgene-specific CD8+ T cells (Blood 136 Supplement 1: 2-3, 2020). In the present study we tested the impact of Kupffer cells depletion on transgene-specific CD8+ T cell response. Since Kupffer cells have been implicated in TLR4-MyD88 mediated secretion of IL-1α in a murine model of acetaminophen induced liver injury (Cell Mol Immunol 15(11): 973, 2018). Therefore, we also explored if innate sensor TLR4 is required for IL-1 mediated CD8+ T cell response to AAV encoded transgene in liver microenvironment. Further, we tested if intrinsic IL-1R1-MyD88 signaling in DCs is essential for these cellular responses. Methods: To study the impact of Kupffer cell depletion on transgene-specific CD8+ T cells we intraperitoneally injected wild-type (WT) C57BL6 mice with 200μg of depleting α-mouse F4/80 antibody (BioXcell) one day prior and 2X/week following vector administered for up to 4 weeks. Groups of C57BL6 WT (control), C57BL6 WT (treated with α-mouse F4/80 antibody) and TLR4-/- mice on the C57BL6 background were intravenously (IV) injected with a predetermined immunogenic dose (1x109vg) of hepatotropic AAV8 vector encoding ovalbumin (OVA) as the transgene (Mol Ther 25(4): 880, 2017). To study the role of MyD88 intrinsic signaling in DCs, we generated mice that lacked adaptor protein MyD88 in DCs (DC-MyD88-/-) using cre-lox system (Blood 129(24): 3184, 2017). Mice were again injected with 1x109vg of AAV8-OVA via IV route. Four weeks after vector administration PBMCs from each group were analyzed for OVA-specific CD8+ T cells using MHC class I tetramer. Results: As expected, ~62% of WT mice developed OVA-specific CD8+ T cell response. Interestingly, only 12% of mice (1 out of 8) treated with α-mouse F4/80 antibody showed CD8+ T cells specific to OVA. Whereas similar to WT mice, 50% of TLR4-/- mice had OVA-specific CD8+ T cell. We conclude that Kupffer cells, which show increased surface expression of IL-1R1 within days after AAV vector administration, are essential for the IL-1R-dependent CD8+ T cells response to the hepatic transgene product but do not function through a mechanism that requires TLR4. This mechanism therefore shares some aspects of acetaminophen-induced liver injury (critical role of KCs and IL-1) but is distinct with regard to TLR involvement. IL-1R1 signaling requires the cytosolic MyD88 adapter molecule. We know from prior studies that MHC I antigen presentation of liver-derived AAV encoded transgene product in lymphoid organs is primarily mediated by cross-presenting XCR1+ DCs. Thus, we tested the requirement of intrinsic MyD88 signaling in DCs. Our results suggest that intrinsic MyD88 signaling in DCs is not required in lymphoid organs, as the CD8+ T cell response in DC-MyD88-/- mice was comparable to that in WT control mice (these mice lack MyD88 in conventional DCs in lymphoid organs but retain MyD88 in hepatic DCs and in pDCs). Conclusions: In summary, we established that Kupffer cells play a central role in elicitation of transgene product-specific CD8+ T cell responses in hepatic AAV gene transfer (through a TLR4-independent mechanisms). While direct sensing of IL-1 by DCs in the hepatic environment may very well play a role, it is not required in lymphoid organs. Following initial priming of CD8+ T cell in the liver, co-stimulation by CD4+ T helper cells may replace IL-1 signaling during the subsequent systemic response.

Chimeric Mice Engrafted With Canine Hepatocytes Exhibits Similar AAV Transduction Efficiency to Hemophilia B Dog.

Adeno-associated virus (AAV) mediated gene therapy has been successfully applied in clinical trials, including hemophilia. Novel AAV vectors have been developed with enhanced transduction and specific tissue tropism. Considering the difference in efficacy of AAV transduction between animal models and patients, the chimeric xenograft mouse model with human hepatocytes has unique advantages of studying AAV transduction efficiency in human hepatocytes. However, it is unclear whether the results in humanized mice can predict AAV transduction efficiency in human hepatocytes. To address this issue, we studied the AAV transduction efficacy in canine hepatocytes in both canine hepatocyte xenografted mice and real dogs. After administration of AAV vectors from different serotypes into canine hepatocyte xenograft mice, AAV8 induced the best canine hepatocyte transduction followed by AAV9, then AAV3, 7, 5 and 2. After administration of AAV/cFIX (cFIX-opt-R338L) vectors in hemophilia B dogs, consistent with the result in chimeric mice, AAV8 induced the highest cFIX protein expression and function, followed by AAV9 and then AAV2. These results suggest that mice xenografted with hepatocytes from different species could be used to predict the AAV liver transduction in real species and highlight this potential platform to explore novel AAV variants for future clinical applications.

Systemic and local immune responses to intraocular AAV vector administration in non-human primates

Positive clinical outcomes in adeno-associated virus (AAV)-mediated retinal gene therapy have often been attributed to the low immunogenicity of AAVs and immune privilege of the eye. However, several recent studies have shown potential for inflammatory responses. The current understanding of the factors contributing to inflammation, such as the pre-existence of serum antibodies against AAVs and their contribution to increases in antibody levels post-injection, is incomplete. The parameters that regulate the generation of new antibodies in response to the AAV capsid or transgene after intraocular injections are also insufficiently described. This study is a retrospective analysis of the pre-existing serum antibodies in correlation with changes in antibody levels after intraocular injections of AAV in non-human primates (NHPs) of the species Macaca fascicularis. In NHP serums, we analyzed the binding antibody (BAB) levels and a subset of these called neutralizing antibodies (NABs) that impede AAV transduction. We observed significantly higher pre-existing serum BABs against AAV8 compared with other serotypes and a dose-dependent increase in BABs and NABs in the serums collected post-injection, irrespective of the serotype or the mode of injection. Lastly, we were able to demonstrate a correlation between the serum BAB levels with clinical grading of inflammation and levels of transgene expression.

Silencing MR-1 Protects against Myocardial Injury Induced by Chronic Intermittent Hypoxia by Targeting Nrf2 through Antioxidant Stress and Anti-Inflammation Pathways.

Background Patients with obstructive sleep apnea hypopnea syndrome (OSAHS) often have cardiac insufficiency mainly due to hypoxia/reperfusion injury caused by chronic intermittent hypoxia (CIH). Inflammation and oxidative stress are involved in the cardiovascular events of OSAHS patients. Studies have found that myofibrillation regulator-1 (MR-1) participates in the pathological process of OSAHS-induced myocardial injury, but the specific mechanism is still unclear. Methods We used a CIH-induced rat model to simulate the process of OSAHS disease. Indices of myocardial injury, inflammation, and oxidative stress were detected using quantitative PCR and enzyme-linked immunosorbent assay (ELISA). After administration of adenoassociated viral vector (AAV) encoding silencing RNA against MR-1, we examined expression of the classic antioxidant stress pathway protein NF-E2-related factor 2 (Nrf2) using western blotting. Results We found that levels of serum inflammatory factors tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 were increased, and we further observed disturbance of the oxidative stress system, in which the content of reactive oxygen species (ROS), superoxide dismutase (SOD), reduced glutathione (GSH), and malondialdehyde (MDA) was enhanced in CIH-induced rats. Subsequently, we detected that expression of Nrf2 and heme oxygenase-1 (HO-1) was slightly increased, while the expression of Kelch-like ECH-associated protein 1 (Keap-1) was significantly increased in the CIH model. Interestingly, after administration of silencing MR-1 AAV, the elevated levels of inflammatory factors were reduced, and the disordered oxidative stress system was corrected. Additionally, the expression of Nrf2 and HO-1 was distinctly increased, but the high expression of Keap-1 was decreased. Conclusions Our research results demonstrate that silencing MR-1 rescued the myocardium the injury from inflammatory and oxidative stress in CIH-induced rats by administration of the Nrf2 signaling pathway.

Repeated Systemic Dosing of Adeno-Associated Virus Vectors in Immunocompetent Mice After Blockade of T Cell Costimulatory Pathways

Neutralizing antibodies (NAbs) strongly limit adeno-associated virus (AAV) vector transduction and repeated administration. Previous studies have shown that NAbs induced by AAVs are associated with T and B cell activation and that the B7/CD28 and CD40/CD40L costimulation signaling pathways are involved. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and CD40 are vital molecules that participate in the costimulatory pathway. In this study, we evaluated CTLA4-Ig and CD40-Ig immunosuppreve efficacies through AAV and investigated their effects on the feasibility for multiple systemic administrations of AAV vectors. The results showed that a single administration of AAV vector carrying either CTLA4-Ig alone or with CD40-Ig could greatly reduce the level of NAbs. An AAV serotype-specific immune tolerance could be successfully established, which enabled repeated, that is, second and third, systemic administration of AAV vectors in the same mice. A combination of CTLA4-Ig and CD40-Ig delivered via AAV vectors significantly inhibited T and B cell activations without affecting the immune response to the total immunoglobulin G production and cytokines. Interestingly, exogenous gene expression significantly improved after multiple administrations of AAV vector in vivo. Our study generates a reliable and effective method for repeated dosing of AAV vectors that is needed on gene therapy.

Amount of Green Fluorescent Protein in the Anterior Chamber after Intravitreal Injection of Triple-Mutated Self-Complementary AAV2 Vectors is Not Affected by Previous Vitrectomy Surgery.

The adeno-associated virus (AAV) vector is a promising vector for ocular gene therapy. Surgical internal limiting membrane peeling before AAV vector administration is useful for efficient retinal transduction. However, no report has investigated localization of AAV vectors after administration into a post-vitrectomy eye. This study investigated the effects of vitrectomy surgery on intravitreal-injected AAV vector-mediated gene expression in the anterior segment and examined the presence of neutralizing antibodies (NAbs) in serum before and after AAV vector administration. Of six eyes from three female cynomolgus monkeys, four were vitrectomized (Group VIT) and two were non-vitrectomized (Group IV). All eyes were injected with 50 μL of triple-mutated self-complementary AAV2 vector (1.9 × 1013 v.g./mL) encoding green fluorescent protein (GFP). NAbs in the serum were examined before administration and at 2 and 6 weeks after administration. GFP expression was analyzed at 19 weeks after administration. Immunohistological analysis showed no GFP expression in the trabecular meshwork in any eye. The GFP genome copy in two slices of the anterior segment was 2.417 (vector genome copies/diploid genome) in Group VIT and 4.316 (vector genome copies/diploid genome) in group IV. The NAb titer was 1:15.9 (geometric mean) before administration, 1:310.7 at 2 weeks after administration, and 1:669.4 at 6 weeks after administration. Previous vitrectomy surgery did not affect gene expression in the anterior segment after intravitreal injection of AAV vectors.

Multicolor sparse viral labeling and 3D digital tracing of enteric plexus in mouse proximal colon using a novel adeno‐associated virus capsid

AbstractBackgroundIntravenous administration of adeno‐associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV‐PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV‐PHP.S with a tunable two‐component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D).MethodsA vector system including a tetracycline inducer with a tet‐responsive element driving three separate fluorophores was packaged in the AAV‐PHP.S capsid. The vectors were injected retro‐orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software.Key ResultsThe transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon–axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size.Conclusions &amp; InferencesThe multicolor AAV‐PHP.S‐packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro‐circuitry.

Long-Term Follow-Up of the First in Human Intravascular Delivery of AAV for Gene Transfer: AAV2-hFIX16 for Severe Hemophilia B

Adeno-associated virus (AAV) vectors are a leading platform for gene-based therapies for both monogenic and complex acquired disorders. The success of AAV gene transfer highlights the need to answer outstanding clinical questions of safety, durability, and the nature of the human immune response to AAV vectors. Here, we present longitudinal follow-up data of subjects who participated in the first trial of a systemically delivered AAV vector. Adult males (n= 7) with severe hemophilia B received an AAV2 vector at doses ranging from 8× 1010 to 2× 1012 vg/kg to target hepatocyte-specific expression of coagulation factor IX; a subset (n= 4) was followed for 12-15 years post-vector administration. No major safety concerns were observed. There was no evidence of sustained hepatic toxicity or development of hepatocellular carcinoma as assessed by liver transaminase values, serum α-fetoprotein, and liver ultrasound. Subjects demonstrated persistent, increased AAV neutralizing antibodies (NAbs) to the infused AAV serotype 2 (AAV2) as well as all other AAV serotypes tested (AAV5 and AAV8) for the duration of follow-up. These data represent the longest available longitudinal follow-up data of subjects who received intravascular AAV and support the preliminary safety of intravascular AAV administration at the doses tested in adults. Data demonstrate, for the first time, the persistence of high-titer, multi-serotype cross-reactive AAV NAbs for up to 15 years post- AAV vector administration. Our observations are broadly applicable to the development of AAV-mediated gene therapy.

Development of AAV Variants with Human Hepatocyte Tropism and Neutralizing Antibody Escape Capacity

Adeno-associated virus (AAV) vectors have been successfully used in patients with bleeding disorders and blindness. For human liver targeting, two major factors restrict effective AAV transduction after systemic administration of AAV vectors: human hepatocyte tropism and neutralizing antibodies (Nabs). In this study, we attempted to isolate AAV variants with the ability to transduce human hepatocytes and escape Nabs using a directed evolution approach in vivo. After four cycles of selection, 14 AAV capsid mutants were identified from a capsid shuffling library selected in the presence of human Intravenous Immunoglobulin (IVIG) and isolated from human hepatocytes xenografted into chimeric mice. AAV neutralization assays using IVIG showed that most of the mutants showed the Nab escape pattern in a manner similar to that of AAV8 or AAV9 and better than that of other AAV serotypes. Different mutants displayed varying capacities to escape Nab activity from individual serum samples collected from healthy subjects or hemophilia patients. The mutant AAV LP2-10 was found in 12 colonies out of 25, which was composed of capsids from AAV serotypes 2, 6, 8, and 9, with VP3 subunits derived from AAV8 swapped with AAV6 from residues 261 to 272. The mutant AAV LP2-10 manifested a higher ability than that of other serotypes to escape Nabs in IVIG and most human serum samples. After injection of AAV vectors encoding a self-complementary GFP cassette into chimeric mice, LP2-10 transduced human hepatocytes with efficiency similar to that of AAV8. In summary, AAV mutants can be isolated in humanized mice with both human hepatocyte tropism and the ability to evade Nab activity.

Timing of Intensive Immunosuppression Impacts Risk of Transgene Antibodies after AAV Gene Therapy in Nonhuman Primates

Adeno-associated virus (AAV) vector gene therapy is a promising treatment for a variety of genetic diseases, including hemophilia. Systemic administration of AAV vectors is associated with a cytotoxic immune response triggered against AAV capsid proteins, which if untreated can result in loss of transgene expression. Immunosuppression (IS) with corticosteroids has limited transgene loss in some AAV gene therapy clinical trials, but was insufficient to prevent loss in other studies. We used a nonhuman primate model to evaluate intensive T cell-directed IS combined with AAV-mediated transfer of the human factor IX (FIX) gene. Early administration of rabbit anti-thymocyte globulin (ATG) concomitant with AAV administration resulted in the development of anti-FIX antibodies, whereas delayed ATG by 5 weeks administration did not. The anti-FIX immune response was associated with increases in inflammatory cytokines, as well as a skewed Th17/regulatory T cell (Treg) ratio. We conclude that the timing of T cell-directed IS is critical in determining transgene-product immunogenicity or tolerance. These data have implications for systemically administered AAV gene therapy being evaluated for hemophilia A and B, as well as other genetic diseases.

Preparation and Administration of Adeno-associated Virus Vectors for Corneal Gene Delivery.

Gene delivery approaches using adeno-associated virus (AAV) vectors are currently the preferred method for human gene therapy applications and have demonstrated success in clinical trials for a diverse set of diseases including retinal blindness. To date, no clinical trials using AAV gene therapy in the anterior eye have been initiated; however, corneal gene delivery appears to be an attractive approach for treating both corneal and ocular surface diseases. Multiple preclinical studies by our lab and others have demonstrated efficient AAV vector-mediated gene delivery to the cornea for immunomodulation, anti-vascularization, and enzyme supplementation. Interestingly, the route of AAV vector administration and nuances such as administered volume influence vector tropism and transduction efficiency. In this chapter, a detailed protocol for AAV vector production and specific approaches for AAV-mediated gene transfer to the cornea via subconjunctival and intrastromal injections are described.

Gene therapy for hemophilia.

Gene therapy offers the potential for a cure for patients with hemophilia by establishing continuous endogenous expression of factor VIII or factor IX (FIX) following transfer of a functional gene to replace the hemophilic patient's own defective gene. The hemophilias are ideally suited for gene therapy because a small increment in blood factor levels (≥5% of normal) is associated with significant amelioration of bleeding phenotype in severely affected patients. In 2011, the St. Jude/UCL phase 1/2 trial was the first to provide clear evidence of a stable dose-dependent increase in FIX levels in patients with severe hemophilia B following a single administration of adeno-associated viral (AAV) vectors. Transgenic FIX expression has remained stable at ∼5% of normal in the high-dose cohort over a 7-year follow-up period, resulting in a substantial reduction in spontaneous bleeding and FIX protein usage without toxicity. This study has been followed by unparalleled advances in gene therapy for hemophilia A and B, leading to clotting factor activity approaching normal or near-normal levels associated with a "zero bleed rates" in previously severely affected patients following a single administration of AAV vectors. Thus, AAV gene therapies are likely to alter the treatment paradigm for hemophilia A and B. This review explores recent progress and the remaining limitations that need to be overcome for wider availability of this novel treatment of inherited bleeding disorders.