Adeno-associated Virus Constructs Research Articles

GO-8: Stable Expression of Factor VIII over 5 Years Following Adeno-Associated Gene Transfer in Subjects with Hemophilia a Using a Novel Human Factor VIII Variant

Background: GO-8 (ClinicalTrials.gov: NCT03001830) is a study of liver-directed adeno-associated virus (AAV) gene therapy for haemophilia A (HA) that uses a factor VIII (FVIII) variant containing a 17 amino-acid peptide comprising six N-linked glycosylation motifs from the human FVIII B-domain (AAV-HLP-hFVIII-V3). In preclinical studies, AAV-HLP-hFVIII-V3 mediated a 3-fold higher FVIII expression when compared to an identical AAV construct encoding the hFVIII-SQ variant used in most HA gene therapy trials. Methods: In a multi-centre, open-label, non-randomised, phase I/II clinical trial, we assessed the safety and efficacy of escalating doses of AAV-HLP-hFVIII-V3 pseudotyped with an AAV8 capsid in adults with severe haemophilia A (FVIII activity ≤1%). All participants received prophylactic glucocorticoids, with or without tacrolimus, with the aim of reducing the risk of vector-related transaminase elevation. The primary endpoints were safety and efficacy. Efficacy was assessed by measuring FVIII activity (FVIII: C) using both chromogenic and one-stage clotting assays and factor consumption pre and post-gene therapy. Results: As of May 31 2023, 12 participants were enrolled sequentially into one of four vector doses: 6×1011 vector genomes (vg)/kg body weight (n=1), 2×1012 vg/kg (n=3), 4×1012 vg/kg (n=3), or 6×1012 vg/kg (n=5). All participants were on FVIII prophylaxis prior to gene therapy. The most common vector-related adverse event was an elevation in liver aminotransferase levels, which occurred in 10 of 12 participants. In 7 of the 8 participants treated at doses ≥4×1012 vg/kg, recurrent elevation in aminotransferase levels was observed during the first 12 months, often associated with tapering of immunosuppression. This resulted in a reduction in transgene expression from peak levels in all participants, with a complete loss of transgenic protein in one participant. Vector-related elevation in aminotransferase was not observed after the 12-month time point in long-term follow-up. Mean chromogenic FVIII: C levels at 12 months after gene therapy were 3 IU/dL in the 6×1011 vg/kg cohort, 13±9IU/dL (range: 2-19 IU/dl) in the 2×1012 vg/kg cohort, 8±1IU/dl in the 4×1012 vg/kg cohort (range: 7-9 IU/dl) and 22±34 IU/dl in the 6×1012 vg/kg cohort (range 1-82 IU/dl). Transgene expression was then stably maintained over a median follow-up of 3 years (range: 0.2-5 years) from the level achieved 1-year post-infusion, best illustrated by the data from the 2×1012 and 4×1012 vg/kg cohorts shown in Figure 1. FVIII: C was, on average 2-fold higher when measured using a one-stage clotting assay compared to the chromogenic method. Nine of the 12 participants remained off prophylaxis after gene therapy for the duration of the follow-up period. Baseline mean and median annualised factor VIII use was 4097 and 4657 IU/kg per year before gene therapy. Following gene therapy, the mean and median annualised factor VIII concentrate use reduced across all participants to 1186 and 61 IU/kg (One sample t-test p=0.0009), respectively. No FVIII inhibitors or thrombotic events were reported for the duration of the study. Conclusion: A single infusion of AAV-HLP-hFVIII-V3 resulted in stable FVIII expression over a follow-up period of up to 5 years in participants with severe haemophilia A. A high rate of liver aminotransferase elevation following gene transfer impacted transgene expression. However, 9 of the 12 participants were able to discontinue FVIII prophylaxis over the duration of the study, resulting in a significant reduction in FVIII concentrate usage.

Second Generation Adeno-Associated Viral Vector Using a Novel Factor VIII Variant with Improved Secretion Achieves Higher Factor VIII Expression in Non-Human Primates

Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in functional factor VIII (FVIII). Adeno-associated virus (AAV)-mediated gene therapy for HA is actively in clinical development, primarily utilizing codon-optimized (CO) wild type (WT) B-domain deleted human FVIII (hFVIII) transgenes. Limitations of these approaches highlight the need to develop novel second generation AAV approaches that improve transgene expression while reducing the vector dose with the fundamental goal of achieving durable levels of FVIII in the normal range while minimizing the potential risk of anti-AAV immune responses and genotoxicity. One of the main challenges of HA gene therapy development is that hFVIII is not expressed efficiently in vitro or in vivo compared to other proteins of similar size. Notably, the FVIII proteins of other species, such as canine and porcine, have superior expression. Previously, we reported that canine FVIII is expressed at higher levels than hFVIII which prompted us to conduct a comparative study between these two proteins (Sabatino 2009). Those studies led us to engineer and characterize a series of FVIII variants around the furin cleavage recognition site (1645-48)(Nguyen 2017) and a secondary cleavage site at 1657-58 in the acidic region 3 (a3). After investigating 15 variants, we identified our best performing variant, hFVIII-Δ3-SP/DE, carrying a deletion of three residues at the furin site (1645-47) (Δ3) (Nguyen 2017) and two amino acid substitutions S1657P and D1658E at the a3 cleavage site. With these minimal modifications, hFVIII-Δ3-SP/DE is secreted significantly more efficiently than WT hFVIII with and without codon-optimization and in conjunction with other improvements in the expression cassette. In the setting of AAV gene therapy, we used AAV8 to deliver WT hFVIII-CO or variant hFVIII-Δ3-SP/DE-CO driven by the modified transthyretin (TTRm) promoter and compared levels of FVIII expression in vivo. Immuno-deficient HA/CD4 KO mice administered AAV8-TTRm-hFVIII-Δ3-SP/DE-CO expressed FVIII levels that were 2-3-fold higher than animals delivered WT FVIII-CO. To investigate this variant in a large animal model, we previously injected AAV8-hFVIII-CO and AAV8-hFVIII-Δ3-SP/DE-CO (2x1012 vg/kg) in an HA dog model tolerized to hFVIII. We observed that the levels of hFVIII expression using the variant were 2 to 4-fold higher than WT hFVIII. Given the success of our variant in HA mice and dogs, we investigated hFVIII-Δ3-SP/DE in non-human primates (NHPs). Animals were negative for neutralizing antibodies to AAV8 prior to vector administration. We delivered AAV8-TTRm-hFVIII-CO or AAV8-TTRm-hFVIII-Δ3-SP/DE-CO to male African green monkeys via intravenous infusion at three vector doses (2x1012, 5x1012, 1x1013 vg/kg)(n=3 per dose per AAV construct). hFVIII antigen levels were measured by hFVIII specific ELISA and are expressed as a percentage of normal levels. Two weeks after AAV administration, NHPs treated with low dose (2x1012 vg/kg) of AAV8-hFVIII-CO expressed a median of 7.4% (range 5.2-8.8%) and AAV8-hFVIII-Δ3-SP/DE-CO expressed 19.2% (range 11.4-72.0%). The mid-dose cohort (5x1012 vg/kg) expressed 17.7% (range 11.8-128.2%) after AAV8-hFVIII-CO and 38.2% (range 12.1-97.0%) after AAV8-hFVIII-Δ3-SP/DE-CO delivery. At 1x1013 vg/kg the AAV8-hFVIII-CO treated NHPs expressed 17.9% (range 12.6-54.3%) while the AAV8-hFVIII-Δ3-SP/DE-CO expressed 114.2% (range 20.2-195.6%). As noted in clinical studies, there is some variability in hFVIII expression in our cohorts. Nevertheless, our data suggests that the delivery of the WT hFVIII transgene did not show a dose response. In contrast, there is a dose-dependent increase in hFVIII-Δ3-SP/DE expression, implying improved secretion correlating with higher levels of FVIII expression. As anticipated, anti-hFVIII antibodies developed to the non-species specific transgene in 16 of 18 animals after 3 weeks post vector administration, leading to a decline in hFVIII expression in most animals. In summary, consistent with our observations in HA mice and dogs, AAV delivery of the Δ3-SP/DE variant in NHPs results in 2 to 5-fold higher dose-dependent levels of FVIII expression than the WT hFVIII. This novel hFVIII variant provides the opportunity to overcome the challenge of inefficient FVIII secretion to improve the efficacy of AAV gene therapy for hemophilia A.

Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina.

Direct reprogramming of retinal Müller glia is a promising avenue for replacing photoreceptors and retinal ganglion cells lost to retinal dystrophies. However, questions have recently been raised about the accuracy of studies claiming efficient glia-to-neuron reprogramming in retina that were conducted using GFAP mini promoter-driven adeno-associated virus (AAV) vectors. In this study, we have addressed these questions using GFAP mini promoter-driven AAV constructs to simultaneously overexpress the mCherry reporter and candidate transcription factors predicted to induce glia-to-neuron conversion, in combination with prospective genetic labeling of retinal Müller glia using inducible Cre-dependent GFP reporters. We find that, while control GFAP-mCherry constructs express faithfully in Müller glia, 5 out of 7 transcription factor overexpression constructs tested are predominantly expressed in amacrine and retinal ganglion cells. These findings demonstrate strong insert-dependent effects on AAV-based GFAP mini promoter specificity that preclude its use in inferring cell lineage relationships when studying glia-to-neuron conversion in retina.

Effect of AAV-mediated overexpression of ATF5 and downstream targets of an integrated stress response in murine skeletal muscle

We previously reported that growth promoter-induced skeletal muscle hypertrophy co-ordinately upregulated expression of genes associated with an integrated stress response (ISR), as well as potential ISR regulators. We therefore used Adeno-Associated Virus (AAV)-mediated overexpression of these genes, individually or in combination, in mouse skeletal muscle to test whether they induced muscle hypertrophy. AAV of each target gene was injected into mouse Tibialis anterior (TA) and effects on skeletal muscle growth determined 28 days later. Individually, AAV constructs for Arginase-2 (Arg2) and Activating transcription factor-5 (Atf5) reduced hindlimb muscle weights and upregulated expression of genes associated with an ISR. AAV-Atf5 also decreased Myosin heavy chain (MyHC)-IIB mRNA, but increased MyHC-IIA and isocitrate dehydrogenase-2 (Idh2) mRNA, suggesting ATF5 is a novel transcriptional regulator of Idh2. AAV-Atf5 reduced the size of both TA oxidative and glycolytic fibres, without affecting fibre-type proportions, whereas Atf5 combined with Cebpg (CCAAT enhancer binding protein-gamma) only reduced the size of glycolytic fibres and tended to increase the proportion of oxidative fibres. It is likely that persistent Atf5 overexpression maintains activation of the ISR, thereby reducing protein synthesis and/or increasing protein degradation and possibly apoptosis, resulting in inhibition of muscle growth, with overexpression of Arg2 having a similar effect.

393-P: Combination Treatment with Lisinopril and Empagliflozin Improves Urine and Histological Markers of Diabetic Kidney Disease in Female Renin-AAV UNx db/db Mice

Introduction: Emerging treatments of diabetic kidney disease (DKD) include SGLT2 inhibitors and GLP-1 receptor agonists that are nephroprotective beyond their blood glucose lowering effects. To confirm the translatability of a mouse model for drug discovery in DKD, we tested the efficacy of an ACE inhibitor and a SGLT2 inhibitor in the reninAAV UNx db/db mouse model. Methods: Female db/db mice were injected with a renin-encoding adeno-associated virus construct (reninAAV) to induce hypertension and uninephrectomized (UNx). At 12 weeks of age, dosing with vehicle, lisinopril, empagliflozin, or the combination (combo) was initiated. Plasma and urine markers were measured after 12 weeks of dosing and terminal kidney samples were collected for 3D light sheet microscopy and 2D histology. Results: In reninAAV UNx db/db mice, treatment with empagliflozin and combo reduced fed BG and HbA1c compared to vehicle. Treatment with lisinopril and combo reduced urine ACR and KIM1-to-creatinine compared to vehicle, while treatment with empagliflozin alone worsened urine ACR. Glomerular hypertrophy as assessed by 3D imaging was reduced in combo treated reninAAV UNx db/db mice compared to vehicle, while treatment with empagliflozin alone worsened glomerular hypertrophy. The total number of glomeruli per kidney was unaffected by treatments. Compared to vehicle treatment, lisinopril and combo treatment reduced the fraction of score 3+4 glomeruli, and glomerulosclerosis index (GSI) was reduced by with lisinopril and combo treatment. Treatment with empagliflozin alone worsened GSI. Morphometric analyses showed that lisinopril and combo treatment reduced kidney CD11b and KIM1 load. Conclusion: Responses to the combination treatment with lisinopril and empagliflozin showed improvement of urine and histological markers of DKD. Together, these data confirm the translatability of the reninAAV UNx db/db mouse model of DKD. Disclosure M. V. Østergaard: None. M. Christensen: None. T. Secher: Employee; Self; Gubra, Employee; Spouse/Partner; Novo Nordisk, Stock/Shareholder; Self; Gubra, Stock/Shareholder; Spouse/Partner; Novo Nordisk. J. L. Skytte: None. U. Roostalu: Employee; Self; Gubra. C. G. Salinas: Employee; Self; Gubra, Employee; Spouse/Partner; Novo Nordisk, Stock/Shareholder; Spouse/Partner; Novo Nordisk. F. E. Sembach: None. L. N. Fink: Employee; Self; Gubra, Stock/Shareholder; Self; Novo Nordisk A/S. N. Vrang: Board Member; Self; Gubra, Employee; Self; Gubra, Stock/Shareholder; Self; Gubra.

MO624COMBINATION TREATMENT WITH LISINOPRIL AND EMPAGLIFLOZIN IMPROVES BIOCHEMICAL AND HISTOLOGICAL MARKERS OF DIABETIC NEPHROPATHY IN HYPERTENSIVE UNINEPHRECTOMIZED DB/DB MICE

Abstract Background and Aims Emerging treatments of diabetic kidney disease (DKD) include SGLT2 inhibitors and GLP-1 receptor agonists that are nephroprotective beyond their blood glucose lowering effects. Despite this progress, patients with diabetes are still at risk of developing DKD, and drug discovery remains impeded by the lack of reproducible rodent models exhibiting features of human DKD. To confirm the translatability of an advanced mouse model, we tested standard of care in the setting of type 2 diabetes and hypertension. Method Female db/db mice were injected with a renin-encoding adeno-associated virus construct (reninAAV) to induce hypertension and uninephrectomized (UNx) at 7-8 weeks of age. At 12 weeks of age, daily dosing with vehicle, lisinopril, empagliflozin, or the combination (combo) was initiated. Blood glucose (BG) was measured every third week, while urine albumin-to-creatinine (ACR) and KIM1 were measured in spot urine samples collected before termination at 24 weeks of age. Cystatin C was measured in terminal plasma, while terminal kidney samples were collected for 3D light sheet microscopy and 2D histology: Glomerulosclerosis scoring was performed by AI-assisted automized scoring, whereby glomeruli were given scores from 0 (normal glomerulus) to 4 (glomerulus with global glomerulosclerosis), and morphometric quantification of kidney collagen 3, CD11b, and KIM1 load was performed. Results In reninAAV UNx db/db mice, 12 weeks treatment with empagliflozin and combo reduced fed BG (12.3±1.5 and 13.1±0.9 mM, vehicle: 18.0±1.9 mM) and HbA1c (5.5±0.2 and 5.8±0.2%, vehicle: 7.4±0.03%). Treatment with lisinopril and combo reduced urine ACR (8110±1320 and 2508±346 µg/mg, vehicle: 26,138±1820 µg/mg) and KIM1-to-creatinine (7.5±1.1 and 3.9±0.6 ng/mg, vehicle: 24.8±3.2 ng/mg), while treatment with empagliflozin alone worsened urine ACR (41,523±4670 µg/mg). Glomerular hypertrophy as assessed by 3D imaging was reduced in reninAAV UNx db/db mice combo treated animals compared to vehicle (glomerular volume: 1.46*105±4.3*103 and 1.65*105±4.3*103 µm3), while treatment with empagliflozin alone worsened glomerular hypertrophy (1.83*105 µm3). The total number of glomeruli per kidney was not affected by treatments. Compared to vehicle treatment, lisinopril and combo treatment reduced the fraction of score 3 + 4 glomeruli (% GS 3 + 4 glomeruli: 0.38±0.06 and 0.27±0.03%, vehicle: 0.54±0.05%) Glomerulosclerosis index (GSI) was also reduced by lisinopril and combo treatment (GSI: 2.29±0.10 and 2.04±0.05, vehicle: 2.62±0.09). Treatment with empagliflozin alone worsened GSI (2.95±0.11). Whereas treatments did not significantly affect collagen 3, lisinopril and combo treatment reduced CD11b (total CD11b mass: 0.25±0.03 and 0.24±0.04 mg, vehicle: 0.57±0.07 mg) and KIM1 (total KIM1 mass: 0.25±0.06 and 0.17±0.09 mg, vehicle: 2.01±0.37 mg), whereas empagliflozin did not affect CD11b and KIM1. Conclusion Responses to the combination treatment with lisinopril and empagliflozin showed improvement of urine and histological markers of DKD. Together, these data confirm the translatability of the ReninAAV UNx db/db mouse model of DKD in type 2 diabetes.

Protection against Doxorubicin-Induced Cardiac Dysfunction Is Not Maintained Following Prolonged Autophagy Inhibition.

Doxorubicin (DOX) is a highly effective chemotherapeutic agent used in the treatment of various cancer types. Nevertheless, it is well known that DOX promotes the development of severe cardiovascular complications. Therefore, investigation into the underlying mechanisms that drive DOX-induced cardiotoxicity is necessary to develop therapeutic countermeasures. In this regard, autophagy is a complex catabolic process that is increased in the heart following DOX exposure. However, conflicting evidence exists regarding the role of autophagy dysregulation in the etiology of DOX-induced cardiac dysfunction. This study aimed to clarify the contribution of autophagy to DOX-induced cardiotoxicity by specifically inhibiting autophagosome formation using a dominant negative autophagy gene 5 (ATG5) adeno-associated virus construct (rAAV-dnATG5). Acute (2-day) and delayed (9-day) effects of DOX (20 mg/kg intraperitoneal injection (i.p.)) on the hearts of female Sprague–Dawley rats were assessed. Our data confirm established detrimental effects of DOX on left ventricular function, redox balance and mitochondrial function. Interestingly, targeted inhibition of autophagy in the heart via rAAV-dnATG5 in DOX-treated rats ameliorated the increase in mitochondrial reactive oxygen species emission and the attenuation of cardiac and mitochondrial function, but only at the acute timepoint. Deviation in the effects of autophagy inhibition at the 2- and 9-day timepoints appeared related to differences in ATG5–ATG12 conjugation, as this marker of autophagosome formation was significantly elevated 2 days following DOX exposure but returned to baseline at day 9. DOX exposure may transiently upregulate autophagy signaling in the rat heart; thus, long-term inhibition of autophagy may result in pathological consequences.

Liver-Directed AAV Gene Therapy for Gaucher Disease

Introduction: Gaucher disease (GD), one of the most common lysosomal storage disorders, is an autosomal recessive condition resulting from mutations in the GBA gene that codes for the b-glucocerebrosidase (GCase) enzyme. Over 90% of patients have type 1 GD, which is characterised by lipid engorged macrophages (known as Gaucher cells) in multiple organs, including spleen, liver and bone marrow, with no overt involvement of the central nervous system (CNS). The current standard of care for type 1 GD patients includes enzyme replacement therapy (ERT), which provides good overall therapeutic benefit. However, ERT is administered intravenously every other week, resulting in a high cumulative cost and a significant treatment burden. Furthermore, disease manifestations, such as pulmonary and skeletal disease, remain unresolved with ERT. Gene therapy is emerging as a very promising avenue of treatment for various monogenic disorders and has the potential to provide sustained levels of GCase enzyme expression after a single treatment. Here we have evaluated liver-directed gene therapy in vitro and in vivo for the treatment of GD. Methods: Adeno-associated virus (AAV) constructs were optimised to express full-length wild-type GCase protein (GBA AAV) and packaged in AAV8 capsids for in vivo mouse studies, or our novel AAVS3 capsid for in vitro studies in a human cell line. GCase activity was determined fluorometrically with 4-Methylumbelliferyl-β-D-glucopyranoside and activity was based on a 4-methylumbelliferone standard curve. Levels of GCase in plasma, and uptake in GD target organs were compared between our GBA AAV optimized construct and ERT treatment with velaglucerase alfa (VPRIV®) in C57BL/6 wild type mice. Doses used ranged from 2x109 to 2x1012 vg/kg for GBA AAV constructs and 60 U/kg for ERT. Results: Our initial proof of concept studies for liver-directed AAV gene therapy of GD used an AAV construct encoding the native full-length human GBA cDNA (RC-04-01). After a single intravenous injection into mice, RC-04-01 led to a dose-dependent expression of GCase in liver and robust levels of enzymatically active GCase in plasma. Based on these preliminary data, 37 GBA AAV constructs with optimisations to the coding sequence and changes to the promoter, signal peptide, and polyA sequences were designed and evaluated in vitro for GCase production. Among them, 6 constructs outperformed RC-04-01 and were further tested in mice. Construct RC-04-26 showed the highest GCase activity levels both in vivo and in vitro. At a dose of 2x1012 vg/kg, RC-04-26 showed plasma active GCase levels up to 9.4-fold higher than RC-04-01. Upon GBA AAV infusion, plasma GCase levels were steady and sustained for the duration of the entire study period (9 months). In addition, RC-04-26 infusion into mice resulted in robust uptake of GCase by cells in spleen, bone marrow and lung, demonstrating that liver-produced GCase is taken up by macrophages present in the GD target organs. Lastly, GCase bioavailability was evaluated after a single administration of RC-04-26 or ERT. With velaglucerase alfa, GCase was rapidly cleared from the bloodstream and tissues. However, RC-04-26 resulted in sustained and steady levels of GCase, with an overall bioavailability of GCase uptake over 170-fold higher than with ERT. Conclusions: Our current data support the hypothesis that a single administration of an optimised liver directed GBA AAV vector results in sustained elevation of GCase in the bloodstream and higher level of GCase bioavailability for uptake into macrophages than velaglucerase alfa. This observation supports further development of AAV gene therapy for Gaucher disease with the potential for enhanced therapeutic benefit from a one-off administration. Disclosures Miranda: Freeline Therapeutics: Employment, Equity Ownership. Canavese:Freeline Therapeutics: Employment, Equity Ownership. Chisari:Freeline Therapeutics: Employment, Equity Ownership. Pandya:Freeline: Employment, Equity Ownership. Cocita:Freeline Therapeutics: Employment, Equity Ownership. Portillo:Freeline: Employment, Equity Ownership. McIntosh:Freeline Therapeutics: Consultancy, Equity Ownership. Kia:Freeline Therapeutics: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Dane:Freeline: Employment, Equity Ownership. Jeyakumar:Freeline Therapeutics: Employment, Equity Ownership. Sheridan:Freeline Therapeutics: Employment, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

MicroRNA-579 upregulation mediates death of human macrophages with mycobacterium tuberculosis infection

Mycobacterium tuberculosis (MTB) infection could induce death of host human macrophages, promoting bacterial spread. In the current study we tested the potential role of microRNA-579 (miR-579) in the death of macrophages infected with MTB. In the primary human macrophages MTB infection induced upregulation of miR-579 but downregulation of its mRNA targets, SIRT1 and PDK1, which were accompanied by significant macrophage death and apoptosis. miR-579 inhibition, by its anti-sense sequence, restored SIRT1-PDK1 expression and significantly attenuated MTB-induced cytotoxicity and apoptosis in human macrophages. Conversely, ectopic overexpression of miR-579 further downregulated SIRT1-PDK1 expression and exacerbated MTB-induced cytotoxicity in human macrophages. Further studies showed that cPWWP2A, the miR-579's endogenous sponge circRNA, was downregulated in MTB-infected macrophages. Conversely, forced overexpression of cPWWP2A, by a recombinant adeno-associated virus construct, reversed MTB-induced miR-579 upregulation and macrophage cytotoxicity. Taken together, our results show that miR-579 upregulation mediates MTB-induced macrophage cytotoxicity. Targeting cPWWP2A-miR-579 axis could be a novel strategy to protect human macrophages from MTB infection.

Multiplexed peroxidase-based electron microscopy labeling enables simultaneous visualization of multiple cell types.

Electron microscopy (EM) is a powerful tool for circuit mapping, but identifying specific cell types in EM datasets remains a major challenge. Here we describe a technique enabling simultaneous visualization of multiple, genetically identified neuronal populations so that synaptic interactions between them can be unequivocally defined. We present 15 AAV constructs and six mouse reporter lines for multiplexed EM labeling in the mammalian nervous system. These reporters feature dAPEX2, which exhibits dramatically improved signal compared to previously described ascorbate peroxidases. By targeting this enhanced peroxidase to different subcellular compartments, multiple orthogonal reporters can be simultaneously visualized and distinguished under EM using a protocol compatible with existing EM pipelines. Proof-of-principle double and triple EM labeling experiments demonstrated synaptic connections between primary afferents, descending cortical inputs, and inhibitory interneurons in the spinal cord dorsal horn. Our multiplexed peroxidase-based EM labeling system should therefore greatly facilitate analysis of connectivity in the nervous system.

Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors.

Immune checkpoint inhibitors (ICIs) can block distinct receptors on T cells or tumor cells thus preventing T cell inactivation and tumor immune escape. While the clinical response to treatment with ICIs in cancer patients is impressive, this therapy is often associated with a number of immune-related adverse events. There is therefore a need to explore innovative strategies of tumor-specific delivery of ICIs. Delivery of therapeutic proteins on a genetic level can be accomplished with viral vectors including those derived from adeno-associated virus (AAV). Here, we assessed the tumor-targeted Her2-AAV, a receptor-targeted AAV vector binding to the tumor antigen Her2/neu for cell entry, as vehicle for ICI gene delivery. Initially, we packaged the coding sequence of a scFv-Fc fusion protein directed against mouse programmed cell death protein-1 (PD-1) into Her2-AAV. Upon transduction of Her2/neu+ RENCA cells, AAV-encoded αPD-1 was readily detectable in the cell culture supernatant and revealed specific binding to its target antigen. In vivo, in BALB/c mice bearing subcutaneous RENCA-Her2/neu tumors, Her2-AAV mediated specific gene delivery into tumor tissue upon intravenous administration as verified by luciferase gene transfer and in vivo imaging thus demonstrating unimpaired tumor-targeting by Her2-AAV vectors in immunocompetent animals. When delivering the αPD-1 gene, levels of ICI were similar in tumor tissue for Her2-AAV and AAV2 but substantially reduced in liver for Her2-AAV. When combined with chemotherapy a tendency for reduced progression of tumor growth was documented for Her2-AAV treated mice. To get closer to the clinical situation, AAV constructs that deliver the complete coding sequence of the therapeutic antibody nivolumab which is directed against human PD-1 were generated next. The AAV-Nivolumab constructs were expressed and released from transduced MDA-MB-453 cells in vitro and from RENCA-Her2/neu cells upon intratumoral as well as intravenous administration in vivo. Antibody processing and expression levels were further improved through optimization of construct design. In conclusion, we provide proof-of-principle for redirecting the biodistribution of ICIs from liver and serum to tumor tissue by the use of engineered AAV vectors. This strategy can be easily combined with other types of immunotherapeutic concepts.

Synergistic Effect of Expressed miR-128 and Puma Protein on Targeted Induction of Tumor Cell Apoptosis.

Background Puma is a highly robust pro-apoptotic protein. The protein becomes activated by p53 ensuing beyond-repair DNA damage. Downregulation of SIRT 1, by miR-128, elevates activated p53 that foment Puma indirectly. Objectives In the present study, we used two-expression Adeno-Associated Virus (AAV) system for co-expression of miR-128 and Puma in order to evaluate apoptotic response; both in the tumor and normal cells, respectively. Materials and Methods Three recombinant AAVs constructs were generated. The First rAAV bearing Puma under the control of hTERT (p-AAV), the second construct designed such that to carry miR-128 downstream of CMV (mi-AAV), and the last construct comprises of the both CMV-miR-128 and hTERT- Puma. Real-Time PCR and western blotting were used to evaluate expression levels of the transduced genes. Results MTT assay and DAPI staining shown suicidal effect of each recombinant AAV vectors. p-AAV cytotoxicity was recorded for 62% of the tumor cells, while for normal cells it was only 20% cytotoxic. The second construct, mi-AAV, was not as potent and selective as p-AAV. This construct was shown to be 27% and 16% cytotoxic for BT-474 and HEK-293 cells, respectively. Co-expression of Puma and miR-128 (p-mi-AAV) was accomplished with a selective cytotoxicity toward BT-474. This construct was 85% toxic for tumor cells, although it was only 25% toxic for the normal cell line (HEK-293). Conclusions In this study, we have shown that not only Puma is able to instigate apoptotic response but also its co-expression along with miR-128 could significantly enhance apoptosis in a synergistic manner.

180. The Role of the Pyruvate Dehydrogenase Kinase 4 (PDK4) Gene Mutation in the Development of Dilated Cardiomyopathy in Dobermanpinschers and Potential Application of Cardiac Gene Therapy

Dilated cardiomyopathy (DCM) is estimated to be the third most common inherited type of heart disease in humans reported to affect 35.6 and 0.57 in 100,000 adults and children, respectively. The Doberman Pinscher (DP) is a canine breed affected with idiopathic, non-ischemic DCM that closely resembles the human counterpart acting as a clinically relevant human model. Recently, a 16 base pair deletion in the 5’ donor splice site of intron 10 of the pyruvate dehydrogenase kinase 4 (PDK4) gene, encoding for a mitochondrial protein involved in energy metabolism, has been identified but the relationship between the PDK4 mutation, mitochondrial function and phenotypic development of DCM is largely unknown. Our research has focused on understanding the relationship between the consequences of the PDK4 mutation on mitochondrial function and phenotypic development of DCM as well as the potential of cardiac gene therapy to correct the phenotype. A total of 64 DP were tested for the PDK4 mutation and screened for DCM. Healthy dogs and dogs diagnosed with DCM were further divided into 3 groups based on the presence of the genetic mutation: Wild-type (PDK4wt/wt), Heterozygous (PDK4wt/del), and Homozygous (PDK4del/del). Preliminary analyses using isolated mitochondria from skin fibroblasts of these dogs demonstrated a lack of detectable PDK4 protein expression in PDK4del/del, and a significant decrease in PDK4wt/del (48% of normal). Phase contrast microscopy and immunofluorescence studies revealed significantly different morpho-phenotypic characteristics and reduced capacity to adapt to unfavorable metabolic conditions as compared to healthy dogs. Assessment of mitochondrial metabolic potential and function (oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)) were performed using an extracellular flux analyzer. Cells were stressed using serum glucose starvation. Compared to healthy dogs, PDK4wt/del and PDK4del/del revealed a much lower basal respiration rate (OCR reduction of 46% and 68% of normal, respectively) and decreased metabolic potential to meet energy demands (85% and 72% of normal, respectively). Maximal mitochondrial respiration and spare respiratory capacity were all significantly lower in the affected dogs (PDK4wt/del 42% and 14%, and PDK4del/del 40% and 1.4% of normal OCR, respectively). Our results indicate that the mitochondria of affected dogs have a lower metabolic capacity and thus exhaust the two major energy-producing pathways, aerobic and glycolytic metabolism, at a much faster rate when under stress compared to healthy control dogs. Although we detected a significant decrease in PDK4 protein levels, PDK4 transcription levels have yet to be determined in these samples. Our immediate future goals include 1) quantitative PCR to compare PDK4 transcript levels, 2) PCR and sequencing to identify abnormalities in PDK4 mRNA splicing in affected dogs, 3) assessments of mitochondria ATP production and PDK4 protein kinase function. Our long-term goal is to perform in vitro experiments to assess if mitochondrial abnormalities can be improved with gene therapy. To this end, we have cloned canine healthy PDK4 into an adeno-associated virus construct. Ultimately, our efforts will lead us to a cardiac gene therapy clinical trial for Doberman PDK4-DCM.

Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease.

Pompe disease is a progressive neuromuscular disorder caused by lysosomal accumulation of glycogen from a deficiency in acid alpha-glucosidase (GAA). Replacement of the missing enzyme is available by repeated protein infusions; however, efficacy is limited by immune response and inability to restore enzymatic function in the central nervous system. An alternative therapeutic option is adeno-associated virus (AAV)-mediated gene therapy, which results in widespread gene transfer and prolonged transgene expression. Both enzyme replacement therapy (ERT) and gene therapy can elicit anti-GAA immune reactions that dampen their effectiveness and pose life-threatening risks to patient safety. To modulate the immune responses related to gene therapy, we show that a human codon-optimized GAA (coGAA) driven by a liver-specific promoter (LSP) using AAV9 is capable of promoting immune tolerance in a Gaa(-/-) mouse model. Copackaging AAV9-LSP-coGAA with the tissue-restricted desmin promoter (AAV9-DES-coGAA) demonstrates the necessary cell autonomous expression in cardiac muscle, skeletal muscle, peripheral nerve, and the spinal cord. Simultaneous high-level expression in liver led to the expansion of GAA-specific regulatory T-cells (Tregs) and induction of immune tolerance. Transfer of Tregs into naïve recipients prevented pathogenic allergic reactions after repeated ERT challenges. Copackaged AAV9 also attenuated preexisting humoral and cellular immune responses, which enhanced the biochemical correction. Our data present a therapeutic design in which simultaneous administration of two copackaged AAV constructs may provide therapeutic benefit and resolve immune reactions in the treatment of multisystem disorders.

Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome.

Mutations in the potassium channel subunit KCNQ1 cause the human severe congenital deafness Jervell and Lange-Nielsen (JLN) syndrome. We applied a gene therapy approach in a mouse model of JLN syndrome (Kcnq1−/− mice) to prevent the development of deafness in the adult stage. A modified adeno-associated virus construct carrying a Kcnq1 expression cassette was injected postnatally (P0–P2) into the endolymph, which resulted in Kcnq1 expression in most cochlear marginal cells where native Kcnq1 is exclusively expressed. We also found that extensive ectopic virally mediated Kcnq1 transgene expression did not affect normal cochlear functions. Examination of cochlear morphology showed that the collapse of the Reissner’s membrane and degeneration of hair cells (HCs) and cells in the spiral ganglia were corrected in Kcnq1−/− mice. Electrophysiological tests showed normal endocochlear potential in treated ears. In addition, auditory brainstem responses showed significant hearing preservation in the injected ears, ranging from 20 dB improvement to complete correction of the deafness phenotype. Our results demonstrate the first successful gene therapy treatment for gene defects specifically affecting the function of the stria vascularis, which is a major site affected by genetic mutations in inherited hearing loss.

Neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma.

Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP), leading to optic nerve head (ONH) cupping, axon loss, and apoptosis of retinal ganglion cells (RGCs), which could ultimately result in blindness. Brn3b is a class-4 POU domain transcription factor that plays a key role in RGC development, axon outgrowth, and pathfinding. Previous studies suggest that a decrease in Brn3b levels occurs in animal models of glaucoma. The goal of this study was to determine if adeno-associated virus (AAV)-directed overexpression of the Brn3b protein could have neuroprotective effects following elevated IOP-mediated neurodegeneration. Intraocular pressure was elevated in one eye of Brown Norway rats (Rattus norvegicus), following which the IOP-elevated eyes were intravitreally injected with AAV constructs encoding either the GFP (rAAV-CMV-GFP and rAAV-hsyn-GFP) or Brn3b (rAAV-CMV-Brn3b and rAAV-hsyn-Brn3b). Retina sections through the ONH were stained for synaptic plasticity markers and neuroprotection was assessed by RGC counts and visual acuity tests. Adeno-associated virus-mediated expression of the Brn3b protein in IOP-elevated rat eyes promoted an upregulation of growth associated protein-43 (GAP-43), actin binding LIM protein (abLIM) and acetylated α-tubulin (ac-Tuba) both posterior to the ONH and in RGCs. The RGC survival as well as axon integrity score were significantly improved in IOP-elevated rAAV-hsyn-Brn3b-injected rats compared with those of the IOP-elevated rAAV-hsyn-GFP- injected rats. Additionally, intravitreal rAAV-hsyn-Brn3b administration significantly restored the visual optomotor response in IOP-elevated rat eyes. Adeno-associated virus-mediated Brn3b protein expression may be a suitable approach for promoting neuroprotection in animal models of glaucoma.

Targeting Sarcoplasmic Reticulum Calcium ATPase by Gene Therapy

Although pharmacologic therapies have provided gains in reducing the mortality of heart failure, the rising incidence of the disease requires new approaches to combat its health burden. Twenty-five years ago, abnormal calcium cycling was identified as a characteristic of failing human myocardium. Sarcoplasmic reticulum calcium ATPase (SERCA2a), the sarcoplasmic reticulum calcium pump, was found to be a key factor in the alteration of calcium cycling. With the advancement of gene vectors, SERCA2a emerged as an attractive clinical target for gene delivery purposes. Using adeno-associated virus constructs, SERCA2a upregulation has been found to improve myocardial function in animal models. The clinical benefits of overexpressing SERCA2a have been demonstrated in the phase I study Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID). This study has demonstrated that a persistent expression of the transgene SERCA2a is associated with a significant improvement in associated biochemical alterations and clinical symptoms of heart failure. In the coming years, additional targets will likely emerge that are amenable to genetic manipulations along with the development of more advanced vector systems with safer delivery approaches.

Evaluation of Lateral Spread of Transgene Expression following Subretinal AAV–Mediated Gene Delivery in Dogs

Dog models with spontaneously occurring mutations in retinal dystrophy genes are an invaluable resource for preclinical development of retinal gene therapy. Adeno-associated virus (AAV) vectors have been most successful; to target the outer retina and RPE they are delivered by subretinal injection, causing a temporary retinal detachment with some potential for retinal morbidity. A recent reporter gene study using an AAV2/8 vector in dogs reported transgene expression beyond the boundary of the subretinal bleb. This could be a desirable feature which increases the area of retina treated while minimizing the retinal detachment and any associated morbidity. We performed a detailed study of the lateral spread of transgene expression beyond the subretinal injection site following subretinally delivered AAV vectors in normal dogs. Vectors expressed green fluorescent protein (GFP) using a small chicken beta-actin promoter. AAV2/2 (quadruple tyrosine to phenylalanine (Y-F) capsid mutant), self-complementary (sc) AAV2/8 (single Y-F capsid mutant) and a scAAV2/5 were used. We found that in all eyes GFP expression involved retina beyond the initial post-injection subretinal bleb boundary. In all eyes there was post-injection spread of the retinal detachment within the first 3 days post procedure and prior to retinal reattachment. In 11/16 eyes this accounted for the entire “lateral spread” of GFP expression while in 5/16 eyes a very slight extension of GFP expression beyond the final boundary of the subretinal bleb could be detected. All 3 AAV constructs induced GFP expression in the nerve fiber layer with spread to the optic nerve. Patients treated by subretinal injection should be monitored for possible expansion of the subretinal injection bleb prior to reattachment. Injections in the para-foveal region may expand to lead to a foveal detachment that may be undesirable. Cell-specific promoters may be required to limit spread of expressed transgene to the brain with these AAV serotypes.

A model of nitric oxide induced α-synuclein misfolding in Parkinson's disease

Inducible nitric oxide synthase (iNOS) upregulation and consequent NO formation are well-recognized neuroinflammatory responses associated with Parkinson's disease (PD). These contribute to nitrosative protein modifications affecting neuronal injury and cell death. Indeed, a pathobiologic signature for PD is Lewy body formation containing misfolded and aggregated forms of alpha-synuclein (α-syn). Moreover, nitration of α-syn promotes protein aggregation in disease. To model such pathological events, we constructed controllable iNOS and bicistronic α-syn-IRES-tTA adeno-associated virus (AAV) expression vectors. Transduction of iNOS and α-syn AAV constructs led to nitration of α-syn in neurons and overexpression of iNOS promoted protein aggregation. We posit that this AAV system mimics critical protein misfolding events associated with the pathogenesis of PD.

P3‐002: Overexpression of tau in the mouse forebrain using adeno‐associated virus (AAV) leads to tau hyperphosphorylation, formation of neurofibrillary tangles and neurodegeneration

The use of viral vectors provides an alternative approach to replicate cardinal features of tauopathies in the mouse brain and complement existing transgenic mouse models. AAV vectors were designed for overexpression of various forms of human tau, and injected in the lateral ventricle to induce tau pathology in the mouse forebrain. We generated AAV constructs encoding various forms of the human tau protein under the control of the mouse PGK-1 promoter. Two wild-type versions of human tau (tau4R0N, tau3R0N) were compared with a tau mutant causing frontotemporal dementia (tau4R0N-P301S), and an aggregation-deficient mutated variant (tau4R0N-I277P/I308P). AAV serotype 6 vectors were bilaterally injected in the lateral ventricles of mouse neonates using a stereotactic system (ICV injections). In order to compare the kinetic of the pathology induced by these different forms of tau, the injected mice were analyzed at 6, 9 and 24 weeks post-injection. We monitored by histological analysis the deposition of phosphorylated and aggregated forms of tau in the mouse cortex and hippocampus, as well as the induced neurodegeneration. Changes in animal motor performance were assessed behaviorally (rotarod test). AAV-injected mice displayed a clear neuronal accumulation of human tau protein in various brain regions including the cerebral cortex, hippocampus and brainstem. Expectedly, high levels of human tau were predominantly found in brain regions lining the ventricular system. At an early time point, we showed significant accumulation of hyperphosphorylated tau protein (AT8 staining) in the neuronal cell bodies as well as in the dendritic compartment. Tau aggregation was revealed by immunohistochemistry using a conformational antibody and confirmed by Gallyas staining at later time points. A significant neurodegeneration was also observed, mostly in the region of the cerebral cortex close to the site of vector injection. In addition, the motor performance of the AAV-injected mice became progressively impaired. ICV delivery of AAV vectors in mouse neonates can efficiently induce neuronal tau expression in the forebrain. The resulting accumulation of human tau leads to a robust neuropathology mainly characterized by tau aggregation. The observed pathology correlates with acute neurodegeneration and progressive impairment of animal motor behavior.