Vector Administration Research Articles

S-50-1: INCREASED FIBROBLAST GROWTH FACTOR-21 IN CHRONIC KIDNEY DISEASE IS A TRADE-OFF BETWEEN SURVIVAL BENEFIT AND BLOOD PRESSURE DYSREGULATION

Circulating levels of fibroblast growth factor-21 (FGF21) start increasing in patients with chronic kidney disease (CKD) since early stages during the cause of disease progression. FGF21 is a liver-derived hormone that induces responses to stress through acting on the suprachiasmatic nucleus in hypothalamus to activate the sympathetic nervous system and the hypothalamus-pituitary-adrenal endocrine axis. However, roles that FGF21 plays in pathophysiology of CKD remains elusive. Here we show in mice that FGF21 is required to survive CKD but responsible for blood pressure dysregulation. CKD was introduced to mice by uninephrectomy followed by high phosphate diet feeding for 6 weeks. FGF21-deficient CKD mice died earlier than wild-type CKD mice. Paradoxically, FGF21-deficeint CKD mice escaped several complications observed in wild-type CKD mice. Wild-type CKD mice exhibited increased variability of systolic blood pressure due to augmentation of blood pressure elevating response during physical activity. The blood pressure dysregulation in wild-type CKD mice was associated with activation of sympathetic nervous activity and suppression of parasympathetic nervous activity during physical activity, and an increase in serum noradrenalin and corticosterone levels. These CKD complications observed in wild-type mice were absent in FGF21-deficeint CKD mice but recapitulated in wild-type non-CKD mice overexpressing FGF21 by administration of an FGF21-expressing adeno-associated virus vector. These findings indicate that FGF21 is necessary and sufficient to induce blood pressure dysregulation. We conclude that increased FGF21 in CKD can be viewed as a survival response at the sacrifice of blood pressure homeostasis.

LncRNA MIAT Upregulates NEGR1 by Competing for miR-150-5p as a Competitive Endogenous RNA in SCIRI Rats.

Spinal cord ischemia-reperfusion injury (SCIRI) can cause a pathological state of irreversible delayed death of neurons in the spinal cord tissue and a range of complications, such as spinal cord dysfunction and motor function impairment. This study aimed to determine whether the long-stranded non-coding ribonucleic acid (lncRNA), myocardial infarction-associated transcript (MIAT), could upregulate neuronal growth regulator 1 (NEGR1) by competing for miR-150-5p as a competitive endogenous RNA in a rat SCIRI model. The MIAT knockdown vector or the corresponding blank vector was injected into the spinal cord of healthy sprague Dawley (SD) rats. Administration of the MIAT knockdown vector led to the establishment of the SCIRI rat model. Basso, Beattie & Bresnahan locomotor rating scale (BBB) assessment of hind limb motion. Pathological changes in the spinal cord were observed via hematoxylin and eosin staining and eosin staining. Quantitative polymerase chain reaction was performed to determine the expression levels of the candidate microRNAs and predicted candidate genes, and the relationship between them. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining was used to detect apoptosis in the spinal cord tissue of rats in each group. Western blotting was performed to determine the expression of the apoptosis-related proteins, caspase-9, caspase-3, and BCL2-Associated X (Bax)/B-cell lymphoma-2 (Bcl-2). The luciferase reporter gene was used to assess the interaction among the lncRNA, MIAT, and miR-150-5, and the interaction between miR-150-5 and NEGR1. The sh-lncRNA, MIAT, improved exercise status, and pathological changes in the spinal cord of SCIRI rats, inhibited apoptosis, increased the expression of miR-150-5p, and reduced the expression of NEGR1. Compared with mimics-NC, the transfection of miR-150-5p significantly decreased the relative fluorescence activity ratio of MIAT 3'-untranslated region (3'-UTR) wild-type Human embryonic kidney cells 293 (HEK-293 cells). Compared with mimics-negative control (NC), the transfection of miR-150-5p significantly decreased the relative fluorescence activity ratio of NEGR1 3'-UTR wild-type HEK-293 cells. MIAT can affect the symptoms of SCIRI in rats. Furthermore, as a competitive endogenous RNA, MIAT upregulates NEGR1 by competing with miR-150-5p in SCIRI rats.

Full-Lung Prophylaxis against SARS-CoV-2 by One-Shot or Booster Intranasal Lentiviral Vaccination in Syrian Golden Hamsters

Following the breakthrough of numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in recent months and the incomplete efficiency of the currently available vaccines, development of more effective vaccines is desirable. Non-integrative, non-cytopathic and non-inflammatory lentiviral vectors elicit sterilizing prophylaxis against SARS-CoV-2 in preclinical animal models and are particularly suitable for mucosal vaccination, which is acknowledged as the most effective in reducing viral transmission. Here, we demonstrate that a single intranasal administration of a vaccinal lentiviral vector encoding a stabilized form of the original SARS-CoV-2 Spike glycoprotein induces full-lung protection of respiratory tracts and strongly reduces pulmonary inflammation in the susceptible Syrian golden hamster model against the prototype SARS-CoV-2. In addition, we show that a lentiviral vector encoding stabilized Spike of SARS-CoV-2 Beta variant (LV::SBeta-2P) prevents pathology and reduces infectious viral loads in lungs and nasal turbinates following inoculation with the SARS-CoV-2 Omicron variant. Importantly, an intranasal boost with LV::SBeta-2P improves cross-seroneutralization much better in LV::SBeta-2P-primed hamsters than in their counterparts primed with an LV-encoding Spike from the ancestral SARS-CoV-2. These results strongly suggest that an immune imprint with the original Spike sequence has a negative impact on cross-protection against new variants. Our results tackle the issue of vaccine effectiveness in people who have already been vaccinated and have vanished immunity and indicate the efficiency of LV-based intranasal vaccination, either as a single dose or as booster.

Significance of Preexisting Vector Immunity and Activation of Innate Responses for Adenoviral Vector-Based Therapy.

An adenoviral (AdV)-based vector system is a promising platform for vaccine development and gene therapy applications. Administration of an AdV vector elicits robust innate immunity, leading to the development of humoral and cellular immune responses against the vector and the transgene antigen, if applicable. The use of high doses (1011-1013 virus particles) of an AdV vector, especially for gene therapy applications, could lead to vector toxicity due to excessive levels of innate immune responses, vector interactions with blood factors, or high levels of vector transduction in the liver and spleen. Additionally, the high prevalence of AdV infections in humans or the first inoculation with the AdV vector result in the development of vector-specific immune responses, popularly known as preexisting vector immunity. It significantly reduces the vector efficiency following the use of an AdV vector that is prone to preexisting vector immunity. Several approaches have been developed to overcome this problem. The utilization of rare human AdV types or nonhuman AdVs is the primary strategy to evade preexisting vector immunity. The use of heterologous viral vectors, capsid modification, and vector encapsulation are alternative methods to evade vector immunity. The vectors can be optimized for clinical applications with comprehensive knowledge of AdV vector immunity, toxicity, and circumvention strategies.

Role of PVAT in obesity-related cardiovascular disease through the buffering activity of ATF3.

Thoracic aortic perivascular adipose tissue (PVAT) is an adipose organ exhibiting similarities to brown adipose tissue (BAT), including cellular morphology and thermogenic gene expression. However, whether the PVAT phenotype is indistinguishable from the BAT phenotype in physiological vasculature remains unclear. We demonstrated that PVAT is distinguishable from classical BAT, given its specific vessel-tone-controlling function. Activating transcription factor 3 (ATF3) is a key factor in hypertension. Compared with wild-type mice, ATF3-deficient (ATF3 -/- ) mice fed a high-fat diet exhibited elevated mean arterial pressure, increased monocyte chemoattractant protein-1 expression and hypertrophy, plus abnormal fatty tissue accumulation in the thoracic aortic PVAT, and enhanced vascular wall tension and vasoconstrictive responses of potassium chloride, U46619, and norepinephrine in isolated aortic rings, which were restored after administration of adeno-associated ATF3 vector. We suggest that PVAT, not BAT, modulates obesity-related vascular dysfunction. ATF3 within PVAT could provide new insights into the pathophysiology of obesity-related cardiovascular diseases.

Affilin-based retargeting of adenoviral vectors to the epidermal growth factor receptor

IntroductionTreatment of head and neck squamous cell carcinomas (HNSCC) by oncolytic adenoviral vectors holds promise as an efficient anti-cancer therapy. The epidermal growth factor receptor (EGFR) represents an attractive target receptor since it is frequently overexpressed in many types of HNSCC. MethodsTo achieve EGFR-specific targeting by human adenovirus type 5 (HAdV-5) based vectors, the EGFR affinity ligand Affilin was covalently attached in a position specific manner either to the fiber or the hexon protein of the vector capsid. In vitro and in vivo studies investigated EGFR-specific cancer cell transduction, susceptibility to natural sequestration mechanisms, pharmacokinetics and biodistribution profiles of Affilin-decorated vectors. ResultsAffilin-decorated vectors showed strongly enhanced and EGFR-specific cancer cell transduction in vitro and less susceptibility to known sequestration mechanisms of HAdV-5 particles. However, in vivo neither systemic nor intratumoral vector administration resulted in an improved transduction of EGFR-positive tumors. Comprehensive analyses indicated hampered EGFR-targeting by Affilin-decorated vectors was caused by rapid vector particle consumption due to binding to the murine EGFR, insufficient tumor vascularization and poor target accessibility for Affilin in the solid tumor caused by a pronounced tumor stroma. ConclusionIn vitro studies yielded proof-of-concept results demonstrating that covalent attachment of a receptor-specific Affilin to the adenoviral capsid provides an effective and versatile tool to address cancer-specific target receptors by adenoviral vectors. Regarding EGFR as the vector target, off-target tissue transduction and low receptor accessibility within the tumor tissue prevented efficient tumor transduction by Affilin-decorated vectors, rendering EGFR a difficult-to-target receptor for adenoviral vectors.

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.

Long-Term Durable FVIII Expression with Improvements in Bleeding Rates Following AAV-Mediated FVIII Gene Transfer for Hemophilia A: Multiyear Follow-up on the Phase I/II Trial of SPK-8011

Background: Hemophilia A (HA) is an inherited blood disorder caused by deficiency or dysfunction of factor (F)VIII. The disease is characterized by recurrent bleeding, particularly into joints, which often results in arthropathy and functional disability. HA is currently managed with intravenous (IV) infusions of replacement FVIII or non-factor replacement therapy such as emicizumab. More recently, gene therapy, involving the use of a recombinant adeno-associated viral (AAV) vector to target hepatocytes, with the aim of safely imparting long-term stable FVIII expression to prevent bleeding, is being investigated for HA. SPK-8011 is an investigational AAV vector optimized to achieve clinically relevant and durable hepatocyte-derived FVIII expression at the lowest possible vector dose. Methods: This Phase I/II trial (NCT03003533/NCT03432520) of SPK-8011 is an open-label, multicenter, non-randomized trial, the full methodology of which has been reported previously (George et al. N Engl J Med 2021). Eligible participants were males ≥18 years of age, with FVIII levels ≤2%, >150 exposure days to FVIII concentrates, and no FVIII inhibitors. All participants were negative for neutralizing antibodies to SPK200. Participants were enrolled into four dose cohorts receiving a single IV dose of either 5×1011, 1×1012, 2×1012, or 1.5×1012 vector genomes (vg) per kilogram of body weight, with a maximum dose based on a BMI of 30 kg/m2. Several immunomodulation (IM) approaches were evaluated. Trial objectives included evaluation of the safety and efficacy of SPK-8011 and the extent and durability of FVIII expression. Results: At data cut-off (30 June 2022), 23 participants were included across dosing cohorts of 5×1011 vg/kg (n=2), 1×1012 vg/kg (n=3), 1.5×1012 vg/kg (n=9), or 2×1012 vg/kg (n=9). Median (range) observation time was 159.3 (6.9-259.9) weeks, based on both the primary study 8011-101 and the long-term follow-up study. A total of 49 treatment-related adverse events (AEs) were reported; 23 were considered vector-related, including one serious adverse event (Grade 2 alanine transaminase [ALT] elevation resulting in elective hospitalization for IV corticosteroid administration), and 26 were related to IM therapies. No FVIII inhibitors or thrombotic events were reported. Transient non-sustained asymptomatic ALT elevation occurred in 11 participants during the expected window of presumed capsid immune response and resolved with use of IM agents. In 16 participants with ≥1 year follow-up, a one-stage FVIII assay showed no apparent decrease in FVIII activity over time, with a majority expressing in the mild HA range (Table). Sustained expression of FVIII was maintained in 21/23 participants with several methods of IM; two participants lost FVIII expression following a presumed capsid immune response (George et al. N Engl J Med 2021). Across all 21 participants with sustained FVIII expression, a 91% (95% CI: 78-97%) reduction in annualized bleeding rate (ABR) for all bleeds was observed, with all-bleed ABR of 11.59 (7.39-18.17) before infusion vs. 0.99 (0.46-2.14) after infusion (Figure). The 16 participants on prophylactic treatment before gene therapy had an 82% (95% CI: 53-93%) reduction in ABR for all bleeds; 6.88 (4.16-11.36) before vs. 1.26 (0.57-2.77) after infusion. ABRs for spontaneous bleeds were 5.20 (2.99-9.04) and 0.52 (0.19-1.39) before and after infusion, respectively. The five participants using on-demand treatment before infusion had a 99% (95% CI: 98-100%) reduction in all-bleed ABR; 27.26 (18.80-39.53) vs. 0.20 (0.09-0.45) before and after infusion, respectively, and 20.98 (12.29-35.82) vs. 0.05 (0.01-0.29) for spontaneous bleeds. In total, 17/21 (81%) participants had an ABR of <1 for treated bleeds and 20/21 (95%) had an ABR of <1 for spontaneous treated bleeds following infusion of SPK-8011. Median (IQR) annualized FVIII infusion rates (AIRs) of 0.2 (0.0-1.2) were reported across all participants 28 days post vector infusion vs. 82.0 (36.0-106.0) before vector administration. Conclusions: With up to 5 years of follow-up, a single infusion of SPK-8011 resulted in durable year-to-year FVIII expression and clinically meaningful reductions in ABR and AIR. No major safety signals were reported, indicating SPK-8011 was well tolerated in this population of people with HA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Effects of Immunomodulation with Corticosteroids to Manage an AAV Capsid Immune response in the Phase I/II Study of SPK-8011

Background: Hemophilia A (HA) is an inherited blood disorder caused by deficiency or dysfunction of coagulation factor VIII (FVIII). SPK-8011 is an adeno-associated viral (AAV) vector gene therapy targeting hepatocyte expression of FVIII, aiming to provide durable FVIII expression and long-term bleed prevention. A Phase I/II trial of SPK-8011 (NCT03003533/NCT03432520) has demonstrated multiyear sustained FVIII expression and notably reduced bleeding in people with HA, with no major safety concerns. There are challenges around preventing and managing cellular immune responses to transduced hepatocytes. Here, we present the outcomes of participants who received corticosteroids following vector administration to prevent and/or treat a presumed AAV capsid immune response. Methods: In this open-label, multicenter, non-randomized, dose-escalation trial, people with HA were infused with SPK-8011 at one of four doses ranging from 5×1011 to 2×1012 vg/kg (full methods published in George et al. New Engl J Med 2021). Participants had baseline FVIII activity of ≤2% of normal levels and were negative for neutralizing antibodies to SPK200. Participants were monitored for 52 weeks after vector administration and enrolled in a 4-year long-term follow-up trial. Corticosteroids were used reactively at the investigators’ discretion upon a presumed capsid immune response, i.e., decrease in FVIII expression, increase in alanine aminotransferase (ALT) level, and/or positive peripheral blood mononuclear cells using interferon-γ enzyme-linked immunospot (ELISpot) to SPK200 peptides. Following loss of FVIII expression in two participants, a prophylactic regimen was explored. Assessments included duration of corticosteroid use and associated adverse events (AEs). Results: Twenty-three males with HA were dosed with SPK-8011 and 17 received corticosteroids (12 reactively, 5 prophylactically); five did not have evidence of a capsid immune response, and one remains in the window of a capsid immune response in the follow-up period (data cut-off: 30 Jun 2022). The median (range) time to initiation of reactive corticosteroids was 9.3 (4.3-12.1) weeks post vector infusion in the 12 participants, with a median (range) duration of treatment of 8.5 (2-18) weeks. Two of these 12 participants completely lost FVIII expression following a presumed capsid immune response (one initiated emicizumab; one resumed FVIII prophylaxis); FVIII expression was maintained in the remaining 10 participants. Corticosteroids were administered prophylactically (2-4 weeks post vector) in five participants, with transgene expression maintained in all five. In four of these five participants, length of exposure was prolonged (31-49 weeks), with attempted corticosteroid tapering resulting in decreased FVIII expression, increased ALT and/or positive ELISpot assays outside the typical window of capsid immune responses. Four participants had AEs related to corticosteroid therapy (Table), with weight gain (8.7%; n=2) and insomnia (8.7%; n=2) being the most common. The occurrence of AEs attributed to corticosteroids increased with duration of use, particularly in those with exposure of >30 weeks. Prophylactic corticosteroids did not consistently prevent a presumed capsid immune response in these five participants and four experienced prolonged courses of corticosteroids necessitating steroid-sparing agents (Figure). All five participants who received prophylactic corticosteroids and four participants who received reactive corticosteroids experienced a recurrence of one or more laboratory trigger(s) potentially linked to a presumed capsid immune response during their corticosteroid taper. Subsequent increased corticosteroid use did not consistently resolve these laboratory changes. Conclusions: Overcoming the challenge of capsid immune responses to achieve long-term stable and predictable FVIII expression remains a goal of AAV gene transfer for HA. Reactive oral corticosteroids were well tolerated but did not prevent loss of FVIII expression in all participants. Early prophylactic corticosteroids did not consistently prevent presumed capsid immune responses and resulted in prolonged immunomodulatory courses with associated AEs. Data so far have permitted refinement of laboratory triggers and investigation is ongoing to identify an alternative, safe and optimal immunomodulatory regimen. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Vector Analysis of Multicentric Lymphoma in a Severe Hemophilia Α Dog after AAV Gene Therapy

Background Adeno-associated viral (AAV) vector gene therapy for severe hemophilia A (HA) is approaching a clinical reality with multiple successful clinical trials. Despite this success, questions remain about the long-term safety of AAV vectors, especially regarding genomic integration and resulting oncogenesis. Studies of neonatal mice have demonstrated AAV integrations that drive hepatocellular carcinoma formation (Donsante et al. Science, 2007). Studies in HA canine models have also demonstrated AAV integrations and clonal expansion of hepatocytes with integrated vector genomes, but no evidence of tumor formation (Nguyen et al. Nat Biotechnol, 2021). Additionally, three tumors have been reported in AAV gene therapy recipients with HA or hemophilia B in clinical trials, without evidence of vector integrations driving oncogenesis. Though overall reassuring, these data highlight that the field needs to remain vigilant about the possibility of AAV integrations driving cancer development. Thus, the assessment of tumors in gene therapy recipients remains important for the AAV field. Aims To determine the role of AAV vector in the development of multicentric lymphoma in a severe HA dog that received liver-directed gene therapy. Methods Vector genome copy numbers in relevant tissues were determined by real-time PCR with primers situated in the 3'-end of the promoter and the 5'- end of the transgene. The vector copy number was calculated by comparing the analyte amplification signal against a standard curve of the plasmid DNA. Results A privately owned mixed-breed dog (PC9) with a history of recurrent spontaneous bleeding events presented with canine factor VIII (cFVIII) levels <1% normal, consistent with severe HA. At 9-months of age, he received liver-directed AAV8 gene therapy with a high-expressing canine factor FVIII (cFVIII-ΔF/V3) variant transgene at a dose of 6 x 1012 vg/kg via intravenous administration. Following gene therapy, his cFVIII levels increased to 2-3% normal and remained stable through duration of follow-up. His annualized bleeding rate decreased from 8 pre-gene therapy to 0 post-gene therapy. At 3.5 years after vector administration, PC9 was diagnosed with a widely disseminated cancer and euthanized due to complications of the disease. At autopsy, he was diagnosed with multicentric lymphoma involving peripheral and visceral lymph nodes, spleen, liver, and lungs. The average age of lymphoma diagnosis in dogs is between 6-9 years of age (Edwards et al. Vet Comp Oncol, 2003), so the development of cancer at an early age in a gene therapy recipient warranted additional study. Vector copy number analysis showed that the liver had the highest levels of AAV vector genomes, as expected. Liver samples showed a heterogenous distribution of vector genomes, consistent with previous studies of AAV8 transduction (Bell et al. Hum Gene Ther, 2011). Splenic samples also had a high level of AAV vector genomes, which again is consistent with known targeting of AAV8. Importantly, the lymph nodes had no detectable AAV vector genomes present, suggesting that vector integration could not contribute to lymphoma development. cFVIII levels remained stable before and after diagnosis of lymphoma, supporting lack of vector integration in the malignant cells. Conclusions AAV gene therapy resulted in a 100% reduction in the annualized bleeding rate in this pet dog with severe HA. Furthermore, the multicentric lymphoma that developed 3.5 years after liver-directed gene therapy is unlikely related to the AAV vector. We are unable to rule out other factors that may have contributed, including environmental influences or genetic predispositions for lymphoma. These results support the ongoing efforts to treat HA with AAV-based gene therapy and reinforce the safety profile of AAV vectors outside of a laboratory environment.

Kinetics and durability of transgene expression after intrastriatal injection of AAV9 vectors.

Understanding the kinetics and durability of AAV-mediated transgene expression in the brain is essential for conducting basic neuroscience studies as well as for developing gene therapy approaches for CNS diseases. Here, we characterize and compare the temporal profile of transgene expression after bilateral injections into the mouse striatum of rAAV9 encoding GFP under the control of either a ubiquitous promoter (CAG), or the neuron-specific human synapsin (hSyn) and CamKII promoters. GFP protein expression with the CAG promoter was highest at 3 weeks, and then decreased to stable levels at 3 and 6 months. Surprisingly, GFP mRNA levels continued to increase from 3 weeks to 3 months, despite GFP protein expression decreasing during this time. GFP protein expression with hSyn increased more slowly, reaching a maximum at 3 months, which was equivalent to protein expression levels from CAG at that time point. Importantly, transgene expression driven by the hSyn promoter at 6 months was not silenced as previously reported, and GFP mRNA was continuing to rise even at the final 6-month time point. Thus, hSyn as a promoter for transgene expression demonstrates long-term durability but may require more time after vector administration to achieve steady-state levels. Because CAG had the highest GFP protein expression in our comparison, which was at 3 weeks post administration, the early kinetics of transgene expression from CAG was examined (1, 2, 5, and 10 days after injection). This analysis showed that GFP protein expression and GFP mRNA increased during the first 3 weeks after administration. Interestingly, vector DNA rapidly decreased 10-fold over the first 3 weeks following injection as it assembled into stable circular episomes and concatemers. Surprisingly, the processing of vector genomes into circular episomes and concatemers was continually dynamic up to 3 months after injection. These results provide novel insight into the dynamic processing of vector genomes and promoter-specific temporal patterns of transgene expression in the brain.

LBSUN178 Teniposide Improves Adeno-associated Virus-mediated Insulin Gene Therapy In Diabetic Mice.

Abstract Type 1 Diabetes Mellitus (T1DM) results from the autoimmune destruction of insulin-producing pancreatic β cells. Despite advances in our understanding of the pathogenesis and management of diabetes, as well as treatment options, which primarily include injectable insulin and whole pancreas transplant, these advancements remain unsatisfactory, with only a small percentage of patients achieving their glycemic goals. Insulin gene therapy refers to the development of β cell substitutes by introducing an insulin producing gene into endogenous cell populations that do not normally produce insulin. This strategy has the promise to overcome the limitations of current treatments. Adeno-associated viral (AAV) vector-based gene therapy holds great promise for the treatment of inherited and many complex acquired disorders. The success of AAV vectors is determined by two major factors: 1) cell transduction efficiency, and 2) persistence of the introduced gene with minimal or no host immune response following vector administration. Previous research has suggested that certain small molecules can improve AAV vector transduction efficiency and lower the vector dose below the immunological response threshold. We explore the use of teniposide, a chemotherapeutic agent, in improving the efficacy of gene therapy using AAV. We co-administered teniposide at a final dosage of 20 mg/kg with an AAV vector encoding an insulin gene controlled by a liver-specific promotor into streptozotocin (STZ) induced diabetic Hsd: ICR mice. Teniposide co-treatment produced up to a 3-fold decrease in AAV dose compared to vector alone while reducing diabetic hyperglycemia to comparable degrees as AAV delivery alone. There was no additional systemic toxicity. While the mechanism of action of teniposide-enhanced viral delivery of gene therapy is unknown, our data suggest that teniposide co-administration could significantly enhance AAV transduction in vivo and provide a novel, useful strategy to improve the clinical efficacy of gene therapy. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Intravenous administration of viral vectors expressing prostate cancer antigens enhances the magnitude and functionality of CD8+ T cell responses

BackgroundThe use of immunotherapeutic vaccination in prostate cancer is a promising approach that likely requires the induction of functional, cytotoxic T cells . The experimental approach described here uses a...

Intravenous immunoglobulin prevents peripheral liver transduction of intrathecally delivered AAV vectors

Gene therapy using neurotropic adeno-associated virus vectors represents an emerging solution for genetic disorders affecting the central nervous system. The first approved central nervous system-targeting adeno-associated virus gene therapy, Zolgensma®, for treating spinal muscular atrophy is administered intravenously at high doses that cause liver-associated adverse events in 20%-30% of patients. Intrathecal routes of vector administration, such as the intra-cisterna magna route, provide efficient gene transduction to central nervous system cells while reducing off-target liver transduction. However, significant levels of liver transduction often occur upon intra-cisterna magna vector delivery in preclinical studies. Using vectors expressing monoclonal antibody transgenes, we examined whether passive transfer of adeno-associated virus-neutralizing antibodies as intravenous immunoglobulin before intrathecal adeno-associated virus delivery improved the safety of viral gene therapy targeting the central nervous system in mice and nonhuman primates. We used intracerebroventricular and intra-cisterna magna routes for vector administration to mice and nonhuman primates, respectively, and evaluated transgene expression and vector genome distribution. Our data indicate that pretreatment with intravenous immunoglobulin significantly reduced gene transduction to the liver and other peripheral organs but not to the central nervous system in both species. With further refinement, this method may improve the safety of adeno-associated virus-based, central nervous system-targeting gene therapies in clinical settings.

Effects of the Cc2d1a/Freud-1 Knockdown in the Hippocampus of BTBR Mice on the Autistic-Like Behavior, Expression of Serotonin 5-HT1A and D2 Dopamine Receptors, and CREB and NF-kB Intracellular Signaling.

The mechanisms of autism are of extreme interest due to the high prevalence of this disorder in the human population. In this regard, special attention is given to the transcription factor Freud-1 (encoded by the Cc2d1a gene), which regulates numerous intracellular signaling pathways and acts as a silencer for 5-HT1A serotonin and D2 dopamine receptors. Disruption of the Freud-1 functions leads to the development of various psychopathologies. In this study, we found an increase in the expression of the Cc2d1a/Freud-1 gene in the hippocampus of BTBR mice (model of autistic-like behavior) in comparison with C57Bl/6J mice and examined how restoration of the Cc2d1a/Freud-1 expression in the hippocampus of BTBR mice affects their behavior, expression of 5-HT1A serotonin and D2 dopamine receptors, and CREB and NF-κB intracellular signaling pathways in these animals. Five weeks after administration of the adeno-associated viral vector (AAV) carrying the pAAV_H1-2_shRNA-Freud-1_Syn_EGFP plasmid encoding a small hairpin RNA (shRNA) that suppressed expression of the Cc2d1a/Freud-1 gene, we observed an elevation in the anxiety levels, as well as the increase in the escape latency and path length to the platform in the Morris water maze test, which was probably associated with a strengthening of the active stress avoidance strategy. However, the Cc2d1a/Freud-1 knockdown did not affect the spatial memory and phosphorylation of the CREB transcription factor, although such effect was found in C57Bl/6J mice in our previous study. These results suggest the impairments in the CREB-dependent effector pathway in BTBR mice, which may play an important role in the development of the autistic-like phenotype. The knockdown of Cc2d1a/Freud-1 in the hippocampus of BTBR mice did not affect expression of the 5-HT1A serotonin and D2 dopamine receptors and key NF-κB signaling genes (Nfkb1 and Rela). Our data suggest that the transcription factor Freud-1 plays a significant role in the pathogenesis of anxiety and active stress avoidance in autism.

Generation of SARS-CoV-2 Mouse Model by Transient Expression of the Human ACE2 Gene Mediated by Intranasal Administration of AAV-hACE2.

One of the most important steps in the development of drugs and vaccines against a new coronavirus infection is their testing on a relevant animal model. The laboratory mouse, with well-studied immunology, is the preferred mammalian model in experimental medicine. However, mice are not susceptible to infection with SARS-CoV-2 due to the lack of human angiotensin-converting enzyme (hACE2), which is the cell receptor of SARS-CoV-2 and necessary for the entry of the virus into the cell. In present work, it was shown that intranasal administration of the adeno-associated vectors AAV9 and AAV-DJ encoding the hACE2 provided a high level of expression of ACE2 gene in the lungs of mice. In contrast, the introduction of the AAV6 vector led to a low level ACE2 expression. Infection with SARS-CoV-2 of mice expressing hACE2 in the lungs led to virus replication and development of bronchopneumonia on the 7th day after infection. Thus, a simple method for delivering the human ACE2 gene to mouse lungs by intranasal administration of the AAV vector has been proposed. This approach enabled rapid generation of mouse model for studying coronavirus infection.

O.09 Gene therapy of spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME)

Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) is an autosomal recessive disorder caused by mutations in the <i>ASAH1</i> gene, which codes for acid ceramidase (ACD), a lysosomal enzyme that catalyses the bioactive lipid ceramide into sphingosine and fatty acid. The disease is progressive, starting during childhood with muscle weakness and/or myoclonic seizures, and leads to death in most cases at teenage. To date, there is no curative treatment and therefore a clear unmet medical need. In the present study, we evaluated a gene therapy approach in a mouse model of acid ceramidase deficiency. We report that intravenous administration of a recombinant AAV9 vector expressing human ACD in diseased <i>Asah1</i>^P361R/P361R mice prolonged survival of all animals until the end of a 6-month study, restored body growth and motor activity, and abolished inflammation in peripheral tissues and the central nervous system. Our findings provide proof-of-concept that systemic AAV-mediated <i>ASAH1</i> gene replacement can correct the severe phenotype of a mouse model of acid ceramidase deficiency, paving the way for clinical translation in patients.

Liver-secreted fluorescent blood plasma markers enable chronic imaging of the microcirculation

SummaryStudying blood microcirculation is vital for gaining insights into vascular diseases. Blood flow imaging in deep tissue is currently achieved by acute administration of fluorescent dyes in the blood plasma. This is an invasive process, and the plasma fluorescence decreases within an hour of administration. Here, we report an approach for the longitudinal study of vasculature. Using a single intraperitoneal or intravenous administration of viral vectors, we express fluorescent secretory albumin-fusion proteins in the liver to chronically label the blood circulation in mice. This approach allows for longitudinal observation of circulation from 2 weeks to over 4 months after vector administration. We demonstrate the chronic assessment of vascular functions including functional hyperemia and vascular plasticity in micro- and mesoscopic scales. This genetic plasma labeling approach represents a versatile and cost-effective method for the chronic investigation of vasculature functions across the body in health and disease animal models.

Hearing disorder following COVID-19 vaccination: A pharmacovigilance analysis using the Vaccine Adverse Event Reporting System.

What is known and objectiveEvidence on whether the coronavirus disease 2019 (COVID‐19) vaccination could cause hearing‐related adverse events is still conflicting. This study aims to access the association between COVID‐19 vaccine and hearing disorder.MethodsThe Vaccine Adverse Event Reporting System (VAERS) was queried between January 2020 to November 2021. The disproportionality pattern for hearing impairment of COVID‐19 vaccine was accessed by calculating the reporting odds ratio (ROR) and proportional reporting ratio (PRR). A further subgroup analysis based on the type of COVID‐19 vaccine and the doses administered was performed. In addition, the disproportionalities for hearing dysfunction between COVID‐19 and influenza vaccines were compared.Results and discussionA total of 14,956 reports of hearing‐related adverse events were identified with COVID‐19 vaccination and 151 with influenza vaccine during the analytic period in VAERS. The incidence of hearing disorder following COVID‐19 vaccination was 6.66 per 100,000. The results of disproportionality analysis revealed that the adverse events of hearing impairment, after administration of COVID‐19 vaccine, was significantly highly reported (ROR 2.38, 95% confidence interval [CI] 2.20–2.56; PRR: 2.35, χ 2 537.58), for both mRNA (ROR 2.37, 95% CI 2.20–2.55; PRR 2.34, χ2 529.75) and virus vector vaccines (ROR 2.50, 95% CI 2.28–2.73; PRR 2.56, χ 2 418.57). While the disproportional level for hearing dysfunction was quite lower in influenza vaccine (ROR 0.36, 95% CI 0.30–0.42; PRR 0.36, χ 2 172.24).What is new and conclusionThis study identified increased risk for hearing disorder following administration of both mRNA and virus vector COVID‐19 vaccines compared to influenza vaccination in real‐world settings.