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Analytical performance and tissue interactions of nitric oxide (NO)-releasing continuous glucose sensors were evaluated over a 28 d study in a diabetic swine model. Interstitial glucose was detected using an implanted needle-type amperometric glucose sensor. Two NO-release durations from the sensor surface were achieved by doping the membranes with nonporous (14 d release) or porous (30 d release) S-nitrosothiol-functionalized silica nanoparticles. Numerical and clinical accuracy of the sensors were assessed at time points (1, 7, 14, 21, and 28 d) following implantation. Nitric oxide-releasing sensors demonstrated accurate glucose detection over a time period directly correlated with the active release of NO. Silica particle-doped sensors that released NO for 30 d showed standard-compliant accuracy (i.e., mean absolute relative difference ≤ 15%) for >3 weeks post-implantation. Histological staining for inflammatory biomarkers suggested that the observed performance improvement was the result of decreased inflammatory cell count and a lower density collagen capsule.

The kidney is an anisotropic organ, with higher elasticity along versus across nephrons. The degree of mechanical anisotropy in the kidney may be diagnostically relevant if properly exploited; however, if improperly controlled, anisotropy may confound stiffness measurements. The purpose of this study is to demonstrate the clinical feasibility of acoustic radiation force (ARF)-induced peak displacement (PD) measures for both exploiting and obviating mechanical anisotropy in the cortex of human kidney allografts, in vivo . Validation of the imaging methods is provided by preclinical studies in pig kidneys, in which ARF-induced PD values were significantly higher ( $p , Wilcoxon) when the transducer executing asymmetric ARF was oriented across versus along the nephrons. The ratio of these PD values obtained with the transducer oriented across versus along the nephrons strongly linearly correlated ( $R^{2} = 0.95$ ) to the ratio of shear moduli measured by shear wave elasticity imaging. On the contrary, when a symmetric ARF was implemented, no significant difference in PD was observed ( $p > 0.01$ ). Similar results were demonstrated in vivo in the kidney allografts of 14 patients. The symmetric ARF produced PD measures with no significant difference ( $p > 0.01$ ) between along versus across alignments, but the asymmetric ARF yielded PD ratios that remained constant over a six-month observation period post-transplantation, consistent with stable serum creatinine level and urine protein-to-creatinine ratio in the same patient population ( $p> 0.01$ ). The results of this pilot in vivo clinical study suggest the feasibility of 1) implementing symmetrical ARF to obviate mechanical anisotropy in the kidney cortex when anisotropy is a confounding factor and 2) implementing asymmetric ARF to exploit mechanical anisotropy when mechanical anisotropy is a potentially relevant biomarker.

Adeno-associated virus (AAV) vectors have been successfully applied in hemophilia clinical trials. However, this approach is limited to patients without AAV-neutralizing antibodies (NAbs). In this study, we explored the feasibility of AAV re-administration in hemophilia A dogs treated initially 8 years ago with AAV8.canine FVIII. After the re-administration in two NAb-negative dogs with AAV8 vectors carrying human factor VIII (hFVIII), along with the proteasome inhibitor bortezomib, we observed a phenotypic improvement in both dogs that persisted in one dog. Phenotypic improvement disappeared at 59 days after re-administration in the other dog, and specific cytotoxic T lymphocytes (CTLs) to the capsid were detected at day 17, but not to hFVIII. hFVIII inhibitors were observed at day 59 and gradually increased. Mechanistic studies demonstrated an increase in pro-inflammatory cytokines, a decrease in immunomodulatory cytokines, as well as lower Tregs after re-administration. These results suggest that hFVIII inhibitor development may contribute to the therapeutic failure via immune response activation. Interestingly, it takes about 30–50 days for AAV NAb titers to decrease by half. Collectively, this study suggests that re-administration of the same AAV serotype after long-term follow-up is feasible and that the study of AAV NAb kinetics will provide important information for predicating the efficacy of re-administration. Keywords: AAV, hemophilia, neutralizing antibodies, NAbs, re-administration, human factor VIII, hFVIII, hFVIII inhibitor

Chronic kidney disease is associated with progressive inflammation and fibrosis, which is conventionally assessed by invasive biopsy. A noninvasive alternative to biopsy may be interrogating elastic and viscous anisotropy. This work evaluates the feasibility of using in vivo Viscoelastic Response (VisR) ultrasound for detecting changes in the elastic and viscous degree of anisotropy (DoA) associated with fibrosis after chronic ischemia-reperfusion injury (IRI) in pig kidney. Histology was used to validate VisR DoA measurements, which were compared to those derived using Shear Wave Elasticity Imaging (SWEI) and Shearwave Dispersion Ultrasound Vibrometry (SDUV). In the injured and contralateral (control) kidneys of 8 pigs, VisR, SWEI, and SDUV-derived elastic DoA, evaluated as the ratio of longitudinal over transverse shear elasticity, was significantly (p

Chronic kidney disease is associated with progressive inflammation, which is conventionally assessed by invasive biopsy. A noninvasive alternative is to interrogate the mechanical property alterations that occur with inflammation. This work evaluates the feasibility of using in vivo Viscoelastic Response (VisR) ultrasound for detecting differences in mechanical degree of anisotropy (DoA) associated with inflammation after ischemia-reperfusion injury (IRI) in pig kidney. VisR measurements were validated by histology, Shear Wave Elasticity Imaging (SWEI), and Shearwave Dispersion Ultrasound Vibrometry (SDUV) derived DoA. In 6 pigs, data were acquired in vivo in the kidney following IRI and in the contralateral kidney, which did not undergo IRI and served as a control. VisR (relative elasticity (RE), relative viscosity (RV), and peak displacement (PD)), SWEI (shear elastic modulus $(\mu))$ , and SDUV (shear elastic $(\mu_{1})$ and viscous $(\mu_{2})$ moduli) outcomes were evaluated alone and as ratios to reflect DoA (longitudinal over transverse). VisR PD and RE, SWEI $\mu$ , and SDUV $\mu_{1}$ demonstrated significantly increased DoA (p $\mu$ in cortex). Notably, while elastic DoA increased in both medulla and cortex, the underlying causes differed (cortex: diminished transverse modulus v. medulla: elevated longitudinal modulus). VisR RV indicated decreased viscous DoA in the medulla after IRI. Spatially matched histology indicated mild inflammation in the injured, but not the control, kidneys. These results suggest that VisR noninvasively detects changes in DoA associated with inflammation induced by IRI in a pig model. VisR may be diagnostically relevant to delineating inflammation in human kidney.

The hemophilia dog models are valuable for evaluating the efficacy of novel hemophilia therapeutics. The hemophilia B dog was predictive of the therapeutic dose of adeno-associated viral (AAV) vector delivery of human factor IX in clinical trials. In addition, it provides an opportunity to study the long-term efficacy and safety after AAV administration. However, there are several challenges in using the hemophilia A (HA) dog model for gene therapy studies. First, canine factor VIII (cFVIII) has higher specific activity and increased rate of secretion compared to human FVIII (hFVIII). This significant difference between cFVIII and hFVIII prevents the use of a species-specific transgene to predict the efficacy of AAV-hFVIII. Second, the HA dogs are immunocompetent and develop an immune response to the xenoprotein, hFVIII, that precludes the ability to measure transgene expression. Therefore, in order to employ this valuable model for gene therapy studies, we generated a unique cohort of HA dogs that are tolerant to B-domain deleted (BDD) hFVIII. We hypothesized that tolerizing dogs to hFVIII will (1) permit accurate evaluation of hFVIII expression and thus predict the therapeutic vector dose and (2) allow the evaluation of the potential immune response to a novel hFVIII variant. To tolerize the dogs to hFVIII, neonatal HA dogs were treated with a retrovirus (RV-hAAT-hFVIII-BDD-WPRE, 3x109 TU/kg) (n=5) on day 2 of life. The hFVIII expression was between 0.3%-6% at 4 weeks after RV delivery and plateaued after 6 months to 0.8% (S28), 0.3% (S29), 0% (V06), 1.5% (V26) and 1.7% (V27) based on Coatest assay. To determine if the dogs were tolerant to hFVIII-BDD, the dogs were challenged with hFVIII-BDD protein at 5-6 months post-RV administration (Xyntha, 25IU/kg per wk x 6 wks, I.V.). Anti-hFVIII antibodies were monitored closely throughout the challenge and up to 8 weeks after the last challenge. In 4 out of 5 dogs, no anti-hFVIII immune response was observed based on IgG1, IgG2, total IgG or Bethesda titer. In contrast, naïve HA dogs (n=2) developed high level anti-hFVIII IgG2 (1.2-3.2 μg/mL), total IgG (3.4-5.0 μg/mL), and Bethesda titer (4.1-67.8 BU/mL) after the same challenge regimen. Interestingly, the hFVIII activity in one RV-treated dog (V06) was undetectable at 6 months post-RV administration. After the challenge, V06 had anti-hFVIII IgG2 (1.7 μg/mL), total IgG (2.6 μg/mL), and a Bethesda titer (9.5 BU/mL), suggesting that FVIII must be maintained to achieve tolerance. These dogs were used to evaluate the efficacy of AAV serotype 8 (AAV8) delivery of a hFVIII-BDD codon-optimized sequence driven by a hepatocyte promoter, modified transthyretin promoter (TTRm). S29 was delivered AAV8-TTRm-hFVIII-CO (2x1012 vg/kg). Prior to AAV delivery, the levels of hFVIII activity were 0.5-1% from the tolerization with the RV. After AAV administration the hFVIII activity was 3.8% at d168 and 4.7% at d387, resulting in a 4% increase in hFVIII expression. No anti-hFVIII antibodies were detected. The annual bleeding rate (ABR) for S29 post-RV delivery was 5 and after AAV delivery was 0, showing an improvement in the bleeding phenotype in contrast to untreated HA dogs (ABR=13, n=11). A hFVIII-tolerized littermate, S28, was recently treated with a hFVIII variant, AAV8-TTRm-hFVIII-CO-Δ3-SP/DE (2x1012 vg/kg). The hFVIII-Δ3-SP/DE variant has a deletion of the furin site (1645-47) and replaces residues SD at 1657-58 with PE. This variant showed higher specific activity (2-fold) in vitro and increased secretion (4-fold) compared to wild type hFVIII-BDD in the setting of AAV delivery in HA mice. Based on the results in S28, we will determine the dosing of V26 and V27. These studies demonstrate that sustained low level hFVIII expression of 0.2-2% up to 4 years post-retroviral delivery were able to induce and maintain tolerance to hFVIII, while allowing for the subsequent assessment of AAV efficacy. A clinically relevant dose of AAV8-TTRm-hFVIII-CO resulted in therapeutic levels of hFVIII expression while ongoing studies will allow investigation of the efficacy of the hFVIII-BDD variant, Δ3-SP/DE, in the setting of AAV administration in a large animal model. Overall, these studies demonstrate that RV-targeting of hFVIII-BDD expression to the liver in neonatal HA dogs leads to tolerance to this xenoprotein and provide a unique large animal model to evaluate both efficacy as well as potential immunogenicity of novel FVIII variants. Disclosures Sabatino: Spark Therapeutics: Patents & Royalties.

The hemophilia A (HA) dogs have been a valuable model for evaluating the efficacy of novel hemophilia therapeutics. These dogs have a mutation that is analogous to the most common mutation in humans with severe disease, the intron 22 inversion. The advantages of the hemophilia dog model include: (1) it was predictive of the therapeutic adeno-associated viral (AAV) vector dose in human factor IX (hFIX) clinical trials, (2) it is an outbred immunocompetent species, (3) it demonstrates a clinical bleeding phenotype consistent with patients, including hemarthrosis, and (4) it permits long-term investigation of both efficacy and safety. Our previous studies delivering AAV-canine factor VIII (cFVIII) in the HA dogs demonstrated long-term (up to 10 years of follow-up) dose-dependent cFVIII expression without evidence of an immune response to the cFVIII protein. In hemophilia B preclinical studies, the comparable biological characteristics between canine FIX and hFIX allows preclinical results using cFIX to be translated to clinical studies. In contrast, for hemophilia A, our studies of recombinant cFVIII and human FVIII (hFVIII) proteins demonstrate that cFVIII is a more stable protein that has higher biological activity and is secreted better than hFVIII (Sabatino et al. 2009). Thus, expression of cFVIII following AAV delivery does not accurately predict the therapeutic dose of AAV-hFVIII which is relevant for translation to clinical trials. The challenge of administering AAV-hFVIII to the HA dogs is that this expressed xenoprotein (hFVIII) results in inhibitor formation that precludes the ability to measure transgene (hFVIII) expression. We hypothesized that tolerizing HA dogs to hFVIII will (1) permit accurate evaluation of hFVIII expression and thus predict the therapeutic vector dose and (2) allow the evaluation of the potential immune response of AAV8-hFVIII versus a novel hFVIII variant that has increased activity and secretion (Nguyen et al. 2017). In this study we used a neonatal retroviral (RV) delivery approach to tolerize the HA dogs (n=5) to B-domain deleted hFVIII (hFVIII-BDD). HA neonatal male dogs (S28, S29, V06, V26, V27) were treated with the retrovirus (RV-hAAT-hFVIII-BDD-WPRE) (3x10e9 TU/kg) on day 2 of life. The levels of hFVIII expression after RV delivery were 0.3-6% 4 weeks after RV delivery and plateaued after 6 months to 0.8% (S28), 0.3% (S29), 0% (V06), 1.5% (V26) and 1.7% (V27) based on Coatest assay. At 5-6 months of life the dogs (S28, S29, V06) were challenged with hFVIII-BDD (Xyntha; 25IU/kg; I.V.) weekly for 6 consecutive weeks. Samples were collected before and 15 minutes after each protein challenge to demonstrate the successful infusion of the protein. At 4 weeks after the final challenge, no anti-hFVIII IgG1 or IgG2 antibodies were detected consistent with no evidence of an inhibitor. At 4.5 years of age, S28 and S29 had stable expression of 0.5-1% of hFVIII and were rechallenged with hFVIII-BDD (Xyntha; 25IU/kg per week x 6 wks). No anti-hFVIII IgG1 or IgG2 antibodies were detected 4 weeks after the final protein challenge. Thus, these data demonstrate that all of the RV-hAAT-hFVIII-BDD-WPRE treated dogs that have been challenged (n=3) have been tolerant to hFVIII-BDD. Since the goal of this study is to generate a cohort of HA dogs tolerant to hFVIII-BDD to address the efficacy and safety of AAV8-hFVIII-BDD versus a hFVIII-BDD variant, we treated the first dog (S29) 12 weeks after the second series of protein challenges with an optimized AAV vector cassette containing a codon-optimized hFVIII sequence with a modified transthyretin (TTRm) promoter, AAV8-TTRm-hFVIII-BDD (2x10e12 vg/kg). Prior to AAV administration the hFVIII activity was 0.2%. At 8 weeks post AAV administration, the hFVIII activity increased to 3.5%. No anti-hFVIII-BDD IgG1 or IgG2 was detected after AAV-hFVIII administration. These data demonstrate that low levels of sustained hFVIII expression of 0.2-2% up to 4 years post-retroviral delivery were able to induce and maintain tolerance to hFVIII. Overall, these studies demonstrate that the neonatal HA dogs treated with a retrovirus targeting hFVIII expression to the liver are tolerant to hFVIII and provide a unique large animal model to evaluate both efficacy as well as potential immunogenicity of our novel FVIII variants. Disclosures Sabatino: Spark Therapeutics: Patents & Royalties.