scholarly journals Generation of a Unique Cohort of Hemophilia A Dogs Tolerant to Human FVIII for Evaluating the Safety and Efficacy of AAV Delivery of Wild Type and Variant Human FVIII

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2453-2453
Author(s):  
Giang N. Nguyen ◽  
Lauren Wimsey ◽  
Elizabeth Merricks ◽  
Katherine P. Ponder ◽  
Timothy C. Nichols ◽  
...  
Keyword(s):  
Immune Response ◽  
Clinical Trials ◽  
Hemophilia A ◽  
Factor Ix ◽  
Common Mutation ◽  
Large Animal ◽  
Efficacy And Safety ◽  
Aav Vector ◽  
Human Factor Ix

Abstract 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.

scholarly journals Hemophilia A Dogs Tolerant to Human Factor VIII Provide a Unique Model to Determine Efficacy and Safety of AAV Delivery of Novel Factor VIII Variants

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3628-3628
Author(s):  
Giang N. Nguyen ◽  
Lauren E. Wimsey ◽  
Elizabeth P. Merricks ◽  
Katherine P. Ponder ◽  
Timothy C. Nichols ◽  
...  
Keyword(s):  
Immune Response ◽  
Animal Model ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Specific Activity ◽  
Large Animal Model ◽  
Large Animal ◽  
Efficacy And Safety ◽  
Therapy Studies

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.

Efficacy and Safety of Continuous Expression of An Improved Variant of FVIIa On Murine Hemostasis with or without Inhibitors to Human Factor IX.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2466-2466
Author(s):  
Elise Roy ◽  
Armida Faella ◽  
Harre Downey ◽  
Lacramioara Ivanciu ◽  
Shangzhen Zhou ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Low Dose ◽  
Human Factor ◽  
Catalytic Domain ◽  
Factor Ix ◽  
High Dose ◽  
Large Animal ◽  
Human Factor Ix ◽  
Inhibitory Antibodies

Abstract Abstract 2466 Poster Board II-443 Recombinant human activated Factor VII (rhFVIIa) is extensively used in the management of hemophilic inhibitor patients. As an alternative, using an adeno-associated viral (AAV) vector as a delivery vehicle for an engineered transgene for activated canine FVII (cFVIIa), our large animal studies in hemophilia A and B dogs demonstrated effective, long-term hemostasis and elimination of the bleeding episodes. However, the relatively high vector doses required for efficacy underscores the need for improvements in the delivery vector and/or the transgene itself. To address this, we decided to investigate whether changes in the catalytic domain of FVIIa can improve its coagulant activity, thus resulting in lowering of the effective vector dose. Using the murine version of our engineered FVIIa (mFVIIa) as a backbone, we generated a variant with four amino-acid substitutions in the catalytic domain of murine FVIIa (L305V/A314E/K337A/I374Y, mFVIIa-VEAY), based on an existing enhanced activity human FVIIa variant. As we have previously described (Amer. Soc. Hematol. Meeting 2006, #3276; Amer. Soc. Gene Cell Ther. Meeting 2008, #127), purified mFVIIa-VEAY exhibited 6-7 fold higher intrinsic coagulant activity than mFVIIa. The hemostatic properties relative to mFVIIa were determined following AAV-mediated gene delivery in hemophilia A (HA) mice. Administration of AAV-mFVIIa-VEAY at 40-100 fold lower vector dose (1.2 - 3E10 vector genomes [vg]/mouse) than AAV-mFVIIa (1.2E12 vg/mouse, High dose) resulted in ∼10-fold lower expression of the mFVIIa-VEAY (based on RNA transcript levels), but was sufficient to result in long-term (>12 weeks) normalization of the hemophilic activated partial thromboplastin time (aPTT), similar to that seen with high dose AAV-mFVIIa (P>0.05). In addition, it improved hemostasis similar to that seen with AAV-mFVIIa following in vivo hemostatic challenges (tail clip assay and ferric chloride carotid artery injury). As an extension of these studies, we have now investigated the ability of low-dose mFVIIa-VEAY gene delivery to improve hemostasis in the presence of inhibitory antibodies to human Factor IX (hFIX). Using an adjuvant, we developed a protocol for generating persistent (>3 months, ongoing) inhibitory antibodies to human Factor IX (hFIX) in hemophilia B (HB) mice. Using the low dose of 3E10 vg/mouse, we administered AAV-mFVIIa-VEAY in HB mice with high titer (7-30 Bethesda units [BU]) to hFIX. In agreement with our previous results, following gene transfer, we observed normalization of the hemophilic aPTT at the expression plateau (4 weeks [ongoing]; P>0.05 vs. wildtype mice; P<0.05 vs. untreated HA mice with similar titers of inhibitory antibodies to hFIX). However, despite the clear efficacy observed in hemophilia mice following low-dose mFVIIa-VEAY gene delivery, our previous observations with mFVIIa-VEAY overexpression (following a 1.2E12 vg/mouse of administered AAV [High dose]) in HA mice indicated a 70% reduction in survival within 6 weeks post vector administration. This coincided with a time-dependent increase in plasma thrombin-antithrombin levels that peaked at 4 weeks post AAV infusion (∼70 ng/ml) and was not observed in untreated HA mice (∼ 20ng/ml, P>0.05), HA mice treated with low-dose mFVIIa-VEAY (∼40ng/ml, P>0.05) or HA mice treated with AAV-mFVIIa (∼35ng/ml, P<0.05). Further extending these studies, histological examination from organs of deceased mice revealed thrombi in the heart as well as gross loss of lung structure. Immunofluoresence microscopy demostrated fibrin deposition in the lung parenchyma, suggesting a compromise of the lung vascular bed in the deceased mice. The identical experiment using hemostatically normal mice resulted in 100% mortality within 6 weeks with similar histological findings compared to HA mice following high dose AAV-mFVIIa-VEAY administration. Overall, our results using a high activity mFVIIa variant, demonstrate similar efficacy to mFVIIa but at a substantially reduced vector dose/expression, in a gene transfer setting for hemophilia with or without inhibitors. This variant thus serves as a potential candidate that can lower the effective vector dose in FVIIa gene-based studies in large animal models of hemophilia. However, the increased mortality observed in mice expressing high levels of mFVIIa-VEAY, warrants further investigation into the long-term safety of coagulation proteases with enhanced activity. Disclosures: High: Novo Nordisk: Grant Review Panel.

The impact of Vaccination worldwide on SARS-CoV-2 infection: A Review on Vaccine Mechanisms, Results of Clinical Trials, Vaccinal Coverage and Interactions with Novel Variants

2021 ◽  
Vol 28 ◽  
Author(s):  
Douglas Henrique Pereira Damasceno ◽  
Arthur Aguiar Amaral ◽  
Cecília Andrade Silva ◽  
Ana Cristina Simões e Silva
Keyword(s):  
Immune Response ◽  
Clinical Trials ◽  
Placebo Group ◽  
Mechanisms Of Action ◽  
Novel Variants ◽  
The Impact ◽  
Positive Results ◽  
Epidemiological Information ◽  
Long Term Studies

Background: The COVID-19 pandemic demanded a global effort towards quickly developing safe and effective vaccines against SARS-CoV-2. Objective: This review aimed to discuss the main vaccines available, their mechanisms of action, results of clinical trials and epidemiological behavior. The implications of viral variants were also debated. Methods: A non-systematic literature review was performed between February and March 2021 by searching the Pubmed, Scopus, and SciELO databases, using different combinations of the following terms: "vaccines", "clinical trials" , "SARS-CoV-2", "Coronavirus", "COVID-19", "mechanisms of action". Data regarding clinical trials of SARS-CoV-2 vaccines and epidemiological information were also searched. Results: The mechanisms of action included vector-virus, mRNA and inactivated virus vaccines. The vaccines showed positive results in phases 2/3 clinical trials. The efficacy of the mRNA 1273 and of mRNA BNT 162b2 vaccines were 94.1% and 95%, respectively. The effectiveness of the ChAdOx1 nCoV-19 vaccine varied according to the scheme, with an overall value of 70.4%. The Gam-COVID-Vac vaccine had an efficacy of 91.6%. Regarding the Ad26.COV2.S vaccine, 99% or more of seroconversion was observed in all subgroups 29 days after vaccination. The CoronaVac vaccine induced an immune response in 92% of the volunteers receiving 3ug and in 98% with 6ug, in comparison to 3% in the placebo group. Conclusion: Global efforts have resulted in vaccines available in record time, with good safety and immunogenicity profile. However, only long-term studies can provide more information on duration of immunity and the need for additional doses.

scholarly journals Association of immune response with efficacy and safety outcomes in adults with phenylketonuria administered pegvaliase in phase 3 clinical trials

EBioMedicine ◽  
2018 ◽  
Vol 37 ◽  
pp. 366-373 ◽  
Author(s):  
Soumi Gupta ◽  
Kelly Lau ◽  
Cary O. Harding ◽  
Gillian Shepherd ◽  
Ryan Boyer ◽  
...  
Keyword(s):  
Immune Response ◽  
Clinical Trials ◽  
Efficacy And Safety ◽  
Phase 3 ◽  
Safety Outcomes

scholarly journals Hemophilia gene therapy: ushering in a new treatment paradigm?

Hematology ◽  
2021 ◽  
Vol 2021 (1) ◽  
pp. 226-233
Author(s):  
Lindsey A. George
Keyword(s):  
Clinical Trial ◽  
Gene Therapy ◽  
Clinical Trials ◽  
Hemophilia A ◽  
Full Potential ◽  
Proof Of Concept ◽  
Aav Vector ◽  
Treatment Paradigm ◽  
Clinical Trial Enrollment ◽  
New Treatment

Abstract After 3 decades of clinical trials, repeated proof-of-concept success has now been demonstrated in hemophilia A and B gene therapy. Current clinical hemophilia gene therapy efforts are largely focused on the use of systemically administered recombinant adeno-associated viral (rAAV) vectors for F8 or F9 gene addition. With multiple ongoing trials, including licensing studies in hemophilia A and B, many are cautiously optimistic that the first AAV vectors will obtain regulatory approval within approximately 1 year. While supported optimism suggests that the goal of gene therapy to alter the paradigm of hemophilia care may soon be realized, a number of outstanding questions have emerged from clinical trial that are in need of answers to harness the full potential of gene therapy for hemophilia patients. This article reviews the use of AAV vector gene addition approaches for hemophilia A and B, focusing specifically on information to review in the process of obtaining informed consent for hemophilia patients prior to clinical trial enrollment or administering a licensed AAV vector.

Long-term Efficacy and Safety of Teriflunomide: An Analysis of Pooled Clinical Trials

2018 ◽  
Vol 26 ◽  
pp. 257
Author(s):  
M.S. Freedman ◽  
J.S. Inshasi ◽  
L. Ramió-torrentà ◽  
M. Zaffaroni ◽  
J. De Seze ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Efficacy And Safety ◽  
Term Efficacy

Long-Term Phenotypic Correction of Hemophilia A Mice Following Intravenous Injection of miRNA-Regulated Lentiviral Vectors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2587-2587
Author(s):  
Hideto Matsui ◽  
Margareth Ozelo ◽  
Carol Hegadorn ◽  
Andrea Labelle ◽  
Erin Burnett ◽  
...  
Keyword(s):  
Immune Response ◽  
Intravenous Injection ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Therapeutic Potential ◽  
Antigen Presenting Cells ◽  
Lentiviral Vectors ◽  
Target Sequence ◽  
Antigen Presenting

Abstract Hemophilia A is an excellent candidate disorder for the use of gene therapy as a treatment modality. To date, although lentiviral delivery of the factor VIII (FVIII) transgene has the potential to provide sustained therapeutic correction of the hemophilia A phenotype, this has not been achieved in adult animals because of the anti-FVIII immune response. We have used lentiviral vectors to deliver the canine FVIII transgene to hemophilia A neonates and although no anti-FVIII immune response occurred, and indeed the treated mice displayed long-term tolerance to the canine FVIII antigen, this strategy did not provide sustained therapeutic levels of plasma FVIII. To overcome these limitations, we modified our lentiviral vector and the protocol for viral delivery to enhance transduction of hepatocytes and direct transgene expression away from antigen presenting cells. We engineered lentiviral vectors that encode the B-domain deleted canine FVIII cDNA under the transcriptional control of either a non-viral ubiquitous promoter or two different liver-restricted promoters. However, no plasma FVIII was detected in any of the adult hemophilia A mice after intravenous injection of the various lentiviral vectors because of an anti-canine FVIII immune response. An alternate pseudotype (GP64) was used to enhance transduction of hepatocytes and a target sequence for a hematopoietic-specific microRNA was incorporated into the transgene to prevent FVIII expression in antigen presenting cells that may arise from promoter trapping. When hemophilia A mice received intravenous infusions of these modified vectors, where the cFVIII trangene is under the control of either of the liver-restricted promoters, all treated mice (n=4) showed sustained FVIII expression (mean FVIII levels 28.2±2.4 mU/mL) for more than 150 days (last time analyzed) without developing anti-FVIII antibodies. Moreover, temporary depletion of Kuppfer cells prior to viral administration resulted in a 3-fold elevation of levels of plasma FVIII (mean FVIII levels 83.3±2.1mU/mL; n=4). Analysis of the biodistribution of the integrated FVIII transgene and expression of canine FVIII mRNA indicate an enhanced restriction of FVIII expression in hepatocytes with the use of the modified lentiviral vectors. These results demonstrate, for the first time, the long-term therapeutic potential of modified lentiviral vectors for treating adult pre-clinical animal models of hemophilia A.

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