Rabbit Anti-Thymocyte Globulin (rATG) Administrated Concomitantly with Liver Delivery of AAV2-hFIX Can Promote Inhibitor Formation In Rhesus Macaques.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3765-3765 ◽  
Author(s):  
Jonathan D. Finn ◽  
Patricia Favaro ◽  
J. Fraser Wright ◽  
Federico Mingozzi ◽  
Katherine A. High ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Neutralizing Antibodies ◽  
Rhesus Macaques ◽  
Rescue Therapy ◽  
Factor Ix ◽  
Large Animal ◽  
Solid Organ ◽  
Large Animal Models ◽  
Human Factor Ix

Abstract Abstract 3765 Adeno-associated viral (AAV) vectors are one of the most extensively studied vector platforms for gene therapy applications. Our group is currently developing AAV vectors for the therapeutic treatment of hemophilia B (HB) in humans. The first clinical trial using an AAV2 vector to express human Factor IX (hFIX) (AAV2-hFIX16) from the liver of HB patients revealed a cytotoxic T lymphocyte (CTL) response directed against AAV capsid that occurred 4–6 weeks following treatment that was associated with a decline in transgene expression. Thus, immunosuppressive (IS) therapies may be required during AAV2 vector administration at high doses to prevent or to halt the immune mediated destruction of transduced hepatocytes. Previous work in murine and non-human primate (NHP) models has shown that sustained AAV-mediated expression of transgenes can induce tolerance, and that this is in part, dependent on CD4+ CD25+ FoxP3+ regulatory T cells (Tregs). Here we investigate the safety of a Treg sparing anti-T cell IS regimen in the context of liver mediated AAV2 gene transfer. Rabbit anti-thymocyte globulin (rATG) is an immune suppressive drug that is used in solid organ transplant and autoimmune disease. rATG has been shown to dramatically deplete the majority of T-cells, however some studies have shown that rATG spares Tregs and can induce tolerance in human T cells. rATG was administered to rhesus macaques (along with an 8-week course of Mycophenolate Mofetil (MMF) and sirolimus) either at the time of AAV vector administration (AAV2-hFIX16), or 5 weeks post-vector administration (rescue therapy). The administration of ATG at week 5 had no detrimental effect on hFIX expression and was not associated with inhibitor formation (n=3) indicating that rATG might be safe to use as an IS ‘rescue' agent, after the detection of an ongoing immune response against transduced cells. Interestingly we observed that early administration of rATG prevented tolerance induction and resulted in inhibitor formation in 2 of 3 animals upon withdrawal of IS. The inhibitor formation was associated with transient elevations in circulating levels of IL-2, IL-4, IL-10 and IFN-g. These results are comparable to previous findings in NHP using an anti-CD25 IS regimen (Daclizumab) at the time of vector administration (Blood 2007, 110(7):2334-41). We conclude that the timing of IS regimens is critical, and that IS regimens that alter the numbers, frequency, and/or function of T-cells at the time of vector administration can result in neutralizing antibodies (inhibitors) to the transgene product (hFIX). These data suggest that there might be multiple mechanisms responsible for maintaining tolerance in this model, and that Tregs alone might not be sufficient. This study highlights the critical need for safety studies in large animal models of potential immune suppressive regimens in the context of gene transfer before translating to the clinic. Disclosures: High: Genzyme, Inc: Consultancy, Patents & Royalties; Third Rock Ventures: Consultancy; Novo-Nordisk: Consultancy; Shire, Inc.: Consultancy.

AAV8-Factor IX Hepatic Gene Transfer with Transient Immunosuppression Is Safe but Is Abrogated by Low Titers of Neutralizing Antibody in Rhesus Macaques.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5532-5532
Author(s):  
Haiyan Jiang ◽  
Susannah Patarroyo-White ◽  
Tongyao Liu ◽  
Bin Yang ◽  
Dea Nagy ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Neutralizing Antibodies ◽  
Rhesus Macaques ◽  
Small Animal ◽  
Neutralizing Antibody ◽  
Cross Reactivity ◽  
Factor Ix ◽  
Post Surgery ◽  
Short Course ◽  
Human Factor Ix

Abstract AAV8 vectors have potential in human gene transfer due to improved transduction efficiencies as demonstrated in small animal models, and are also reported to show less cross-reactivity to AAV2 neutralizing antibodies prevalent in humans. To assess this hypothesis, the efficacy and safety of AAV8-human Factor IX (hFIX) was evaluated in rhesus macaques. At doses of 5–20x1012 vg/kg, AAV8-hFIX achieved therapeutic to supraphysiological levels of circulating human FIX via intra-hepatic artery delivery in AAV8-naïve macaques. However, transduction was abrogated in macaques with pre-existing AAV8 neutralizing antibodies at titers as low as 1:5. Intrahepatic delivery of AAV8-hFIX resulted in gene transfer primarily in liver, and was well distributed among individual hepatic lobes. Two macaques that experienced traumatic catheterization developed immediate and delayed (week 4–6) transient transaminitis post surgery. Nevertheless, a significant percentage of AAV8-hFIX transduced hepatocytes persisted despite the liver damage and partial loss of hFIX expression. Transduction was well tolerated in all other macaques. Transient immunosuppression with tacrolimus and mycophenolate mofetil did not impair AAV8-hFIX transduction. The results [1] support AAV8-mediated gene transfer in humans but indicate that both the efficacy achieved and the resistance to pre-existing neutralizing antibodies of AAV8-hFIX are unlikely to be significantly enhanced over AAV2 vector, [2] suggest that a peri-operative insult and likely triggering of innate immunity may result in a later inflammatory response that results in a decrease in transgene protein production, and [3] provide evidence that a short course of immunosuppression does not alter liver transduction by AAV vectors.

scholarly journals Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3321-3328 ◽  
Author(s):  
Haiyan Jiang ◽  
Linda B. Couto ◽  
Susannah Patarroyo-White ◽  
Tongyao Liu ◽  
Dea Nagy ◽  
...  
Keyword(s):  
Clinical Study ◽  
T Cell ◽  
Gene Transfer ◽  
Neutralizing Antibodies ◽  
Rhesus Macaques ◽  
T Cell Response ◽  
Factor Ix ◽  
Target Tissue ◽  
Human Factor Ix

Abstract In a clinical study of recombinant adeno-associated virus-2 expressing human factor IX (AAV2-FIX), we detected 2 impediments to long-term gene transfer. First, preexisting anti-AAV neutralizing antibodies (NABs) prevent vector from reaching the target tissue, and second, CD8+ T-cell responses to hepatocyte-cell surface displayed AAV-capsid–terminated FIX expression after several weeks. Because the vector is incapable of synthesizing viral proteins, a short course of immunosuppression, until AAV capsid is cleared from the transduced cells, may mitigate the host T-cell response, allowing long-term expression of FIX. To evaluate coad-ministration of immunosuppression, we studied AAV8 vector infusion in rhesus macaques, natural hosts for AAV8. We administered AAV8-FIX in 16 macaques via the hepatic artery and assessed the effects of (1) preexisting anti-AAV8 NABs, (2) a standard T-cell immunosuppressive regimen, and (3) efficacy and safety of AAV8-FIX. We found that low titers (1:5) of preexisting NABs abrogate transduction, whereas animals with undetectable NABs are safely and effectively transduced by AAV8-FIX. Coadministration of mycophenolate mofetil and tacrolimus with vector does not induce toxicity and does not impair AAV transduction or FIX synthesis. These findings enable a clinical study to assess the effects of immunomodulation on long-term FIX expression in patients with hemophilia B.

A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia

Blood ◽  
2003 ◽  
Vol 102 (7) ◽  
pp. 2403-2411 ◽  
Author(s):  
Anja Ehrhardt ◽  
Hui Xu ◽  
Aaron M. Dillow ◽  
Dwight A. Bellinger ◽  
Timothy C. Nichols ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Adenoviral Vector ◽  
High Efficiency ◽  
Liver Toxicity ◽  
Normal Liver ◽  
Adenoviral Vectors ◽  
Factor Ix ◽  
Hemophilia B ◽  
Large Animal ◽  
Large Animal Models

Abstract Many approaches for treating hemophilia via gene transfer have been attempted in large animal models but all have potential drawbacks. Recombinant adenoviral vectors offer high-efficiency transfer of an episomal vector but have been plagued by the cytotoxicity/immunogenicity of early-generation vectors that contain viral genes. In our current study, we have used a nonintegrating helper-dependent (HD) adenoviral vector for liver-directed gene transfer to achieve hemostatic correction in a dog with hemophilia B. We measured plasma canine factor IX (cFIX) concentrations at a therapeutic range for up to 2.5 months and normalization of the whole blood clotting time (WBCT) for about a month. This was followed by a decrease and stabilized partial correction for 4.5 months. Hepatic gene transfer of a slightly lower dose of the HD vector resulted in WBCTs that were close to normal for 2 weeks, suggesting a dose threshold effect in dogs. In sharp contrast to other studies using first- or second-generation adenoviral vectors, we observed no vector-related elevation of liver enzymes, no fall in platelet counts, and normal liver histology. Taken together, this study demonstrates that injection of an adenoviral HD vector results in complete but transient phenotypic correction of FIX deficiency in canine models with no detectable toxicity. (Blood. 2003;102:2403-2411)

Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic factor IX in mice

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3700-3709 ◽  
Author(s):  
Antonia Follenzi ◽  
Manuela Battaglia ◽  
Angelo Lombardo ◽  
Andrea Annoni ◽  
Maria Grazia Roncarolo ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Immune Responses ◽  
Transgene Expression ◽  
Factor Ix ◽  
Stable Gene ◽  
Human Factor Ix ◽  
Immunocompetent Mice

Abstract Stable gene replacement by in vivo administration of lentiviral vectors (LVs) has therapeutic potential for metabolic disorders and other systemic diseases. We studied the expression of intracellular and secreted proteins by LVs in immunocompetent mice. Liver, spleen, and bone marrow cells were efficiently transduced. However, transgene expression, driven by a ubiquitous promoter, was limited by transgene-specific cellular and humoral immune responses, leading to the clearance of transduced cells. After green fluorescent protein (GFP) gene transfer, the liver showed infiltration of CD8+ cytotoxic T cells, and GFP-specific CD8+ T cells were isolated from the spleen. After human factor IX (hF.IX) gene transfer, anti-hF.IX antibodies were induced. These immune responses were not detected in mice injected with heat-inactivated or genome-lacking LVs or in GFP-transgenic mice, indicating that they were specifically triggered by transgene expression in vivo. Intriguingly, selective targeting of LV expression to hepatocytes limited the immune responses to the transgenes. By this approach, high levels of hF.IX, potentially in the therapeutic range, were reached and maintained long term in immunocompetent mice, without inducing antibody formation. These results prompt further studies in relevant animal models to explore the potential of in vivo LV administration for the gene therapy of hemophilias and other liver-based diseases.

scholarly journals Hemophilia gene therapy comes of age

2017 ◽  
Vol 1 (26) ◽  
pp. 2591-2599 ◽  
Author(s):  
Lindsey A. George
Keyword(s):  
Clinical Trial ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Factor Ix ◽  
Large Animal Model ◽  
Large Animal ◽  
Adeno Associated Virus ◽  
Target Disease ◽  
American Society ◽  
Clinical Trial Results

Abstract Concurrent with the development of recombinant factor replacement products, the characterization of the F9 and F8 genes over 3 decades ago allowed for the development of recombinant factor products and made the hemophilias a target disease for gene transfer. The progress of hemophilia gene therapy has been announced in 3 American Society of Hematology scientific plenary sessions, including the first “cure” in a large animal model of hemophilia B in 1998, first in human sustained vector-derived factor IX activity in 2011, and our clinical trial results reporting sustained vector-derived factor IX activity well into the mild or normal range in 2016. This progression to clinically meaningful success combined with numerous ongoing recombinant adeno-associated virus (rAAV)–mediated hemophilia gene transfer clinical trials suggest that the goal of gene therapy to alter the paradigm of hemophilia care may soon be realized. Although several novel therapeutics have recently emerged for hemophilia, gene therapy is unique in its potential for a one-time disease-altering, or even curative, treatment. This review will focus on the prior progress and current clinical trial investigation of rAAV-mediated gene transfer for hemophilia A and B.

scholarly journals Large Animal Models of Vascularized Composite Allotransplantation: A Review of Immune Strategies to Improve Allograft Outcomes

2021 ◽  
Vol 12 ◽  
Author(s):  
Abraham J. Matar ◽  
Rebecca L. Crepeau ◽  
Gerhard S. Mundinger ◽  
Curtis L. Cetrulo ◽  
Radbeh Torabi
Keyword(s):  
Animal Models ◽  
Ex Vivo ◽  
Solid Organ Transplantation ◽  
Tolerance Induction ◽  
Large Animal ◽  
Solid Organ ◽  
Large Animal Models ◽  
Large Animals ◽  
And Function ◽  
Made In

Over the past twenty years, significant technical strides have been made in the area of vascularized composite tissue allotransplantation (VCA). As in solid organ transplantation, the allogeneic immune response remains a significant barrier to long-term VCA survival and function. Strategies to overcome acute and chronic rejection, minimize immunosuppression and prolong VCA survival have important clinical implications. Historically, large animals have provided a valuable model for testing the clinical translatability of immune modulating approaches in transplantation, including tolerance induction, co-stimulation blockade, cellular therapies, and ex vivo perfusion. Recently, significant advancements have been made in these arenas utilizing large animal VCA models. In this comprehensive review, we highlight recent immune strategies undertaken to improve VCA outcomes with a focus on relevant preclinical large animal models.

scholarly journals Development of Monospecific Antibodies to Human Factor IX

1977 ◽  
Author(s):  
Cheryl Y. Tiarks ◽  
Chin-Hai Chang ◽  
Liberto Pechet
Keyword(s):  
Neutralizing Antibodies ◽  
Human Factor ◽  
Human Albumin ◽  
Factor Ix ◽  
Hemophilia B ◽  
Red Cross ◽  
Factor X ◽  
Normal Plasma ◽  
Human Factor Ix ◽  
Monospecific Antibodies

The purpose of this research was to develop neutralizing and precipitating antibodies to factor IX. Human factor IX, purified by the method of Rosenberg et.al. (J. Biol. Chem. 250:8883, 1975), was electrophoresed on acrylamide gel. Two major bands migrating adjacently were eluted. They contained factor IX activity only. The eluates and their homogenized gel segments 7 and 8 were injected separately into two rabbits, Rl and R2, respectively. On immunodiffusion the antiserum Rl showed one precipitating line with normal plasma. It neutralized human factor IX (20 Bethesda units) and also slightly neutralized factor X. It had no effect on factors II and VII. Following absorption of this antiserum with purified factor X it neutralized factor IX only. With continuous immunization, however, this antiserum revealed two new precipitating contaminants. The antiserum R2 neutralized only factor IX; it reached 220 Bethesda inhibitory units. On immunodiffusion it showed two precipitating lines, one of which disappeared after absorption with human albumin. On immunodiffusion and Laurell immunoelectrophoresis, the albumin-absorbed R2 antiserum showed one precipitin line of identity, or one rocket, with normal plasma, a Red Cross factor IX preparation (rich in factors IX, II and X), the original eluates 7 and 8, and a Hemophilia-B antigen-positive plasma. No line or rocket developed with normal plasma absorbed with aluminum hydroxide or with antigen-negative Hemophilia-B plasma. We conclude that the antisera Rl and R2 contain factor IX neutralizing antibodies and that albumin-absorbed R2 has monospecific precipitating antibodies to human non-activated factor IX.

scholarly journals Induction and role of regulatory CD4+CD25+ T cells in tolerance to the transgene product following hepatic in vivo gene transfer

Blood ◽  
2007 ◽  
Vol 110 (4) ◽  
pp. 1132-1140 ◽  
Author(s):  
Ou Cao ◽  
Eric Dobrzynski ◽  
Lixin Wang ◽  
Sushrusha Nayak ◽  
Bethany Mingle ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Antibody Formation ◽  
Coagulation Factor ◽  
Gene Replacement ◽  
Factor Ix ◽  
In Vivo Gene Transfer

Abstract Gene replacement therapy is complicated by the risk of an immune response against the therapeutic transgene product, which in part is determined by the route of vector administration. Our previous studies demonstrated induction of immune tolerance to coagulation factor IX (FIX) by hepatic adeno-associated viral (AAV) gene transfer. Using a regulatory T-cell (Treg)–deficient model (Rag-2−/− mice transgenic for ovalbumin-specific T-cell receptor DO11.10), we provide first definitive evidence for induction of transgene product-specific CD4+CD25+ Tregs by in vivo gene transfer. Hepatic gene transfer–induced Tregs express FoxP3, GITR, and CTLA4, and suppress CD4+CD25− T cells. Tregs are detected as early as 2 weeks after gene transfer, and increase in frequency in thymus and secondary lymphoid organs during the following 2 months. Similarly, adoptive lymphocyte transfers from mice tolerized to human FIX by hepatic AAV gene transfer indicate induction of CD4+CD25+GITR+ that suppresses antibody formation to FIX. Moreover, in vivo depletion of CD4+CD25+ Tregs leads to antibody formation to the FIX transgene product after hepatic gene transfer, which strongly suggests that these regulatory cells are required for tolerance induction. Our study reveals a crucial role of CD4+CD25+ Tregs in preventing immune responses to the transgene product in gene transfer.

scholarly journals Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates

2019 ◽  
Vol 3 (17) ◽  
pp. 2632-2641 ◽  
Author(s):  
David Salas ◽  
Karin L. Kwikkers ◽  
Nerea Zabaleta ◽  
Andrea Bazo ◽  
Harald Petry ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Neutralizing Antibodies ◽  
Nonhuman Primates ◽  
Human Subjects ◽  
Factor Ix ◽  
Target Cells ◽  
Targeted Gene Delivery ◽  
Human Factor Ix ◽  
Relevant Animal Model ◽  
Liver Gene

Abstract Adeno-associated virus (AAV)–based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.

Neonatal gene transfer with a retroviral vector results in tolerance to human factor IX in mice and dogs

Blood ◽  
2004 ◽  
Vol 103 (1) ◽  
pp. 143-151 ◽  
Author(s):  
Jun Zhang ◽  
Lingfei Xu ◽  
Mark E. Haskins ◽  
Katherine Parker Ponder
Keyword(s):  
Gene Transfer ◽  
Antibody Formation ◽  
Human Factor ◽  
Retroviral Vector ◽  
Factor Ix ◽  
High Dose ◽  
Human Factor Ix ◽  
B Mice ◽  
Different Strains ◽  
Induce Antibody

Abstract The effect of neonatal gene transfer on antibody formation was determined using a retroviral vector (RV) expressing human factor IX (hFIX). Normal mice from different strains injected intravenously with RV as newborns achieved therapeutic levels of hFIX without antibody production and were tolerant as adults to challenge with hFIX. Neonatal hemophilia B mice that received different amounts of RV achieved stable and dose-related expression of hFIX without anti-hFIX antibody formation. After protein challenge, antibody formation was markedly reduced for animals that expressed hFIX at levels higher than 14 ng/mL (0.3% of normal). However, antibodies developed for animals that received the lowest dose of RV and expressed hFIX at approximately 2 ng/mL before protein challenge. In dogs, neonatal injection of a high dose of RV resulted in 500 ng/mL hFIX in plasma without antibody formation. We conclude that neonatal gene transfer with RV does not induce antibody responses to hFIX in mice or dogs and that mice achieving levels greater than 3 × 10–10 M hFIX are usually tolerant to protein injection as adults. Low-dose gene therapy or frequent protein injections in the neonatal period might induce tolerance to subsequent injections of protein with a low risk for adverse effects.

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