Transient Blockade of ICOS Co-Stimulatory Pathways Induces Long-Term Tolerance to Factor VIII Following Nonviral Gene Transfer of Factor VIII Plasmid into Hemophilia A Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3283-3283
Author(s):  
Baowei Peng ◽  
Peiqing Ye ◽  
Bruce R. Blazer ◽  
Hans D. Ochs ◽  
Carol H. Miao
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Gene Transfer ◽  
Factor Viii ◽  
Combination Treatment ◽  
Hemophilia A ◽  
Inhibitory Antibody ◽  
Nonviral Gene Transfer ◽  
Inhibitory Antibodies ◽  
High Level

Abstract Formation of inhibitory antibodies is a significant problem encountered in the treatment of hemophilia by replacement therapy. Nonviral gene transfer of a factor VIII plasmid into hemophilia mice induced strong humoral responses through predominantly TH2 signals. The plasmid-treated mice produced persistent, high-level inhibitory antibody specifically against FVIII, representing a unique and useful model for testing various immunomodulation strategies. It was previously demonstrated by our group that transient immunosuppression by CTLA4-Ig and anti-CD40L (MR1) can prevent inhibitory antibody formation following nonviral gene transfer of FVIII plasmid into hemophilia A mice. In this study, we tested if blockade of inducible costimulator (ICOS)-ICOS ligand (ICOSL) pathway in combination with or without agents blocking other co-stimulatory pathways can modulate the immune response following gene therapy treatment. Three groups of mice (n=5/group) were subjected to administration of FVIII plasmid via hydrodynamics-based tail-vein injection, and transient immunosuppressive regimens including anti-ICOS (8 treatment in 2 week period), combination of anti-ICOS (same dose) and CTLA4-Ig (2 treatment at day 0 and 2), and combination of anti-ICOS (same dose) and MR1 (5 treatment in 2 week period). 2 mice from anti-ICOS only group, 3 mice from combination treatment of anti-ICOS and CTLA4-Ig group, and 2 mice from combination treatment of anti-ICOS and MR1 group developed inhibitors at 2 weeks post treatment. The rest of the mice did not develop inhibitors. These results imply that neither synergistic nor additional modulation was achieved by combining CTLA4-Ig or MR1 with anti-ICOS compared to anti-ICOS alone. Subsequently a more frequent and longer anti-ICOS treatment (16 treatment in 4 week period) was administered in two separate groups of FVIII plasmid-treated mice (n=5 and 11 per group, respectively). All the treated mice did not produce inhibitory antibodies against FVIII and produced persistent, high-level (100–300 μg/ml) FVIII gene expression for at least 150 days (experimental period). The CD4+ T cells isolated from the spleen of tolerized mice did not proliferate in response to FVIII stimulation in vitro. Furthermore, higher population of CD4+CD25+ regulatory T cells were detected in peripheral blood in the tolerized mice compared to untreated and plasmid-treated mice. Adoptive transfer of CD4+ T cells isolated from tolerized mice is performed to test if these cells can protect the recipient mice from developing inhibitory antibodies against FVIII. Anti-ICOS treatment has the potential for a new immunomodulatory strategy for preventing the formation of inhibitory antibodies against FVIII following gene therapy.

Reduction of Inhibitory Anti-FVIII Antibody Titer by Using a B Domain Variant FVIII/N6 cDNA for Nonviral Gene Therapy in Hemophilia a Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3537-3537 ◽  
Author(s):  
Dominika Jirovska ◽  
Peiqing Ye ◽  
Steven W. Pipe ◽  
Carol H. Miao
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Hemophilia A ◽  
High Titer ◽  
Specific Gene ◽  
Nonviral Gene Transfer ◽  
Inhibitory Antibodies ◽  
Domain Variant

Abstract Due to the large size of FVIII, a B-domain deleted FVIII (BDD-FVIII) cDNA is usually used for developing gene therapy protocols for treating hemophilia A. Inefficient transcription of wild- type FVIII cDNA can be overcome by deletion of the heavily glycosylated B-domain encoding portion of the gene. BDD-FVIII is as clinically efficacious and not more immunogenic than full-length recombinant FVIII. More recently, it was demonstrated that a partial deletion of the B-domain leaving an N-terminal 226 amino acid stretch containing 6 putative asparagine-linked glycosylation sites intact (FVIII/N6) was able to increase in vitro and in vivo secretion of FVIII by 10–15 fold. We have inserted this B domain variant FVIII/N6 cDNA into our liver-specific gene expression vector. The resulting construct, FVIII/N6 plasmid was delivered into the hemophilia A mouse liver by the hydrodynamic method. In control mice treated with BDD-FVIII plasmid (n=5/group), FVIII expression dropped to undetectable levels at 2 weeks post injection and high-titer anti-FVIII antibodies were generated in all the plasmid-treated mice. However, in mice treated with FVIII/N6 plasmid (n=5/group), one out of five mice never developed inhibitory antibodies and still had some FVIII gene expression (~10%) at 8 weeks post gene transfer. Three FVIII/N6 plasmid-treated mice developed anti-FVIII antibodies with significantly reduced inhibitor titer and only one mouse developed high-titer inhibitory antibodies. The CD4+ T cells isolated from the spleen of mice injected with FVIII/N6 constructs proliferated less in response to FVIII stimulation than those from mice injected with BDD-FVIII. These results indicate that FVIII/N6 protein is less immunogenic than BDD-FVIII. Interestingly, both BDD-FVIII and FVIII/N6 constructs produced similar levels of FVIII gene expression (100–300%) initially following nonviral gene transfer. However this could be due to saturation of the ER to Golgi transport apparatus for FVIII by the initial high-level gene expression. Gene expression levels produced by using reduced dosages of BDD-FVIII and FVIII/N6 plasmids are currently being evaluated and compared. These findings suggest that use of a FVIII/N6 construct decreases transgene-specific immune responses following nonviral gene transfer and facilitates long-term gene expression.

Dependence of Vector Dose and Age of Mice in the Induction of Immune Response Against Factor VIII Following Liver-Directed Nonviral Gene Transfer.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3186-3186
Author(s):  
Peiqing Ye ◽  
Carol H. Miao
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Factor Viii ◽  
Plasmid Dna ◽  
Hemophilia A ◽  
High Titer ◽  
Activated T Cells ◽  
Human Factor Viii ◽  
Plasmid Injection ◽  
Inhibitory Antibodies

Abstract Formation of inhibitory antibodies to transgene product may limit the success of gene therapy especially for the treatment of hemophilia A. The risk of forming inhibitory antibodies against factor VIII depends on multiple factors. Previously we have shown that following naked gene transfer of fifty micrograms of a liver-specific, high-expressing factor VIII plasmid, pBS-HCRHPI-FVIIIA into hemophilia A mice (at least 60 days old), a robust humoral response was induced two weeks post plasmid injection despite of initial high-level gene expression of factor VIII (Ye et al. (2004) Mol. Ther. 10, 117–126). This response completely inhibited the activity of circulating factor VIII although factor VIII was persistently expressed in the liver. In this study, the cytokine production was characterized in human factor VIII-activated T cells from plasmid-treated and untreated hemophilic A mice, consistent with a response predominantly mediated by Th2-induced signals. Injection of plasmid DNA into 4 groups of hemophilia A mice (n=5, 60 days old) with 4 different doses (0.4, 2, 10, & 50 microgram per animal) resulted in vector dose-dependent expression of factor VIII. In addition, the two groups of mice with lower doses of plasmid DNA (0.4 & 2 microgram per animal) did not elicit any antibody response against factor VIII, whereas the two groups of mice with higher doses of plasmid DNA (10 & 50 microgram per animal) induced inhibitory antibody formation. Nevertheless, when the two groups of animals (n=4) with lower doses were treated with second injection of fifty microgram of factor VIII plasmid 180 days post plasmid delivery, all mice developed inhibitors suggesting no immune tolerance was induced by first injection of plasmids. Furthermore, fifty micrograms of factor VIII plasmids were injected into 4 groups of hemophilia A mice (n=5) of 4 different age groups (36, 48, 60 & 72 days). It was found that none of the mice with age 36 days at the time of plasmid injection developed inhibitors, 1/5 mice with age 48 days developed inhibitors, whereas the two groups of mice with age 60 & 72 days all developed high-titer inhibitors. These results indicate that induction of anti-factor VIII antibody following gene therapy is strongly dependent upon the vector dose and age of the animals, which has important implication for developing immunomodulation or other strategies to avoid/eliminate antibody responses.

scholarly journals Anti-CD3 antibodies modulate anti–factor VIII immune responses in hemophilia A mice after factor VIII plasmid-mediated gene therapy

Blood ◽  
2009 ◽  
Vol 114 (20) ◽  
pp. 4373-4382 ◽  
Author(s):  
Baowei Peng ◽  
Peiqing Ye ◽  
David J. Rawlings ◽  
Hans D. Ochs ◽  
Carol H. Miao
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Growth Factor ◽  
Immune Responses ◽  
Factor Viii ◽  
Transforming Growth Factor ◽  
Hemophilia A ◽  
Transforming Growth Factor Β ◽  
Inhibitory Antibodies

Abstract One major obstacle in gene therapy is the generation of immune responses directed against transgene product. Five consecutive anti-CD3 treatments concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic levels of FVIII gene expression in treated hemophilia A mice. Repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+ and CD8+ T cells, whereas increased transforming growth factor-β levels in plasma and the frequency of both CD4+CD25+FoxP3+ and CD4+CD25−Foxp3+ regulatory T cells in the initial few weeks after treatment. Although prior depletion of CD4+CD25+ cells did not abrogate tolerance induction, adoptive transfer of CD4+ cells from tolerized mice at 6 weeks after treatment protected recipient mice from anti-FVIII immune responses. Anti-CD3–treated mice mounted immune responses against both T-dependent and T-independent neo-antigens, indicating that anti-CD3 did not hamper the immune systems in the long term. Concomitant FVIII plasmid + anti-CD3 treatment induced long-term tolerance specific to FVIII via a mechanism involving the increase in transforming growth factor-β levels and the generation of adaptive FVIII-specific CD4+Foxp3+ regulatory T cells at the periphery. Furthermore, anti-CD3 can reduce the titers of preexisting anti-FVIII inhibitory antibodies in hemophilia A mice.

Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system

Blood ◽  
2005 ◽  
Vol 105 (7) ◽  
pp. 2691-2698 ◽  
Author(s):  
John R. Ohlfest ◽  
Joel L. Frandsen ◽  
Sabine Fritz ◽  
Paul D. Lobitz ◽  
Scott G. Perkinson ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Protein Product ◽  
Sleeping Beauty ◽  
Molecular Evidence ◽  
Inhibitory Antibody ◽  
Nonviral Gene Transfer ◽  
Sleeping Beauty Transposon ◽  
High Level

AbstractHemophilia A is a lead candidate for treatment by gene therapy because small increments in the missing secreted protein product, coagulation factor VIII (FVIII), would result in substantial clinical amelioration. Clinically relevant therapy might be achieved by stably delivering a human FVIII cDNA to correct the bleeding disorder. We used the Sleeping Beauty (SB) transposon, delivered as naked plasmid DNA by tail-vein injection, to integrate B-domain–deleted FVIII genes into the chromosomes of hemophilia A mice and correct the phenotype. Since FVIII protein is a neoantigen to these mice, sustaining therapeutic plasma FVIII levels was problematic due to inhibitory antibody production. We circumvented this problem by tolerizing 82% of neonates by a single facial-vein injection of recombinant FVIII within 24 hours of birth (the remaining 18% formed inhibitors). Achievement of high-level (10%-100% of normal) FVIII expression and phenotypic correction required co-injection of an SB transposase-expressing plasmid to facilitate transgene integration in immunotolerized animals. Linker-mediated polymerase chain reaction was used to clone FVIII transposon insertion sites from liver genomic DNA, providing molecular evidence of transposition. Thus, SB provides a nonviral means for sustained FVIII gene delivery in a mouse model of hemophilia A if the immune response is prevented.

Reduction of the inhibitory antibody response to human factor VIII in hemophilia A mice by mutagenesis of the A2 domain B-cell epitope

Blood ◽  
2004 ◽  
Vol 104 (3) ◽  
pp. 704-710 ◽  
Author(s):  
Ernest T. Parker ◽  
John F. Healey ◽  
Rachel T. Barrow ◽  
Heather N. Craddock ◽  
Pete Lollar
Keyword(s):  
Antibody Response ◽  
Factor Viii ◽  
B Cell ◽  
Human Factor ◽  
Hemophilia A ◽  
Cell Epitope ◽  
Inhibitory Antibody ◽  
B Cell Epitope ◽  
Human Factor Viii ◽  
Inhibitory Antibodies

AbstractApproximately 25% of patients with hemophilia A develop inhibitory antibodies after treatment with factor VIII. Most of the inhibitory activity is directed against epitopes in the A2 and C2 domains. Anti-A2 inhibitory antibodies recognize a 25-residue segment bounded by R484-I508. Several antigenic residues in this segment have been identified, including R484, R489, and P492. The immunogenicity of purified recombinant B domain–deleted (BDD) human factor VIII molecules containing mutations at R484A/R489A or R484A/R489A/P492A was studied in hemophilia A mice. Inhibitory antibody titers in mice receiving the R484A/R489A/P492A mutant, but not the R484A/R489A mutant, were significantly lower than in mice receiving control human BDD factor VIII. The specific coagulant activity and the in vivo clearance and hemostatic efficacy in hemophilia A mice of the R484A/R489A/P492A mutant were indistinguishable from human BDD factor VIII. Thus, the inhibitory antibody response to human factor VIII can be reduced by mutagenesis of a B-cell epitope without apparent loss of function, suggesting that this approach may be useful for developing a safer form of factor VIII in patients with hemophilia A.

scholarly journals 78. Regulation of Immune Responses Against Factor VIII Following Nonviral Gene Transfer in Hemophilia A and FOXP3 Transgenic Mouse Models

2006 ◽  
Vol 13 ◽  
pp. S33 ◽  
Author(s):  
Benjamin R. Harmeling ◽  
Steven Ziegler ◽  
Troy Torgerson ◽  
Liping Chen ◽  
Hans D. Ochs ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Immune Responses ◽  
Factor Viii ◽  
Transgenic Mouse ◽  
Mouse Models ◽  
Hemophilia A ◽  
Transgenic Mouse Models ◽  
Nonviral Gene Transfer

Development And Application Of A Minimal-Adenoviral Vector System For Gene Therapy Of Hemophilia A

1999 ◽  
Vol 82 (08) ◽  
pp. 562-571 ◽  
Author(s):  
Steven Josephs ◽  
Jiemin Zhou ◽  
Xiangming Fang ◽  
Ramón Alemany ◽  
Cristina Balagué ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Vector System ◽  
High Level Expression ◽  
Viral Particles ◽  
Human Factor Viii ◽  
High Level

IntroductionHemophilia A and B are the most common bleeding disorders caused by deficiencies of clotting factors VIII and IX, respectively, both of which are X-linked with a recessive heredity.1 Replacement of the deficient factors with frequent intravenous injections of plasma concentrates or recombinant proteins is the standard treatment for these diseases.2 Great efforts have been made for nearly a decade toward developing experimental gene therapy for these diseases and aiming at the development of a medical intervention that is more effective and convenient than the currently available replacement therapies.3 Hemophilia is a suitable clinical model for the development of gene therapy products and has a number of advantages: 1) there is a simple and well defined cause-and-effect relationship between the protein deficiencies and bleeding symptoms; 2) tissue-specific expression and precise regulation of the transgenes are not necessary; 3) well characterized animal models are available for preclinical studies; 4) an unequivocal endpoint for product efficacy can be assessed in clinical trials; and 5) even 1% to 5% of the normal physiological levels of the proteins is therapeutic.For gene therapy of hemophilia, the most challenging hurdle, with respect to the long-term expression of the deficient proteins at adequate levels, is the development of a suitable gene delivery system. Technologies have been evolving from ex vivo to in vivo approaches, from initial use of retroviral vector to recent application of adenviral (Ad) or adeno-associated virus (AAV) vector, demonstrating progress from early results of transient low-level expression to more sustained high-level expression.3 For hemophilia A treatment, Ad vectors are particularly useful, since the liver naturally produces factor VIII, and following intravenous (i.v.) injection, Ad vectors concentrate in the liver. This makes the gene transduction efficiency to liver very high. Adenovirus vectors have been developed for gene therapy due to their high titer, broad infectivity, potential for large payload, and in vivo gene delivery capacity.4 Although the immunogenicity and cytotoxicity associated with the early-generation Ad vectors have been a concern with respect to their clinical application, newly developed vectors, in which the viral coding sequences have been deleted, have significantly reduced the side effects associated with the vectors. The “gutless” Ad vector, or so called helper-dependent, large-capacity, or mini- Ad vectors are the representative examples of these new-generation Ad vectors.5-15 The mini-Ad vector system described in this report was developed based on two major research findings. First, an Ad- SV40 hybrid virus discovered during attempts to grow human Ad in non-permissive monkey COS-7 cells.16 The hybrid virus had a genome structure in which only both ends of the Ad sequences were retained and almost all coding sequences of the Ad genome were replaced by symmetric, tandemly repeated SV40 genomes. The hybrid viruses replicated and were packaged in the presence of a wild-type Ad as a helper. This finding implied that total replacement of the Ad genome was possible to form a mini-Ad vector as long as proper helper function and selective pressure was provided. Secondly, it was discovered that Ad packaging can be attenuated by deleting portions of the packaging signal.17 This finding provided a means to put selective pressure on the helper Ad (referred to as ancillary Ad) by specifically limiting its packaging process and allowing a preferential packaging of the mini-Ad. The system, therefore, is designed to have three main components: the mini-Ad vector, the E1-deleted ancillary Ad, and a production cell line that provides AdE1 complementation.Based on the mini-Ad vector system, MiniAdFVIII was developed. The MiniAdFVIII vector carries a 27 kb expression cassette, in which the full-length human factor VIII cDNA is flanked by a human albumin promoter and cognate genomic sequences. Infection of MiniAdFVIII in vitro showed that the vector mediated expression of functional human factor VIII at levels of 100-200 ng/106 cells per 24 hours in HepG2 and 293 cells. With single-dose intravenous injection of 1011 viral particles in hemophilic mice, MiniAdFVIII produced a sustained high-level expression of human factor VIII (at 100-800 ng/ml for up to 369 days) that corrected the factor VIII-deficient phenotype. Safety studies of MiniAdFVIII showed that there were no significant toxicities in mice and dogs after a single intravenous dose of up to 3×1011 and 6×1012 viral particles, respectively. In this report, other studies for developing the MiniAdFVIII vector with a site-specific integration capability and the development of a human factor VIII-tolerized mouse model for preclinical studies of MiniAdFVIII are described.

Sustained Phenotypic Correction of Murine Hemophilia A with Pre-Existing Anti-FVIII Immunity Using Lentivirus-Mediated Platelet-Specific FVIII Gene Transfer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 29-29 ◽  
Author(s):  
Qizhen Shi ◽  
Erin L. Kuether ◽  
Brian C. Cooley ◽  
Scot A. Fahs ◽  
Jocelyn A. Schroeder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Real Time Pcr ◽  
Hemophilia A ◽  
Hematopoietic Stem ◽  
Fviii Activity ◽  
Inhibitory Antibodies ◽  
Tail Clip

Abstract Abstract 29 The development of inhibitory antibodies to exogenous factor VIII (FVIII) is considered a severe and important complication of FVIII infusion in hemophilia A patients. Gene therapy of hemophilia A with inhibitors is especially challenging because functional FVIII activity may be inactivated by circulating inhibitory antibodies if transgene protein is constitutively secreted into the blood circulation. Our previous studies have demonstrated that syngeneic transplantation of hematopoietic stem cells from 2bF8 transgenic mice that express platelet-specific FVIII can efficiently restore hemostasis to hemophilic mice with pre-existing inhibitory antibodies. In the current study, we assessed whether lentivirus-mediated 2bF8 gene transfer could efficiently introduce 2bF8 transgene expression and ameliorate the hemorrhagic phenotype in hemophilic mice with pre-existing immunity. To mimic the clinical situation of an autologous transplant in an inhibitor patient, both donor and recipient FVIIInull mice were immunized with recombinant human B-domain deleted FVIII to induce inhibitory antibody development. Platelet-derived FVIII expression in FVIIInull mice was introduced by 2bF8 lentiviral-mediated bone marrow (BM) transduction and syngeneic transplantation. Following BM reconstitution, mice were analyzed by PCR, quantitative real-time PCR, platelet lysate FVIII activity assay, and inhibitor assay. Phenotypic correction was assessed by tail clip survival test and electrolytic-induced thrombus formation. Expression of the 2bF8 product was detected in all recipients that received 2bF8 lentivirus transduced BM cells, indicating viable engraftment of BM genetically modified with the 2bF8 lentivirus transfer vector. Functional platelet-FVIII activity levels in the transduced mice with pre-existing immunity ranged from 0.36 to 6.18 mU/108 platelets (mean 1.56 ± 1.76 mU/108 platelets, n = 10), which was not significantly different from the levels obtained from a parallel non-inhibitor model (1.46 ± 0.87 mU/108 platelets, n = 4). Real-time PCR demonstrated that there was an average of 0.17 ± 0.05 LV DNA copies per cell in peripheral white blood cells from transduced mice. FVIII inhibiter titer gradually declined with the time, indicating that transduced platelet FVIII is well protected from exposure to the immune system, avoiding activation of a memory response. The tail clip survival test showed that 90% of mice survived tail clip challenge. The electrolytic injury model demonstrated that hemostasis was improved in recipients that received 2bF8 lentivirus-transduced BM cells. Furthermore, BM transferred from the primary transplant recipients into immunized FVIIInull secondary recipients demonstrated sustained platelet-FVIII expression, resulting in the correction of the hemophilia A phenotype with pre-existing immunity. This shows that gene transfer has occurred within long-term repopulating hematopoietic stem cells even in the presence of inhibitory antibodies. These results demonstrate that lentivirus-mediated bone marrow transduction/transplantation can provide sustained improvement of hemostasis in hemophilic mice with pre-existing immunity, indicating that this approach may be a promising strategy for gene therapy of hemophilia A with inhibitory antibodies in humans. Disclosures: Montgomery: GTI Diagnostics: Consultancy; Baxter: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 1662-1672 ◽  
Author(s):  
Baowei Peng ◽  
Peiqing Ye ◽  
Bruce R. Blazar ◽  
Gordon J. Freeman ◽  
David J. Rawlings ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Monoclonal Antibody ◽  
T Cells ◽  
Factor Viii ◽  
Cd4 T Cells ◽  
Hemophilia A ◽  
Term Therapy ◽  
Antibody Treatment ◽  
Inducible Costimulator ◽  
Human Factor Viii

Abstract Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid–treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS+CD4+ T cells and activation of CD25+Foxp3+ Tregs in the peripheral blood, spleen, and lymph nodes. CD4+ T cells from anti-ICOS–treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-β. Moreover, CD4+CD25+ Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS–treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Φx 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

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