Genetically-Engineered Endothelial Cells Implanted Into the Omentum of Hemophilia A Dogs Provides Long-Term Circulating FVIII Resulting From Sustained FVIII Expression and Persistent Cell Viability.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3578-3578 ◽  
Author(s):  
Margareth C Ozelo ◽  
Hideto Matsui ◽  
Carol A. Hegadorn ◽  
Lori Harpell ◽  
Sandra Powell ◽  
...  
Keyword(s):  
Immune Response ◽  
Endothelial Cells ◽  
Growth Factors ◽  
Cell Viability ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Autologous Blood ◽  
Cell Transplant ◽  
Therapy Strategy

Abstract Abstract 3578 Poster Board III-515 Ex vivo delivery of therapeutic transgenes provides an additional level of safety as it avoids systemic administration of viral vectors. Our group has shown that autologous blood outgrowth endothelial cells (BOECs) transduced with a lentiviral vector delivery system containing the FVIII transgene is a promising gene therapy strategy for hemophilia A. We have shown that subcutaneous implantation of factor (F) VIII-expressing BOECs in a murine model of hemophilia A can produce therapeutic levels of FVIII that are sustained for more than 6 months. However, to improve the levels of FVIII expression and cell viability we wanted to evaluate the omentum as an alternative site for BOEC implantation. Initially this strategy was evaluated in two normal dogs. One and three months after delivery of the cells, immunostaining of biopsies from the injection sites showed the presence of viable cells expressing FVIII and evidence of neovascularization. To evaluate the efficacy of this strategy, a hemophilia A dog received 5×108 transduced autologous BOECs that expressed high levels of FVIII in vitro (1.5 IU/106 cells/24hrs). We used autologous fibrinogen as a vehicle for the cells along with canine endothelial growth factors (VEGF and bFGF). For the implantation procedure the dog received prophylaxis with canine cryoprecipitate transfusions. FVIII antigen levels (FVIII:Ag) of between 20 and 50 ng/mL continue to be detected in the plasma 8 months post-implantation, indicating that these cells remain viable and express persistent high levels of FVIII over an extended period of time. However, two weeks after the procedure, the dog developed an anti-FVIII immune response comprising both inhibitory and non-inhibitory antibodies, and therefore no FVIII coagulant activity (FVIII:C) was detected. With a view to preventing the development of the anti-FVIII immune response, we used immunosuppression with cyclophosphamide in two additional hemophilia A dogs. Each of these dogs received 5 × 108 transduced autologous BOECs. In place of fibrinogen, these cells were delivered in a gel comprised of synthetic, heparin-binding peptide-amphiphiles (HBPA) and heparan sulfate, along with canine VEGF and bFGF. The peptide gel prolongs the activity of these growth factors and protects them from proteolysis, enhancing their angiogenic activity. HBPA gel has been shown to increase vascularization of cell transplant sites, which should improve BOEC survival in the omentum. The procedure of implanting the genetically modified BOECs was completed without complications in all hemophilia A dogs and we are continuing to evaluate the efficacy of this strategy. Disclosures: Hulvat: Nanotope Inc.: Employment.

Ex Vivo Gene Trasfer for Hemophilia A That Enhances Safety and Results in Sustained In Vivo Factor VIII Expression from Lentivirally-Engineered Endothelial Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3251-3251
Author(s):  
Hideto Matsui ◽  
Masaru Shibata ◽  
Brian Brown ◽  
Andrea Labelle ◽  
Carol Hegadorn ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Endothelial Cell ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Scid Mice ◽  
Anatomic Site ◽  
Cmv Promoter ◽  
Endothelial Progenitors ◽  
Therapy Strategy

Abstract Hemophilia is an excellent candidate disorder for the use of gene therapy as a treatment modality. However, significant obstacles have been encountered with systemic delivery of viral vectors that have prevented sustained expression of the therapeutic protein. Investigation of alternative gene therapy strategies for hemophilia that enhance safety and facilitate long-term, therapeutic levels of the transgene product is imperative. In this study, we evaluated an ex vivo gene therapy strategy for hemophilia A. Circulating endothelial cell progenitors (blood outgrowth endothelial cells - BOECs) were isolated from canine and mouse blood and transduced with a third generation self-inactivating lentiviral vector encoding the canine FVIII transgene under the transcriptional control of either the CMV promoter or an endothelial cell-specific regulatory element. Transduced BOECs were injected either intravenously (IV) or subcutaneously mixed with Matrigel (SC+Matrigel) into NOD/SCID mice. Canine FVIII antigen levels were assayed at weekly intervals using an Asserachrom VIII:Ag ELISA that detects canine FVIII against a background of normal murine FVIII levels in the NOD/SCID mice. The mean FVIII antigen levels in mice injected with BOECs at 3 weeks following treatment were 37.5 mU/mL and 105.8mU/mL, for IV and SC+Matrigel administration, respectively. These FVIII antigen levels were sustained up to 12 weeks at therapeutic levels (21.3mU/mL and 21.7mU/mL, for IV and SC+Matrigel administration respectively). To evaluate if the observed loss of FVIII expression by 12 weeks post-treatment resulted from transcriptional silencing of the viral promoter, the CMV promoter was replaced with the endothelial cell-specific thrombomodulin (TM) promoter and transduced BOECs were implanted SC with Matrigel. In contrast to results from the CMV-regulated transgene, sustained therapeutic levels of FVIII have been documented for the duration of the study with the TM-regulated construct (34.3 mU/mL at 3 weeks and 22.5 mU/mL at 20 weeks) Immunostaining at 18 weeks after SC implantation of the transduced BOECs, shows that these cells still express FVIII and von Willebrand Factor. Biodistribution analysis by flow cytometry and quantitative PCR demonstrated that SC-implanted BOECs were retained inside the scaffold and were not detected at any other anatomic site. These results indicate that genetically-modified endothelial progenitors implanted in a SC scaffold can provide sustained therapeutic levels of FVIII and are a promising safe delivery vehicle for gene therapy of hemophilia. Currently, these engineered cells have been implanted into immunocompetant mice and FVIII levels are being assessed.

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.

Targeting FVIII Expression to Platelets Induces Immune Tolerance in Hemophilia A Mice with or without Pre-Existing Anti-FVIII Immunity,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4170-4170
Author(s):  
Yingyu Chen ◽  
Erin L. Kuether ◽  
Jocelyn A. Schroeder ◽  
Robert R. Montgomery ◽  
David W. Scott ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Immune Tolerance ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
High Titer ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Therapy Strategy ◽  
Inhibitory Antibodies

Abstract Abstract 4170 Our previous studies have shown that targeting FVIII expression to platelets (2bF8) can correct murine hemophilia A phenotype even in the presence of inhibitory antibodies. In the present study, we wanted to explore 1) whether platelets containing FVIII can act as an immunogen; and 2) whether platelet-derived FVIII can induce immune tolerance in a hemophilia A mouse model. To investigate whether platelets containing FVIII can act as an immunogen in hemophilia A mice, we infused transgenic mouse platelets with a level of platelet-FVIII of 6 mU/108 platelets to naïve FVIIInull mice weekly for 8 weeks. These platelets were between 30 to 50% of total platelets upon infusion and the levels of platelet-FVIII in the infused animals were 0.11 ± 0.01 mU/108 platelets (n = 6) one week after infusion. No anti-FVIII inhibitory antibodies were detected in the infused mice during the study course. All animals developed inhibitors following further challenged with recombinant human FVIII (rhFVIII) at a dose of 50 U/kg by intravenous injection weekly for 4 weeks, indicating that infusion of platelets containing FVIII does not trigger immune response in hemophilia A mice. To explore whether platelet-derived FVIII will act as an immunogen in the presence of primed spleen cells (from mice already producing inhibitory antibody), we co-transplanted splenocytes from highly immunized FVIIInull mice and bone marrow (BM) cells from 2bF8 transgenic mice into 400 cGy sub-lethal irradiated FVIIInull recipients. We monitored the levels of inhibitory antibodies in recipients for up to 8 weeks and found that inhibitor titers declined with time after transplantation. We then challenged co-transplantation recipients with rhFVIII and found that inhibitor titers in the control group co-transplantat of FVIIInull BM cells increased 103.55 ± 64.83 fold (n = 4), which was significantly more than the group receiving 2bF8 transgenic BM cells (14.34 ± 18.48, n = 5) (P <.05). The inhibitor titers decreased to undetectable in 40% of 2bF8 transgenic BM cells co-transplantation recipients even after rhFVIII challenge, indicating immune tolerance was induced in these recipients. To further explore the immune response in the lentivirus-mediated platelet-derived FVIII gene therapy of hemophilia A mice, we transduced hematopoietic stem cells from pre-immunized FVIIInull mice with 2bF8 lentivirus (LV) followed by syngeneic transplantation into pre-immunized lethally irradiated FVIIInull recipients and monitored the levels of inhibitor titers in recipients. After full BM reconstitution, platelet-FVIII expression was sustained (1.56 ± 0.56 mU/108platelets, n = 10), but inhibitor titers declined with time, indicating that platelet-derived FVIII does not provoke a memory response in FVIIInullmice that had previously mounted an immune response to rhFVIII. The t1/2 of inhibitor disappearance in 2bF8 LV-transduced recipients (33.65 ± 11.12 days, n = 10) was significantly shorter than in untransduced controls (66.43 ± 22.24 days, n = 4) (P <.01). We also transplanted 2bF8 LV-transduced pre-immunized HSCs into 660 cGy sub-lethal irradiated naïve FVIIInull mice. After BM reconstituted, recipients were assessed by platelet lysate FVIII:C assay and tail clip survival test to confirm the success of genetic therapy. Animals were then challenged with rhFVIII. Only 2 of 7 2bF8 LV-transduced recipients developed inhibitory antibodies (55 and 87 BU/ml), while all untransduced control developed high titer of inhibitors (735.50 ± 94.65 BU/ml, n = 4). In conclusion, our results demonstrate that 1) platelets containing FVIII are not immunogenic in hemophilia A mice; and 2) platelet-derived FVIII may induce immune tolerance in hemophilia A mice with or without pre-existing inhibitory antibodies. This tolerance induction would add an additional significant benefit to patients with platelet-derived FVIII gene therapy strategy because protein infusion could be administered in some special situations (e.g. surgery in which a greater levels of FVIII may be required) with minimized risk of inhibitor development. Disclosures: No relevant conflicts of interest to declare.

Combination of the IGF-1 Receptor Inhibitor Picropodophylin and the BH3 Mimetic ABT-737 Has Synergistic Anti-Myeloma Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4010-4010
Author(s):  
Liesbeth Bieghs ◽  
Ken Maes ◽  
Els Van Valckenborgh ◽  
Eline Menu ◽  
Hans Erik Johnsen ◽  
...  
Keyword(s):  
Growth Factors ◽  
Cell Viability ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Differential Sensitivity ◽  
Expression Ratio ◽  
Therapy Strategy ◽  
Induced Apoptosis ◽  
Bh3 Mimetic

Abstract Abstract 4010 Despite intensive research multiple myeloma (MM) is still an incurable disease. MM cells are strongly dependent on the BM micro-environment where growth factors are secreted. IGF-1 is one of the most important growth factors in MM and thus forms an attractive target for anti-cancer therapy. Previously, we demonstrated that picropodophylin (PPP), an IGF-1R kinase inhibitor, indeed has potent anti-MM effects both on human cells and in the 5T2MM and 5T33MM murine models. However, mice eventually relapsed and showed signs of morbidity. Therefore it would be an attractive approach to combine PPP with other cytotoxic drugs. ABT-737 is a BH3 mimetic that binds with high affinity to Bcl-xL, Bcl-2 and Bcl-w, but not Mcl-1. In MM, elevated expression of the Bcl-2 pro-survival family of proteins, especially Mcl-1 and to a lesser extent Bcl-2, has extensively been shown to cause resistance to drug induced apoptosis in MM cells. Consequently, ABT-737 was shown to have potent anti-MM activity but only on a subset of human cell lines. Only very recently, it was shown that the ABT-737 sensitivity appears to be determined both by the Bcl-2/Mcl-1 expression ratio and the interaction of these anti-apoptotic proteins with Bim. Interestingly, we demonstrated that IGF-1R inhibition reduces the expression of Mcl-1 and Bcl-xL and that IGF-1 down-regulates the expression of Bim. In addition, a protective effect of IL-6 and BMSC against ABT-737 has been reported. Together, all the above suggests that it would be beneficial to combine ABT-737 with agents that target growth factors, like PPP. Here, we investigated the potential synergistic anti-MM effects of PPP and ABT-737 and studied the underlying mechanisms using two human myeloma cell lines (OPM-2 and RPMI-8226) and the murine 5T33MM model. Both PPP and ABT-737 (kindly provided by Abbott Laboratories) alone were found to significantly decrease cell viability and induce apoptosis dose and time dependently as evidenced by a decrease in ATP levels and an increase in the number of AnnexV/7'AAD positive cells. However, in agreement with previous reported data, we observed differential sensitivity to ABT-737 between the cell lines used. Nevertheless, treatment with PPP/ABT-737 synergistically decreased cell viability and induced apoptosis in all cell lines. In addition, by western blot analysis we could observe increased cleavage of caspase- 3,- 9 and PARP. Mechanistically, PPP was found to circumvent the adverse effect of ABT-737 by blocking the ABT-737 induced Mcl-1 expression and increasing the expression of Noxa. Interestingly, while CD138+ 5T33MM cells were more sensitive to PPP and the CD138- cells more sensitive to ABT-737, treatment with PPP/ABT-737 targeted both MM cell subpopulations to an equal extent. Finally, we tested the combination of PPP and ABT-737 in the 5T33MM model in a prophylactic setting. Whereas, vehicle and ABT-737 treated mice exhibited progressive MM growth, PPP, and to a significant greater extent, PPP/ABT-737 reduced the tumor burden and prolonged overall survival (p≤0.001). In conclusion, PPP combined with ABT-737 appears to have synergistic anti-MM activity and might thus be a novel and promising therapy strategy for MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Unexpected enhancement of FVIII immunogenicity by endothelial expression in lentivirus-transduced and transgenic mice

2020 ◽  
Vol 4 (10) ◽  
pp. 2272-2285
Author(s):  
Qizhen Shi ◽  
Christopher V. Carman ◽  
Yingyu Chen ◽  
Peter T. Sage ◽  
Feng Xue ◽  
...  
Keyword(s):  
Immune Response ◽  
Hemophilia A ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Robust Immune Response ◽  
Tfh Cells ◽  
Expression Site

Abstract Factor VIII (FVIII) replacement therapy for hemophilia A is complicated by development of inhibitory antibodies (inhibitors) in ∼30% of patients. Because endothelial cells (ECs) are the primary physiologic expression site, we probed the therapeutic potential of genetically restoring FVIII expression selectively in ECs in hemophilia A mice (FVIIInull). Expression of FVIII was driven by the Tie2 promoter in the context of lentivirus (LV)-mediated in situ transduction (T2F8LV) or embryonic stem cell–mediated transgenesis (T2F8Tg). Both endothelial expression approaches were associated with a strikingly robust immune response. Following in situ T2F8LV-mediated EC transduction, all FVIIInull mice developed inhibitors but had no detectable plasma FVIII. In the transgenic approach, the T2F8Tg mice had normalized plasma FVIII levels, but showed strong sensitivity to developing an FVIII immune response upon FVIII immunization. A single injection of FVIII with incomplete Freund adjuvant led to high titers of inhibitors and reduction of plasma FVIII to undetectable levels. Because ECs are putative major histocompatibility complex class II (MHCII)-expressing nonhematopoietic, “semiprofessional” antigen-presenting cells (APCs), we asked whether they might directly influence the FVIII immune responses. Imaging and flow cytometric studies confirmed that both murine and human ECs express MHCII and efficiently bind and take up FVIII protein in vitro. Moreover, microvascular ECs preconditioned ex vivo with inflammatory cytokines could functionally present exogenously taken-up FVIII to previously primed CD4+/CXCR5+ T follicular helper (Tfh) cells to drive FVIII-specific proliferation. Our results show an unanticipated immunogenicity of EC-expressed FVIII and suggest a context-dependent role for ECs in the regulation of inhibitors as auxiliary APCs for Tfh cells.

The Immunogenicity of Platelet-Derived FVIII in Hemophilia a Mice with or without Pre-Existing Anti-FVIII Immunity

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2809-2809
Author(s):  
Yingyu Chen ◽  
Jocelyn Schroeder ◽  
Juan Chen ◽  
Xiaofeng Luo ◽  
Christina Baumgartner ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
High Titer ◽  
Hematopoietic Stem ◽  
Inhibitory Antibodies ◽  
Platelet Infusion

Abstract Recent studies from our group and others have demonstrated that FVIII ectopically targeted to platelets under control of the platelet-specific αIIb promoter (2bF8) can efficiently restore hemostasis in hemophilia A mice even in the presence of high-titer inhibitory antibodies directed against FVIII (inhibitors). Our studies have demonstrated that platelet-targeted FVIII gene therapy can not only correct the hemophilic phenotype, but also induce FVIII-specific immune tolerance. In the platelet gene therapy model, hematopoietic stem cells (HSCs) are ex vivo transduced with lentivirus carrying 2bF8 and transplanted into the recipient. Sufficient preconditioning has to be employed to create space for therapeutic engraftment of the transduced HSCs. It is not clear whether preconditioning affects the potential for an immune response in the context of platelet-derived FVIII. Furthermore, if current efforts to generate platelets in vitro succeed, genetically manipulated platelets containing FVIII may be used therapeutically, as potential transfusion alternative, in hemophilia A patients even with inhibitors. One important question that has not been explored, however, is the immunogenicity of platelet-derived FVIII. To investigate whether platelet-derived FVIII can act as an immunogen in hemophilia A mice, we infused transgenic mouse platelets with a level of platelet-FVIII of 6 mU/108 platelets into naïve FVIIInull mice without any preconditioning weekly for 8 weeks. These platelets were transfused to a level between 20 to 57% of total platelets upon infusion, and all animals survived the tail-clip survival test 13-hours after platelet infusion. The level of platelet-FVIII in the infused animals was 0.11 ± 0.01 mU/108 platelets (n = 6) even one week after infusion. Neither inhibitory nor non-inhibitory anti-FVIII antibodies were detected in the infused mice during the study course (n = 9). All animals developed inhibitors following further challenge with recombinant human FVIII (rhF8) at a dose of 50 U/kg by intravenous injection weekly for 4 weeks, indicating that infusion of platelets containing FVIII does not trigger an immune response in hemophilia A mice. We then explored whether platelets containing FVIII can act as an immunogen in FVIIInull mice with pre-existing anti-FVIII immunity. FVIIInull mice were immunized with rhF8 to induce anti-FVIII antibodies. Four week after the last immunization, 2bF8 transgenic platelets were transfused into rhF8-primed FVIIInull mice (n = 4) weekly for 4 weeks and anti-FVIII antibody titers were monitored. There was not significant augmentation of FVIII-specific antibodies as determined by Bethesda assay for inhibitory antibodies and ELISA assay for total anti-FVIII IgG, indicating that infusion of platelets containing FVIII does not stimulate an anti-FVIII memory response in the inhibitor model. To investigate whether preconditioning affects the anti-FVIII immune response, animals were pre-conditioned with a sub-lethal 660 cGy total body irradiation (TBI) followed by 2bF8 transgenic platelet infusion weekly for 8 weeks. No anti-FVIII antibodies were detected in recipients (n = 6) after 2bF8 transgenic platelet infusion. Following further challenge with rhF8, the inhibitor titer in this group was significantly lower (75 ± 42 BU/ml) than in the naïve FVIIInull mice without preconditioning when the same infusion protocol was employed (270 ± 76 BU/ml), indicating that 660 cGy TBI plus 2bF8 transgenic platelet infusion may suppress anti-FVIII immune response. In conclusion, our data demonstrate that infusion of platelets containing FVIII triggers neither primary nor memory anti-FVIII immune response in hemophilia A mice. Disclosures No relevant conflicts of interest to declare.

Development of an Anti-HTLV-1 Vaccine for the Treatment of Adult T-Cell Leukemia/Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4010-4010 ◽  
Author(s):  
Déborah Revaud ◽  
Ana Bejanariu ◽  
Lamya Loussaief ◽  
Emeline Sarry ◽  
Abdel Zemmar ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Cellular Immune Response ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Cell Leukemia ◽  
Adult T Cell Leukemia ◽  
New Treatments ◽  
Cellular Immune ◽  
Stimulation Of

Abstract Human T-cell Lymphotrophic Virus 1 (HTLV-1) is the etiologic agent of Adult-T cell Leukemia/Lymphoma (ATL). Therapeutic options for ATL patients are very limited and in aggressive forms of the disease survival rate is only 10% to 30% with conventional chemotherapies and bone marrow transplantation. Although some clinical trials gave encouraging results regarding the efficacy of new treatments, most of them are lifelong, aggressive and failed to achieve a significant impact on long-term survival. Consequently, new treatments for ATL patients are needed to limit relapses and side effects. Specific HTLV-1 cellular immune response is dramatically impaired in ATL patients, which could favor the initiation and the progression of the disease. Hence, stimulating immune responses against HTLV-1 can be an appropriate therapeutic option to treat ATL. THERAVECTYS has developed an anti-HTLV-1 vaccine, based on its lentiviral vector technology inducing a broad, intense and long-lasting cellular immune response after intra-muscular injection. THERAVECTYS was the first company to have launched a clinical trial based on lentiviral vectors technology with the THV01 vaccine for the treatment of HIV (NCT02054286). Results obtained demonstrated both safety and immunogenicity of THV01 in human, with polyfunctional and multi-specific CD4 and CD8 T-cells responses. The anti-HTLV-1 lentiviral vector, THV02 vaccine, encodes for a unique polypeptide derived from Tax, HBZ, p12I and p30II proteins, involved in HTLV-1 pathogenicity and known to be recognized by the immune system of HTLV-1 infected patients. Our preclinical results have demonstrated that THV02 can induce a cellular immune response in C57Bl/6j and BalbC mice and in Sprague Dawley rats, as demonstrated by IFN-γ Elispot. Safety of the THV02 vaccine has been demonstrated during carcinogenicity and regulatory GLP preclinical toxicity studies. Biodistribution and shedding studies demonstrated the very limited diffusion of THV02 after injection, its fast clearance and a non-dissemination in body fluids. As no relevant ATL immunocompetent animal model is available to assess the anti-tumor effect of THV02, THERAVECYTS is developing an ex-vivo efficacy model using blood samples of ATL patients. Briefly, monocyte-derived dendritic cells (MDDC) from blood of ATL patients are purified by isolation of CD14 positive cells from PBMC and differentiation in the presence of IL4 and GM-CSF. MDDC are then transduced with lentiviral vectors encoding for the anti-HTLV-1 antigen and maturation is induced upon TNFa and PGE2 exposure before the co-culture with autologous CD8+ T-cells for stimulation of the cellular immune response. Then, stimulated CD8+ are co-cultured with autologous CD4+ CD25+ ATL cells and the cytotoxic activity is monitored by flow cytometry. Preliminary results demonstrated that MDDC from a chronic ATL patient can be efficiently transduced and matured as attested by the CD40, CD86, HLA-DR, -A, -B and C markers on their surface. In addition, we have observed a specific stimulation of the CD8+, ie an increase of IFNg, TNFa, IL2 and perforin in the media of the co-culture of CD8+ with MDDC expressing anti-HTLV-1 antigen. These data are very encouraging and demonstrate for the first time the feasibility to develop an ex vivo model to assess vaccine efficacy using ATL blood sample. The development of this model is ongoing using several ATL donors representing the different subtypes of the disease and will be presented at the meeting. Regarding the indication and the safety profile of THV02, THERAVECTYS plans to begin a clinical trial in Q4 2015. This assay will be an open-label, dose escalation phase I/II study to assess the safety and the immunogenicity (cellular immune response) of the THV02 vaccination as a treatment of ATL patients. All ATL subtypes will be considered since THV02 vaccine can be combined with conventional ATL treatments. In addition, as the THV02 antigen contains peptides derived from Tax but also HBZ, p12I and p30II viral proteins, all ATL patients can be treated whatever the status of Tax expression. As secondary objectives, both humoral immune response and clinical effect will be assessed. HTLV-1 RNA expression and clonality of HTLV-1 infected cells will be studied as exploratory objectives. Finally, up to 16 patients will be enrolled in France, UK, French Guiana, Martinique and Guadeloupe before doing a phase of extension cohort in US. Disclosures No relevant conflicts of interest to declare.

scholarly journals Distinct Roles of Adenovirus Vector-Transduced Dendritic Cells, Myoblasts, and Endothelial Cells in Mediating an Immune Response against a Transgene Product

2002 ◽  
Vol 76 (6) ◽  
pp. 2899-2911 ◽  
Author(s):  
Stéphanie Mercier ◽  
Hanne Gahéry-Segard ◽  
Martine Monteil ◽  
Renée Lengagne ◽  
Jean-Gérard Guillet ◽  
...  
Keyword(s):  
Immune Response ◽  
Endothelial Cells ◽  
Dendritic Cells ◽  
Recombinant Adenovirus ◽  
Ex Vivo ◽  
Adenovirus Vector ◽  
Cell Types ◽  
T Cell Response ◽  
Specific Response ◽  
Melanoma Tumor

ABSTRACT Adenovirus-mediated gene delivery via the intramuscular route efficiently promotes an immune response against the transgene product. In this study, a recombinant adenovirus vector encoding β-galactosidase (AdβGal) was used to transduce dendritic cells (DC), which are antigen-presenting cells, as well as myoblasts and endothelial cells (EC), neither of which present antigens. C57BL/6 mice received a single intramuscular injection of AdβGal-transduced DC, EC, or myoblasts and were then monitored for anti-β-galactosidase (anti-β-Gal) antibody production, induction of gamma interferon-secreting CD8+ T cells, and protection against melanoma tumor cells expressing β-Gal. While all transduced cell types were able to elicit an antibody response against the transgene product, the specific isotypes were distinct, with exclusive production of immunoglobulin G2a (IgG2a) antibodies following injection of transduced DC and EC versus equivalent IgG1 and IgG2a responses in mice inoculated with transduced myoblasts. Transduced DC induced a strong ex vivo CD8+ T-cell response at a level of 50% of the specific response obtained with the AdβGal control. In contrast, this response was 6- to 10-fold-lower in animals injected with transduced myoblasts and EC. Accordingly, only animals injected with transduced DC were protected against a β-Gal tumor challenge. Thus, in order to induce a strong and protective immune response to an adenovirus-encoded transgene product, it is necessary to transduce cells of dendritic lineage. Importantly, it will be advantageous to block the transduction of DC for adenovirus-based gene therapy strategies.

The Effect of VAS972 on Allergic Contact Hypersensitivity

2000 ◽  
Vol 4 (3) ◽  
pp. 132-137 ◽  
Author(s):  
Gulnar M. Shivji ◽  
Hirotake Suzuki ◽  
Arkady Sh. Mandel ◽  
Anthony E. Bolton ◽  
Daniel N. Sauder
Keyword(s):  
Immune Response ◽  
Ex Vivo ◽  
Autologous Blood ◽  
Immunosuppressive Agents ◽  
Lymphocytic Infiltration ◽  
Contact Hypersensitivity ◽  
Controlled Exposure ◽  
Preliminary Study ◽  
Allergic Contact ◽  
Cellular Components

Background: Contact hypersensitivity (CHS) is a Th1-mediated immune response that can be down-regulated by immunosuppressive agents such as cyclosporine and environmental stimuli such as ultraviolet light. Recently, an immunomodulation therapy, VAS972, has been developed which is believed to down-regulate the Th1 arm of the immune response. This VAS972 involves modifying autologous blood by controlled exposure to the oxidizing agent ozone and UVC light, at an elevated temperature ex vivo. The processed blood is then administered by intramuscular injection. Objective: To further evaluate the immune modulating effect of VAS972. Methods: We examined the effect of VAS972 treatment on CHS. Contact hypersensitivity was induced with dinitro-fluorobenzene (DNFB) in animals receiving VAS972-processed blood, control blood, or saline. A preliminary study was also conducted to evaluate the effect of plasma and cellular fractions of processed blood. Results: Mice injected with VAS972-processed blood demonstrated a significantly lower (46%) CHS response than controls. Histologic examination of challenged ear skin from control mice displayed edema with a significant lymphocytic infiltration, whereas animals administered processed blood demonstrated a reduction in lymphocytic infiltration. Mice injected with either plasma or the cellular fraction of the VAS972-treated blood also demonstrated a significant suppression (49% and 41%, respectively). Conclusion: The results of this study demonstrated that VAS972 suppresses CHS and cellular infiltration. Furthermore, the plasma and cellular components of the VAS972 treatment were also able to induce immunosuppression. This further supports the hypothesis that VAS972 down-regulates the Th1 arm of the immune response.

Correction of Fanconi Anemia Group A in Primary Human and Murine Hematopoietic Progenitors with a Clinical Lentiviral Vector

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2357-2357
Author(s):  
Jason Taylor ◽  
Grant D Trobridge ◽  
Martin Wohlfahrt ◽  
Sylvia Chien ◽  
Xin Zhao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Bone Marrow ◽  
Fanconi Anemia ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Hematopoietic Progenitors ◽  
Cell Clusters ◽  
Group A

Abstract Fanconi Anemia (FA) is a rare genetic disorder of DNA repair that typically manifests with bone marrow failure. Allogeneic stem cell transplant is the only cure known for the bone marrow disorder. Unfortunately, many patients do not have matched family donors, and alternative donor transplant has been associated with considerable morbidity and mortality. For individuals without an appropriate donor, ex vivo genetic modification of autologous stem cells is a potential therapeutic strategy. However, one of the major hurdles in gene therapy for this condition is the increased sensitivity of FA stem cells to free-radical induced DNA damage during ex vivo culture and manipulation. To minimize this damage we have developed a brief transduction procedure for lentivirus vector mediated gene transfer for Fanconi Anemia complementation group A (FancA), and evaluated this method in bone marrow progenitors from patients with FA, and murine progenitors from FancA deficient mice. The lentiviral vector, RSCPFancA-sW (FancA-sW), was specifically developed for clinical studies, and has a synthetic wpre which has been modified for safety (does not express a partial woodchuck hepatitis virus X protein open reading frame) and most of the 3′ untranslated region of the FancA gene was removed. FancA −/− mice were preconditioned with cyclophosphamide (120 mg/kg) prior to injection of transduced syngeneic bone marrow. After two subsequent rounds of the same dose of cyclophosphamide, 3/5 mice demonstrated mitomycin C (MMC) resistance in cells from bone marrow or spleen via colony assay. Two mice had peripheral blood marking with the vector seen by quantitative PCR. In an initial mouse experiment using similar conditions, the estimated transduction efficiency by colony PCR was determined to be 31% with the average number of integrations per cell determined to be 1.8 by LAM-PCR. Bone marrow mononuclear cells (BM MNCs) from a patient with FancA were also transduced with FancA-sW. Very few colonies were obtained on methylcellulose, although cell clusters were observed in MMC-treated cultures derived from FancA-sW transduced BM MNCs, while no clusters or colonies formed from cells transduced with the control vector. In an attempt to increase progenitor survival by reducing oxidative stress, primary human FA bone marrow was transduced and placed in 5% oxygen in the presence of 1 nM NAC (N-Acetyl-L-Cysteine). Cells plated in methylcellulose under these conditions had 2–3 fold increased colony formation compared to conditions without NAC or in 21% oxygen (P &lt; 0.03). FancA-sW transduced BM MNCs from a second FancA patient were plated under these conditions formed colonies and numerous cell clusters and demonstrated resistance to MMC compared to GFP transduced controls (P &lt; 0.007). Furthermore, transduced BM MNCs in culture had increased survival in culture when exposed to MMC ( P &lt; 0.004). In summary, a lentiviral vector with a functional FancA transgene was developed which achieves phenotypic correction of human and murine hematopoietic progenitors with the brief period of prestimulation and transduction that would be required to maintain survival of FA cells ex vivo in culture. Reduction of oxidative stress may enhance the viability and engraftment of fragile FA stem cells.

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