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Yanjun Liu - One of the best experts on this subject based on the ideXlab platform.
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Human CD8+CD28- T suppressor cells expanded by Common Gamma Chain (γc) cytokines retain steady allospecific suppressive capacity in vivo.
BMC immunology, 2020Co-Authors: Guihuan Liu, Fu Feng, Ping Zhu, Hua Zhang, Danni Zhang, Xiaoqiang Feng, Zedan Zhang, Yanjun LiuAbstract:Background CD8+CD28- T suppressor (Ts) cells play critical role in transplant tolerance. Our previous study has generated CD8+CD28- Ts cells in vitro which exert robust allospecific suppressive capacity in vitro. Results CD8+CD28- Ts cells were expanded by stimulating human CD8+ T cells with allogeneic antigen presenting cells in the presence of the Common Gamma Chain cytokines IL-2, IL-7 and IL-15 in vitro, and were further verified in vitro through day 7 to 11 for their persistency of the allospecific suppressive capacity. When CD8+CD28- Ts cells were adoptively transferred into NOG mice, their capacity to inhibit CD4+ T cell proliferation in allospecific manner remained potent on 11 days after their injection. The mechanisms for expansion of CD8+CD28- Ts cells by the Common Gamma Chain cytokines were investigated. These included promoting CD8+CD28- T cells proliferation, converting CD8+CD28+ T cells to CD8+CD28- T cells and decreasing CD8+CD28- T cell death. Furthermore, the expanded CD8+CD28- Ts cells showed upregulation of the co-inhibitory molecule Tim-3 and down-regulation of the cytotoxic molecule granzyme B. Conclusions In summary, these results demonstrated that the in vitro-expanded human CD8+CD28- T cells retained potent allospecific suppressive capacity in vivo and depicted multiple mechanisms for the expansion of Ts cells, which might promote further bench to clinic research.
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human cd8 cd28 t suppressor cells expanded by Common Gamma Chain γc cytokines retain steady allospecific suppressive capacity in vivo
BMC Immunology, 2020Co-Authors: Guihuan Liu, Fu Feng, Ping Zhu, Hua Zhang, Danni Zhang, Xiaoqiang Feng, Zedan Zhang, Yanjun LiuAbstract:Background CD8+CD28- T suppressor (Ts) cells play critical role in transplant tolerance. Our previous study has generated CD8+CD28- Ts cells in vitro which exert robust allospecific suppressive capacity in vitro. Results CD8+CD28- Ts cells were expanded by stimulating human CD8+ T cells with allogeneic antigen presenting cells in the presence of the Common Gamma Chain cytokines IL-2, IL-7 and IL-15 in vitro, and were further verified in vitro through day 7 to 11 for their persistency of the allospecific suppressive capacity. When CD8+CD28- Ts cells were adoptively transferred into NOG mice, their capacity to inhibit CD4+ T cell proliferation in allospecific manner remained potent on 11 days after their injection. The mechanisms for expansion of CD8+CD28- Ts cells by the Common Gamma Chain cytokines were investigated. These included promoting CD8+CD28- T cells proliferation, converting CD8+CD28+ T cells to CD8+CD28- T cells and decreasing CD8+CD28- T cell death. Furthermore, the expanded CD8+CD28- Ts cells showed upregulation of the co-inhibitory molecule Tim-3 and down-regulation of the cytotoxic molecule granzyme B. Conclusions In summary, these results demonstrated that the in vitro-expanded human CD8+CD28- T cells retained potent allospecific suppressive capacity in vivo and depicted multiple mechanisms for the expansion of Ts cells, which might promote further bench to clinic research.
Guihuan Liu - One of the best experts on this subject based on the ideXlab platform.
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Human CD8+CD28- T suppressor cells expanded by Common Gamma Chain (γc) cytokines retain steady allospecific suppressive capacity in vivo.
BMC immunology, 2020Co-Authors: Guihuan Liu, Fu Feng, Ping Zhu, Hua Zhang, Danni Zhang, Xiaoqiang Feng, Zedan Zhang, Yanjun LiuAbstract:Background CD8+CD28- T suppressor (Ts) cells play critical role in transplant tolerance. Our previous study has generated CD8+CD28- Ts cells in vitro which exert robust allospecific suppressive capacity in vitro. Results CD8+CD28- Ts cells were expanded by stimulating human CD8+ T cells with allogeneic antigen presenting cells in the presence of the Common Gamma Chain cytokines IL-2, IL-7 and IL-15 in vitro, and were further verified in vitro through day 7 to 11 for their persistency of the allospecific suppressive capacity. When CD8+CD28- Ts cells were adoptively transferred into NOG mice, their capacity to inhibit CD4+ T cell proliferation in allospecific manner remained potent on 11 days after their injection. The mechanisms for expansion of CD8+CD28- Ts cells by the Common Gamma Chain cytokines were investigated. These included promoting CD8+CD28- T cells proliferation, converting CD8+CD28+ T cells to CD8+CD28- T cells and decreasing CD8+CD28- T cell death. Furthermore, the expanded CD8+CD28- Ts cells showed upregulation of the co-inhibitory molecule Tim-3 and down-regulation of the cytotoxic molecule granzyme B. Conclusions In summary, these results demonstrated that the in vitro-expanded human CD8+CD28- T cells retained potent allospecific suppressive capacity in vivo and depicted multiple mechanisms for the expansion of Ts cells, which might promote further bench to clinic research.
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human cd8 cd28 t suppressor cells expanded by Common Gamma Chain γc cytokines retain steady allospecific suppressive capacity in vivo
BMC Immunology, 2020Co-Authors: Guihuan Liu, Fu Feng, Ping Zhu, Hua Zhang, Danni Zhang, Xiaoqiang Feng, Zedan Zhang, Yanjun LiuAbstract:Background CD8+CD28- T suppressor (Ts) cells play critical role in transplant tolerance. Our previous study has generated CD8+CD28- Ts cells in vitro which exert robust allospecific suppressive capacity in vitro. Results CD8+CD28- Ts cells were expanded by stimulating human CD8+ T cells with allogeneic antigen presenting cells in the presence of the Common Gamma Chain cytokines IL-2, IL-7 and IL-15 in vitro, and were further verified in vitro through day 7 to 11 for their persistency of the allospecific suppressive capacity. When CD8+CD28- Ts cells were adoptively transferred into NOG mice, their capacity to inhibit CD4+ T cell proliferation in allospecific manner remained potent on 11 days after their injection. The mechanisms for expansion of CD8+CD28- Ts cells by the Common Gamma Chain cytokines were investigated. These included promoting CD8+CD28- T cells proliferation, converting CD8+CD28+ T cells to CD8+CD28- T cells and decreasing CD8+CD28- T cell death. Furthermore, the expanded CD8+CD28- Ts cells showed upregulation of the co-inhibitory molecule Tim-3 and down-regulation of the cytotoxic molecule granzyme B. Conclusions In summary, these results demonstrated that the in vitro-expanded human CD8+CD28- T cells retained potent allospecific suppressive capacity in vivo and depicted multiple mechanisms for the expansion of Ts cells, which might promote further bench to clinic research.
Andrew M. Scharenberg - One of the best experts on this subject based on the ideXlab platform.
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284. Long-Term Therapeutic Immune Reconstitution in XSCID Canine Model via In Vivo Foamy Virus Delivery of Common Gamma Chain
Molecular Therapy, 2016Co-Authors: Frieda Chan, Christopher R. Burtner, Patricia O'donnell, Nicholas Hubbard, Daniel Humphrys, Jennifer E. Adair, Grant D. Trobridge, Olivier Humbert, Troy Torgerson, Andrew M. ScharenbergAbstract:X-linked combined immunodeficiency disease (XSCID) is caused by mutation in the Common Gamma Chain, γC (interleukin-2 receptor subunit Gamma, IL2RG) in both humans and canines. It is characterized by the inability of T-cell development leading to absence of T-cells in peripheral blood, lack of T-cell mediated immune response, low IgA and IgG levels, and early infant mortality. In the 1990s, human XSCID clinical trials utilizing Gamma-retroviral vectors to deliver the IL2RG gene caused leukemia in 5 out of 20 patients due to vector integration in or near proto-oncogenes. Recent studies showed Foamy virus based vectors as an excellent alternative for in vivo gene-therapy because it is non-pathogenic in humans while exhibiting increased serum stability and favorable integration pattern. Previously, we have demonstrated CD3+ T-cell reconstitution in the canine model via intravenous injection of foamy virus expressing human elongation factor-1 alpha promoter (Ef1α)-yC. Unfortunately, the treated animals contained a low number of gene corrected progenitors at a sub-therapeutic level. Here, we achieved long-term therapeutic immune-reconstitution by intravenous delivery of a human phosphoglycerate kinase promoter (Pgk)-mediated γC foamy viral vector into XSCID neonatal canines. Long-term (2 years) post-injection follow-up demonstrated therapeutic levels of CD3+ T-cell expansion. Within the T-cell population, gene correction with Pgk-γC stabilized at ~80%. We validated T-cell functionality by using spectratyping analysis, which exhibited a diverse repertoire of receptor gene rearrangement. Retroviral integration site analysis (RIS) indicated polyclonal contribution to the reconstituted T-cells. Immunoglobulin ELISA assays showed that IgA and IgG levels in peripheral blood are comparable to normal healthy controls. We immunized the gene-corrected canine recipients with bacteriophage ϕx174 and confirmed production of specific IgG antibodies, showing the ability for isotype switching in B-lymphocytes. Currently, the gene-corrected canines exhibit comparable health and physical attributes to normal controls. Furthermore, semen from the gene-corrected male canine was used via artificial insemination to produce a litter of viable offsprings. In summary, our data demonstrate that Pgk-γC foamy viral vector delivered long-term therapeutic gene correction in a large-animal model for XSCID gene therapy. Most importantly, these results indicate that in vivo Pgk-γC foamy vector administration is a viable option for long-term immune reconstitution in future XSCID human clinical trials.
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Pgk-Mediated Expression of Common Gamma Chain Is More Effective Than EF1a for Therapeutic Immune Reconstitution of X-SCID Dogs after In Vivo Gene Therapy with Foamy Virus Vector
Blood, 2015Co-Authors: Olivier Humbert, Christopher R. Burtner, Patricia O'donnell, Nicholas Hubbard, Daniel Humphrys, Jennifer E. Adair, Grant D. Trobridge, Troy R. Torgerson, Frieda Chan, Andrew M. ScharenbergAbstract:In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy because they are non-pathogenic in humans, they exhibit increased serum stability and they integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human Common Gamma Chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of functional CD3+ lymphocytes in canine X-SCID (Burtner CR et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood. 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy that include different promoters and different fluorophores. We performed a head-to-head comparison of two promoters, our previously used EF1α promoter and the human phosphoglycerate kinase (PGK) promoter, by simultaneously injecting three X-SCID pups with equal titers of two therapeutic, human γC-encoding FV vectors. These vectors expressed the fluorophores GFP or mCherry to allow for tracking of transduced cells. Each dog received between 3 and 4 x 108 infectious units of each FV vector. In all treated dogs, lymphocyte marking in the PGK arm reached 50% between day 60 and day 110 post-injection and continued to expand over time, while the EF1α arm peaked at day 42 and never expanded above 10% (Fig 1A). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the EF1α arm, suggesting competition within the expanding T cell niche. The development of total CD3+ T cells achieved therapeutic levels (1000 cells/μL of blood) in all three dogs between day 70 and day 130 post-treatment (Fig 1B). We further validated the functionality of these cells by showing that they express a diverse T cell receptor repertoire using spectratyping analysis. In addition, peripheral blood mononuclear cells from the treated animals could be activated in vitro by exposure to the mitogen Phytohemagglutinin A at a level comparable to normal cells. Immunization of the treated dogs with bacteriophage ΦX174 showed production of specific IgG antibodies, suggesting the ability of B lymphocytes to undergo isotype switching. Finally, retroviral integration site analysis revealed polyclonal contribution to the reconstituting T cells. In summary, our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. The outcome in dogs was substantially improved compared to our previous study using EF1α, where robust lymphocyte marking was achieved in only 2 of 5 dogs, and where clonal dominance was observed. Ongoing work will determine whether the superior performance of the PGK vector is due to higher γC expression in PGK vs. EF1α corrected cells. Disclosures No relevant conflicts of interest to declare.
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441. Direct Comparison of EF1α and PGK Promoters Reveals Superior Performance of the PGK Promoter for Expression of the Common Gamma Chain in a Canine Model of In Vivo Foamy Virus Gene Therapy for Severe Combined Immunodeficiency
Molecular Therapy, 2015Co-Authors: Christopher R. Burtner, Humbert Olivier, Patricia O'donnell, Nicholas Hubbard, Daniel Humphrys, Jennifer E. Adair, Grant D. Trobridge, Troy R. Torgerson, Andrew M. Scharenberg, David J. RawlingsAbstract:Foamy viruses (FV) are ideal vectors for in vivo gene therapy because FV infection is non-pathogenic in people, FV are more resistant to serum inactivation than lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and FV exhibit favorable proviral integration patterns compared to Gammaretroviruses. In vivo gene therapy with a FV vector expressing the human Common Gamma Chain (γC) under the short EF1α promoter results in a functional immune reconstitution in X-SCID dogs.
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Robust Therapeutic Expression of the Common Gamma Chain with the Human Pgk Promoter Using Foamy Virus in Vivo Gene Therapy in a Canine Model of Severe Combined Immunodeficiency
Blood, 2014Co-Authors: Christopher R. Burtner, Patricia O'donnell, Nicholas Hubbard, Daniel Humphrys, Jennifer E. Adair, Grant D. Trobridge, Troy R. Torgerson, Andrew M. Scharenberg, Olivier Humbert, David J. RawlingsAbstract:In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy for 3 primary reasons: (1) FV are non-pathogenic in humans, (2) they exhibit enhanced serum stability as compared to lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and (3) FV integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human Common Gamma Chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of CD3+ lymphocytes in canine X-SCID, which undergo T cell receptor rearrangement and exhibit a functional signaling response to T cell activating mitogens (Burtner CR, Beard BC, Kennedy DR, et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood . 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy, including different promoters, using injections of vectors marked with different fluorophores. Preliminary data indicated that ex vivo transduction of canine CD34+ cells with a FV vector expressing human γC and a fluorescent reporter under the human phosphoglycerate kinase (PGK) promoter resulted in higher transduction efficiencies and increased mean fluorescence intensity, compared to that of an identical vector containing the EF1α promoter. We therefore performed a head-to-head comparison of the two promoters by simultaneously injecting X-SCID pups with equal titers of 2 therapeutic, human γC-encoding FV vectors that differed only in the promoter used to drive human γC expression and in the fluorophore color to distinguish gene-marked cells (GFP and mCherry). Each dog received 4 x 108 infectious units of each FV vector. A significant population of gene-marked lymphocytes appeared in the PGK arm 42 days post in vivo gene therapy, which continued to expand over the next two months of follow-up ( Fig 1A ). By 84 days post injection, lymphocyte gene marking in the competitive PGK arm reached 60% in both dogs. For comparison, this robust level of lymphocyte gene marking was achieved in only 2 of 5 dogs after 122 and 160 days, respectively, in our previous EF1α virus treated cohort. In contrast, the EF1α arm peaked at 42 days after in vivo gene therapy and never expanded above 10% ( Fig 1A ). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the by the EF1α arm, suggesting competition within the expanding T cell niche. The expansion of gene-marked lymphocytes was followed by the development of CD3+ T cells, leading to a therapeutic level of CD3+ cells (1000 cells/μl of blood) in both dogs ( Fig 1B ). Additionally, our data indicate low but persistent gene marking in other blood cells, including granulocytes and B cells, with B cell marking in one animal exceeding 2% in the PGK arm. Our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. These data also highlight the utility of the in vivo approach to explore key parameters of vector design in competitive repopulation experiments that may be useful for other diseases. ![Figure 1][1] Figure 1 Disclosures No relevant conflicts of interest to declare. [1]: pending:yes
R M Gorczynski - One of the best experts on this subject based on the ideXlab platform.
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TCR diversity in γδTCR+ hybridomas derived from mice given portal vein donor-specific pre-immunization and skin allografts
Immunology Letters, 1998Co-Authors: Y Sun, Z Chen, H Zeng, Stephen Chung, R M GorczynskiAbstract:Portal venous (p.v.) immunization with multiple minor histoincompatible cells leads to antigen-specific increased skin allograft survival. GammadeltaTCR+ hybridoma cells, prepared from mesenteric lymphocytes of p.v. immunized animals, can adoptively transfer this increased graft survival to naive animals. We have analyzed VGammaVdelta gene usage, and TCR Gamma-Chain junctional diversity in GammadeltaTCR+ hybridomas from mice immunized with different antigen combinations by p.v. or conventional lateral tail vein (i.v.) immunization. Following p.v. immunization two independent sets of hybridoma cells were derived, one expressing a Common Gamma-Chain junctional sequence which was also found in > 85% of the hybridomas derived following i.v. immunization, while the other set showed remarkable Gamma-Chain junctional sequence diversity. The diversity seen in these latter hybridomas was associated with the antigen specificity of the hybridoma cells. Cells expressing these 'unique' TCR junctional sequences were stimulated to produce cytokines both by hsp and by minor-histocompatibility-specific irradiated peritoneal cells. Cells expressing TCR with a Common Gamma-Chain junctional sequence were stimulated to cytokine production by MHC-matched but minor-histocompatibility mismatched (as well as matched) peritoneal cells, but not by hsp. We suggest that p.v. immunization results in stimulation of both antigen-specific and non-specific regulatory GammadeltaTCR+ cells, which can be distinguished by Gamma-Chain TCR sequence diversity.
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TCR diversity in GammadeltaTCR+ hybridomas derived from mice given portal vein donor-specific pre-immunization and skin allografts.
Immunology letters, 1998Co-Authors: Y Sun, Z Chen, S W Chung, H Zeng, R M GorczynskiAbstract:Portal venous (p.v.) immunization with multiple minor histoincompatible cells leads to antigen-specific increased skin allograft survival. GammadeltaTCR+ hybridoma cells, prepared from mesenteric lymphocytes of p.v. immunized animals, can adoptively transfer this increased graft survival to naive animals. We have analyzed VGammaVdelta gene usage, and TCR Gamma-Chain junctional diversity in GammadeltaTCR+ hybridomas from mice immunized with different antigen combinations by p.v. or conventional lateral tail vein (i.v.) immunization. Following p.v. immunization two independent sets of hybridoma cells were derived, one expressing a Common Gamma-Chain junctional sequence which was also found in > 85% of the hybridomas derived following i.v. immunization, while the other set showed remarkable Gamma-Chain junctional sequence diversity. The diversity seen in these latter hybridomas was associated with the antigen specificity of the hybridoma cells. Cells expressing these 'unique' TCR junctional sequences were stimulated to produce cytokines both by hsp and by minor-histocompatibility-specific irradiated peritoneal cells. Cells expressing TCR with a Common Gamma-Chain junctional sequence were stimulated to cytokine production by MHC-matched but minor-histocompatibility mismatched (as well as matched) peritoneal cells, but not by hsp. We suggest that p.v. immunization results in stimulation of both antigen-specific and non-specific regulatory GammadeltaTCR+ cells, which can be distinguished by Gamma-Chain TCR sequence diversity.
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Specificity for in vivo graft prolongation in Gamma delta T cell receptor+ hybridomas derived from mice given portal vein donor-specific preimmunization and skin allografts.
Journal of immunology (Baltimore Md. : 1950), 1997Co-Authors: R M Gorczynski, Z Chen, H ZengAbstract:Gamma delta TCR+ hybridoma cells prepared from mesenteric lymph node cells of animals receiving donor-specific immunization via the portal vein can adoptively transfer this increased graft survival to naive animals. Analysis of TCR Gamma-Chain junctional sequence diversity suggested that some 40 to 50% of the hybridomas expressed Gamma-Chain junctional sequence diversity and were stimulated to produce cytokines both by heat shock proteins and by minor histocompatibility Ag-specific irradiated peritoneal cells. The remaining Gamma delta TCR+ hybridoma cells expressed TCR with a Common Gamma-Chain junctional sequence and were stimulated to cytokine production by MHC-matched, but minor histocompatibility Ag-mismatched (as well as matched), peritoneal cells, but not by heat shock proteins. We have compared the effectiveness of representative hybridomas expressing unique Gamma-Chain junctional sequences or Common Gamma-Chain junctional sequences for prolongation of donor-specific or third-party (MHC-matched or MHC-mismatched) skin grafts. Our data show a good correlation between the specificity for stimulation for cytokine production in vitro and efficacy in graft prolongation assays in vivo. Hybridoma cells expressing unique Gamma-Chain junctional sequences that showed Ag-specific stimulation of cytokine production in vitro and skin graft survival in vivo augmented survival of third-party skin grafts if simultaneously transplanted with both Ag-specific and third-party skin grafts. Graft prolongation in vivo using cells from either population of Gamma delta TCR+ hybridomas was decreased by infusion of anti-IL-10 mAb and abolished when both anti-IL-10 and anti-TGF-beta Abs were used together.
Christopher R. Burtner - One of the best experts on this subject based on the ideXlab platform.
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284. Long-Term Therapeutic Immune Reconstitution in XSCID Canine Model via In Vivo Foamy Virus Delivery of Common Gamma Chain
Molecular Therapy, 2016Co-Authors: Frieda Chan, Christopher R. Burtner, Patricia O'donnell, Nicholas Hubbard, Daniel Humphrys, Jennifer E. Adair, Grant D. Trobridge, Olivier Humbert, Troy Torgerson, Andrew M. ScharenbergAbstract:X-linked combined immunodeficiency disease (XSCID) is caused by mutation in the Common Gamma Chain, γC (interleukin-2 receptor subunit Gamma, IL2RG) in both humans and canines. It is characterized by the inability of T-cell development leading to absence of T-cells in peripheral blood, lack of T-cell mediated immune response, low IgA and IgG levels, and early infant mortality. In the 1990s, human XSCID clinical trials utilizing Gamma-retroviral vectors to deliver the IL2RG gene caused leukemia in 5 out of 20 patients due to vector integration in or near proto-oncogenes. Recent studies showed Foamy virus based vectors as an excellent alternative for in vivo gene-therapy because it is non-pathogenic in humans while exhibiting increased serum stability and favorable integration pattern. Previously, we have demonstrated CD3+ T-cell reconstitution in the canine model via intravenous injection of foamy virus expressing human elongation factor-1 alpha promoter (Ef1α)-yC. Unfortunately, the treated animals contained a low number of gene corrected progenitors at a sub-therapeutic level. Here, we achieved long-term therapeutic immune-reconstitution by intravenous delivery of a human phosphoglycerate kinase promoter (Pgk)-mediated γC foamy viral vector into XSCID neonatal canines. Long-term (2 years) post-injection follow-up demonstrated therapeutic levels of CD3+ T-cell expansion. Within the T-cell population, gene correction with Pgk-γC stabilized at ~80%. We validated T-cell functionality by using spectratyping analysis, which exhibited a diverse repertoire of receptor gene rearrangement. Retroviral integration site analysis (RIS) indicated polyclonal contribution to the reconstituted T-cells. Immunoglobulin ELISA assays showed that IgA and IgG levels in peripheral blood are comparable to normal healthy controls. We immunized the gene-corrected canine recipients with bacteriophage ϕx174 and confirmed production of specific IgG antibodies, showing the ability for isotype switching in B-lymphocytes. Currently, the gene-corrected canines exhibit comparable health and physical attributes to normal controls. Furthermore, semen from the gene-corrected male canine was used via artificial insemination to produce a litter of viable offsprings. In summary, our data demonstrate that Pgk-γC foamy viral vector delivered long-term therapeutic gene correction in a large-animal model for XSCID gene therapy. Most importantly, these results indicate that in vivo Pgk-γC foamy vector administration is a viable option for long-term immune reconstitution in future XSCID human clinical trials.
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Pgk-Mediated Expression of Common Gamma Chain Is More Effective Than EF1a for Therapeutic Immune Reconstitution of X-SCID Dogs after In Vivo Gene Therapy with Foamy Virus Vector
Blood, 2015Co-Authors: Olivier Humbert, Christopher R. Burtner, Patricia O'donnell, Nicholas Hubbard, Daniel Humphrys, Jennifer E. Adair, Grant D. Trobridge, Troy R. Torgerson, Frieda Chan, Andrew M. ScharenbergAbstract:In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy because they are non-pathogenic in humans, they exhibit increased serum stability and they integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human Common Gamma Chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of functional CD3+ lymphocytes in canine X-SCID (Burtner CR et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood. 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy that include different promoters and different fluorophores. We performed a head-to-head comparison of two promoters, our previously used EF1α promoter and the human phosphoglycerate kinase (PGK) promoter, by simultaneously injecting three X-SCID pups with equal titers of two therapeutic, human γC-encoding FV vectors. These vectors expressed the fluorophores GFP or mCherry to allow for tracking of transduced cells. Each dog received between 3 and 4 x 108 infectious units of each FV vector. In all treated dogs, lymphocyte marking in the PGK arm reached 50% between day 60 and day 110 post-injection and continued to expand over time, while the EF1α arm peaked at day 42 and never expanded above 10% (Fig 1A). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the EF1α arm, suggesting competition within the expanding T cell niche. The development of total CD3+ T cells achieved therapeutic levels (1000 cells/μL of blood) in all three dogs between day 70 and day 130 post-treatment (Fig 1B). We further validated the functionality of these cells by showing that they express a diverse T cell receptor repertoire using spectratyping analysis. In addition, peripheral blood mononuclear cells from the treated animals could be activated in vitro by exposure to the mitogen Phytohemagglutinin A at a level comparable to normal cells. Immunization of the treated dogs with bacteriophage ΦX174 showed production of specific IgG antibodies, suggesting the ability of B lymphocytes to undergo isotype switching. Finally, retroviral integration site analysis revealed polyclonal contribution to the reconstituting T cells. In summary, our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. The outcome in dogs was substantially improved compared to our previous study using EF1α, where robust lymphocyte marking was achieved in only 2 of 5 dogs, and where clonal dominance was observed. Ongoing work will determine whether the superior performance of the PGK vector is due to higher γC expression in PGK vs. EF1α corrected cells. Disclosures No relevant conflicts of interest to declare.
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441. Direct Comparison of EF1α and PGK Promoters Reveals Superior Performance of the PGK Promoter for Expression of the Common Gamma Chain in a Canine Model of In Vivo Foamy Virus Gene Therapy for Severe Combined Immunodeficiency
Molecular Therapy, 2015Co-Authors: Christopher R. Burtner, Humbert Olivier, Patricia O'donnell, Nicholas Hubbard, Daniel Humphrys, Jennifer E. Adair, Grant D. Trobridge, Troy R. Torgerson, Andrew M. Scharenberg, David J. RawlingsAbstract:Foamy viruses (FV) are ideal vectors for in vivo gene therapy because FV infection is non-pathogenic in people, FV are more resistant to serum inactivation than lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and FV exhibit favorable proviral integration patterns compared to Gammaretroviruses. In vivo gene therapy with a FV vector expressing the human Common Gamma Chain (γC) under the short EF1α promoter results in a functional immune reconstitution in X-SCID dogs.
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Robust Therapeutic Expression of the Common Gamma Chain with the Human Pgk Promoter Using Foamy Virus in Vivo Gene Therapy in a Canine Model of Severe Combined Immunodeficiency
Blood, 2014Co-Authors: Christopher R. Burtner, Patricia O'donnell, Nicholas Hubbard, Daniel Humphrys, Jennifer E. Adair, Grant D. Trobridge, Troy R. Torgerson, Andrew M. Scharenberg, Olivier Humbert, David J. RawlingsAbstract:In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy for 3 primary reasons: (1) FV are non-pathogenic in humans, (2) they exhibit enhanced serum stability as compared to lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and (3) FV integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human Common Gamma Chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of CD3+ lymphocytes in canine X-SCID, which undergo T cell receptor rearrangement and exhibit a functional signaling response to T cell activating mitogens (Burtner CR, Beard BC, Kennedy DR, et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood . 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy, including different promoters, using injections of vectors marked with different fluorophores. Preliminary data indicated that ex vivo transduction of canine CD34+ cells with a FV vector expressing human γC and a fluorescent reporter under the human phosphoglycerate kinase (PGK) promoter resulted in higher transduction efficiencies and increased mean fluorescence intensity, compared to that of an identical vector containing the EF1α promoter. We therefore performed a head-to-head comparison of the two promoters by simultaneously injecting X-SCID pups with equal titers of 2 therapeutic, human γC-encoding FV vectors that differed only in the promoter used to drive human γC expression and in the fluorophore color to distinguish gene-marked cells (GFP and mCherry). Each dog received 4 x 108 infectious units of each FV vector. A significant population of gene-marked lymphocytes appeared in the PGK arm 42 days post in vivo gene therapy, which continued to expand over the next two months of follow-up ( Fig 1A ). By 84 days post injection, lymphocyte gene marking in the competitive PGK arm reached 60% in both dogs. For comparison, this robust level of lymphocyte gene marking was achieved in only 2 of 5 dogs after 122 and 160 days, respectively, in our previous EF1α virus treated cohort. In contrast, the EF1α arm peaked at 42 days after in vivo gene therapy and never expanded above 10% ( Fig 1A ). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the by the EF1α arm, suggesting competition within the expanding T cell niche. The expansion of gene-marked lymphocytes was followed by the development of CD3+ T cells, leading to a therapeutic level of CD3+ cells (1000 cells/μl of blood) in both dogs ( Fig 1B ). Additionally, our data indicate low but persistent gene marking in other blood cells, including granulocytes and B cells, with B cell marking in one animal exceeding 2% in the PGK arm. Our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. These data also highlight the utility of the in vivo approach to explore key parameters of vector design in competitive repopulation experiments that may be useful for other diseases. ![Figure 1][1] Figure 1 Disclosures No relevant conflicts of interest to declare. [1]: pending:yes
