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Retroviral Vector-Mediated Neonatal Gene Therapy Results in Long-Term and Therapeutic Levels of Canine Factor VIII Expression in Hemophilia A Mice and Dogs
INBORN ERRORS OF METABOLISM: HEMOPHILIA AND OTHER DISORDERS 32. Efficacy and Safety of Mouse Coagulation Factor VIIa Gene Transfer in Hemophilia B Mice Majed N. Aljamali,1 Paris Margaritis,1 Valder R. Arruda,1,2 Alexander Schlachterman,1 Rodney M. Camire,1,2 Katherine A. High.1,2,3 1 Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA; 2Pediatrics, University of Pennsylvania, Philadelphia, PA; 3 Howard Hughes Medical Institute, Philadelphia, PA. One major complication of replacement therapy with clotting factors in patients with hemophilia A and B is the development of inhibitory antibodies that neutralize and inactivate the administered protein. Infusion of recombinant human factor VIIa (NovoSeven) into hemophilic patients bypasses the risk of antibody formation and provides a successful alternative approach to achieving normal hemostasis. We generated a recombinant adeno-associated virus (AAV) vector delivering an engineered mouse factor VII gene under the control of liver-specific promoter. The gene product is cleaved intracellularly and secreted as an activated mFVIIa. The levels of mFVII, measured by our in house ELISA, were about two to three fold of the baseline in HB mice, four and eight weeks after AAV injection into their spleen. This was accompanied by shortening of clotting times measured in the plasma of these mice (PTs and aPTTs). Additionally, in vivo tail clip assay showed a partial correction of bleeding in AAV treated HB mice compared to non-treated HB mice. Thrombin-antithrombin (TAT) levels did not change significantly in the treated HB mice up to 6 months post injection at 1.2 X 1012 vg/mouse dose, although an increase was seen at a higher dose. The survival rate was also similar between the treated and non-treated HB mice. In a follow-up study, we have generated transgenic mice with a range of levels of mFVIIa to analyze levels of expression that afford optimal efficacy and safety. We conclude that elevated and effective levels of activated murine factor VII can be achieved by AAV-mediated, liver-directed transgene delivery. This novel approach improves hemostasis in hemophilic mice with apparently no risk for thrombosis at this dose.
33. Induction of Antigen-Specific T Cell Anergy and Deletion by Hepatic AAV Gene Transfer Eric Dobrzynski,1 Lixin Wang,1 Ou Cao,1 Federico Mingozzi,1 Roland W. Herzog.1 1 Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA. Gene therapy for genetic disease may be limited by immune responses because of a lack of tolerance to the therapeutic gene product. Therefore, it is desirable that expression of a transgene product representing a neo-antigen induces immune tolerance. In published experiments, we have shown that adeno-associated virus (AAV)-mediated, hepatic in vivo gene transfer of a coagulation factor IX (F.IX) transgene can induce tolerance to the transgene product in adult immunocompetent mice (JCI 111:1347). Tolerized animals failed to form anti-F.IX even after challenge with F.IX in adjuvant, and F.IX-specific lymphocyte proliferation was substantially reduced. However, direct evidence for induction of T cell tolerance was elusive because of the low number of antigen-specific T cells and inability to physically identify these cells. Here, we demonstrate induction of antigen-specific CD4+ T cell tolerance in ovalbuminspecific T cell receptor (TCR) transgenic mice by hepatic AAVmediated gene transfer of ovalbumin (ova), a secreted protein. Transduced BALB/c mice and BALB/c mice transgenic for MHC class II-restricted ova-specific DO11.10 TCR maintained stable, vector dose-dependent systemic ova levels without evidence for immune responses. Infusion of 3x1012 vg of AAV2-EF1α-ova into the portal circulation of DO11.10 transgenics resulted in levels of S14
∼50 ng/ml plasma at day 10 and 100-200 ng/ml at days 30 and 60 after gene transfer. Lymph node (LN) cells and splenocytes were hypo-responsive to ova as early as day 10 after gene transfer, as demonstrated by reduced IL-2 cytokine release compared to controls. Dual color flow cytometry showed that numbers of TCR+CD4+ cells were reduced in secondary lymphoid organs and in the thymus by 1-2 months after vector administration (up to 2.5-fold). At 2 months, numbers of CD4+ cell lacking DO11.10 TCR expression were increased, consistent with a central deletion mechanism. The remaining TCR+CD4+ cell population was anergic to ova antigen in vitro. Splenocytes and LN cells showed markedly reduced IL-2 secretion and proliferation after stimulation with ova. Proliferation could largely be restored by addition of murine IL-2 to conditioned media, indicating T cell anergy. At day 60, percentage of CD25+ cells among TCR+CD4+ cells was increased in all examined lymphoid organs. CD4+CD25+ splenocytes isolated from hepatic AAV-ova transduced DO11.10 mice failed to express IL-2 in vitro after stimulation with ova, and suppressed IL-2 expression of CD4+CD25- splenocytes from naïve DO11.10 mice in a cell dosedependent manner, supporting the hypothesis that hepatic gene transfer promotes regulatory CD4+ T cell responses. These data provide direct evidence that transgene expression following in vivo viral gene transfer can induce CD4+ T cell tolerance to the transgene product, involving anergy and deletion mechanisms. Increase in regulatory T cell proportion likely enhances T cell anergy among the remaining ova-specific CD4+ T cell population. Induction of T cell tolerance correlated with absence of B cell responses, as AAVova transduced mice failed to form anti-ova IgG after subcutaneous injection of ova in complete Freund’s adjuvant. This immunization protocol caused IgG1 anti-ova formation in naïve control DO11.10 transgenics.
34. Retroviral Vector-Mediated Neonatal Gene Therapy Results in Long-Term and Therapeutic Levels of Canine Factor VIII Expression in Hemophilia A Mice and Dogs Lingfei Xu,1 Timothy C. Nichols,2 Rita Sarkar,3 Stephanie McCorquodale,2 Dwight Bellinger,2 Katherine P. Ponder.1 1 Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO; 2Department of Laboratory Medicine and Pathology, University of North Carolina, Chapel Hill, NC; 3 Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA. Hemophilia A is a common severe bleeding disorder due to a deficiency of clotting Factor VIII (FVIII). Affected individuals are at risk for spontaneous bleeding, which can be life-threatening. It has been difficult to achieve fully therapeutic levels of expression of FVIII by gene therapy in large animal models. The hypothesis of this study is that neonatal gene therapy with a retroviral vector (RV) could produce a sustained correction of the hemophilic coagulopathy in hemophilia A, and might prevent the development of inhibitory antibodies. A Moloney murine leukemia (MLV)-based RV that contains the liver-specific human α1-antitrypsin promoter, the canine B-domain deleted Factor VIII (cFVIII) cDNA, and the Woodchuck hepatitis virus post-transcriptional regulatory element was generated. Neonatal hemophilia A mice were injected with 1x1010 transducing units (TU)/kg of RV via the temporal vein at birth (N=10). Plasma cFVIII activity was determined by chromogenic substrate assay (COATEST FVIII) using normal canine plasma as the standards. In RV-treated hemophilia A mice, cFVIII activity was 92.5 +/-20.1% of normal at 1 month after transduction, and remained high at 109.4+/-10.8% of normal at 11 months thus far. The cFVIII was also functional in an aPTT assay with 23.5% of normal cFVIII levels, and allowed all treated mice to survive after tail-clip. A previous study showed that 50% of mice produced Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
INBORN ERRORS OF METABOLISM: HEMOPHILIA AND OTHER DISORDERS inhibitors after neonatal transduction of a vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped RV expressing human FVIII. However, in our study, none of 12 mice that were transduced as neonates produced cFVIII inhibitors, which was determined by Bethesda assay. To verify the capability of making inhibitors in hemophilia A mice, a single dose of RV was given IV into 6-week old mice at 3.3x109 TU/kg (N=3). All three mice made cFVIII inhibitors at 2-3 weeks after transduction. We conclude that completely therapeutic levels of cFVIII can be achieved in mice after neonatal transduction, and neonatal gene therapy with an amphotropic RV expressing cFVIII is not immunogenic. Based upon the above results, we performed neonatal RV transduction in two Chapel Hill hemophilia A dogs. RV was given IV at 3 days after birth at 9x109 TU/kg and 7x109 TU/kg, respectively. The whole blood clotting time (WBCT) in both dogs has been normalized since day 4 after RV transduction, and remained in the normal range for 4 months thus far. The plasma cFVIII activity in one RV-treated dog was 417% of normal by COATEST assay and 430% of normal by aPTT assay, and the aPTT time was normalized in this dog. The plasma cFVIII activity in another dog was 353% of normal by COATEST assay and 145% of normal by aPTT assay, and the aPTT time was shortened, but not normalized, in this dog. The plasma cFVIII activities have remained stable in both dogs for 4 months thus far. No cFVIII inhibitors were detected. No bleeding has occurred. We conclude that stable expression of therapeutic levels of cFVIII have been achieved in hemophilia A mice and dogs with neonatal transduction of an MLV-based RV.
35. Downregulation of Immune Responses Induced by Oral DNA Administration Andrew Gomez Vargas,1 Gonzalo Hortelano.1,2 1 Pathology & Molecular Medicine, McMaster University, Hamilton, ON, Canada; 2Research & Development, Canadian Blood Services. Protein replacement treatments often trigger unwanted immune responses in the host. Induction of immune tolerance would be a desirable goal to mitigate such events, and would eliminate the need for life-long immunosuppressive therapy in affected individuals. Hemophilia B is an X-linked bleeding disorder caused by blood coagulation factor IX (FIX) deficiency. Some hemophiliacs develop neutralizing antibodies to FIX (inhibitors) that render the infused FIX ineffective. In this study we evaluated whether oral delivery of hFIX-DNA nanoparticles can downregulate the immune response against hFIX in immunized mice. A group of C57BL/6 mice was immunized with recombinant hFIX in Freund’s adjuvant followed by three boost at day 7, 14, and 21. As expected, all mice developed high titer of anti-hFIX antibodies as measured by day 28. At this time, half the mice were treated with a single oral dose of plasmid DNA nanoparticles containing hFIX cDNA under the control of the b -actin promoter. The other half was left untreated, and served as control. By ELISA, we detected circulating levels of hFIX in the treated mice by day 3 after the oral treatment. In contrast, control mice had undetectable levels of circulating hFIX. Importantly, the titer of inhibitors against hFIX was measured by a Bethesda assay. Control mice had inhibitors, on average, of 40 BU/ml on day 21 after the oral administration of DNA. In contrast, the level of inhibitors became undetectable in the treated group by day 21. The cellular immune response was also assessed. Stimulation of splenocytes from orally treated mice with hFIX resulted in increased secretion of transforming growth factor-b , but no detectable interferon-g production. In contrast, stimulation of splenocytes from immunized control mice with hFIX showed an increase in the production of interferon-g , and no secretion of transforming growth factor-b . Additionally, a hFIX-specific cytotoxic T-lymphocyte (CTL) response was detected in the control, but not in the orally treated Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene Therapy
mice. Thus, our results suggest that the oral delivery of DNA nanoparticles downregulated the pre-existing immune responses against human FIX. This downregulation may result from the induction of a shift from a proinflammatory T helper cell type 1 to an anti-inflammatory T helper cell type 2 immune response. In conclusion, we have shown that pre-existing immune responses against hFIX can be overcome. This strategy may facilitate the delivery of proteins in a pre-sensitized host. Similar approaches may have potential applications for the induction of immune tolerance in autoimmune diseases. This strategy may also have implications for the prevention of unwanted immune responses elicited by the expression of novel transgenes. The authors have a significant financial interest in a company commercializing this technology.
36. Permanent Phenotypic Correction of Haemophilia B in Immunocompetent Mice by Prenatal Gene Therapy Simon N. Waddington, Megha S. Nivsarkar, Ajay R. Mistry, Suzanne M. K. Buckley, Geoffrey Kemball-Cook, Karen L. Mosley, Kyriacos Mitrophanous, Pippa Radcliffe, Maxine V. Holder, Mairi Brittan, Tassos Georgiadis, Faisal Al-Allaf, Brian W. Bigger, Lisa G. Gregory, Terence H. Cook, Robin R. Ali, Adrian Thrasher, Edward G. D. Tuddenham, Mike Themis, Charles Coutelle. Haemophilia B arises from mutations in the factor IX gene. Its treatment in humans, by recombinant protein substitution, is expensive thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of this disease without development of immune reactions by introduction of HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent haemophiliac and normal outbred mice. Plasma factor IX persists at therapeutic concentrations in all treated mice to date (ten months). Functional normalisation of the blood clotting has been achieved in two haemophiliac mice and significant correction in a third. In comparison, administration of an adenoviral vector encoding human factor IX resulted in transient and diminishing factor IX expression which was nearly undetectable by 8 months. Following lentiviral administration, no anti-factor IX antibodies were detected by ELISA and no cellular immunity was detected by immunohistochemical staining of macrophages, neutrophils, CD4-positive or CD8-positive cells. There was no elevation of serum liver enzymes or evidence of vector spread to the maternal circulation. No vector spread to sperm was detected by TaqMan analysis. This is the first demonstration of complete phenotypic correction of a severe genetic disease by in utero gene therapy with no evidence of toxicity.
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33. Induction of Antigen-Specific T Cell Anergy and Deletion by Hepatic AAV Gene Transfer Eric Dobrzynski,1 Lixin Wang,1 Ou Cao,1 Federico Mingozzi,1 Roland W. Herzog.1 1 Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA. Gene therapy for genetic disease may be limited by immune responses because of a lack of tolerance to the therapeutic gene product. Therefore, it is desirable that expression of a transgene product representing a neo-antigen induces immune tolerance. In published experiments, we have shown that adeno-associated virus (AAV)-mediated, hepatic in vivo gene transfer of a coagulation factor IX (F.IX) transgene can induce tolerance to the transgene product in adult immunocompetent mice (JCI 111:1347). Tolerized animals failed to form anti-F.IX even after challenge with F.IX in adjuvant, and F.IX-specific lymphocyte proliferation was substantially reduced. However, direct evidence for induction of T cell tolerance was elusive because of the low number of antigen-specific T cells and inability to physically identify these cells. Here, we demonstrate induction of antigen-specific CD4+ T cell tolerance in ovalbuminspecific T cell receptor (TCR) transgenic mice by hepatic AAVmediated gene transfer of ovalbumin (ova), a secreted protein. Transduced BALB/c mice and BALB/c mice transgenic for MHC class II-restricted ova-specific DO11.10 TCR maintained stable, vector dose-dependent systemic ova levels without evidence for immune responses. Infusion of 3x1012 vg of AAV2-EF1α-ova into the portal circulation of DO11.10 transgenics resulted in levels of S14
∼50 ng/ml plasma at day 10 and 100-200 ng/ml at days 30 and 60 after gene transfer. Lymph node (LN) cells and splenocytes were hypo-responsive to ova as early as day 10 after gene transfer, as demonstrated by reduced IL-2 cytokine release compared to controls. Dual color flow cytometry showed that numbers of TCR+CD4+ cells were reduced in secondary lymphoid organs and in the thymus by 1-2 months after vector administration (up to 2.5-fold). At 2 months, numbers of CD4+ cell lacking DO11.10 TCR expression were increased, consistent with a central deletion mechanism. The remaining TCR+CD4+ cell population was anergic to ova antigen in vitro. Splenocytes and LN cells showed markedly reduced IL-2 secretion and proliferation after stimulation with ova. Proliferation could largely be restored by addition of murine IL-2 to conditioned media, indicating T cell anergy. At day 60, percentage of CD25+ cells among TCR+CD4+ cells was increased in all examined lymphoid organs. CD4+CD25+ splenocytes isolated from hepatic AAV-ova transduced DO11.10 mice failed to express IL-2 in vitro after stimulation with ova, and suppressed IL-2 expression of CD4+CD25- splenocytes from naïve DO11.10 mice in a cell dosedependent manner, supporting the hypothesis that hepatic gene transfer promotes regulatory CD4+ T cell responses. These data provide direct evidence that transgene expression following in vivo viral gene transfer can induce CD4+ T cell tolerance to the transgene product, involving anergy and deletion mechanisms. Increase in regulatory T cell proportion likely enhances T cell anergy among the remaining ova-specific CD4+ T cell population. Induction of T cell tolerance correlated with absence of B cell responses, as AAVova transduced mice failed to form anti-ova IgG after subcutaneous injection of ova in complete Freund’s adjuvant. This immunization protocol caused IgG1 anti-ova formation in naïve control DO11.10 transgenics.
34. Retroviral Vector-Mediated Neonatal Gene Therapy Results in Long-Term and Therapeutic Levels of Canine Factor VIII Expression in Hemophilia A Mice and Dogs Lingfei Xu,1 Timothy C. Nichols,2 Rita Sarkar,3 Stephanie McCorquodale,2 Dwight Bellinger,2 Katherine P. Ponder.1 1 Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO; 2Department of Laboratory Medicine and Pathology, University of North Carolina, Chapel Hill, NC; 3 Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA. Hemophilia A is a common severe bleeding disorder due to a deficiency of clotting Factor VIII (FVIII). Affected individuals are at risk for spontaneous bleeding, which can be life-threatening. It has been difficult to achieve fully therapeutic levels of expression of FVIII by gene therapy in large animal models. The hypothesis of this study is that neonatal gene therapy with a retroviral vector (RV) could produce a sustained correction of the hemophilic coagulopathy in hemophilia A, and might prevent the development of inhibitory antibodies. A Moloney murine leukemia (MLV)-based RV that contains the liver-specific human α1-antitrypsin promoter, the canine B-domain deleted Factor VIII (cFVIII) cDNA, and the Woodchuck hepatitis virus post-transcriptional regulatory element was generated. Neonatal hemophilia A mice were injected with 1x1010 transducing units (TU)/kg of RV via the temporal vein at birth (N=10). Plasma cFVIII activity was determined by chromogenic substrate assay (COATEST FVIII) using normal canine plasma as the standards. In RV-treated hemophilia A mice, cFVIII activity was 92.5 +/-20.1% of normal at 1 month after transduction, and remained high at 109.4+/-10.8% of normal at 11 months thus far. The cFVIII was also functional in an aPTT assay with 23.5% of normal cFVIII levels, and allowed all treated mice to survive after tail-clip. A previous study showed that 50% of mice produced Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
INBORN ERRORS OF METABOLISM: HEMOPHILIA AND OTHER DISORDERS inhibitors after neonatal transduction of a vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped RV expressing human FVIII. However, in our study, none of 12 mice that were transduced as neonates produced cFVIII inhibitors, which was determined by Bethesda assay. To verify the capability of making inhibitors in hemophilia A mice, a single dose of RV was given IV into 6-week old mice at 3.3x109 TU/kg (N=3). All three mice made cFVIII inhibitors at 2-3 weeks after transduction. We conclude that completely therapeutic levels of cFVIII can be achieved in mice after neonatal transduction, and neonatal gene therapy with an amphotropic RV expressing cFVIII is not immunogenic. Based upon the above results, we performed neonatal RV transduction in two Chapel Hill hemophilia A dogs. RV was given IV at 3 days after birth at 9x109 TU/kg and 7x109 TU/kg, respectively. The whole blood clotting time (WBCT) in both dogs has been normalized since day 4 after RV transduction, and remained in the normal range for 4 months thus far. The plasma cFVIII activity in one RV-treated dog was 417% of normal by COATEST assay and 430% of normal by aPTT assay, and the aPTT time was normalized in this dog. The plasma cFVIII activity in another dog was 353% of normal by COATEST assay and 145% of normal by aPTT assay, and the aPTT time was shortened, but not normalized, in this dog. The plasma cFVIII activities have remained stable in both dogs for 4 months thus far. No cFVIII inhibitors were detected. No bleeding has occurred. We conclude that stable expression of therapeutic levels of cFVIII have been achieved in hemophilia A mice and dogs with neonatal transduction of an MLV-based RV.
35. Downregulation of Immune Responses Induced by Oral DNA Administration Andrew Gomez Vargas,1 Gonzalo Hortelano.1,2 1 Pathology & Molecular Medicine, McMaster University, Hamilton, ON, Canada; 2Research & Development, Canadian Blood Services. Protein replacement treatments often trigger unwanted immune responses in the host. Induction of immune tolerance would be a desirable goal to mitigate such events, and would eliminate the need for life-long immunosuppressive therapy in affected individuals. Hemophilia B is an X-linked bleeding disorder caused by blood coagulation factor IX (FIX) deficiency. Some hemophiliacs develop neutralizing antibodies to FIX (inhibitors) that render the infused FIX ineffective. In this study we evaluated whether oral delivery of hFIX-DNA nanoparticles can downregulate the immune response against hFIX in immunized mice. A group of C57BL/6 mice was immunized with recombinant hFIX in Freund’s adjuvant followed by three boost at day 7, 14, and 21. As expected, all mice developed high titer of anti-hFIX antibodies as measured by day 28. At this time, half the mice were treated with a single oral dose of plasmid DNA nanoparticles containing hFIX cDNA under the control of the b -actin promoter. The other half was left untreated, and served as control. By ELISA, we detected circulating levels of hFIX in the treated mice by day 3 after the oral treatment. In contrast, control mice had undetectable levels of circulating hFIX. Importantly, the titer of inhibitors against hFIX was measured by a Bethesda assay. Control mice had inhibitors, on average, of 40 BU/ml on day 21 after the oral administration of DNA. In contrast, the level of inhibitors became undetectable in the treated group by day 21. The cellular immune response was also assessed. Stimulation of splenocytes from orally treated mice with hFIX resulted in increased secretion of transforming growth factor-b , but no detectable interferon-g production. In contrast, stimulation of splenocytes from immunized control mice with hFIX showed an increase in the production of interferon-g , and no secretion of transforming growth factor-b . Additionally, a hFIX-specific cytotoxic T-lymphocyte (CTL) response was detected in the control, but not in the orally treated Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene Therapy
mice. Thus, our results suggest that the oral delivery of DNA nanoparticles downregulated the pre-existing immune responses against human FIX. This downregulation may result from the induction of a shift from a proinflammatory T helper cell type 1 to an anti-inflammatory T helper cell type 2 immune response. In conclusion, we have shown that pre-existing immune responses against hFIX can be overcome. This strategy may facilitate the delivery of proteins in a pre-sensitized host. Similar approaches may have potential applications for the induction of immune tolerance in autoimmune diseases. This strategy may also have implications for the prevention of unwanted immune responses elicited by the expression of novel transgenes. The authors have a significant financial interest in a company commercializing this technology.
36. Permanent Phenotypic Correction of Haemophilia B in Immunocompetent Mice by Prenatal Gene Therapy Simon N. Waddington, Megha S. Nivsarkar, Ajay R. Mistry, Suzanne M. K. Buckley, Geoffrey Kemball-Cook, Karen L. Mosley, Kyriacos Mitrophanous, Pippa Radcliffe, Maxine V. Holder, Mairi Brittan, Tassos Georgiadis, Faisal Al-Allaf, Brian W. Bigger, Lisa G. Gregory, Terence H. Cook, Robin R. Ali, Adrian Thrasher, Edward G. D. Tuddenham, Mike Themis, Charles Coutelle. Haemophilia B arises from mutations in the factor IX gene. Its treatment in humans, by recombinant protein substitution, is expensive thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of this disease without development of immune reactions by introduction of HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent haemophiliac and normal outbred mice. Plasma factor IX persists at therapeutic concentrations in all treated mice to date (ten months). Functional normalisation of the blood clotting has been achieved in two haemophiliac mice and significant correction in a third. In comparison, administration of an adenoviral vector encoding human factor IX resulted in transient and diminishing factor IX expression which was nearly undetectable by 8 months. Following lentiviral administration, no anti-factor IX antibodies were detected by ELISA and no cellular immunity was detected by immunohistochemical staining of macrophages, neutrophils, CD4-positive or CD8-positive cells. There was no elevation of serum liver enzymes or evidence of vector spread to the maternal circulation. No vector spread to sperm was detected by TaqMan analysis. This is the first demonstration of complete phenotypic correction of a severe genetic disease by in utero gene therapy with no evidence of toxicity.
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