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

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

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

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

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

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

Endothelial and Platelet FVIII/VWF Expression - Divergence in Clinical Effect in Murine Models of Hemophilia A with and without FVIII Inhibitory Antibodies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3286-3286 ◽  
Author(s):  
Qizhen Shi ◽  
Scot A. Fahs ◽  
David A. Wilcox ◽  
Hartmut Weiler ◽  
Sandra L. Haberichter ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Endothelial Cells ◽  
Transgenic Mice ◽  
Transgenic Mouse ◽  
Clinical Efficacy ◽  
Hemophilia A ◽  
High Titer ◽  
Local Delivery ◽  
Specific Gene ◽  
Inhibitory Antibodies

Abstract Our laboratory has had a longstanding interest in the association of FVIII with its carrier protein, VWF, - both in plasma and within platelets and endothelial cells where there is co-storage with endogenous VWF when FVIII expression is induced. Recently we have demonstrated clinical efficacy of FVIII ectopically expressed and stored within platelets even though FVIII remains undetectable in plasma in the platelet-specific FVIII (2bF8) transgenic mice. This correction is maintained even in the presence of high titer inhibitors (JCI, 2006). Since VWF is also synthesized and stored in endothelial cells, FVIII could also be supplied together with VWF using cell-specific expression within a second vascular cell, providing local as well as systemic levels of FVIII. We developed another transgenic mouse in which FVIII was driven by the endothelial cell-specific Tie2 promoter (Tie2-F8) and contrasted it with the 2bF8 transgenic mouse previously described. When bred into the FVIIInull mouse, homozygous Tie2F8 mice maintained normal plasma FVIII levels (1.15 ± 0.16 U/ml) and 50% levels in Tie2F8+/− mice (0.56 ± 0.16 U/ml). Both Tie2F8+/− and Tie2F8+/+ phenotypes effectively abrogate the bleeding phenotype in FVIIInull mice. Since both cell types could be advantageous in the local delivery of FVIII at sites of injury, we explored the phenotypic correction of our transgenic models in the presence of FVIII inhibitory antibodies (Ab) that were generated by either transplantation of spleen cells from highly immunized FVIIInull mice or in transgenic mice immunized with recombinant FVIII with adjuvant. In marked contrast to the platelet 2bF8 mice, the Tie2F8 mice’ ability to survive a minor tail wound was significantly decreased (21/27 vs 3/10, P < 0.01) in the presence of FVIII inhibitory Ab. After one immunization, the inhibitor titers in Tie2F8 mice were much higher (850 – 3,000 BU/ml) than in 2bF8 mice (10 – 150 BU/ml). Furthermore, in the presence of Ab, circulating FVIII levels in whole blood were maintained in the platelets of 2bF8 mice, but dropped to undetectable levels in the Tie2F8 mice. While both platelets and endothelial cells could provide local FVIII at sites of injury, in the presence of inhibitory Ab, endothelial delivery was not clinically efficacious and appeared to rely on the plasma FVIII levels it could maintain for its clinical efficacy. Thus, local delivery of FVIII by platelets concentrates its effect at sites of vascular injury, and storage within platelets protects FVIII from inactivation by Abs. This effect is a unique property of platelet delivery and is not maintained with endothelial FVIII delivery. Our further studies demonstrated when the lentivirus-mediated gene transfer system was used to introduce FVIII expression in either platelets or endothelial cells by transplantation of bone marrow cells transduced with 2bF8 lentivirus or systemic transduction of endothelial cells with Tie2F8 lentivirus, the outcome was different. Sustained phenotypic correction without Ab development was achieved in lentivirus-mediated platelet-specific gene therapy. In contrast, all endothelial-specific Tie2F8 lentivirus treated mice developed inhibitory Ab (50 ± 14 BU/ml) with no detectable plasma FVIII. Thus, we have demonstrated that platelets are potentially the optimal target for gene therapy of hemophilia A with inhibitors.

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.

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

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

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

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.

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

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

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

scholarly journals Ultrasound Combined with Microbubbles Mediated Factor VIII Plasmid Gene Therapy in Hemophilia a Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2039-2039
Author(s):  
Shuxian Song ◽  
James Harrang ◽  
Bryn Smith ◽  
Carol H. Miao
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Liver Perfusion ◽  
Transfer Efficiency ◽  
Clotting Factor ◽  
Gene Transfer Efficiency ◽  
Fviii Activity

Abstract Hemophilia A is a genetic bleeding disorder resulted from a deficiency of blood clotting factor VIII. In order to develop the efficient approach to gene therapy for hemophilia A, we previously explored reporter gene transfer mediated by ultrasound (US) combined with microbubbles (MBs). It was demonstrated that US/MB can significantly enhance gene transfer efficiency and serve as an efficient non-viral physical delivery strategy. In this study, we further delivered a therapeutic FVIII plasmid into the livers of hemophilia A (HA) mice. In consideration of FVIII synthesis from multiple tissues/cell lines, we first explored the distribution of gene expression using a pGL4.13 [luc2/SV40] luciferase plasmid driven by a ubiquitous promoter. One day following gene transfer, hepatocytes and endothelia cells were isolated from treated lobes by liver perfusion and centrifuge method. Evaluation of luciferase levels in two cell populations indicated that luciferase predominantly expressed in hepatocytes (5.35´104 RLU/107 cells vs. 1.46´103 RLU/107 cells in endothelia cells). Furthermore, gene transfer of pGFP (driven by a ubiquitous CMV promoter) mediated by US/MB also showed fluorescence distribution mostly in hepatocytes. These results indicate that hepatocyte is the predominant site of gene expression following US/MB mediated gene transfer into the liver. Based on these results, a hepatocyte-specific human FVIII plasmid (pBS-HCRHP-hFVIII/N6A) was used for US/MB mediated gene transfer in HA mice. In the short-term experiment, FVIII activity levels of treated HA mice ranged from 4-40% of normal FVIII activity. To follow FVIII expression for longer term, HA mice were pretreated with IL-2/IL-2 mAb (JES6-1) complexes on day −5, −4, and −3 to prevent immune response. In addition, the mice were infused with normal mouse plasma and human FVIII protein prior to gene transfer to maintain hemostasis. Subsequently, FVIII plasmids and 5 Vol% NUVOX MBs were injected into the mouse liver under simultaneous US exposure (1.1MHz transducer H158A driven by a pulse generator and high-power radio frequency amplifier capable of generating up to 1000W). Blood and liver samples were collected at serial time points after treatment to determine FVIII activity in plasma and liver damage. Following gene transfer, 10-30% of FVIII activity was achieved on day 4 and persisted in the average level of 20% by day 28. In a separate long-term follow-up experiment (n=3), 2 of 3 mice still maintained 10-30% activity after 120 days. Both transaminase levels (alanine aminotransferase and aspartate aminotransferase) and histological examination showed that the procedure of plasmid/MBs portal-vein injection and pulse-train acoustic exposure produced transiently localized liver damages however the damages were repaired and the liver recovered rapidly. Phenotypic correction of HA mice was further examined by tail clip assay. Blood loss of US/MB treated mice was significantly reduced compared with naive HA mice. Furthermore, a novel plasmid encoding a B domain-deleted FVIII variant containing mutations of 10 amino acids in the A1 domain (BDDFVIII-X10, a kind gift from Weidong Xiao) was constructed. Preliminary results from ongoing study showed that the gene transfer efficiency could be further improved with better plasmid and more efficient immune modulation. Together all the results indicate that US/MB mediated gene transfer is highly promising for efficient and safe gene therapy of hemophilia A. Disclosures No relevant conflicts of interest to declare.

Platelet-Derived Factor VIII (FVIII) Corrects the Murine Hemophilia A Phenotype Even in the Presence of FVIII Inhibitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 457-457
Author(s):  
Qizhen Shi ◽  
David A. Wilcox ◽  
Scot A. Fahs ◽  
Brian C. Cooley ◽  
Drashti Desai ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgenic Mice ◽  
Spleen Cell ◽  
Cell Transplantation ◽  
Hemophilia A ◽  
High Titer ◽  
Local Concentration ◽  
Inhibitory Antibody ◽  
Inhibitory Antibodies ◽  
Tail Clip

Abstract Inhibitory antibody development is not only the most severe and important complication of FVIII infusion in hemophilia A patients, but also a major potential concern in patients following hemophilia A gene therapy. We have developed a transgenic model in which human B-domain deleted FVIII (hFVIII) expression is controlled by the platelet-specific αIIb promoter (2bF8) and results in FVIII storage together with VWF in platelets. The transgenic mice were then bred into FVIII knockout mice (FVIIInull) so that the only FVIII in peripheral blood is derived in platelets. The bleeding phenotype was corrected in the 2bF8/FVIIInull transgenic mice, even though FVIII was not detectable in plasma. We further hypothesize that platelet-derived FVIII in a complex with VWF will 1) protect FVIII from inactivation by circulating FVIII-specific antibodies and 2) release FVIII in sufficiently high local concentration at sites of vessel damage so as to overcome immediate inactivation by FVIII inhibitors. To address our hypothesis, we used three strategies -1) an acute model infusing plasma from highly immunized FVIIInull mice, 2) immunization of 2bF8 transgenic mice with human FVIII, and 3) a model using transplantation of spleen cells from highly immunized mice into sub-lethally irradiated 2bF8 transgenic mice. Thirteen mice were infused with plasma containing 48-64 BU and 12 survived following tail clipping using a 1.59 mm template. FVIII-immunized transgenic mice (given adjuvant) generated anti-FVIII inhibitory antibodies as high as 350 BU after a single immunization. The level of platelet-FVIII and the platelet number in these mice were not significantly different from pre-immunization levels and 13 of 15 survived tail clipping. In the 3rd group, spleen cell transplantation resulted it titers of >5000 BU and 8 of 12 transgenic mice survived tail clip. In contrast, only 2 of 7 wild type mice survived tail clip with inhibitor titers >5000 BU after spleen cell transplantation. None of 9 untransplanted FVIIInull mice survived. These results demonstrate that platelet-derived FVIII is protected from inhibitor inactivation and correct the phenotype of murine hemophilia A even in the presence of a high-titer anti-FVIII antibody. This approach may be useful not only for preventing inhibitor formation in hemophilia A gene therapy but also for gene therapy in the case of pre-existing inhibitors.

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

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