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.

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.

Neonatal Gene Transfer Induces Tolerance to Human Factor VIII in Mice and Cats.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 215-215
Author(s):  
Lingfei Xu ◽  
Manxue Mei ◽  
Mark E. Haskins ◽  
Karyn Cullen ◽  
Katherine P. Ponder
Keyword(s):  
Gene Therapy ◽  
Immune System ◽  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Large Animal ◽  
Human Factor Viii ◽  
C3h Mice ◽  
Low Levels ◽  
Medium Dose

Abstract Hemophilia A is a bleeding disorder due to Factor VIII (FVIII) deficiency. FVIII is a very immunogenic protein, as approximately 30% of patients develop inhibitory antibodies (inhibitors) after FVIII protein replacement therapy. In addition, inhibitors often develop after gene therapy for hemophilia A. Identification of a treatment that could prevent inhibitor formation would be important. In this study, we explored the possibility of inducing tolerance to FVIII by neonatal gene transfer. An amphotropic gamma retroviral vector (RV) expressing human B domain-deleted FVIII (hFVIII) from the human α1-antitrypsin promoter was used to define the level of hFVIII that is necessary to achieve tolerance. Hemophilia A mice were injected with 1010 (high), 109 (medium) or 108 (low) transducing units (TU)/kg of RV at 2 to 3 days after birth, which resulted in expression of 63%, 7.3% or <2% of normal hFVIII antigen, respectively. Animals that received the high or medium dose of RV achieved hemostasis in vivo, although those that received the low dose did not. None of the mice produced antibodies to hFVIII. Similar results were also achieved in normal C3H mice, although some animals with low expression (2% of normal) developed low levels of anti-hFVIII antibodies. In contrast, hemophilia A and C3H mice that were challenged with hFVIII protein made potent inhibitors. Thus, neonatal gene transfer does not induce antibodies to hFVIII if the level of expression achieved is sufficiently high (>2x10−9 M). Mice that are tolerant to gene transfer are being challenged with recombinant BDD-hFVIII protein to determine if they are truly tolerant. Although induction of tolerance with neonatal gene transfer in mice is encouraging, tolerance has been more difficult to achieve in large outbred animals because their immune systems are more mature. Cats appear to have a much more mature immune system at birth than mice, as they mount a potent cytotoxic T lymphocyte response to canine iduronidase after neonatal gene therapy. Cats were therefore chosen as a large animal model in which to study tolerance induction with neonatal gene transfer. Neonatal cats were injected IV with 8.5x108 TU/kg of RV (medium dose) at day 5 after birth. Four cats achieved 37±3 ng/ml hFVIII (19% of normal) at 2 weeks after RV transduction. Three cats maintained hFVIII expression for 4 months. The two cats with the highest level of expression (~40 ng/ml) never developed antibodies, while the cat with a medium level of expression (30 ng/ml) developed only very low levels of anti-hFVIII antibodies. The cat with the lowest level of expression (20 ng/ml) lost expression at 2 months after RV transduction, and developed high titer inhibitors. We conclude that neonatal gene transfer does not induce antibodies to hFVIII in most cats. However, a high level of hFVIII expression may be necessary to achieve tolerance after neonatal gene transfer in cats. RV-treated cats will be challenged with hFVIII protein to determine if they are truly tolerant. In addition, normal cats will be challenged with hFVIII protein to determine the frequency of inhibitor formation in cats. Since the cat immune system is relatively mature at birth, these data are encouraging that neonatal gene or protein therapy might induce tolerance to hFVIII in humans. (This project is supported by National Hemophilia Foundation and the Bayer Hemophilia Awards Program.)

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.

Induction of tolerance to human factor VIII in mice

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3311-3312 ◽  
Author(s):  
Hengjun Chao ◽  
Christopher E. Walsh
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
High Titer ◽  
Inhibitory Antibody ◽  
Adeno Associated Virus ◽  
Immunodeficient Mice ◽  
Human Factor Viii ◽  
Induction Of Tolerance

Abstract This paper reports loss of human factor VIII (hFVIII) inhibitory antibody in immunocompetent C57BL/6 mice. High-titer anti-hFVIII antibody developed in the mice within 7 to 14 days of intraportal administration of adeno-associated virus (AAV) carrying FVIII that coincided with a reduction in plasma hFVIII antigen. Bethesda titers (&gt; 100 units) persisted relatively unchanged for 9 to 10 months. Unexpectedly, at 10 months after injection of the virus, hFVIII protein (up to 59 ng/mL) was detected in 3 mice at the same time as disappearance of hFVIII inhibitor. The level of hFVIII was similar to that found in immunodeficient mice receiving the same dose of recombinant AAV carrying hFVIII without hFVIII inhibitor. These results suggest that tolerance to hFVIII can be induced by sustained expression of hFVIII in a mouse model. Further elucidation of this observation may affect use of FVIII gene transfer in the treatment of inhibitor-positive patients with hemophilia A.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Differential Immunodominance of Human and Porcine Factor VIII in Hemophilia A Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 40-40 ◽  
Author(s):  
John F. Healey ◽  
Ernest T. Parker ◽  
Rachel T. Barrow ◽  
Pete Lollar
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
High Titer ◽  
Domain Specificity ◽  
Chi Square ◽  
C2 Domains ◽  
Intravenous Injections ◽  
Domain Specific ◽  
Human Factor Viii ◽  
Chi Square Test

Abstract Hemophilia A inhibitor patients and patients with acquired hemophilia A recognize immunodominant epitopes in the A2 and C2 domains of human factor VIII (fVIII). Hemophilia A mice also recognize A2 and C2 domain epitopes when immunized with human fVIII using a dosing schedule that mimics clinical use. We compared the immune responses of hemophilia A mice to human and porcine fVIII using a domain specific ELISA. In this assay, monoclonal antibodies are tested against a panel of six single human fVIII domain hybrid human/porcine fVIII molecules that contain the human A1, A2, ap, A3, C1 or C2 domains. With anti-human antibodies, a positive signal with one of the single human domain proteins identifies domain specificity, whereas loss of signal indicates domain specificity of anti-porcine fVIII antibodies. Exon16 (E16) - disrupted hemophilia A mice (n = 3) received six weekly μ10 g/kg intravenous injections of recombinant B-domain deleted human fVIII and a final 25 μg/kg boost. To obtain comparable inhibitor titers, E16 mice (n = 3) received six weekly injections of μ40 g/kg of recombinant B-domain deleted porcine fVIII. Spleens from high titer mice were fused with NS1 mouse myeloma cells and 485 of the resulting hybridomas were analyzed for fVIII domain specificity (Table). Only two hybridomas secreted antibodies specific for the ap domain. Human fVIII elicited a significantly greater number of antibodies to the A2 domain, whereas porcine fVIII elicited a significantly greater number of antibodies to the A1 and A3 domains (p < 0.01, chi square test). The greater number of anti-C2 antibodies to human fVIII was not significant at the 95% confidence level (p = 0.08). The differential immunodominance of human and porcine fVIII epitopes suggests that it may be possible to design a recombinant hybrid human/porcine fVIII molecule that is less immunogenic than human fVIII in the treatment of patients with hemophilia A. Domain Specificity of Anti-FVIII MAbs Mouse ID: Immunogen No. of MAbs A1 A2 A3 C1 C2 CR & MD CR: Cross Reactive MD: Multidomain 1- Human fVIII 95 2 16 2 7 21 23 & 24 2- Human fVIII 126 13 23 1 2 27 39 & 21 3- Human fVIII 54 1 15 2 1 10 9 & 15 4- Porcine fVIII 123 39 7 19 8 16 33 & 0 5- Porcine fVIII 27 13 5 0 0 4 2 & 3 6- Porcine fVIII 60 9 6 12 1 9 13 & 10

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.

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

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

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

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

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

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

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

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

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

Sign in / Sign up

Export Citation Format

Share Document