Sustained Human Factor VIII Expression in Hemophilia A Mice Following Systemic Delivery of a Gutless Adenoviral Vector

Deficiency of coagulation factor VIII (FVIII) results in hemophilia A, a common hereditary bleeding disorder. Using a human FVIII-encoding adenoviral vector, Av1ALAPH81, we have demonstrated expression of therapeutic levels of human FVIII in mice sustained for more than 5 months after vector administration. Administration of a high dose (4 x 10(9) plaque-forming units [pfu]) of Av1ALAPH81 to mice resulted in a peak expression of 2,063 ng/mL of human FVIII in the mouse plasma, with levels decreasing to background by weeks 15 to 17. Normal FVIII levels in humans range from 100 to 200 ng/mL and therapeutic levels are as low as 10 ng/mL. Alternatively, administration of 8- to 80-fold lower vector doses (5 x 10(8) pfu to 5 x 10(7) pfu) to normal adult mice resulted in expression of FVIII at therapeutic levels sustained for at least 22 weeks. Detailed analysis of vector toxicity indicated that the high vector dose caused a dramatic elevation of liver-specific enzyme levels, whereas an eight-fold lower vector dose was significantly less hepatotoxic. The data presented here demonstrate that administration of lower, less toxic vector doses allow long-term persistence of FVIII expression.

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Evaluation of an Adenoviral Vector Encoding Full-Length Human Factor VIII in Hemophiliac Mice

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614449 ◽

1999 ◽

Vol 81 (02) ◽

pp. 234-239 ◽

Cited By ~ 19

Author(s):

Sheila Connelly ◽

Julie Andrews ◽

Angela Gallo-Penn ◽

Luigina Tagliavacca ◽

Randal Kaufman ◽

...

Keyword(s):

Factor Viii ◽

Adenoviral Vector ◽

Human Factor ◽

Hemophilia A ◽

Adenoviral Vectors ◽

Full Length ◽

Human Factor Viii ◽

Immunohistochemical Analyses

SummaryAdenoviral vectors provide a promising gene therapy system for the treatment of hemophilia A. Potent vectors encoding a human factor VIII (FVIII) cDNA were developed that mediated sustained FVIII expression in normal and hemophiliac mice and complete phenotypic correction of the bleeding disorder in hemophiliac mice and dogs (Connelly and Kaleko, Haemophilia 1998; 4: 380-8). However, these studies utilized vectors encoding a truncated version of the human FVIII cDNA lacking the B-domain (BDD FVIII). In this work, an adenoviral vector encoding the human full-length (FL) FVIII cDNA was generated and characterized. While functional FL FVIII was secreted in vitro, expression of the FL protein was not detected in the plasma of vector-treated hemophiliac mice. Unexpectedly, the FL FVIII vector-treated animals demonstrated phenotypic correction of the bleeding defect as measured by a tail-clip survival study. FL FVIII protein was visualized in the mouse livers using human FVIII-specific immunohistochemical analyses. These data demonstrate that adenoviral vector-mediated in vivo expression of BDD FVIII is more efficient than that of the FL protein and that phenotypic correction can occur in the absence of detectable levels of FVIII.

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Evaluation of the Duration of Human Factor VIII Expression in Nonhuman Primates After Systemic Delivery of an Adenoviral Vector

Human Gene Therapy ◽

10.1089/104303402760128559 ◽

2002 ◽

Vol 13 (11) ◽

pp. 1331-1336 ◽

Cited By ~ 12

Author(s):

Julie L. Andrews ◽

Pamela S. Shirley ◽

William O. Iverson ◽

Alfred D. Sherer ◽

Judit E. Markovits ◽

...

Keyword(s):

Factor Viii ◽

Adenoviral Vector ◽

Nonhuman Primates ◽

Human Factor ◽

Systemic Delivery ◽

Human Factor Viii

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Neonatal helper-dependent adenoviral vector gene therapy mediates correction of hemophilia A and tolerance to human factor VIII

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1015571108 ◽

2011 ◽

Vol 108 (5) ◽

pp. 2082-2087 ◽

Cited By ~ 38

Author(s):

Chuhong Hu ◽

Racel G. Cela ◽

Masataka Suzuki ◽

Brendan Lee ◽

Gerald S. Lipshutz

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Adenoviral Vector ◽

Human Factor ◽

Hemophilia A ◽

Human Factor Viii

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Mechanism of the Immune Response to Human Factor VIII in Murine Hemophilia A

Thrombosis and Haemostasis ◽

10.1055/s-0037-1612915 ◽

2001 ◽

Vol 85 (01) ◽

pp. 125-133 ◽

Cited By ~ 55

Author(s):

Huiyun Wu ◽

Mark Reding ◽

Jiahua Qian ◽

David Okita ◽

Ernie Parker ◽

...

Keyword(s):

Immune Response ◽

Factor Viii ◽

Human Factor ◽

Hemophilia A ◽

Th2 Cells ◽

Tween 20 ◽

Sorbitan Monolaurate ◽

Human Factor Viii ◽

Ifn Γ ◽

Th1 And Th2

SummaryMice genetically deficient in factor VIII (fVIII) are a model of hemophilia A. As a first step to reproduce in this mouse model what occurs over time in hemophilia A patients treated with human fVIII (hfVIII), we have investigated the time course and the characteristics of their immune response to hfVIII, after multiple intravenous injections. Anti-hfVIII antibodies appeared after four to five injections. They were IgG1 and to a lesser extent IgG2, indicating that they were induced by both Th2 and Th1 cells. Inhibitors appeared after six injections. CD4+ enriched splenocytes from hfVIII-treated mice proliferated in response to fVIII and secreted IL-10: in a few mice they secreted also IFN-γ and in one mouse IL-4, but never IL-2. A hfVIII-specific T cell line derived from hfVIII-treated mice secreted both IL-4 and IFN-γ, suggesting that it included both Th1 and Th2 cells. CD4+ enriched splenocytes of hfVIII-treated mice recognized all hfVIII domains. Thus, hemophilic mice develop an immune response to hfVIII administered intravenously similar to that of hemophilia A patients. Their anti-hfVIII antibodies can be inhibitors and belong to IgG subclasses homologous to those of inhibitors in hemophilic patients; their anti-hfVIII CD4+ cells recognize a complex repertoire and both Th1 and Th2 cytokines, and especially IL-10, may drive the antibody synthesis. Abbreviations used: antibodies, Ab; antigen presenting cells, APC; Arbitrary Units, AU; enzyme-linked immunosorbant assay, ELISA; factor VIII, fVIII; human factor VIII, hf VIII; intravenous, i.v.; optical density, OD; polymerase chain reaction, PCR; phosphate buffered saline solution, PBS; PBS containing 3% bovine serum albumin, PBS/BSA; PBS containing 0.05% polyoxyethylene sorbitan monolaurate, PBS/Tween-20; phytohemoagglutinin, PHA; stimulation index, SI

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Identification of a Novel Missense Mutation in Exon 4 of the Human Factor VIII Gene Associated with Sever Hemophilia a Patient

Pakistan Journal of Biological Sciences ◽

10.3923/pjbs.2007.4299.4302 ◽

2007 ◽

Vol 10 (23) ◽

pp. 4299-4302 ◽

Cited By ~ 3

Author(s):

Habib Onsori ◽

Mohammad Ali Hossein . ◽

Sheideh Montaser-Kou . ◽

Mohammad Asgharzadeh . ◽

Abbas Ali Hosseinpou .

Keyword(s):

Factor Viii ◽

Missense Mutation ◽

Human Factor ◽

Hemophilia A ◽

Factor Viii Gene ◽

Human Factor Viii ◽

Exon 4

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Adenoviral Vector-Mediated Expression of Physiologic Levels of Human Factor VIII in Nonhuman Primates

Human Gene Therapy ◽

10.1089/10430349950016401 ◽

1999 ◽

Vol 10 (18) ◽

pp. 2999-3011 ◽

Cited By ~ 26

Author(s):

Terrence Brann ◽

Dawn Kayda ◽

Russette M. Lyons ◽

Pamela Shirley ◽

Soumitra Roy ◽

...

Keyword(s):

Factor Viii ◽

Adenoviral Vector ◽

Nonhuman Primates ◽

Human Factor ◽

Human Factor Viii

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

Blood ◽

10.1182/blood-2003-11-3891 ◽

2004 ◽

Vol 104 (3) ◽

pp. 704-710 ◽

Cited By ~ 37

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.

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