Evaluation of the Duration of Human Factor VIII Expression in Nonhuman Primates After Systemic Delivery of an 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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In vivo evaluation of a novel epitope-tagged human factor VIII-encoding adenoviral vector

Haemophilia ◽

10.1046/j.1365-2516.1999.00310.x ◽

1999 ◽

Vol 5 (5) ◽

pp. 340-348 ◽

Cited By ~ 7

Author(s):

Roy ◽

Shirley ◽

Connelly ◽

Andrews ◽

Kayda ◽

...

Keyword(s):

Factor Viii ◽

Adenoviral Vector ◽

Human Factor ◽

In Vivo Evaluation ◽

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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Efficient adenoviral vector transduction and expression of functional human factor VIII in cultured primary human hepatocytes

Haemophilia ◽

10.1046/j.1365-2516.1999.00304.x ◽

1999 ◽

Vol 5 (3) ◽

pp. 160-168 ◽

Cited By ~ 2

Author(s):

Andrews ◽

Weaver ◽

Kaleko ◽

Connelly

Keyword(s):

Factor Viii ◽

Adenoviral Vector ◽

Human Factor ◽

Human Hepatocytes ◽

Primary Human Hepatocytes ◽

Human Factor Viii ◽

Vector Transduction

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Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant

Blood ◽

10.1182/blood-2012-10-462200 ◽

2013 ◽

Vol 121 (17) ◽

pp. 3335-3344 ◽

Cited By ~ 141

Author(s):

Jenny McIntosh ◽

Peter J. Lenting ◽

Cecilia Rosales ◽

Doyoung Lee ◽

Samira Rabbanian ◽

...

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Nonhuman Primates ◽

Human Factor ◽

Hemophilia A ◽

Raav Vector ◽

Peripheral Vein ◽

Human Factor Viii ◽

Key Points

Key Points Novel, more potent codon-optimized human FVIII variant (codop-hFVIII-V3). Codop-hFVIII-V3 is safe and efficacious in mice and nonhuman primates, thus improving the prospects of gene therapy for hemophilia A.

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The Fractionation Properties of Human Factor VIII (Antihaemophilic Factor)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654503 ◽

1960 ◽

Vol 04 (02) ◽

pp. 253-260 ◽

Cited By ~ 1

Author(s):

Franco Gobbi

Keyword(s):

Factor Viii ◽

Human Factor ◽

Maximum Yield ◽

Factor Vii ◽

Precipitation Method ◽

Salting Out ◽

Human Factor Viii ◽

Plasma Factor ◽

Two Factors ◽

Thromboplastic Activity

SummaryThe fractionation properties of human Factor VIII (antihaemophilic factor, AHF, antihaemophilic globulin) have been studied using a plasma of congenital afibrinogenaemia as a starting material.From a fibrinogen-free plasma, Factor VIII does not precipitate with ethanol at a final concentration of 8%; on the contrary the maximum yield is reached at an ethanol concentration of 25%.With a precipitation method carried out by a one to ten dilution of plasma with distilled water and acidification by N/10 hydrochloric acid to a pFI 5.2, Factor VIII does not precipitate with the euglobulin fraction; when normal plasma is used, such a precipitation is almost complete.With the salting-out fractionation method by ammonium sulphate, Factor VIII precipitates at a concentration between 25 and 33% of saturation either from fibrinogen-free and from normal human plasma.A non-specific thromboplastic activity appears in the fractions prepared by every method. This activity, which is probably due to the activation of seric accelerators, is easily removed by Al(OH)s adsorption. Thus, in order to insure the specificity of Factor VIII assays, the preliminary adsorption of the fractions is indispensable before testing their antihaemophilic activity.Fibrinogen and Factor VIII have different and definite precipitation patterns. When these two factors are associated the fractionation properties of AHF appear quite modified, showing a close similarity to those of fibrinogen. This fact can explain the technical difficulties encountered in the attempt to purify the antihaemophilic factor, and the lack of reproducible procedures for removing fibrinogen without affecting Factor VII.

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Impaired Prothrombin Consumption in Bernard-Soulier Syndrome Is Corrected In Vitro by Human Factor VIII

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655972 ◽

1997 ◽

Vol 77 (02) ◽

pp. 383-386 ◽

Cited By ~ 8

Author(s):

S Bellucci ◽

J P Girma ◽

M Lozano ◽

D Meyer ◽

J P Caen

Keyword(s):

Factor Viii ◽

Human Factor ◽

Platelet Membrane ◽

Giant Platelets ◽

Prolonged Bleeding Time ◽

Human Factor Viii ◽

Von Willebrand ◽

Willebrand Factor ◽

Bernard Soulier Syndrome

SummaryThe Bernard-Soulier syndrome (BSS) is characterized by thrombocytopenia with giant platelets, a prolonged bleeding time with defective platelet adhesion to the subendothelium related to a defect in platelet membrane glycoprotein lb (GPIb) and a decreased prothrombin consumption. The mechanism of the latter abnormality remains unknown. In this study, we showed that this defect was corrected by the addition of purified human factor VIII (FVIII) to blood from four patients with BSS. The correction of prothrombin consumption was almost complete at concentrations between 1.5 and 3 IU/ml of FVIII procoagulant activity (VIII.'C) and partially abolished by a monoclonal antibody which neutralizes VIII:C. This correction was specific for FVIII and was not observed after addition of purified human FIX. It was obtained, in the same magnitude range, with FVIII complexed to von Willebrand factor (vWF) but not with free vWF. These data provide a new insight into the knowledge of the physiological interaction between the platelet membrane and the vWF-FVIII complex facilitating plasma coagulation activation and may lead to helpful therapeutic advances.

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