Factor VIII Knockout Mice Research Articles

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

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.

Correction of bleeding diathesis without liver toxicity using arenaviral-pseudotyped HIV-1-based vectors in hemophilia A mice.

Hemophilia A is an inheritable X-linked bleeding disorder most frequently occurring as a consequence of genetic alterations within the factor VIII (FVIII) gene. In the present study, pseudotyped human immunodeficiency virus type 1 (HIV-1)-derived lentivectors expressing hFVIII were assessed for the ability to correct the hemophilia A phenotype in FVIII knockout mice. Therapeutic levels of plasma hFVIII (1-7 ng/mL) were detected in C57B1/6 mice (4-5 weeks old) after portal vein administration of hFVIII-expressing lentivectors pseudotyped with the rhabdoviral vesicular stomatitis viral G protein (VSV-G). More importantly, transduction of hemophilia A mice with FVIII expressing lentivectors resulted in transient correction of the bleeding diathesis phenotype. Moreover, the use of alternate viral pseudotypes based on the lymphocytic choriomeningitis virus (LCMV) resulted in similar circulating levels of FVIII. Interestingly, similar doses of LCMV-pseudotyped lentiviral vectors resulted in minimal systemic or hepatic injury as measured by plasma alanine transferase (ALT), aspartate transferase (AST), and tumor necrosis factor (TNF)-alpha compared to the more commonly used envelope, VSV-G. In summary, these studies demonstrated both the potential merit of lentivectors in terms of correcting monogenic inherited disorders, and also the importance of using alternate pseudotypes, such as LCMV, to safely transfer therapeutic genes in vivo without producing adverse effects.

Efficient production of human FVIII in hemophilic mice using lentiviral vectors

Lentiviral vectors (LV) have the ability to integrate their proviral DNA containing a therapeutic gene into the host cell's genome. Therefore, these vectors have a great potential for gene therapy especially in the treatment of hereditary diseases like hemophilia A, which require lifelong expression of the transgene. We constructed an HIV-1-based LV containing human B-domain-deleted factor VIII (FVIII) cDNA under the control of a promoter consisting of the chicken β-actin promoter, CMV enhancers, and a large synthetic intron (CAG), which is a robust transcription promoter. High levels of FVIII expression from this vector could be demonstrated in vitro in 293T cells, primary liver cells, and hematopoietic progenitor cells. To test whether this viral vector was able to correct the bleeding disorder of C57BL/6 FVIII knockout mice, we transduced these mice with the FVIII LV either by intraperitoneal injection or by transplantation with transduced syngeneic bone marrow. FVIII production was analyzed in the blood plasma for a period of 3 months; however, only low levels of FVIII (<50 mU), which were below 5% of normal FVIII levels of 1000 mU, could be detected. Further analysis revealed that the low levels of FVIII activity present in the blood plasma were due to the presence of neutralizing antibodies to FVIII and not due to lack of expression of FVIII from the viral vector. FVIII expression could be detected in the tissues of the transduced mice by Western blot analysis and in ex vivo cultures. These data demonstrate that LVs are able to produce therapeutic levels of FVIII in knockout mice when administered by ip infection or by transduced hematopoietic cells. The challenge is to overcome the immune barriers to the therapeutic gene product.

Characterization of Antibodies Induced by Human Factor VIII in a Murine Knockout Model of Hemophilia A

To investigate the usefulness of factor VIII (FVIII) knockout mice as an animal model of hemophilia A, we characterized the antibody response in FVIII knockout mice to recombinant human FVIII, administered intravenously or subcutaneously with or without adjuvant, and compared results to those in normal mice. Anti-factor VIII antibodies were detected after both intravenous and subcutaneous administration, with the highest titers after subcutaneous administration plus adjuvant. Depending on the administration strategy. knockout mice formed antibodies more rapidly and developed higher titers of inhibitory antibodies (Bethesda) than normal mice, suggesting differences in epitope specificity. Blotting thrombin cleavage products separated by gel electrophoresis showed that both strains developed antibodies against the nonfunctional B domain as well as against functional domains of factor VIII. The antibodies were mainly of the IgG1 subclass and resembled type I antibodies in hemophilia A.