Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Hemophilia is a genetically inherited bleeding disorder caused by a deficiency of the blood clotting factors VIII (hemophilia A) or IX (hemophilia B). Hemophiliacs suffer prolonged bleeding which can be life threatening and often leads to chronic disabilities. Current hemophilia treatment involves infusions of plasma-derived or recombinant clotting factor in response to bleeding crises. Prophylactic treatment is not available and current treatments remain problematic. The development of a gene therapy for hemophilia has been under investigation for the past decade. An overview is presented of the initial efforts using retroviral and adenoviral vectors for ex vivo and in vivo gene delivery strategies, respectively. Recent progress in developing FIX and FVIII adeno-associated virus vectors is reviewed. Sustained expression of therapeutic levels of FIX and FVIII have been demonstrated in mice. Phenotypic correction of hemophilia B has been shown in the murine and dog models of disease. A phase I human clinical trial has been initiated involving intramuscular injection of FIX. Prospectsfor hemophilia gene therapy look bright and the hopefor a cure has now moved from the realm of the possible to the probable.
Curr Opin Mol Ther 1999 Aug
PMID:Gene therapy for hemophilia. 1171 65

Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of <2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes 1 day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.
Mol Ther 2002 Jan
PMID:Sustained human factor VIII expression in hemophilia A mice following systemic delivery of a gutless adenoviral vector. 1178 47

Deficiency in a coagulation factor VIII (FVIII) causes a genetic disorder hemophilia A, which is treated by repeated infusions of expensive FVIII products. Recombinant FVIII (rFVIII), the culmination of years of extensive international research, is an important alternative to plasma-derived FVIII (pdFVIII) and is considered to have a higher margin of safety. Advances in biotechnology allowed production of rFVIII at industrial scale, which significantly improved treatment of hemophilia A patients. We review the contemporary methods used for FVIII expression in mammalian cell culture systems and discuss the factors responsible for insufficient recoveries of rFVIII, such as inefficient accumulation of FVIII mRNA in the cell, complexity of the mechanisms of FVIII secretion, and instability of secreted FVIII. The approaches to improve the yield of rFVIII in cell culture systems include genetic engineering of B-domain-deleted FVIII, introduction of introns into FVIII cDNA constructs for more efficient processing and accumulation of FVIII mRNA, and introduction of mutations into chaperone-binding sites of FVIII to improve its secretion. Design of FVIII with prolonged half-life in vivo is considered as another promising direction in improving rFVIII protein and efficiency of hemophilia A therapy. As an alternative to expression of rFVIII in cell culture systems, we discuss production of rFVIII in transgenic animals, where high levels of rFVIII have been successfully secreted into milk. We also pay attention to the major limitations of this approach, such as safety issues associated with potential transmission of animal pathogens. Finally, we present a brief characterization of commercial recombinant FVIII products currently available on the market for hemophilia A treatment.
Blood Cells Mol Dis
PMID:Expression of factor VIII in recombinant and transgenic systems. 1206 19

In an earlier study, a site directed mutant rFVIII (rFVIII(m), Arg(336) --> Gln(336)) expressed in baculovirus-insect cell (Sf9) system was found to sustain high level activity during incubation at 37 degrees celsius for 24 h while the cofactor activity of normal plasma was declined steadily. In this study, a mutant B-domain deleted rFVIII(m), Arg(336) --> Gln(336) expressed in baculovirus-insect cell (Sf9) system was characterized for its enzymatic and chemical properties. The expressed rFVIII(m) and plasma FVIII (pFVIII) were purified by immunoaffinity column chromatography and identified by Western blot analysis. The partially purified rFVIII(m) exhibited cofactor specific activity of 2.01 x 10(3)units/mg protein. The molecular weight of rFVIII(m) ranged between 40 to 150 kDa with a major band at 150 kDa. Treatment of both rFVIII(m) and pFVIII with thrombin increased their cofactor activity in a similar pattern. Treatment of both the activated rFVIII(m) and native FVIII with APC decreased their cofactor activities, however, the former exhibited a slower decrease than the latter, although no significant difference was present. rFVIII(m) formed a complex with vWF, resulting in a stabilized form, and the lag period of thrombin-mediated activating was extended by vWF association. These results implicated that rFVIII(m) expressed in baculovirus-insect cell system had a comparable capacity as FVIII cofactor activity and might be a good candidate for the FVIII replacement therapy for hemophilia A patients.
Exp Mol Med 2002 Jul 31
PMID:Expression and characterization of a mutant recombinant blood coagulation factor VIII (rFVIII (m)). 1221 15

Hemophilia results from a deficiency of coagulation Factor VIII or IX and manifests clinically as spontaneous bleeding into the large joints and soft tissue. Current treatment relies on the intravenous infusion of recombinant or purified Factor proteins. Factor infusion is effective, but transient due to the short half-life of Factor proteins. Recent developments in gene transfer technology have led to new strategies using molecular therapeutics as permanent treatment for bleeding disorders. This review describes recent novel molecular strategies for the treatment of the hemophilias.
Curr Opin Mol Ther 2002 Oct
PMID:Hemophilia gene therapy: novel rAAV vectors and RNA repair strategy. 1243 52

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 beta-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.
Mol Ther 2003 May
PMID:Efficient production of human FVIII in hemophilic mice using lentiviral vectors. 1271 5

Hemophilia A, which results in defective or deficient factor VIII (FVIII) protein, is one of the genetic diseases that has been addressed through gene therapy trials. FVIII synthesis does not occur in normal megakaryocytes. In hemophilia patients who have inhibitors to FVIII activity, megakaryocytes could be a protected site of FVIII synthesis and subsequent release. Since von Willebrand factor (VWF) is a carrier protein for FVIII, we hypothesize that by directing FVIII synthesis to megakaryocytes, it would traffick together with VWF to storage in megakaryocyte alpha-granules and the platelets derived from these cells. Such synthesis would establish a protected, releasable alpha-granule pool of FVIII together with VWF. When platelets are activated in a region of local vascular damage, FVIII and VWF could potentially be released together to provide improved local hemostatic effectiveness. To direct FVIII expression to the megakaryocyte lineage, we designed a FVIII expression cassette where the human B-domain deleted FVIII cDNA was placed under the control of the megakaryocytic/platelet-specific glycoprotein IIb (alphaIIb) promoter. We demonstrated by means of a functional FVIII activity assay that the biosynthesis of FVIII occurred normally in Dami cells transfected with FVIII. FVIII production was higher when driven by the alphaIIb promoter compared to the CMV promoter, and was increased about 8-fold following PMA treatment of the transfected Dami cells. Immunofluorescence staining of the transfected cells showed that FVIII stored together with VWF in the granules. The data indicate that the megakaryocytic compartment of hematopoietic cells may represent a potential target of gene therapy for hemophilia A-especially in those patients who have developed inhibitors to plasma FVIII.
Mol Genet Metab 2003 May
PMID:Expression of human factor VIII under control of the platelet-specific alphaIIb promoter in megakaryocytic cell line as well as storage together with VWF. 1276 43

Molecular medicine is a new research field underlain by achievements of the Human Genome Project. The review considers the contribution of the Laboratory of Prenatal Diagnostics of the Ott Institute of Obstetrics and Gynecology to the development of molecular medicine in Russia. Special emphasis is placed on molecular diagnostics, predictive medicine, and gene therapy. The lab obtained priority results in devising and promoting methods of molecular diagnostics of the most common severe hereditary disorders such as cystic fibrosis, Duchenne muscular dystrophy, hemophilia A, and fragile X syndrome. Owing to the Russian program Human Genome, St. Petersburg researchers laid the foundations for theoretical and applied predictive medicine, which is aimed at identifying and analyzing the genes associated with predisposition to high-incidence multifactorial disorders. Experiments with mdx mice providing a model of Duchenne muscular dystrophy were carried out to select the optimal way of delivering a transgene (cDNA of the dystrophin gene) contained in various constructs for the purpose of gene therapy.
Mol Biol (Mosk)
PMID:[Genomic and molecular medicine]. 1504 41

The adhesion receptor P-selectin has long been known to support leukocyte rolling and emigration at sites of inflammation. Recently, P-selectin was also revealed to be a key molecule in hemostasis and thrombosis, mediating platelet rolling, generating procoagulant microparticles containing active tissue factor and enhancing fibrin deposition. Elevated levels of plasma P-selectin are indicative of thrombotic disorders and predictive of future cardiovascular events. Because the interaction between P-selectin and its receptor P-selectin glycoprotein ligand-1 (PSGL-1) represents an important mechanism by which P-selectin induces the formation of procoagulant microparticles and recruits the microparticles to thrombi, anti-thrombotic strategies are currently aimed at inhibiting this interaction. Recent developments also suggest that the procoagulant potential of P-selectin could be used to treat coagulation disorders such as hemophilia A.
Trends Mol Med 2004 Apr
PMID:A new role in hemostasis for the adhesion receptor P-selectin. 1505 9

The development of antibodies to a previously unexpressed protein product may limit the success of human gene therapy approaches. We inserted B-domain-deleted factor VIII (FVIII) cDNA of human, canine, or murine origin into the multiple cloning site of a liver-specific vector, pBS-HCRHPI-A, to yield plasmids pBS-HCRHPI-FVIIIA, pBS-HCRHPI-cFVIIIA, and pBS-HCRHPI-mFVIIIA, respectively. Fifty micrograms of each plasmid in 2 ml of solution was rapidly injected into the tail vein of three groups of hemophilia A mice. Factor VIII levels ranging from 3 to 12 IU/ml were obtained from all three groups (normal is 1 IU/ml in human plasma) 3 days after treatment. These initial very high levels of functional human, canine, or murine factor VIII, however, fell gradually to undetectable levels within 2-3 weeks, and their disappearance correlated with the generation of high-titer, inhibitory anti-FVIII antibodies. Notably, this immune response occurred independent of the species of origin of the exogenous factor VIII. Antibody titers to factor VIII were detected beginning at 2 weeks, reached a plateau and remained at high levels for over 6 months. The majority of anti-hFVIII IgG was IgG1 isotype specific, suggesting a humoral response mediated by Th2-induced signals. Consistent with this idea, in a separate group of mice treated with pBS-HCRHPI-FVIIIA, transient immunosuppression by cyclophosphamide significantly delayed (5/6) or abolished (1/6) inhibitory antibody formation against the transgene.
Mol Ther 2004 Jul
PMID:Naked DNA transfer of Factor VIII induced transgene-specific, species-independent immune response in hemophilia A mice. 1523 48


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