A case of Behçet's-like disease associated with trisomy 8-positive myelodysplastic syndrome carrying MEFV E148Q variant presented with periodic fever.

Behçet's-like disease, which incompletely fulfils the criteria of Behçet's disease, is often associated with trisomy 8-positive myelodysplastic syndrome (MDS). We report a case of an 82-year-old man with these conditions carrying the E148Q variant of MEFV gene who presented with periodic fever. The patient presented with joint pain, muscle pain, and episodes of periodic fever every 2 weeks for the past 3 months. On admission, painful erythema and fever were observed. Colonoscopy revealed erosion in the caecum and ascending colon. The patient had bicytopenia, and a bone marrow biopsy showed findings compatible with trisomy 8-positive unclassifiable MDS. Because the patient incompletely fulfilled the criteria for Behçet's disease, he was diagnosed with Behçet's-like disease associated with trisomy 8-positive MDS. Positron emission tomography-computed tomography performed during the fever revealed multiple muscle lesions consistent with the sites of pain. To examine the cause of the periodic fever attacks, MEFV gene was analysed, and the results revealed an E148Q variant. Steroids were ineffective against periodic fever attacks. A daily dose of 0.5 mg colchicine was prescribed, but the effect was minimal, probably, because of the insufficient dose due to renal dysfunction. Based on the diagnosis of atypical familial Mediterranean fever, canakinumab was added, which partially mitigated the periodic fever. This case suggests the importance of ruling out MDS when physicians see an elderly patient who present with Behçet's-like disease. Although the significance of the E148Q variant in the pathogenesis of periodic fever remains controversial, it may act as a disease modifier in accordance with trisomy 8-positive MDS.

Overexpression of factor VII ameliorates bleeding diathesis of factor VIII-deficient mice with inhibitors.

INTRODUCTION: Factor VIII (FVIII) treatment for hemophilia A has difficulties in correcting bleeding diathesis in the presence of inhibitors. MATERIALS AND METHODS: An adeno-associated virus type 8 (AAV8) vector containing the factor VII (FVII) gene or the activated factor VII (FVIIa) gene was used to investigate the therapeutic effect of FVII or FVIIa overexpression in FVIII-deficient mice with inhibitors. RESULTS: Following repeated human FVIII injection, FVIII-deficient mice developed anti-human FVIII antibodies that cross-reacted with mouse FVIII. High transgene expression of murine FVII or murine FVIIa was achieved using the AAV8 vector and resulted in increased blood FVII activity greater than 800% of normal murine FVII levels in vector-injected FVIII-deficient mice. Thromboelastography analysis showed significant improvements in clotting time, clot formation time, α angle, and mean clot firmness in AAV8 vector-injected FVIII-deficient mice with inhibitors. Overexpression of FVIIa ameliorated the bleeding phenotype of FVIII-deficient mice with inhibitors and significantly increased the survival rate after tail clipping. In addition, overexpression of FVII increased the survival rate of FVIII-deficient mice with inhibitors after tail clipping though it was not as efficient as FVIIa overexpression. CONCLUSIONS: These data suggest that FVII overexpression is an alternative strategy for the treatment of hemophilia A with inhibitors.

Minimizing the inhibitory effect of neutralizing antibody for efficient gene expression in the liver with adeno-associated virus 8 vectors.

Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Evading the inhibitory effects of antibodies against AAV vectors is necessary for efficient transfer of therapeutic genes clinically. For this purpose, we tested the efficacy of saline flushing in order to avoid contact of vectors with NAbs present in blood. Direct injection of the AAV8 vector carrying the factor IX (FIX) gene into the portal vein of macaques using saline flushing achieved transgene-derived FIX expression (4.7 ± 2.10-10.1 ± 5.45% of normal human FIX concentration) in the presence of NAbs. Expression was as efficient as that (5.43 ± 2.59-12.68 ± 4.83%) in macaques lacking NAbs. We next tested the efficacy of saline flushing using less invasive balloon catheter-guided injection. This approach also resulted in efficient expression of transgene-derived FIX (2.5 ± 1.06-9.0 ± 2.37%) in the presence of NAbs (14-56× dilutions). NAbs at this range of titers reduced the efficiency of transduction in the macaque liver by 100-fold when the same vector was injected into mesenteric veins without balloon catheters. Our results suggest that portal vein-directed vector delivery strategies with flushing to remove blood are efficacious for minimizing the inhibitory effect of anti-AAV antibodies.

Porcine model of hemophilia A.

Hemophilia A is a common X chromosome-linked genetic bleeding disorder caused by abnormalities in the coagulation factor VIII gene (F8). Hemophilia A patients suffer from a bleeding diathesis, such as life-threatening bleeding in the brain and harmful bleeding in joints and muscles. Because it could potentially be cured by gene therapy, subhuman animal models have been sought. Current mouse hemophilia A models generated by gene targeting of the F8 have difficulties to extrapolate human disease due to differences in the coagulation and immune systems between mice and humans. Here, we generated a porcine model of hemophilia A by nuclear transfer cloning from F8-targeted fibroblasts. The hemophilia A pigs showed a severe bleeding tendency upon birth, similar to human severe hemophiliacs, but in contrast to hemophilia A mice which rarely bleed under standard breed conditions. Infusion of human factor VIII was effective in stopping bleeding and reducing the bleeding frequency of a hemophilia A piglet but was blocked by the inhibitor against human factor VIII. These data suggest that the hemophilia A pig is a severe hemophilia A animal model for studying not only hemophilia A gene therapy but also the next generation recombinant coagulation factors, such as recombinant factor VIII variants with a slower clearance rate.

Local regulation of neutrophil elastase activity by endogenous α1-antitrypsin in lipopolysaccharide-primed hematological cells.

Neutrophil elastase released from activated neutrophils contributes in combating bacterial infection. While chronic inflammation results in anemia and decreased bone marrow activities, little is known about the effect of neutrophil elastase on hematological cell growth in severe inflammatory states. Here, we demonstrated that α1-antitrypsin, a physiological inhibitor of neutrophil elastase, functions as a regulator for cell growth by neutralizing neutrophil elastase activity in lipopolysaccharide-primed hematological cells. HL-60 cells were resistant to neutrophil elastase, as they also expressed α1-antitrypsin. The growth of HL-60 cells transduced with a LentiLox-short hairpin α1-antitrypsin vector was significantly suppressed by neutrophil elastase or lipopolysaccharide. When CD34(+) progenitor cells were differentiated towards a granulocytic lineage, they concomitantly expressed neutrophil elastase and α1-antitrypsin and prevented neutrophil elastase-induced growth inhibition. These results suggest that granulocytes might protect themselves from neutrophil elastase-induced cellular damage by efficiently neutralizing its activity through the simultaneous secretion of endogenous α1-antitrypsin.

Degradation of cross-linked fibrin by leukocyte elastase as alternative pathway for plasmin-mediated fibrinolysis in sepsis-induced disseminated intravascular coagulation.

An alternative pathway for fibrinolysis that comprises leukocyte elastase and its interaction with the plasminogen activator-plasmin system has been suggested. Plasma levels of cross-linked fibrin degradation product by leukocyte elastase (e-XDP) were significantly increased in patients with sepsis induced disseminated intravascular coagulation (DIC) compared with healthy subjects (18.6±19.9 vs 0.58±0.47U/mL, p<0.001). Twenty seven unique spots were identified from e-XDP dominant patients by immune-purification and two-dimensional difference gel electrophoresis, and they contained fibrinogen Bß-chain derived fragments Bß Asp-164, Ser-200, Gln-301, Ala-354, Ile-484 and γ-chain derivatives γ Val-274 at their amino-termini by acquired and processed tandem mass spectrometer. The Sequential Organ Failure Assessment Scores in patients with e-XDPs levels 3-10U/mL were significantly lower than those with e-XDPs levels -3U/mL, 10-30U/mL, and 30- U/mL. The adjusted odds for 28-day mortality rate in patients with e-XDP levels less than 3U/mL (hazard ratio, 4.432; 95% CI, 1.557-12.615 [p=0.005]) were significantly higher than those in patients with e-XDP levels of 3-10U/mL. These data suggest that leukocyte elastase might contribute to the degradation of cross-linked fibrin in sepsis-induced DIC.

Vinculin activates inside-out signaling of integrin αIIbß3 in Chinese hamster ovary cells.

Although vinculin is used frequently as a marker for integrin-mediated focal adhesion complexes, how it regulates the activation of integrin is mostly unknown. In this study, we examined whether vinculin would activate integrin in Chinese hamster ovary (CHO) cells expressing human integrin αIIbß3. Silencing of vinculin by lentiviral transduction with a short hairpin RNA sequence affected the binding of PAC-1 (an antibody recognizing activated human αIIbß3) to a constitutively active form of αIIbß3 (α6Bß3) expressed on CHO cells, while its inhibitory effects were much weaker than those of talin-1. Overexpression of an active form of vinculin without intramolecular interactions, but not the full length one, induced PAC-1 binding to native αIIbß3 expressed on CHO cells in a manner dependent on talin-1. On the other hand, silencing of talin-1, but not vinculin, failed to induce cell spreading of α6Bß3-CHO cells on fibrinogen, even in the presence of PT 25-2, a monoclonal antibody that activates αIIbß3. Thus, an active form of vinculin could induce αIIbß3 inside-out signaling through the actions of talin-1, while vinculin was dispensable for outside-in signaling.

Vinculin is indispensable for repopulation by hematopoietic stem cells, independent of integrin function.

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function, little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit(+)Sca1(+)Lin(-) HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages, to migrate toward stromal cell-derived factor 1α, and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However, the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit(+)Sca1(+)Lin(-) HSCs. In contrast, adhesion to the extracellular matrix was inhibited by silencing of talin-1, but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity, independent of integrin functions.

Mutant macaque factor IX T262A: a tool for hemophilia B gene therapy studies in macaques.

INTRODUCTION: Gene therapy is expected to be the next generation therapy for hemophilia, and a good animal model is required for hemophilia gene therapy preclinical studies. METHODS: Taking advantage of the human factor IX (FIX) specificity of monoclonal antibody 3A6, the epitope of which resides in the amino acid polypeptide segment including Ala 262 of human FIX, mutant macaque FIX with an amino acid substitution of Thr 262 to Ala (macaque FIX T262A) was generated and its reactivity to monoclonal antibody 3A6, biological activity and expression in vivo were studied. RESULTS: Enzyme-linked immunosorbent assays (ELISAs) and Western blot analyses showed that monoclonal antibody 3A6 bound to human FIX and macaque FIX T262A but not to wild-type macaque FIX. Recombinant macaque FIX T262A exhibited a comparable coagulation activity to wild-type macaque FIX and human FIX. High expression of macaque FIX T262A was achieved in mice by injection of AAV8 vectors carrying the macaque FIX T262A gene and reached levels of up to 31.5microg/mL (1050% of the normal human FIX concentration). Macaque FIX T262A expressed in the liver of mice was as biologically active as that expressed in vitro. In addition, the macaque FIX T262A concentrations determined by a 3A6-based ELISA were not influenced by the presence of normal macaque plasma. CONCLUSIONS: The results of the present study suggest that macaque FIX T262A may be processed appropriately in vivo and that the macaque FIX T262A concentration in the macaque circulation can be quantified precisely by a monoclonal antibody 3A6-based ELISA.

Impact of acute cellular rejection on coagulation and fibrinolysis biomarkers within the immediate post-operative period in pediatric liver transplantation.

We studied restoration of the coagulation and fibrinolysis system in pediatric patients following liver transplantation and biomarkers of blood coagulation and fibrinolysis for suspecting the occurrence of acute cellular rejection. Coagulation activity recovered rapidly within two days following transplantation, but it took approximately 21-28 days for full recovery of the coagulation and fibrinolysis factors synthesized in the liver. PAI-1 levels were significantly higher in patients at the time of acute cellular rejection compared with levels after control of AR, and levels on days 14 and 28 in patients without AR. Plasma protein C and plasminogen levels at the time of rejection were significantly lower than those on day 14 in patients without AR. Statistical analysis suggested that an increase in plasma PAI-1 at a single time point in the post-operative period is a reliable marker among the coagulation and fibrinolysis factors for suspecting the occurrence of acute cellular rejection. These data suggested that appropriate anticoagulation may be required for 14 days after liver transplantation in order to avoid vascular complications and measurement of plasma PAI-1 levels may be useful for suspecting the occurrence of acute cellular rejection in pediatric patients following liver transplantation.

Liver-restricted expression of the canine factor VIII gene facilitates prevention of inhibitor formation in factor VIII-deficient mice.

BACKGROUND: Gene therapy for hemophilia A with adeno-associated virus (AAV) vectors involves difficulties in the efficient expression of factor VIII (FVIII) and in antibody formation against transgene-derived FVIII. METHODS: AAV8 vectors carrying the canine B domain deleted FVIII (cFVIII) gene under the control of the ubiquitous beta-actin promoter, the liver-specific human alpha1 anti-trypsin promoter (HAAT) and the liver-specific hepatic control region (HCR) enhancer/human alpha1 anti-trypsin promoter complex (HCRHAAT) were used for the expression of cFVIII in FVIII deficient (fviii(-/-)) mice. RESULTS: Addition of the hepatic control region enhancer element to the HAAT promoter successfully augmented HAAT promoter activity without loss of liver-specificity in vivo. Using this enhancer/promoter complex, a high cFVIII transgene expression was achieved, resulting in increased blood cFVIII activities to more than 100% of the normal canine FVIII levels in fviii(-/-) mice at a 1 : 10 lower dose of the AAV8 vector carrying the cFVIII gene driven by the HAAT promoter. Under short-term immunosuppression, neutralizing antibodies against cFVIII developed in only one out of six mice when the HAAT promoter was used for cFVIII expression, whereas all the mice developed neutralizing antibodies against cFVIII when the beta-actin promoter was used for cFVIII expression. No neutralizing antibodies against cFVIII developed in fviii(-/-) mice that received the AAV8 vector carrying the cFVIII gene driven by the HCRHAAT enhancer/promoter complex without immunosuppression. CONCLUSIONS: These data suggest that AAV8 vector-mediated liver-restricted cFVIII gene expression is sufficient for immune hypo-responsiveness to transgene-derived cFVIII in fviii(-/-) mice.

Phenotypic correction of hemophilia A by ectopic expression of activated factor VII in platelets.

Platelets are receiving much attention as novel target cells to secrete a coagulation factor for hemophilia gene therapy. In order to extend the application of platelet-directed gene therapy, we examined whether ectopic expression of activated factor VII (FVIIa) in platelets would result in an efficient bypass therapy to induce sufficient thrombin generation on platelet surfaces in mice with hemophilia A. Transduction of bone marrow cells with a simian immunodeficiency virus (SIV)-based lentiviral vector harboring the platelet-specific GPIb alpha promoter resulted in efficient transgene expression in platelets. FVIIa antigen was expressed in platelets by this SIV system; FVII transgene products were found to localize in the cytoplasm and translocate toward the sub-membrane zone and cell surface after activation. Although FVII antigen levels in platelets did not reach the therapeutic levels seen with FVIIa infusion therapy, whole-blood coagulation, as assessed by thromboelastography, was significantly improved in mice with hemophilia A. Further, we observed correction of the bleeding phenotype in mice with hemophilia A after transplantation, even in the presence of FVIII-neutralizing antibodies. Our results demonstrate that FVIIa-expressing platelets can strengthen hemostatic function and may be useful in treating hemophilia and other inherited bleeding disorders. These findings are comparable to the proven therapeutic effects of FVIIa infusion.

Unbalanced expression of ADAMTS13 and von Willebrand factor in mouse endotoxinemia.

INTRODUCTION: Secondary ADAMTS13 deficiency may occur in septic patients. The expression of ADAMTS13 in mouse endotoxinemia was studied. METHODS: The blood and mRNA expression levels of ADAMTS13 and von Willebrand factor were measured in lipopolysaccharide-injected mice. RESULTS: The plasma ADAMTS13 activity in wild-type mice was significantly decreased at 2 h after lipopolysaccharide injection, and this decrease in ADAMTS13 activity preceded the decrease in ADAMTS13 mRNA expression in the liver and continued for 24 h. However, no decreases in the plasma ADAMTS13 activity after lipopolysaccharide injection were observed in mice pretreated with a neutrophil elastase inhibitor or in plasminogen-deficient mice, suggesting that the decrease in ADAMTS13 activity was processed efficiently by the coordinated actions of plasmin and neutrophil elastase. von Willebrand factor mRNA was abundantly expressed in the lung and moderately in the kidney, but showed relatively low expression in the liver without lipopolysaccharide injection. However, von Willebrand factor mRNA expression in the liver was significantly increased after lipopolysaccharide injection and this high expression level continued for 24 h after the injection. The von Willebrand factor and ADAMTS13 mRNA expression levels in these organs changed in the opposite manners following lipopolysaccharide administration. Furthermore, the blood von Willebrand factor level increased after lipopolysaccharide administration, in contrast to the decrease in the blood ADMTS13 level after lipopolysaccharide administration. CONCLUSION: These data suggest that imbalance between the blood von Willebrand factor and ADAMTS13 levels may occur in endotoxinemia, and that this may partly contribute to the thrombotic state associated with endotoxinemia.

Silencing of a targeted protein in in vivo platelets using a lentiviral vector delivering short hairpin RNA sequence.

OBJECTIVE: Because platelets are anucleate cells having a limited life span, direct gene manipulation cannot in principle be used to investigate the involvement of a specific signal transduction pathway in platelet activation. In this study, we examined whether the expression of a short hairpin RNA (shRNA) sequence in hematopoietic stem cells is maintained during megakaryocyte differentiation, thus resulting in inhibition of targeted protein in platelets. METHODS AND RESULTS: To identify platelets derived from transduced stem cells, we generated a lentiviral vector that simultaneously expresses the shRNA sequence driven by the U6 promoter and GFP under the control of the glycoprotein (GP) Ib alpha promoter. Transplantation of mouse bone marrow cells transduced with the vector facilitated specifically mark platelets derived from the transduced cells. Transplantation of cells transduced with shRNA sequence targeting integrin alphaIIb caused a significant reduction of integrin alphaIIb beta3 (alphaIIb beta3) expression in GFP-positive platelets. It also inhibited alphaIIb beta3 activation assessed by the binding of JON/A, an antibody that recognizes activated alphaIIb beta3. Talin-1 silencing by the same method resulted in normal alphaIIb beta3 expression but deficient inside-out alphaIIb beta3 signaling. CONCLUSIONS: shRNA expression driven by the U6 promoter is preserved during megakaryopoiesis. This method facilitates functional analysis of targeted protein in platelet activation.

Efficient expression of a transgene in platelets using simian immunodeficiency virus-based vector harboring glycoprotein Ibalpha promoter: in vivo model for platelet-targeting gene therapy.

Platelets release several mediators that modify vascular integrity and hemostasis. In the present study, we developed a technique for efficient transgene expression in platelets in vivo and examined whether this targeted-gene-product delivery system using a platelet release reaction could be exploited for clinical applications. Analysis of luciferase reporter gene constructs driven by platelet-specific promoters (the GPIIb, GPIbalpha, and GPVI) revealed that the GPIbalpha promoter was the most potent in the megakaryoblastic cell line UT-7/TPO and human CD34+-derived megakaryocytes. Transduction of UT-7/TPO; CD34+-derived megakaryocytes; and c-Kit+, ScaI+, and Lineage- (KSL) murine hematopoietic stem cells with a simian immunodeficiency virus (SIV)-based lentiviral vector carrying eGFP resulted in efficient, dose-dependent expression of eGFP, and the GPIbalpha promoter seemed to bestow megakaryocytic-specific expression. Transplantation of KSL cells transduced with SIV vector containing eGFP into mice showed that there was preferable expression of eGFP in platelets driven by the GPIbalpha promoter [7-11% for the cytomeglovirus (CMV) promoter, 16-27% for the GPIbalpha promoter]. Furthermore, transplantation of ex vivo-transduced KSL cells by SIV vector carrying human factorVIII (hFVIII) driven by the GPIbalpha promoter induced the production of detectable transcripts of the hFVIII gene and the hFVIII antigen in bone marrow and spleen for at least 90 days and partially corrected the hemophilia A phenotype. Platelet-targeting gene therapy using SIV vectors appears to be promising for gene therapy approaches toward not only inherited platelet diseases but also other hemorrhagic disorders such as hemophilia A.

Severe secondary deficiency of von Willebrand factor-cleaving protease (ADAMTS13) in patients with sepsis-induced disseminated intravascular coagulation: its correlation with development of renal failure.

Deficiency of ADAMTS13 is found in patients with thrombotic thrombocytopenic purpura (TTP), and the genetic defects in the ADAMTS13 gene or the autoantibody against ADAMTS13 is thought to be responsible for the development of TTP. The clinical correlation and mechanisms of secondary ADAMTS13 deficiency in other disease states were investigated. In addition to TTP, ADAMTS13 levels were severely decreased in patients with sepsis-induced disseminated intravascular coagulation (DIC). The incidence of acute renal failure and serum creatinine levels in patients with ADAMTS13 activity levels lower than 20% (incidence, 41.2%; creatinine, 160 +/- 150 microM [1.81 +/- 1.70 mg/dL]) (P < .05) were significantly higher than they were in patients with ADAMTS13 activity levels higher than 20% (incidence, 15.4%; creatinine, 84 +/- 67 microM [0.95 +/- 0.76 mg/dL]) (P < .01). Additionally, unusually large von Willebrand factor multimers were detected in 26 (51.0%) of 51 patients with ADAMTS13 activity levels lower than 20%. Lower molecular weight forms of ADAMTS13 were found in the plasma of patients with sepsis-induced DIC, suggesting that the deficiency of ADAMTS13 was partially caused by its cleavage by proteases in addition to decreased synthesis in the liver. These data suggested that severe secondary ADAMTS13 deficiency can be associated with sepsis-induced DIC and may contribute to the development of renal failure.

Phenotype correction of hemophilia A mice with adeno-associated virus vectors carrying the B domain-deleted canine factor VIII gene.

Adeno-associated virus (AAV) vectors carrying the B domain-deleted canine FVIII (BDD cFVIII) gene utilizing the beta-actin minimum promoter (167b) pseudotyped with serotype 1 (AAV1-beta-actin-cFVIII) and serotype 8 (AAV8-beta-actin-cFVIII) were developed to express cFVIII in hemophilia A mice. FVIII clotting activities measured by the APTT method increased in hemophilia A mice with intramuscular injection of AAV1-beta-actin-cFVIII in a dose-dependent manner. Therapeutic FVIII levels (2.9+/-1.0%) in hemophilia A mice with the AAV1-beta-actin-cFVIII dose of 1 x 10(12) gc/body were achieved, suggesting partial correction of the phenotype with AAV1-beta-actin-cFVIII vectors. FVIII clotting activity levels in hemophilia A mice with intravenous injection of AAV8-beta-actin-cFVIII also were increased dose-dependently, achieving therapeutic FVIII levels (5-90%) in hemophilia A mice with the AAV8-beta-actin-cFVIII doses of 1-3 x 10(11) gc/body and supernormal FVIII levels (180-670%) in hemophilia A mice with the AAV8-beta-actin-cFVIII dose of 1 x 10(12) gc/body. Transduction of the liver with AAV8-beta-actin-cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding the FVIII production and antibody formation. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.

Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice.

BACKGROUND: Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34+ cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). METHODS: CD34+ cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34+ cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34+ cells and expression of transgenes were studied. RESULTS: We could efficiently transduce CD34+ cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 +/- 0.1%) at MOI of 5 x 10(3) vector genome/cell. After transducing CD34+ cells with SIVhFVIII, hFVIII was produced (274.3 +/- 20.1 ng) from 10(6) CD34+ cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34+ cells (5-10 x 10(5)) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34+ cells and production of hFVIII (minimum 1.2 +/- 0.9 ng/mL, maximum 3.6 +/- 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. CONCLUSIONS: Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients.

Thrombophilic dysfibrinogen Tokyo V with the amino acid substitution of gammaAla327Thr: formation of fragile but fibrinolysis-resistant fibrin clots and its relevance to arterial thromboembolism.

Thrombophilic dysfibrinogen Tokyo V was identified in a 43-year-old man with recurrent thromboembolism. Based on analyses of the patient fibrinogen genes, the amino acid sequence of the aberrant fibrinogen peptide, and deglycosylation experiments, fibrinogen Tokyo V was shown to have an amino acid substitution of gamma Ala327Thr and possibly extra glycosylation at gamma Asn325 because the mutation confers the N-linked glycosylation consensus sequence Asn-X-Thr. The mutation resulted in impaired function and hypofibrinogenemia (hypodysfibrinogen). Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium, resulting in very low clottability. Additionally, a large amount of soluble cross-linked fibrin was formed upon thrombin treatment in the presence of factor XIII and calcium. However, Tokyo V-derived fibrin was resistant to degradation by tissue plasminogen activator (tPA)-catalyzed plasmin digestion. The structure of Tokyo V fibrin appeared severely perturbed, since there are large pores inside the tangled fibrin networks and fiber ends at the boundaries. Taken together, these data suggest that Tokyo V fibrin clots are fragile, so that fibrinolysis-resistant insoluble fibrin and soluble fibrin polymers may be released to the circulation, partly accounting for the recurrent embolic episodes in the patient.