Strategies to Enhance the Efficacy of Platelet-Derived Factor (F) VIII: Studies with Inactivation Resistant FVIII (IR8) and Canine FVIII in Hemophilia A Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3496-3496
Author(s):  
Teshell K. Greene ◽  
Cheng Wang ◽  
Jessica Hirsch ◽  
Hongzhi Miao ◽  
Steven W. Pipe ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Carotid Artery ◽  
Hemophilia A ◽  
Specific Activity ◽  
Proximal Promoter ◽  
Severe Bleeding ◽  
Platelet Factor ◽  
Laser Injury ◽  
Adenoviral Delivery ◽  
Injury Model

Abstract Abstract 3496 Poster Board III-433 Hemophilia A is the most common, inherited severe bleeding diathesis and is due to deficient FVIII. We and others have shown that targeted delivery of ectopic human B-domainless (hBD) FVIII from within platelet α-granules is effective at improving clotting in several injury models using FVIIInull mice. Importantly, platelet-derived (p) hBDFVIII is ∼100-fold more effective than comparable levels of plasma hFVIII against circulating inhibitors, a significant problem in the hemophilia A population. However, we have also shown in both a cuticular injury model and an in situ cremaster laser arteriole/venule injury model that there are certain limitations to pFVIII therapy, especially concerns of clot stability and risks of clot embolization. In the present study, we examined the efficacy of platelet-delivered inactivation resistant FVIII (IR8), resistant to inactivation by thrombin and activated protein C, and canine (c) BDFVIII, a species of FVIII that has 3-5-fold greater specific activity than hBDFVIII. Both FVIIIs were expressed in platelets using both a transgenic mouse approach and lentiviral gene therapy, and compared to similarly expressed phBDFVIII mice. Multiple founder lines of mice were generated for pIR8 and pcBDFVIII using the platelet-specific glycoprotein Ibα proximal promoter to drive platelet-specific expression. Lentiviral studies involved transducing FVIIInull murine hematopoietic cells with an HIV-1 based self-inactivating lentivirus encoding the previously mentioned FVIIIs driven by either a ubiquitin promoter or by the platelet-specific platelet factor 4 (PF4) proximal promoter; lethally irradiated FVIIInull mice were rescued by injection of virally transduced bone marrow. FVIII antigenic levels in both platelet lysates and releasates were measured, and free, plasma FVIII levels were detected by a FVIII ELISA. Maximum pIR8 levels achieved were comparable to those seen with hBDFVIII in spite of the fact that IR8 binds poorly to von Willebrand factor (vWF), supporting prior observations that pFVIII is stored in α-granules independent of the presence of vWF in α-granules. Surprisingly, maximal pcBDFVIII levels were 1/3rd of those of phBDFVIII. The basis for this observation is presently being determined. The ability of the pFVIII to correct hemophilia A in vivo was tested by a FeCl3 carotid artery injury model and a cuticular bleeding model. In addition, laser injury studies to cremaster arterioles and venules are on-going. FeCl3 carotid artery and cuticular studies demonstrated that both pIR8 and pcBDFVIII were more potent than phBDFVIII in improving outcome in FVIIInull mice. For pcBDFVIII, this occurred despite the low levels of pcBDFVIII. In the cremaster laser injury model, lentiviral-based gene therapy using the three FVIII variants, markedly improved clot formation compared to FVIIInull mice, but further studies are needed to define whether a specific FVIII variant is particularly efficacious. Prior studies of plasma IR8 correction using an adenoviral delivery system showed no improved outcome relative to hBDFVIII. We propose that this difference for IR8 between plasma and platelet expression is due to IR8 binding vWF poorly and that vWF binding is important for maintaining plasma FVIII levels. The higher specific activity of pcBDFVIII appears to more than compensate for its lower level in circulating platelets. We now intend to define the molecular basis for the greater efficacy of pIR8 and pcBDFVIII compared to phBDFVIII and to use these insights to further optimize the efficacy and safety of this delivery strategy in the care of patients with hemophilia A. Disclosures: Poncz: Diagnostica Stago: Patents & Royalties.

Characterizing Factor VIII Inhibitor Resistance of Platelet-Delivered Factor VIII in the Treatment of Hemophilia A.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 781-781 ◽  
Author(s):  
Jamie E. Gewirtz ◽  
Lubica Rauova ◽  
Mortimer Poncz
Keyword(s):  
Carotid Artery ◽  
Factor Viii ◽  
Hemophilia A ◽  
Thrombus Formation ◽  
Low Flow ◽  
Factor Viii Inhibitor ◽  
Carotid Artery Injury ◽  
Artery Injury ◽  
Injury Model ◽  
The Difference

Abstract Gene therapy strategies directed at expressing Factor (F) VIII in megakaryocytes may have several potential advantages in the treatment of severe hemophilia A. Among these is that platelet (p) FVIII may be protected from circulating inhibitors. We have previously described a murine transgenic line that expressed human B-domainless FVIII in a megakaryocyte-specific fashion and that this pFVIII was localized to within alpha granules. We also showed that these platelets contained FVIII equivalent to an infusion of 9% human FVIII into FVIIInull mice. We further showed that these pFVIII mice, on a FVIIInull murine background, formed stable clots in a FeCl3 carotid artery injury model. We then tested the ability of infused anti-human FVIII inhibitors in this setting. Using up to 100 μL of ESH8 monoclonal antibody (Ab) to the FVIII C2 light chain (1 μg/mL), anti-human polyclonal Ab (11 mg/mL) or a monoclonal Ab to the A2 domain (5 mg/mL), we were unable to alter thrombus formation in the carotid artery model. However, by using a 1:1:1 mixture of these inhibitors, we were able to show a dose-response curve. None of these mice developed thrombocytopenia suggesting that pFVIII is not exposed on the surface of circulating platelets. We then compared these studies to an acute infusion of the same inhibitor mixture in a FVIIInull mice receiving a 25% hFVIII correction. These studies showed that pFVIII/FVIIInull mice were ∼100-fold more resistant to inhibitors than plasma FVIII infusion into a FVIIInull mice in the carotid artery injury model. Since we had shown in the pFVIII mice that the FVIII is stored in alpha-granules, which can also store circulating Ab, we wondered whether the pFVIII/FVIIInull mice would be more sensitive to inhibitors when exposed in a chronic model where animals receive repeat doses of the inhibitor mixture. We therefore infused 3 doses of the inhibitor over 10 days, measured plasma and platelet inhibitor levels, and found that despite detectable stores of inhibitor within their platelets, these mice still demonstrated a comparable ability to form thrombosis as mice in the acute model with comparable plasma inhibitor levels. These studies suggest that pFVIII provides limited improved protection in mice with inhibitors comparable to <6 Bethesda Units (BU) in both acute and chronic models of inhibitor. Also, the presence of inhibitors within alpha-granules does not significantly inhibit the ability of these mice to form a clot. Our findings differ from a recent report of the efficacy of pFVIII in a tail vein survival model where pFVIII effectively protected mice from exsanguinations even in the presence of >100 BU/mL. We propose that the difference in outcome is due to the tail model being extremely sensitive to even low levels of pFVIII as exsanguinated, hypovolemic mice likely shunt blood away from their tail veins, and platelet activation and granular release are occurring in a low flow setting, while the FeCl3 model used in this report requires a plasma equivalency of >3–5% human FVIII to show even partial correction.

The Light Chain of Rat Factor VIII Confers Its Superior Cofactor Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2038-2038
Author(s):  
Qizhao Wang ◽  
Jenni Firrman ◽  
Katie A Pokiniewski ◽  
Wenjing Cao ◽  
Hongying Wei ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Specific Activity ◽  
Genetic Defect ◽  
High Dose ◽  
Coagulation Activity ◽  
Fviii Activity ◽  
Cofactor Activity

Abstract Hemophilia A is caused by genetic defect of human coagulation factor VIII (hFVIII) and patients have to take lifelong replacement therapy to prevent excessive bleedings upon hemostatic challenges. Due to the short half-life of hFVIII, replacement treatment has to be given frequently and inhibitors against infused hFVIII can be developed in about 20-30% of patients. These shortcomings have generated tremendous interest in developing HA gene therapies which is more efficient and long-lasting. However, early preclinical studies have shown FVIII activities were still limited after vector delivery. A Modified hFVIII with higher specific activity and pharmacodynamics properties is highly desirable to overcome the disadvantages of current protein replacement and gene therapy strategies. In the current study, we successfully constructed a B-domain deleted rat FVIII(rBDDF8) that contained a PACE/furin recognition site (RHQR) within a 14 amino acid linker between A2 and A3 domains. The rBDDF8 displayed significantly higher coagulation activity(~2.5-fold) than hBDDF8 after transfection into HEK 293 cells. In order to explore the mechanism for the observed superior cofactor activity, we constructed heavy chain(rHC) and light chain(rLC) of rFVIII. The rHC and rLC are able to reconstitute 5 times more FVIII activity than their human counter parts. However, when rHC is associated with human FVIII light chain (hLC), the reconstituted FVIII activity is lower that from hHC and hLC, suggesting that high coagulation activity of rFVIII is not mediated by its HC. On the contrary, when FVIII is constituted by hHC with rLC, we found that the activity is increased by 3~5-fold as against hHC and hLC. The hHC antigen level of FVIII reconstituted from hHC and rLC was 1.5-fold higher than that of hHC and hLC, suggesting that higher activity of FVIII with hHC and rLC is not through increased secretion. The specific activity deduced from activity/antigen ratio showed that FVIII with rLC is 3 times higher more than FVIII with hLC. To investigate the potential application of rFVIII in gene therapy, rBDDF8 was delivered in hemophilia A mouse model using AAV8 vectors. The high dose rBDDF8(4*1011 vg/mouse) resulted 2.5U FVIII activity at week 17, which is much higher(about 10-fold) than that of hBDDF8. When the rFVIII was delivered by dual chains strategy, i.e, administering vectors carrying only LC or HC simultaneously, it also showed 2-4 fold increased in FVIII activity. Interestingly, the combination of hHC and rLC also generated similar FVIII activity as rHC and rLC, further proving the rLC is the major contributor to the superior coagulation activity of rFVIII. Our results showed that the rFVIII has higher cofactor activity conferred by its LC. Our results suggest that rFVIII can be further exploited to make an ideal candidate for hemophilia gene therapy using AAV vectors. Disclosures No relevant conflicts of interest to declare.

A Novel Deletion in the Fviii B-Domain That Reduces Transgene Size While Preserving FVIII Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3182-3182
Author(s):  
Yi-Lin Liu ◽  
Hua Zhu ◽  
Alexander Schlachterman ◽  
Heesoon Chang ◽  
Rodney M. Camire ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Dose Group ◽  
Hemophilia A ◽  
Specific Activity ◽  
Post Injection ◽  
Vector Genome ◽  
Fviii Activity

Abstract Hemophilia A is an inherited X-linked bleeding disorder caused by a deficiency in Factor VIII (FVIII). Clinically significant improvement of hemophilia phenotype can be achieved with low circulating factors, thus makes it a good target disease for gene therapy. Adeno-associated virus (AAV) vectors have proven successful for the delivery of the factor IX gene in humans with hemophilia B. For the treatment of hemophilia A, a problem in the packaging of the rFVIII cDNA or various B-domainless derivatives (i.e. rFVIII-SQ) in AAV vectors is the large size of the insert, which combined with required elements, can exceed the packaging capacity of AAV (~5 kb). This difficulty limits the choice of both promoter and regulatory elements when designing an expression cassette for AAV vectors. Here we developed strategies to overcome these limitations by (1) development of a novel FVIII B-domain deleted molecule (2) construction of a short liver-specific promoter. We further tested these vectors in a series of in vitro and in vivo experiments. Factor VIII-SQ is a well-characterized derivative of FVIII and has been used by several groups in a gene therapy setting; the recombinant protein is used clinically to treat hemophilia A. We have constructed a shorter version of FVIII-SQ, by deleting the entire B-domain. In addition, we have engineered this FVIII to be intracellularly processed using a PACE-furin recognition site such that the protein is secreted from cells as two chains (FVIII-RKR; fully processed heavy and light chains). This FVIII-RKR along with FVIII-SQ was transiently expressed in COS-1 cells and conditioned media was collected at 24, 48 and 72 hrs post transfection. Using a combination of ELISA and functional assays we were able to demonstrate that FVIII-RKR was efficiently secreted from these cells. The data also revealed that FVIII-RKR has a 4–8-fold increase in specific activity compared to FVIII-SQ. We further tested whether FVIII-RKR could function in an in vivo setting. Plasmid DNA (50μg) containing FVIII-RKR or FVIII-SQ with liver-specific mouse transthyretin (mTTR) promoter were introduced into hemophilia A (HA) mice hydrodynamically via tail vein. Two out of four mice in the SQ group and three out of four mice in the RKR group had significant shortening of the clotting time at days 1 and 3 post injection, indicating that this shortened version of FVIII is functional in vivo. To address FVIII long-term expression we synthesized AAV vectors and delivered to immuno-deficient HA mice through hepatic portal vein. AAV vectors containing an expression cassette of mTTR promoter and FVIII-SQ have been administered. Expression of physiological FVIII levels was observed in high dose group (4.0E+12 vector genome per animal, n=4). FVIII activity averages 1.88 U/ml by Coamatic assay or 0.81 U/ml by aPTT assay at 12 weeks post injection. In low dose group (1.0E+12 vector genome per animal, n=5) therapeutic level of FVIII is achieved, 0.59 U/ml by Coamatic assay or 0.23 U/ml by aPTT assay at 12 weeks post injection. Finally, AAV vectors with FVIII-RKR have been produced and shown to have similar packaging efficiency to AAV-FVIII-SQ. Studies are currently underway with AAV-FVIII-RKR to evaluate the ability of this vector to drive long-term expression of functional protein. In summary, we developed a novel FVIII molecule that has high specific activity and is suitable for efficiently packaging in the AAV vectors.

scholarly journals Analysis of the spatial and temporal characteristics of platelet-delivered factor VIII–based clots

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1101-1108 ◽  
Author(s):  
Michael Neyman ◽  
Jamie Gewirtz ◽  
Mortimer Poncz
Keyword(s):  
Gene Therapy ◽  
Hemophilia A ◽  
Temporal And Spatial Distribution ◽  
Laser Injury ◽  
Therapy Strategy ◽  
Spatial Differences ◽  
Gene Therapy Strategy ◽  
Platelet Accumulation ◽  
Temporal And Spatial ◽  
Spatial And Temporal Characteristics

Abstract Normally factor (F) VIII is not expressed in megakaryocytes, but when human FVIII was transgenically expressed in murine megakaryocytes, it was stored in platelet α-granules and released at sites of injury. This platelet FVIII (pFVIII) is effective in correcting hemostasis, even in the presence of circulating inhibitors, so it offers a potential gene therapy strategy for hemophilia A. To understand clot development by pFVIII, we have examined clot response to laser injury in both cremaster arterioles and venules in FVIIInull mice either infused with FVIII or transgenic for pFVIII. In both sets of vessels, pFVIII is at least as effective as infused FVIII. However, there are temporal and spatial differences in fibrin and platelet accumulation within clots depending on how FVIII is delivered. These differences may be related to the temporal and spatial distribution of the α-granular–released FVIII within the developing clot, and may explain the increased frequency and size of embolic events seen with pFVIII. These observations may not only have implications for the use of pFVIII in gene therapy for hemophilia A, but may also have physiologic consequences, explaining why many procoagulant factors are delivered both in the plasma and in platelet α-granules.

Understanding Ectopically Expressed Factor VIII (F8) In Megakaryocytes: Implications for Optimum Platelet-Delivered F8 Activity for Gene Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2205-2205
Author(s):  
Teshell K Greene ◽  
Denise E. Sabatino ◽  
Nicholas P Iacobelli ◽  
Li Zhai ◽  
Steven W. Pipe ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Board Of Directors ◽  
Hemophilia A ◽  
Specific Activity ◽  
Mrna Levels ◽  
Single Chain ◽  
Advisory Committees ◽  
Platelet Counts ◽  
Laser Injury ◽  
Normal Hemostasis

Abstract Abstract 2205 F8 ectopically expressed during megakaryopoiesis is stored in platelet (p) a-granules and released at sites of injury. This F8 is effective in hemostasis even in the presence of circulating inhibitors so that pF8 represents a novel strategy for therapy for patients with hemophilia A with inhibitors. However, cremaster laser injury studies showed that pF8 release from a-granules has a distinct temporal and spatial availability that leads to clot instability. We proposed that F8s, such as canine (c) B-domainless (B) F8, with greater specific activity than human (h) BF8, would prevent pF8 clot instability. We confirmed our thesis; however at the same time, pcBF8 levels were only ∼30% of phBF8 whether studied in transgenic mice models or by lentiviral/bone marrow transplantation (lenti/BMT) into F8null mice. This was surprising because in baby hamster kidney cells, cBF8 was secreted into the media at three times greater than hBF8. This lower level in murine megakaryocytes (Megs) was not due to mRNA levels as shown by qRT-PCR analysis. Using cultured marrow from transgenic mice expressing hBF8 or cBF8, there was a marked decrease in relative number of Megs in both pF8s compared to wildtype (WT) Megs, with the pcBF8 cells showing a greater decrease (49 ± 5% for cBF8 Megs vs. 36 ± 8% for phBF8, p < 0.02). Cultured Megs from pcBF8 and phBF8 mice both showed increased numbers of small, low ploidy Megs, and this was again higher for pcBF8 Megs (58 ± 9% for cBF8 Megs vs. 32 ± 6% for phBF8, p < 0.02). TUNEL studies as an indication of apoptosis showed that Megs expressing either F8s showed significant increased apoptosis than WT Megs with pcBF8 showing 37 ± 22% vs. 19 ± 11% in phBF8. The above data show that pcBF8 is deleterious to Meg development and was supported by a retrospective analysis of lenti/BMT pF8 platelet counts where recipient mice platelets made up a higher % of recovered platelet counts (17% ± 3% for pcBF8 vs. 4 ± 1% for phBF8, p < 0.05). We then tested whether we can separate the greater specific activity affect of pcBF8 from its deleterious affect on megakaryopoiesis. Our group has previously shown that cBF8 may have increased stability because it is predominantly expressed as a single chain, likely involving an R1645H (RH) substitution at a conserved PACE/furin site in most F8 species. Lenti/BMT pF8 studies expressing phBF8RH showed that this pF8 was expressed at the same level in reconstituted mice as phBF8, but was more efficacious in several bleeding models, including near-normal hemostasis in the cremaster laser injury model in F8null recipient mice, thus becoming the first lenti/BMT pF8-expressing F8null mouse with near-normal hemostasis in a F8null setting. Preliminary Meg count and apoptosis studies show that phBF8RH is no more deleterious to Megs than phBF8. Thus, our studies point out that F8 is deleterious to Megs with some species being more deleterious than others. This apoptosis limits F8 levels in Megs, and this deleterious effect can influence post-BMT outcome. We also present a model of how one can take advantage of a F8 variant that had a high specific activity while avoiding its low expression levels in Megs. Thus our studies provide important new insights into the biology of pF8 that may be important in developing this platelet-delivery strategy for the treatment of hemophilia A. Disclosures: Pipe: Baxter BioScience: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; Inspiration Biopharmaceuticals: Honoraria, Research Funding; CSL Behring: Honoraria.

Platelet Factor VIII-Induced Megakaryocyte Apoptosis: Implications for Hemophilia A Gene Therapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2051-2051
Author(s):  
Teshell K Greene ◽  
Li Zhai ◽  
Beatrice Razzo ◽  
Karen Vo ◽  
Denise E. Sabatino ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cell Lines ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Clotting Factor ◽  
Mrna Levels ◽  
Single Chain ◽  
Platelet Factor ◽  
Platelet Counts

Abstract Abstract 2051 Hemophilia A is an X-chromosome-linked bleeding disorder due to a deficiency in clotting factor (F) VIII, affecting ∼1:5000 males. Inhibitor development to circulating FVIII is not an uncommon complication for patients receiving protein replacement therapy. We had proposed that the ectopic expression of FVIII in platelet alpha-granules will be targeted to sites of vascular injury, and be protected from circulating FVIII inhibitors. We and others have confirmed that this platelet (p)FVIII is in fact effective in a number of hemostatic models and is protected from circulating inhibitors. However, pFVIII has different temporal and spatial availability from plasma FVIII that may underlie the limited efficacy of pFVIII observed in some hemostatic models using FVIIInull mice. No group has been able to achieve levels greater than the equivalent of ∼200 mU/ml of whole blood pFVIII levels using standard human (h) B-domainless FVIII (hBFVIII). We therefore sought to improve pFVIII efficacy by expressing higher levels and/or by using pFVIII species with increased activity. We tested canine (c) BFVIII because of its reported higher efficacy in vitro and in mice, and because it is secreted at 1.5–2 fold HIGHER levels than hBFVIII in cell lines. We found that the level of pcBFVIII protein in Megs was actually ∼3-fold LOWER than phBFVIII. In spite of this lower level, pcBFVIII corrected the bleeding diathesis in FVIIInull mice more effectively than phBFVIII. Using Megs from transgenic FVIIInull mice that expressed pcBFVIII or phBFVIII, we found that mRNA levels of the FVIIIs were comparable. However, compared to littermate pFVIIInull mice, FVIIInull mice that expressed either pFVIII had lower ploidy Megs and these Megs showed higher levels of apoptosis. This apoptosis was enhanced by increasing the pFVIII level in the Megs by exposure to sodium butyrate. These effects were more marked in the pcBFVIII- than phBFVIII-expressing Megs. We then tested whether this phenomenon was limited to murine Megs, but found that lentiviral expression of pBFVIII increased Meg apoptosis whether the Megs were derived from mice, canine or human marrow, and that in each, levels of pcBFVIII were ∼3-fold lower than phBFVIII. In vivo studies support these in vitro apoptotic effects of pFVIII on Megs: In transgenic mice, steady-state platelet counts were normal in pFVIII mice, but TPO levels were 4-times higher in the phBFVIII and 8-times higher in the pcBFVIII compared to littermate controls (p<0.02 and p<0.0002 for phBFVIII/FVIIInull and pcBFVIII/FVIIInull vs. FVIIInull, respectively). In FVIII lentiviral transfected marrow/bone marrow transplant (lenti/BMT) studies, platelet counts did not differ in recovering recipient mice to negative controls, but both pBFVIII-expressing recipient lenti/BMT mice showed a peak in recipient platelets contributing to the total platelet mass at 4 weeks that decreased over the following month. This was especially true for the pcBFVIII-expressing lenti/BMT mice wherein >12% of the platelets were recipient-derived at 4 weeks compared to <2% in mice receiving a control lentivirus-transfected marrow (p<0.002). Thus, these studies suggest that there is a limit as to how much pFVIII can be stored in platelets, so efforts should be focused on increasing pFVIII activity. As an example of this approach, we had identified an R1645H Furin/PACE cleavage site difference between hBFVIII and cBFVIII that increases the level of single-chain to two-chain FVIII released from cell lines. The more single-chain FVIII, the slower the loss of FVIII activity, and the more overall activity was seen. We introduced this R1645H substitution into phBFVIII Megs and show antigen levels of this phBFVIIIR1645H comparable to phBFVIII in vitro and in lenti/BMT-reconstituted FVIIInull mice. Excitingly, these phBFVIIIR1645H lenti/BMT FVIIInull mice demonstrated normalization of hemostasis in several hemostatic models. Since pFVIII tends to be released deep inside a growing thrombus from degranulating platelets with limited washout, a more stable version of FVIII such as FVIIIR1645H may be particularly efficacious as pFVIII, and we intend to pursue this hFVIII variant in larger animal studies as it may be especially useful for clinical gene therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Complete short-term correction of canine hemophilia A by in vivo gene therapy

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3846-3853 ◽  
Author(s):  
S Connelly ◽  
J Mount ◽  
A Mauser ◽  
JM Gardner ◽  
M Kaleko ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Hemophilia A ◽  
Specific Inhibitor ◽  
Canine Model ◽  
Clotting Factor ◽  
Severe Bleeding ◽  
Short Term ◽  
Dog Plasma

Hemophilia A is a severe bleeding disorder caused by a deficiency in clotting factor VIII (FVIII). A canine model that closely mimics the human disease was used to determine if an adenoviral vector expressing a human FVIII cDNA could be used to correct the hemophilia A phenotype. Within 48 hours after peripheral vein administration of the vector to FVIII-deficient dogs, the hemophilic phenotype was corrected, based on determination of the activated clotting time, the activated partial thromboplastin time, and the cuticle bleeding time. Direct measurement of human FVIII in the dog plasma showed FVIII expression at amounts well above the human therapeutic level. FVIII expression in treated dogs was short-term, lasting 1 to 2 weeks, due to the development of a human FVIII-specific inhibitor antibody response. These data provide the first demonstration of in vivo gene therapy of hemophilia A.

Coagulation Disorders in Thrombocythaemia, a Study of Seven Cases

1962 ◽  
Vol 07 (01) ◽  
pp. 114-128 ◽  
Author(s):  
Stefan Niewiarowski ◽  
Halina Zywicka ◽  
Zbigniew Latałło
Keyword(s):  
Liver Parenchyma ◽  
Platelet Factor 4 ◽  
Coagulation System ◽  
Severe Bleeding ◽  
Clotting Factors ◽  
Platelet Factor ◽  
Antiheparin Activity ◽  
Thromboplastic Activity ◽  
Bleeding Episodes ◽  
Routine Coagulation

SummaryThe blood coagulation system has been studied in 7 patients with thrombocythaemia. 4 of these patients had thrombocythaemia after splenectomy, 2 of them had thrombocythaemia associated with myeloid leukemia, and 1 thrombocythaemia associated with polycythaemia. Severe bleeding episodes were noted in 5 cases, 2 patients had only mild bleeding symptoms.Each patient was examined several times. The period of observations varied from 2 months to 3 years. Platelet count varied from 350 000 to 3 800 000 per mm3.Bleeding time and tourniquet test were normal in all cases. Routine coagulation and fibrinolysis studies did not reveale characteristic abnormalities in plasma clotting factors. A decrease of prothrombin complex components was observed in 4 cases. This disturbance was due to the coexisting injury of liver parenchyma or myeloid changes but not to an increase of platelets or to the abnormalities in the platelet system.An increase of antiheparin activity was found in the plasma of 4 patients. This activity is probably due to the escape of platelet factor 4 from destroyed or qualitatively changed platelets into plasma.Platelet clotting factors were investigated in isolated platelet suspensions, A significant decrease of platelet factor 1 was observed in all patients and a decrease of platelet factor 4 in 5 patients. In 2 cases platelet factor 4 increased. Platelet thromboplastic activity showed a great variety of disturbances in conformity with other workers observations.Recent views on the pathogenesis of bleedings in thrombocythaemia are discussed. On the basis of their own investigations the authors suggest that the significant disturbances of platelet function may contribute to the development of bleeding, and that the increase of antiheparin activity in plasma may produce hypercoagulability and favorize the formation of thrombi.

Sign in / Sign up

Export Citation Format

Share Document