Use of blood outgrowth endothelial cells for gene therapy for hemophilia A

Blood ◽  
2002 ◽  
Vol 99 (2) ◽  
pp. 457-462 ◽  
Author(s):  
Yi Lin ◽  
Liming Chang ◽  
Anna Solovey ◽  
John F. Healey ◽  
Pete Lollar ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Endothelial Cells ◽  
Hemophilia A ◽  
Scale Up ◽  
Coagulation Factor ◽  
Green Fluorescence Protein ◽  
Cell Dose ◽  
Effective Therapeutic Strategy ◽  
Fluorescence Protein

Abstract A culture of human blood outgrowth endothelial cells (BOECs) was established from a sample of peripheral blood and was transfected using a nonviral plasmid carrying complementary DNA for modified human coagulation factor VIII (B domain deleted and replaced with green fluorescence protein). BOECs were then chemically selected, expanded, cryopreserved, and re-expanded in culture. Stably transfected BOECs were administered intravenously daily for 3 days to NOD/SCID mice at 4 cell dose levels (from 5 × 104 to 40 × 104 cells per injection). In 156 days of observation, mice showed levels of human FVIII that increased with cell dose and time. Mice in all cell dose groups achieved therapeutic levels (more than 10 ng/mL) of human FVIII, and mice in the 3 highest dose groups acquired levels that were normal (100-200 ng/mL) or even above the normal range (highest observed value, 1174 ng/mL). These levels indicate that the BOECs expanded in vivo after administration. When the mice were killed, it was found that BOEC accumulated only in bone marrow and spleen and that these cells retained endothelial phenotype and transgene expression. Cell doses used here would make scale-up to humans feasible. Thus, the use of engineered autologous BOECs, which here resulted in sustained and therapeutic levels of FVIII, may comprise an effective therapeutic strategy for use in gene therapy for hemophilia A.

Bioengineering of coagulation factor VIII for improved secretion

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3412-3419 ◽  
Author(s):  
Hongzhi Z. Miao ◽  
Nongnuch Sirachainan ◽  
Lisa Palmer ◽  
Phillip Kucab ◽  
Michael A. Cunningham ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Mrna Levels ◽  
Golgi Transport ◽  
Recombinant Fviii ◽  
Therapy Strategies

Abstract Factor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation. Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A. Expression of FVIII (domain structure A1-A2-B-A3-C1-C2) in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)-FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport we engineered several asparagine-linked oligosaccharides within a short B-domain spacer within BDD-FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals. (Blood. 2004;103: 3412-3419)

Use of Engineered Autologous BOEC for Gene Therapy of Canine Hemophilia A.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1281-1281
Author(s):  
Robert P. Hebbel ◽  
Liming Milbauer ◽  
Mark Roney ◽  
David Lillicrap ◽  
Jan Voorberg ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Hemophilia A ◽  
Ex Vivo ◽  
Insertion Site ◽  
Scale Up ◽  
Novel Approach ◽  
Fviii Activity ◽  
Initial Scale ◽  
Original Donor

Abstract Hemophilia A is an attractive candidate disease for corrective gene therapy because relatively small amounts of the missing protein, FVIIII, have a significant biological effect. We previously described a novel approach: intravenous administration of ex vivo expanded BOEC (blood outgrowth endothelial cells) engineered to express FVIII. Robust therapeutic results were obtained using NOD/SCID mice and human BOEC stably transfected to express human FVIII (Blood99:457, 2002). We are now conducting scale-up studies in the canine hemophilia A model. Based on the previous mouse data, we estimated that 400 million cells would be required to achieve a therapeutic effect in the dog. First, two normal dogs were infused with autologous unmanipulated BOEC, which revealed removal from the circulation via single-pass kinetics. Then, BOEC cultures were obtained from three hemophilia A dogs, and an onco-retroviral vector (MIRG/cFVIII) was used to obtain transduced BOEC producing B-domainless canine FVIII. Then, for each treatment, autologous engineered BOEC were given IV to the original donor dogs, with dose divided over 3 daily infusions, and whole blood clotting time (WBCT, in minutes) was followed. Dog Date # cells infused pre Rx WBCT effect on WBCT D28 12/04 274x106 >60 nadir 36 at day 14, Still 39 at day 185 D28 6/05 386x106 39 nadir 24.5 at day 23, now at 30 at day 44, being followed E64 7/04 220x106 >60 nadir 34.5 at day 3, >60 min by day 38 E64 12/04 274x106 >60 still 37.5 at day 189 E64 6/05 185x106 37.5 nadir 34.5 at day 2, at 36 on day 32, being followed H17 6/05 272x106 >60 nadir 29 at day 44, being followed WBCT (nl = 8-12 min) needs to be <20 min to reflect >0.1% FVIII activity; but the observed WBCT are clearly improved compared to baseline. One dog (E64) was treated for a mouth bleeds on d37 in cycle 2, and d36 in cycle 3. One animal (E64) developed hypotension and tachypnea during one of the infusions (probably because cells were not kept sufficiently in suspension) but recovered. Four of the infusions were associated with mild transient thrombocytopenia. Based on these initial scale-up experiments, it appears that use of engineered BOEC for gene therapy is an approach worthy of continued study, as cell doses can be increased and other improvements in the method are readily envisioned. It has a number of advantages, including use of autologous carrier cells with which any expression vector can be paired, ex vivo exposure to vector rather than in vivo, use of BOEC expansion in culture to simultaneously achieve vector expansion, and the ability to chose a clone of engineered cells having a single, known (and studied) insertion site.

scholarly journals Sustained Phenotypic Correction of Murine Hemophilia A by In Vivo Gene Therapy

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3273-3281 ◽  
Author(s):  
Sheila Connelly ◽  
Julie L. Andrews ◽  
Angela M. Gallo ◽  
Dawn B. Kayda ◽  
Jiahua Qian ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Small Animal ◽  
Canine Model ◽  
Pharmaceutical Products ◽  
Biologically Active ◽  
Mouse Strains ◽  
Therapy Studies

Abstract Hemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been widely discussed as a candidate for gene therapy. While the natural canine model of hemophilia A has been valuable for the development of FVIII pharmaceutical products, the use of hemophiliac dogs for gene therapy studies has several limitations such as expense and the long canine generation time. The recent creation of two strains of FVIII-deficient mice provides the first small animal model of hemophilia A. Treatment of hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII at levels, which declined, but remained above the human therapeutic range for over 9 months. The duration of expression and FVIII plasma levels achieved were similar in both hemophiliac mouse strains. Treated mice readily survived tail clipping with minimal blood loss, thus showing phenotypic correction of murine hemophilia A by in vivo gene therapy.

DDAVP-Induced Increase of Factor VIII Activity in Blood Is Likely Due To Release of Factor VIII That Is Synthesized by Endothelial Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 692-692 ◽  
Author(s):  
Lingfei Xu ◽  
Timothy C. Nichols ◽  
Stephanie McCorquodale ◽  
Aaron Dillow ◽  
Elizabeth Merricks ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Endothelial Cells ◽  
Factor Viii ◽  
Hemophilia A ◽  
Fold Increase ◽  
Von Willebrand Disease ◽  
Major Site ◽  
Von Willebrand

Abstract Desmopressin (1-deamino-8-D-arginine vasopressin, DDAVP) is commonly used as a nonreplacement therapy for mild von Willebrand disease (VWD) and hemophilia A. In humans, IV injection of 0.3 μg/kg of DDAVP induces a rapid 2 to 5-fold increase in plasma levels of both von Willebrand factor (VWF) and Factor VIII (FVIII) within 30–60 minutes, which is due to release from Wiebel-Palade bodies (WPBs) in endothelial cells. The stored FVIII may be synthesized by endothelial cells, which express FVIII in vitro. However, hepatoma cells can also express FVIII in vitro, and liver transplantation can correct hemophilia A. Thus, the liver may be the major site of production of FVIII in vivo, thus, an alternative explanation is that endothelial cells take up FVIII from blood and store it in WPBs with VWF, which can be released after DDAVP. DDAVP is effective in humans and dogs, but not in mice. In this study, we tested the effect of DDAVP on hemophilia A dogs after neonatal hepatic gene therapy with a retroviral vector (RV) expressing canine FVIII (cFVIII). With this gene therapy approach, canine hepatocytes express high levels of a reporter gene from an RV, but no expression is observed in endothelial cells. Thus, the major site of FVIII synthesis is the hepatocyte in this model. Our hypothesis is that if DDAVP increases FVIII levels in this dog model, it would indicate that the FVIII increase is due to uptake from blood by endothelial cells. Alternatively, if no increase in FVIII occurs after DDAVP stimulation, it would suggest that the increase in normal dogs is due to synthesis of FVIII by endothelial cells. An RV that contains the liver-specific human α1-antitrypsin promoter and the canine B-domain deleted FVIII cDNA was generated. RV was given IV to two hemophilia A dogs at 8x109 transducing units (TU)/kg at 3 days after birth. The whole blood clotting time (WBCT) and APTT time in both dogs have been normalized, and the plasma cFVIII COATEST activity has been maintained at 100–200% of normal for 11 months to date. DDAVP was injected IV at 0.5 μg/kg into RV-treated hemophilia A dogs at 7 months of age. Two separate doses of DDAVP were given with an interval of one week. The same dose of DDAVP was given to normal dogs as controls (N=4). In normal dogs, both VWF and FVIII levels increased 40% and 50% between 15 to 60 minutes after DDAVP, respectively. However, FVIII levels were not changed in RV-treated dogs, although VWF levels increased 150% or 60%. Thus, our data suggest that the normal FVIII increase after DDAVP administration is due to release of FVIII that is synthesized by endothelial cells. These data also demonstrate that DDAVP will not be effective at increasing FVIII activity in patients that receive liver-directed gene therapy and only achieve partial correction. Such patients would need to be treated with factor replacement if bleeding episodes occur.

scholarly journals Sustained Phenotypic Correction of Murine Hemophilia A by In Vivo Gene Therapy

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3273-3281 ◽  
Author(s):  
Sheila Connelly ◽  
Julie L. Andrews ◽  
Angela M. Gallo ◽  
Dawn B. Kayda ◽  
Jiahua Qian ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Small Animal ◽  
Canine Model ◽  
Pharmaceutical Products ◽  
Biologically Active ◽  
Mouse Strains ◽  
Therapy Studies

Hemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been widely discussed as a candidate for gene therapy. While the natural canine model of hemophilia A has been valuable for the development of FVIII pharmaceutical products, the use of hemophiliac dogs for gene therapy studies has several limitations such as expense and the long canine generation time. The recent creation of two strains of FVIII-deficient mice provides the first small animal model of hemophilia A. Treatment of hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII at levels, which declined, but remained above the human therapeutic range for over 9 months. The duration of expression and FVIII plasma levels achieved were similar in both hemophiliac mouse strains. Treated mice readily survived tail clipping with minimal blood loss, thus showing phenotypic correction of murine hemophilia A by in vivo gene therapy.

scholarly journals Characterization and visualization of murine coagulation factor VIII-producing cells in vivo

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Morisada Hayakawa ◽  
Asuka Sakata ◽  
Hiroko Hayakawa ◽  
Hikari Matsumoto ◽  
Takafumi Hiramoto ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Fluorescent Protein ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Genetically Engineered ◽  
Coagulation Factor Viii ◽  
Liver Sinusoidal Endothelial Cells ◽  
Genetically Engineered Mice

AbstractCoagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of the F8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31high, CD146high, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)+. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin− and C-type lectin-like receptor-2 (CLEC-2)+. In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31high, CD146high, Lyve1+, CLEC-2+, and podoplanin− in liver sinusoidal endothelial cells.

Abstract 229: The Novel Angiogenic Cytokine Secretoneurin promotes Angiogenesis, Arteriogenesis and Vasculogenesis in the Hind-Limb Ischemia Model

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Wilfried Schgoer ◽  
Margot Egger ◽  
Arno Peer ◽  
Johannes Jeschke ◽  
Ivan Tancevski ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Green Fluorescence Protein ◽  
Limb Ischemia ◽  
Promoter Sequence ◽  
The Novel ◽  
Gene Therapy Vector ◽  
Adductor Muscles

Introduction - Secretoneurin (SN) represents a sensory, inflammatory neuropeptide which was recently demonstrated to act as an angiogenic and vasculogenic cytokine in vitro and in vivo. The present study was conducted to test the hypothesis that SN may be implicated in reparative angiogenesis. Furthermore, we challenged the healing potential of SN applied as a newly generated SN gene therapy vector in the setting of limb ischemia. Methods and Results - We cloned the human SN coding sequence into the pAAV plasmid containing a cytomegalovirus enhancer/promoter sequence. Bioactivity of recombinant SN was shown by proliferative and chemotactic activity on endothelial cells in vitro. Unilateral limb ischemia was induced in C57/bl mice by femoral artery resection. By Real Time PCR, Western Blotting, SN-specific RIA and Immunhistochemistry, we documented that SN is up-regulated in ischemic muscles. Next, we tested whether SN gene therapy may exert curative effects in this ischemia model. Injection of the SN plasmid into ischemic adductor muscles increased capillary (0.67 vs. 0.35, n = 24, p = 0.02) and arteriole (0.16 vs. 0.8, n = 24, p = 0.04) density, reduced endothelial cell apoptosis, and accelerated perfusion recovery as shown by Laser Doppler Perfusion Index (LDPI ratio ischemic/control leg after 28 days of ischemia 1.1 vs. 0.7, n = 24, p < 0.01) in comparson to pAAV-GFP (green-fluorescence protein) treated mice. Furthermore, SN gene therapy significantly reduced toe necrosis of ischemic limbs compared to control animals (26% vs. 50%, n = 24, p < 0.05). In bone marrow transplantation models, increased vascularity of ischemic hind-limbs after SN gene therapy was shown to be mediated, at least in part, by enhanced recruitment of bone marrow-derived endothelial progenitor cells. Conclusions -These results suggest that the novel angiogenic cytokine Secretoneurin is up-regulated by ischemia in skeletal muscle cells. Furthermore, results from gene therapy in this ischemia model suggest that Secretoneurin represent a promising new substance for therapeutic angiogenesis.

Animal Testing of Retroviral-Mediated Gene Therapy for Factor VIII Deficiency

1999 ◽  
Vol 82 (08) ◽  
pp. 555-561 ◽  
Author(s):  
Douglas Jolly ◽  
Judith Greengard
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Joint Damage ◽  
Coagulation Factor ◽  
The United States ◽  
Severe Hemophilia ◽  
Factor Viii Gene ◽  
Bleeding Episodes

IntroductionHemophilia A results from the plasma deficiency of factor VIII, a gene carried on the X chromosome. Bleeding results from a lack of coagulation factor VIII, a large and complex protein that circulates in complex with its carrier, von Willebrand factor (vWF).1 Severe hemophilia A (<1% of normal circulating levels) is associated with a high degree of mortality, due to spontaneous and trauma-induced, life-threatening and crippling bleeding episodes.2 Current treatment in the United States consists of infusion of plasma-derived or recombinant factor VIII in response to bleeding episodes.3 Such treatment fails to prevent cumulative joint damage, a major cause of hemophilia-associated morbidity.4 Availability of prophylactic treatment, which would reduce the number and severity of bleeding episodes and, consequently, would limit such joint damage, is limited by cost and the problems associated with repeated venous access. Other problems are associated with frequent replacement treatment, including the dangers of transmission of blood-borne infections derived from plasma used as a source of factor VIII or tissue culture or formulation components. These dangers are reduced, but not eliminated, by current manufacturing techniques. Furthermore, approximately 1 in 5 patients with severe hemophilia treated with recombinant or plasma-derived factor VIII develop inhibitory humoral immune responses. In some cases, new inhibitors have developed, apparently in response to unnatural modifications introduced during manufacture or purification.5 Gene therapy could circumvent most of these difficulties. In theory, a single injection of a vector encoding the factor VIII gene could provide constant plasma levels of factor in the long term. However, long-term expression after gene transfer of a systemically expressed protein in higher mammals has seldom been described. In some cases, a vector that appeared promising in a rodent model has not worked well in larger animals, for example, due to a massive immune response not seen in the rodent.6 An excellent review of early efforts at factor VIII gene therapy appeared in an earlier volume of this series.7 A summary of results from various in vivo experiments is shown in Table 1. This chapter will focus on results pertaining to studies using vectors based on murine retroviruses, including our own work.

VEGF-induced mobilization of caveolae and increase in permeability of endothelial cells

2002 ◽  
Vol 282 (5) ◽  
pp. C1053-C1063 ◽  
Author(s):  
Jun Chen ◽  
Filip Braet ◽  
Sergey Brodsky ◽  
Talia Weinstein ◽  
Victor Romanov ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Electrical Resistance ◽  
Polyclonal Antibodies ◽  
Endothelial Permeability ◽  
Green Fluorescence Protein ◽  
Vascular Endothelial ◽  
Transmission Electron ◽  
Glomerular Epithelial Cells ◽  
Fluorescence Protein ◽  
Endothelial Growth Factor

Glomerular epithelial cells (GEC) are a known site of vascular endothelial growth factor (VEGF) production. We established immortalized rat GEC, which retained the ability to produce VEGF. The isoforms expressed by GEC were defined as VEGF-205, -188, -120, and -164. The electrical resistance of endothelial cells cultured on GEC-conditioned matrix, an indicator of the permeability of monolayers to solutes, was significantly increased by the treatment with the neutralizing polyclonal antibodies to VEGF and decreased by VEGF-165. Transfection of endothelial cells with green fluorescence protein-caveolin construct and intravital confocal microscopy showed that VEGF results in a rapid appearance of transcellular elongated structures decorated with caveolin. Transmission electron microscopy of endothelial cells showed that caveolae undergo rapid internalization and fusion 30 min after application of VEGF-165. Later (36 h), endothelial cells pretreated with VEGF developed fenestrae and showed a decrease in electrical resistance. Immunoelectron microscopy of glomeruli confirmed VEGF localization to podocytes and in the basement membrane. In summary, immortalized GEC retain the ability to synthesize VEGF. Matrix-deposited and soluble VEGF leads to the enhancement of caveolae expression, their fission and fusion, formation of elongated caveolin-decorated structures, and eventual formation of fenestrae, both responsible for the increase in endothelial permeability.

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