|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
GENE THERAPY
From the Department of Medicine, University of
Minnesota Medical School, Minneapolis, and the Winship Cancer Center,
Emory University, Atlanta, GA.
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.
(Blood. 2002;99:457-462) Hemophilia A is an inherited X-linked disorder
caused by a deficiency of coagulation factor VIII
(FVIII).1 This defect results in a severe bleeding
phenotype leading to death, significant illness, and enormous use of
health system resources. The available standard therapy, intravenous
infusion of FVIII, is expensive. Aside from certain specific situations
such as preoperative factor coverage, FVIII therapy is usually used to
treat acute bleeding rather than to provide prophylaxis. Furthermore,
FVIII has a relatively short half-life of approximately 12 hours,
mandating repeated and prolonged administration. In addition,
historical disasters have occurred, such as the emergence of human
immunodeficiency virus in this patient population because of viral
contamination of commercial factor products.
Gene therapy for hemophilia A would be of tremendous
benefit,2 and many investigators have actively pursued
this goal. An attractive aspect of FVIII therapy is that only small
amounts of FVIII are required to shift hemophilia phenotype from severe to mild. For example, patients with FVIII levels greater than 5% of
normal (ie, greater than 5-10 ng/mL) have a mild phenotype; patients
whose levels are lower than 1% have a severe phenotype. Unfortunately,
numerous impediments hamper achieving a sustained, therapeutically
useful elevation in circulating FVIII through a gene therapy strategy.
The FVIII gene is large and suppresses its own expression. This problem
has been ameliorated by deleting the large, centrally located FVIII B
domain, which is not necessary for function.3-6 Various
vector systems have been designed for expression of FVIII in a gene
therapy setting,7-12 and encouraging results have been
observed in mice subjected to intravenous administration of recombinant
adenoviral,13,14 rAAV,11
lentiviral,12 and retroviral vectors.15 Each
of these approaches has entailed the potential disadvantage of systemic
exposure to viral vectors. Notably, viral vectors carry potential
risks, including untoward inflammatory reactions, germline
transmission, and carcinogenesis.
The current work is based on the premise that, regardless of which
expression vector ultimately proves to be optimal for the production of
FVIII, a novel delivery vehicle for that vector might be useful. We
propose that blood outgrowth endothelial cells (BOECs) can comprise
such a vehicle. Monolayer cultures of BOECs can be reproducibly
established from human peripheral blood buffy coat mononuclear
cells.16 Outgrowth BOECs have typical endothelial cobblestone morphology, take up acetylated low-density lipoprotein (LDL), contain Weibel-Palade bodies, and express multiple endothelial cell markers: CD34, von Willebrand factor (VWF), P1H12,
vascular-endothelial (VE)-cadherin, flk-1, CD36, thrombomodulin, and
platelet/endothelial cell adhesion molecule
(PECAM).16 BOECs can be expanded from 20 cells at
the start of culture to 1019 cells by 65 days.16 Thus, a virtually unlimited number of BOECs can be
available, offering a potential advantage for gene transfer protocols
that require chemical selection, because it may overcome the
endothelial cell's relative intolerance of growth at low density
(that typically results from chemical selection).
BOEC culture
Construction of chimeric eGFP-FVIII expression vector
We digested plasmid HSQ-eGFP/ReNeo with XhoI and
NotI to yield a 5.4-kb fragment containing the cDNA for the
chimeric eGFP-FVIII protein. We then ligated this fragment into the
Neo-containing plasmid pcDNA3.1( Gene transfer to BOECs We exposed BOECs (at 75% confluence in a 10-cm dish prepared as above) to 25 µg Fugene6 and 10 µg plasmid pcF8G in a culture medium composed of MCDB-131 base medium-supplemented 20% heat-inactivated male human serum, 0.246 mg/mL dibutyl cyclic adenosine monophosphate, 0.04 mg/mL heparin, 1% PenStrepFungizone (Gibco Invitrogen, Carlsbad, CA), 1.5 mg/mL L-glutamine, 1 µg/mL hydrocortisone, and 10 µg/mL endothelial cell growth factors (ECGF) (Clonetics).22 Cells were exposed in this manner for 72 hours at 37°C in a humidified environment with 5% CO2. We added fresh medium at 24 hours without removing the old medium. Thereafter, we cultured the transfected BOECs in EBM-2/EGM-2 medium (Clonetics) containing 50 µg/mL G418 (Gibco-BRL) for 10 days. They were cultured in this medium without G418 for 45 days, after which G418 was again added (at 50 µg/mL) for 14 days.Supernates from 5 × 105 transfected BOECs were tested after 72 hours and after final G418 selection for content of human FVIII. We obtained several positive clones, as evidenced by enzyme-linked immunosorbent assay (ELISA) for human FVIII on culture supernatants (see below) and flow cytometry detection of the chimeric eGFP-FVIII protein (not shown). We chose one BOEC clone, pcF8G-6, producing 22 ng/mL FVIII into 72-hour-conditioned culture medium as the stably transfected BOEC (tBOEC) for use in this study. For one type of control BOEC, we used vector pLEIN (Clontech), which is the retroviral vector pLXIN with an eGFP sequence inserted in the multiple cloning site. PA317 cells were used in the packaging. Particles of replication-incompetent virus were used to transduce human BOECs; 1 × 105 BOECs, prepared and seeded as above, were exposed at 75% confluence to virus-containing medium in combination with 4 µg/mL polybrene. Fresh medium was added at 24 hours. After this, cells were selected as described above. Characterization of tBOECs To demonstrate the presence of the transgene, we isolated genomic DNA from wild-type BOEC (wtBOEC) and tBOEC (clone pcF8G-6) using DNAeasy tissue kit (Qiagen, Valencia, CA). Five hundred nanograms genomic DNA from wtBOEC or tBOEC or 50 pg plasmid pcF8G (positive control) was subjected to PCR using specific primers. The sense primer (5'-GTC TCC ACC CCA TTG-3') was located in the cytomegalovirus (CMV) promoter region of the pcF8G expression vector; the antisense primer (5'-TGG CAC TCT AGG AGG-3') was located in the A domain of the human FVIII cDNA in the expression vector. The expected size of the PCR product was 415 base pair (bp). Thermocycling parameters were 31 cycles of incubation at 94°C (45 seconds), 55°C (30 seconds), and 72°C (45 seconds), followed by a final 10-minute extension at 72°C. PCR products were visualized by electrophoresis on ethidium bromide-stained 0.8% agarose gel. The gel was subjected to Southern blot analysis using a 415-bp digoxigenin-labeled PCR probe spanning the junction between sequences for CMV promoter and human FVIII.To estimate the number of transgene copies per cell, we took 0.5 pg to 100 pg pcF8G plasmid DNA and 3 µg tBOEC genomic DNA and subjected them to dot blot analysis using the DIG system29; 415 bp digoxigenin-labeled PCR product was used as the probe. The transgene copy number in tBOEC was estimated by comparison of the dot intensities on the dot blot, compared to standards, where 1 pg pcF8G plasmid DNA equals 1 copy of the transgene per cell in 600 ng tBOEC genomic DNA. Phenotype of tBOEC was determined as previously described16 for wtBOEC based on morphology, uptake of acetylated LDL, expression of the transgene human FVIII, expression of endothelial markers (VE-cadherin, thrombomodulin, VWF, flk-1, vascular cell adhesion molecule 1 [VCAM-1], PECAM-1, CD34, P1H12, and CD36), and expression of makers of monocytes (CD14), hematopoietic cells (CD45), and putative endothelial progenitors (AC133). Each fluorescence activated cell sorting (FACS) analysis used unlabeled cells and cells with isotype control antibody as controls. Because these 2 controls were always identical in these studies, presentation of the results illustrates the unlabeled cell control only. ELISA for human FVIII and eGFP We measured the level of human FVIII in BOEC supernates or in murine platelet-poor plasma using an ELISA kit that detects human, but not murine, FVIII (American Diagnostica, Greenwich, CT). We verified the lack of cross-reactivity by examining dilutions of mouse plasma (with or without added human FVIII). The presence of any mouse plasma added a nonspecific background signal, but this did not increase as the amount of mouse plasma increased from 10% to 100%. Each animal's own prestudy background signal was subtracted from each subsequent measurement. In individual mice monitored sequentially day to day, the level of background signal varied by ± 5 ng/mL. All measurements of FVIII were made in duplicate. For this ELISA, the average coefficient of variation of duplicate measurements was 49.5% for FVIII levels lower than 30 ng/mL, 17.6% for those between 30 and 100 ng/mL, and 3.3% for FVIII levels greater than 100 ng/mL.To detect the eGFP component of the eGFP-FVIII chimeric gene product in murine plasma, we similarly used an ELISA with rabbit anti-GFP antibody (Clontech). The average coefficient of variation of replicate measurements in this ELISA was 8.9%. Cell administration and animal monitoring The NOD/SCID mice used in these studies weighed 35 g. Although they exhibit a greatly truncated lifespan, they have a severe immunodeficiency so there could be no immunologic reactivity against the human BOEC and human FVIII used for the experiment.23,24 We suspended BOEC in 500 µL mouse saline (330 mOsm/L) and injected them by tail vein once a day for 3 consecutive days. Mice received either 5, 10, 20, or 40 × 104 BOECs per injection (ie, total dose from 4.3 × 106 to 3.4 × 107 cells per kg). Control mice received no cells (injection of saline only), or they received unmanipulated wtBOEC or BOEC transduced to express eGFP only (gfpBOEC) at the dose level of 20 × 104 cells per injection. We obtained blood from the retro-orbital plexus at multiple time points after BOEC administration for ELISA measurement of human FVIII, as above.We performed histologic examination of available (deceased or killed)
animals to localize tBOECs. We sampled multiple tissues (heart, lung,
liver, spleen, kidney, bone marrow, abdominal lymph nodes, and gut
wall) and probed fixed sections with a goat antibody to human
To identify the phenotype of tBOEC detected in murine marrows, we took
frozen sections of marrow at day 156 and fixed them with 4%
paraformaldehyde. BOECs were characterized by double staining for
To determine the percentage of nucleated cells in murine marrow that
were human BOECs, we evaluated marrow sections stained for human
We established a culture of BOECs from a healthy human volunteer and expanded them approximately 106-fold to obtain approximately 107 cells. After confirming that these BOECs had the endothelial markers we previously observed for BOECs,16 we transfected them using a nonviral plasmid vector (pcF8G) containing a cDNA encoding a modified form of human FVIII. In this cDNA, the unnecessary B domain was replaced by sequence encoding eGFP. We selected a clone of stably transfected BOECs (clone pcF8G-6) and expanded them another 106-fold before use in the current experiments. Thus, this clone of tBOEC was analyzed and used after the cells had undergone an overall 3.4 × 1012-fold expansion since establishment of the initial BOEC culture. In these tBOECs, we demonstrated the presence of the transgene by PCR
and Southern blot analysis by probing for the junction between the CMV
promoter and the A domain of FVIII. tBOECs were positive, and wtBOECs
were negative (data not shown). We also obtained an estimate of
transgene copy number of 1.5 per cell from dot blot intensities (data
not shown). This clone of cells produced 22 ng/mL FVIII into culture
medium (supernate from 5 × 105 cells) in 72 hours (Table
1). Phenotypic characterization of the
tBOECs (Figure 1) showed that they were
somewhat more elongated than the starting wtBOECs, they took up
acetylated LDL, and they retained their endothelial markers
(VE-cadherin, thrombomodulin, von Willebrand factor, flk-1, PECAM,
CD34, P1H12, and CD36). They were negative for activation antigen
VCAM-1 but expressed it when deliberately stimulated in vitro (data not
shown). In addition, they were negative for monocyte marker CD14
(Figure 1) and negative for hematopoietic marker CD45 and endothelial
progenitor marker AC133 (data not shown). Thus, the tBOECs retained the
general phenotype of wtBOECs (ie, that of differentiated, quiescent,
microvascular endothelial cells).
We gave BOECs by tail vein to NOD-SCID mice, each of which received 5, 10, 20, or 40 × 104 eGFP-FVIII tBOECs each day for 3 consecutive days. Our 3 types of control animals received 3 injections of saline vehicle, 20 × 104 unmanipulated wtBOEC, or 20 × 104 BOECs transduced with retroviral vector pLEIN to express eGFP only (gfpBOEC). We then obtained retro-orbital plexus blood from each experimental animal at 9, 28, 47, 97, and 156 days for an ELISA that detected human, but not murine, FVIII. Each measurement was reported after subtraction of the baseline, background signal obtained from blood of the same mouse 3 days before BOEC infusion. After the administration of tBOEC, levels of human FVIII increased with
cell dose and elapsed time (Table 2,
Figure 2). All mice that received
eGFP-FVIII tBOECs, even those given the lowest cell dose, achieved a
therapeutic level of FVIII (greater than 10 ng/mL). Mice in the 3 highest dose groups achieved levels that are normal (100-200 ng/mL) or
even substantially higher than normal (highest observed level was 1174 ng/mL at 156 days). Even with this small number of experimental
animals, FVIII levels reached statistically significant levels compared
to baseline for mice in the highest cell-dose treatment groups
(analysis by analysis of variance; Table 2). In mice at 156 days, our
evaluation by separate ELISAs for human FVIII and for eGFP showed a
strong positive correlation between these 2 independent measures for
presence of expressed gene product in murine plasma
(r = 0.903; n = 10; P < .001).
Although control animals given saline only showed no appearance of human FVIII, animals given control BOEC (either wtBOEC or gfpBOEC) developed detectable amounts of human FVIII. Levels in these control animals were low compared with those generated by eGFP-FVIII tBOEC, but they did eventually become transiently therapeutic themselves. This occurrence is consistent with our observation in preliminary experiments that unmanipulated BOECs may produce small amounts of FVIII in culture (Table 1). This possibility will have to be confirmed by separate study of a larger number of animals given control BOECs. Histologic examination of mice after tBOEC administration revealed
detectable accumulations of BOEC only in bone marrow and in spleen
(Figure 3). Otherwise, we rarely observed
a single cell in other locations. As assessed by double staining, 100%
of the human tBOECs detected in murine marrow at 156 days were also
positive for human VE-cadherin, indicating preservation of a
differentiated endothelial phenotype. Similarly, virtually all (more
than 97%) tBOECs were positive for human FVIII, indicating the
preservation of transgene product production up to that time point.
Conversely, the tBOECs detected in murine marrow were uniformly
negative for hematopoietic marker CD45.
We were able to analyze several mice to determine the percentages of
nucleated cells in the marrow or spleen that were composed of human
BOECs. The limited data available (Table
3) allowed a direct comparison only for
the 20 × 104 cell dose group at day 9 versus day 156, but they seem to confirm a substantial increase in tBOEC cell numbers
in marrow over time after the initial administration (see
"Discussion").
During this study, 4 mice died of unknown causes (1 of 2 no-cell control mice and 3 of 17 mice that received some form of BOEC). This degree of animal loss was within the limits of expected mortality for these NOD/SCID mice that were older than 3 months at the start of this study, and it paralleled mortality in the parent colony from which these animals were derived. Careful observation of multiple random sections prepared from liver, heart, lung, spleen, and kidney of 6 animals (1 at 9 days, 2 at 28 days, and 3 at 156 days) showed no evidence of thrombosis, vascular occlusion, or infarction. Thus, even animals with the very highest FVIII levels (approximately 1000 ng/mL) had no evidence of thrombosis.
We have shown that the administration of engineered human BOECs can be used to attain sustained (up to 156 days, the longest time point examined) and therapeutic (even normal or supranormal) levels of FVIII in immunodeficient mice. This experiment thus demonstrates the feasibility of using BOECs as a delivery system for the administration of FVIII expression vectors, and we have done so using a nonviral plasmid vector. This therapeutic strategy would use autologous cells that obviate concerns about immunologic incompatibility or pathogenic viral infection. Growth characteristics of BOECs in culture allow for chemical selection of engineered cells in vitro and, thus, avert the need for systemic exposure to transducing agents in vivo. The specific plasmid construct used is not necessarily the preferable choice for this purpose. After further development of vector science in general, and as it applies to the expression of FVIII in particular, better expression systems than the one used here likely will be identified. An attraction of our strategy is that any viral or nonviral expression system presumably could be applied using the BOEC delivery system. Several aspects of our results deserve specific comment. The fact that
human FVIII levels in murine plasma increased over time suggests that
BOECs expanded more than 100-fold in vivo after their administration.
To arrive at this, we used the conservative assumptions that the
half-life of human FVIII in our mice was the same as previously
reported for human FVIII in immunodeficient mice (1 hour)25
and that all administered BOECs remained viable. The latter assumption
is almost certainly incorrect, which means that the degree of expansion
is even greater. The alternative explanation Detailed histologic examination revealed accumulations of tBOEC only in spleen and marrow. This suggests a seeding specificity consistent with a homing mechanism involving specific adhesion receptors, analogous to that of hematopoietic stem cells, though this, too, will have to be defined by further studies. It is important to note that analysis of the tBOECs found in murine marrow revealed that the endothelial phenotype was preserved; they were positive for VE-cadherin and negative for CD45. Most important, the tBOECs in murine marrow were still virtually all positive for the transgene product even at 156 days, consistent with the persistence of human FVIII in murine plasma observed here. Sustained transgene expression, specificity for seeding marrow and spleen, and proliferation of tBOECs in vivo may all be unique aspects of BOEC biologic function. This could account for the current result being particularly encouraging, compared to other attempts to use ex vivo gene transfer and subsequent administration of autologous cells for hemophilia therapy. Chuah et al26 used the retroviral expression of FVIII in bone marrow stromal cells given to mice and found only low-level and transient FVIII expression. Recently, Roth et al27 gave engineered autologous fibroblasts to humans with severe hemophilia A. Although results were disappointing, this was only a phase 1 trial, and the usefulness of the approach remains to be seen. It is encouraging that Qiu et al28 had earlier reported better success using that same strategy for hemophilia B. Whether engineered autologous BOECs truly present a unique system with an advantageous biologic function for gene therapy can only be answered by continued studies. Nevertheless the results presented here predict that this strategy can, indeed, be scaled up to support preclinical trials in dogs and clinical trials in humans. For example, the second highest cell dose used here, 6 × 105 cells (ie, 3 doses of 20 × 104 cells) is equivalent to 4 × 108 BOECs given to an adult dog on a weight-equivalent basis. An additional 4-fold scale up to humans would require 1.6 × 109 cells. This is not a trivial number of cells, but this degree of cell expansion is certainly achievable for human BOECs, which easily expand to 1019 cells.16 There are at least 2 reasons to assume that efficacy of the approach in humans would be even better than observed here. First, the half-life of human FVIII in the human patient should be substantially longer than in the mouse,25 and this should allow an order of magnitude fewer cells per kilogram to be used. In addition, because FVIII levels apparently rise with time, it may be that clinical efficacy could be achieved using the lower range of cell doses. Therefore, we believe this approach should be considered for the treatment of hemophilia A. Similarly, it could be a useful therapy for other disorders in which the generation of a gene product into the bloodstream would be beneficial. Hemophilia B is but one example.
We thank Ann Kerimo for technical assistance.
Submitted January 29, 2001; accepted August 31, 2001.
Supported by National Institutes of Health grants HL30160 and HL62931 (R.P.H.) and HL40921 (P.L.).
P.L. has a financial interest in a company that has licensed the technology reported here. P.L. and R.P.H. receive research support from this company.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Robert P. Hebbel, Dept of Medicine, University of Minnesota Medical School, Mayo Mail Code 480, 420 Delaware St SE, Minneapolis, MN 55455; e-mail hebbe001{at}tc.umn.edu.
1.
Hoyer LW.
Hemophilia A.
N Engl J Med.
1994;330:38-47
2.
Kay MA, High K.
Gene therapy for hemophilias.
Proc Natl Acad Sci U S A.
1999;96:9973-9975
3.
Burke RL, Pachl C, Quiroga M, et al.
The functional domains of coagulation factor VIII:C.
J Biol Chem.
1986;261:12574-12578 4. Eaton DL, Wood WI, Eaton D, et al. Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule. Biochemistry. 1986;25:8343-8347[CrossRef][Medline] [Order article via Infotrieve].
5.
Toole JJ, Pittmann DD, Orr EC, Murtha P, Wasley LC, Kaufmann RJ.
A large region (95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity.
Proc Natl Acad Sci U S A.
1986;83:5939-5942 6. Lind P, Larsson K, Spira J, et al. Novel forms of B-domain-deleted recombinant factor VIII molecules: construction and biochemical characterization. Eur J Biochem. 1995;232:19-27[Medline] [Order article via Infotrieve]. 7. Hortelano G, Chang PL. Gene therapy for hemophilia. Artif Cells Blood Substit Immobil Biotechnol. 2000;28:1-24[Medline] [Order article via Infotrieve]. 8. Greengard JS, Jolly DJ. Animal testing of retroviral-mediated gene therapy for factor VIII deficiency. Thromb Haemost. 1999;82:555-561[Medline] [Order article via Infotrieve]. 9. Zhang W-W, Josephs SF, Zhou J, et al. Development and application of a minimal-adenoviral vector system for gene therapy of hemophilia A. Thromb Haemost. 1999;82:562-571[Medline] [Order article via Infotrieve]. 10. Gnatenko DV, Saenko EL, Jesty J, Cao L-X, Bahou WF. Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to haemophilia A gene therapy. Br J Haematol. 1999;104:27-36[CrossRef][Medline] [Order article via Infotrieve].
11.
Chao H, Mao L, Bruce AT, Walsh CE.
Sustained expression of human factor VIII in mice using a parvovirus-based vector.
Blood.
2000;95:1594-1599
12.
Park F, Ohashi K, Kay MA.
Therapeutic levels of human factor VIII and IX using HIV-1-based lentiviral vectors in mouse liver.
Blood.
2000;96:1173-1176 13. Connelly S, Gardner JM, McClelland A, Kaleko M. High-level tissue-specific expression of functional human factor VIII in mice. Hum Gene Ther. 1996;7:183-195[Medline] [Order article via Infotrieve]. 14. Connelly S, Smith TA, Dhir G, et al. In vivo gene delivery and expression of physiological levels of functional human factor VIII in mice. Hum Gene Ther. 1995;6:185-193[Medline] [Order article via Infotrieve]. 15. Lynch CM, Israel DI, Kaufman RJ, Miller AD. Sequences in the coding region of clotting factor VIII act as dominant inhibitors of RNA accumulation and protein production. Hum Gene Ther. 1993;4:259-272[Medline] [Order article via Infotrieve]. 16. Lin Y, Weisdorf DJ, Solovey A, Hebbel RP. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest. 2000;105:1-77.
17.
Barrow RT, Healey JF, Jacquemin MG, Saint-Remy J-MR, Lollar P.
Antigenicity of putative phospholipid membrane-binding residues in factor VIII.
Blood.
2001;97:169-174 18. Vehar GA, Keyt B, Eaton D, et al. Structure of human factor VIII. Nature. 1984;312:337-342[CrossRef][Medline] [Order article via Infotrieve]. 19. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989;77:51-59[CrossRef][Medline] [Order article via Infotrieve].
20.
Healey JF, Barrow RT, Tamim HM, et al.
Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII.
Blood.
1998;92:3701-3709 21. Bowie EJW, Owen CA. The clinical and laboratory diagnosis of hemorrhagic disorders. In: Ratnoff OD,Forbes CD, eds. Disorders of Hemostasis. Orlando, FL: Gruen & Stratton; 1984:43-72. 22. Gupta K, Ramakrishnan S, Browne PV, Solovey A, Hebbel RP. A novel technique for culture of human dermal microvascular endothelial cells under either serum-free or serum-supplemented conditions: isolation by panning and stimulation with vascular endothelial growth factor. Exp Cell Res. 1997;230:244-251[CrossRef][Medline] [Order article via Infotrieve].
23.
Prochazka M, Gaskins HR, Shultz LD, Leiter EH.
The nonobese diabetic scid mouse: model for spontaneous thymomagenesis associated with immunodeficiency.
Proc Nat Acad Sci U S A.
1992;89:3290-3294 24. Schultz LD, Schweitzer PA, Christianson SW, et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol. 1995;154:180-191[Abstract].
25.
Dwarki VJ, Belloni P, Nijjar T, et al.
Gene therapy for hemophilia A: production of therapeutic levels of human factor VIII in vivo in mice.
Proc Natl Acad Sci U S A.
1995;92:1023-1027 26. Chuah MK, Van Damme A, Zwinnen H, et al. Long-term persistence of human bone marrow stromal cells transduced with factor VIII-retroviral vectors and transient production of therapeutic levels of human factor VIII in nonmyeloablated immunodeficient mice. Hum Gene Ther. 2000;11:729-738[CrossRef][Medline] [Order article via Infotrieve].
27.
Roth DA, Tawa NE, O'Brien JM, Treco DA, Selden RF.
Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A.
N Engl J Med.
2001;344:1735-1742 28. Qiu X, Lu D, Zhou J, et al. Implantation of autologous skin fibroblast genetically modified to secret clotting factor IX partially corrects the hemorrhagic tendencies in two hemophilia B patients. Chin Med J. 1996;109:832-839[Medline] [Order article via Infotrieve]. 29. The DIG System User's Guide for Filter Hybridization. Mannheim, Germany: Boehringer Mannheim.
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  S. F. De Meyer, H. Deckmyn, and K. Vanhoorelbeke von Willebrand factor to the rescue Blood, May 21, 2009; 113(21): 5049 - 5057. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. van den Biggelaar, E. A.M. Bouwens, N. A. Kootstra, R. P. Hebbel, J. Voorberg, and K. Mertens Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells Haematologica, May 1, 2009; 94(5): 670 - 678. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Chang Milbauer, P. Wei, J. Enenstein, A. Jiang, C. A. Hillery, J. P. Scott, S. C. Nelson, V. Bodempudi, J. N. Topper, R.-B. Yang, et al. Genetic endothelial systems biology of sickle stroke risk Blood, April 1, 2008; 111(7): 3872 - 3879. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Matsui, M. Shibata, B. Brown, A. Labelle, C. Hegadorn, C. Andrews, R. P. Hebbel, J. Galipeau, C. Hough, and D. Lillicrap Ex Vivo Gene Therapy for Hemophilia A That Enhances Safe Delivery and Sustained In Vivo Factor VIII Expression from Lentivirally Engineered Endothelial Progenitors Stem Cells, October 1, 2007; 25(10): 2660 - 2669. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Othman, A. Labelle, I. Mazzetti, H. S. Elbatarny, and D. Lillicrap Adenovirus-induced thrombocytopenia: the role of von Willebrand factor and P-selectin in mediating accelerated platelet clearance Blood, April 1, 2007; 109(7): 2832 - 2839. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. V. Pislaru, A. Harbuzariu, R. Gulati, T. Witt, N. P. Sandhu, R. D. Simari, and G. S. Sandhu Magnetically Targeted Endothelial Cell Localization in Stented Vessels J. Am. Coll. Cardiol., November 7, 2006; 48(9): 1839 - 1845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Pan, X. Shen, A. Jiang, and R. P. Hebbel Semi-supervised learning via penalized mixture model with application to microarray sample classification Bioinformatics, October 1, 2006; 22(19): 2388 - 2395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. F. De Meyer, K. Vanhoorelbeke, M. K. Chuah, I. Pareyn, V. Gillijns, R. P. Hebbel, D. Collen, H. Deckmyn, and T. VandenDriessche Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor Blood, June 15, 2006; 107(12): 4728 - 4736. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Fernandez-L, F. Sanz-Rodriguez, F. J. Blanco, C. Bernabeu, and L. M. Botella Hereditary Hemorrhagic Telangiectasia, a Vascular Dysplasia Affecting the TGF-{beta} Signaling Pathway. Clin. Med. Res., March 1, 2006; 4(1): 66 - 78. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Rohde, C. Malischnik, D. Thaler, T. Maierhofer, W. Linkesch, G. Lanzer, C. Guelly, and D. Strunk Blood Monocytes Mimic Endothelial Progenitor Cells Stem Cells, February 1, 2006; 24(2): 357 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. C. Isenberg, C. Williams, and R. T. Tranquillo Small-Diameter Artificial Arteries Engineered In Vitro Circ. Res., January 6, 2006; 98(1): 25 - 35. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Stachelek, I. Alferiev, J. M. Connolly, M. Sacks, R. P. Hebbel, R. Bianco, and R. J. Levy Cholesterol-Modified Polyurethane Valve Cusps Demonstrate Blood Outgrowth Endothelial Cell Adhesion Post-Seeding In Vitro and In Vivo Ann. Thorac. Surg., January 1, 2006; 81(1): 47 - 55. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Liu, G. Pernod, S. Dunoyer-Geindre, R. J. Fish, H. Yang, H. Bounameaux, and E. K. O. Kruithof Promoter Dependence of Transgene Expression by Lentivirus-Transduced Human Blood-Derived Endothelial Progenitor Cells Stem Cells, January 1, 2006; 24(1): 199 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. P. Hebbel, L. Milbauer, M. Roney, D. Lillicrap, J. Voorberg, and T. C. Nichols Use of Engineered Autologous BOEC for Gene Therapy of Canine Hemophilia A. Blood (ASH Annual Meeting Abstracts), November 16, 2005; 106(11): 1281 - 1281. [Abstract]
|
|
|
|
 |
|
 A. Fernandez-L, F. Sanz-Rodriguez, R. Zarrabeitia, A. Perez-Molino, R. P. Hebbel, J. Nguyen, C. Bernabeu, and L.-M. Botella Blood outgrowth endothelial cells from Hereditary Haemorrhagic Telangiectasia patients reveal abnormalities compatible with vascular lesions Cardiovasc Res, November 1, 2005; 68(2): 235 - 248. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Xu, T. C. Nichols, R. Sarkar, S. McCorquodale, D. A. Bellinger, and K. P. Ponder Absence of a desmopressin response after therapeutic expression of factor VIII in hemophilia A dogs with liver-directed neonatal gene therapy PNAS, April 26, 2005; 102(17): 6080 - 6085. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Ohlfest, J. L. Frandsen, S. Fritz, P. D. Lobitz, S. G. Perkinson, K. J. Clark, G. Nelsestuen, N. S. Key, R. S. McIvor, P. B. Hackett, et al. Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system Blood, April 1, 2005; 105(7): 2691 - 2698. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Pearson Using Endothelial Progenitor Cells for Gene Therapy Arterioscler. Thromb. Vasc. Biol., December 1, 2003; 23(12): 2117 - 2118. [Full Text] [PDF]
|
|
|
|
|
|
C. Herder, T. Tonn, R. Oostendorp, S. Becker, U. Keller, C. Peschel, M. Grez, and E. Seifried Sustained Expansion and Transgene Expression of Coagulation Factor VIII-Transduced Cord Blood-Derived Endothelial Progenitor Cells Arterioscler. Thromb. Vasc. Biol., December 1, 2003; 23(12): 2266 - 2272. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. V. Yarovoi, D. Kufrin, D. E. Eslin, M. A. Thornton, S. L. Haberichter, Q. Shi, H. Zhu, R. Camire, S. S. Fakharzadeh, M. A. Kowalska, et al. Factor VIII ectopically expressed in platelets: efficacy in hemophilia A treatment Blood, December 1, 2003; 102(12): 4006 - 4013. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Rick, C. E. Walsh, and N. S. Key Congenital Bleeding Disorders Hematology, January 1, 2003; 2003(1): 559 - 574. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
ex 488 nm,
) (Invitrogen, Carlsbad, CA) that had
been predigested with XhoI and NotI. The
resultant plasmid is referred to as pcF8G.
2-microglobulin, which did not cross-react with murine tissues (Accurate Chemical and Scientific, Westbury, NY). After blocking endogenous peroxidase activity and nonspecific biotin-avidin binding (with H2O2 and Avidin/Biotin Blocking
Kit [Vector Laboratories, Burlingame, CA], respectively), we used a
biotin-SP-conjugated donkey anti-goat immunoglobulin secondary
antibody (Jackson ImmunoResearch, West Grove, PA) and the R.T.U.
Vectastain Elite ABC reagent and peroxidase substrate DAB (both from
Vector Laboratories) to visualize human BOECs in murine tissues. To
assess nonspecific background staining, we omitted the primary
antibody, and we used tissues from untreated mice as negative controls.
marrow from a NOD-SCID
mouse that did not receive BOEC. (D) Positive control