U.S. patent application number 10/948344 was filed with the patent office on 2006-01-19 for transgenic circulating endothelial cells.
This patent application is currently assigned to Regents of the University of Minnesota. Invention is credited to Robert P. Hebbel, Yi Lin, John S. Lollar.
Application Number | 20060013805 10/948344 |
Document ID | / |
Family ID | 22328998 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060013805 |
Kind Code |
A1 |
Hebbel; Robert P. ; et
al. |
January 19, 2006 |
Transgenic circulating endothelial cells
Abstract
A process is provided for expanding the population of
endothelial cells obtained from peripheral blood which can be
transformed with a vector comprising a DNA sequence encoding a
preselected bioactive polypeptide. The resulting transgenic
endothelial cells are useful to biocompatibilize implantable
medical devices or can be used directly, as for gene therapy.
Inventors: |
Hebbel; Robert P.; (North
Oaks, MN) ; Lin; Yi; (St. Paul, MN) ; Lollar;
John S.; (Decatur, GA) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG, WOESSNER & KLUTH
1600 TCF TOWER
121 SOUTH EIGHT STREET
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Regents of the University of
Minnesota
Emory University
|
Family ID: |
22328998 |
Appl. No.: |
10/948344 |
Filed: |
September 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09865022 |
May 24, 2001 |
6852537 |
|
|
10948344 |
Sep 23, 2004 |
|
|
|
60109687 |
Nov 24, 1998 |
|
|
|
Current U.S.
Class: |
424/93.21 ;
424/423; 435/372; 435/6.16 |
Current CPC
Class: |
A61L 31/005 20130101;
A61P 7/04 20180101; A61K 48/00 20130101; C12N 5/0692 20130101; C12N
2501/115 20130101; A61L 27/3895 20130101; A61K 35/12 20130101; C12N
2501/165 20130101; C12N 2510/00 20130101; A61L 27/3808 20130101;
C12N 2501/39 20130101; A61L 27/3839 20130101; A61L 27/3843
20130101 |
Class at
Publication: |
424/093.21 ;
435/372; 424/423; 435/006 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 5/08 20060101 C12N005/08; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A process for expanding the population of endothelial cells
obtained from peripheral blood comprising culturing, in contact
with a collagen I-coated surface, buffy coat cells obtained from
peripheral mammalian blood in the presence of a cell culture medium
containing an effective amount of vascular endothelial growth
factor (VEGF), and which medium is free of bovine brain extract, so
as to expand the population of endothelial cells in said buffy coat
cells.
2. The process of claim 1 wherein the blood is human blood.
3. The process of claim 1 wherein said cell culture medium
comprises heparin, dextran sulfate or mixtures thereof.
4. The process of claim 1 wherein the buffy coat cells are obtained
by washing cells from a buffy coat layer obtained from human blood
in cell culture medium comprising 20% human male serum.
5. The process of claim 1 wherein the cell culture medium comprises
human basic fibroblast growth factor.
6. The process of claim 1 or 5 wherein the cell culture medium
comprises insulin-like growth factor.
7. The process of claim 1 or 5 wherein the cell culture medium
contains human epidermal growth factor.
8. The process of claim 1 wherein the cell culture medium comprises
about 0.5-10 vol-% fetal bovine serum and about 95-99.5 vol-% of a
cell culture medium.
9. The process of claim 1 wherein the cultured cells are
trypsinized at about 10.sup.3-fold expansion, collected by
centrifugation, resuspended in cell culture medium, and subjected
to continued culture in contact with a fibronectin/gelatin-coated
surface.
10. The process of claim 1 wherein the cultured cells are subjected
to cryopreservation.
11. The process of claim 10 wherein the cells are frozen in a
cryopreservation medium comprising fetal calf serum containing an
effective amount of dimethylsulfoxide.
12. The process of claim 10 or 11 wherein the cryopreserved cells
are thawed and culturing is resumed in said cell culture
medium.
13. The process of claim 1 wherein the expanded population
comprises microvascular endothelial cells.
14. The process of claim 13 wherein the microvascular endothelial
cells are CD34.sup.+, CD36.sup.+ and express the P1H1 antigen.
15. A transgenic mammalian endothelial cell comprising an isolated
DNA sequence encoding a recombinant Factor VIII protein.
16. The transgenic endothelial cell of claim 15 wherein the Factor
VIII protein is a hybrid human/porcine protein.
17. A transgenic endothelial cell which is prepared by a process
comprising stably transforming a population of circulating human
endothelial cells outgrown from blood with a vector comprising an
isolated DNA sequence encoding a preselected protein operably
linked to a promoter functional in human endothelial cells.
18. The transgenic endothelial cell of claim 17 wherein the DNA
sequence encodes a Factor VIII protein.
19. The transgenic endothelial cell of claim 17 wherein the DNA
sequence comprises SEQ ID NO:1 or SEQ ID NO:2.
20. The transgenic endothelial cell of claim 18 wherein the Factor
VIII protein is a hybrid human/porcine protein.
21. The transgenic endothelial cell of claim 18 wherein the Factor
VIII protein is a chimeric human protein.
22. The transgenic endothelial cell of claim 17 wherein the DNA
sequence further comprises a selectable marker gene or a reporter
gene.
23. The transgenic endothelial cell of claim 17 which is prepared
by lipofection.
24. A pharmaceutical composition comprising a population of the
transgenic endothelial cells of claim 15 or 17 in combination with
a pharmaceutically acceptable carrier.
25. A method of treating hemophilia comprising introducing an
amount of the endothelial cells of claim 15 or 17 into the
bloodstream of a mammal afflicted with hemophilia so that an
effective amount of a Factor VIII protein is secreted into the
bloodstream of said mammal.
26. The method of claim 25 wherein the mammal is a human.
27. An implantable medical prosthetic device comprising a surface
coated with the endothelial cells of claim 17 wherein the protein
is expressed in an amount effective to increase the
biocompatibility of said device upon implantation into a
mammal.
28. The device of claim 27 which comprises a plastic surface, a
metal surface or a laminate surface.
29. The device of claim 28 which is a vascular graft.
30. The device of claim 28 which is a shunt or a stent.
31. The device of claim 28 which is a heart valve.
32. The device of claim 28 which is a controlled drug release
depot.
33. A diagnostic method comprising: detecting or determining
whether an expanded population of endothelial cells obtained from
peripheral blood of a test mammal has an acquired or genetic
indication or disease relative to a control expanded population of
endothelial cells obtained from a mammal not at risk of the
acquired or genetic indication or disease, wherein the expanded
cells are obtained by culturing, in contact with a collagen
I-coated surface, mononuclear cells from a buffy coat layer
obtained from the peripheral blood of the test mammal in the
presence of a cell culture medium containing an effective amount of
vascular endothelial growth factor (VEGF), which medium is free of
bovine brain extract, so as to yield an expanded population of
endothelial cells.
34. The method of claim 33 wherein the indication or disease is a
clotting disorder.
35. The method of claim 33 wherein the indication or disease is
associated with a reduction in the activity of an enzyme.
36. The method of claim 33 wherein the indication or disease is
acquired.
37. The method of claim 33 wherein the indication or disease is
associated with expression of a mutant gene in endothelial
cells.
38. The method of claim 33 wherein the indication or disease is
associated with altered expression of a gene in endothelial cells
of the test mammal relative to the control.
39. The method of claim 33 wherein the mammal is a human.
40. The method of claim 33 wherein the cultured cells were
subjected to cryopreservation.
41. The method of claim 33 wherein the expanded population
comprises microvascular endothelial cells.
42. The method of claim 41 wherein the microvascular endothelial
cells are CD34.sup.+, CD36.sup.+ and express the P1H1 antigen.
43. The method of claim 33 wherein antibodies were not employed to
obtain the buffy coat mononuclear cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 09/865,022, filed May 24, 2001, which is a
national stage filing of PCT/US99/28033, filed Nov. 24, 1999, which
claims the benefit of the filing date of U.S. application Ser. No.
60/109,687, filed Nov. 24, 1998, the disclosures of which are
incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] The endothelial cell participates in numerous functions of
vascular physiology. Many factors, such as cytokines, can alter the
surface of the endothelial cell and thereby modulate the role of
the endothelium in coagulation, inflammation, vaso-regulation, and
adhesion. See, for example, R. P. Hebbel et al., J. Lab. Clin.
Med., 129, 288 (1997); J. S. Pober, Am. J. Path., 133, 426 (1988);
E. J. Favaloro, Immunol. Cell. Biol., 71, 571 (1993). The
endothelial cell may also have a key role in the vascular pathology
of sickle cell anemia, including the vaso-occlusions that cause
acute painful crises. However, research in this area has been
hindered by the inaccessibility of vascular endothelium in
patients. For example, E. M. Levine et al. (U.S. Pat. No.
5,132,223) disclosed cloning and serial cultivation of adult human
endothelial cells derived from brain-dead, but heart-beating
cadaver organs. K. Gupta et al., Exp. Cell. Res., 230, 244 (1997)
reported the culture of microvascular endothelial cells derived
from newborn human foreskin. Thus, circulating endothelial cells
might provide useful material for the study of vascular
pathologies, for gene therapy, and for biomedical engineering
applications. In previous investigations increased numbers of
circulating endothelial cells have been found in sickle cell anemia
and other conditions associated with vascular injury, such as that
due to cytomegalovirus infection, rickettsial infection, myocardial
infarction, intravascular instrumentation, and endotoxinemia. See,
for example, F. George et al., Blood, 80, Suppl: 12a, abstract
(1992); E. Percivalle et al., J. Clin. Invest., 92, 663 (1993), F.
George et al., Blood, 82, 2109 (1993); C. A. Bouvier et al., Thomb.
Diath. Haemorrh. Suppl., 40, 163 (1970); F. George et al., J.
Immunol. Meth., 139, 65 (1991) and R. G. Gerrity et al., Exp. Mol.
Pathol., 24, 59 (1976).
[0003] However, in normal donors, there are only about 2-3
circulating endothelial cells per ml of blood; they have a
quiescent phenotype, and about 50% of them are microvascular as
evidenced by CD36 positivity. See, A. Solovey et al., New Engl. J.
Med., 337, 1584 (1997), who reported using the methodology of Gupta
et al., cited above, to coculture viable circulating endothelial
cells identified in the blood of patients with sickle cell anemia
with primary microvascular endothelial cells (MVEC). T. Asahara et
al., Science, 275, 964 (1997) isolated putative endothelial cell
(EC) progenitors from human peripheral blood after CD34.sup.+
enrichment by magnetic bead selection on the basis of cell surface
antigen expression. The cells were cultured on fibronectin-coated
wells in modified M-199 medium containing bovine brain extract and
20% fetal bovine serum. Q. Shi et al., Blood, 92, 362 (1998)
characterized bone marrow-derived precursor endothelial cells by
isolating CD34.sup.+ cells derived from peripheral blood using
murine anti-CD34.sup.+ antibody binding followed by exposure to
anti-mouse immunomagnetic beads. The cells were cultured in gelatin
or fibronectin-coated plastic wells in M199 medium containing VEGF,
FBS, bFGF and IGF.
[0004] However, due to the low concentration of CEC in blood, a
need exists for a culture method and medium that will permit the
rapid expansion of CEC from blood, without the attendant
difficulties of isolation discussed above.
SUMMARY OF THE INVENTION
[0005] The present invention provides a process for expanding the
population of endothelial cells (EC) present in an aliquot of
peripheral mammalian blood, i.e., blood obtained from an animal or
human patient. The process comprises culturing buffy coat
mononuclear cells, which are readily obtained from peripheral
mammalian blood. The buffy coat mononuclear cells are cultured in
contact with a surface coated with collagen I, such as a coated
plastic culture well, wall of a culture flask or bioreactor, in a
culture medium containing an effective amount of vascular
endothelial growth factor (VEGF), which medium is free of bovine
brain extract. Optionally, the buffy coat mononuclear cells can
thereafter be cultured in contact with a surface coated with
fibronectin/gelatin. The medium can also comprise heparin and/or
dextran sulfate, and other growth factors conventionally employed
in endothelial cell culture media. The present method accomplishes
the rapid and extensive expansion of the initial population of
endothelial cells and any endothelial progenitor cells present in
the population of buffy coat mononuclear cells. For example, the
present method typically results in an outgrowth of endothelial
cells that is at least a billion-fold greater than the 20-30
endothelial cells identifiable on culture day #2, when endothelial
cells are first counted. The endothelial outgrowth includes the
expansion of two populations. There is a limited expansion of the
mature endothelial cells of vessel wall origin that are found in
fresh blood, as well as a delayed, but greater, expansion of a
rarer population of marrow-derived cells, e.g., endothelial
progenitor cells or angioblasts, that are also found in fresh
blood.
[0006] Unexpectedly, the cultured endothelial cells have been found
to be amenable to cryopreservation in conventional cryopreservation
media, followed by thawing and continued culture/expansion. The
present method is thus more convenient and less complex than
methods based on processing and culturing adult vessel human
endothelial cells, as described, for example, in E. M. Levine et
al. (U.S. Pat. No. 5,132,223). It does not comprise selection of
sub-populations of EC-enriched hematopoietic cells using antibodies
and/or magnetic bead technology.
[0007] The present invention also comprises an isolated, purified
transgenic mammalian endothelial cell comprising a recombinant DNA
sequence encoding at least one biologically active preselected
protein or polypeptide, such as a Factor VIII protein, and
optionally, a selectable marker gene or reporter gene. Preferably,
the transgenic endothelial cell is prepared by stably transforming
(transfecting/transducing) a population of circulating endothelial
cells outgrown from blood in accord with the present method, with a
vector comprising an isolated DNA sequence encoding the
protein/polypeptide of interest, operably linked to a promoter
functional in said endothelial cells.
[0008] A population of said transgenic cells can be formulated into
a pharmaceutical composition and administered to a mammal, such as
a human patient afflicted with hemophilia, preferably in
combination with a pharmaceutically acceptable carrier. The carrier
may be a liquid carrier (for example, a saline solution) or a solid
carrier; e.g., an implant. In employing a liquid carrier, the
engineered cells may be introduced, e.g., intravenously,
sub-cutaneously, intramuscularly, intraperitoneally,
intralesionally, and the like. The polypeptide, such as Factor VIII
protein or proteins are expressed in situ, i.e., in the bloodstream
of said mammal in an amount effective to treat (alleviate) said
hemophilia. Since the transgenic endothelial cells will also
migrate to the bone marrow, they are also useful for many aspects
of gene therapy, apart from treating circulatory pathologies. Other
uses of these transgenic EC, such as in biocompatibilization of
implantable devices, diagnostics, and local drug delivery, are
discussed hereinbelow.
[0009] Novel vectors useful for transforming the expanded
circulating endothelial cells of the invention are also within the
scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a graph plotting the outgrowth of endothelial
cells in accord with the method of the invention. Arrows show times
at which cultures were passaged. All data points are plotted as
mean.+-.SD. Data are shown for n=5 through culture passage 6, and
n=4 for subsequent passages.
[0011] FIG. 2 is a graph plotting the outgrowth of endothelial
cells that were subjected to cryopreservation.
[0012] FIG. 3 depicts the DNA sequence of HSQ/eGFP (SEQ ID NO:1).
(HFVIII/SQ/egfp) which comprises original HB with B-domain SQ
insert based on Lind et al., Eur. J. Biochem., 232; and p21 primer
sequence containing eGFP protein sequence.
[0013] FIG. 4 shows the DNA sequence (SEQ ID NO:2) of HSQRENeo.
[0014] FIG. 5 shows the results of G418 selection (shaded area) of
outgrowth endothelial cells stably transfected with a vector
encoding eGFP.
[0015] FIG. 6 shows factor VIII activity in conditioned media from
cells transfected with various constructs (see Example 7).
[0016] FIG. 7 depicts the results of a RT-PCR analysis for human
FVIII mRNA in cells transfected with various constructs.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The endothelial cell culture medium used in the process of
this invention comprises any of the media conventionally employed
for the culture of this type of cell, as modified in accord with
the present method, to include an effective amount of endothelial
cell growth factor (ECG or VEGF), and to exclude bovine brain
extract. The exclusion of bovine brain extract is advantageous both
from a standpoint of consistency of formulation, and from a health
standpoint, in that cells are not subjected to the risk of
contamination with infectious agents (viruses, prions, etc.) which
may be present in such extracts.
[0018] Preferably, the medium contains heparin, dextran sulfate or
a combination thereof. These materials are described in detail in
U.S. Pat. No. 5,132,223. Of the various basal endothelial cell
growth media, EGM.RTM.-2 (Clonetics, Inc., San Diego, Calif.) is
particularly preferred. It is based on CCMD.RTM. 130 culture medium
plus human epidermal growth factor (hEGF), human basic fibroblast
growth factor (hFGF-B), human recombinant insulin-like growth
factor (Long R3-IGF-1). FGF can be present at about 0.5-5 ng/ml,
EGF can be present at about 0.5-10 ng/ml and IGF can be present at
about 1-7.5 ng/ml of the culture medium.
[0019] The medium can also comprise hydrocortisone (0.1-2
.mu.g/ml), heparin (1-20 .mu.g/ml), gentamicin, amphotericin-B
(0.1-0.5 mg/ml) and fetal bovine serum. Vascular endothelial growth
factor (VEGF) is present at about 1-100 ng/ml, preferably about
5-25 ng/ml. It is included within this concentration range in some
commercially available media or supplements or is available from
Collaborative Research, Inc. VEGF can also be prepared as described
by Maciag et al., PNAS USA, 76, 5674 (1979).
[0020] A medium that is effective to disperse and pre-wash the
buffy coat mononuclear cells prior to culturing, has been described
by K. Gupta et al., Exp. Cell Res., 230, 244 (1997). This
endothelial culture medium consists of MCDB 131 medium supplemented
with 1 .mu.g/ml hydrocortisone acetate, 5.times.10.sup.-4 M
bibutyryl cAMP, 1.6 nM L-glutamine, 100 U/ml penicillin, 100 U/ml
streptomycin, 0.25 mg/ml amphotericin B, 0.004% heparin and 20%
human male serum. VEGF (0.01-100 ng/ml) can also be added to this
medium.
[0021] Circulating endothelial cells can be isolated from whole
blood via isolation of the buffy coat mononuclear cells by
methodologies well-known to the art. For example, see A. Solovey et
al., New Engl. J. Med., 337, 1584 (1997). Whole blood is
anti-coagulated and diluted with a physiological salt solution
containing EDTA and BSA. The diluted blood is layered onto
Histopaque.RTM. 1077 (Sigma Chem. Co.) and centrifuged at ambient
temperature. The buffy coat mononuclear cells are collected and
washed by centrifuging with the medium described in K. Gupta et
al., Exp. Cell. Research, 230, 244 (1977), described hereinabove,
i.e., supplemented MCDB 131 medium.
[0022] The buffy coat mononuclear cells are resuspended in the
endothelial cell culture medium (i.e., EGM.RTM.-2) and held in a
suitable vessel having the walls thereof coated with collagen I.
Type I collagen is commercially available from Sigma Chem. Co. (St.
Louis, Mo.) and is obtainable from bovine Achilles tendon, calf
skin, rat tail, and human placenta. See, for example, Niyiloizi et
al., J. Biol. Chem., 259, 14170 (1984). On day two of culture,
unattached buffy coat mononuclear cells and cell debris are removed
when the medium is changed, and the small number of adhered cells
exhibit endothelial cell morphology, and stain positive with mAb
P1H12.
[0023] Cultured cells were evaluated by inverted phase-contrast
microscopy to confirm the characteristic cobblestone morphology of
the endothelial cells and to examine for the presence of cells
other than endothelial cells. Cells were also analyzed for von
Willebrand factor (vWF) and CD36 expression by immunofluorescence
and flow cytometry. The presence of cell surface CD36 has been used
as a marker for human microvascular endothelial cells (HDMEC), as
it has been shown that most MEC express CD36, but endothelial cells
lining large vessels do not. Briefly, cells in suspension were
fixed with paraformaldehyde, cytospun onto glass slides, washed,
and incubated with either rabbit anti-human vWF (1:400 dilution) or
anti-CD36 mAb FA6-152 (5 .mu.g/ml) (Immunotech, Westbrook, Me.).
Slides were then washed and incubated with secondary antibodies
conjugated to rhodamine or fluorescein, washed, and viewed under a
fluorescent microscope.
[0024] To identify endothelial cells, the antibody P1H12 was also
used. This murine IgG1 monoclonal antibody was obtained by
immunizing mice with HUVEC, generating a hybridoma line, and
separating IgG from supernatants of hybridoma cell cultures with a
protein G column. For some studies, fluorescein
isothiocyanate-labeled P1H12 was used, prepared with the Fluoro Tag
FITC Conjugation kit (Sigma).
[0025] P1H12 reacts specifically with endothelial cells in blood.
It stains primary HUVEC and MVEC cultures and the endothelial cells
of all vessels in frozen sections of human skin, intestine, ovary,
tonsil, lymph node, lung, and kidney. It does not stain any other
type of cell in those tissues. It does not stain carcinoma cell
lines HT-29 and COLO205, melanoma cells lines A-375 and M21, the
T-cell lines Jurkat and HuT78, fibroblasts, HL-60 or
Chinese-hamster-ovary cells, or Epstein-Barr virus-transformed
B-cell lines. It does not stain monocytes, granulocytes, red cells,
platelets, T cells, or B cells from marrow or peripheral blood; nor
does it react with marrow megakaryocytes or the megakaryoblast line
HU3. The peripheral blood cells that do stain with P1H12 are also
positive for both von Willebrand factor and thrombomodulin (the
combined expression of which is limited to endothelium), and they
stain for fit and flk (receptors for the endothelial-specific
vascular endothelial growth factor). Subgroups of P1H12-positive
blood cells also stain for CD34 and two endothelial-specific
activation markers (VCAM and E-selectin).
[0026] After good EC growth is obtained, preferably after about
10.sup.3-fold expansion, or at 15-25 days, the cells are
trypsinized and isolated from the supernatant by centrifugation.
The cells are then suspended in the initial culture medium in a
flask coated with fibronectin/gelatin for continued culture. Clones
can be derived from secondary cultures and seeded at about 10
cells/cm.sup.2 of flask surface. The clones are then serially
propagated in the culture medium.
[0027] It was also found that EC cultured in accord with the
present method can be cryopreserved and then thawed and returned to
culture without significant loss of their capacity to proliferate.
To cryopreserve the cells, the cultured cells can be detached as
described hereinabove and resuspended in suitable cryopreservation
medium, i.e., containing a cryopreservation agent such as sugar(s),
BSA, dimethylsulfoxide (DMSO), glycerol, glycerol esters and the
like.
[0028] Cryopreservation of hematopoietic cells, such as bone marrow
and peripheral blood fractions enriched in progenitor stem cells,
has become standard clinical practice for autologous bone marrow
transplantation (Areman et al., Bone Marrow and Stem Cell
Processing: A Manual of Current Techniques, F. A. Davis,
Philadelphia, Pa., 1st edition (1992)). Two basic techniques are
used to cryopreserve hematopoietic cells. The most commonly used
technique in clinical laboratories, uses tissue culture medium
combined with 95% fetal calf serum ("FCS"), and 5 v/v %
dimethylsulfoxide ("DMSO"). After suspension in the
cryopreservation medium, the cells are placed on ice for about 5
minutes, then at -70.degree. C. overnight and finally in liquid
nitrogen. This technique was developed in the early 1960s by
Ashwood-Smith (see Sputtek et al., Clinical Applications of
Cryobiology, Chapter 5, 127-147 (CRC Press, Boca Raton, Fla.,
1991)), and has been the predominant technique used in clinical
practice. In early 1983, Stiff and colleagues developed a modified
method for the preservation of stem cells (Stiff et al.,
Cryobiology, 20, 17-24 (1983)). In this method, the concentration
of DMSO was reduced to 5% and an additional cryoprotective agent,
hydroxyethyl starch ("HES"), was added to the solution. The cells
could be frozen using a controlled rate freezer or in a mechanical
freezer at -80.degree. C.
[0029] In present clinical practice, the base of most
cryopreservation solutions is a tissue culture medium, potentially
containing many different components. Typical tissue culture media
include RPMI 1640, IMDM, AIM-5, X-VIVO 10, or .alpha.MEM.
[0030] The cell suspension is then frozen, i.e., at liquid nitrogen
temperatures in a suitable container and stored until needed. The
frozen suspension is then thawed, by immersing the container in a
warm water bath and culturing can be resumed. The resulting EC
growth is similar to that of cells that were not subjected to
cryopreservation.
[0031] The cultured EC may also be used in gene therapy wherein a
gene producing a protein polypeptide, enzyme or other product is
inserted into the DNA of the EC. The transgenic EC are then
administered to a mammal, e.g., by infusion into a patient's
bloodstream. See, for example, B. P. Luskey et al., Annals. N. Y.
Acad. Sci., 612, 398 (1990), B. A. Naughton et al. (U.S. Pat. No.
4,721,096), and Anderson et al. (U.S. Pat. No. 5,399,346).
[0032] The gene carried by the EC can be any gene which allows the
blood cells to exert a therapeutic effect that they would not
ordinarily have, such as a gene encoding a clotting factor, such as
Factor VIII, useful in the treatment of hemophilia. The gene can
encode one or more products having therapeutic effects. Examples of
suitable genes include those that encode cytokines such as TNF,
interleukins (interleukins 1-12), interferons (.alpha., .beta.,
.gamma.-interferons), T-cell receptor proteins and Fc receptors for
antigen-binding domains of antibodies, such as immunoglobulins.
[0033] Additional examples of suitable genes include genes that
modify EC to "target" a site in the body to which the blood cells
would not ordinarily "target," thereby making possible the use of
the blood cell's therapeutic properties at that site. In this
fashion, blood cells such as TIL can be modified, for example, by
introducing a Fab portion of a monoclonal antibody into the cells,
thereby enabling the cells to recognize a chosen antigen. Likewise,
blood cells having therapeutic properties can be used to target,
for example, a tumor, that the blood cells would not normally
target. Other genes useful in cancer therapy can be used to encode
chemotactic factors which cause an inflammatory response at a
specific site, thereby having a therapeutic effect. Other examples
of suitable genes include genes encoding soluble CD4 which is used
in the treatment of HIV infection and genes encoding
.alpha.-antitrypsin, which is useful in the treatment of emphysema
caused by .alpha.-antitrypsin deficiency, or genes encoding factors
which promote bone growth such as bone morphogenic proteins (BMPs)
or other factors in the BMP pathway.
[0034] The gene therapy of the present invention is useful in the
treatment of a variety of diseases including but not limited to
adenosine deaminase deficiency, sickle cell anemia, thalassemia,
hemophilia, diabetes, .alpha.-antitrypsin deficiency, brain
disorders such as Alzheimer's disease, and other illnesses such as
growth disorders and heart diseases, for example, those caused by
alterations in the way cholesterol is metabolized and defects for
the immune system, as well as to repair bone fractures or treat or
prevent osteoporosis.
[0035] In still another embodiment, there is provided a method of
detecting the presence of human CEC present in a patient,
comprising: (i) inserting into an expanded population of human CEC
removed from the patient a DNA segment encoding the detectable
marker under conditions such that the marker is expressed in the
CEC; (ii) introducing cells resulting from step (i) into the
patient; (iii) removing from the patient an aliquot of tissue
(which can be, for example, normal tissue, cancerous tissue,
vascular tissue, blood, lymph nodes, etc.) including cells
resulting from step (ii) and their progeny; and (iv) detecting or
determining the quantity of the cells resulting from step (ii) and
their progeny, in said aliquot.
[0036] To effect gene therapy with a substantially pure population
of human EC, the following general method may be used to insert a
gene into these EC. For a review of transformation methodologies,
see Friedman, Science, 244, 1275 (1989) and Lancet, Jun. 4, 1988,
p. 1271. In order to introduce a normal structural gene, to correct
a genetic error, a gene can first be isolated from the cells of a
donor. The cells may be isolated from tissue(s), blood or other
body fluids. To find a gene coding for the defective protein, DNA
from the donor cell is isolated and cleaved by enzymatic digestion
into segments of varying length by means known to those skilled in
the art. The segments of DNA then may be inserted individually into
vectors containing the appropriate regulatory sequences for
expression of a gene product. The vectors then can be screened by
conventional means such as Northern blotting, if the sequence for
the normal gene is known, or the expression product can be screened
by Western blotting.
[0037] Alternatively, if the DNA sequence of the desired gene or
the sequence of the normal protein is known, the gene can be made
by synthetic methods such as by using a DNA synthesizer (Applied
Biosystems). In any case, the method of isolation or construction
of the gene sequence can yield a "normal" gene that codes for the
desired gene product. Once isolated, a functional structural gene,
such as a DNA sequence coding for a factor VIII protein, human
factor VIII, porcine factor VIII and the like can be modified for
expression in vivo by linkage to suitable control regions, such as
promoters. Hybrid or modified genes can also be constructed, such
as DNA sequences encoding factor VIII molecules, modified to reduce
their antigenicity and immunogenicity. DNA methods can be used to
substitute elements of animal factor VIII for the corresponding
elements of human factor VIII, resulting in hybrid human/animal
factor VIII molecules. The preparation of transformation vectors
comprising DNA sequences encoding chimeric hybrid Factor VIII
molecules is disclosed in Lollar et al. (U.S. Pat. No. 5,744,446).
DNA methods may also be used to substitute amino acid residues at
selected sites, such as epitopes, to yield factor VIII constructs
having improved therapeutic attributes such as reduced antigenicity
or immunogenicity or greater potency in comparison to unmodified
human factor VIII.
[0038] In a preferred embodiment, a hybrid human/porcine cDNA
encoding factor VIII, in which the porcine sequence encodes a
domain or part domain, such as the A2 domain or part domain, is
inserted in a mammalian expression vector, such as ReNeo, to form a
hybrid factor VIII construct. Preliminary characterization of the
hybrid factor VIII is accomplished by insertion of the hybrid cDNA
into the ReNeo mammalian expression vector and transient expression
of the hybrid protein in COS-7 cells. A determination of whether
active hybrid protein is expressed can then be made. The expression
vector construct is used further to stably transfect cells in
culture, such as baby hamster kidney cells, using methods that are
routine in the art, such as liposome-mediated transfection
(Lipofectin.TM., Life Technologies, Inc.). Expression of
recombinant hybrid factor VIII protein can be confirmed, for
example, by sequencing, Northern and Western blotting, or
polymerase chain reaction (PCR). Hybrid factor VIII protein in the
culture media in which the transfected cells stably expressing the
protein are maintained can be precipitated, pelleted, washed, and
resuspended in an appropriate buffer, and the recombinant hybrid
factor VIII protein purified by standard techniques, including
immunoaffinity chromatography using, for example, monoclonal
anti-A2-Sepharose.TM..
[0039] In a further embodiment, the hybrid factor VIII comprising
subunit, domain, or amino acid sequence substitutions is expressed
as a fusion protein from a recombinant molecule in which a sequence
encoding a protein or peptide that enhances, for example,
stability, secretion, detection, isolation, or the like is inserted
adjacent to the factor VIII encoding sequence. Established
protocols for use of homologous or heterologous species expression
control sequences including, for example, promoters, operators, and
regulators, in the preparation of fusion proteins are known and
routinely used in the art. See Current Protocols in Molecular
Biology (Ausubel, F. M. et al., eds.), Wiley Interscience, New
York.
[0040] The purified hybrid factor VIII or fragment thereof can be
assayed for coagulation activity by standard assays including, for
example, the plasma-free factor VIII assay, the one-stage clotting
assay, and the enzyme-linked immunosorbent assay using purified
recombinant human factor VIII as a standard. Antigenicity and
immunogenicity of factor VIII constructs may be tested in vitro
using the standard Bethesda inhibitor assay, or in vivo using a
knockout hemophilic mouse model.
[0041] Other vectors, including both plasmid and eukaryotic viral
vectors, may be used to express a recombinant gene construct in
eukaryotic cells depending on the preference and judgment of the
skilled practitioner. Other vectors and expression systems,
including bacterial, yeast, and insect cell systems, can be used
but are not preferred due to differences in, or lack of,
glycosylation.
[0042] The same methods employed for preparing hybrid human/porcine
factor VIII having subunit, domain, or amino acid sequence
substitution can be used to prepare other recombinant hybrid factor
VIII protein and fragments thereof and the nucleic acid sequences
encoding these hybrids, such as human/non-human, non-porcine
mammalian or animal/animal. As used herein the term "Factor VIII
protein" includes any of these materials that are biologically
active as determined by the assays listed above, i.e., that possess
coagulation activity when assayed in vitro. The murine and part of
the porcine factor VIII cDNA have been cloned. Factor VIII
sequences of other species for use in preparing a hybrid
human/animal or animal/animal factor VIII molecule can be obtained
using the known human and porcine DNA sequences as a starting
point. Other techniques that can be employed include PCR
amplification methods with animal tissue DNA, and use of a cDNA
library from the animal to clone the factor VIII sequence.
[0043] Once the DNA containing the gene is prepared, the DNA can be
inserted into the population of EC isolated and expanded as above.
The DNA can be inserted by 1) physical methods such as
coprecipitation with calcium phosphate, electroporation,
lipofection or microinjection (e.g., U.S. Pat. No. 4,873,191),
and/or by 2) the use of viral vectors such as adenoviruses, if the
DNA is less than approximately 7-8 kB, or retroviruses for longer
segments of DNA. In the latter case, the DNA of the retrovirus is
cut with a restriction enzyme and the human DNA containing the
desired sequence is inserted and ligated. The retrovirus containing
the insertion then is transfected into the EC. The EC then can be
assayed for production of the desired protein. See, e.g., U.S. Pat.
Nos. 4,902,783 and 5,681,746.
[0044] In general, molecular DNA cloning methods are well known in
the art and are not limiting in the practice of this invention. For
a further description of similar methods, see Friedmann, Science,
244, 1275 (1989) and Molecular Cloning: A Laboratory Manual (2nd
ed.), Cold Spring Harbor Laboratory Press, Sambrook, Fritsch and
Maniatis eds. (1989).
[0045] Transgenic, i.e., transduced, endothelial cells stably
incorporating and expressing heterologous DNA or RNA and
therapeutic uses therefore, are described in Mulligan et al. (U.S.
Pat. No. 5,674,722). In particular, retroviral vectors have been
used to stably transduce endothelial cells with genetic material
which includes genetic material encoding a polypeptide or protein
of interest not normally expressed at biologically or
therapeutically significant levels in endothelial cells. The
genetic material introduced in this manner can also include genetic
material encoding a dominant selectable marker, such as antibiotic
or herbicide resistance. Genetic material including DNA encoding a
polypeptide of interest alone, such as a Factor VIII protein, or
DNA encoding a polypeptide of interest and a dominant selectable
marker can be introduced into cultured endothelial cells.
Expression of these genes by the endothelial cells into which they
have been incorporated (i.e., endothelial cells transduced by the
use of retroviral vectors) has also been demonstrated.
[0046] Endothelial cells transduced in vitro with the genetic
material can then be transplanted using one of a variety of known
methods. Such methods include, but are not limited to, the
transplantation of synthetic vessels or prosthetic valves lined
with transduced endothelial cells or the transplantation of a
device or matrix designed to house transduced endothelial
cells.
[0047] To administer the EC containing the desired gene, the cells
may simply be introduced into the bloodstream of the patient by
conventional means, such as of intravenous infusion over a period
of time.
[0048] Endothelial cells which have been transduced in vitro are
particularly useful for improving prosthetic implants (e.g.,
vessels made of synthetic materials such as Dacron.RTM.,
Gortex.RTM. and other plastics or metal-plastic laminates),
including shunts, stents and grafts, which are used in vascular
reconstructive surgery. For example, prosthetic arterial grafts are
often used to replace diseased arteries which perfuse vital organs
or limbs. However, the currently available grafts are usually made
of synthetic material and are subject to many complications, the
worst of which is a high rate of thrombosis or occlusion. Animal
studies suggest that lining the graft with autologous endothelial
cells prior to implantation may decrease, but not prevent, graft
reocclusion with its attendant morbid consequences.
[0049] However, endothelial cells can be modified according to the
method of the present invention in a way that improves their
performance in the context of an implanted graft or provides a
means for local drug delivery. Examples include local delivery to
the interior of the lumen of antirestenotic, antiproliferative, or
thrombolytic agents to prevent intraluminal clot formation,
secretion of an inhibitor of smooth muscle proliferation to prevent
luminal stenosis due to smooth muscle hypertrophy, and expression
and/or secretion of an endothelial cell mitogen or autocrine factor
to stimulate endothelial cell proliferation and improve the extent
or duration of the endothelial cell lining of the graft lumen. The
latter agents are termed "biocompatibilization" agents
(polypeptides or proteins).
[0050] For a similar application, endothelial cells of the present
invention can also be used to cover the surface of prosthetic heart
valves to decrease the risk of the formation of emboli by making
the valve surface less thrombogenic.
[0051] Endothelial cells transduced by the method of the subject
invention or a vascular implant lined with transduced endothelial
cells can also be used to provide constitutive synthesis and
delivery of polypeptides or proteins, which are useful in
prevention or treatment of disease, such as Factor VIII proteins,
to treat hemophilia. In this way, the polypeptide is secreted
directly into the bloodstream of the individual, from circulating
cells, over an extended period of time. Currently available
methods, in contrast, involve parenteral administration of the
desired polypeptide.
[0052] In addition, there is no need for extensive purification of
the polypeptide before it is administered to an individual, as is
generally necessary with an isolated polypeptide (e.g., insulin).
Endothelial cells modified according to the present invention
produce the polypeptide hormone as it would normally be
produced.
[0053] Another advantage to the use of genetically engineered
endothelial cells is that one can target the delivery of
therapeutic levels of a secreted product to a specific organ or
limb. For example, a vascular implant lined with endothelial cells
transduced in vitro can be grafted into a specific organ or limb;
or the endothelial cells of a particular limb, organ or vessel can
be transduced in vivo. The secreted product of the transduced
endothelial cells will be delivered in high concentrations to the
perfused tissue, thereby achieving a desired effect to a targeted
anatomical location. This product will then be diluted to
nontherapeutic levels in the venous circulation during its return
to the heart.
[0054] Another important advantage of the delivery system of this
invention is that because it is a continuous delivery system, the
short half lives of hormone polypeptides is not a limitation. For
example, the half life of human growth hormone (HGH) is
approximately 19 minutes and parathyroid hormone, approximately
21/2 to 5 minutes.
[0055] The invention will be further described by reference to the
following detailed examples.
EXAMPLE 1
Culture of Endothelial Cells from Peripheral Blood
[0056] One hundred ml of fresh venous blood anti-coagulated with
either heparin or citrate was diluted 1:2 with Hanks' Balanced Salt
Solution (HBSS) containing 1 mM EDTA and 0.5% bovine serum albumin,
carefully layered on an equal volume of Histopaque.RTM.-1077 (Sigma
Chemical Co.) without disruption of the interface, and centrifuged
at 400.times.G for 30 minutes at room temperature. The layer
containing the mononuclear cells was collected, and the cells were
washed 3 times by centrifuging at 250.times.G for 10 minutes, using
the previously described MEC media (Gupta et al., Exp. Cell Res.,
230, 244-251 (1997)) modified to contain 10% human male serum, to
exclude the ECGS and comprising 1 .mu.g/ml hydrocortisone. Washing
with this culture medium is preferred over washing with buffer
because the medium maintains higher levels of cell viability.
[0057] The buffy coat mononuclear cells were resuspended in
EGM.RTM.-2 medium (Clonetics), and all buffy coat mononuclear cells
from 100 ml of starting blood were placed into one well of a 6-well
plate coated with collagen I (Becton-Dickinson). This is defined as
day #1, on which the plate is placed into an incubator at a
controlled temperature (37.degree. C.) and humidified environment
(5% CO.sub.2). The EGM.RTM.-2 medium is changed daily. On day #2,
most of the buffy coat mononuclear cells have remained unattached
and they and cell debris were removed at the time of culture medium
change. This left on the bottom of the culture well a small number
of cells that have typical endothelial cell morphology and which
stain positively with anti-endothelial monoclonal antibody P1H12.
Typically, there were about 20-30 such cells identifiable on day
#2, along with 100-200 other mononuclear cells.
[0058] After good cell growth was established (typically at about
20 days, when cells may or may not be confluent), they are passed
into a fibronectin/gelatin-coated T25 flask, and then into
fibronectin/gelatin-coated T75 flasks. To pass the cells, the wells
are washed twice with calcium-free HBSS and then once with
0.5.times. trypsin (i.e., 50% of the concentration for the Gibco
BRL trypsin product recommended for cell lifting, because it is
less harsh than the full-strength product) plus 1 mM EDTA. Cells
detach after 2 minutes. Trypsin is neutralized by adding an equal
volume of human serum or MEC media containing 20% human serum.
Cells were collected from the solution by centrifugation at
250.times.G for 5 minutes, and resuspended in EGM.RTM.-2 medium for
continued culture.
[0059] FIG. 1 is plot of the outgrowth of endothelial cells
expanded using this method. The mean.+-.SD of five different
culture experiments using five different blood donors was plotted
up until culture passage 5, after which four different cultures
were plotted. Error bars are not evident on the graph because of
the high reproducibility of the method. As shown, the method of the
invention can result in an outgrowth, and a 10.sup.18 fold
expansion, of endothelial cells far greater than the 20-30
endothelial cells identifiable on culture day #2 (the day the
unattached mononuclear cells and cell debris from the buffy coat
are removed and any EC can be first identified).
[0060] It should be noted that this fold expansion is not a maximal
expansion. It is just the degree of expansion allowed before cells
are used for further experiments. The maximal cap (if any) in
expandability of these cells, therefore, has not yet been
established or reached in these experiments.
[0061] The outgrowth cells have the characteristics of quiescent,
microvascular EC. They have typical "cobblestone" endothelial
morphology; they exhibit positive expression for von Willebrand
Factor, P1H12 antigen, ICAM1, .alpha..sub.V.beta..sub.3,
.beta..sub.2-microglobulin, thrombomodulin, flk-1 (a VEGF
receptor), and CD34; they are uniformly CD36-positive; they take up
acetylated-LDL; they do not constitutively express tissue factor or
VCAM, but do express both upon appropriate stimulation.
EXAMPLE 2
Cyropreservation of Cultured Endothelial Cells
[0062] Vigorous EC outgrowth can be obtained even after
cryopreservation. This is important because the utility of the
present method is greater if cultured EC can be cryopreserved for
later use.
[0063] To show this, EC were cryopreserved after their outgrowth
had reached the capacity of two T75 flasks. To do this, the cells
were detached as above and washed twice with HBSS. Cells were
resuspended in 100% fetal calf serum at a concentration of
one-million cells per 950 .mu.l serum, and then 50 .mu.l
dimethylsulfoxide was added. The suspension was mixed quickly and
placed on ice for 5 minutes. Afterward, the cells were stored at
-70.degree. C. overnight, and they were then transferred to liquid
nitrogen.
[0064] After six weeks of storage, the cells were thawed and one
million cells were plated onto T75 flask with excess (20 ml) of
EGM.RTM.-2 medium. Medium was changed again four hours after
initial plating. The cells were then grown as in Example 1.
[0065] The resulting growth is similar to that of EC that were not
subjected to cryopreservation, as shown in FIG. 2. Again, data are
shown as mean.+-.SD for three experiments. Cell count at origin
indicates the number of cells thawed and started with. These data
suggest that the endothelial outgrowth cells expanded from blood
resume their vigorous growth rate after cryopreservation.
EXAMPLE 3
Study of Origin of CEC in Blood
[0066] The origin of the CEC identifiable in fresh blood is not
known, nor is the relationship between CEC and the endothelial
outgrowth from cultured blood. To answer this, 4 adults were
studied who had undergone allogeneic marrow transplantation (for
malignancy) 5-20 months earlier using opposite-gender donors (three
female donors to three male recipients and one male donor to one
female recipient). All 4 subjects are disease-free and have
peripheral blood and/or marrow aspirates that are 100% donor by
RFLP; two also had marrow cytogenetics showing 100% donor.
[0067] The CEC in fresh blood, plus the peripheral blood
endothelial outgrowth from buffy coat mononuclear cells cultured
with endothelial growth factors was studied using the culture
conditions of Ex. 1. MAb P1H12 was used to identify cells as being
endothelial, and fluorescence in situ hybridization was used to
identify cells as having XX or XY genotype.
[0068] CEC in fresh blood were almost exclusively of recipient
genotype (95, 100, 100, 88%), arguing for their origin from the
vessel wall. However, the blood endothelial outgrowth at 26-28 days
(after 100-fold expansion) in culture was mostly donor genotype
(82, 86, 77, 85%), revealing predominant origination from a
marrow-derived cell. Remarkably, endothelial outgrowth at only 9
days (after 5-fold expansion) in contemporaneous culture was still
largely recipient genotype (78, 88%). These data indicate that the
CEC detected in fresh blood are derived from vessel wall and have
some, but limited, expansion potential. Conversely, blood contains
transplantable marrow-derived cells that take longer to expand but
have greater proliferative potential. Thus, the CEC comprise a more
mature and differentiated cell population, while there also are
circulating marrow-derived endothelial-generating cells that
comprise a more primitive progenitor population (putative
angioblasts).
EXAMPLE 4
Transfection of CEC with Factor VIII Expression Vectors
[0069] Endothelial cells were expanded from blood, as described in
Example 1. For these transfection experiments, they were used after
4-5 passages. They were plated in a 6 well plate at density of
2.times.10.sup.5/well, and allowed to attach overnight. Then, cells
in different wells were transfected by lipofection with one of four
vectors as summarized in Table 1, below. TABLE-US-00001 TABLE 1
Plasmid Vectors for CEC Transfection A. Two vectors from Octagen
with B-domainless FVIII replaced with green fluorescent protein
(GFP): #1 HSQ/eGFP/ReNeo #2 HSQ/eGFP/CP B. Control vector from
Octagen with GFP but no factor VIII: #3 pEGFP-N1 C. Vector
constructed at University of Minnesota, Department of Medicine with
the FVIII/GFP from Octagen inserted between XhoI and NotI sites in
commercial vector pcDNA3.1(-): #4 pcHSQ/eGFP
[0070] All the transfection vectors contain the neomycin resistance
gene (ReNeo) as a selection marker.
[0071] Vectors 1, 2 and 4 contain an HSQ/eGFP insert which codes
for a fVIII-dGFP fusion protein. This insert comprises DNA that
codes in order of sequence, 1) the human fVIII human activation
peptide, 2) the human fVIII A1 domain, 3) the human fVIII A2
domain, 4) the first five amino acids of the SQ B domain linker
peptide, 5) the enhanced green fluorescent protein (eGFP), 6) the
last nine amino acids of the SQ linker peptide, 7) the human fVIII
light chain activation peptide, 8) the human fVIII A3 domain, 9)
the human fVIII C1 domain, and 10) the human fVIII C2 domain.
[0072] The DNA sequence of HSQ/eGFP is depicted in FIG. 3 (SEQ ID
NO:1). The DNA sequence contains 5094 bases, encoding the peptides
listed on Table 2. TABLE-US-00002 TABLE 2 HSO/eGFP Peptides SEQ ID
NO:1 1-57 signal peptide 58-1173 A1 domain 1174-2274 A2 domain
2275-2292 SQ sequence (first part) 2293-3012 eGFP sequence
3013-3039 SQ sequence (second part) 3040-3162 light chain
activation peptide 3163-4152 A3 domain 4153-4611 C1 domain
4612-5091 C2 domain 5092-5094 stop codon
[0073] ReNeo is a mammalian expression vector (non-proprietary).
The second plasmid, HSQ/eGFP/CP, contained the same fVIII construct
but the vector is the same one used to express eGFP (see below).
The eGFP protein was removed from p-EGFP-N1 and replaced with the
HSQ/eGFP construct. The third plasmid is p-EGFP-N1 from Clontech,
which expresses only eGFP.
[0074] The transfection protocol was based on the instructions
supplied by Life Technologies Co. with the lipofectamine. Briefly,
2 micrograms of plasmid DNA was mixed gently with 15 microliters of
lipofectamine in 200 microliters of E-STIM.RTM. basal medium
(Becton Dickinson) without antibiotics. The mixture was allowed to
stand at room temperature for 30 minutes. Expanded endothelial
cells in 6-well plates were washed twice with E-STIM.RTM. basal
medium before adding the DNA-liposome complex. Dropwise, 1.8 ml of
E-STIM.RTM. was added to the DNA-liposome complex, then the mixture
was overlaid on the endothelial cells. After 5 hours incubation,
the DNA-liposome mixture was replaced with the endothelial culture
medium of Example 1. Then, 2 days after transfection, the cells
from one culture well were subcultured into one 10 cm dish in
endothelial culture medium containing 50-100 .mu.g/ml of Geneticin
(Life Technologies Co.). This selection medium was changed every
other day.
[0075] Transfection of endothelial cells derived from the blood of
two donors has been accomplished to date. The number of endothelial
colonies (derived from the transfection of greater than
2.times.10.sup.5 cells) was checked on day 20 in selection medium
for two donors, as shown on Table 3. TABLE-US-00003 TABLE 3 Number
of Colonies After 20 Days in Selection Medium Vector # Donor 1
Donor 2 1 3 9 2 2 8 3 4 7 4 1 8
[0076] On day 20, fluorescence microscopy showed all selected
colonies to be positive for green fluorescent protein (GFP). On day
20, the colonies were pooled and transferred to the regular
endothelial growth condition.
[0077] In the past, many basic and applied studies had to be
performed on endothelial cells from other animal species because
existing culture techniques permitted only restricted proliferation
of human endothelial cells. The present method accomplishes at
least a 10.sup.18-fold expansion of circulating human EC. This
method will permit peripheral blood from living donors to be used
for the generation of large numbers of cultured endothelial cells.
Thus, problems of human pathology involving the endothelium now can
be approached directly by employing a human endothelial cell model.
In addition, expanded mammalian EC should prove valuable for
various clinical applications, such as in vitro testing of
vasoactive agents, the coating of artificial graft materials and
gene therapy designed to treat vascular pathologies and genetic
disorders, for local drug delivery and enhanced
biocompatibility.
EXAMPLE 5
Use of Endothelial Cell Culture Methodology in Disease
Diagnostics
[0078] Currently, there is no direct or non-invasive method to
examine a patient's endothelium to assess it for either acquired or
genetic defects that contribute to disease. A 25 year old male
patient had been evaluated for the presence of a hypercoagulability
disorder. The patient had a history of multiple thromboses, and a
family history suggesting a familial pre-disposition to thrombosis.
No positive diagnosis was obtained after extensive evaluation for
known genetic and predisposing causes. Therefore, the patient was
evaluated for a defect in thrombomodulin. Thrombomodulin is an
enzyme that is expressed on the endothelial cell surface and is an
anti-thrombotic defense. A patient having a mutation in
thrombomodulin that leads to poor function would have an increased
risk for thrombosis.
[0079] Endothelial cells were expanded from the blood of the
patient and three normal control donors using the present method.
Thrombomodulin activity was assessed in a standard biochemical
assay. Briefly, a known number of endothelial cells is incubated
with bovine protein C (100 .mu.L per well of a 1 .mu.M solution)
and human recombinant thrombin (10 .mu.L of 10 nM). It is incubated
at 37.degree. C. for 2.5 hours. The reaction is terminated by
adding EDTA amd I-2581 (Chromogenix AB, Molndal, Sweden), and the
chromatographic substrate S-2366 (DiaPharma Group, Inc., Franklin,
Ohio) is added. Supernatant absorbance is read at 405 nm.
[0080] The results are given in OD units per minute per 50,000
endothelial cells. The value for 18 samples from the three normal
donors was 53.+-.7, while the value for six samples from the
patient was 41.+-.5, a significant difference (p=0.0034). Thus, the
expanded endothelial cells from the patient had a significantly
lower thrombomodulin activity than those of control patients.
Therefore, a genetic mutation in thrombomodulin resulting in a
thrombomodulin deficiency may be the underlying basis for this
patient's disorder. Molecular biological analyses are employed to
confirm the genetic basis for the thrombomodulin deficiency.
EXAMPLE 6
Stable Transfection, Selection and Expansion
[0081] The plasmid pLE (Clontech), which contains sequence for
enhanced green fluorescent protein (eGFP), was transfected into
line PA317. Virus from one of the resulting cloned transfectants,
pLE9, was employed to infect endothelial cells.
[0082] A culture of blood buffy coat mononuclear cells was set up
according to the method of the invention. When cells had reached
passage 4 and about a 10.sup.6 fold expansion, they were seeded at
10.sup.5 cells/dish, with the 10 cm dishes coated with 6
microgram/cm.sup.2 of type 1 collagen and 250 microgram/dish of
fibronectin in 1% gelatin. After two days of growth, the cells were
exposed to conditioned medium from the packaging cell line in
combination with 4 microgram/ml of polybrene to dishes that were
70% confluent. After three days exposure, cells were split 1:2 into
a 10 cm dish (coated as above), and new culture medium added. Then
they were subjected to selection in media containing G418 for 19
days.
[0083] As shown in FIG. 5, cultured endothelial cells prepared
according to the method of the invention can survive chemical
selection and be expanded thereafter, and be stably transfected to
express a foreign (non-selected) gene. Moreover, the outgrowth
cells have preserved normal endothelial cell morphology.
EXAMPLE 7
Transfection of CEC with Human Factor VIII (hFVIII) Expression
Vectors
Materials and Methods
Transfection
[0084] Ten cm dishes and T75 flasks were coated with 6 .mu.g/ml of
type I collagen and 50 .mu.g/ml of fibronectin. Passage 4 or 5
outgrowth endothelial cells were obtained as described in Example
1. They were seeded into the dish or flasks and allowed to grow to
40% confluence. Then the cells were transfected with hFVIII vectors
(Table 4) using transfection reagent Fugene6 (Roche Molecular
Biochemicals) following the manufacturer's protocol. Briefly,
Fugene6 was added to each vector at a ratio of 2.5:1. Ten .mu.g of
DNA was used for transfection of cells in 10 cm dishes; and 15
.mu.g of DNA was used for cells in T75 flasks.
[0085] The Fugene6-DNA mixture was added to the cells in media
containing 10% human serum and incubated for 3 days. Cells reached
100% confluence after 3 days. TABLE-US-00004 TABLE 4 Plasmid
Vectors pTracerHSQ: has HSQ (the B-domainless hFVIII without the
eGFP insert) cloned into pTracer-CMV (Invitrogen) pcF8G: has
HSQ/eGFP (eGFP replaces the B domain of hFVIII) cloned into plasmid
pCDNA 3.1 (Invitrogen) pcHSQ: has HSQ cloned into plasmid pCDNA 3.1
(Invitrogen) pTracerCMV: obtained from Invitrogen HSQ/GFP/ReNeo:
has HSQ/eGFP cloned into pRENeo (Biogen)
Transient Expression
[0086] At the end of 3 days, the conditioned media from T75 flasks
were collected and centrifuged. The presence of hFVIII in the
supernatant was detected using an ELISA kit (American Diagnostics).
Cells from T75 flask were harvested, lysed and processed (RT-PCR,
Titan One Tube RT-PCR System, Roche Molecular Biochemicals) for
detection of hFVIII mRNA. A reverse transcriptase reaction was
conducted at 50.degree. C. for 45 minutes, then the samples were
subject to 94.degree. C., for 2 minutes; 10 cycles of 94.degree. C.
for 30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for
1 minute; 20 cycles of 94.degree. C. for 30 seconds, 55.degree. C.
for 30 seconds, and 72.degree. C. for 1 minute, plus cycle
elongation of 5 seconds for each cycle elongation at 72.degree. C.
for 7 minutes. The primer pair employed was hFVIIIC-S (5' GCC CTT
TTC TTG GAT CAA GGT GG3'; SEQ ID NO: 3) and hFVIIIC-AS (5' CTC CCT
GAG TAG TTA CTC CTG TG3'; SEQ ID NO: 4).
Results
[0087] Three days after infection, HFVIII was detected in the
supernatant of transfected cells (FIG. 6) but not in control cells.
FIG. 7 shows the results of a RT-PCR analysis of RNA from those
cells. The expression of hFVIII mRNA is increased in transfected
cells relative to control cells.
[0088] At the end of 3 days, cells from 10 cm dishes were split
into 2 10 cm dishes coated with 6 .mu.g/ml of type I collagen and
50 .mu.g/ml of fibronectin. The following day, cells were exposed
to selection medium EGM-2 (Clonectis) containing 50 to 100 .mu.g/ml
of G418 or 50 .mu.g/ml of Zeocin or both (Zeocin was used for
selection of pTracerHSQ; G418 for pcFVIIIG and HSQ/GFP/ReNeo; and
both Zeocin and G418 were used for cells subjected to
cotransfection). Stable expression of hFVIII is detected in these
cells after chemical selection.
[0089] All patents, patent applications and publications cited
hereinabove, are incorporated by reference herein. While a
preferred embodiment of the present invention has been described,
it should be understood that various changes, adaptations and
modifications may be made therein without departing from the spirit
of the invention and the scope of the appended claims.
Sequence CWU 1
1
4 1 5094 DNA Artificial Sequence The DNA sequence of HSQ/eGFP. 1
atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc
60 accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca
aagtgatctc 120 ggtgagctgc ctgtggacgc aagatttcct cctagagtgc
caaaatcttt tccattcaac 180 acctcagtcg tgtacaaaaa gactctgttt
gtagaattca cggttcacct tttcaacatc 240 gctaagccaa ggccaccctg
gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300 gatacagtgg
tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360
ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg
420 gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg
gcaggtcctg 480 aaagagaatg gtccaatggc ctctgaccca ctgtgcctta
cctactcata tctttctcat 540 gtggacctgg taaaagactt gaattcaggc
ctcattggag ccctactagt atgtagagaa 600 gggagtctgg ccaaggaaaa
gacacagacc ttgcacaaat ttatactact ttttgctgta 660 tttgatgaag
ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720
gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct
780 ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat
tggaatgggc 840 accactcctg aagtgcactc aatattcctc gaaggtcaca
catttcttgt gaggaaccat 900 cgccaggcgt ccttggaaat ctcgccaata
actttcctta ctgctcaaac actcttgatg 960 gaccttggac agtttctact
gttttgtcat atctcttccc accaacatga tggcatggaa 1020 gcttatgtca
aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080
gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat
1140 gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca
tcctaaaact 1200 tgggtacatt acattgctgc tgaagaggag gactgggact
atgctccctt agtcctcgcc 1260 cccgatgaca gaagttataa aagtcaatat
ttgaacaatg gccctcagcg gattggtagg 1320 aagtacaaaa aagtccgatt
tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380 attcagcatg
aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440
ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact
1500 gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt
gaaggatttt 1560 ccaattctgc caggagaaat attcaaatat aaatggacag
tgactgtaga agatgggcca 1620 actaaatcag atcctcggtg cctgacccgc
tattactcta gtttcgttaa tatggagaga 1680 gatctagctt caggactcat
tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740 agaggaaacc
agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800
aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg
1860 cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa
tggctatgtt 1920 tttgatagtt tgcagttgtc agtttgtttg catgaggtgg
catactggta cattctaagc 1980 attggagcac agactgactt cctttctgtc
ttcttctctg gatatacctt caaacacaaa 2040 atggtctatg aagacacact
caccctattc ccattctcag gagaaactgt cttcatgtcg 2100 atggaaaacc
caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160
atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac
2220 agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga
acctaggagc 2280 ttctctcaga atatggtgag caagggcgag gagctgttca
ccggggtggt gcccatcctg 2340 gtcgagctgg acggcgacgt aaacggccac
aagttcagcg tgtccggcga gggcgagggc 2400 gatgccacct acggcaagct
gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 2460 ccctggccca
ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 2520
gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
2580 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
gaagttcgag 2640 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
acttcaagga ggacggcaac 2700 atcctggggc acaagctgga gtacaactac
aacagccaca acgtctatat catggccgac 2760 aagcagaaga acggcatcaa
ggtgaacttc aagatccgcc acaacatcga ggacggcagc 2820 gtgcagctcg
ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 2880
cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
2940 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
catggacgag 3000 ctgtacaagt atccaccagt cttgaaacgc catcaacggg
aaataactcg tactactctt 3060 cagtcagatc aagaggaaat tgactatgat
gataccatat cagttgaaat gaagaaggaa 3120 gattttgaca tttatgatga
ggatgaaaat cagagccccc gcagctttca aaagaaaaca 3180 cgacactatt
ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 3240
catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc
3300 caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact
aaatgaacat 3360 ttgggactcc tggggccata tataagagca gaagttgaag
ataatatcat ggtaactttc 3420 agaaatcagg cctctcgtcc ctattccttc
tattctagcc ttatttctta tgaggaagat 3480 cagaggcaag gagcagaacc
tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 3540 ttttggaaag
tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 3600
gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt
3660 ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac
agtacaggaa 3720 tttgctctgt ttttcaccat ctttgatgag accaaaagct
ggtacttcac tgaaaatatg 3780 gaaagaaact gcagggctcc ctgcaatatc
cagatggaag atcccacttt taaagagaat 3840 tatcgcttcc atgcaatcaa
tggctacata atggatacac tacctggctt agtaatggct 3900 caggatcaaa
ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3960
attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg
4020 tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa
agctggaatt 4080 tggcgggtgg aatgccttat tggcgagcat ctacatgctg
ggatgagcac actttttctg 4140 gtgtacagca ataagtgtca gactcccctg
ggaatggctt ctggacacat tagagatttt 4200 cagattacag cttcaggaca
atatggacag tgggccccaa agctggccag acttcattat 4260 tccggatcaa
tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 4320
ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc
4380 ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg
gcagacttat 4440 cgaggaaatt ccactggaac cttaatggtc ttctttggca
atgtggattc atctgggata 4500 aaacacaata tttttaaccc tccaattatt
gctcgataca tccgtttgca cccaactcat 4560 tatagcattc gcagcactct
tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 4620 atgccattgg
gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 4680
tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg
4740 agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt
ggacttccag 4800 aagacaatga aagtcacagg agtaactact cagggagtaa
aatctctgct taccagcatg 4860 tatgtgaagg agttcctcat ctccagcagt
caagatggcc atcagtggac tctctttttt 4920 cagaatggca aagtaaaggt
ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4980 tctctagacc
caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 5040
cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta ctga 5094 2
12445 DNA Artificial Sequence The DNA sequence of HSQRENeo. 2
gaattccgga attccagctt gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
60 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
aaccaggtgt 120 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
gcatgcatct caattagtca 180 gcaaccatag tcccgcccct aactccgccc
atcccgcccc taactccgcc cagttccgcc 240 cattctccgc cccatggctg
actaattttt tttatttatg cagaggccga ggccgcctcg 300 gcctctgagc
tattccagaa gtagtgagga ggcttttttg gaggggtcct cctcgtatag 360
aaactcggac cactctgaga cgaaggctcg cgtccaggcc agcacgaagg aggctaagtg
420 ggaggggtag cggtcgttgt ccactagggg gtccactcgc tccagggtgt
gaagacacat 480 gtcgccctct tcggcatcaa ggaaggtgat tggtttatag
gtgtaggcca cgtgaccggg 540 tgttcctgaa gggggggtat aaaagggggt
gggggcgcgt tcgtcctcac tctcttccgc 600 atcgctgtct gcgagggcca
gctgttgggc tcgcggttga ggacaaactc ttcgcggtct 660 ttccagtact
cttggatcgg aaacccgtcg gcctccgaac ggtactccgc caccgaggga 720
cctgagcgag tccgcatcga ccggatcgga aaacctctcg agccaccatg caaatagagc
780 tctccacctg cttctttctg tgccttttgc gattctgctt tagtgccacc
agaagatact 840 acctgggtgc agtggaactg tcatgggact atatgcaaag
tgatctcggt gagctgcctg 900 tggacgcaag atttcctcct agagtgccaa
aatcttttcc attcaacacc tcagtcgtgt 960 acaaaaagac tctgtttgta
gaattcacgg ttcacctttt caacatcgct aagccaaggc 1020 caccctggat
gggtctgcta ggtcctacca tccaggctga ggtttatgat acagtggtca 1080
ttacacttaa gaacatggct tcccatcctg tcagtcttca tgctgttggt gtatcctact
1140 ggaaagcttc tgagggagct gaatatgatg atcagaccag tcaaagggag
aaagaagatg 1200 ataaagtctt ccctggtgga agccatacat atgtctggca
ggtcctgaaa gagaatggtc 1260 caatggcctc tgacccactg tgccttacct
actcatatct ttctcatgtg gacctggtaa 1320 aagacttgaa ttcaggcctc
attggagccc tactagtatg tagagaaggg agtctggcca 1380 aggaaaagac
acagaccttg cacaaattta tactactttt tgctgtattt gatgaaggga 1440
aaagttggca ctcagaaaca aagaactcct tgatgcagga tagggatgct gcatctgctc
1500 gggcctggcc taaaatgcac acagtcaatg gttatgtaaa caggtctctg
ccaggtctga 1560 ttggatgcca caggaaatca gtctattggc atgtgattgg
aatgggcacc actcctgaag 1620 tgcactcaat attcctcgaa ggtcacacat
ttcttgtgag gaaccatcgc caggcgtcct 1680 tggaaatctc gccaataact
ttccttactg ctcaaacact cttgatggac cttggacagt 1740 ttctactgtt
ttgtcatatc tcttcccacc aacatgatgg catggaagct tatgtcaaag 1800
tagacagctg tccagaggaa ccccaactac gaatgaaaaa taatgaagaa gcggaagact
1860 atgatgatga tcttactgat tctgaaatgg atgtggtcag gtttgatgat
gacaactctc 1920 cttcctttat ccaaattcgc tcagttgcca agaagcatcc
taaaacttgg gtacattaca 1980 ttgctgctga agaggaggac tgggactatg
ctcccttagt cctcgccccc gatgacagaa 2040 gttataaaag tcaatatttg
aacaatggcc ctcagcggat tggtaggaag tacaaaaaag 2100 tccgatttat
ggcatacaca gatgaaacct ttaagactcg tgaagctatt cagcatgaat 2160
caggaatctt gggaccttta ctttatgggg aagttggaga cacactgttg attatattta
2220 agaatcaagc aagcagacca tataacatct accctcacgg aatcactgat
gtccgtcctt 2280 tgtattcaag gagattacca aaaggtgtaa aacatttgaa
ggattttcca attctgccag 2340 gagaaatatt caaatataaa tggacagtga
ctgtagaaga tgggccaact aaatcagatc 2400 ctcggtgcct gacccgctat
tactctagtt tcgttaatat ggagagagat ctagcttcag 2460 gactcattgg
ccctctcctc atctgctaca aagaatctgt agatcaaaga ggaaaccaga 2520
taatgtcaga caagaggaat gtcatcctgt tttctgtatt tgatgagaac cgaagctggt
2580 acctcacaga gaatatacaa cgctttctcc ccaatccagc tggagtgcag
cttgaggatc 2640 cagagttcca agcctccaac atcatgcaca gcatcaatgg
ctatgttttt gatagtttgc 2700 agttgtcagt ttgtttgcat gaggtggcat
actggtacat tctaagcatt ggagcacaga 2760 ctgacttcct ttctgtcttc
ttctctggat ataccttcaa acacaaaatg gtctatgaag 2820 acacactcac
cctattccca ttctcaggag aaactgtctt catgtcgatg gaaaacccag 2880
gtctatggat tctggggtgc cacaactcag actttcggaa cagaggcatg accgccttac
2940 tgaaggtttc tagttgtgac aagaacactg gtgattatta cgaggacagt
tatgaagata 3000 tttcagcata cttgctgagt aaaaacaatg ccattgaacc
taggagcttc tctcagaatc 3060 caccagtctt gaaacgccat caacgggaaa
taactcgtac tactcttcag tcagatcaag 3120 aggaaattga ctatgatgat
accatatcag ttgaaatgaa gaaggaagat tttgacattt 3180 atgatgagga
tgaaaatcag agcccccgca gctttcaaaa gaaaacacga cactatttta 3240
ttgctgcagt ggagaggctc tgggattatg ggatgagtag ctccccacat gttctaagaa
3300 acagggctca gagtggcagt gtccctcagt tcaagaaagt tgttttccag
gaatttactg 3360 atggctcctt tactcagccc ttataccgtg gagaactaaa
tgaacatttg ggactcctgg 3420 ggccatatat aagagcagaa gttgaagata
atatcatggt aactttcaga aatcaggcct 3480 ctcgtcccta ttccttctat
tctagcctta tttcttatga ggaagatcag aggcaaggag 3540 cagaacctag
aaaaaacttt gtcaagccta atgaaaccaa aacttacttt tggaaagtgc 3600
aacatcatat ggcacccact aaagatgagt ttgactgcaa agcctgggct tatttctctg
3660 atgttgacct ggaaaaagat gtgcactcag gcctgattgg accccttctg
gtctgccaca 3720 ctaacacact gaaccctgct catgggagac aagtgacagt
acaggaattt gctctgtttt 3780 tcaccatctt tgatgagacc aaaagctggt
acttcactga aaatatggaa agaaactgca 3840 gggctccctg caatatccag
atggaagatc ccacttttaa agagaattat cgcttccatg 3900 caatcaatgg
ctacataatg gatacactac ctggcttagt aatggctcag gatcaaagga 3960
ttcgatggta tctgctcagc atgggcagca atgaaaacat ccattctatt catttcagtg
4020 gacatgtgtt cactgtacga aaaaaagagg agtataaaat ggcactgtac
aatctctatc 4080 caggtgtttt tgagacagtg gaaatgttac catccaaagc
tggaatttgg cgggtggaat 4140 gccttattgg cgagcatcta catgctggga
tgagcacact ttttctggtg tacagcaata 4200 agtgtcagac tcccctggga
atggcttctg gacacattag agattttcag attacagctt 4260 caggacaata
tggacagtgg gccccaaagc tggccagact tcattattcc ggatcaatca 4320
atgcctggag caccaaggag cccttttctt ggatcaaggt ggatctgttg gcaccaatga
4380 ttattcacgg catcaagacc cagggtgccc gtcagaagtt ctccagcctc
tacatctctc 4440 agtttatcat catgtatagt cttgatggga agaagtggca
gacttatcga ggaaattcca 4500 ctggaacctt aatggtcttc tttggcaatg
tggattcatc tgggataaaa cacaatattt 4560 ttaaccctcc aattattgct
cgatacatcc gtttgcaccc aactcattat agcattcgca 4620 gcactcttcg
catggagttg atgggctgtg atttaaatag ttgcagcatg ccattgggaa 4680
tggagagtaa agcaatatca gatgcacaga ttactgcttc atcctacttt accaatatgt
4740 ttgccacctg gtctccttca aaagctcgac ttcacctcca agggaggagt
aatgcctgga 4800 gacctcaggt gaataatcca aaagagtggc tgcaagtgga
cttccagaag acaatgaaag 4860 tcacaggagt aactactcag ggagtaaaat
ctctgcttac cagcatgtat gtgaaggagt 4920 tcctcatctc cagcagtcaa
gatggccatc agtggactct cttttttcag aatggcaaag 4980 taaaggtttt
tcagggaaat caagactcct tcacacctgt ggtgaactct ctagacccac 5040
cgttactgac tcgctacctt cgaattcacc cccagagttg ggtgcaccag attgccctga
5100 ggatggaggt tctgggctgc gaggcacagg acctctactg agggcggccg
ctgcagcacc 5160 tgccactgcc gtcacctctc cctcctcagc tccagggcag
tgtccctccc tggcttgcct 5220 tctacctttg tgctaaatcc tagcagacac
tgccttgaag cctcctgaat taactatcat 5280 cagtcctgca tttctttggt
ggggggccag gagggtgcat ccaatttaac ttaactctta 5340 cctattttct
gcagctgctc ccagattact ccttccttcc aatataacta ggcaaaaaga 5400
agtgaggaga aacctgcatg aaagcattct tccctgaaaa gttaggcctc tcagagtcac
5460 cacttcctct gttgtagaaa aactatgtga tgaaactttg aaaaagatat
ttatgatgtt 5520 aacatttcag gttaagcctc atacgtttaa aataaaactc
tcagttgttt attatcctga 5580 tcaagcatgg aacaaagcat gtttcaggat
cagatcaata caatcttgga gtcaaaaggc 5640 aaatcatttg gacaatctgc
aaaatggaga gaatacaata actactacag taaagtctgt 5700 ttctgcttcc
ttacacatag atataattat gttatttagt cattatgagg ggcacattct 5760
tatctccaaa actagcattc ttaaactgag aattatagat ggggttcaag aatccctaag
5820 tcccctgaaa ttatataagg cattctgtat aaatgcaaat gtgcattttt
ctgacgagtg 5880 tccatagata tgggacatat gacgtgagct cagatctttg
tgaaggaacc ttacttctgt 5940 ggtgtgacat aattggacaa actacctaca
gagatttaaa gctctaaggt aaatataaaa 6000 tttttaagtg tataatgtgt
taaactactg attctaattg tttgtgtatt ttagattcca 6060 acctatggaa
ctgatgaatg ggagcagtgg tggaatgcct ttaatgagga aaacctgttt 6120
tgctcagaag aaatgccatc tagtgatgat gaggctactg ctgagtgtga acattctact
6180 cctccaaaaa agaagagaaa ggtagaagac cccaaggact ttccttcaga
attgctaagt 6240 tttttgagtc atgctgtgtt tagtaataga actcttgctt
gctttgctat ttacaccaca 6300 aaggaaaaag ctgcactgct atacaagaaa
attatggaaa aatattctgt aacctttata 6360 agtaggcata acagttataa
tcataacata ctgttttttc ttactccaca caggcataga 6420 gtgtctgcta
ttaataacta tgctcaaaaa ttgtgtacct ttagcttttt aatttgtaaa 6480
ggggttaata aggaatattt gatgtatagt gccttgacta gagatcataa tcagccatac
6540 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
tgaacctgaa 6600 acataaaatg aatgcaattg ttgttgttaa cttgtttatt
gcagcttata atggttacaa 6660 ataaagcaat agcatcacaa atttcacaaa
taaagcattt ttttcactgc attctagttg 6720 tggtttgtcc aaactcatca
atgtatctta tcatgtctgg atcctctacg ccggacgcat 6780 cgtggccggc
atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac 6840
cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
6900 ggtggcaggc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc
accattcctt 6960 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
gcttcctaat gcaggagtcg 7020 cataagggag agcgtcgaaa ttctcatgtt
tgacagctta tcatcggcgc agcaccatgg 7080 cctgaaataa cctctgaaag
aggaacttgg ttaggtacct tctgaggcgg aaagaaccag 7140 ctgtggaatg
tgtgtcagtt agggtgtgga aagtccccag gctggggagc aggcagaagt 7200
atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
7260 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
cccgccccta 7320 actccgccca tcccgcccct aactccgccc agttccgccc
attctccgcc ccatggctga 7380 ctaatttttt ttatttatgc agaggccgag
gccgcctcgg cctctgagct attccagccg 7440 tagtgaggag gcttttttgg
aggcctaggc ttttgcaaaa agcttcacgc tgccgcaagc 7500 actcagggcg
caagggctgc taaaggaagc ggaacacgta gaaagccagt ccgcagaaac 7560
ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg
7620 caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac
tgggcggttt 7680 tatggacagc aagcgaaccg gaattgccag ctggggcgcc
ctctggtaag gttgggaagc 7740 cctgcaaagt aaactggatg gctttcttgc
cgccaaggat ctgatggcgc aggggatcaa 7800 gatctgatca agagacagga
tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 7860 caggttctcc
ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 7920
tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
7980 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
cggctatcgt 8040 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
cgttgtcact gaagcgggaa 8100 gggactggct gctattgggc gaagtgccgg
ggcaggatct cctgtcatct caccttgctc 8160 ctgccgagaa agtatccatc
atggctgatg caatgcggcg gctgcatacg cttgatccgg 8220 ctacctgccc
attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 8280
aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
8340 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
gtgacccatg 8400 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
cttttctgga ttcatcgact 8460 gtggccggct gggtgtggcg gaccgctatc
aggacatagc gttggctacc cgtgatattg 8520 ctgaagagct tggcggcgaa
tgggctgacc gcttcctcgt gctttacggt atcgccgctc 8580 ccgattcgca
gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct 8640
ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
8700 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
gctggatgat 8760 cctccagcgc ggggatctca tgctggagtt cttcgcccac
cccgggctcg atcccctcgc 8820 gagttggttc agctgctgcc tgaggctgga
cgacctcgcg gagttctacc ggcagtgcaa 8880 atccgtcggc atccaggaaa
ccagcagcgg ctatccgcgc atccatgccc ccgaactgca 8940 ggagtgggga
ggcacgatgg ccgctttggt cccggatctt tgtgaaggaa ccttacttct 9000
gtggtgtgac ataattggag aaactaccta cagagattta aagctctaag gtaaatataa
9060 aatttttaag tgtataatgt gttaaactac tgattctaat tgtttgtgta
ttttagattc 9120 caacctatgg aactgatgaa tgggagcagt ggtggaatgc
ctttaatgag gaaaacctgt 9180 tttgctcaga agaaatgcca tctagtgatg
atgaggctac tgctgactct caacattcta 9240 ctcctccaaa aaagaagaga
aaggtagaag accccaagga ctttccttca gaattgctaa 9300 gttttttgag
tcatgctgtg tttagtaata gaactcttgc ttgctttgct atttacacca 9360
caaaggaaaa agctgcactg ctatacaaga aaattatgga aaaatattct gtaaccttta
9420 taagtaggca taacagttat aatcataaca tactgttttt tcttactcca
cacaggcata 9480 gagtgtctgc tattaataac tatgctcaaa aattgtgtac
ctttagcttt ttaatttgta 9540 aaggggttaa taaggaatat ttgatgtata
gtgccttgac tagagatcat aatcagccat 9600 accacatttg tagaggtttt
acttgcttta aaaaacctcc cacacctccc cctgaacctg 9660 aaacataaaa
tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac 9720
aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
9780 tgtggtttgt ccaaactcat caatggtatc ttatcatgtc tggatctcga
ccgagccctt 9840
gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
9900 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
cgctctgggt 9960 cattttcggc gaggaccgct ttcgctggag cgcgacgatg
atcggcctgt cgcttgcggt 10020 attcggaatc ttgcacgccc tcgctcaagc
cttcgtcact ggtcccgcca ccaaacgttt 10080 cggcgagaag caggccatta
tcgccggcat ggcggccgac gcgctgggct acgtcttgct 10140 ggcgttcgcg
acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg 10200
catcgggatg cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg
10260 acagcttcaa ggatcgctcg cggctcttac cagcctaact tcgatcactg
gaccgctgat 10320 cgtcacggcg atttatgccg cctcggcgag cacatggaac
gggttggcat ggattgtagg 10380 cgccgcccta taccttgtct gcctccccgc
gttgcgtcgc ggtgcatgga gccgggccac 10440 ctcgacctga atggaagccg
gcggcacctc gctaacggat tcaccactcc aagaattgga 10500 gccaatcaat
tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc 10560
atcgcgtccg ccatctccag cagccgcacg cggcgcatct cgggccgcgt tgctggcgtt
10620 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
gtcagaggtg 10680 gcgaaacccg acaggactat aaagatacca ggcgtttccc
cctggaagct ccctcgtgcg 10740 ctctcctgtt ccgaccctgc cgcttaccgg
atacctgtcc gcctttctcc cttcgggaag 10800 cgtggcgctt tctcaatgct
cacgctgtac ctatctcagt tcggtgtacc tcgttcgctc 10860 caagctgggc
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 10920
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
10980 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
agtggtggcc 11040 taactacggc tacactagaa ggacagtatt tggtatctgc
gctctgctga agccagttac 11100 cttcggaaaa agagttggta gctcttgatc
cggcaaacaa accaccgctg gtagcggtgg 11160 tttttttgtt tgcaagcagc
agattacgcg cagaaaaaaa ggatctcaag aagatccttt 11220 gatcttttct
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 11280
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
11340 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
taatcagtga 11400 ggcacctatc tcagcgatct gtctatttcg ttcatccata
gttgcctgac tccccgtcgt 11460 gtagataact acgatacggg agggcttacc
atctggcccc agtgctgcaa tgataccgcg 11520 agacccacgc tcaccggctc
cagatttatc agcaataaac cagccagcca gaagggccga 11580 gcgcagaagt
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 11640
agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg
11700 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
cccaacgatc 11760 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
gttagctcct tcggtcctcc 11820 gatcgttgtc agaagtaagt tggccgcagt
gttatcactc atggttatgg cagcactgca 11880 taattctctt actgtcatgc
catccgtaag atgcttttct gtgactggtg agtactcaac 11940 caagtcattc
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg 12000
ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
12060 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
aacccactcg 12120 tgcacccaac tgatcttcag catcttttac tttcaccagc
gtttctgggt gagcaaaaac 12180 aggaaggcaa aatgccgcaa aaaagggaat
aagggcgaca cggaaatgtt gaatactcat 12240 actcttcctt tttcaatatt
attgaagcat ttatcagggt tattgtctca tgagcggata 12300 catatttgaa
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 12360
agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
12420 tatcacgagg ccctttcgtc ttcaa 12445 3 23 DNA Homo sapiens 3
gcccttttct tggatcaagg tgg 23 4 23 DNA Homo sapiens 4 ctccctgagt
agttactcct gtg 23
* * * * *