U.S. patent application number 10/701331 was filed with the patent office on 2004-07-01 for delivery vehicle for recombinant proteins.
Invention is credited to Cines, Douglas B., Poncz, Mortimer.
Application Number | 20040126885 10/701331 |
Document ID | / |
Family ID | 32659305 |
Filed Date | 2004-07-01 |
United States Patent
Application |
20040126885 |
Kind Code |
A1 |
Cines, Douglas B. ; et
al. |
July 1, 2004 |
Delivery vehicle for recombinant proteins
Abstract
Recombinant nucleic acid molecules are constructed with a first
sequence encoding a transgene under the control of regulatory
sequences that direct expression of the transgene product in a
hematopoietic stem cell, or a progenitor cell therefrom or cell
differentiated therefrom. In one embodiment, the cell which
expresses the transgene is a secretory cell. The cell is a
megakaryotic progenitor cell, or a cell further differentiated
therefrom, such as a platelet. The cell is a granulocyte/macrophage
progenitor cell or a cell further differentiated therefrom, such as
a mast cell or neutrophils. Such host cells containing the molecule
or the molecule itself are employed in methods for treating or
preventing infection, inflammation or vascular injuries or any
disorders involving or mediated by cells of the hematopoietic
lineage.
Inventors: |
Cines, Douglas B.;
(Wynnewood, PA) ; Poncz, Mortimer; (Wynnewood,
PA) |
Correspondence
Address: |
HOWSON AND HOWSON
ONE SPRING HOUSE CORPORATION CENTER
BOX 457
321 NORRISTOWN ROAD
SPRING HOUSE
PA
19477
US
|
Family ID: |
32659305 |
Appl. No.: |
10/701331 |
Filed: |
November 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60424234 |
Nov 5, 2002 |
|
|
|
Current U.S.
Class: |
435/455 ;
435/372; 514/44R; 536/23.1 |
Current CPC
Class: |
A01K 2267/03 20130101;
C07K 14/721 20130101; A01K 2267/0381 20130101; A61K 2035/124
20130101; C12N 5/0644 20130101; A61K 48/00 20130101; C07K 14/522
20130101; C12N 2517/02 20130101; A01K 2217/05 20130101; A01K
2227/105 20130101; C12N 2830/85 20130101; C12N 2830/008 20130101;
C12N 15/8509 20130101; A01K 67/0275 20130101 |
Class at
Publication: |
435/455 ;
435/372; 514/044; 536/023.1 |
International
Class: |
C12N 005/08; C12N
015/85 |
Goverment Interests
[0002] The present invention was supported, in part, by the
National Institutes of Health, Grant Nos. HL54749, HL64190,
HL60169, HL66442, HL47826 and HL63194. The United States government
has an interest in this invention.
Claims
1. A recombinant nucleic acid molecule comprising a first sequence
encoding a transgene under the control of regulatory sequences that
direct expression of said transgene in a hematopoietic stem cell, a
progenitor cell or cell differentiated therefrom.
2. The molecule according to claim 1, wherein said differentiated
cell is a secretory cell.
3. The molecule according to claim 1, wherein said progenitor cell
is selected from the group consisting of common lymphoid
progenitor, common myeloid progenitor, megakaryotic/erythrocyte
progenitor and granulocytes/macrophage progenitor.
4. The molecule according to claim 3, wherein said cells
differentiated from megakaryotic/erythrocyte progenitor cells are
selected from the group consisting of platelets, megakaryocytes and
erythrocytes.
5. The molecule according to claim 3, wherein said cells
differentiated from granulocyte/macrophage progenitor cells are
selected from the group consisting of neutrophils, eosinophils,
monocytes, basophils and immature dendritic cells.
6. The molecule according to claim 3, wherein said cells
differentiated from said lymphoid progenitors are natural killer
cells.
7. The molecule according to claim 5, wherein said cells
differentiated from said monocytes are selected from the group
consisting of mast cells, macrophages and dendritic cells.
8. The molecule according to claim 1, wherein said nucleic acid
molecule is a viral or non-viral vector.
9. The molecule according to claim 1, wherein said regulatory
sequence is a platelet-specific promoter.
10. The molecule according to claim 9, wherein said promoter is
selected from the group consisting of the Platelet factor 4
promoter, the glycoprotein IIb promoter, the glycoprotein IIIa
promoter, and the glycoprotein VI promoter.
11. The molecule according to claim 1, wherein said regulatory
sequence is a neutrophil-specific promoter.
12. The molecule according to claim 11, wherein said promoter is
selected from the group consisting of DEFA1 human neutrophil alpha
defensin promoter, the DEFA2 human neutrophil alpha defensin
promoter, DEFA3 human neutrophil alpha defensin promoter, and the
DEFA4 human neutrophil alpha defensin promoter.
13. The molecule according to claim 1, wherein said regulatory
sequence is a natural killer cell-specific promoter.
14. The molecule according to claim 13, wherein said promoter is
the human perforin gene promoter.
15. The molecule according to claim 1, wherein said regulatory
sequence is an eosinophil-specific promoter.
16. The molecule according to claim 15, wherein said promoter is
selected from the group consisting of the human eotaxin gene
promoter and the eosinophil peroxidase gene promoter.
17. The molecule according to claim 1, wherein said regulatory
sequence is an erythrocyte-specific promoter.
18. The molecule according to claim 17 wherein said promoter is the
human RhD gene promoter.
19. A hematopoietic stem cell transformed, transduced, infected or
transfected with a nucleic acid molecule of claim 1.
20. A host cell differentiated from a hematopoietic stem cell
transformed, transduced, infected or transfected with a nucleic
acid molecule of claim 1.
21. The host cell according to claim 20, selected from the group
consisting of a common lymphoid progenitor cell, a common myeloid
progenitor cell, a megakaryotic/erythrocyte progenitor cell, a
granulocyte/macrophage progenitor cell, platelets, megakaryocytes,
neutrophils, eosinophils, monocytes, basophils, dendritic cells,
mast cells, macrophages, dendritic cells, erythrocytes, and natural
killer cells.
22. A platelet transformed, transduced, infected or transfected
with a nucleic acid molecule comprising a first sequence encoding a
transgene under the control of regulatory sequences that direct
expression of said transgene in said platelet.
23. A method for generating a modified hematopoietic stem cell,
modified progenitor cell or a modified cell differentiated from
said stem cell or progenitor cell comprising the step of
transferring a nucleic acid molecule of claim 1 into said cell via
transformation, transduction, infection or transfection.
24. The method according to claim 23, further comprising the step
of harvesting said stem cells or progenitor cells of the
hematopoietic lineage from bone marrow of a mammal prior to said
transferring step, wherein said transferring step occurs in vitro
or ex vivo.
25. The method according to claim 23, further comprising the step
of harvesting said cells differentiated from said hematopoietic
stem cells or progenitor cells from peripheral blood of a mammal
prior to said transferring step, wherein said transferring step
occurs in vitro or ex vivo.
26. A method for treating a disorder in a mammal comprising the
steps of delivering to said mammal a recombinant nucleic acid
molecule comprising a first sequence comprising a transgene
encoding a product under the control of regulatory sequences that
direct expression of the product of said transgene in a
hematopoietic stem cell, a progenitor cell of the hematopoietic
lineage, or a cell differentiated therefrom.
27. The method according to claim 26, wherein a differentiated cell
produces said product at a suitable site in said mammal.
28. The method according to claim 26, wherein delivering step
comprises (a) harvesting said stem cells or progenitor cells from
bone marrow of said mammal; and (b) transferring said nucleic acid
molecule into said cells.
29. The method according to claim 28 further comprising reinfusing
said cells into the bone marrow of said mammal.
30. The method according to claim 26, wherein delivering step
comprises (a) harvesting said differentiated cells from peripheral
blood of said mammal; and (b) transferring said nucleic acid
molecule into said differentiated cells.
31. The method according to claim 30, further comprising reinfusing
said cells into the blood of said mammal.
32. The method according to claim 26, wherein said delivering step
comprises administering said nucleic acid molecule directly into
the mammal.
33. A method for treating or preventing thrombus formation in a
mammal comprising delivering to a mammalian patient a recombinant
nucleic acid molecule comprising a first sequence comprising a
transgene encoding a fibrinolytic protein under the control of
regulatory sequences that direct expression of the product of said
transgene in a platelet.
34. The method according to claim 33, wherein said fibrinolytic
protein is selected from the group consisting of u-PA, Factor VIIa,
Factor VIII, Factor IX and fibrinogen.
35. The method according to claim 33, wherein said nucleic acid
molecule is present in a platelet and said delivering step
comprises administering to said patient said platelet.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the priority of U.S.
Provisional Patent Application No. 60/424,234, filed Nov. 5,
2002.
BACKGROUND OF THE INVENTION
[0003] Hematopoietic stem cells are pluripotent cells present in
the bone marrow, which divide to produce more specialized
progenitor stem cells, i.e., lymphoid progenitors and myeloid
progenitors. Cells that are differentiated from the lymphoid
progenitors in the bone marrow and that are found in the peripheral
blood include B cells and T cells. From B cells are generated
plasma cells; from T cells are generated activated T cells.
Similarly, the common myeloid progenitor stem cells produce the
granulocytes/macrophage progenitor cells and the
megakaryocyte/erthyrocyt- e progenitor cells in the bone marrow.
Cells differentiated from the granulocyte/macrophage progenitors
that are present in the blood include neutrophils, eosinophils,
basophils, monocytes and immature dendritic cells. Monocytes
further give rise to mast cells, macrophages and dendritic cells
that are present in tissue and lymph nodes. Cells differentiated
from the megakaryocyte/erthrocyte progenitors include
megakaryocytes and erythroblasts, which further differentiate into
platelets and erythrocytes (red blood cells) in the blood.
[0004] A number of these hematopoietic lineage cells are secretory
cells upon activation. For example, platelets, the smallest
corpuscular components of human blood, are characterized by a
diameter of about 2-4 micrometers, the absence of a nucleus, and a
physiological number varying from 150,000 to 300,000 per cubic
millimeter of blood. Platelets contribute to the complex,
multistep, and highly regulated process of thrombus formation and
arterial occlusive disorders, a leading cause of human morbidity.
Platelets target and adhere to sites of vascular injury. At the
sites of vascular injury, the platelets are activated and form
aggregates that provide a provisional seal. Platelets
preferentially release their granular contents at the site of
injury, e.g., contributing to the subsequent growth and stability
of thrombi in part through the release of von Willebrand factor
(vWF), fibrinogen, and other coagulation proteins such as Factor V
(Holt J. C., and Niewiarowski, S. 1985 Sem. Hematol. 22:151-163)
from their alpha-granules. Activated platelets also release
proteins that inhibit thrombolysis, chief among which is
plasminogen activator inhibitor-1 (PAI-1). Over 90% of the
circulating PAI-1 is stored in platelet alpha-granules (Booth, N. A
et al, 1988 Brit. J. Haematol. 70:327-333), although much of this
is in an inactive form (Declerck, P. J et al, 1988 Blood
71:220-225; Kruithof, E. K et al, 1987 Blood 70:1645-1653).
Nonetheless, this pool of PAI-1 is thought to be one of the main
reasons why platelet-rich thrombi are especially resistant to
thrombolytic therapy (Booth, N. A et al, 1992 Ann. N. Y. Acad. Sci.
667:70-80; Fay, W. P et al, 1994 Blood 83:351-356).
[0005] Paradoxically, platelets also contain or can bind small
amounts of plasma-derived profibrinolytic proteins, including
urokinase-type plasminogen activator (u-PA) and plasminogen (Fay,
W. P et al, 1994 cited above; Lenich, C et al, 1997 Blood
90:3579-3586; Jiang, Y et al. 1996 Blood 87:2775-2781; Holt, J. C.,
and Niewiarowski, S. 1980 Circulation 62:342a). However, these
proteins are found at very low levels, and their activity is
overwhelmed by the large amounts of PAI-1, which helps to stabilize
nascent thrombi.
[0006] Recently, the effect of changing this balance in platelet
fibrinolytic proteins has been described. Quebec Platelet Disorder
(QPD) is a rare bleeding disorder not responsive to platelet
transfusion, but responsive to anti-fibrinolytic agents, such as
tranexamic acid (Hayward, C. P. et al, 1997 Blood 89:1243-1253;
Hayward, C. P. et al, 1996 Blood 87:4967-4978; Hayward, C. P. et
al, 1997 Brit. J. Haematol. 97:497-503). The etiology of QPD has
been ascribed recently to ectopic expression of an excess of u-PA
in megakaryocytes and platelets (Kahr, W. H. et al., 2001 Blood
98:257-265). QPD platelets contain predominantly activated
two-chain urokinase (tcu-PA). The etiology for the bleeding
diathesis may in part be due to local release of activated u-PA
within thrombi leading to premature lysis. However, degradation of
multiple platelet alpha-granular proteins, including vWF and Factor
V, presumably by plasmin generated as a result of urokinase, may
interfere with thrombus development as well.
[0007] There remains a need in the art for methods for harnessing
the cellular mechanisms of platelets as well as other cells
differentiated from hematopoietic progenitor cells for therapeutic,
diagnostic and research purposes.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention provides a recombinant nucleic
acid molecule comprising a first sequence encoding a transgene
under the control of regulatory sequences that direct expression of
the transgene product in a hematopoietic progenitor cell or a cell
differentiated therefrom In one embodiment, the cell that expresses
the transgene is a secretory cell. In another embodiment, the cell
is a lymphoid progenitor cell, a myeloid progenitor cell or a cell
further differentiated therefrom, such as a platelet.
[0009] In another aspect, the invention provides a hematopoietic
progenitor, lymphoid progenitor, or nyeloid progenitor host cell
transformed, transduced, infected or transfected with an
above-described nucleic acid molecule.
[0010] In still another aspect, the invention provides a host cell
differentiated from a progenitor cell that is transformed,
transduced, infected or transfected with an above-described nucleic
acid molecule.
[0011] In yet a further aspect, the invention provides a platelet
transformed, transduced, infected or transfected with a nucleic
acid molecule comprising a first sequence encoding a transgene,
which is optionally a fibrinolytic protein, under the control of
regulatory sequences that direct expression of the transgene in the
platelet.
[0012] In a further aspect, the invention provides a method for
generating a modified hematopoietic stem cell, a progenitor cell,
or a modified cell differentiated from the hematopoietic stem cell
and/or progenitor cell. The method involves transferring a nucleic
acid molecule as described above into the cell via transformation,
transduction, infection or transfection.
[0013] In yet another aspect, the invention provides methods for
treating or preventing certain disorders, diseases, symptoms or
injuries in which cells of the hematopoietic lineage are involved,
by delivering to a mammalian patient a recombinant nucleic acid
molecule described above or a suitable host cell described above,
that contain a first sequence comprising a transgene encoding a
product under the control of regulatory sequences that direct
expression of the product of the transgene in the host cell. A
differentiated cell produces the product at a suitable in the
mammal.
[0014] In still a further aspect, the invention provides a method
for preventing unwanted thrombus formation in a mammal by
administering an above-described molecule or platelet containing
same, in which the transgene is a fibrinolytic protein and the
regulatory sequences direct expression of the product of the
transgene in a platelet. The platelet produces the product at the
site of the thrombus formation.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 is a schematic representation of the 10.2 kb Sac II
insert obtained from the transgenic mice expressing u-PA in their
platelets. The insert contains the 1.2 kb murine Xba I/Kpn I PF4
promoter (open box) plus a 5' untranslated region (5'-UTR; light
gray box) followed by the 11-exon murine u-PA gene (Heckel, J. L.
et al, 1990 Cell 62:447-456, black boxes) and ending with the hGH
3'-UTR and polyadenylation sequence (dark gray box). The 2.8 kb Bgl
II fragment containing the transgene was detected in a genomic
Southern blot (not shown).
[0016] FIG. 2 is a graph of a pulmonary microemboli lysis study.
Solid circles show residual labeled microemboli remaining in the
lungs of wildtype mice at the indicated times (n=6). Open squares
show the residual radioactivity in the lungs of line #19 mice
studied in parallel (n=6). Mean.+-.1 SD is shown.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention provides novel nucleic acid molecules,
host cells containing the molecules, and methods for delivery of
recombinant transgenes using modified cells of the hematopoietic
stem cell lineage. Methods for treatment and prophylaxis using
these cells are disclosed for a variety of disorders involving or
mediated by the cells of the hematopoietic lineage. Such disorders
can include inflammations, infections, tissue injuries, vascular
injuries, and the like.
I. NUCLEIC ACID MOLECULES OF THE INVENTION
[0018] One aspect of this invention includes isolated, synthetic or
recombinant nucleic acid molecules and sequences comprising a first
sequence encoding a transgene under the control of regulatory
sequences that direct expression of the transgene in a cell of the
hematopoietic lineage. Such nucleic acid molecules are used to
express the transgene product in vitro or ex vivo, or to permit
expression of the transgene product in vivo in a mammal.
[0019] As used herein, the term "isolated nucleotide molecule or
sequence" refers to a nucleic acid segment or fragment which is
free from contamination with other biological components that are
associated with the molecule or sequence in its natural
environment. For example, one embodiment of an isolated nucleotide
molecule or sequence of this invention is a sequence separated from
sequences which flank it in a naturally occurring state, e.g., a
DNA fragment which has been removed from the sequences which are
normally adjacent to the fragment, such as the sequences adjacent
to the fragment in a genome in which it naturally occurs. Further,
the nucleotide sequences and molecules of this invention have been
altered to encode a selected transgene product. Thus, the term
"isolated nucleic acid molecule or sequence" also applies to
nucleic acid sequences or molecules that have been substantially
purified from other components that naturally accompany the
unmutagenized nucleic acid, e.g., RNA or DNA, or proteins, in the
cell. An isolated nucleotide molecule or sequence of this invention
also encompasses sequences and molecules that have been prepared by
other conventional methods, such as recombinant methods, synthetic
methods, e.g., mutagenesis, or combinations of such methods. The
nucleotide sequences or molecules of this invention should not be
construed as being limited solely to the specific nucleotide
sequences presented herein, but rather should be construed to
include any and all nucleotide sequences which share homology
(i.e., have sequence identity) with the nucleotide sequences
presented herein.
[0020] By the term "promoter" or "regulatory sequence" is meant a
DNA sequence required to direct expression of a nucleic acid
operably linked thereto in a cell of hematopoietic lineage.
Preferably the promoter/regulatory sequence is positioned at the 5'
end of the transgene coding sequence such that it drives expression
of the transgene-encoded protein in a cell. In the molecules of
this invention, the promoter/regulatory sequence may also include
an enhancer sequence and other regulatory elements that are
required for expression in these hematopoietic cells.
[0021] Suitable regulatory sequences/promoters for use in the
present invention are readily selected from among regulatory
sequences that express the selected transgene in the hematopoietic
stem cell itself, or in one of the progenitor cells of the
hematopoietic lineage, including the common lymphoid progenitor,
the common myeloid progenitor, the megakaryotic/erythrocyte
progenitor or the granulocyte/macrophage progenitor. In one
embodiment the regulatory sequences are able to direct expression
of the transgene in a cell that is further differentiated from
these progenitor cells. For example, suitable cells differentiated
from the megakaryotic/erythrocyte progenitor cells are platelets,
megakaryocytes or erythrocytes. Suitable cells differentiated from
granulocyte/macrophage progenitor cells are neutrophils,
eosinophils, monocytes, basophils and immature dendritic cells.
Still other suitable cells differentiated from monocytes are mast
cells, macrophages and dendritic cells. Suitable cells
differentiated from said lymphoid progenitors are natural killer
cells. Promoters capable of directing expression of a transgene in
any of these cells of the hematopoietic lineage are useful as
regulatory sequences in the nucleic acid molecules of this
invention. In one embodiment, the promoter expresses the gene in
secretory cells of the hematopoietic lineage, i.e., cells that
release the contents of their granules upon activation, e.g.,
platelets, mast cells, neutrophils, eosinophils, etc. See, e.g.,
IMMUNOBIOLOGY, THE IMMUNE SYSTEM IN HEALTH AND DISEASE, 5.sup.th
edit., C. Janeway et al., Ed., Garland Publishing, New York, N.Y.:
2001.
[0022] In one embodiment, a platelet-specific promoter is used as a
regulatory sequence to direct expression of the transgene in a
plasmid. Among suitable promoters are, without limitation, the
Platelet factor 4 (PF4) promoter, the glycoprotein IIb promoter,
the glycoprotein IIIa promoter, and the glycoprotein VI promoter.
In another embodiment, the regulatory sequence is a
neutrophil-specific promoter, such as one or more of the human
neutrophil alpha defensin promoters DEFA1, DEFA2, DEFA3, and DEFA4,
among others. Where the transgene is desirably expressed in a NK
cell, the regulatory sequence is a natural killer cell-specific
promoter, for example, the human perform gene promoter. In still
another embodiment, the regulatory sequence useful in the nucleic
acid molecule of this invention is an eosinophil-specific promoter.
Examples of eosinophil-specific promoters include, without
limitation, the human eotaxin gene promoter and the eosinophil
peroxidase gene promoter. In another embodiment, a suitable
regulatory sequence is an erythrocyte-specific promoter, such as
the human RhD gene promoter.
[0023] Depending upon the use for which the nucleic acid molecule
is constructed, the transgene is any peptide, polypeptide or
protein useful for the treatment of a disorder or reduction or
prevention of a symptom in which cells of the hematopoietic system
are involved. For example, a non-exclusive list of products
includes those encoded by therapeutic transgenes for the treatment
of a variety of inflammatory conditions, microbial or parasitic
infections, injuries, such as vascular injuries and other
hematopoietic cell-involved disorders. In one embodiment, such
products include fibrinolytic proteins, including without
limitation, urokinase-type plasminogen activator, plasminogen
activator inhibitor-1 (PAI-1), von Willebrand factor, fibrinogen,
Factor V, and plasminogen for use in altering the hemostatic
balance at sites of thrombosis. Suitable products also include,
without limitation, hormones and growth and differentiation factors
including, without limitation, insulin, glucagon, growth hormone
(GH), parathyroid hormone (PTH), growth hormone releasing factor
(GHRF), follicle stimulating hormone (FSH), luteinizing hormone
(LH), human chorionic gonadotropin (hCG), vascular endothelial
growth factor (VEGF), angiopoietins, angiostatin, granulocyte
colony stimulating factor (GCSF), erythropoietin (EPO), connective
tissue growth factor (CTGF), basic fibroblast growth factor (bFGF),
acidic fibroblast growth factor (aFGF), epidermal growth factor
(EGF), transforming growth factor a (TGF.alpha.), platelet-derived
growth factor (PDGF), insulin growth factors I and II (IGF-I and
IGF-II), any one of the transforming growth factor .beta.
superfamily, including TGF .beta., activins, inhibins, or any of
the bone morphogenic proteins (BMP) including BMPs 1-15, any one of
the heregluin/neuregulin/ARIA/neu differentiation factor (NDF)
family of growth factors, nerve growth factor (NGF), brain-derived
neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary
neurotrophic factor (CNTF), glial cell line derived neurotrophic
factor (GDNF), neurturin, agrin, any one of the family of
semaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth
factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine
hydroxylase.
[0024] Other useful transgene products include proteins that
regulate the immune system including, without limitation, cytokines
and lymphokines such as thrombopoietin (TPO), interleukins (IL)
IL-1 through IL-25 (including, IL-2, IL-4, IL-12, and IL-18),
monocyte chemoattractant protein, leukemia inhibitory factor,
granulocyte-macrophage colony stimulating factor, Fas ligand, tumor
necrosis factors .alpha. and .beta., interferons .alpha., .beta.,
and .gamma., stem cell factor, flk-2/flt3 ligand. Gene products
produced by the immune system are also useful in the invention.
These include, without limitation, immunoglobulins IgG, IgM, IgA,
IgD and IgE, chimeric immunoglobulins, humanized antibodies, single
chain antibodies, T cell receptors, chimeric T cell receptors,
single chain T cell receptors, class I and class II MHC molecules,
as well as engineered immunoglobulins and MHC molecules. Useful
gene products also include complement regulatory proteins, membrane
cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2
and CD59.
[0025] Still other useful gene products include any one of the
receptors for the hormones, growth factors, cytokines, lymphokines,
regulatory proteins and immune system proteins. The invention
encompasses receptors for cholesterol regulation, including the low
density lipoprotein (LDL) receptor, high density lipoprotein (HDL)
receptor, the very low density lipoprotein (VLDL) receptor, and the
scavenger receptor. The invention also encompasses gene products
such as members of the steroid hormone receptor superfamily
including glucocorticoid receptors and estrogen receptors, Vitamin
D receptors and other nuclear receptors. In addition, useful gene
products include transcription factors such as jun, fos, max, mad,
serum response factor (SRF), AP-1, AP2, myb, MyoD, myogenin,
ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4,
ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding proteins,
interferon regulation factor (IRF-1), Wilms tumor protein,
ETS-binding protein, STAT, GATA-box binding proteins, e.g., GATA-3,
and the forkhead family of winged helix proteins.
[0026] Other useful gene products include, carbamoyl synthetase I,
ornithine transcarbamylase, arginosuccinate synthetase,
arginosuccinate lyase, arginase, fumarylacetacetate hydrolase,
phenylalanine hydroxylase, alpha-1 antitrypsin,
glucose-6-phosphatase, porphobilinogen deaminase, factor VIII,
factor IX, cystathione beta-synthase, branched chain ketoacid
decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA
carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase,
insulin, beta-glucosidase, pyruvate carboxylate, hepatic
phosphorylase, phosphorylase kinase, glycine decarboxylase,
H-protein, T-protein, a cystic fibrosis transmembrane regulator
(CFTR) sequence, and a dystrophin cDNA sequence.
[0027] Still other useful gene products include enzymes such as are
useful in enzyme replacement therapy, which is useful in a variety
of conditions resulting from deficient activity of enzyme.
[0028] Further useful gene products include non-naturally occurring
polypeptides, such as chimeric or hybrid polypeptides having a
non-naturally occurring amino acid sequence containing insertions,
deletions or amino acid substitutions. For example, single-chain
engineered immunoglobulins could be useful in certain
immunocompromised patients. Other types of non-naturally occurring
gene sequences include antisense molecules and catalytic nucleic
acids, such as ribozymes, which could be used to reduce
overexpression of a target.
[0029] Reduction and/or modulation of expression of a gene are
particularly desirable for treatment of hyperproliferative
conditions characterized by hyperproliferating cells, as are
cancers and psoriasis. Some polypeptides, which are produced
exclusively or at higher levels in hyperproliferative cells as
compared to normal cells. The compositions of this invention are
employed to express as transgene products polypeptides that can
reduce the activity or inactivate oncogenes such as myb, myc, fyn,
and the translocation gene bcr/abl, ras, src, P53, neu, trk and
EGRF. Anti-cancer treatments and protective regimens include
transgene products directed to inactivate or reduce the activity of
variable regions of antibodies made by B cell lymphomas and
variable regions of T cell receptors of T cell lymphomas which, in
some embodiments, are also used as target antigens for autoimmune
disease. Other tumor-associated polypeptides can be used as target
polypeptides such as polypeptides, which are found at higher levels
in tumor cells including the polypeptide recognized by monoclonal
antibody 17-1A and folate binding polypeptides.
[0030] Other suitable therapeutic polypeptides and proteins include
those which are useful for treating individuals suffering from
autoimmune diseases and disorders by conferring a broad based
protective immune response against targets that are associated with
autoimmunity including cell receptors and cells which produce
"self"-directed antibodies. T cell mediated autoimmune diseases
include Rheumatoid arthritis (RA), multiple sclerosis (MS),
Sjogren's syndrome, sarcoidosis, insulin dependent diabetes
mellitus (IDDM), autoimmune thyroiditis, reactive arthritis,
ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis,
psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease
and ulcerative colitis. Each of these diseases is characterized by
T cell receptors (TCRs) that bind to endogenous antigens and
initiate the inflammatory cascade associated with autoimmune
diseases.
[0031] Still other suitable transgenes encode fibrinolytic proteins
and peptides suitable for delivery by transgene expression in
platelets, such as illustrated in the below-noted examples. The
selection of the transgene sequence, or other molecule carried by
the nucleic acid molecule, is not a limitation of this invention.
Choice of a transgene sequence is within the skill of the artisan
in accordance with the teachings of this application.
[0032] The terms "homology" or "similarity," when referring to a
nucleic acid or fragment thereof, indicate that, when optimally
aligned with appropriate nucleotide insertions or deletions with
another nucleic acid (or its complementary strand), there is
nucleotide sequence identity in at least about 70% of the
nucleotide bases, as measured by any well-known algorithm of
sequence identity, such as FASTA, a program in GCG Version 6.1. The
term "homologous" as used herein, refers to the sequence similarity
between two polymeric molecules, e.g., between two nucleic acid
molecules, e.g., two DNA molecules, two RNA molecules, or two
polypeptide molecules. When a nucleotide or amino acid position in
both of the two molecules is occupied by the same monomeric
nucleotide or amino acid, e.g., if a position in each of two DNA
molecules is occupied by adenine, then they are homologous at that
position. The homology between two sequences is a direct function
of the number of matching or homologous positions, e.g. if half
(e.g., five positions in a polymer ten subunits in length) of the
positions in two compound sequences are homologous, then the two
sequences are 50% homologous. If 90% of the positions, e.g, 9 of
10, are matched or homologous, the two sequences share 90%
homology. By way of example, the DNA sequences 3'ATTGCC5' and
3'TATGCG5' share 50% homology. By the term "substantially
homologous" as used herein, is meant DNA or RNA which is about 70%
homologous, more preferably about 80% homologous, and most
preferably about 90% homologous to the desired nucleic acid.
[0033] When referring to the transgenes or the regulatory sequences
disclosed above, the invention is also directed to an isolated
nucleotide molecule comprising a nucleic acid sequence that is at
least 70%, 80% or 90% homologous to a nucleic acid sequence
encoding a naturally occurring transgene-encoded protein or a
polypeptide that has similar biological effect as the native
transgene product. Furthermore, due to the degeneracy of the
genetic code, any three-nucleotide codon that encodes a mutant or
substituted amino acid residue of a transgene-encoded protein,
described herein is within the scope of the invention.
[0034] Where, as discussed herein, proteins, and/or DNA sequences
encoding them, or other sequences useful in nucleic acid molecules
or compositions described herein are defined by their percent
homologies or identities to identified sequences, the algorithms
used to calculate the percent homologies or percent identities
include the following: the Smith-Waterman algorithm (J. F. Collins
et al, 1988, Comput. Appl. Biosci., 4:67-72; J. F. Collins et al,
Molecular Sequence Comparison and Alignment, (M. J. Bishop et al,
eds.) In Practical Approach Series: Nucleic Acid and Protein
Sequence Analysis XVIII, IRL Press: Oxford, England, UK (1987)
pp.417), and the BLAST and FASTA programs (E. G. Shpaer et al,
1996, Genomics, 38:179-191). These references are incorporated
herein by reference.
[0035] By describing two DNA sequences as being "operably linked"
as in the relationship between the transgene and the regulatory
sequences used herein, is meant that a single-stranded or
double-stranded DNA comprises two DNA sequences and that the two
DNA sequences are arranged within the molecule or sequence in such
a manner that at least one of the DNA sequences is able to exert a
physiological effect by which it is characterized upon the
other.
[0036] Preferably, each protein encoding transgene sequence and
necessary regulatory sequences of this invention are present in a
separate viral or non-viral recombinant vector (including non-viral
methods of delivery of a nucleic acid molecule into a cell).
Alternatively, two or more of these nucleic acid sequences encoding
duplicate copies of a the transgene-encoded protein or encoding
multiple different therapeutic proteins of this invention are
contained in a polycistronic transcript, i.e., a single molecule
designed to express multiple gene products.
[0037] The isolated nucleic acid of this invention is desirably a
recombinant vector, particularly a plasmid, containing isolated and
purified DNA sequences comprising DNA sequences that encode a
selected therapeutic protein. By the term "vector" as used herein,
is meant a DNA molecule derived from viral or non-viral, e.g.,
bacterial, species that has been designed to encode an exogenous or
heterologous nucleic acid sequence. Thus, the term includes
conventional bacterial plasmids. Such plasmids or vectors can
include plasmid sequences from viruses or phages. Such vectors
include chromosomal, episomal and virus-derived vectors, e.g.,
vectors derived from bacterial plasmids, bacteriophages, yeast
episomes, yeast chromosomal elements, and viruses. Vectors may also
be derived from combinations thereof, such as those derived from
plasmid and bacteriophage genetic elements, cosmids, and phagemids.
The term also includes non-replicating viruses that transfer a gene
from one cell to another. The term should also be construed to
include non-plasmid and non-viral compounds which facilitate
transfer of nucleic acid into cells, such as, for example,
polylysine compounds and the like.
[0038] The nucleic acid molecules of the invention include
non-viral vectors or methods for delivery of the sequences encoding
the therapeutic protein to a host cell according to this invention.
A variety of non-viral vectors are known in the art and may
include, without limitation, plasmids, bacterial vectors,
bacteriophage vectors, "naked" DNA and DNA condensed with cationic
lipids or polymers.
[0039] Examples of bacterial vectors include, but are not limited
to, sequences derived from bacille Calmette Gurin (BCG),
Salmonella, Shigella, E. coli, and Listeria, among others. Suitable
plasmid vectors include, for example, pBR322, pBR325, pACYC177,
pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pK37, pKC101,
pAC105, pVA51, pKH47, pUB110, pMB9, pBR325, Co1 E1, pSC101, pBR313,
pML21, RSF2124, pCR1, RP4, pBAD18, and pBR328. Examples of suitable
inducible Escherichia coli expression vectors include pTrc (Amann
et al., 1988 Gene, 69:301-315), the arabinose expression vectors
(e.g., pBAD18, Guzman et al, 1995 J. Bacteriol., 177:4121-4130),
and pETIId (Studier et al., 1990 Methods in Enzymology,
185:60-89).
[0040] Another type of useful vector is a single or double-stranded
bacteriophage vector. For example, a suitable cloning vector
includes, but is not limited to the vectors such as bacteriophage
.lambda. vector system, .lambda.gt11, .mu.gt .mu.WES.tB, Charon 4,
.lambda.gt-WES-.lambda.B, Charon 28, Charon 4A,
.lambda.gt-1-.lambda.BC, .lambda.gt-1-.lambda.B, M13mp7, M13mp8, or
M13mp9, among others.
[0041] In yet another embodiment, a mammalian expression vector is
used for expression of the selected transgene in mammalian cells of
the hematopoietic lineage. Examples of mammalian expression vectors
include pCDM8 (Seed, 1987 Nature, 329:840-842) and pMT2PC (Kaufman
et al., 1987 EMBO J., 6(1):187-93). When used in mammalian cells,
the expression vector's control functions are often provided by
viral regulatory elements other than the promoters specified
above.
[0042] One type of recombinant vector is a recombinant single or
double-stranded RNA or DNA viral vector. A variety of viral vector
systems are known in the art. Examples of such vectors include,
without limitation, recombinant adenoviral vectors, herpes simplex
virus (HSV)-based vectors, adeno-associated viral (AAV) vectors,
hybrid adenoviral/AAV vectors, recombinant retroviruses or
lentiviruses, recombinant poxvirus vectors, recombinant vaccinia
virus vectors, SV-40 vectors, insect viruses such as baculoviruses,
and the like that are constructed to carry or express a selected
nucleic acid composition of interest.
[0043] Retrovirus vectors that can be employed include those
described in EP 0 415 731; International Patent Publication Nos. WO
90/07936; WO 94/03622; WO 93/25698; and WO 93/25234; U.S. Pat. No.
5,219,740; International Patent Publication Nos. WO 93/11230 and WO
93/10218; Vile and Hart, 1993 Cancer Res. 53:3860-3864; Vile and
Hart, 1993 Cancer Res. 53:962-967; Ram et al., 1993 Cancer Res.
53:83-88; Takamiya et al., 1992 J. Neurosci. Res. 33:493-503; Baba
et al., 1993 J. Neurosurg. 79:729-735; U.S. Pat. No. 4,777,127; GB
Pat. No. 2,200,651; and EP 0 345 242. Examples of suitable
recombinant retroviruses include those described in International
Patent Publication No. WO 91/02805. See also, D. A. Wilcox et al,
2000 Blood, 95 (12):3645-3652.
[0044] Alphavirus-based vectors may also be used as the nucleic
acid molecule encoding the therapeutic protein. Such vectors can be
constructed from a wide variety of alphaviruses, including, for
example, Sindbis virus vectors, Semliki forest virus (ATCC VR-67;
ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and
Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250;
ATCC VR 1249; ATCC VR-532). Representative examples of such vector
systems include those described in U.S. Pat. Nos. 5,091,309;
5,217,879; and 5,185,440; and International Patent Publication Nos.
WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; and WO
95/07994.
[0045] Examples of adenoviral vectors include those described by
Berkner, 1988 Biotechniques 6:616-627; Rosenfeld et al., 1991
Science 252:431-434; International Patent Publication No. WO
93/19191; Kolls et al., 1994 PNAS 91:215-219; Kass-Eisler et al.,
1993 PNAS 90:11498-11502; Guzman et al., 1993 Circulation
88:2838-2848; Guzmanetal., 1993 Cir. Res. 73:1202-1207;Zabner et
al., 1993 Cell 75:207-216; Li et al, 1993 Hum. Gene Ther.
4:403-409; Cailaud et al., 1993 Eur. J. Neurosci. 5:1287-1291;
Vincent et al., 1993 Nat. Genet. 5:130-134; Jaffe et al., 1992 Nat.
Genet. 1:372-378; and Levrero et al., 1991 Gene 101: 195-202.
Exemplary adenoviral vectors include those described in
International Patent Publication Nos. WO 94/12649; WO 93/03769; WO
93/19191; WO 94/28938; WO 95/11984 and WO 95/00655. Other
adenoviral vectors include those derived from chimpanzee
adenoviruses, such as those described in U.S. Pat. No.
6,083,716.
[0046] Another viral vector is based on a parvovirus such as an
adeno-associated virus (AAV). Representative examples include the
AAV vectors described in International Patent Publication No. WO
93/09239, Samulski et al., 1989 J. Virol. 63:3822-3828; Mendelson
et al., 1988 Virol. 166:154-165; and Flotte et al., 1993 PNAS
90:10613-10617. Other particularly desirable AAV vectors include
those based upon AAV1; see, International Patent Publication No. WO
00/28061, published May 18, 2000. Other desirable AAV vectors
include those which are pseudotyped, i.e., contain a minigene
composed of AAV 5' ITRs, a transgene, and AAV 3' ITRs packaged in a
capsid of an AAV serotype heterologous to the AAV ITRs. Methods of
producing such pseudotyped AAV vectors are described in detail in
International Patent Publication No. WOO1/83692.
[0047] In an embodiment in which the nucleic acid molecule of the
invention is "naked DNA", it is combined with polymers including
traditional polymers and non-traditional polymers such as
cyclodextrin-containing polymers and protective, interactive
noncondensing polymers, among others. The "naked" DNA and DNA
condensed with cationic lipids or polymers are typically delivered
to the cells using chemical methods. A number of chemical methods
are known in the art for cell delivery and include using lipids,
polymers, or proteins to complex with DNA, optionally condensing
the same into particles, and delivering to the cells. Another
non-viral chemical method includes using cations to condense DNA,
which is then placed in a liposome and used according to the
present invention. See, C. Henry, 2001 Chemical and Engineering
News, 79(48):35-41.
[0048] The nucleic acid molecule encoding the selected therapeutic
protein is introduced directly into the cells of the hematopoietic
lineage either as "naked" DNA (U.S. Pat. No. 5,580,859) or
formulated in compositions with agents that facilitate direct
immunization, such as bupivicaine and other local anesthetics (U.S.
Pat. No. 6,127,170).
[0049] All components of the viral and non-viral vectors above are
readily selected from among known materials in the art and
available from the pharmaceutical industry. Selection of the vector
components other than the regulatory sequences are not considered a
limitation on this invention. Each nucleic acid sequence encoding a
protein according to this invention is preferably under the control
of regulatory sequences that direct the replication and generation
of the product of each nucleic acid sequence, preferably
ectopically, in a mammalian hematopoietic lineage, progenitor cell
or differentiated cell.
[0050] Additional regulatory sequences for inclusion in a nucleic
acid sequence, molecule or vector of this invention include,
without limitation, one or more of an enhancer sequence, a
polyadenylation sequence, a splice donor sequence and a splice
acceptor sequence, a site for transcription initiation and
termination positioned at the beginning and end, respectively, of
the polypeptide to be translated, a ribosome binding site for
translation in the transcribed region, an epitope tag, a nuclear
localization sequence, an IRES element, a Goldberg-Hogness "TATA"
element, a restriction enzyme cleavage site, a selectable marker
and the like. Enhancer sequences include, e.g., the 72 bp tandem
repeat of SV40 DNA or the retroviral long terminal repeats or LTRs,
etc. and are employed to increase transcriptional efficiency.
Selection of other non-promoter common vector elements is
conventional and many such sequences are available with which to
design the nucleotide molecules and vectors useful in this
invention. See, e.g., Sambrook et al, Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory, New York, (1989)
and references cited therein at, for example, pages 3.18-3.26 and
16.17-16.27 and Ausubel et al., Current Protocols in Molecular
Biology, John Wiley & Sons, New York (1989). One of skill in
the art may readily select from among such known non-promoter
sequences to prepare nucleic acid molecules of this invention. The
selection of such non-promoter sequences is not a limitation of
this invention.
II. METHODS OF PRODUCING THE NUCLEIC ACID MOLECULES
[0051] The preparation or synthesis of the nucleotide sequences and
nucleotide molecules of this invention is well within the ability
of the person having ordinary skill in the art using available
material. The synthesis methods are not a limitation of this
invention. The examples below detail presently preferred
embodiments of synthesis of sequences encoding an exemplary
transgene useful in this invention for the construction of a
transgenic animal model. However, similar methods are employed to
produce nucleic acid molecules for the generation of therapeutic or
prophylactic compositions of this invention.
[0052] The nucleotide molecules and sequences of this invention are
produced by chemical synthesis methods. For example, the nucleotide
sequences useful in the invention are prepared conventionally by
resort to known chemical synthesis techniques, e.g., solid-phase
chemical synthesis, such as described by Merrifield, 1963 J. Amer.
Chem. Soc., 85:2149-2154; J. Stuart and J. Young, Solid Phase
Peptide Synthesis, Pierce Chemical Company, Rockford, Ill. (1984);
Matteucci et al., 1981 J. Am. Chem. Soc., 103:3185; Alvarado-Urbina
et al., 1980 Science, 214:270; and Sinha, N. D. et al., 1984 Nucl.
Acids Res., 13:4539, among others. See, also, e.g.,
PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York, 1993; Wold, F.,
"Posttranslational Protein Modifications: Perspectives and
Prospects", pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF
PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983;
Seifter et al., 1990 Meth. Enzymol., 182:626-646, and Rattan et
al., 1992 Ann. N.Y Acad. Sci., 663:48-62.
[0053] Alternatively, the nucleic acid molecules of this invention
are constructed recombinantly using conventional molecular biology
techniques, site-directed mutagenesis, genetic engineering or
polymerase chain reaction, such as, by cloning and expressing a
nucleotide molecule encoding a desired therapeutic protein with
optional other proteins within a host cell of the hematopoietic
lineage, etc. utilizing the information provided herein (See, e.g.,
Sambrook et al., cited above; Ausubel et al. cited above). Coding
sequences for the transgenes and the regulatory sequences can be
prepared synthetically (W. P. C. Stemmer et al, 1995 Gene,
164:49).
[0054] In general, recombinant DNA techniques involve obtaining by
synthesis or isolation a DNA sequence that encodes the desired
therapeutic protein as described above, and introducing it into an
appropriate vector where it is expressed preferably under the
control of the selected promoter that can direct expression in a
cell of the hematopoietic lineage.
[0055] Any of the methods described for the insertion of DNA into a
vector is used to ligate the selected promoter and other regulatory
control elements into specific sites within the selected
recombinant vector to generate the nucleic acid molecule.
III. HOST CELLS OF THE INVENTION
[0056] In another aspect of this invention, a cell of the
hematopoietic lineage is manipulated to contain a nucleic acid
molecule described above.
[0057] In one embodiment, the nucleic acid molecule generated as
described above is transferred into an isolated hematopoietic stem
cell, a common lymphoid progenitor cell, a common myeloid
progenitor cell, a megakaryotic/erythrocyte progenitor cell or a
granulocyte/macrophage progenitor cell. The hematopoietic stem
cells or progenitor cells of the hematopoietic lineage are isolated
from bone marrow of a suitable human or non-human mammal. For
example, the cells are isolated from a mammalian patient into whom
the manipulated cells are re-introduced. Alternatively, the mammal
providing the host cells is a different mammal, for either
introduction into another mammal or for research or laboratory use.
Methods for isolating such cells from bone marrow are well known.
See, for example, the Stem Cell Database of Princeton University;
Phillips, R L et al, 2000 Science, 288:1635-1640 and references
cited therein.
[0058] In another embodiment, the host cells of the hematopoietic
lineage are those cells found in the peripheral blood or tissue,
such as platelets, megakaryocytes, neutrophils, eosinophils,
monocytes, basophils, dendritic cells, mast cells, macrophages,
dendritic cells, erythrocytes, and natural killer cells. These
cells must be isolated from the peripheral blood or tissue of a
suitable human or non-human mammal. For example, the cells are
isolated from a mammalian patient into whom the manipulated cells
would be re-introduced. Alternatively, the mammal providing the
host cells is a different mammal, for either introduction into
another mammal or for research or laboratory use. Methods for
isolating such cells from peripheral blood or tissue are well
known. The introduction of a platelet expressing a suitable
transgene under the platelet specific PF4 regulatory sequence is
exemplified in the examples below.
[0059] Once the suitable host cell is isolated, the nucleic acid
molecule or vector is transferred therein in vitro or ex vivo by a
conventional technique such as transformation, transduction,
infection or transfection. The selection of other suitable methods
for transferring the nucleic acid molecule or the vector into an
isolated cell of the hematopoietic lineage can be performed by one
of skill in the art by reference to known techniques. See, e.g.,
Gething and Sambrook, 1981 Nature, 293:620-625, among others.
[0060] If necessary, such host cells are cultured. Culture
conditions are well known in the art and include ionic composition
and concentration, temperature, pH and the like. Typically,
transfected cells are maintained under culture conditions in a
culture medium. Suitable media for various cell types are well
known in the art. In a preferred embodiment, the temperature is
from about 20.degree. C. to about 50.degree. C., more preferably
from about 30.degree. C. to about 40.degree. C. and, even more
preferably about 37.degree. C.
[0061] The pH is preferably from about a value of 6.0 to a value of
about 8.0, more preferably from about a value of about 6.8 to a
value of about 7.8 and, most preferably about 7.4. Osmolality is
preferably from about 200 milliosmols per liter (mosm/L) to about
400 mosm/l and, more preferably from about 290 mosm/L to about 310
mosm/L. Other biological conditions needed for transfection of a
vector are well known in the art.
IV. METHODS OF U.S.E. OF THE NUCLEIC ACID COMPOSITION AND HOST
CELLS OF THE INVENTION
[0062] Once transfected, the host cells of the hematopoietic
lineage are employed in pharmaceutical or prophylactic compositions
and methods for the treatment of a variety of disorders in human or
non-human mammals. Such treatment may include enhancement of a
biological activity or reduction or a disadvantageous biological
activity occurring in the body. Similarly, the nucleic acid
molecules themselves are used in direct treatment of disorders such
as inflammatory disorders, microbial or parasitic infection,
vascular or hemorrhagic disorders, and the like in which the
hematopoietic cells, their progenitors or differentiated cells are
implicated. In other embodiments, the host cells (preferably
platelets) are employed to prevent formation of stable, occlusive
thrombus development.
[0063] One of skill in the art may readily identify a number of
such disorders. Among such disorders are included without
limitation, coagulation disorders (either an insufficiency or
excess thereof), acute lung injury and sepsis, helminth infection,
asthma or other allergic reactions, viral infections, bacterial
infections, etc. For example, the compositions of this invention
are useful in the prevention and/or treatment of disease(s) caused
by microbial infections including, without limitation, Haemophilus
influenzae (both typable and nontypable), Haemophilus somnus,
Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus
pyogenes, Streptococcus agalactiae, Streptococcus faecalis,
Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae,
Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci,
Bordetella pertussis, Alloiococcus otiditis, Salmonella typhi,
Salmonella typhimurium, Salmonella choleraesuis, Escherichia coli,
Shigella, Vibrio cholerae, Corynebacterium diphtheriae,
Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium
intracellulare complex, Proteus mirabilis, Proteus vulgaris,
Staphylococcus aureus, Staphylococcus epidermidis, Clostridium
tetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurella
haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae
and Mycoplasma gallisepticum.
[0064] The compositions of this invention are useful in the
prevention and/or treatment of disease caused by, without
limitation, Respiratory syncytial virus, Parainfluenza virus types
1-3, Human metapneumovirus, Influenza virus, Herpes simplex virus,
Human cytomegalovirus, Human immunodeficiency virus, Simian
immunodeficiency virus, Hepatitis A virus, Hepatitis B virus,
Hepatitis C. virus, Human papillomavirus, Poliovirus, rotavirus,
caliciviruses, Measles virus, Mumps virus, Rubella virus,
adenovirus, rabies virus, canine distemper virus, rinderpest virus,
avian pneumovirus (formerly turkey rhinotracheitis virus), Hendra
virus, Nipah virus, coronavirus, parvovirus, infectious
rhinotracheitis viruses, feline leukemia virus, feline infectious
peritonitis virus, avian infectious bursal disease virus, Newcastle
disease virus, Marek's disease virus, porcine respiratory and
reproductive syndrome virus, equine arteritis virus and various
Encephalitis viruses.
[0065] The compositions of this invention are useful in enhancing
response against fungal pathogens such as Aspergillis, Blastomyces,
Candida, Coccidiodes, Cryptococcus and Histoplasma or against
parasites including Leishmania major, Ascaris, Trichuris, Giardia,
Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma gondii and
Pneumocystis carinii.
[0066] Compositions of this invention may also be useful for the
prevention and/or treatment of disease(s), without limitation, such
as autoimmune disease, such as multiple sclerosis, lupus and
rheumatoid arthritis and others, asthma, atherosclerosis, Alzheimer
disease, amyloidosis or amyloidogenic disease, and cancers.
Clotting disorders and other vascular injuries caused by other
infections, injury, aging, thrombocytopenia, inappropriate thrombus
formation, myelodysplasia, AML, and the like may also be treated
according to the methods of this invention. These compositions and
methods can be useful to treat allergic reactions to allergens such
as pollen, insect venoms, animal dander, fungal spores and drugs
(such as penicillin). Other conditions that are treated by the
methods of this invention included disease characterized by
unwanted thrombus formation, amyloid deposition, diabetes, and
gastroesophageal reflux disease, among others. The methods of this
invention may also be useful in the enhancement of wound healing.
The selection of the disorder to be treated by the compositions and
methods of this invention is not a limitation of this invention.
One of skill in the art may readily include other disorders
suitable for the treatment described herein.
[0067] One method for treating a disorder in a mammal according to
this invention includes the step of delivering to a mammal a
recombinant nucleic acid molecule comprising a first sequence
comprising a transgene encoding a product under the control of
regulatory sequences that direct expression of the product of the
transgene in a hematopoietic stem cell, a progenitor cell of the
hematopoietic lineage, or a cell differentiated therefrom.
Depending upon the identity of the regulatory sequence and the
transgene, the method permits a differentiated cell of a
hematopoietic lineage to express the product of the selected
transgene at a suitable site in the mammal. Where the
differentiated cell is secretory, the cell may express the
transgene produce ectopically and target the expression to a
particular site. For example, a plasmid expressing a transgene will
target to the site of vascular injury or thrombus formation.
[0068] In one embodiment, therefore, the method of the invention
involves harvesting stem cells or progenitor cells from bone marrow
of a patent, transferring the nucleic acid molecule into the cells
ex vivo and reinfusing the cells into the bone marrow or peripheral
blood of the mammalian patient. Alternatively, the method of
treatment can involve infusing or injecting into the patients bone
marrow or blood a non-self transfected host cell.
[0069] In another embodiment, the method of the invention involves
harvesting differentiated cells of the hematopoietic lineage from
peripheral blood of a mammalian patient; transferring the nucleic
acid molecule into the differentiated cells; and
[0070] returning these cells into the blood of the patient.
Alternatively, the method of treatment can involve infusing or
injecting into the patient's blood a non-self transfected
differentiated host cell.
[0071] In still another embodiment of this invention, a method of
treatment can involve directly administering to a mammalian
patient, simply the nucleic acid molecule, i.e., as naked DNA. The
regulatory sequences should aid in the uptake of the molecule by
the appropriate differentiated cells of the hematopoietic
lineage.
[0072] Thus, as one specific embodiment, the invention provides a
method for enhancing coagulation in a patient by delivering to the
mammalian patient with an insufficient clotting mechanism a
recombinant nucleic acid molecule (or a platelet containing the
molecule) comprising a first sequence comprising a transgene
encoding a product under the control of regulatory sequences that
direct expression of the product of the transgene in a platelet.
Preferably the regulatory sequence would be a platelet-specific
sequence mentioned above. Examples of suitable transgenes for this
method are transgenes encoding one or more of Factor VIIa, Factor
VIII, Factor IX or fibrinogen.
[0073] Another specific embodiment involves a method for preventing
or reducing coagulation in a mammalian patient, where needed.
According to this method, the patient is administered a recombinant
nucleic acid molecule (or a platelet containing the molecule)
comprising a first sequence comprising a transgene encoding a
product under the control of regulatory sequences that direct
expression of the product of the transgene in a platelet.
Preferably the regulatory sequence would be a platelet-specific
sequence mentioned above. Examples of suitable transgenes for this
method are transgenes encoding one or more of urokinase plasminogen
activator, plasminogen, tissue plasminogen activator, and tissue
factor pathway inhibitor.
[0074] Another example of a method of this invention is a method
for enhancing coagulation in a mammalian patient by delivering to
the patient a recombinant nucleic acid molecule (or an erythrocyte
containing the molecule) comprising a first sequence comprising a
transgene encoding a product under the control of regulatory
sequences that direct expression of the product of the transgene in
an erythrocyte. Preferably the regulatory sequence would be an
erythrocyte-specific sequence mentioned above. Examples of suitable
transgenes for this method are transgenes encoding a urokinase
plasminogen activator receptor, preferably expressed on the cell
surface.
[0075] Still another example of this invention is a method for
treating acute lung injury and sepsis in a mammalian patient. This
method includes delivering to the patient a recombinant nucleic
acid molecule (or neutrophils containing the molecule) comprising a
first sequence comprising a transgene encoding a product under the
control of regulatory sequences that direct expression of the
product of the transgene in a neutrophil. Preferably the regulatory
sequence would be a neutrophil-specific sequence mentioned above.
An example of a suitable transgene for this method is a transgene
encoding activated Protein C.
[0076] The methods and compositions of this invention may also be
employed in a method for treating parasitic helminth infection of a
mammalian human or non-human patient. This method involves
delivering to a mammal a recombinant nucleic acid molecule (or
eosinophils containing the molecule) comprising a first sequence
comprising a transgene encoding a product under the control of
regulatory sequences that direct expression of the product of the
transgene in eosinophils. Preferably the regulatory sequence would
be an eosinophil-specific sequence mentioned above. An example of a
suitable transgene for this method is a transgene encoding a
protein toxic to a helminth.
[0077] Another method according to this invention involves treating
asthma or allergic responses in a mammalian patient. This method
involves delivering to a mammal a recombinant nucleic acid molecule
(or an eosinophils containing same) comprising a first sequence
comprising a transgene encoding a product under the control of
regulatory sequences that direct expression of the product of the
transgene in an eosinophil. Preferably the regulatory sequence
would be an eosinophil-specific sequence mentioned above. Examples
of a suitable transgene for this method are transgenes encoding one
or more of human TSG6, an antibody to IL-1 receptor alpha, and an
anti-inflammatory protein.
[0078] The invention also includes a method for treating a viral
infection in a mammal comprising delivering to a mammal a
recombinant nucleic acid molecule (or NK cell containing same)
comprising a first sequence comprising a transgene encoding a
product under the control of regulatory sequences that direct
expression of the product of the transgene in a natural killer
cell. Preferably the regulatory sequence would be an NK
cell-specific sequence mentioned-above. Examples of a suitable
transgene for this method is a transgene encoding a neutralizing
antibody against a viral coat protein, e.g., anti-HIV gag protein,
anti-HPV proteins, anti-HIV proteins, etc.
[0079] Finally, as demonstrated specifically by the examples below,
the present invention provides a method for the treatment and
prevention of undesirable thrombus development in a mammalian
patient by administering to the patient a recombinant nucleic acid
molecule (or platelet containing same) comprising a first sequence
comprising a transgene encoding a product under the control of
regulatory sequences that direct expression of the product of the
transgene in a platelet. As illustrated below, a platelet that
ectopically expressed u-PA under the control of the platelet
specific PF4 promoter was able to control thrombus formation in a
patient. See the Examples below.
[0080] The nucleic acid molecules or host cells are employed in
compositions containing a physiologically acceptable diluent or a
pharmaceutically acceptable carrier, such as sterile water or
sterile isotonic saline. The compositions may also be mixed with
such diluents or carriers in a conventional manner. As used herein
the language "pharmaceutically acceptable carrier" is intended to
include any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with administration to
humans or other vertebrate hosts. The appropriate carrier will be
evident to those skilled in the art and will depend in large part
upon the route of administration.
[0081] The compositions may also include, but are not limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles,
pastes, and implantable sustained-release or biodegradable
formulations. Such formulations may further comprise one or more
additional ingredients including, but not limited to, suspending,
stabilizing, or dispersing agents. In one embodiment of a
formulation for parenteral administration, the active ingredient is
provided in dry (i.e., powder or granular) form for reconstitution
with a suitable vehicle (e.g., sterile pyrogen-free water) prior to
parenteral administration of the reconstituted composition. Other
formulations may optionally include a liposomal preparation, or as
a component of a biodegradable polymer system Compositions for
sustained release or implantation may comprise pharmaceutically
acceptable polymeric or hydrophobic materials such as an emulsion,
an ion exchange resin, a sparingly soluble polymer, or a sparingly
soluble salt.
[0082] Still additional components that are present are adjuvants,
preservatives, chemical stabilizers, or other proteins. Typically,
stabilizers, adjuvants, and preservatives are optimized to
determine the best formulation for efficacy in the target human or
animal. Suitable exemplary preservatives include chlorobutanol,
potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the
parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
Suitable stabilizing ingredients that are used include, for
example, casanino acids, sucrose, gelatin, phenol red, N-Z amine,
monopotassium diphosphate, lactose, lactalbumin hydrolysate, and
dried milk.
[0083] Various cytokines and lymphokines are also suitable for
inclusion in the compositions of this invention or administration
therewith. One such cytokine is granulocyte-macrophage colony
stimulating factor (GM-CSF), which has a nucleotide sequence as
described in U.S. Pat. No. 5,078,996, which is hereby incorporated
by reference. A plasmid containing GM-CSF cDNA has been transformed
into E coli and has been deposited with the American Type Culture
Collection (ATCC), 10801 University Boulevard, Manassas, Va.
20110-2209, under Accession Number 39900. The cytokine
Interleukin-12 (IL-12) is described in U.S. Pat. No. 5,723,127,
which is hereby incorporated by reference (available from Genetics
Institute, Inc., Cambridge, Ma.). Other cytokines or lymphokines
include but are not limited to, the interleukins 1-.alpha.,
1-.beta., through 24, the interferons-.alpha., .beta. and .gamma.,
granulocyte colony stimulating factor, and the tumor necrosis
factors .alpha. and .beta..
[0084] Still other suitable optional components of the compositions
of this invention include, but are not limited to: surface active
substances (e.g., hexadecylamine, octadecylamine, octadecyl amino
acid esters, lysolecithin, dimethyl-dioctadecylammonium bromide),
methoxyhexadecylgylcerol, and pluronic polyols; polyamines, e.g.,
pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl
dipeptide, dimethylglycine, tuftsin; oil emulsions; and mineral
gels, e.g., aluminum phosphate, etc. and immune stimulating
complexes, liposomes, polysaccharides, lipopolysaccharides and/or
other polymers.
[0085] In addition to a carrier as described above, compositions
composed of polynucleotide molecules desirably contain optional
polynucleotide facilitating agents or "co-agents", such as a local
anesthetic, a peptide, a lipid including cationic lipids, a
liposome or lipidic particle, a polycation such as polylysine, a
branched, three-dimensional polycation such as a dendrimer, a
carbohydrate, a cationic amphiphile, a detergent, a benzylammonium
surfactant, or another compound that facilitates polynucleotide
transfer to cells. Such a facilitating agent includes bupivicaine
(see U.S. Pat. No. 5,593,972, which is hereby incorporated by
reference). Other non-exclusive examples of such facilitating
agents or co-agents useful in this invention are described in U.S.
Pat. Nos. 5,703,055; 5,739,118; 5,837,533; International Patent
Publication No. W096/10038, published Apr. 4, 1996; and
International Patent Publication No W094/16737, published Aug. 8,
1994, which are each incorporated herein by reference.
[0086] Most preferably, the local anesthetic is present in an
amount that forms one or more complexes with the nucleic acid
molecules. When the local anesthetic is mixed with nucleic acid
molecules or plasmids of this invention, it forms a variety of
small complexes or particles that pack the DNA and are homogeneous.
Thus, in one embodiment of the compositions of this invention, the
complexes are formed by mixing the local anesthetic and at least
one plasmid of this invention. Any single complex resulting from
this mixture may contain a variety of combinations of the different
plasmids. Alternatively, in another embodiment of the compositions
of this invention, the local anesthetic is pre-mixed with each
plasmid separately, and then the separate mixtures combined in a
single composition to ensure the desired ratio of the plasmids is
present in a single composition, if all plasmids are to be
administered in a single bolus administration. Alternatively, the
local anesthetic and each plasmid are mixed separately and
administered separately to obtain the desired ratio. When the
facilitating agent used is a local anesthetic, preferably
bupivacaine, an amount of from about 0.1 weight percent to about
1.0 weight percent based on the total weight of the polynucleotide
composition is preferred. See, also, International Patent
Publication No. W099/21591, which is hereby incorporated by
reference, and which teaches the incorporation of benzylammonium
surfactants as co-agents, preferably administered in an amount of
between about 0.001-0.03 weight %. According to the present
invention, the amount of local anesthetic is present in a ratio to
the nucleic acid molecules of 0.01-2.5% w/v local anesthetic to
1-10 .mu.g/ml nucleic acid. Another such range is 0.05-1.25% w/v
local anesthetic to 100 .mu.g/ml to 1 ml/ml nucleic acid.
[0087] As used, such a polynucleotide composition may express the
transgene product on a transient basis in vivo; no genetic material
is inserted or integrated into the chromosomes of the host.
[0088] The compositions may also contain other additives suitable
for the selected mode of administration of the composition. The
composition of the invention may also involve lyophilized
polynucleotides, which can be used with other pharmaceutically
acceptable excipients for developing powder, liquid or suspension
dosage forms. See, e.g., Remington: The Science and Practice of
Pharmacy, Vol. 2, 19.sup.th edition (1995), e.g., Chapter 95
Aerosols; and International Patent Publication No. W099/45966, the
teachings of which are hereby incorporated by reference. Routes of
administration for these compositions are combined, if desired, or
adjusted.
[0089] These nucleic acid molecule-containing compositions can
contain additives suitable for administration via any conventional
route of administration. In some embodiments, administration is
intravenous or directly into the bone marrow. In some embodiments,
the composition of the invention is prepared for administration to
human subjects in the form of, for example, liquids, powders,
aerosols, tablets, etc.
[0090] The compositions of the present invention (whether host
cell-containing or nucleic acid molecule-containing compositions),
as described above, are not limited by the selection of the
conventional, physiologically acceptable, carriers, adjuvants, or
other ingredients useful in pharmaceutical preparations of the
types described above. The preparation of these pharmaceutically
acceptable compositions, from the above-described components,
having appropriate pH isotonicity, stability and other conventional
characteristics is within the skill of the art.
[0091] Suitable routes of administration include, but are not
limited to, intranasal, oral, vaginal, rectal, parenteral,
intradermal, transdermal, intramuscular, intraperitoneal,
subcutaneous, intravenous and intraarterial. The appropriate route
is selected depending on the nature of the composition used, and an
evaluation of the age, weight, sex and general health of the
patient and the components present in the immunogenic composition,
and similar factors by an attending physician.
[0092] Similarly, suitable doses of compositions of this invention
are readily determined by one of skill in the art, depending upon
the condition being treated, the health, age and weight of the
veterinary or human patient, and other related factors, and the
other characteristics of the composition, e.g., nucleotide
molecule, vector or host cell. In general, selection of the
appropriate "effective amount" or dosage for the composition(s) of
the present invention will also be based upon the form of the
composition, the identity of the transgene and host cell, as well
as the physical condition of the subject. The method and routes of
administration and the presence of additional components in the
compositions may also affect the dosages and amounts of the
compositions. Such selection and upward or downward adjustment of
the effective dose is within the skill of the art. The amount of
composition required to produce a suitable response in the patient
without significant adverse side effects varies depending upon
these factors. Suitable doses are readily determined by persons
skilled in the art.
[0093] For example, the amounts of nucleotide molecules in the DNA
and vector compositions are selected and adjusted by one of skill
in the art. In one embodiment, each dose will comprise between
about 50 .mu.g to about 1 mg of nucleic acid molecule, e.g., DNA
plasmid, per mL of a sterile solution. Generally, a suitable dose
where the vector is a viral vector is in the range of 10.sup.3 to
10.sup.18, preferably about 10.sup.5 to 10.sup.16 transducing units
(TU) per dose, and most preferably, about 10.sup.7 to 10.sup.9 TU
for an adult human having a weight of about 80 kg. Transducing
Units (TU) represents the number of infectious particles and is
determined by evaluation of transgene expression upon infection of
host cells. Generally, when used for ex vivo therapy, the host
cells are infected with 10.sup.5 TU to 10.sup.10 TU viral vectors
for each 10.sup.1 to 10.sup.10 cells in a population. However,
other suitable ex vivo dosing levels are readily selected by one of
skill in the art. This dose is formulated in a pharmaceutical
composition, as described above (e.g., suspended in about 0.01 mL
to about 1 mL of a physiologically compatible carrier) and
delivered by any suitable means.
[0094] The number of doses and the dosage regimen for the
composition are also readily determined by persons skilled in the
art. The intended therapeutic or prophylactic effect is conferred
by a single dose of the composition or may require the
administration of several doses, in addition to booster doses at
later times. The dose is repeated, as needed or desired, daily,
weekly, monthly, or at other selected intervals.
V. EXAMPLES OF THE INVENITON
[0095] As illustrated in the examples below, the methods of this
invention were employed to modify the biological behavior of
platelets by causing these hematopoietic lineage cells to
ectopically express a protein of interest at a site of vascular
injury.
[0096] A. Transgenic Animal Model
[0097] A murine urokinase-type plasminogen activator (u-PA)
transgene was expressed transgenically in mice under the regulatory
control of a platelet factor 4 promoter that directed expression to
platelets. The resultant transgenic animals have altered platelet
biology; i.e., they express and store u-PA selectively in their
platelet alpha-granules, in the absence of systemic fibrinolysis
and bleeding characteristic of transgenic over-expression of u-PA
in the liver (Heckel, J. L. et al, 1990 Cell 62:447-456). These
transgenic mice also had a characteristic bleeding diathesis
similar to that seen in patients with Quebec Platelet Disorder
(QPD) that is predominantly at the time of parturition and that can
be controlled by tranexamic acid.
[0098] More importantly, these mice were resistant to the
development of occlusive carotid arterial thrombi in the absence of
systemic fibrinolysis. The mice rapidly lysed pulmonary venous
thrombi or emboli. Moreover, transfusion of small numbers of
urokinase-expressing platelets into wildtype recipients prevented
formation of stable, occlusive carotid arterial thrombi.
[0099] Thus, this animal model confirms that ectopic expression of
u-PA in platelets is the etiology of inherited QPD, provides new
insights into the contribution of activated platelets to thrombus
stability, and provides a new method for preventing inopportune
thrombus development. The transgenic model shows that developing
megakaryocytes are genetically altered in such a way that platelet
function is effectively tipped from pro-thrombotic to
anti-thrombotic. These examples provide a evidence that ectopic
u-PA PA expression in platelets can be achieved.
[0100] B. Summary of the Examples
[0101] The present invention involves the discovery that ectopic
expression of proteins in platelets are useful to favorably alter
the hemostatic balance at sites of thrombosis. Fibrinolytic agents
can be delivered to sites of incipient thrombus formation through
selective storage in platelets, a new method to prevent thrombosis
and hemorrhage. As demonstrated in the Examples below, ectopically
expressed proteins carrying a signal peptide are stored within
platelets and released specifically at sites of injury. By use of
this method, one may alter the prothrombotic role of platelets as a
means to modify pathological thrombus development.
[0102] The method of this invention has clinical application in
situations where re-thrombosis or the prevention of extension of a
previous thrombosis is desirable for an extended period of time.
For example, transient expression of u-PA in megakaryocyte
progenitors harvested from peripheral blood and treated with
present-day retroviral gene-transfer vectors are a potent mechanism
to prevent post-angioplasty restenosis for several weeks to months
with low risk of long-term alteration of the recipient's stem cell
genome. Untoward bleeding associated with the therapy can be
controlled by treatment with an anti-fibrinolytic agent such as
tranexamic acid. Lastly, the methods of this invention enable
delivery of pro-hemostatic proteins to sites of vascular
interruption in patients with diverse hemorrhagic disorders.
[0103] To determine whether developing megakaryocytes could be
altered to express either anti-thrombotic or pro-hemostatic
proteins, which would be stored in platelet and be released in a
concentrated fashion directly at a site of injury, a transgenic
model was developed. In this model, the expression of u-PA was
directed to platelets of transgenic mice using the PF4 promoter,
which is known to drive megakaryocyte-specific expression (Ravid,
K. et al, 1991 Proc. Nat. Acad. Sci. U.S.A. 88:1521-1525). It was
found that platelet u-PA was stored in platelets and was released
within developing thrombi when platelets were activated. The u-PA
was partially counter-balanced by PAI-1, which is present in
platelet alpha-granules of both human and mice platelets (Ngo, T.
H., and Declerck, P. J. 1999 Thromb. Haemost. 82:1510-1515). This
method tipped the balance sufficiently to lessen the resistance of
clots, especially in the arterial circulation where platelet
activation is more intense, to commonly employed agents.
[0104] The "line #19" transgenic animals, described in the examples
below, had u-PA mRNA, u-PA-like protease activity and u-PA protein
detectable in their platelets. Several of the constituent platelet
alpha-granular proteins, including vWF and fibrinogen, were
partially degraded, probably due to plasmin-mediated proteolysis.
However, unlike the previously described murine u-PA
over-expressors driven by a liver-specific promoter (Heckel, J. L.
et al, cited above), these animals did not demonstrate systemic
fibrinolysis. Few of the transgenic adults developed spontaneous
hemorrhage despite life-long expression of elevated levels of
platelet-uPA. The platelet counts were normal, and the red cells
had normal morphology. Plasma fibrinogen was intact and D-dimers
were not detected in the plasma of these mice.
[0105] Megakaryocyte-expressed u-PA was not secreted to a
considerable extent, but rather was preferentially stored in the
circulating platelets, where it led to the digestion of
alpha-granular proteins, as in the human disorder termed QPD. The
u-PA within the alpha-granules was predominantly in the form of
enzymatically active tcu-PA, as determined by immunoblotting.
Platelets are believed to endocytose sufficient plasminogen from
plasma (Holt, J. C., and Niewiarowski, S. cited above) into their
alpha-granules to form plasmin, as suggested by the capacity of
u-PA platelets to digest exogenously added Factor V. Plasmin
formation is initiated by the low level of intrinsic activity of
scu-PA (Lenich, C. et al, 1997 Blood 90:3579-3586) or by the
activation of scu-PA by u-PAR (Higazi, A. et al, 1995 J. Biol.
Chem. 270:17375-17380) or, less likely, by another platelet
granular enzyme. It is likely that plasmin, once formed, then
converts additional scu-PA to tcu-PA within the granules
themselves, although the role of other proteases again cannot be
excluded. Little tcu-PA was found as high molecular weight
complexes with PAI-1. Murine platelets may contain less PAI-1 than
human platelets, or the latent state of PAI-1 within the granules
(Booth, N. A. et al, 1988 Brit. J. Haematol. 70:327-333; Lang, I.
M. et al, 1992 Blood 80:2269-2274) may limit its binding to u-PA,
or complex formation is inhibited by u-PAR (Higazi, A. A.-R et al,
1996 Blood 87:3545-3549), the pH or other components of the
alpha-granules, or, more likely, most of the PAI-1 was degraded by
the u-PA.
[0106] Aside from peripartum deaths, adult transgenic mice had few
spontaneous mucosal bleeding episodes, had normal bleeding times,
and exhibited normal platelet aggregation in vitro. However, these
animals clearly were resistant to developing occlusive thrombi in a
ferric chloride-induced carotid artery thrombosis model. Only 5% of
the u-PA expressing animals developed complete arterial occlusion
by the end of the study (60 minutes) as opposed to 85% of their
wildtype littermates (Table 1). It appears that the thrombi that
did form were far more friable and transitory in nature. Moreover,
lysis of preformed, fibrin microemboli targeted to the lungs
occurred far more rapidly in the u-PA expressing mice than in their
wildtype counterparts. These data suggest that platelet activation
initiated by fibrin contributes to thrombus growth or stability on
the venous side of the circulation (Bdeir, K. et al, cited above)
and that local release of u-PA causes rapid lysis of the nascent
thrombi.
[0107] Recent studies using combined platelet glycoprotein IIb/IIIa
inhibitors and agents (Murciano, J. C. et al, 2002 Am. J. Physiol.
Lung Cell. Mol. Physiol. 282:L529-539), and studies using
pharmacological inhibition of PAI-1 (Rupin, A. et al, 2001 Thromb.
Haemost. 86:1528-1531) are consistent with these conclusions.
[0108] Transfusion of u-PA platelets equivalent to 10% of the
recipient's platelet mass potently inhibited thrombus development
in wildtype recipients. The ability of the transgenic platelets to
disrupt thrombus development is more likely to be due to the
released u-PA than to degradation of hemostatic alpha-granular
proteins for several reasons. First, the near total loss of
granular proteins, as in the Gray Platelet Disorder, is associated
with little or no bleeding (Rao, A. K, 1998 Am. J. Med Sci.
316:69-76, 1998). Second, arterial thrombi are enriched with PAI-1.
The absence of PAI-1 in mice results in a failure of thrombus
formation in a chemically-induced carotid artery thrombosis model
(Eitzman, D. T. et al, 2000 Blood 95:577-580). Third, transfusion
of transgenic platelets into wildtype recipients that had been
drinking water containing tranexamic acid reversed the effect of
platelet-associated u-PA on thrombus formation (Table 2),
notwithstanding the persistence of degraded alpha-granular proteins
in the transfused platelets. These results suggest that it is the
u-PA that is the major cause for the defective hemostasis in these
mice, although a contribution from degraded alpha-granular proteins
is by no means excluded.
[0109] Based on an analysis of the data generated by these examples
similar to that of (Kahr, W. H. et al., cited above), line #19
platelets were estimated to contain .about.20 pg of u-PA per .mu.g
of total platelet protein. Moreover, there are striking
similarities between the phenotype of the QPD patients and these
transgenic mice. This provides additional support for the
conclusion that this syndrome results from the ectopic expression
of u-PA in platelets. Both are dominantly inherited disorders. Both
QPD and the transgenic mice ectopically express u-PA in the
platelets, which also contain many degraded alpha-granular
proteins. In both QPD and the transgenic mice, the ectopic
expression of u-PA is essentially confined to the platelets, and
insufficient u-PA is secreted into the plasma to cause disseminated
fibrinolysis. The phenotype of neither is improved by platelet
infusions, but is improved by use of an anti-fibrinolytic agent,
such as tranexamic acid.
[0110] In summary, these studies support the etiology of QPD as
being due to ectopic expression of u-PA in platelets. Second, they
highlight the importance of the balance between platelet-dependent
coagulation and fibrinolysis during thrombus growth on venous as
well as on the arterial side of the circulation. Third, they show
that the ectopic expression of u-PA in platelets is a valid method
for preventing untoward thrombus development.
[0111] The following examples are provided to illustrate
construction and use of the recombinant vectors and compositions of
the invention and do not limit the scope thereof. One skilled in
the art will appreciate that although specific elements, reagents
and conditions are outlined in the following examples,
modifications can be made which are meant to be encompassed by the
spirit and scope of the invention.
Example 1
Establishment of Transgenic Mice Expressing u-PA Message
[0112] A transgene was designed as illustrated schematically in
FIG. 1. 1.2 kb of the 129 SV murine platelet factor 4 (PF4)
proximal promoter region plus its 5'-untranslated region (UTR)
(Zhang, C. et al, 2001 Blood 98:610-607) was PCR amplified with an
artificial upstream Xba I site and a downstream Kpn I site added.
The inventors found that this promoter could also drive
megakaryocyte-specific expression of LacZ in transgenic mice (data
not shown). This promoter was inserted in place of the albumin
enhancer/promoter immediately upstream of a mouse u-PA minigene
construct that contained a 3'-UTR and poly-adenylation sequence
from the human growth hormone (hGH) gene. This construct was
previously described in Heckel, J. L. et al, 1990 Cell 62:447-456,
which is incorporated by reference herein.
[0113] A 10.2 kb Sac II fragment containing this construct was used
to create transgenic mice by pronuclear injections following
standard methods at the University of Pennsylvania Transgenic Mice
Core Facility. Seven transgenic founders were obtained with copy
numbers ranging from 1 to >20 copy per haploid genome.
[0114] Genomic DNA was isolated from mouse tails using a QIAamp.TM.
DNA Mini Kit (Qiagen, Valencia, Calif.). Positive founder lines
were detected by genomic Southern blot analysis (Tunnacliffe, A. et
al, 1992 Blood 79: 2896-2900). Genomic Southern blot analysis was
made of Bgl II-digested DNA from a wildtype animal and the three
lines that had offspring with the fewest copy numbers of the
transgene (Lines #13, #19 and #49). After digestion, genomic DNA
was separated on a 1% (wt/vol) agarose gel. The probe used was the
mouse PF4 proximal promoter from -680 to -360 bp upstream of the
transcriptional start site (Zhang, C. et al, cited above), and the
final probed membrane was exposed on a PhosphorImaging screen
(Amersham Biosciences, Sunnyvale, Calif.). The 2.8 kb Bgl II
fragment was detected for the transgene in the genomic Southern
blot.
[0115] The transgene copy number per haploid genome was determined
by phosphorimaging (not shown). The intensity of bands on film was
analyzed using the Imagequant PhosphorImager software (Amersham
Biosciences). The copy number was determined by comparing the
intensity of the 2.8 kb transgene PF4 band to the native genomic
1.2 kb PF4 band.
[0116] Three male founders had offspring with the fewest copy
number of transgene (i.e., line #13, copy number 1, line #19, copy
number 2 and line #49, copy number 3), although the line #49
transgene did not transmit well (see below), and these mice were
not available for most studies. All three female founders had high
transgene copy number, and when-pregnant, died peripartum from
uterine hemorrhage (see below). The final male founder was high
copy number (>20) and never produced offspring.
[0117] Founder animals and their offspring were also characterized
by genomic PCR analysis using a mouse PF4 5'-UTR
(5'-CACTTAAGAGCCCTAGACCCATT- TCC-3'; SEQ ID NO: 1) sense primer and
a mouse u-PA exon 2 (5'-TTCAGAGTTTTT CACCACCAA-3'; SEQ ID NO: 2)
antisense primer, which generates a 479 bp genomic and a 114 bp
cDNA band. RT-PCR analysis of total platelet RNA was performed on
lines #13, #19 and #49 for the 114 bp transgenic u-PA message that
extended from the 5'-UTR of PF4 into the murine u-PA 2.sup.nd exon
in their platelets. The 185 bp PF4 message was used as a positive
control of the platelet nature of the RNA. PCR was performed to
confirm founder lines using primers complementary to the mouse PF4
5'-UTR and exon 2 of the murine u-PA gene (data not shown).
[0118] Direct sequence analysis of this RT-PCR product for all
three lines confirmed that the 1.sup.st intron was appropriately
spliced out (data not shown). When RT-PCR was performed on multiple
tissues, e.g., spleen, liver, lung, heart, kidney, adrenal, tongue,
brain, and bone marrow, only bone marrow tested positive for the
114 bp transgenic-derived u-PA mRNA band. All samples showed the
249 bp HPRT positive control cDNA band. Immunohistochemistry of the
bone marrow showed detectable u-PA only in morphologically
recognizable megakaryocytes in the transgenic animals, but not in
wild-type marrow.
[0119] All biological studies described below were done with
transgenic animals that had been backcrossed at least 4 generations
onto a C57BL6 background. Wildtype littermates served as controls.
Cardiac or portal vein blood was drawn from mice into 1/10.sup.th
volume of 3.8% sodium citrate. All studies were approved by the
Animal Care and Use Committee of the Children's Hospital of
Philadelphia.
Example 2
Transgenic u-PA Message Detection
[0120] The animals of Example 1 were then examined to determine
whether they expressed u-PA in their platelets. Murine platelet RNA
was isolated using RNA STAT-60.TM. (Tel-Test, Friendswood, Tex.) as
previously described (Zhang, C. et al, cited above). Tissues
(.about.100 mg each) from these animals were collected, rinsed
vigorously with saline, disaggregated in 500 .mu.l of RNA
STAT-60.TM., and RNA was isolated (Zhang, C. et al, cited above).
Some platelet RNA samples were pretreated with DNase free RNase (1
U/10 .mu.l reaction, Sigma, St. Louis, Mo.) or RNase free DNase (1
U/10 .mu.l reaction, Life Technologies, Gaithersburg, Md.) for 1
hour at 37.degree. C.
[0121] Reverse transcription was performed using the SuperScript II
Reverse Transcriptase Kit.TM. (Life Technologies) as per the
manufacturer's instructions. PCR amplification of the transgenic
u-PA cDNA was accomplished using the two primers discussed above.
Platelet-specific control RT-PCR primers for PF4 message were sense
5'-AATTCTCGGGATCTGGGT-3' SEQ ID NO: 3 and antisense
5'-CTGGGCTCTAGACAGCAGT-3 SEQ ID NO: 4 (Eisman, R. et al, 1990 Blood
76:336-344), with an expected cDNA product of 185 bp. RT-PCR for
the housekeeping gene human phosphoribosyltransferase (HPRT) used
primers 5'-GCTGGTGAAAAGGACCT CT-3' SEQ ID NO: 5 and
5'-CACAGGACTAGAACACCTGC-3' SEQ ID NO: 6, with an expected cDNA
product of 249 bp (Jackson, C. L. et al, 1984 Proc. Nat. Acad. Sci.
U.S.A. 81:2945-2949).
[0122] Platelet murine transgenic u-PA cDNA band was isolated using
a QIAkwik.TM. Gel Extraction Kit (Qiagen) and directly sequenced
using an ABI 373A automated sequencer (ABI Instruments, Foster
City, Calif.).
Example 3
Immunohistochemical Staining for Murine Urokinase
[0123] Spleen and bone marrow aspirates from wildtype and murine
u-PA transgenic mice were stained for murine u-PA expression using
a mouse monoclonal anti-murine u-PA antibody (A10, Molecular
Innovations, Inc., Southfield, Mich.) as the primary antibody and a
biotinylated anti-mouse immunoglobulin (ARK detections system,
DAKO, Caprinteria, Calif.) as the secondary antibody. Specifically,
formalin-fixed, paraffin-embedded 5 .mu.m sections were
deparaffinized in xylene and rehydrated. Endogenous peroxidase
activity was quenched with 0. 9% peroxide in methanol for a total
of 20 minutes. Slides were then treated with trypsin (1 mg/ml in
PBS) for 10 minutes at 37.degree. C. After incubation of the
monoclonal anti-murine u-PA antibody (0.5 .mu.g/ml) with
biotinylated ARK reagent (according to manufacturer's instructions)
for 15 minutes at room temperature, the slides were stained at room
temperature for 2 hours. Slides were washed and incubated with
Streptavidin-HRP (DAKO) for 15 minutes at room temperature. Slides
were again washed DAB reagent (DAKO) was applied for 5 minutes at
room temperature. Slides were counterstained with dilute (1:10)
hematoxylin for 30 seconds.
[0124] Immunohistochemistry of the bone marrow in wildtype and line
#19 mice showed detectable u-PA only in morphologically
recognizable megakaryocytes m the transgenic animals, but not in
wildtype marrow.
Example 4
u-PA Activity in the Platelets--Zymogram and Immunoblot Studies
[0125] A. Zymogram Studies
[0126] Platelet-rich plasma (PRP) was obtained as described in
Zhang, cited above. Platelet counts were determined using a HemaVet
counter (Triad Associates, Concord, Calif.). The platelets were
pelleted at 800 g for 5 minutes and resuspended immediately in
1.times. NuPage Sample Buffer (Invitrogen, Carlsbad, Calif.). Total
platelet protein and platelet releasates (0.5 to 10 .mu.g/lane)
were separated by size under non-reducing conditions on a 12%
SDS-polyacrylamide gel (SDS-PAGE) with 0.4% nonfat dry milk, e.g.,
casein (Carnation Instant Skim Milk Powder, Nestle, Fulton, N.Y.)
with or without supplemental 20 .mu.g/ml human plasminogen
(American Diagnostica, Greenwich, Conn.), and then renatured with
0.5% Triton X-100 in PBS, pH 7.4, for 1 hour as previously
described (Heckel, J. L. et al, cited above). The gel was incubated
at 37.degree. C. for 3 hours. A control lane of 0.1 ng of human
2-chain urokinase (American Diagnostica) was included in each
gel.
[0127] Zymograms of two separate preparations of lysates from line
#19 and #13 platelets and wildtype platelets (10 .mu.g/lane) reveal
a prominent band likely representing tcu- at .about.45 kDa was seen
with platelets from line #19 (data not shown). This is similar to
what has been previously described for murine u-PA (Heckel, J. L.
et al, cited above), consistent with the fact that mouse u-PA is
not glycosylated. Line #13 and wildtype lanes had no detectable
zones of lysis, although twice as much platelet protein was loaded
compared to the #19 line lanes. Omission of plasminogen from the
zymogram or the inclusion of the selective u-PA inhibitor amiloride
in the zymogram (data not shown) abolished lysis induced by
transgenic platelet lysates and the human tcu-PA control. These
results are similar to those reported for QPD platelets, although
the lower band in the human disorder was .about.10% as intense as
the upper band, rather than .about.30% as seen in lysates from the
transgenic mouse platelets.
[0128] B. Immunoblots
[0129] To confirm the presence of u-PA in the transgenic platelets,
total platelet proteins were separated by size on a non-reducing
Western gel and then immunoblotted with A10, an anti-murine u-PA
mAb. Platelet immunoblots were performed as previously described
(Zhang, C. et al, cited above), except that 4-8% gradient gels were
used (NuPAGE Novex Bis-Tris Gels, Invitrogen, Carlsbad, Calif.).
Gels were run under non-reducing conditions, except when studying
platelet fibrinogen, where 1 .mu.l of reducing agent (NuPAGE Sample
Reducing Agent) was added per sample. Mouse u-PA was detected with
a murine anti-mouse u-PA monoclonal antibody (A10, Molecular
Innovations, Southfield, Mich.) added at a 1:75 dilution and
detected with a biotinylated anti-mouse monoclonal antibody
(Molecular Innovations) followed by peroxidase-conjugated
streptavidin (StreptABComplex/HRP, DAKO, Carpinteria, Calif.).
Murine vWF was detected using a 1:200 dilution of a horse radish
peroxidase (HRP)-conjugated rabbit anti-human vWF polyclonal
antibody (DAKO), and murine fibrinogen was detected using a 1:100
dilution of an HRP-conjugated goat anti-human fibrinogen polyclonal
antibody (Rockland Immunochemicals, Gilbertsville, Pa.).
[0130] The Western blot of platelet lysates (10 .mu.g/lane) for
lines #19 and #13, wildtype, and renal extract (30 .mu.g/lane)
revealed a major doublet at .about.45 kDa in line #19; total
platelet lysate that was not seen in wildtype littermate platelet
lysate (data not shown). The major component in the doublet appears
to be tcu-PA and co-migrates with the renal extract control. The
higher, less intense band is likely to be single chain uPA (scu-PA)
or an otherwise modified tcu-PA as previously described (Franco, P.
et al, 1997 J. Cell Biol. 137:779-791) and migrates similar to a
band in renal extract. Thus, as with QPD platelet lysates, the
major form of u-PA in the transgenic platelets appears to be tcu-PA
(Kahr, W. H. et al., cited above).
[0131] Also, as with QPD platelet lysates, a few high molecular
weight species were observed that likely include covalent complexes
between PAI-1 and both tcu-PA and low molecular weight proteolytic
derivatives of u-PA (Jiang, Y et al. 1996 Blood 87:2775-2781). The
typical low molecular weight u-PA species commonly observed in
biological specimens (.about.30 kD), was not a feature of platelet
lysates. Rather, as observed with QPD platelets, a somewhat smaller
low molecular weight u-PA species was observed in transgenic mouse
platelet lysates. None of these mouse u-PA bands was detected when
a comparable amount of platelet lysate from wildtype and line #13
mice were studied. This lack of detectable u-PA in Line #13 is
consistent with the lack of u-PA activity seen in the
above-described zymogram and in the clinical course of these
animals (discussed below).
Example 5
Alpha-Granular Proteins
[0132] Several alpha-granular proteins, including vWF, fibrinogen
and Factor V undergo proteolysis in QPD platelets. Proteolysis of
these proteins has been attributed to the ectopically expressed
u-PA (Kahr, W. H. et al., cited above). To determine whether the
presence of mouse u-PA in platelets led to a similar expression of
proteolytic activity and study the in vitro digestion of Factor V
by platelet lysate, 10 ng of plasma-derived human Factor V (Rodney
Camire, Children's Hospital of Philadelphia) was digested with
either human tcu-PA (10 pg, American Diagnostica) or with 2.5 .mu.g
of total murine platelet lysate prepared from
1.3.times.10.sup.6/.mu.l platelets in the presence or absence of
supplemental plasminogen (1 .mu.g). The resultant digest was then
separated by SDS-PAGE. Factor V was detected using a 1:100 dilution
of an HRP-conjugated sheep anti-human Factor V polyclonal antibody
(Affinity Biologicals, Inc., Hamilton, ON, Canada) as the primary
antibody.
[0133] In a Western blot of platelet lysate (2.5 .mu.g/lane) from
lines #13 and #19 and wildtype mice immunoblotted with a rabbit
anti-human vWF polyclonal antibody (not shown), vWF underwent
extensive digestion in the line #19 platelets, compared with the
intact high molecular weight vWF observed in the littermate
wildtype sample. Platelet fibrinogen immunoblots of line #19 lysate
consistently contained high molecular weight complexes that entered
the gel poorly. These high molecular weight complexes are absent on
a reduced gel, suggesting that they represent disulfide bond,
cross-linked fibrinogen-derived products generated by u-PA.
Consistent with the immunoblot for mouse u-PA, no degradation of
vWF was seen in platelets from line #13 mice.
[0134] In a Western blot of reduced gel of platelet lysates as
above or plasma (3 .mu.g/lane for mouse and 6 .mu.g/lane for human)
proteins (not shown) both murine and human fibrinogen were detected
using a goat anti-human fibrinogen polyclonal antibody. Platelet
alpha-granular fibrinogen was also degraded in transgenic line #19,
but not in line #13 and in wildtype platelets, as assessed on the
non-reducing SDS-PAGE gel. In contrast, plasma fibrinogen was not
degraded in line #19 mice.
[0135] This finding clearly contrasts with the systemic
fibrinolysis that develops in the previously described u-PA
over-expressing transgenic mice that were generated using a
liver-specific promoter (Heckel, J. L. et al, cited above).
Platelet fibrinogen immunoblots of line #19 lysate consistently
contained high molecular weight complexes that entered the gel
poorly. These high molecular weight complexes were absent on a
reduced gel, suggesting that they represent disulfide-bonded,
cross-linked, fibrinogen-derived products generated by u-PA and
plasmin.
[0136] In another test to determine whether platelet releasate from
the transgenic mice could proteolyze human Factor V in a manner
similar to that seen in QPD, platelet lysate was incubated with
human Factor V for up to 4 hours. Factor V (FV) (10 ng/lane) was
digested with 2.5 .mu.g/lane of the indicated platelet lysate or
human u-PA (5 .mu.g/lane) at room temperature, in the absence or
presence of 100 .mu.g/lane of plasminogen per lane.
[0137] Platelet lysate from line #19 digested the Factor V in a
rapid fashion, with degradation nearly complete by 4 hours, giving
a similar pattern to that seen with exogenously added human u-PA
(data not shown). Platelets from wildtype littermates also degraded
Factor V, but at a much slower rate. The addition of exogenous
plasminogen to the platelet releasate enhanced Factor V digestion
by line #19 platelet lysate, but had little effect on the rate of
digestion by wildtype platelets. This suggests that the amount of
plasminogen available in alpha-granules in the transgenic line #19
platelets is rate limiting for maximal protein degradation.
Example 6
Clinical Course and Hematologic Studies
[0138] A. Clinical Course
[0139] Fewer line #19 and #49 transgenic mice were born than
expected. Assuming that 50% of the offspring from a cross between a
hemizygous animal and a wildtype animal should be transgenic, line
#19 showed a 68% mortality rate (57 transgenic animals vs. 175
wildtype littermates at weaning) and line #49 showed an 85%
mortality rate (2 transgenic and 12 wildtype). Line #13 had a very
low mortality rate, consistent with little platelet u-PA expression
(71 transgenic and 80 wildtype). At day 16.5 of gestation, line #19
embryos looked normal and appeared at the expected frequency (10
transgenic and 11 wildtype), indicating that transgenic animals
were lost peripartum Surviving transgenic animals were normal for
weight and growth, although occasional spontaneous deaths occurred
among the adults secondary to subcutaneous or internal hemorrhage.
Autopsies on two such adult line #19 mice expressing u-PA in their
platelets showed examples of spontaneous hemorrhage: one mouse had
free blood in the opened peritoneum and the other showed free blood
filling a small intestinal loop.
[0140] However, when ten line #19 animals and ten littermate
wildtype controls were specifically observed for >11 months, no
deaths were observed in either group, suggesting a low rate of
death in the line #19 animals.
[0141] As noted above, none of the female founder lines survived
birthing. This was also true for pregnant line #19 and #43
transgenic females. In line #19, none of nine pregnant females
survived giving birth, exsanguinating from uterine bleeding at
birth (data not shown). Litter sizes were normal, with embryos
containing equal numbers of wildtype and transgenic animals, and
all embryos appeared normal in size (data not shown).
[0142] Three pregnant line #19 females were given tranexamic acid
(20 mg/ml), an inhibitor of plasminogen activator, in their
drinking water (Hattori, N. et al, 2000 J. Clin. Invest.
106:1341-50) during the last week of pregnancy and all survived.
One lost her pups at birth, and the other two had small litters,
consisting of 3 pups each (2 of the 6 were transgenic).
[0143] B. Blood Counts
[0144] Complete blood counts were performed on line #19 adult mice
and wildtype littermate controls (n=10, each). Blood counts were
measured using a Hemavet 850 (CDC Technologies, Inc, Oxford, Conn.)
calibrated for murine blood. Dried blood smears were stained using
Wright-Giemsa reagent (EM Science, Gibson, N.J.) and the red cell
and platelet morphology was examined. Fibrin D-dimers were measured
in plasma samples obtained according to manufacturer's instructions
(Asserachrom D-Di, American Bioproducts Co., Diagnostica Stago,
Asnieres, France).
[0145] Results of the counts showed nearly identical platelet
counts and hemoglobin levels for the line #19 and wildtype controls
(data not shown). Peripheral blood smears were normal, including
normal platelet numbers and appearance and no evidence of red cell
schistocytes or spherocytes (data not shown).
[0146] C. Platelet Aggregation
[0147] Platelet aggregation studies were performed as described
previously (Kowalska, M. A. et al, 2000 Blood 96:50-57).
Aggregation and dense granule ATP release were measured in a
lumi-aggregometer (Chrono-Log, Havertown, Pa.). Agonists studied
included collagen (1-5 .mu.g/ml), ADP (1-5 .mu.M), epinephrine (50
.mu.M), and thrombin (0.1-1 U/ml) (Bachem Torrance, Calif.). Marrow
samples from wildtype and transgenic mice were stained for mouse
u-PA expression using the murine anti-mouse u-PA A10 primary
antibody as described previously (Zhang, C. et al, cited
above).
[0148] Platelet aggregation in response to collagen (1-5 .mu.g/ml),
ADP (1-5 .mu.M), epinephrine (50 .mu.M), and thrombin (0.1-1 U/ml)
were also normal (data not shown). D-dimer measurements were
negative in the line #19 mice (data not shown). Together with the
measurements of plasma fibrinogen discussed above, these studies
confirm that line #19 mice do not exhibit systemic
fibrinolysis.
Example 7
Carotid Artery Thrombosis Model
[0149] Bleeding times were normal in line #19 mice when compared
with wildtype littermate controls (5.8.+-.3.2 mins versus
5.1.+-.3.4 (n=7, each), respectively), but this test has previously
proven to be an insensitive measurement of thrombotic tendency
(Mayadas, T. N. et al, 1993 Cell 74:541-554). Therefore, the
carotid artery injury thrombosis model was employed. This model has
been used successfully to demonstrate a bleeding diathesis in
diverse mouse backgrounds. This approach also permits study of the
effect of ectopic expression of u-PA in platelets on thrombus
development and stability.
[0150] Ferric chloride-induced arterial injury was performed
according to published procedures (Fay, W. P et al, 1994 cited
above; Fay, W. P. et al, 1999 Blood 93:1825-1830) in 6-8 week old
animals. Briefly, the right common carotid artery was exposed by
blunt dissection, and a miniature Doppler flow probe (Model 0.5VB,
Transonic Systems, Ithaca, N.Y.) was positioned around the artery.
A 1.times.2 mm.sup.2 strip of Number 1 Whatman filter paper (Fisher
Scientific, Pittsburgh, Pa.) soaked in 10% ferric chloride was then
applied to the adventitial surface of the artery for 2 min. The
field was flushed with saline, and blood flow was continuously
monitored for 30 minutes. The time to the initial complete
occlusion and the presence or absence of arterial occlusion at 30
min was recorded.
[0151] To study the effects of a platelet transfusion,
1.2-1.5.times.10.sup.8 gel-filtered platelets in 300 .mu.l of
gel-filtering buffer (4 mM NaH.sub.2PO.sub.4; 5mM Hepes; 137 mM
NaCl; 2.6 mM KCl; 5 mM glucose; 1 mM MgCl.sub.2) was prepared as
previously described (Kowalska, M. A. et al, cited above) and
infused into the left jugular vein immediately before the ferric
chloride patch was applied. Platelets were used within 2 hours of
collection. Total blood counts were measured immediately before and
2 minutes after platelet infusion.
[0152] Unlike their wildtype littermates, few transgenic animals
expressing platelet u-PA formed completely occlusive coronary
artery thrombi after ferric chloride-induced injury (Table 1), and
those that occluded tended to reopen rapidly. At 30 minutes, only
5% of occlusive thrombi formed in the u-PA mice remained as opposed
to the >85% of thrombi formed in controls (Table 1).
Supplementing the transgenic animals with 20 mg/ml tranexamic acid,
a small molecule inhibitor of plasminogen activation, reversed this
protection from occlusive coronary artery thrombosis. Line #13 mice
occluded normally.
1TABLE 1 Ferric chloride thrombosis model. Time to initial Occluded
at Founder line occlusion (mins) Initial occlusion (5) 30 mins (%)
WT 8.8 .+-. 4.3 31/34 (91%) 29/34 (85%) #13 6.8 .+-. 3.3 9/10 (90%)
9/10 (90%) #19 10.3 .+-. 4.8 4/20 (20%).sup..dagger-dbl. 1/20
(5%).sup..dagger-dbl. WT + TA 8.3 .+-. 1.1 5/5 (100%) 4/5 (80%) #19
+ TA 9.4 .+-. 1.6 5/5 (100%) 5/5 (100%) .sup..dagger-dbl.= p >
0.0001 compared to wildtype. Initial occlusion refers to the time
to first occlusion in those animals that developed any occlusive
thrombi. WT = wildtype littermates combined for lines #13 and #19.
TA = drinking water contained tranexamic acid (20 mg/ml).
Example 8
Pulmonary Microemboli Model
[0153] The following experiment was performed to determine whether
the u-PA containing platelets are also effective on the venous side
of the circulation and would lead to rapid dissolution of pulmonary
microemboli. Previously, the lysis of .sup.125I-labeled, fibrin
microparticles was shown to depend on u-PA expression in the
recipient mice as an intravenous infusion of u-PA into u-PA null
mice normalized the rate of fibrinolysis (Bdeir, K. et al, cited
above). This study determined whether u-PA-containing platelets
similarly enhance fibrin clot breakdown.
[0154] .sup.125I-labeled human microemboli, 1.5-3.5.mu..sup.3 in
size, were prepared as previously described (Bdeir, K. et al, 2000
Blood 96:1820-1826). Transgenic and wildtype animals were injected
with these particles within 48 hours of preparation. At various
time points (2-60 minutes) the animals were euthanized, the lungs
removed, washed free of blood, and the amount of .sup.125I activity
measured using a ZM Coulter Counter (Coulter Electronics, Hialeah,
Fla.). In other experiments, autoradiograms of lungs were taken
from wildtype and transgenic mice 30 minutes after injection of the
microemboli by exposing the lungs to X-OMAT film (Kodak, Rochester
N.Y.).
[0155] FIG. 2 shows residual labeled microemboli remaining in the
lungs of wildtype mice over time and residual radioactivity in the
lungs of line #19 mice studied in parallel (n=6). Fibrinolysis was
dramatically accelerated in line #19 mice compared to wildtype
controls. Differences were already evident at two minutes. By 10
minutes, at a time when there was minimal or no fibrinolysis in
wildtype mice, >80% of the clot burden had already been cleared
from the lungs of the line #19 mice.
Example 9
Platelet Transfusion/Carotid Artery Studies
[0156] Based on the prior experiments that showed that
platelet-expressed u-PA both prevented untoward arterial thrombosis
and accelerated lysis of venous emboli, this experiment
investigated whether transfused u-PA-containing platelets would
interfere with thrombus development and stability in wildtype
animals.
[0157] Platelet transfusions equivalent to .about.10% of the total
circulating platelets in an animal were given from line #19 animals
to wildtype littermate controls prior to exposing the carotid
artery to a ferric chloride injury. Surprisingly, wildtype animals
that received u-PA-containing platelets were protected against the
development of thrombosis to the same extent as line #19 mice
themselves (Table 2). Transfusion of buffer only or wildtype
platelets into wildtype animals did not prevent the development of
occlusive thrombi. As anticipated from the above data, giving
wildtype platelets to line #19 mice did not confer resistance to
arterial thrombosis.
2TABLE 2 Platelet transfusion ferric chloride thrombosis model.
Founder Line Time to initial Initial occlusion Occluded at
Recipient Donor occlusion (mins) (%) 30 mins (%) WT Buffer 9.3 .+-.
1.2 5/5 (100%) 5/5 (100%) WT WT 8.2 .+-. 1.4 5/5 (100%) 5/5 (100%)
WT #19 12.5 1/10 (10%).sup..dagger-dbl. 1/10 (0%).sup..dagger-dbl.
WT/TA #19 8.3 .+-. 1.9 5/5 (100%) 4/5 (80%) WT #19/TA 10.5, 22.5
2/3 (67%) 0/3 (0%) #19 WT -- 0/3 (0%) 0/3 (0)% .sup..dagger-dbl.= p
< 0.0001 compared to wildtype receiving wildtype platelets. Time
to initial occlusion, WT and TA are defined in Table 1. Buffer =
gel-filtering buffer.
[0158] To test whether protection from thrombosis seen in wildtype
animals receiving line #19 platelets was attributable to the
released u-PA or to the released degraded alpha-granular proteins,
wildtype animals were placed on drinking water containing
tranexamic acid. A week later, carotid artery injury was performed
on these wildtype mice after they had received an infusion of
platelets from the line #19 animals. It was anticipated that
platelet-associated plasminogen activation would be inhibited in
tranexamic acid-treated recipients leading to loss of protection
from thrombosis if released u-PA is the primary mechanism by which
line #19 platelets interfere with normal thrombus development. As
hypothesized, recipient wildtype animals that had received
tranexamic acid lost their resistance to arterial occlusion
notwithstanding transfusion of the transgenic platelets, suggesting
that the released u-PA contributes significantly to the observed
anti-thrombotic effect of the line #19 platelets. The converse
experiment, infusing platelets from a line #19 animal that had been
on tranexamic acid for one week to wildtype littermates showed that
the infused platelets conferred resistance to arterial thrombosis.
These data support the conclusion that line #19 platelets interfere
with thrombosis in wildtype animals primarily because ectopically
expressed u-PA is released from the donor platelets.
[0159] Applicants specifically incorporate by reference D. Kufrin
et al, 2003 Blood, 102(3):926-933. All publications cited in this
specification are incorporated herein by reference. While the
invention has been described with reference to a particularly
preferred embodiment, it will be appreciated that modifications can
be made without departing from the spirit of the invention. Such
modifications are intended to fall within the scope of the appended
claims.
Sequence CWU 1
1
6 1 26 DNA Artificial mouse PF4 5'UTR sense primer 1 cacttaagag
ccctagaccc atttcc 26 2 21 DNA Artificial mouse u-PA exon 2
antisense primer 2 ttcagagttt ttcaccacca a 21 3 18 DNA Artificial
mouse PF4 5'UTR sense primer 3 aattctcggg atctgggt 18 4 19 DNA
Artificial mouse u-PA exon 2 antisense primer 4 ctgggctcta
gacagcagt 19 5 17 DNA Artificial primer 5 gctggtgaaa aggacct 17 6
20 DNA Artificial primer 6 cacaggacta gaacacctgc 20
* * * * *