Endothelial cell tropic compositions and methods of making and using the same

An isolated nucleic acid molecule comprising a modified Murine Leukemia Virus Long Terminal Repeat sequence that is capable of tropically regulating expression of operably linked coding sequences in a vessel endothelial cell is disclosed. A method of producing a non-Murine Leukemia Virus protein in a vessel endothelial cell is disclosed. The method comprises the step of introducing into the cell a nucleic acid molecule that comprises a modified Murine Leukemia Virus Long Terminal Repeat sequences operably linked to a nucleotide sequence which encodes a non-Murine Leukemia Virus protein, the modified Murine Leukemia Virus Long Terminal Repeat sequences being capable of tropically regulating expression in vessel endothelial cells.

FIELD OF THE INVENTION 
The present invention relates to genetic regulatory sequences that direct 
vessel endothelial cell specific expression of genes, chimeric genes 
comprising the regulatory sequence linked to coding sequences and to 
methods of producing proteins in endothelial cells using the chimeric 
genes. 
BACKGROUND OF THE INVENTION 
Advances in molecular biology over the past few decades have created new 
technologies for combating human disease. In particular, the transduction 
of foreign genes into somatic cells, or gene therapy, has shown great 
promise as a powerful clinical tool. Gene therapy has been used 
experimentally to correct disorders such as ADA deficiency and new trials 
are underway for treating other genetic diseases such as cystic fibrosis 
and muscular dystrophy. Most gene therapy studies to date have focused on 
correcting single gene defects by introducing the wild type gene into 
somatic cells. The product of the newly transduced gene then restores the 
normal phenotype. Other recent application include targeting tumor cells 
with neo-antigens for enhanced recognition by the immune system. 
Additionally, biologically active proteins may be delivered to an 
individual by the introduction and expression of foreign genes encoding 
such proteins. 
Many studies have used bone marrow or bone marrow derived cells as targets 
for gene therapy. However, there has been a recent surge of interest in 
using other cell types, particularly endothelial cells. Endothelial cells 
represent an ideal target for gene therapy in many diseases. Endothelial 
cells are particularly useful in the treatment of hematological and 
vascular disorders as well as any disease that requires the systemic 
deliver of therapeutic factors. 
Broader applicability of gene therapy has been hindered due to technical 
limitations. Most studies thus performed have been so using infected 
tissue ex vivo and then reconstituting the host with the genetically 
altered cells. While this practice is acceptable for tissues that can be 
harvested and re-implanted with relative ease, many desired target cells 
do not fall into this category. For this reason, the search for and/or 
creation of vectors that are expressed by specific tissues is an area of 
intense research interest. There is a need for regulatory elements which 
can be used to direct expression of foreign genes and endothelial cells. 
There is a need to create tissue specific vectors for in vivo gene 
therapy. There is a need to provide gene therapy constructs with 
regulatory elements that will direct tissue specific expression of the 
foreign genes. 
SUMMARY OF THE INVENTION 
The present invention relates to isolated nucleic acid molecules that 
comprise a modified Murine Leukemia Virus Long Terminal Repeat which 
comprises SEQ ID NO:4. In some embodiments, such nucleic acid molecules 
may comprise SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1. 
The present invention relates to isolated nucleic acid molecules that 
comprise a modified Murine Leukemia Virus Long Terminal Repeat which 
comprises SEQ ID NO:4 and is operably linked to a nucleotide sequence that 
encodes a non-Murine Leukemia Virus. In some embodiments, such nucleic 
acid molecules may comprise SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1. In 
some embodiments, the non-Murine Leukemia Virus protein is a human 
protein, such as for example Factor VII protein, Factor IX protein, von 
Willdebrand factor, complement proteins, insulin, cytokines, tissue 
plasminogen activator, alpha-L-iduronidase, iduronate sulfatase, heparin, 
N-sulfatase and alpha 1 antitrypsin. 
The present invention relates to isolated nucleic acid molecules that 
comprise a modified Murine Leukemia Virus Long Terminal Repeat which: a) 
comprises SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1; b) is 
operably linked to a nucleotide sequence that encodes a non-Murine 
Leukemia Virus and c) is encapsulated within a liposome or a viral coat. 
The viral coat may be that of an infectious, replicating viral particle 
such as a retrovirus or a viral coat of an infectious, non-replicating 
viral package particle. 
The present invention relates to methods of producing a non-Murine Leukemia 
Virus protein in an endothelial cell that comprises the step of 
introducing into said endothelial cell, nucleic acid molecule comprising a 
modified Murine Leukemia Virus Long Terminal Repeat which comprises SEQ ID 
NO:4, SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1 operably linked to a 
nucleotide sequence that encodes a non-Murine Leukemia Virus. In some 
embodiments, the cell is a human vessel endothelial cell. In some 
embodiments, the protein is a human protein such as for example: Factor 
VII protein, Factor IX protein, von Willdebrand factor, complement 
proteins, insulin, cytokines, tissue plasminogen activator, 
alpha-L-iduronidase, iduronate sulfatase, heparin, N-sulfatase and alpha 1 
antitrypsin. In some embodiments, the nucleic acid molecule is 
encapsulated within a liposome or a viral coat. In some embodiments, the 
nucleic acid molecule is encapsulated within a viral coat of an 
infectious, replicating viral particle such as retrovirus particle. In 
some embodiments, the nucleic acid molecule is encapsulated within a viral 
coat of an infectious, non-replicating viral package particle. In some 
embodiments, the human vessel endothelial cells are in an individual. In 
some embodiments, the human vessel endothelial cells are in an individual 
and the nucleic acid molecule is delivered intravenously to the 
individual. In some embodiments, the human vessel endothelial cells are in 
an individual, the nucleic acid molecule is delivered intravenously to the 
individual and the nucleic acid molecule comprises a modified Murine 
Leukemia Virus Long Terminal Repeat which: a) comprises SEQ ID NO:4, SEQ 
ID NO:3, SEQ ID NO:2 or SEQ ID NO:1; b) is operably linked to a nucleotide 
sequence that encodes a non-Murine Leukemia Virus and c) is encapsulated 
within a liposome or a viral coat. The viral coat may be that of an 
infectious, replicating viral particle such as a retrovirus or a viral 
coat of an infectious, non-replicating viral package particle.

DETAILED DESCRIPTION OF THE INVENTION 
As used herein, the term "tropism" is meant to refer to gene expression 
which takes place in a cell specific manner. In particular, the term 
"endothelial cell tropism" is meant to refer to expression of coding 
sequences of nucleic acid molecules which occurs in endothelial cells 
only. Thus, the term "endothelial cell tropic compositions" refers to 
nucleic acid molecules which are specifically expressed in endothelial 
cells to the exclusion of other cells. 
It has been discovered that modified forms of the long terminal repeat 
(LTR) of murine leukemia virus (MuLV) provides vessel endothelial cell 
tropic gene expression. According to the invention, MuLV LTR sequences 
which contain specific nucleotide sequences confer vessel endothelial cell 
tropism for expression of non-MuLV coding sequences operably linked to 
such modified LTR sequences. When operably linked to the modified LTRs of 
the invention, nucleotide sequences that encode non-MuLV proteins will be 
expressed at a high level in vessel endothelial cells but not in other 
cell types. 
In previously studied systems, MuLV LTRs that have been observed to 
regulate expression in endothelial do not do so exclusively of expression 
in other cell types. That is, endothelial cell expression is not exclusive 
but rather is just one of many cell types in which the regulatory elements 
are functional. The present invention allows for gene expression 
specifically and exclusively in vessel endothelial cells. According to the 
present invention, modified MuLV LTRs of the invention, such as the MuLV 
LTR molecular clone TR1.3, may be used to express non-MuLV proteins in 
vessel endothelial cells but not in other cell types which have the 
MuLV/non-MuLV constructs. According to the present invention, the modified 
MuLV LTRs may be used in genetic constructs useful in gene therapy which 
are particularly directed at expressing genes in endothelial cells. 
Regardless of what other cell types take up the constructs of the 
invention, only the vessel endothelial cells which take up the construct 
will be capable of expressing the non-MuLV protein encoded by the sequence 
operably linked to the vessel endothelial cell tropic LTR of the 
invention. 
According to some preferred embodiments, the modified MuLV LTR is a MuLV 
LTR in which nucleotides 131-200 of the LTR are SEQ ID NO:4. According to 
some preferred embodiments, the modified MuLV LTR is a Friend MuLV LTR in 
which nucleotides 131-200 of the LTR are SEQ ID NO:4. 
According to some preferred embodiments, the modified MuLV LTR is a MuLV 
LTR in which nucleotides 101-200 of the LTR are SEQ ID NO:3. According to 
some preferred embodiments, the modified MuLV LTR is a Friend MuLV LTR in 
which nucleotides 101-200 of the LTR are SEQ ID NO:3. 
According to some preferred embodiments, the modified MuLV LTR is a MuLV 
LTR in which the U3 region is SEQ ID NO:2. According to some preferred 
embodiments, the modified MuLV LTR is a Friend MuLV LTR in which the U3 
region is SEQ ID NO:2. 
According to some preferred embodiments, the modified MuLV LTR is a MuLV 
LTR in which nucleotides 1-415 of the LTR are SEQ ID NO:2. According to 
some preferred embodiments, the modified MuLV LTR is a Friend MuLV LTR in 
which nucleotides 1-415 of the LTR are SEQ ID NO:2. 
According to some preferred embodiments, the modified MuLV LTR is a 
modified Friend MuLV LTR having SEQ ID NO:1. SEQ ID NO:1 is the LTR from 
the molecular clone TR1.3, which is a Friend MuLV. 
LTRs of retroviruses function as both a promoter/enhancer element as well 
as a polyadenylation signal. Accordingly, LTRs may be used upstream and 
downstream of the coding sequence to provide the necessary regulatory 
sequences for transcription. According to the present invention, LTRs are 
operably linked upstream and/or downstream of the coding sequence. In 
genetic contructs which are delivered as linear constructs, the construct 
contains a modified LTR sequence of the invention upstream of and operably 
linked to a coding sequence that encodes a non-MuLV protein. In genetic 
contructs which are delivered as circular constructs, the construct 
contains a modified LTR sequence of the invention downstream of and 
operably linked to a coding sequence that encodes a non-MuLV protein. In 
both linear and circular constructs, the modified LTR of the invention as 
insered is operably linked to and upstream of the a coding sequence that 
encodes a non-MuLV protein. Those of ordinary skill in the art can 
freadily design vectors and constructs whereby the modified LTR of the 
invention is operably linked to and upstream of the a coding sequence that 
encodes a non-MuLV protein when the non-MuLV protein is being expressed 
tropically in vessel endothelial cells. 
According to some preferred embodiments, the modified MuLV LTR is linked to 
the coding sequence that encodes a non-MuLV protein at a location 3' of 
the coding sequence. According to some preferred embodiments, two modified 
MuLV LTRs are linked to the coding sequence that encodes a non-MuLV 
protein; one at a location 3' of the coding sequence, and one at a 
location 5' of the coding sequence. 
SEQ ID NO:1 contains a nucleotide sequence of a modified MuLV LTR, 
specifically the LTR from MuLV TR1.3. This nucleotide sequence is 
presented as a cDNA sequence of the genomic RNA of the virus. The modified 
LTR disclosed in SEQ ID NO:1 lacks three binding sites, FVb1, FVa and 
FVb2, as compared with the LTR of wild type Friend MuLV which replicates 
poorly or not at all in endothelial cells. The modified LTRs according to 
the present invention include MuLV LTRs which contain deletions, 
insertions and substitutions and which are capable of regulating 
endothelial cell specific expression of coding sequences operably linked 
thereto. Examples include SEQ ID NO:1. Modified LTRs according to the 
present invention include fragments of SEQ ID NO:1 which are capable of 
regulating endothelial cell specific expression of coding sequences 
operably linked thereto. SEQ ID NO:2 provides nucleotides 1-415 of SEQ ID 
NO:1. This sequence is the U3 region of the LTR and contains the portion 
of the LTR which is responsible for the vessel endothelial cell tropism 
which render the LTR particularly useful. SEQ ID NO:3 provides nucleotides 
101-200 of SEQ ID NO:1. This sequence contains the portion of the LTR 
which is responsible for the vessel endothelial cell tropism which render 
the LTR particularly useful. SEQ ID NO:4 provides nucleotides 131-200 of 
SEQ ID NO:1. This sequence contains the portion of the LTR which is 
responsible for the vessel endothelial cell tropism which render the LTR 
particularly useful. 
One having ordinary skill in the art can determine whether or not a 
modified MuLV LTR is capable of vessel endothelial cell tropic expression 
of coding sequences operably linked thereto using standard techniques and 
readily available starting materials without undue experimentation. For 
example, the nucleic acid molecule can be constructed that comprises 
modified LTRs operably linked to a reporter gene. A reporter gene is a 
gene which encodes a protein product that can be detected. Examples of 
common reporter genes include beta-galactosidase or chloramphenicol acetyl 
transferase. Simple assays are available to detect the presence of the 
protein product of these reporter genes. The nucleic acid molecule is 
transfected into endothelial cells and detection of the reporter gene 
product indicates that the LTRs are functioning in the cells. The nucleic 
acid molecule is transfected into non-endothelial cells. If the reporter 
gene product is not detected, the LTRs are not functioning in those cells. 
Primary endothelial cell cultures are best suited for in vitro expression 
of foreign genes. These cultures are easily obtained from human umbilical 
cord using standard techniques. 
Modified LTRs, such as the modified LTR disclosed in SEQ ID NO:1, or a 
modified MuLV which comprises SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a 
functional fragment thereof, may be operably linked to nucleotide 
sequences which encode non-MuLV proteins to form a functional chimeric 
gene. When introduced into endothelial cells, the chimeric gene can be 
expressed to produce the protein in the endothelial cells. In particular, 
one or two copies of a modified MuLV LTRs such as that which is disclosed 
in SEQ ID NO:1 or a modified MuLV which comprises SEQ ID NO:2, SEQ ID 
NO:3, SEQ ID NO:4 or a functional fragment thereof may be operably linked 
to a nucleotide sequence that encodes a human protein in order to express 
the human protein in human vessel endothelial cells. In some embodiments, 
one copy is provided and it is linked upstream of the coding sequence. In 
some embodiments, one copy is provided and it is linked downstream of the 
coding sequence. In some embodiments, two copies are provided, one is 
linked upstream of the coding sequence and one copy is linked downstream. 
In addition to a vessel endothelial tropic modified MuLV LTR operable 
linked to a coding sequence encoding a non-MuLV protein, gene constructs 
according to the invention may additionally comprise nucleotide sequences 
encoding signal sequences and other amino acid sequences linked to the 
non-MuLV protein sequence to direct secretion or intracellular location of 
the protein. Signal sequences which direct secretion of protein by 
endothelial cells are well known and those having ordinary skill in the 
art can readily design gene construct to express non-MuLV proteins in 
vessel endothelial cells by placing the coding sequence under the 
regulatory control of a modified MuLV LTR and further directing secretion 
of the non-MuLV protein so expressed in the vessel endothelial cells. 
Secretion by vessel endothelial cells can be specifically directed to 
result in luminal or abluminal secretion. Further, gene constructs can be 
designed so that the non-MuLV protein contains a transmembrane amino acid 
sequence. Such proteins will be anchored into the cell membrane of the 
vessel endothelial cell. 
In some embodiments, gene constructs provide for delivering coding 
sequences that encode non-MuLV proteins into primary cells to express such 
proteins in primary vessel endothelial cells only. In some embodiments, 
the non-MuLV protein confers antibiotic resistance. Delivery of the gene 
construct to primary cell cultures results in expression of the non-MuLV 
protein in vessel endothelial cells only. Culturing the cells in selection 
medium, i.e. medium that contains the antibiotic will result in the death 
of all cells except those expressing the resistance gene. Thus, only the 
vessel endothelial cells will survive and the resulting culture will be 
pure primary vessel endothelial cells. The present invention is thus 
useful for expressing proteins in primary vessel endothelial cells and in 
some embodiments, for selectively culturing such cells. Transport and 
secretion of proteins in endothelial cells and the signals and peptide 
domains associated therewith are discussed in Rodriguez-Boulan, E. and C. 
Zurzolo 1993 J. Cell Sci. Suppl. 17:9-12 and Rodriguez-Boulan, E. and S. 
K. Powell 1992 Annu. Rev. Cell Biol. 8:395-427, each of which is 
incorporated herein by reference. 
In some embodiments, construct may be useful in gene therapy applications 
in which gene expression and protein production in vessel endothelial 
cells is desired. The non-MuLV proteins which may be produced in 
endothelial cells according to the present invention include human and 
non-human proteins. Examples of human proteins which may be produced in 
endothelial cells according to the present invention include, but are not 
limited to Factor VII protein, Factor IX protein, von Willdebrand factor, 
complement proteins, insulin, cytokines, tissue plasminogen activator, 
alpha-L-iduronidase, iduronate sulfatase, heparin, N-sulfatase and alpha 1 
antitrypsin. Factor VII protein is useful to treat individuals suffering 
from Hemophilia A. Factor IX protein is useful to treat individuals 
suffering from Hemophilia B. von Willdebrand factor is useful to treat 
individuals suffering from von Willdebrand's disease. Complement proteins 
are useful to treat individuals suffering from complement related 
immunodeficiencies. Insulin is useful to treat individuals suffering from 
diabetes. Cytokines are useful to treat individuals suffering from cancer, 
HIV infection and hereditary anemias among other conditions. Tissue 
plasminogen activator is useful in fibrinolytic therapy and in the 
prevention of stroke due to emboli. Alpha-L-iduronidase, iduronate 
sulfatase, heparin, N-sulfatase are useful to treat individuals suffering 
from mucopolysacharidoses and CNS and related disorders. Alpha 1 
antitrypsin is useful to treat individuals suffering from alpha 1 
antitrypsin deficiency and lung and related disorders. 
According to some preferred embodiments, the coding sequence for human 
nerve growth factor is provided in gene construct. Delivery of the gene 
construct results in production of human NGF in vessel endothelial cells, 
facilitating renervation when delivered at or near wound sites. 
According to some preferred embodiments, the coding sequence for human 
Factor IX is provided in gene construct. Delivery of the gene construct, 
particularly intravenously to individual suffering from hemophilia, 
results in production of Factor IX in vessel endothelial cells. Signal 
sequences may be provided to direct the Factor IX thus expressed to be 
secreted. Secretion of the Factor IX protein provides the individual with 
the necessary proteins for effective clotting. 
According to other contemplated embodiments, nucleotide sequences encoding 
proteases, digestive enzymes and the like delivered are provided in gene 
construct. Delivery of the gene construct, particularly intravenously to 
an individual suspected to be at risk of suffering from or being 
susceptible to vessel occlusion results in production of the proteins in 
vessel endothelial cells. Transmembrane sequences may be provided to 
direct the location of the protein as being anchored in the blood vessel 
wall. The protein provides the individual with the necessary proteins to 
effectively prevent or reduce occlusion. 
The ability to construct nucleic acid molecules, including the genomes of 
vectors, that exhibit tissue specific tropism will greater facilitate the 
applicability of human gene therapy. The ability to construct nucleic acid 
molecules, including the genomes of vectors, that can be transcriptional 
active in vessel endothelial cells exclusively are particularly useful for 
delivering proteins systemically through vascular vessel endothelial 
tissue or proteins directed at hematological or vascular disorders. 
According to the present invention, vessel endothelial cells can be used to 
produce proteins while other cell types in which the gene constructs are 
also delivered do not express the protein. Vessel endothelial cells in the 
brain are particularly useful for expressing chimeric genes which comprise 
modified MuLV LTRs according to the invention. 
According to one aspect of the present invention, non-MuLV proteins may be 
produced in vessel endothelial cells by introducing into the vessel 
endothelial cells a chimeric gene which includes a modified LTR operably 
linked to a nucleotide sequence which encodes the non-MuLV protein 
operably linked to a modified LTR. Introduction of the chimeric gene into 
the vessel endothelial cell will result in the expression of the 
nucleotide sequence that encodes the non-MuLV protein and, accordingly, 
the production of the non-MuLV protein. 
According to some aspects of the present invention, the human vessel 
endothelial cells are targeted for production of non-MuLV proteins that 
are useful as therapeutics. In particular, the chimeric gene which 
includes modified LTRs operably linked to the nucleotide sequence that 
encodes the non-MuLV protein is delivered to the vessel endothelial cells 
of an individual by way of a vector or other vehicle. 
For example, the chimeric gene may be part of a viral vector genome. Such 
viral vector genomes include those of retroviruses and DNA viruses which 
comprise the chimeric gene. An example of a viral vector system is Miller 
et al. 1993 Methods of Enzymol. 217:581-599, which is incorporated herein 
by reference. The nucleic acid sequence of such viral vectors are 
encapsulated within a viral particle made up of viral proteins. The viral 
particle is used to facilitate entry of the nucleic acid molecule that 
comprises the chimeric gene into the endothelial cell. Once inside the 
cell, the chimeric gene is expressed and the protein is produced. 
Other methods of introducing nucleic acid molecules into cells are 
well-known to those having ordinary skill in the art. For example, nucleic 
acid molecules encapsulated within liposomes may be used to facilitate 
entry of the nucleic acid molecule into an endothelial cells. Liposomes 
are particularly useful because they have been shown to specifically 
target endothelial cells for the delivery of nucleic acid molecules 
encapsulated therein. Accordingly, the combination of liposomes with the 
chimeric gene comprises a modified MuLV LTR is particularly useful for 
delivering chimeric nucleic acid molecules to endothelial cells and 
expressing the coding sequences of chimeric genes. The production of 
liposome encapsulated gene constructs and there administration in vivo as 
well as the specific utility of liposomes for delivering nucleic acid 
molecules to endothelial cells has been pointed out in Zhu, N. et al. 
(1993) SCIENCE 261:209-211, which is incorporated herein by reference. 
Additionally, Debs, R. J. et al. (1990) J. Biol. Chem. 
265(18):10189-10192, which has been incorporated herein by reference, 
teaches liposome production and administration of liposome encapsulated 
compositions. 
The gene constructs may be prepared as plasmid DNA, linear DNA or RNA, as 
part of viral genomes, or as nucleic acid, molecules within viral 
packages. Viral packaging is well known and refers to a particular type of 
viral vector which is a infectious, non-replicating agent comprising a 
nucleic acid molecule such as a gene construct within a viral coat. Viral 
packages thus provide a means of delivery gene constructs into cells by 
providing viral-like particles which attach and introduce nucleic acid 
molecules into cells. However, instead of delivering a viral genome 
capable of directing viral replication, the nucleic acid molecule 
delivered by the particle is a gene construct which, in the present 
invention, encodes a non-MuLV protein whose expression is directed and 
regulated by the modified MuLV LTR. In packaging systems, cells express 
the proteins which make up the viral coat and additionally comprise 
genetic information to replicate the gene construct. The copies of the 
gene construct which are produced are packaged into the viral coat, 
yielding particles useful to deliver the gene construct into cells of an 
individual. However, once introduced into the cells, the particles cannot 
replicate. Examples of viral packaging systems include Markowitz et al. 
1988 J. Virol. 62:1120-1124, which is incorporated herein by reference. 
In some preferred embodiments, the gene construct is packaged in an 
amphotropic line. An amphotropic line will deliver gene constructs into 
all cells. The nature of viral coats is that they require a cellular 
receptor for which to attach. Amphotropic packaging lines produce viral 
particles which can attach to most or all cells. In some preferred 
embodiments, the gene construct is packaged in an amphotropic line in 
which the glycoprotein and env proteins of AM12 are expressed. 
In some preferred embodiments, the gene constructs are administered 
intravenously. In some preferred embodiments, the gene constructs are 
packaged in an amphotropic line and delivered intravenously, In some 
preferred embodiments, 10.sup.4 to 10.sup.6 viral package particles are 
administered to an individual. In some preferred embodiments, about 
10.sup.5 viral package particles are administered to an individual. 
In one embodiment of the present invention a chimeric gene is constructed 
with comprises the modified MuLV LTR as disclosed in SEQ ID NO:1 operably 
linked to a nucleic acid sequence which encodes human Factor VII protein. 
The nucleic acid sequence which encodes human Factor VII protein is 
disclosed in O'Hara, P. J.. et al. (1987) Proc. Natl. Acad. Sci. USA 
84:5158-5162, which is incorporated herein by reference. 
In one embodiment of the present invention a chimeric gene is constructed 
with comprises the modified MuLV LTR as disclosed in SEQ ID NO:1 operably 
linked to a nucleic acid sequence which encodes human Factor IX protein. 
The nucleic acid sequence which encodes human Factor IX protein is 
disclosed in Yao, S. et al. (1991) Proc. Natl. Acad. Sci. USA 
88:8101-8105, which is incorporated herein by reference. 
In some embodiments of the invention, the chimeric gene is inserted into 
retroviral vectors using a complementation system using vectors such as 
SV-psi.sup.- -env.sup.- -MLV and SV-psi.sup.- -A-MLV. These vectors are 
disclosed in Landau, N. R. and D. R. Littman (1992) J. Virol. 
66(8):5110-5114, which is incorporated herein by reference. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 574 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AATGAAAGACCCCACCAAATTGCTTAGCCTGATAGCCGCAGTAACGCCATTTTGCAAGGC60 
ATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACACGAAAACA120 
GCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGCGAA180 
GAACAGATGGTCACCGCAGTTCGGCCCCGGCCCGGGCGAAGAACAGATGGTCCCCAGATA240 
TGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGAC300 
CTGAAATGACCCTGTGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCG360 
CGCGCTTCTGCTTCCCTAGCCCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGT420 
CCTCCGACAGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAATCCTCTTGCTGTTGCA480 
TCCGACTCGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCAGAGTGATTGACTACCCGTC540 
TCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGAT574 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 415 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
AATGAAAGACCCCACCAAATTGCTTAGCCTGATAGCCGCAGTAACGCCATTTTGCAAGGC60 
ATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACACGAAAACA120 
GCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGCGAA180 
GAACAGATGGTCACCGCAGTTCGGCCCCGGCCCGGGCGAAGAACAGATGGTCCCCAGATA240 
TGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGAC300 
CTGAAATGACCCTGTGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCG360 
CGCGCTTCTGCTTCCCTAGCCCTATAAAAGAGCTCACAACCCCTCACTCGGCGCG415 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 100 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AGGGCGGGTACACGAAAACAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTT60 
TCGGCCCCGGCCCGGGCGAAGAACAGATGGTCACCGCAGT100 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 70 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGCGAAGAACAGATGG60 
TCACCGCAGT70 
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