Adenovirus vectors containing cell status-specific response elements and methods of use thereof
Application no:09392822 -
Filed date:1999-09-09 -
The present invention provides adenoviral vectors comprising cell status-specific transcriptional regulatory elements which confer cell status-specific transcriptional regulation on an adenoviral gene. A “cell status” is generally a reversible physiological and/or environmental state. The invention further provides compositions and host cells comprising the vectors, as well as methods of using the vectors.
No:435 - Chemistry: molecular biology and microbiology / No:320.1 - VECTOR, PER SE (E.G., PLASMID, HYBRID PLASMID, COSMID, VIRAL VECTOR, BACTERIOPHAGE VECTOR, ETC.)
No:435 - Chemistry: molecular biology and microbiology / No:69.1 - Recombinant DNA technique included in method of making a protein or polypeptide
No:435 - Chemistry: molecular biology and microbiology / No:325 - ANIMAL CELL, PER SE (E.G., CELL LINES, ETC.); COMPOSITION THEREOF; PROCESS OF PROPAGATING, MAINTAINING OR PRESERVING AN ANIMAL CELL OR COMPOSITION THEREOF; PROCESS OF ISOLATING OR SEPARATING AN ANIMAL CELL OR COMPOSITION THEREOF; PROCESS OF PREPARING A CO
What is claimed is:
1. A replication-competent adenovirus vector for selective cytolysis of a target cell comprising, a hypoxia responsive element (HRE) operably linked to an adenovirus gene essential for replication selected from the group consisting of E1A, E1B and E4, wherein said HRE comprises a binding site for hypoxia inducible factor-1 is activated and the vector effects selective cytolysis of said target cell under hypoxic conditions.
2. The adenovirus vector of claim 1, wherein the HRE is human.
3. The adenovirus vector of claim 1, wherein said adenovirus gene essential for replication is operably linked to a composite regulatory element comprising said HRE and a tumor cell-specific transcriptional regulatory element (TRE).
4. The adenovirus vector of claim 3, wherein said tumor cell-specific TRE comprises a promoter.
5. The adenovirus vector of claim 3, wherein said tumor cell-specific TRE comprises an enhancer.
6. The adenovirus vector of claim 3, wherein said tumor cell-specific TRE comprises a prostate specific promoter and enhancer.
7. The adenovirus vector of claim 3, wherein said tumor cell-specific transcriptional regulatory element (TRE) is selected from the group consisting of a prostate-specific TRE (PSA-TRE), a glandular kallikrein-1 TRE (hKLK2-TRE), a probasin TRE (PB-TRE), an .alpha.-fetoprotein TRE (AFP TRE) and a carcinoembryonic antigen TRE (CEA TRE).
8. A composition comprising: a replication-competent adenovirus vector of claim 1 and a pharmaceutically acceptable excipient.
9. An isolated host cell comprising the adenovirus vector of claim 1.
10. A method of propagating adenovirus in vitro, the method comprising: introducing into a cell an adenovirus vector comprising a hypoxia responsive element (HRE) operably linked to an adenovirus gene essential for replication selected from the group consisting of E1A, E1B and E4, wherein said HRE comprises a binding site for hypoxia inducible factor-1 wherein said cell is maintained under hypoxic conditions in vitro, thereby expressing said adenovirus gene essential for replication; wherein said adenovirus is propagated.
11. The method of claim 10, wherein said propagating of said adenovirus is cytotoxic to said cell.
12. The method of claim 11, wherein said cell is a tumor cell.
13. A replication-competent adenovirus vector for selective cytolysis of a target cell with a disrupted RB function, comprising: an E2F-1 transcriptional regulatory element (TRE) operably linked to an adenovirus gene essential for replication selected from the group consisting of E1A, E1B and E4, wherein said vector effects selective cytolysis due to selective replication in a target cell with disrupted RB function.
14. The adenovirus vector of claim 13, wherein the E2F-1 TRE is human.
15. The adenovirus vector of claim 14, wherein said E2F-1 TRE comprises the nucleotide sequence set forth in SEQ ID NO:2.
16. The adenovirus vector of claim 13, wherein said E2F-1 TRE comprises a nucleotide sequence having at least 80% sequence identity with the sequence set forth in SEQ ID NO:2.
17. The adenovirus vector of claim 13, wherein said adenovirus gene essential for replication is operably linked to a composite regulatory element comprising said E2F-1 transcriptional regulatory element and a cell-type specific transcriptional regulatory element (TRE).
18. The adenovirus vector of claim 17, wherein said tumor cell-specific transcriptional regulatory element (TRE) is selected from the group consisting of a prostate-specific TRE (PSA-TRE), a glandular kallikrein-1 TRE (hKLK2-TRE), a probasin TRE (PB-TRE), an .alpha.-fetoprotein TRE (AFP TRE) and a carcinoembryonic antigen TRE (CEA TRE).
19. A composition comprising: a replication competent adenovirus vector of claim 13 and a pharmaceutically acceptable excipient.
20. An isolated host cell comprising the adenovirus vector of claim 13.
21. A method of propagating adenovirus in vitro, the method comprising: a replication competent adenovirus vector for selective cytolysis of a target cell, comprising an E2F-1 transcriptional regulatory element (TRE) operably linked to an adenovirus gene essential for replication selected from the group consisting of E1A, E1B and E4 wherein said cell is maintained under cell cycling conditions in vitro, thereby expressing said adenovirus gene essential for replication; wherein said adenovirus is propagated.
22. The method of claim 21, wherein said propagating of said adenovirus is cytotoxic to said cell.
23. The method of claim 21, wherein said cell is a tumor cell.
24. A replication-competent adenovirus vector for selective cytolysis of a target cell comprising, a hypoxia responsive element (HRE) comprising a binding site for hypoxia inducible factor-1 operably linked to a first adenovirus gene essential for replication and a transcriptional regulatory element (TRE) comprising a heterologous promoter or enhancer operably linked to a second adenoviral gene essential for replication wherein said first and second adenoviral genes essential for replication are selected from the group consisting of E1A, E1B and E4 wherein said adenovirus vector results in cytolysis due to selective replication in a target cell in which a hypoxia inducible factor-1 is present.
25. The replication-competent adenovirus vector of claim 24, wherein said transcriptional regulatory element (TRE) linked to said second adenoviral gene essential for replication is a cell status-specific transcriptional regulatory element (TRE).
26. The replication-competent adenovirus vector of claim 24, wherein said transcriptional regulatory element (TRE) linked to said second adenoviral gene essential for replication is a cell type-specific transcriptional regulatory element (TRE).
27. A replication-competent adenovirus vector for selective cytolysis of a target cell, comprising an E2F-1 transcriptional regulatory element (TRE) operably linked to a first adenovirus gene essential for replication and a transcriptional regulatory element (TRE) comprising a heterologous promoter or enhancer operably linked to a second adenoviral gene essential for replication wherein said first and second adenoviral genes essential for replication are selected from the group consisting of E1A, E1B and E4 wherein said adenovirus vector results in cytolysis due to selective replication in a target cell in which RB function is disrupted.
28. The replication-competent adenovirus vector of claim 27, wherein said transcriptional regulatory element (TRE) linked to said second adenoviral gene essential for replication is a cell status-specific transcriptional regulatory element (TRE).
29. The replication-competent adenovirus vector of claim 27, wherein said transcriptional regulatory element (TRE) linked to said second adenoviral gene essential for replication is a cell type-specific transcriptional regulatory element (TRE).
30. A replication-competent adenovirus vector for selective cytolysis of a target cell comprising, a hypoxia responsive element (HRE) operably linked to an adenovirus gene essential for replication selected from the group consisting of E1A, E1B and E4, wherein said HRE comprises a binding site for hypoxia inducible factor-1 wherein target cell, said adenovirus vector results in cytolysis due to selective replication in a tumor cell in which a hypoxia inducible factor-1 is present.
31. A replication-competent adenovirus vector for selective cytolysis of a tumor target cell, comprising: an E2F-1 transcriptional regulatory element (TRE) operably linked to an adenovirus gene essential for replication selected from the group consisting of E1A, E1B and E4 wherein said adenovirus vector results in cytolysis due to selective replication in a tumor target cell to which E2F-1 is present.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER
FEDERALLY SPONSORED RESEARCH
This invention relates to cell transfection using adenoviral vectors. More specifically, it relates to cell status-specific replication of adenovirus vectors in cells, regardless of tissue or cell type.
In spite of numerous advances in medical research, cancer remains the second leading cause of death in the United States. In the industrialized nations, roughly one in five persons will die of cancer. Traditional modes of clinical care, such as surgical resection, radiotherapy and chemotherapy, have a significant failure rate, especially for solid tumors. Neoplasia resulting in benign tumors can usually be completely cured by removing the mass surgically. If a tumor becomes malignant, as manifested by invasion of surrounding tissue, it becomes much more difficult to eradicate. Once a malignant tumor metastasizes, it is much less likely to be eradicated.
Excluding basal cell carcinoma, there are over one million new cases of cancer per year in the United States alone, and cancer accounts for over one half million deaths per year in this country. In the world as a whole, the five most common cancers are those of lung, stomach, breast, colon/rectum, and uterine cervix, and the total number of new cases per year is over 6 million.
Lung cancer is one of the most refractory of solid tumors because inoperable cases are up to 60% and the 5-year survival is only 13%. In particular, adenocarcinomas, which comprise about one-half of the total lung cancer cases, are mostly chemo-radioresistant. Colorectal cancer is the third most common cancer and the second leading cause of cancer deaths. Pancreatic cancer is virtually always fatal. Thus, current treatment prospects for many patients with these carcinomas are unsatisfactory, and the prognosis is poor.
Hepatocellular carcinoma (HCC or malignant hepatoma) is one of the most common cancers in the world, and is especially problematic in Asia. Treatment prospects for patients with hepatocellular carcinoma are dim. Even with improvements in therapy and availability of liver transplant, only a minority of patients are cured by removal of the tumor either by resection or transplantation. For the majority of patients, the current treatments remain unsatisfactory, and the prognosis is poor.
Breast cancer is one of the most common cancers in the United States, with an annual incidence of about 182,000 new cases and nearly 50,000 deaths. In the industrial nations, approximately one in eight women can expect to develop breast cancer. The mortality rate for breast cancer has remained unchanged since 1930. It has increased an average of 0.2% per year, but decreased in women under 65 years of age by an average of 0.3% per year. See e.g., Marchant (1994) Contemporary Management of Breast Disease II: Breast Cancer, in: Obstetrics and Gynecology Clinics of North America 21:555-560; and Colditz (1993) Cancer Suppl. 71:1480-1489.
Despite ongoing improvement in the understanding of the disease, breast cancer has remained resistant to medical intervention. Most clinical initiatives are focused on early diagnosis, followed by conventional forms of intervention, particularly surgery and chemotherapy. Such interventions are of limited success, particularly in patients where the tumor has undergone metastasis. There is a pressing need to improve the arsenal of therapies available to provide more precise and more effective treatment in a less invasive way.
Prostate cancer is the fastest growing neoplasm in men with an estimated 244,000 new cases in the United States being diagnosed in 1995, of which approximately 44,000 deaths will result. Prostate cancer is now the most frequently diagnosed cancer in men. Prostate cancer is latent; many men carry prostate cancer cells without overt signs of disease. It is associated with a high morbidity. Cancer metastasis to bone (late stage) is common and is almost always fatal.
Current treatments include radical prostatectomy, radiation therapy, hormonal ablation and chemotherapy. Unfortunately, in approximately 80% of cases, diagnosis of prostate cancer is established when the disease has already metastasized to the bones, thus limiting the effectiveness of surgical treatments. Hormonal therapy frequently fails with time with the development of hormone-resistant tumor cells. Although chemotherapeutic agents have been used in the treatment of prostate cancer, no single agent has demonstrated superiority over its counterparts, and no drug combination seems particularly effective. The generally drug-resistant, slow-growing nature of most prostate cancers makes them particularly unresponsive to standard chemotherapy.
A major, indeed the overwhelming, obstacle to cancer therapy is the problem of selectivity; that is, the ability to inhibit the multiplication of tumor cells, while leaving unaffected the function of normal cells. For example, in prostate cancer therapy, the therapeutic ratio, or ratio of tumor cell killing to normal cell killing of traditional tumor chemotherapy, is only 1.5:1. Thus, more effective treatment methods and pharmaceutical compositions for therapy and prophylaxis of neoplasia are needed.
Solid tumors frequently contain regions that are poorly vascularized, partly because the tumor cells grow faster than the endothelial cells that make up the blood vessels. Tumor cells can remain viable in such hypoxic conditions and are often refractory to chemotherapy and radiation therapy. In a recent study of cervical cancer, the oxygen status of a tumor was shown to be the single most important prognostic factor, ahead of age of patient, menopausal status, clinical stage, size and histology. Hoeckel et al. (1996) Semin. Radiat. Oncol. 6:1-8.
Of particular interest is development of more specific, targeted forms of cancer therapy, especially for cancers that are difficult to treat successfully. In contrast to conventional cancer therapies, which result in relatively non-specific and often serious toxicity, more specific treatment modalities attempt to inhibit or kill malignant cells selectively while leaving healthy cells intact. Radioresistant and chemoresistant tumors present particular challenges, and there is a need for methods of treating these types of tumors.
One possible treatment approach for many of these cancers is gene therapy, whereby a gene of interest is introduced into the malignant cell. Various viral vectors, including adenoviral vectors, have been developed as vehicles for gene therapy. The virtually exclusive focus in development of adenoviral vectors for gene therapy is use of adenovirus merely as a vehicle for introducing the gene of interest, not as an effector in itself. Replication of adenovirus has been viewed as an undesirable result, largely due to the host immune response. In the treatment of cancer by replication-defective adenoviruses, the host immune response limits the duration of repeat doses at two levels. First, the capsid proteins of the adenovirus delivery vehicle itself are immunogenic. Second, viral late genes are frequently expressed in transduced cells, eliciting cellular immunity. Thus, the ability to repeatedly administer cytokines, tumor suppressor genes, ribozymes, suicide genes, or genes which convert prodrug to an active drug has been limited by the immunogenicity of both the gene transfer vehicle and the viral gene products of the transfer vehicle as well as the transient nature of gene expression.
Use of adenoviral vectors as therapeutic vehicles for cancer has been reported. Some of these approaches utilize tissue (i.e., cell type) specific transcriptional regulatory elements to selectively drive adenoviral replication (and thus cytotoxcity). U.S. Pat. No. 5,698,443; see also WO 95/11984; WO 96/17053; WO 96/34969; WO 98/35028. While useful and promising, there remain other treatment contexts for which tissue specific replication may be insufficient.
Besides cancerous cells, it is often desirable to selectively destroy certain unwanted cells or tissues. Besides surgery, however, which is invasive, there is a dearth of methods available, particularly non-invasive methods, which would allow such selective cytotoxicity and/or suppression.
There is a need for vector constructs that are capable of eliminating essentially all cancerous cells in a minimum number of administrations before specific immunological response against the vector prevents further treatment and which are suitable for use in specific, focused cancer ablation treatments. There is also a need for an ability to selectively destroy, or impair, unwanted cells, regardless of cell type and/or regardless of anatomical location.
SUMMARY OF THE INVENTION
Replication-competent adenoviral vectors specific for cells in a given, or particular, physiological state that permits or induces expression of polynucleotides under transcriptional control of a cell status-specific TRE, and methods for their use are provided. In these replication-competent adenovirus vectors, one or more adenoviral genes is under transcriptional control of an cell status-specific transcriptional regulatory element (TRE). Preferably, the adenoviral gene under transcriptional control of a cell status-specific TRE is one that is essential for adenoviral propagation. A transgene under control of the cell status-specific TRE may also be present. For the adenoviral vectors of the present invention, a cell status-specific TRE is active in a cell existing in a particular, reversible, physiological state, which may be an aberrant physiological state, i.e., a physiological state that deviates from the typical, or normal, physiological state of that same cell when in a non-dividing or regulated dividing state under normal, physiological conditions.
Accordingly, in one aspect, the invention provides an adenovirus vector comprising an adenovirus gene, wherein said adenovirus gene is under transcriptional control of a cell status-specific TRE. In another embodiment, a cell status-specific TRE is human. In another embodiment, a cell status-specific TRE comprises a cell status-specific promoter and enhancer. In yet another embodiment, a cell status-specific TRE is juxtaposed with a cell type-specific TRE, and together the two elements control expression of an adenovirus gene. Thus, the invention provides adenovirus vectors comprising a TRE comprising a cell status-specific TRE and a cell type-specific TRE.
In some embodiments, the adenovirus gene under transcriptional control of a cell status-specific TRE is an adenovirus gene essential for replication. In some embodiments, the adenoviral gene essential for replication is an early gene. In another embodiment, the early gene is E1A. In another embodiment, the early gene is E1B. In yet another embodiment, both E1A and E1B are under transcriptional control of a cell status-specific TRE. In other embodiments, the adenovirus gene essential for replication is a late gene.
In another embodiment, the cell status-specific TRE comprises a hypoxia responsive element. In another embodiment, the cell status-specific TRE comprises the nucleotide sequence of SEQ ID NO:1.
In another embodiment, the cell status-specific TRE comprises a cell cycle-specific TRE. The cell cycle-specific TRE can be derived from the E2F1 5' flanking region. In one embodiment, the cell cycle-specific TRE comprises the nucleotide sequence depicted in SEQ ID NO:2.
In other embodiments, the adenovirus vector can further comprise a transgene, wherein said transgene is under transcriptional control of an cell status-specific TRE. In some embodiments, the transgene is a cytotoxic gene.
In other embodiments, the adenoviral vector comprises an adenoviral gene essential for adenoviral replication under control of a first cell status-specific TRE, and a second adenoviral gene essential for adenoviral replication under control of a second cell status-specific TRE. The first and the second cell status-specific TREs can be identical, substantially identical, or different from, one another.
In other embodiments, the adenoviral vector comprises an adenoviral gene essential for adenoviral replication under control of a first cell status-specific TRE, and a transgene under control of a second cell status-specific TRE. The first and the second cell status-specific TREs can be substantially identical to, or different from, one another.
In other embodiments, the adenovirus vector comprises an adenovirus gene under transcriptional control of a cell status-specific TRE, and a second adenovirus gene under transcriptional control of a cell type-specific TRE. In other embodiments, the adenovirus vector comprises an adenovirus gene under transcriptional control of a cell status-specific TRE, and a transgene under transcriptional control of a cell type-specific TRE.
In another aspect, the invention provides a host cell comprising the adenovirus vector(s) described herein.
In another aspect, the invention provides pharmaceutical compositions comprising an adenovirus vector(s) described herein.
In another aspect, the invention provides kits which contain an adenoviral vector(s) described herein.
In another aspect, methods are provided for conferring selective cytoxicity in target cells (i.e., cells which permit or induce a cell status-specific TRE to function), comprising contacting the cells with an adenovirus vector(s) described herein, whereby the vector enters the cell.
Another embodiment of the invention is an adenovirus which replicates preferentially in mammalian cells whose cell status permits or induces the function of a cell status-specific TRE.
In another aspect, methods are provided for propagating an adenovirus specific for mammalian cells whose cell status permits the function of a cell status-specific TRE, said method comprising combining an adenovirus vector(s) described herein with mammalian cells whose cell status permits the function of a cell status-specific TRE, whereby said adenovirus is propagated.
The invention further provides methods of suppressing tumor cell growth, more particularly a target tumor cell (i.e., a tumor cell that permits or induces a cell status-specific TRE to function), comprising contacting a tumor cell with an adenoviral vector of the invention such that the adenoviral vector enters the tumor cell and exhibits selective cytotoxicity for the tumor cell.
In another aspect, methods are provided for detecting cells whose cell status permits the function of a cell status-specific TRE in a biological sample, comprising contacting cells of a biological sample with an adenovirus vector(s) described herein, and detecting replication of the adenovirus vector, if any.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of adenovirus vector CN796, in which the E1A gene is under transcriptional control of an HRE and a PSA-TRE, as described in Example 1.
FIG. 2 shows the nucleotide sequence of the 5' flanking region of a human E2F1 gene (SEQ ID NO: 1). The asterisk indicates the transcription start site.
FIG. 3 depicts a nucleotide sequence of a prostate-specific antigen TRE.
FIG. 4 depicts a nucleotide sequence of a carcinoembryonic antigen TRE.
FIG. 5 depicts a nucleotide sequence of a human glandular kallikrein TRE.
FIG. 6 depicts a nucleotide sequence of a mucin TRE.
FIG. 7 depicts a nucleotide sequence of a rat probasin TRE.
FIG. 8 depicts a nucleotide sequence and translated amino acid sequence of an adenovirus death protein.
MODES FOR CARRYING OUT THE INVENTION
We have discovered and constructed replication-competent adenovirus vectors which contain an adenoviral gene under transcriptional control of a cell status-specific transcriptional response element (TRE) such that the adenovirus gene is transcribed preferentially in cells whose cell status permit the function of the cell status-specific TRE, and have developed methods using these adenovirus vectors. In some preferred embodiments, the adenovirus vectors of this invention comprise at least one adenovirus gene necessary for adenoviral replication, preferably at least one early gene, under the transcriptional control of a TRE specifically regulated by binding of transcriptional factor(s) and/or co-factor(s) necessary for transcription regulated by the cell status-specific TRE. By providing for cell status-specific transcription of at least one adenovirus gene required for replication, the invention provides adenovirus vectors that can be used for specific cytotoxic effects due to selective replication and/or selective transcription. This is especially useful in the cancer context, in which targeted cell killing is desirable. This is also useful for targeted cytotoxic effects in other, non-tumor cells, when selective destruction and/or suppression of these cells is desirable. The vectors can also be useful for detecting the presence of cells whose cell status permits function of a cell status-specific TRE in, for example, an appropriate biological (such as clinical) sample. Further, the adenovirus vector(s) can optionally selectively produce one or more proteins of interest in a target cell by using a cell status-specific TRE.
We have found that adenovirus vectors of the invention replicate and/or express an adenoviral gene operably linked to a cell status-specific TRE preferentially in cells whose status permits the function of a cell status-specific TRE. In contrast to previously-described adenoviral vectors designed to replicate preferentially in specific, differentiated cell types, the adenovirus vectors of the present invention comprise regulatory elements that are not cell type-specific. Rather, they confer cell status-specific adenoviral replication and/or cell status-specific expression of an operably linked adenoviral gene and/or transgene.
The adenovirus vectors of the present invention comprise a cell status-specific TRE which is functional in a cell which exhibits a particular physiological (i.e., environmental or metabolic) characteristic which is reversible and/or progressive. The target cell may exhibit an aberrant physiological state, such as low oxygen tension, or may be subjected to an aberrant environmental condition, such as heat or ionizing radiation, in order for the cell-status TRE to function. The replication preference of these vectors is indicated by comparing the level of replication (i.e., titer) in cells in a requisite physiological state or condition (for example, an aberrant physiological state) to the level of replication in cells not exhibiting the requisite physiological state (for example, under normal physiological conditions). Thus, the invention also uses and takes advantage of what has been considered an undesirable aspect of adenoviral vectors, namely, their replication and possibly concomitant immunogenicity. The probability of runaway infection is significantly reduced due to the cell status-specific requirements for viral replication. Without wishing to be bound by any particular theory, the inventors note that production of adenovirus proteins can serve to activate and/or stimulate the immune system, generally and/or specifically toward target cells producing adenoviral proteins, which can be an important consideration in the cancer context, where patients are often moderately to severely immunocompromised.
The adenovirus vectors of the present invention find particular utility in specific treatment regimens, in which the treatment is highly focused toward, for example, a particular cancer which might otherwise be inoperable or untreatable. An important feature of the invention is that the vectors are useful in these treatments regardless of the tissue or cell type of the cancer, and yet their cytotoxicity can be targeted to certain locations.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sanbrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D. M. Wei & C. C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991).
For techniques related to adenovirus, see, inter alia, Felgner and Ringold (1989) Nature 337:387-388; Berkner and Sharp (1983) Nucl. Acids Res. 11:6003-6020; Graham (1984) EMBO J. 3:2917-2922; Bett et al. (1993) J. Virology 67:5911-5921; Bett et al. (1994) Proc. Natl. Acad. Sci. USA 91:8802-8806.
As used herein, a "transcription response element" or "transcriptional regulatory element", or "TRE" is a polynucleotide sequence, preferably a DNA sequence, which increases transcription of an operably linked polynucleotide sequence in a host cell that allows that TRE to function. A TRE can comprise an enhancer and/or a promoter.
As used herein, the term "cell status-specific TRE" is one that confers transcriptional activation on an operably linked polynucleotide in a cell which allows a cell status-specific TRE to function, i.e., a cell which exhibits a particular physiological condition, including, but not limited to, an aberrant physiological state. "Cell status" thus refers to a given, or particular, physiological state (or condition) of a cell, which is reversible and/or progressive. The physiological state may be generated internally or externally; for example, it may be a metabolic state (such as low oxygen), or it may be generated due to heat or ionizing radiation. "Cell status" is distinct from a "cell type", which relates to a differentiation state of a cell, which under normal conditions is irreversible. Generally (but not necessarily), as discussed herein, a cell status is embodied in an aberrant physiological state, examples of which are given below.
A "normal cell status" or "normal physiological state" is the status of a cell which exists in normal physiological conditions and which is non-dividing or divides in a regulated manner, i.e., a cell in a normal physiological state.
The terms "aberrant cell status" and "aberrant physiological state", used interchangeably herein, intend a condition of a cell which is a response to, a result of, or is influenced by, an aberrant physiological condition. An aberrant cell status is neither cell type-specific nor tissue type-specific. An aberrant cell status is defined in relation to a cell of the same type which is in a non-dividing/regulated dividing state and under normal physiological conditions.
"Normal physiological conditions" are known to those skilled in the art. These conditions may vary, depending on a cell's location in the body. For example, oxygen tension can vary from tissue to tissue. For in vitro analyses for the purposes of determining whether a TRE is responsive to deviations from normal physiological conditions, these conditions generally include an oxygen concentration of about 20% O.sub.2, and a temperature of about 37.degree. C. "Regulated cell division" is a term well understood in the art and refers to the normal mitotic activity of a cell. Those skilled in the art understand that normal mitotic activity varies from cell type to cell type. For example, many terminally differentiated cells in tissues exhibit little or no mitotic activity, while hematopoietic cells are generally mitotically active.
An "aberrant physiological condition" or "aberrant physiological state", as used herein, intends a condition which deviates from normal physiological conditions, and includes, but is not limited to, a physiological condition that is characterized by alterations in oxygen concentration, such as hypoxic conditions; temperatures which deviate from physiological temperatures; a condition that triggers apoptosis; radiation, including ionizing radiation and UV irradiation; de-regulated cell division, resulting for example, from a lack of, or insufficient amounts of, or inactivity of, a factor which controls cell division, such as, for example, retinoblastoma protein (Rb); variations in timing of cell cycle; infection with a pathogen; exposure to a chemical substance; or a combination of the above-listed conditions. Another example is a mutation that could, or does, exist in any cell type, i.e., its existence does not depend on, or is not related to, the differentiation state of the cell.
A "target cell", as used herein, is one that permits or induces the function of a cell status-specific TRE such that it effects transcriptional activation of an operably linked polynucleotide. A target cell is one which exhibits a requisite physiological (or environmental) state, which may be an aberrant physiological state. Preferably, a target cell is a mammalian cell, preferably a human cell. A target cell may or may not be neoplastic. By transcriptional activation, it is intended that transcription is increased in the target cell above the levels in a control cell (e.g., a that cell when not exhibiting a requisite physiological state (generally a normal physiological state) by at least about 2 fold, preferably at least about 5 fold, preferably at least about 10 fold, more preferably at least about 20 fold, more preferably at least about 50 fold, more preferably at least about 100 fold, more preferably at least about 200 fold, even more preferably at least about 400 fold to about 500 fold, even more preferably at least about 1000 fold. The normal levels are generally the level of activity (if any) in a cell as tested under conditions that activate the cell status-specific TRE, or the level of activity (if any) of a reporter construct lacking a cell status-specific TRE as measured in a cell exhibiting the requisite physiological condition.
A "functionally-preserved" variant of a cell status-specific TRE is a cell status-specific TRE which differs from another cell status-specific TRE, but still retains cell status cell-specific transcription activity. The difference in an cell status-specific TRE can be due to differences in linear sequence, arising from, for example, single base mutation(s), addition(s), deletion(s), and/or modification(s) of the bases. The difference can also arise from changes in the sugar(s), and/or linkage(s) between the bases of a cell status-specific TRE.
An "adenovirus vector" or "adenoviral vector" (used interchangeably) comprises a polynucleotide construct of the invention. A polynucleotide construct of this invention may be in any of several forms, including, but not limited to, DNA, DNA encapsulated in an adenovirus coat, DNA packaged in another viral or viral-like form (such as herpes simplex, and AAV), DNA encapsulated in liposomes, DNA complexed with polylysine, complexed with synthetic polycationic molecules, conjugated with transferrin, and complexed with compounds such as PEG to immunologically "mask" the molecule and/or increase half-life, and conjugated to a nonviral protein. Preferably, the polynucleotide is DNA. As used herein, "DNA" includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides. For purposes of this invention, adenovirus vectors are replication-competent in a target cell.
The terms "polynucleotide" and "nucleic acid", used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be a oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-8; Chaturvedi et al. (1996) Nucleic Acids Res. 24: 2318-23; Schultz et al. (1996) Nucleic Acids Res. 24: 2966-73. A phosphorothiate linkage can be used in place of a phosphodiester linkage. Braun et al. (1988) J. Immunol. 141: 2084-9; Latimer et al. (1995) Mol. Immunol. 32: 1057-1064. In addition, a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.
The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support. Preferably, the polynucleotide is DNA. As used herein, "DNA" includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides.
A polynucleotide or polynucleotide region has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. A preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania), preferably using default parameters.
"Under transcriptional control" is a term well-understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operably (operatively) linked to an element which contributes to the initiation of, or promotes, transcription. "Operably linked" refers to a juxtaposition wherein the elements are in an arrangement allowing them to function.
"Replication" and "propagation" are used interchangeably and refer to the ability of a polynucleotide construct of the invention to reproduce, or proliferate. This term is well understood in the art. For purposes of this invention, replication involves production of adenovirus proteins and is generally directed to reproduction of adenovirus. Replication can be measured using assays standard in the art and described herein, such as a burst assay, plaque assay, or a one-step growth curve assay.
As used herein, "cytotoxicity" is a term well understood in the art and refers to a state in which a cell's usual biochemical or biological activities are compromised (i.e., inhibited). These activities include, but are not limited to, metabolism; cellular replication; DNA replication; transcription; translation; uptake of molecules. "Cytotoxicity" includes cell death and/or cytolysis. Assays are known in the art which indicate cytotoxicity, such as dye exclusion, .sup.3 H-thymidine uptake, and plaque assays.
The term "selective cytotoxicity", as used herein, refers to the cytotoxicity conferred by an adenovirus vector of the present invention on a cell which allows or induces a cell status-specific TRE to function (a target cell) when compared to the cytotoxicity conferred by an adenoviral vector of the present invention on a cell which does not allow a cell status-specific TRE to function (a non-target cell). Such cytotoxicity may be measured, for example, by plaque assays, by reduction or stablization in size of a tumor comprising target cells, or the reduction or stabilization of serum levels of a marker characteristic of the tumor cells, or a tissue-specific marker, e.g., a cancer marker, such as prostate specific antigen.
In the context of adenovirus, a "heterologous polynucleotide" or "heterologous gene" or "transgene" is any polynucleotide or gene that is not present in wild-type adenovirus. Preferably, the transgene will also not be expressed or present in the target cell prior to introduction by the adenovirus vector. Examples of preferred transgenes are provided below.
In the context of adenovirus, a "heterologous" promoter or enhancer is one which is not associated with or derived from an adenovirus gene.
In the context of adenovirus, an "endogenous" promoter, enhancer, or TRE is native to or derived from adenovirus.
In the context of a cell status-specific TRE, a "heterologous" promoter or enhancer is one which is not normally associated in a cell with or derived from a cell status-specific TRE. Examples of a heterologous promoter or enhancer are the albumin promoter or enhancer and other viral promoters and enhancers, such as SV40, or cell type-specific TREs such as a prostate-specific TRE.
A "cell type-specific TRE" is preferentially functional in a specific type of cell relative to other types of cells. In contrast to cell status, "cell type" is a reflection of a differentiation state of a cell which is irreversible. For example, a prostate-specific antigen is expressed in prostate cells, but is not substantially expressed in other cell types such as hepatocytes, astrocytes, cardiocytes, lymphocytes, etc. Generally, a cell type-specific TRE is active in only one cell type. When a cell type-specific TRE is active in more than one cell type, its activity is restricted to a limited number of cell types, i.e., it is not active in all cell types. A cell type-specific TRE may or may not be tumor cell specific.
"Suppressing" tumor growth indicates a growth state that is curtailed when compared to growth without contact with, i.e., transfection by, an adenoviral vector described herein. Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a .sup.3 H-thymidine incorporation assay, or counting tumor cells. "Suppressing" tumor cell growth means any or all of the following states: slowing, delaying, and stopping tumor growth, as well as tumor shrinkage.
As used herein, the terms "neoplastic cells", "neoplasia", "tumor", "tumor cells", "cancer" and "cancer cells", (used interchangeably) refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation (i.e., de-regulated cell division). Neoplastic cells can be malignant or benign.
A "host cell" includes an individual cell or cell culture which can be or has been a recipient of an adenoviral vector(s) of this invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with an adenoviral vector of this invention.
"Replication" and "propagation" are used interchangeably and refer to the ability of an adenovirus vector of the invention to reproduce or proliferate. These terms are well understood in the art. For purposes of this invention, replication involves production of adenovirus proteins and is generally directed to reproduction of adenovirus. Replication can be measured using assays standard in the art and described, herein, such as a burst assay or plaque assay. "Replication" and "propagation" include any activity directly or indirectly involved in the process of virus manufacture, including, but not limited to, viral gene expression; production of viral proteins, nucleic acids or other components; packaging of viral components into complete viruses; and cell lysis.
An "ADP coding sequence" is a polynucleotide that encodes ADP or a functional fragment thereof. In the context of ADP, a "functional fragment" of ADP is one that exhibits cytotoxic activity, especially cell lysis, with respect to adenoviral replication. Ways to measure cytotoxic activity are known in the art and are described herein.
A polynucleotide that "encodes" an ADP polypeptide is one that can be transcribed and/or translated to produce an ADP polypeptide or a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.
An "ADP polypeptide" is a polypeptide containing at least a portion, or region, of the amino acid sequence of an ADP (see, for example, SEQ ID NO:5), and which displays a function associated with ADP, particularly cytotoxicity, more particularly, cell lysis. As discussed herein, these functions can be measured using techniques known in the art. It is understood that certain sequence variations may be used, due to, for example, conservative amino acid substitutions, which may provide ADP polypeptides.
A polynucleotide sequence that is "depicted in" a SEQ ID NO means that the sequence is present as an identical contiguous sequence in the SEQ ID NO. The term encompasses portions, or regions of the SEQ ID NO as well as the entire sequence contained within the SEQ ID NO.
A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term "biological sample" encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
An "individual" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, rodents, primates, and pets.
An "effective amount" is an amount sufficient to effect beneficial or desired results, which may include clinical results. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of an adenoviral vector is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.
Adenoviral Vectors Comprising a Cell Status-Specific TRE
The present invention provides adenoviral vector constructs which comprise an adenovirus gene under transcriptional control of a cell status-specific TRE. Preferably, the adenovirus gene contributes to cytotoxicity (whether direct and/or indirect), more preferably one that contributes to or causes cell death, even more preferably is essential for advenoviral replication. Examples of a gene that contributes to cytotoxicity include, but are not limited to, adenovirus death protein (ADP; discussed below). When the adenovirus vector(s) is selectively (i.e., preferentially) replication competent for propagation in target cells, i.e., cells which permit or induce a cell-status TRE to function, these cells will be preferentially killed upon adenoviral proliferation. Once the target cells are destroyed due to selective cytotoxic and/or cytolytic replication, the adenovirus vector replication is significantly reduced, thus lessening the probability of runaway infection and undesirable bystander effects. In vitro cultures may be retained to monitor the mixture (such as, for example, a biopsy or other appropriate biological sample) for occurrence (i.e., presence) and/or recurrence of the target cell, e.g., a neoplastic cell or other undesired cell. To further ensure cytotoxicity, one or more transgenes having a cytotoxic effect may also be present and under selective transcriptional control. In this embodiment, one may provide higher confidence that the target cells will be destroyed. Additionally, or alternatively, an adenovirus gene that contributes to cytotoxicity and/or cell death (such as ADP) may be included in the adenoviral vector, either free of, or under, selective transcriptional control.
Cell status-specific TREs for use in the adenoviral vectors of the present invention can be derived from any species, preferably a mammal. A number of genes have been described which are expressed in response to, or in association with, a cell status. Any of these cell status-associated genes may be used to generate a cell status-specific TRE.
An example of a cell status is cell cycle. An exemplary gene whose expression is associated with cell cycle is E2F-1, a ubiquitously expressed, growth-regulated gene, which exhibits peak transcriptional activity in S phase. Johnson et al. (1994) Genes Dev. 8:1514-1525. The RB protein, as well as other members of the RB family, form specific complexes with E2F-1, thereby inhibiting its ability to activate transcription. Thus, E2F-1-responsive promoters are down-regulated by RB. Many tumor cells have disrupted RB function, which can lead to de-repression of E2F-1-responsive promoters, and, in turn, de-regulated cell division.
Accordingly, in one embodiment, the invention provides an adenoviral vector in which an adenoviral gene (preferably a gene necessary for replication) is under transcriptional control of a cell status-specific TRE, wherein the cell status-specific TRE comprises a cell cycle-activated, or cell-cycle specific, TRE. In one embodiment, the cell cycle-activated TRE is an E2F1 TRE. In one embodiment, this TRE comprises the sequence depicted in FIG. 3 and SEQ ID NO:2.
Another group of genes which are regulated by cell status are those whose expression is increased in response to hypoxic conditions. Bunn and Poyton (1996) Physiol. Rev. 76:839-885; Dachs and Stratford (1996) Br. J. Cancer 74:5126-5132; Guillemin and Krasnow (1997) Cell 89:9-12. Many tumors have insufficient blood supply, due in part to the fact that tumor cells typically grow faster than the endothelial cells that make up the blood vessels, resulting in areas of hypoxia in the tumor. Folkman (1989) J. Natl Cancerinst. 82:4-6; and Kallinowski (1996) The Cancer J. 9:37-40. An important mediator of hypoxic responses is the transcriptional complex HIF-1, or hypoxia inducible factor-1, which interacts with a hypoxia-responsive element (HRE) in the regulatory regions of several genes, including vascular endothelial growth factor, and several genes encoding glycolytic enzymes, including enolase-1. Murine HRE sequences have been identified and characterized. Firth et al. (1994) Proc. Natl. Acad. Sci. USA 91:6496-6500. An HRE from a rat enolase-1 promoter is described in Jiang et al. (1997) Cancer Res. 57:5328-5335.
Accordingly, in one embodiment, an adenovirus vector comprises an adenovirus gene, preferably an adenoviral gene essential for replication, under transcriptional control of a cell status-specific TRE comprising an HRE.
Other cell status-specific TREs include heat-inducible (i.e., heat shock) promoters, and promoters responsive to radiation exposure, including ionizing radiation and UV radiation. For example, the promoter region of the early growth response-1 (Egr-1) gene contains an element(s) inducible by ionizing radiation. Hallahan et al. (1995) Nat. Med. 1:786-791; and Tsai-Morris et al. (1988) Nucl. Acids. Res. 16:8835-8846. Heat-inducible promoters, including heat-inducible elements, have been described. See, for example Welsh (1990) in "Stress Proteins in Biology and Medicine", Morimoto, Tisseres, and Georgopoulos, eds. Cold Spring Harbor Laboratory Press; and Perisic et al. (1989) Cell 59:797-806. Accordingly, in some embodiments, the cell status-specific TRE comprises an element(s) responsive to ionizing radiation. In one embodiment, this TRE comprises a 5' flanking sequence of an Egr-1 gene. In other embodiments, the cell status-specific TRE comprises a heat shock responsive, or heat-inducible, element.
A cell status-specific TRE can also comprise multimers. For example, an HRE can comprise a tandem series of at least two, at least three, at least four, or at least five hypoxia-responsive elements. These multimers may also contain heterologous promoter and/or enhancer sequences.
A cell status-specific TRE may or may not lack a silencer. The presence of a silencer (i.e., a negative regulatory element) may assist in shutting off transcription (and thus replication) in non-permissive cells (i.e., cell in a normal cell state). Thus, presence of a silencer may confer enhanced cell status-specific replication by more effectively preventing adenoviral vector replication in non-target cells. Alternatively, lack of a silencer may assist in effecting replication in target cells, thus conferring enhanced cell status-specific replication due to more effective replication in target cells.
In other embodiments, the adenoviral vector comprises an adenoviral gene essential for adenoviral replication under control of a first cell status-specific TRE, and a second adenoviral gene essential for adenoviral replication under control of a second cell status-specific TRE. The first and the second cell status-specific TREs may or may not be identical, and may or may not be substantially identical to one another. By "substantially identical" is meant a requisite degree of sequence identity between the two TREs. The degree of sequence identity between these TREs is at least about 80%, preferably at least about 85%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 98%, and most preferably 100%. Sequence identity can be determined by a sequence comparison using, i.e., sequence alignment programs that are known in the art, such as those described in Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1 A preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania), preferably using default parameters. Alternatively, hybridization under stringent conditions can also indicate degree of sequence identity. Stringent conditions are known in the art; an example of a stringent condition is 80.degree. C. (or higher temperature) and 6.times. SSC (or less concentrated SSC). Other hybridization conditions and parameters (in order of increasing stringency) are: incubation temperatures of 25.degree. C., 37.degree. C., 50.degree. C., and 68.degree. C.; buffer concentrations of 10.times. SSC, 6.times. SSC, 1.times. SSC, 0.1.times. SSC (where 1.times. SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalents using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from about 24 hours about 5 minutes; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6.times. SSC, 1.times. SSC, 0.1.times. SSC, or deionized water.
Adenoviral constructs in which the first and second cell status-specific TREs are identical or substantially identical, particularly if these TREs control transcription of early genes (such as E1A and E1B), may display an instability which may be desirable in certain contexts, such as when an automatic "self-destruction" property can shut down the virus, thereby controlling the degree of propagation. Accordingly, in some embodiments, the first and second cell status-specific TRE, or the first and second TRE (one of which is a cell-status-specific TRE) are sufficiently identical to confer instability when compared to two TREs which are less identical with respect to each other (i.e., have more sequence divergence or dissimilarity). Preferred embodiments are those in which the two TREs control E1A and E1B respectively. "Instability" means that the structural integrity of the adenoviral vectors is not preserved as the virus replicates in cells, and can be measured using standard methods in the art, such as Southern analysis. In other embodiments, the first and second TREs are sufficiently divergent and/or placed in the vector such that the vector is stable (i.e., the structural integrity of the adenoviral vector is preserved).
In other embodiments, the adenoviral vector comprises an adenoviral gene essential for adenoviral replication under control of a first cell status-specific TRE, and a transgene under control of a second cell status-specific TRE. The first and the second cell status-specific TREs may or may not be substantially identical to one another.
In some embodiments, a cell status-specific TRE can be juxtaposed with another TRE, such as a different cell status-specific TRE, or, alternatively, a cell type-specific TRE. "Juxtaposed" means a cell status-specific TRE and the second TRE transcriptionally control the same gene, or at least are proximate with respect to the same gene. For these embodiments, the cell status-specific TRE and the second TRE may be in any of a number of configurations, including, but not limited to, (a) next to each other (i.e., abutting); (b) both 5' to the gene that is transcriptionally controlled (i.e., may have intervening sequences between them); (c) one TRE 5' and the other TRE 3' to the gene. For example, as described in Example 1 and shown in FIG. 1, a cell type-specific TRE can be juxtaposed with a cell status-specific TRE to control transcription of an operably linked adenoviral gene. Such "composite" TREs can be used to confer both cell status- and cell type-specific expression of an operably linked polynucleotide, and thus may confer significantly greater specificity and/or efficacy. Examples of cell type-specific TREs are provided below. Alternatively, "composite" TREs can be used to confer different, and possibly synergistic, cell status specificity. For example, a composite TRE could confer specificity to hypoxia and heat shock.
Example 1 provides a description of an adenovirus construct in which a composite TRE upstream of E1A consisting of an HRE and a prostate-specific TRE, PSA-TRE (which consists of enhancer sequences -5322 to -3738 fused to PSA promoter sequence -541 to +12; see U.S. Pat. Nos. 5,871,726; 5,648,478). Accordingly, in some embodiments, the invention provides an adenovirus vector comprising an adenovirus gene essential for replication, preferably an early gene, preferably E1A or E1B, under transcriptional control of a TRE comprising an HRE and a prostate cell specific TRE, preferably comprising a PSA enhancer (preferably -5322 to -3738; or about 503 to about 2086 of SEQ ID NO:3 (bases about 503 to about 2086 of FIG. 4), and a promoter, preferably comprising a PSA enhancer and a PSA promoter (about 5285 to about 5836 of SEQ ID NO:3).
As is readily appreciated by one skilled in the art, a cell status-specific TRE is a polynucleotide sequence, and, as such, can exhibit function over a variety of sequence permutations. Methods of nucleotide substitution, addition, and deletion are known in the art, and readily available functional assays (such as the CAT or luciferase reporter gene assay) allow one of ordinary skill to determine whether a sequence variant exhibits requisite cell status-specific transcription function. Hence, the invention also includes functionally-preserved variants of the nucleic acid sequences disclosed herein, which include nucleic acid substitutions, additions, and/or deletions. While not wishing to be bound by a single theory, the inventors note that it is possible that certain modifications will result in modulated resultant expression levels, including enhanced expression levels. Achievement of modulated resultant expression levels, preferably enhanced expression levels, may be especially desirable in the case of certain, more aggressive forms of cancer, or when a more rapid and/or aggressive pattern of cell killing is warranted (due to an immunocompromised condition of the individual, for example).
As an example of how cell status-specific TRE activity can be determined, a polynucleotide sequence or set of such sequences can be generated using methods known in the art, such as chemical synthesis, site-directed mutagenesis, PCR, and/or recombinant methods. The sequence(s) to be tested is inserted into a vector containing an appropriate reporter gene, including, but not limited to, chloramphenicol acetyl transferase (CAT), .beta.-galactosidase (encoded by the lacZ gene), luciferase (encoded by the luc gene), green fluorescent protein, alkaline phosphatase, and horse radish peroxidase. Such vectors and assays are readily available, from, inter alia, commercial sources. Plasmids thus constructed are transfected into a suitable host cell to test for expression of the reporter gene as controlled by the putative cell status-specific TRE using transfection methods known in the art, such as calcium phosphate precipitation, electroporation, liposomes (lipofection), and DEAE-dextran. Suitable host cells include any cell type, including but not limited to, Hep3B, Hep G2, HuH7, HuH1/C12, LNCaP, HBL-100, Chang liver cells, MCF-7, HLF, HLE, 3T3, HUVEC, and HeLa. Host cells transfected with the TRE-reporter gene construct to be tested are subjected to conditions which result in a change in cell status (for example, one which result in an aberrant physiological state). The same cells not subjected to these conditions, i.e., which are under normal physiological conditions and therefore in a normal physiological state, serve as controls. Results are obtained by measuring the level of expression of the reporter gene using standard assays. Comparison of expression between cells in a particular state and control indicates presence or absence of transcriptional activation. "Transcriptional activation" has been defined above.
Comparisons between or among various cell status-specific TREs can be assessed, for example, by measuring and comparing levels of expression within a single cell line under normal and aberrant physiological conditions. These comparisons may also be made by measuring and comparing levels of expression within a single cell line subjected to reversible environmental conditions (such as heat) and cells not subjected to such conditions. It is understood that absolute transcriptional activity of an cell status-specific TRE will depend on several factors, such as the nature of the target cell, delivery mode and form of the cell status-specific TRE, and the coding sequence that is to be selectively transcriptionally activated. To compensate for various plasmid sizes used, activities can be expressed as relative activity per mole of transfected plasmid. Alternatively, the level of transcription (i.e., mRNA) can be measured using standard Northern analysis and hybridization techniques. Levels of transfection (i.e., transfection efficiencies) are measured by co-transfecting a plasmid encoding a different reporter gene under control of a different TRE, such as the cytomegalovirus (CMV) immediate early promoter. This analysis can also indicate negative regulatory regions, i.e., silencers.
As an example of how hypoxia induction can be measured, one can use an assay such as that described in Jiang et al. (1997) Cancer Research 57:5328-5335 or Dachs et al. (1997) Nature Med. 3:515-520. For example, a construct comprising a putative HRE, or multiple tandem copies thereof, together with a minimal promoter element, operably linked and controlling transcription of a polynucleotide which encodes a protein which is detectable or can be used to give a detectable signal, is introduced into host cells. The host cells are then subjected to conditions of normoxia (e.g., 20% O.sub.2), and varying degrees of hypoxia, such as 5%, 2%, 1%, 0.1%, or less, O.sub.2. The expression product of the operably linked polynucleotide (reporter gene) is then measured.
Alternatively a putative cell status-specific TRE can be assessed for its ability to confer adenoviral replication preference for cells exhibiting the requisite physiological state, such as heat or ionizing radiation. For this assay, constructs containing an adenovirus gene essential to replication operably linked to a putative cell status-specific TRE are transfected into cells which exhibit the requisite physiological state. Viral replication in those cells is compared, for example, to viral replication by the construct in cells under normal physiological conditions (i.e., not exhibiting the requisite physiological state).
Any of the various serotypes of adenovirus can be used, such as Ad2, Ad5, Ad12 and Ad40. For purposes of illustration, serotype Ad5 will be exemplified herein.
When a cell status-specific TRE is used with an adenovirus gene that is essential for propagation replication competence is preferentially achievable in the target cell expressing cell status. Preferably, the gene is an early gene, such as E1A, E1B, E2, or E4. (E3 is not essential for viral replication.) More preferably, the early gene under cell status-TRE control is E1A and/or E1B. More than one early gene can be placed under control of an cell status-specific TRE. Example 1 provides a more detailed description of such constructs.
The E1A gene is expressed immediately after viral infection (0-2 hours) and before any other viral genes. E1A protein acts as a trans-acting positive-acting transcriptional regulatory factor, and is required for the expression of the other early viral genes E1B, E2, E3, E4, and the promoter-proximal major late genes. Despite the nomenclature, the promoter proximal genes driven by the major late promoter are expressed during early times after Ad5 infection. Flint (1982) Biochem. Biophys. Acta 651:175-208; Flint (1986) Advances Virus Research 31:169-228; Grand (1987) Biochem. J. 241:25-38. In the absence of a functional E1A gene, viral infection does not proceed, because the gene products necessary for viral DNA replication are not produced. Nevins (1989) Adv. Virus Res. 31:35-81. The transcription start site of Ad5 E1A is at 498 and the ATG start site of the E1A protein is at 560 in the virus genome.
The E1B protein functions in trans and is necessary for transport of late mRNA from the nucleus to the cytoplasm. Defects in E1B expression result in poor expression of late viral proteins and an inability to shut off host cell protein synthesis. The promoter of E1B has been implicated as the defining element of difference in the host range of Ad40 and Ad5: clinically Ad40 is an enterovirus, whereas Ad5 causes acute conjunctivitis. Bailey, Mackay et al. (1993) Virology 193:631; Bailey et al. (1994) Virology 202:695-706). The E1B promoter of Ad5 consists of a single high-affinity recognition site for Spl and a TATA box.
The E2 region of adenovirus codes for proteins related to replication of the adenoviral genome, including the 72 kDa DNA-binding protein, the 80 kD precursor terminal protein and the viral DNA polymerase. The E2 region of Ad5 is transcribed in a rightward orientation from two promoters, termed E2 early and E2 late, mapping at 76.0 and 72.0 map units, respectively. While the E2 late promoter is transiently active during late stages of infection and is independent of the E1A transactivator protein, the E2 early promoter is crucial during the early phases of viral replication.
The E2 late promoter overlaps with the coding sequences of a gene encoded by the counterstrand and is therefore not amenable to genetic manipulation. However, the E2 early promoter overlaps only for a few base pairs with sequences coding for a 33 kD protein on the counterstrand. Notably, the SpeI restriction site (Ad5 position 27082) is part of the stop codon for the above mentioned 33 kD protein and conveniently separates the major E2 early transcription initiation site and TATA-binding protein site from the upstream transcription factor biding sites E2F and ATF. Therefore, insertion of a cell status-TRE having SpeI ends into the SpeI site in the +-strand would disrupt the endogenous E2 early promoter of Ad5 and should allow cell status-restricted expression of E2 transcripts.
The E4 gene has a number of transcription products. The E4 region codes for two polypeptides which are responsible for stimulating the replication of viral genomic DNA and for stimulating late gene expression. The protein products of open reading frames (ORFS) 3 and 6 can both perform these functions by binding the 55 kD protein from E1B and heterodimers of E2F-1 and DP-1. The ORF 6 protein requires interaction with the E1B 55 kD protein for activity while the ORF 3 protein does not. In the absence of functional protein from ORF 3 and ORF 6, plaques are produced with an efficiency less than 10.sup.-6 that of wild type virus. To further restrict viral replication to cells exhibiting a requisite physiological condition or state, E4 ORFs 1-3 can be deleted, making viral DNA replication and late gene synthesis dependent on E4 ORF 6 protein. By combining such a mutant with sequences in which the E1B region is regulated by a cell status-specific TRE, a virus can be obtained in which both the E1B function and E4 function are dependent on a cell status-specific TRE driving E1B.
The major late genes relevant to the subject invention are genes L1, L2, L3, L4, and L5 which encode proteins of the adenovirus virion. All of these genes (typically coding for structural proteins) are probably required for adenoviral replication. The late genes are all under the control of the major late promoter (MLP), which is located in Ad5 at +5986 to +6048.
In addition to conferring selective cytotoxic and/or cytolytic activity by virtue of preferential replication competence in cells exhibiting a requisite physiological state (for example, an aberrant physiological state such as low oxygen conditions), the adenovirus vectors of this invention can further include a heterologous gene (transgene) under the control of a cell status-specific TRE. In this way, various genetic capabilities may be introduced into target cells, particularly cancer cells. For example, in certain instances, it may be desirable to enhance the degree and/or rate of cytotoxic activity, due to, for example, the relatively refractory nature or particular aggressiveness of the cancerous target cell. This could be accomplished by coupling the cell status-specific replicative cytotoxic activity with cell-specific expression of, for example, HSV-tk and/or cytosine deaminase (cd), which renders cells capable of metabolizing 5-fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil (5-FU). Using these types of transgenes may also confer a bystander effect.
Other desirable transgenes that may be introduced via an adenovirus vector(s) include genes encoding cytotoxic proteins, such as the A chains of diphtheria toxin, ricin or abrin (Palmiter et al. (1987) Cell 50: 435; Maxwell et al. (1987) Mol. Cell. Biol. 7: 1576; Behringer et al. (1988) Genes Dev. 2: 453; Messing et al. (1992) Neuron 8: 507; Piatak et al. (1988) J. Biol. Chem. 263: 4937; Lamb et al. (1985) Eur. J. Biochem. 148: 265; Frankel et al. (1989) Mol. Cell. Biol. 9: 415), genes encoding a factor capable of initiating apoptosis, sequences encoding antisense transcripts or ribozymes, which among other capabilities may be directed to mRNAs encoding proteins essential for proliferation, such as structural proteins, or transcription factors; viral or other pathogenic proteins, where the pathogen proliferates intracellularly; genes that encode an engineered cytoplasmic variant of a nuclease (e.g. RNase A) or protease (e.g. awsin, papain, proteinase K, carboxypeptidase, etc.), or encode the Fas gene, and the like. Other genes of interest include cytokines, antigens, transmembrane proteins, and the like, such as IL-1, -2, -6, -12, GM-CSF, G-CSF, M-CSF, IFN-.alpha., -.beta., -.gamma., TNF-.alpha., -.beta., TGF-.alpha., -.beta., NGF, and the like. The positive effector genes could be used in an earlier phase, followed by cytotoxic activity due to replication.
In one embodiment, the adenovirus death protein (ADP), encoded within the E3 region, is maintained in the adenovirus vector. The ADP gene, under control of the major late promoter (MLP), appears to code for a protein (ADP) that is important in expediting host cell lysis. Tollefson et al. (1996) J. Virol. 70(4):2296; Tollefson et al. (1992) J. Virol. 66(6):3633. Thus, adenoviral vectors containing the ADP gene may render the adenoviral vector more potent, making possible more effective treatment and/or a lower dosage requirement.
Accordingly, the invention provides an adenoviral vector as described herein that further includes a polynucleotide sequence encoding an ADP. A DNA sequence encoding an ADP and the amino acid sequence of an ADP are depicted FIG. 9. Briefly, an ADP coding sequence is obtained preferably from Ad2 (since this is the strain in which ADP has been more fully characterized) using techniques known in the art, such as PCR. Preferably, the Y leader (which is an important sequence for correct expression of late genes) is also obtained and ligated to the ADP coding sequence. The ADP coding sequence (with or without the Y leader) can then be introduced into the adenoviral genome, for example, in the E3 region (where the ADP coding sequence will be driven by the MLP). The ADP coding sequence could also be inserted in other locations of the adenovirus genome, such as the E4 region. Alternatively, the ADP coding sequence could be operably linked to a heterologous promoter (with or without enhancer(s)), including, but not limited to, another viral promoter, a cell status-specific TRE such as a hypoxia responsive element, or a cell type-specific TRE such as those derived from carcinoembryonic antigen (CEA), mucin, and rat probasin genes.
Adenoviral Vectors of the Invention Further Comprising a Cell Type Specific Element
In addition to conferring selective cytotoxic and/or cytolytic activity by virtue of preferential replication competence and/or by preferential transcription of a gene encoding a cytotoxic factor in cells exhibiting a requisite physiological state, the adenovirus vectors of this invention can further include an adenovirus gene and/or a heterologous gene (transgene) under the control of a cell type-specific TRE. In this way, cytotoxicity is further limited to a particular cell type.
For example, TREs that function preferentially in prostate cells include, but are not limited to, TREs derived from the prostate-specific antigen gene (PSA-TRE) (U.S. Pat. No. 5,648,478), the glandular kallikrein-1 gene (from the human gene, hKLK2-TRE), and the probasin gene (PB-TRE) (International Patent Application No. PCT/US98/04132). All three of these genes are preferentially expressed in prostate cells and the expression is androgen-inducible. Generally, expression of genes responsive to androgen induction requires the presence of an androgen receptor (AR).
PSA is synthesized exclusively by normal, hyperplastic, and malignant prostatic epithelia; hence, its tissue-specific expression has made it an excellent biomarker for benign prostatic hyperplasia (BPH) and prostatic carcinoma (CaP). Normal serum levels of PSA are typically below 5 ng/ml, with elevated levels indicative of BPH or CaP. Lundwall et al. (1987) FEBS Lett. 214: 317; Lundwall (1989) Biochem. Biophys. Res. Comm. 161: 1151; and Riegmann et al. (1991) Molec. Endocrin. 5:1921.
The region of the PSA gene that is used to provide cell specificity dependent upon androgens, particular in prostate cells, involves approximately 6.0 kilobases. Schuur et al. (1996) J. Biol. Chem. 271:7043-7051. An enhancer region of approximately 1.5 kb in humans is located between nt -5322 and nt -3738, relative to the transcription start site of the PSA gene. The PSA promoter consists of the sequence from about nt -540 to nt +12 relative to the transcription start site. Juxtapositioning of these two genetic elements yield a fully functional, minimal prostate-specific enhancer/promoter (PSE) TRE. Other portions of the approximately 6.0 kb region of the PSA gene can be used in the present invention, as long as requisite functionality is maintained. In Example 1, adenoviral vector CN796 is described which comprises a composite TRE comprising an HRE and a PSA-TRE, the PSA-TRE comprising a PSA enhancer from -5322 to -3738 fused to a PSA promoter from -541 to +12. This PSA-TRE is derived from adenoviral vector CN706. Rodriguez et al. (1997) Cancer Research 57:2559-2563. Accordingly, in one embodiment an adenoviral vector comprises and adenovirus E1A gene under transcriptional control of a composite TRE comprising the cell status-specific TRE, HRE, and a cell type-specific TRE, a PSA-TRE.
The PSE and PSA TRE used in the present invention are derived from sequences depicted in FIG. 4 (SEQ ID NO:3). The enhancer element is nucleotides about 503 to about 2086 of FIG. 4 (SEQ ID NO:3). The promoter is nucleotides about 5285 to about 5836 of FIG. 4 (SEQ ID NO:3). Accordingly, in some embodiments, the composite TRE comprises an HRE and a PSA-TRE comprises nucleotides about 503 to about 2086 of SEQ ID NO:3. In other embodiments, the composite TRE comprises an HRE and a PSA-TRE comprises nucleotides about 503 to about 2086 of SEQ ID NO:3 and nucleotides about 5285 to about 5836 of SEQ ID NO:3. As described above, these composite (HRE/PSA) TREs may be operably linked to an adenovirus gene essential for replication, especially an early gene such as E1A or E1B. Example 1 describes such a construct.
In the present invention, replication-competent adenovirus vectors comprising a cell status-specific TRE and a cell type-specific TRE may employ cell type-specific TREs that are preferentially functional in particular tumor cells. Non-limiting examples of tumor cell-specific TREs, and non-limiting examples of respective potential target cells, include TREs from the following genes: .alpha.-fetoprotein (AFP) (liver cancer), mucin-like glycoprotein DF3 (MUC1) (breast carcinoma), carcinoembryonic antigen (CEA) (colorectal, gastric, pancreatic, breast, and lung cancers), plasminogen activator urokinase (uPA) and its receptor gene (breast, colon, and liver cancers), HER-2/neu (c-erbB2/neu) (breast, ovarian, stomach, and lung cancers).
Other cell type-specific TREs may be derived from the following exemplary genes (cell type in which the TREs are specifically functional are in parentheses): vascular endothelial growth factor receptor (endothelium), albumin (liver), factor VII (liver), fatty acid synthase (liver), von Willebrand factor (brain endothelium), alpha-actin and myosin heavy chain (both in smooth muscle), synthetase I (small intestine), Na--K--Cl transporter (kidney). Additional cell type-specific TREs are known in the art.
AFP is an oncofetal protein, the expression of which is primarily restricted to developing tissues of endodermal origin (yolk sac, fetal liver, and gut), although the level of its expression varies greatly depending on the tissue and the developmental stage. AFP is of clinical interest because the serum concentration of AFP is elevated in a majority of hepatoma patients, with high levels of AFP found in patients with advanced disease. The serum AFP levels in patients appear to be regulated by AFP expression in hepatocellular carcinoma but not in surrounding normal liver. Thus, the AFP gene appears to be regulated to hepatoma cell-specific expression.
Cell type-specific TREs from the AFP gene have been identified. For example, the cloning and characterization of human AFP-specific enhancer activity is described in Watanabe et al. (1987) J. Biol. Chem. 262:4812-4818. The entire 5' AFP flanking region (containing the promoter, putative silencer, and enhancer elements) is contained within approximately 5 kb upstream from the transcription start site.
The AFP enhancer region in human is located between about nt -3954 and about nt -3335, relative to the transcription start site of the AFP gene. The human AFP promoter encompasses a region from about nt -174 to about nt +29. Juxtapositioning of these two genetic elements yields a fully functional AFP-TRE. Ido et al. (1995) describe a 259 bp promoter fragment (nt -230 to nt +29) that is specific for HCC. Cancer Res. 55:3105-3109. The AFP enhancer contains two regions, denoted A and B, located between nt -3954 and nt -3335 relative to the transcription start site. The promoter region contains typical TATA and CAAT boxes. Preferably, the AFP-TRE contains at least one enhancer region. More preferably, the AFP-TRE contains both enhancer regions.
Suitable target cells for adenoviral vectors containing AFP-TREs are any cell type that allow an AFP-TRE to function. Preferred are cells that express, or produce, AFP, including, but not limited to, tumor cells expressing AFP. Examples of such cells are hepatocellular carcinoma cells, gonadal and other germ cell tumors (especially endodermal sinus tumors), brain tumor cells, ovarian tumor cells, acinar cell carcinoma of the pancreas (Kawanoto et al. (1992) Hepatogastroenterology 39:282-286), primary gall bladder tumor (Katsuragi et al. (1989) Rinsko Hoshasen 34:371-374), uterine endometrial adenocarcinoma cells (Koyama et al. (1996) Jpn. J. Cancer Res. 87:612-617), and any metastases of the foregoing (which can occur in lung, adrenal gland, bone marrow, and/or spleen). In some cases, metastatic disease to the liver from certain pancreatic and stomach cancers produce AFP. Especially preferred are hepatocellular carcinoma cells and any of their metastases. AFP production can be measured using assays standard in the art, such as RIA, ELISA or Western blots (immunoassays) to determine levels of AFP protein production or Northern blots to determine levels of AFP mRNA production. Alternatively, such cells can be identified and/or characterized by their ability to activate transcriptionally an AFP-TRE (i.e., allow an AFP-TRE to function).
The protein urokinase plasminogen activator (uPA) and its cell surface receptor, urokinase plasminogen activator receptor (uPAR), are expressed in many of the most frequently occurring neoplasia and appear to represent important proteins in cancer metastasis. Both proteins are implicated in breast, colon, prostate, liver, renal, lung and ovarian cancer. Transcriptional regulatory elements that regulate uPA and uPAR transcription have been extensively studied. Riccio et al. (1985) Nucleic Acids Res. 13:2759-2771; Cannio et al. (1991) Nucleic Acids Res. 19:2303-2308.
CEA is a 180,000-Dalton glycoprotein tumor-associated antigen present on endodermally-derived neoplasia of the gastrointestinal tract, such as colorectal, gastric (stomach) and pancreatic cancer, as well as other adenocarcinomas such as breast and lung cancers. CEA is of clinical interest because circulating CEA can be detected in the great majority of patients with CEA-positive tumors. In lung cancer, about 50% of total cases have circulating CEA, with high concentrations of CEA (greater than 20 ng/ml) often detected in adenocarcinomas. Approximately 50% of patients with gastric carcinoma are serologically positive for CEA.
The 5' upstream flanking sequence of the CEA gene has been shown to confer cell-specific activity. The CEA promoter region, approximately the first 424 nucleotides upstream of the translational start site in the 5' flanking region of the gene, was shown to confer cell-specific activity when the region provided higher promoter activity in CEA-producing cells than in non-producing HeLa cells. Schrewe et al. (1990) Mol. Cell. Biol. 10:2738-2748. In addition, cell-specific enhancer regions have been found. WO/95/14100. The entire 5' CEA flanking region (containing the promoter, putative silencer, and enhancer elements) appears to be contained within approximately 14.5 kb upstream from the transcription start site. Richards et al. (1995); WO 95/14100. Further characterization of the 5' flanking region of the CEA gene by Richards et al. (1995) indicated two upstream regions, -13.6 to -10.7 kb or -6.1 to -4.0 kb, when linked to the multimerized promoter resulted in high-level and selective expression of a reporter construct in CEA-producing LoVo and SW1463 cells. Richards et al. (1995) also localized the promoter region to nt -90 and nt +69 relative to the transcriptional start site, with region nt -41 to nt -18 as essential for expression. WO95/14100 describes a series of 5' flanking CEA fragments which confer cell-specific activity, such as about nt -299 to about nt +69; about nt -90 to about nt +69; nt -14,500 to nt -10,600; nt -13,600 to nt -10,600, nt -6100 to nt -3800. In addition, cell specific transcription activity is conferred on an operably linked gene by the CEA fragment from nt -402 to nt +69, depicted in (SEQ ID NO:6). Any CEA-TREs used in the present invention are derived from mammalian cells, including but not limited to, human cells. Thus, any of the CEA-TREs may be used in the invention as long as requisite desired functionality is displayed in the adenovirus vector. The cloning and characterization of CEA sequences have been described in the literature and are thus made available for practice of this invention and need not be described in detail herein.
The protein product of the MUC1 gene (known as mucin or MUC1 protein; episialin; polymorphic epithelial mucin or PEM; EMA; DF3 antigen; NPGP; PAS-O; or CA15.3 antigen) is normally expressed mainly at the apical surface of epithelial cells lining the glands or ducts of the stomach, pancreas, lungs, trachea, kidney, uterus, salivary glands, and mammary glands. Zotter et al. (1988) Cancer Rev. 11-12: 55-101; and Girling et al. (1989) Int. J. Cancer 43: 1072-1076. However, mucin is overexpressed in 75-90% of human breast carcinomas. Kufe et al. (1984) Hybridoma 3: 223-232. For reviews, see Hilkens (1988) Cancer Rev. 11-12: 25-54; and Taylor-Papadimitriou, et al. (1990) J. Nucl. Med. Allied Sci. 34: 144-150. Mucin protein expression correlates with the degree of breast tumor differentiation. Lundy et al. (1985) Breast Cancer Res. Treat. 5: 269-276. This overexpression appears to be controlled at the transcriptional level.
Overexpression of the MUC1 gene in human breast carcinoma cells MCF-7 and ZR-75-1 appears to be regulated at the transcriptional level. Kufe et al. (1984); Kovarik (1993) J. Biol. Chem. 268:9917-9926; and Abe et al. (1990) J. Cell. Physiol. 143: 226-231. The regulatory sequences of the MUC1 gene have been cloned, including the approximately 0.9 kb upstream of the transcription start site which contains a TRE that appears to be involved in cell-specific transcription. Abe et al. (1993) Proc. Natl. Acad. Sci. USA 90: 282-286; Kovarik et al. (1993); and Kovarik et al. (1996) J. Biol. Chem. 271:18140-18147.
Any MUC1-TREs used in the present invention are derived from mammalian cells, including but not limited to, human cells. Preferably, the MUC1-TRE is human. In one embodiment, the MUC1-TRE may contain the entire 0.9 kb 5' flanking sequence of the MUC1 gene. In other embodiments, the MUC1-TREs comprise the following sequences (relative to the transcription start site of the MUC1 gene): about nt -725 to about nt +31, nt -743 to about nt +33, nt -750 to about nt +33, and nt -598 to about nt +485 (operably-linked to a promoter).
The c-erbB2/neu gene (HER-2/neu or HER) is a transforming gene that encodes a 185 kD epidermal growth factor receptor-related transmembrane glycoprotein. In humans, the c-erbB2/neu protein is expressed during fetal development, however, in adults, the protein is weakly detectable (by immunohistochemistry) in the epithelium of many normal tissues. Amplification and/or over-expression of the c-erbB2/neu gene has been associated with many human cancers, including breast, ovarian, uterine, prostate, stomach and lung cancers. The clinical consequences of the c-erbB2/neu protein over-expression have been best studied in breast and ovarian cancer. c-erbB2/neu protein over-expression occurs in 20 to 40% of intraductal carcinomas of the breast and 30% of ovarian cancers, and is associated with a poor prognosis in subcategories of both diseases. Human, rat and mouse c-erbB2/neu TREs have been identified and shown to confer c-erbB2/neu expressing cell specific activity. Tal et al. (1987) Mol. Cell. Biol. 7:2597-2601; Hudson et al. (1990) J. Biol. Chem. 265:43894393; Grooteclaes et al. (1994) Cancer Res. 54:41934199; Ishii et al. (1987) Proc. Natl. Acad. Sci. USA 84:4374-4378; Scott et al. (1994) J. Biol. Chem. 269:19848-19858.
The cell type-specific TREs listed above are provided as non-limiting examples of TREs that would function in the instant invention. Additional cell-specific TREs are known in the art, as are methods to identify and test cell specificity of suspected TREs.
Using the Adenoviral Vectors of the Invention
The adenoviral vectors can be used in a variety of forms, including, but not limited to, naked polynucleotide (usually DNA) constructs; polynucleotide constructs complexed with agents to facilitate entry into cells, such as cationic liposomes or other cationic compounds such as polylysine; packaged into infectious adenovirus particles (which may render the adenoviral vector(s) more immunogenic); packaged into other particulate viral forms such as HSV or AAV; complexed with agents (such as PEG) to enhance or dampen an immune response; complexed with agents that facilitate in vivo transfection, such as DOTMA.TM., DOTAP.TM., and polyamines. Thus, the invention also provides an adenovirus capable of replicating preferentially in cell status-producing cells. "Replicating preferentially" means that the adenovirus replicates more in cell exhibting a requisite physiological state than a cell not exhbiting that state. Preferably, the adenovirus replicates at least about 2-fold higher, preferably at least about 5-fold higher, more preferably at least about 10-fold higher, still more preferably at least about 50-fold higher, even more preferably at least about 100-fold higher, still more preferably at least about 400-fold to about 500-fold higher, still more preferably at least about 1000-fold higher, most preferably at least about 1.times.10.sup.6 higher. Most preferably, the adenovirus replicates solely in cells exhibiting a requisite physiological state (that is, does not replicate or replicates at very low levels in cells not exhibiting the requisite physiological state).
If an adenoviral vector is packaged into an adenovirus, the adenovirus itself may also be selected to further enhance targeting. For example, adenovirus fibers mediate primary contact with cellular receptor(s) aiding in tropism. See, e.g., Amberg et al. (1997) Virol. 227:239-244. If a particular subgenus of an adenovirus serotype displayed tropism for a target cell type and/or reduced affinity for non-target cell types, such subgenus (or subgenera) could be used to further increase cell-specificity of cytoxicity and/or cytolysis.
The adenoviral vectors may be delivered to the target cell in a variety of ways, including, but not limited to, liposomes, general transfection methods that are well known in the art (such as calcium phosphate precipitation or electroporation), direct injection, and intravenous infusion. The means of delivery will depend in large part on the particular adenoviral vector (including its form) as well as the type and location of the target cells (i.e., whether the cells are in vitro or in vivo).
If used as a packaged adenovirus, adenovirus vectors may be administered in an appropriate physiologically acceptable carrier at a dose of about 10.sup.4 to about 10.sup.14. The multiplicity of infection will generally be in the range of about 0.001 to 100. If administered as a polynucleotide construct (i.e., not packaged as a virus) about 0.01 .mu.g to about 1000 .mu.g of an adenoviral vector can be administered. The adenoviral vector(s) may be administered one or more times, depending upon the intended use and the immune response potential of the host, and may also be administered as multiple, simultaneous injections. If an immune response is undesirable, the immune response may be diminished by employing a variety of immunosuppressants, so as to permit repetitive administration, without a strong immune response. If packaged as another viral form, such as HSV, an amount to be administered is based on standard knowledge about that particular virus (which is readily obtainable from, for example, published literature) and can be determined empirically.
Host Cells Comprising the Adenoviral Vectors of the Invention
The present invention also provides host cells comprising (i.e., transformed with) the adenoviral vectors described herein. Both prokaryotic and eukaryotic host cells can be used as long as sequences requisite for maintenance in that host, such as appropriate replication origin(s), are present. For convenience, selectable markers are also provided. Prokaryotic host cells include bacterial cells, for example, E. coli and mycobacteria. Among eukaryotic host cells are yeast, insect, avian, plant and mammalian. Host systems are known in the art and need not be described in detail herein.
Compositions of the Invention
The present invention also provides compositions, including pharmaceutical compositions, containing the adenoviral vectors described herein. Such compositions (especially pharmaceutical compositions) are useful for administration in vivo, for example, when measuring the degree of transduction and/or effectiveness of cell killing in an individual. Pharmaceutical compositions, comprised an adenoviral vector of this invention in a pharmaceutically acceptable excipient (generally an effective amount of the adenoviral vector), are suitable for systemic administration to individuals in unit dosage forms, sterile parcnteral solutions or suspensions, sterile non-parenteral solutions or oral solutions or suspensions, oil in water or water in oil emulsions and the like. Formulations for parenteral and nonparenteral drug delivery are known in the art and are set forth in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing (1995). Pharmaceutical compositions also include lyophilized and/or reconstituted forms of the adenoviral vectors (including those packaged as a virus, such as adenovirus) of the invention.
Other compositions are used, and are useful for, detection methods described herein. For these compositions, the adenoviral vector usually is suspended in an appropriate solvent or solution, such as a buffer system. Such solvent systems are well known in the art.
Kits of the Invention
The present invention also encompasses kits containing an adenoviral vector(s) of this invention. These kits can be used for diagnostic and/or monitoring purposes, preferably monitoring. Procedures using these kits can be performed by clinical laboratories, experimental laboratories, medical practitioners, or private individuals. Kits embodied by this invention allow someone to detect the presence of cell status-producing cells in a suitable biological sample, such as biopsy specimens.
The kits of the invention comprise an adenoviral vector described herein in suitable packaging. The kit may optionally provide additional components that are useful in the procedure, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information.
Preparation of the Adenovirus Vectors of the Invention
The adenovirus vectors of this invention can be prepared using recombinant techniques that are standard in the art. Generally, a cell status-specific TRE is inserted 5' to the adenoviral gene of interest, preferably one or more early genes (although late gene(s) may be used). A cell status-specific TRE can be prepared using oligonucleotide synthesis (if the sequence is known) or recombinant methods (such as PCR and/or restriction enzymes). Convenient restriction sites, either in the natural adeno-DNA sequence or introduced by methods such as oligonucleotide directed mutagenesis and PCR, provide an insertion site for a cell status-specific TRE. Accordingly, convenient restriction sites for annealing (i.e., inserting) a cell status-specific TRE can be engineered onto the 5' and 3' ends of a cell status-specific TRE using standard recombinant methods, such as PCR Polynucleotides used for making adenoviral vectors of this invention may be obtained using standard methods in the art, such as chemical synthesis, by recombinant methods, and/or by obtaining the desired sequence(s) from biological sources.
Adenoviral vectors are conveniently prepared by employing two plasmids, one plasmid providing for the left hand region of adenovirus and the other plasmid providing for the right hand region, where the two plasmids share at least about 500 nt of middle region for homologous recombination. In this way, each plasmid, as desired, may be independently manipulated, followed by cotransfection in a competent host, providing complementing genes as appropriate, or the appropriate transcription factors for initiation of transcription from a cell status-specific TRE for propagation of the adenovirus. Plasmids are generally introduced into a suitable host cell such as 293 cells using appropriate means of transduction, such as cationic liposomes. Alternatively, in vitro ligation of the right and left-hand portions of the adenovirus genome can also be used to construct recombinant adenovirus derivative containing all the replication-essential portions of adenovirus genome. Berkner et al. (1983) Nucleic Acid Research 11: 6003-6020; Bridge et al. (1989) J. Virol. 63: 631-638.
For convenience, plasmids are available that provide the necessary portions of adenovirus. Plasmid pXC.1 (McKinnon (1982) Gene 19:33-42) contains the wild-type left-hand end of Ad5. pBHG10 (Bett et al. (1994) Proc. Natl. Acad. Sci USA 91:8802-8806; Microbix Biosystems Inc., Toronto) provides the right-hand end of Ad5, with a deletion in E3. The deletion in E3 provides room in the virus to insert a 3 kb cell status-TRE without deleting the endogenous enhancer/promoter. Bett et al. (1994). The gene for E3 is located on the opposite strand from E4 (r-strand). pBHG11 provides an even larger E3 deletion (an additional 0.3 kb is deleted). Bett et al. (1994).
For manipulation of the early genes, the transcription start site of Ad5 E1A is at 498 and the ATG start site of the E1A protein is at 560 in the virus genome. This region can be used for insertion of an cell status-specific TRE. A restriction site may be introduced by employing polymerase chain reaction (PCR), where the primer that is employed may be limited to the Ad5 genome, or may involve a portion of the plasmid carrying the Ad5 genomic DNA. For example, where pBR322 is used, the primers may use the EcoRI site in the pBR322 backbone and the Xbal site at 1339 of Ad5. By carrying out the PCR in two steps, where overlapping primers at the center of the region introduce a 30 sequence change resulting in a unique restriction site, one can provide for insertion of heterologous TRE at that site.
A similar strategy may also be used for insertion of a heterologous TRE to regulate E1B. The E1B promoter of Ad5 consists of a single high-affinity recognition site for Spl and a TATA box. This region extends from 1636 to 1701. By insertion of a heterologous TRE in this region, one can provide for target cell-specific transcription of the E1B gene. By employing the left-hand region modified with a heterologous TRE regulating E1A as the template for introducing a heterologous TRE to regulate E1B, the resulting adenovirus vector will be dependent upon the cell status-specific transcription factors for expression of both E1A and E1B.
Similarly, a cell status-specific TRE can be inserted upstream of the E2 gene to make its expression cell status specific. The E2 early promoter, mapping in Ad5 from 27050-27150, consists of a major and a minor transcription initiation site, the latter accounting for about 5% of the E2 transcripts, two non-canonical TATA boxes, two E2F transcription factor binding sites and an ATF transcription factor binding site. For a detailed review of the E2 promoter architecture see Swaminathan et al., Curr. Topics in Micro. and Imm. (1995) 199 (part 3):177-194.
For E4, one must use the right hand portion of the adenovirus genome. The E4 transcription start site is predominantly at 35609, the TATA box at 35638 and the first ATG/CTG of ORF 1 is at 35532. Virtanen et al. (1984) J. Virol. 51: 822-831. Using any of the above strategies for the other genes, a cell status-specific TRE may be introduced upstream from the transcription start site. For the construction of mutants in the E4 region, the co-transfection and homologous recombination are performed in W162 cells (Weinberg et al. (1983) Proc. Natl. Acad. Sci. 80:5383-5386) which provide E4 proteins in trans to complement defects in synthesis of these proteins. Alternatively, these constructs can be produced by in vitro ligation.
Methods Using the Adenovirus Vectors of the Invention
The adenoviral vectors of the invention can be used for a wide variety of purposes, which will vary with the desired or intended result. Accordingly, the present invention includes methods using the adenoviral vectors described above.
In one embodiment, methods are provided for conferring selective cytoxicity in target cells (i.e., cells exhibiting a requisite physiological state which allows a cell status-specific TRE to function), generally but not necessarily in an individual (preferably human), comprising contacting the cells with an adenovirus vector described herein, such that the adenovirus vector enters the cell. Cytotoxicity can be measured using standard assays in the art, such as dye exclusion, .sup.3 H-thymidine incorporation, and/or lysis.
In another embodiment, methods are provided for propagating an adenovirus specific for mammalian cells which allow a cell status-specific TRE to function. These methods entail combining an adenovirus vector with mammalian cells, whereby said adenovirus is propagated.
The invention further provides methods of suppressing tumor cell growth, generally but not necessarily in an individual (preferably human), comprising contacting a tumor cell with an adenoviral vector of the invention such that the adenoviral vector enters the tumor cell and exhibits selective cytotoxicity for the tumor cell. Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a H-thymidine incorporation assay, or counting tumor cells.
The invention also includes methods for detecting target cells (i.e., cells which permit or induce a cell status-specific TRE to function) in a biological sample. These methods are particularly useful for monitoring the clinical and/or physiological condition of an individual (i.e., mammal), whether in an experimental or clinical setting. For these methods, cells of a biological sample are contacted with an adenovirus vector, and replication of the adenoviral vector is detected. A suitable biological sample is one in which cells exhibiting a requisite physiological (and/or environmental) state, for example, an aberrant physiological state (such as cells in hypoxic conditions and exhibiting a phenotype characteristic of cells in hypoxic conditions, such as expression of HIF-1) may be or are suspected to be present. Generally, in mammals, a suitable clinical sample is one in which cancerous cells exhbiting a requisite physiological state, such as cells within a solid tumor which are under hypoxic conditions, are suspected to be present. Such cells can be obtained, for example, by needle biopsy or other surgical procedure. Cells to be contacted may be treated to promote assay conditions, such as selective enrichment, and/or solubilization. In these methods, target cells can be detected using in vitro assays that detect adenoviral proliferation, which are standard in the art. Examples of such standard assays include, but are not limited to, burst assays (which measure virus yield) and plaque assays (which measure infectious particles per cell). Propagation can also be detected by measuring specific adenoviral DNA replication, which are also standard assays.
The following examples are provided to illustrate but not limit the invention.
Adenovirus Vector Comprising E1A Under Transcriptional Control of a Hypoxia Responsive Element and a PSA-TRE
A human embryonic kidney cell line, 293, efficiently expresses E1A and E1B genes of Ad5 and exhibits a high transfection efficiency with adenovirus DNA. To generate recombinant adenovirus, 293 cells were co-transfected with one left end Ad5 plasmid and one right end Ad5 plasmid. Homologous recombination generates adenoviruses with the required genetic elements for replication in 293 cells which provide E1A and E1B proteins in trans to complement defects in synthesis of these proteins.
The plasmids to be combined were co-transfected into 293 cells using cationic liposomes such as Lipofectin (DOTMA:DOPE.TM., Life Technologies) by combining the two plasmids, then mixing the plasmid DNA solution (10 .mu.g of each plasmid in 500 .mu.l of minimum essential medium (MEM) without serum or other additives) with a four-fold molar excess of liposomes in 200 .mu.l of the same buffer. The DNA-lipid complexes were then placed on the cells and incubated at 37.degree. C., 5% CO.sub.2 for 16 hours. After incubation the medium was changed to MEM with 10% fetal bovine serum and the cells are further incubated at 37.degree. C., 5% CO.sub.2, for 10 days with two changes of medium. At the end of this time the cells and medium were transferred to tubes, freeze-thawed three times, and the lysate was used to infect 293 cells at the proper dilution to detect individual viruses as plaques.
Plaques obtained were plaque purified twice, and viruses were characterized for presence of desired sequences by PCR and occasionally by DNA sequencing. For further experimentation, the viruses were purified on a large scale by cesium chloride gradient centrifugation.
Adenovirus Vectors in which E1A is Under Transcriptional Control of a Cell Status-Specific TRE
An adenovirus vector containing a hypoxia response element (HRE) was generated. CN796, an adenovirus vector in which E1A is under the control of a composite TRE consisting of an HRE and a PSA-TRE, was made by co-transfecting CN515 with pBHG10. CN515 was constructed by inserting a 67 base pair fragment from HRE enol (Jiang et al. (1997) Cancer Research 57:5328-5335) into CN65 at the BglII site. CN65 is a plasmid containing an enhancer and promoter from the human PSA gene, consisting of an enhancer from -5322 to -3738 fused to a PSA promoter from -541 to +12. This is the PSA-TRE contained within plasmid CN706. Rodriguez et al. (1997) Cancer Res. 57:2559-2563.
Virus growth in vitro
Growth selectivity of recombinant adenovirus is analyzed in plaque assays in which a single infectious particle produces a visible plaque by multiple rounds of infection and replication. Virus stocks are diluted to equal pfu/ml, then used to infect monolayers of cells for 1 hour. The inoculum is then removed and the cells are overlayed with semisolid agar containing medium and incubated at 37.degree. C. for 10 days. Plaques in the monolayer are then counted and titers of infectious virus on the various cells are calculated. The data are normalized to the titer of CN702 (wild type) on 293 cells.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160> NUMBER OF SEQ ID NOS: 8 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 1 <211> LENGTH: 1519 <212> TYPE: DNA <213> ORGANISM: H. sapiens <400> SEQUENCE: 1 gggcccaaaa ttagcaagtg accacgtggt tctgaagcca gtggcctaag gaccaccctt 60 gcagaaccgt ggtctccttg tcacagtcta ggcagcctct ggcttagcct ctgtttcttt 120 cataaccttt ctcagcgcct gctctgggcc agaccagtgt tgggaggagt cgctactgag 180 ctcctagatt ggcaggggag gcagatggag aaaaggagtg tgtgtggtca gcattggagc 240 agaggcagca gtgggcaata gaggaagtga gtaaatcctt gggagggctc cctagaagtg 300 atgtgttttc tttttttgtt ttagagacag gatctcgctc tgtcgcccag gctggtgtgc 360 agtggcatga tcatagctca ctgcagcctc gacttctcgg gctcaagcaa tcctcccacc 420 tcagcctccc aagtagctgg gactacgggc acacgccacc atgcctggct aatttttgta 480 ttttttgtag agatgggtct tcaccatgtt gatcaggctg gtctcgaact cctgggctca 540 tgcgatccac cccgccagct gattacaggg attccggtgg tgagccaccg cgcccagacg 600 ccacttcatc gtattgtaaa cgtctgttac ctttctgttc ccctgtctac tggactgtga 660 gctccttagg gccacgaatt gaggatgggg cacagagcaa gctctccaaa cgtttgttga 720 atgagtgagg gaatgaatga gttcaagcag atgctatacg ttggctgttg gagattttgg 780 ctaaaatggg acttgcagga aagcccgacg tccccctcgc catttccagg caccgctctt 840 cagcttgggc tctgggtgag cgggataggg ctgggtgcag gattaggata atgtcatggg 900 tgaggcaagt tgaggatgga agaggtggct gatggctggg ctgtggaact gatgatcctg 960 aaaagaagag gggacagtct ctggaaatct aagctgaggc tgttgggggc tacaggttga 1020 gggtcacgtg cagaagagag gctctgttct gaacctgcac tatagaaagg tcagtgggat 1080 gcgggagcgt cggggcgggg cggggcctat gttcccgtgt ccccacgcct ccagcagggg 1140 acgcccgggc tgggggcggg gagtcagacc gcgcctggta ccatccggac aaagcctgcg 1200 cgcgccccgc cccgccattg gccgtaccgc cccgcgccgc cgccccatcc cgcccctcgc 1260 cgccgggtcc ggcgcgttaa agccaatagg aaccgccgcc gttgttcccg tcacggccgg 1320 ggcagccaat tgtggcggcg ctcggcggct cgtggctctt tcgcggcaaa aaggatttgg 1380 cgcgtaaaag tggccgggac tttgcaggca gcggcggccg ggggcggagc gggatcgagc 1440 cctcgccgag gcctgccgcc atgggcccgc gccgccgccg ccgcctgtca cccgggccgc 1500 gcgggccgtg agcgtcatg 1519 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2 <211> LENGTH: 5836 <212> TYPE: DNA <213> ORGANISM: H. sapiens <400> SEQUENCE: 2 aagcttctag ttttcttttc ccggtgacat cgtggaaagc actagcatct ctaagcaatg 60 atctgtgaca atattcacag tgtaatgcca tccagggaac tcaactgagc cttgatgtcc 120 agagattttt gtgttttttt ctgagactga gtctcgctct gtgccaggct ggagtgcagt 180 ggtgcaacct tggctcactg caagctccgc ctcctgggtt cacgccattc tcctgcctca 240 gcctcctgag tagctgggac tacaggcacc cgccaccacg cctggctaat ttttttgtat 300 ttttagtaga gatggggttt cactgtgtta gccaggatgg tctcagtctc ctgacctcgt 360 gatctgccca ccttggcctc ccaaagtgct gggatgacag gcgtgagcca ccgcgcctgg 420 ccgatatcca gagatttttt ggggggctcc atcacacaga catgttgact gtcttcatgg 480 ttgactttta gtatccagcc cctctagaaa tctagctgat atagtgtggc tcaaaacctt 540 cagcacaaat cacaccgtta gactatctgg tgtggcccaa accttcaggt gaacaaaggg 600 actctaatct ggcaggatat tccaaagcat tagagatgac ctcttgcaaa gaaaaagaaa 660 tggaaaagaa aaagaaagaa aggaaaaaaa aaaaaaaaaa gagatgacct ctcaggctct 720 gaggggaaac gcctgaggtc tttgagcaag gtcagtcctc tgttgcacag tctccctcac 780 agggtcattg tgacgatcaa atgtggtcac gtgtatgagg caccagcaca tgcctggctc 840 tggggagtgc cgtgtaagtg tatgcttgca ctgctgaatg cttgggatgt gtcagggatt 900 atcttcagca cttacagatg ctcatctcat cctcacagca tcactatggg atgggtatta 960 ctggcctcat ttgatggaga aagtggctgt ggctcagaaa ggggggacca ctagaccagg 1020 gacactctgg atgctgggga ctccagagac catgaccact caccaactgc agagaaatta 1080 attgtggcct gatgtccctg tcctggagag ggtggaggtg gaccttcact aacctcctac 1140 cttgaccctc tcttttaggg ctctttctga cctccaccat ggtactagga ccccattgta 1200 ttctgtaccc tcttgactct atgaccccca ctgcccactg catccagctg ggtcccctcc 1260 tatctctatt cccagctggc cagtgcagtc tcagtgccca cctgtttgtc agtaactctg 1320 aaggggctga cattttactg acttgcaaac aaataagcta actttccaga gttttgtgaa 1380 tgctggcaga gtccatgaga ctcctgagtc agaggcaaag gcttttactg ctcacagctt 1440 agcagacagc atgaggttca tgttcacatt agtacacctt gcccccccca aatcttgtag 1500 ggtgaccaga gcagtctagg tggatgctgt gcagaagggg tttgtgccac tggtgagaaa 1560 cctgagatta ggaatcctca atcttatact gggacaactt gcaaacctgc tcagcctttg 1620 tctctgatga agatattatc ttcatgatct tggattgaaa acagacctac tctggaggaa 1680 catattgtat cgattgtcct tgacagtaaa caaatctgtt gtaagagaca ttatctttat 1740 tatctaggac agtaagcaag cctggatctg agagagatat catcttgcaa ggatgcctgc 1800 tttacaaaca tccttgaaac aacaatccag aaaaaaaaag gtgttgctgt ctttgctcag 1860 aagacacaca gatacgtgac agaaccatgg agaattgcct cccaacgctg ttcagccaga 1920 gccttccacc cttgtctgca ggacagtctc aacgttccac cattaaatac ttcttctatc 1980 acatcctgct tctttatgcc taaccaaggt tctaggtccc gatcgactgt gtctggcagc 2040 actccactgc caaacccaga ataaggcagc gctcaggatc ccgaaggggc atggctgggg 2100 atcagaactt ctgggtttga gtgaggagtg ggtccaccct cttgaatttc aaaggaggaa 2160 gaggctggat gtgaaggtac tgggggaggg aaagtgtcag ttccgaactc ttaggtcaat 2220 gagggaggag actggtaagg tcccagctcc cgaggtactg atgtgggaat ggcctaagaa 2280 tctcatatcc tcaggaagaa ggtgctggaa tcctgagggg tagagttctg ggtatatttg 2340 tggcttaagg ctctttggcc cctgaaggca gaggctggaa ccattaggtc cagggtttgg 2400 ggtgatagta atgggatctc ttgattcctc aagagtctga ggatcgaggg ttgcccattc 2460 ttccatcttg ccacctaatc cttactccac ttgagggtat caccagccct tctagctcca 2520 tgaaggtccc ctgggcaagc acaatctgag catgaaagat gccccagagg ccttgggtgt 2580 catccactca tcatccagca tcacactctg agggtgtggc cagcaccatg acgtcatgtt 2640 gctgtgacta tccctgcagc gtgcctctcc agccacctgc caaccgtaga gctgcccatc 2700 ctcctctggt gggagtggcc tgcatggtgc caggctgagg cctagtgtca gacagggagc 2760 ctggaatcat agggatccag gactcaaaag tgctagagaa tggccatatg tcaccatcca 2820 tgaaatctca agggcttctg ggtggagggc acagggacct gaacttatgg tttcccaagt 2880 ctattgctct cccaagtgag tctcccagat acgaggcact gtgccagcat cagccttatc 2940 tccaccacat cttgtaaaag gactacccag ggccctgatg aacaccatgg tgtgtacagg 3000 agtagggggt ggaggcacgg actcctgtga ggtcacagcc aagggagcat catcatgggt 3060 ggggaggagg caatggacag gcttgagaac ggggatgtgg ttgtatttgg ttttctttgg 3120 ttagataaag tgctgggtat aggattgaga gtggagtatg aagaccagtt aggatggagg 3180 atcagattgg agttgggtta gataaagtgc tgggtatagg attgagagtg gagtatgaag 3240 accagttagg atggaggatc agattggagt tgggttagag atggggtaaa attgtgctcc 3300 ggatgagttt gggattgaca ctgtggaggt ggtttgggat ggcatggctt tgggatggaa 3360 atagatttgt tttgatgttg gctcagacat ccttggggat tgaactgggg atgaagctgg 3420 gtttgatttt ggaggtagaa gacgtggaag tagctgtcag atttgacagt ggccatgagt 3480 tttgtttgat ggggaatcaa acaatggggg aagacataag ggttggcttg ttaggttaag 3540 ttgcgttggg ttgatggggt cggggctgtg tataatgcag ttggattggt ttgtattaaa 3600 ttgggttggg tcaggttttg gttgaggatg agttgaggat atgcttgggg acaccggatc 3660 catgaggttc tcactggagt ggagacaaac ttcctttcca ggatgaatcc agggaagcct 3720 taattcacgt gtaggggagg tcaggccact ggctaagtat atccttccac tccagctcta 3780 agatggtctt aaattgtgat tatctatatc cacttctgtc tccctcactg tgcttggagt 3840 ttacctgatc actcaactag aaacagggga agattttatc aaattctttt tttttttttt 3900 ttttttttga gacagagtct cactctgttg cccaggctgg agtgcagtgg cgcagtctcg 3960 gctcactgca acctctgcct cccaggttca agtgattctc ctgcctcagc ctcctgagtt 4020 gctgggatta caggcatgca gcaccatgcc cagctaattt ttgtattttt agtagagatg 4080 gggtttcacc aatgtttgcc aggctggcct cgaactcctg acctggtgat ccacctgcct 4140 cagcctccca aagtgctggg attacaggcg tcagccaccg cgcccagcca cttttgtcaa 4200 attcttgaga cacagctcgg gctggatcaa gtgagctact ctggttttat tgaacagctg 4260 aaataaccaa ctttttggaa attgatgaaa tcttacggag ttaacagtgg aggtaccagg 4320 gctcttaaga gttcccgatt ctcttctgag actacaaatt gtgattttgc atgccacctt 4380 aatctttttt tttttttttt taaatcgagg tttcagtctc attctatttc ccaggctgga 4440 gttcaatagc gtgatcacag ctcactgtag ccttgaactc ctggccttaa gagattctcc 4500 tgcttcggtc tcccaatagc taagactaca gtagtccacc accatatcca gataattttt 4560 aaattttttg gggggccggg cacagtggct cacgcctgta atcccaacac catgggaggc 4620 tgagatgggt ggatcacgag gtcaggagtt tgagaccagc ctgaccaaca tggtgaaact 4680 ctgtctctac taaaaaaaaa aaaaatagaa aaattagccg ggcgtggtgg cacacggcac 4740 ctgtaatccc agctactgag gaggctgagg caggagaatc acttgaaccc agaaggcaga 4800 ggttgcaatg agccgagatt gcgccactgc actccagcct gggtgacaga gtgagactct 4860 gtctcaaaaa aaaaaaattt tttttttttt tttgtagaga tggatcttgc tttgtttctc 4920 tggttggcct tgaactcctg gcttcaagtg atcctcctac cttggcctcg gaaagtgttg 4980 ggattacagg cgtgagccac catgactgac ctgtcgttaa tcttgaggta cataaacctg 5040 gctcctaaag gctaaaggct aaatatttgt tggagaaggg gcattggatt ttgcatgagg 5100 atgattctga cctgggaggg caggtcagca ggcatctctg ttgcacagat agagtgtaca 5160 ggtctggaga acaaggagtg gggggttatt ggaattccac attgtttgct gcacgttgga 5220 ttttgaaatg ctagggaact ttgggagact catatttctg ggctagagga tctgtggacc 5280 acaagatctt tttatgatga cagtagcaat gtatctgtgg agctggattc tgggttggga 5340 gtgcaaggaa aagaatgtac taaatgccaa gacatctatt tcaggagcat gaggaataaa 5400 agttctagtt tctggtctca gagtggtgca gggatcaggg agtctcacaa tctcctgagt 5460 gctggtgtct tagggcacac tgggtcttgg agtgcaaagg atctaggcac gtgaggcttt 5520 gtatgaagaa tcggggatcg tacccacccc ctgtttctgt ttcatcctgg gcatgtctcc 5580 tctgcctttg tcccctagat gaagtctcca tgagctacaa gggcctggtg catccagggt 5640 gatctagtaa ttgcagaaca gcaagtgcta gctctccctc cccttccaca gctctgggtg 5700 tgggaggggg ttgtccagcc tccagcagca tggggagggc cttggtcagc ctctgggtgc 5760 cagcagggca ggggcggagt cctggggaat gaaggtttta tagggctcct gggggaggct 5820 ccccagcccc aagctt 5836 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 15056 <212> TYPE: DNA <213> ORGANISM: H. sapiens <400> SEQUENCE: 3 aagcttttta gtgctttaga cagtgagctg gtctgtctaa cccaagtgac ctgggctcca 60 tactcagccc cagaagtgaa gggtgaagct gggtggagcc aaaccaggca agcctaccct 120 cagggctccc agtggcctga gaaccattgg acccaggacc cattacttct agggtaagga 180 aggtacaaac accagatcca accatggtct ggggggacag ctgtcaaatg cctaaaaata 240 tacctgggag aggagcaggc aaactatcac tgccccaggt tctctgaaca gaaacagagg 300 ggcaacccaa agtccaaatc caggtgagca ggtgcaccaa atgcccagag atatgacgag 360 gcaagaagtg aaggaaccac ccctgcatca aatgttttgc atgggaagga gaagggggtt 420 gctcatgttc ccaatccagg agaatgcatt tgggatctgc cttcttctca ctccttggtt 480 agcaagacta agcaaccagg actctggatt tggggaaaga cgtttatttg tggaggccag 540 tgatgacaat cccacgaggg cctaggtgaa gagggcagga aggctcgaga cactggggac 600 tgagtgaaaa ccacacccat gatctgcacc acccatggat gctccttcat tgctcacctt 660 tctgttgata tcagatggcc ccattttctg taccttcaca gaaggacaca ggctagggtc 720 tgtgcatggc cttcatcccc ggggccatgt gaggacagca ggtgggaaag atcatgggtc 780 ctcctgggtc ctgcagggcc agaacattca tcacccatac tgacctccta gatgggaatg 840 gcttccctgg ggctgggcca acggggcctg ggcaggggag aaaggacgtc aggggacagg 900 gaggaagggt catcgagacc cagcctggaa ggttcttgtc tctgaccatc caggatttac 960 ttccctgcat ctacctttgg tcattttccc tcagcaatga ccagctctgc ttcctgatct 1020 cagcctccca ccctggacac agcaccccag tccctggccc ggctgcatcc acccaatacc 1080 ctgataaccc aggacccatt acttctaggg taaggagggt ccaggagaca gaagctgagg 1140 aaaggtctga agaagtcaca tctgtcctgg ccagagggga aaaaccatca gatgctgaac 1200 caggagaatg ttgacccagg aaagggaccg aggacccaag aaaggagtca gaccaccagg 1260 gtttgcctga gaggaaggat caaggccccg agggaaagca gggctggctg catgtgcagg 1320 acactggtgg ggcatatgtg tcttagattc tccctgaatt cagtgtccct gccatggcca 1380 gactctctac tcaggcctgg acatgctgaa ataggacaat ggccttgtcc tctctcccca 1440 ccatttggca agagacataa aggacattcc aggacatgcc ttcctgggag gtccaggttc 1500 tctgtctcac acctcaggga ctgtagttac tgcatcagcc atggtaggtg ctgatctcac 1560 ccagcctgtc caggcccttc cactctccac tttgtgacca tgtccaggac cacccctcag 1620 atcctgagcc tgcaaatacc cccttgctgg gtgggtggat tcagtaaaca gtgagctcct 1680 atccagcccc cagagccacc tctgtcacct tcctgctggg catcatccca ccttcacaag 1740 cactaaagag catggggaga cctggctagc tgggtttctg catcacaaag aaaataatcc 1800 cccaggttcg gattcccagg gctctgtatg tggagctgac agacctgagg ccaggagata 1860 gcagaggtca gccctaggga gggtgggtca tccacccagg ggacaggggt gcaccagcct 1920 tgctactgaa agggcctccc caggacagcg ccatcagccc tgcctgagag ctttgctaaa 1980 cagcagtcag aggaggccat ggcagtggct gagctcctgc tccaggcccc aacagaccag 2040 accaacagca caatgcagtc cttccccaac gtcacaggtc accaaaggga aactgaggtg 2100 ctacctaacc ttagagccat caggggagat aacagcccaa tttcccaaac aggccagttt 2160 caatcccatg acaatgacct ctctgctctc attcttccca aaataggacg ctgattctcc 2220 cccaccatgg atttctccct tgtcccggga gccttttctg ccccctatga tctgggcact 2280 cctgacacac acctcctctc tggtgacata tcagggtccc tcactgtcaa gcagtccaga 2340 aaggacagaa ccttggacag cgcccatctc agcttcaccc ttcctccttc acagggttca 2400 gggcaaagaa taaatggcag aggccagtga gcccagagat ggtgacaggc agtgacccag 2460 gggcagatgc ctggagcagg agctggcggg gccacaggga gaaggtgatg caggaaggga 2520 aacccagaaa tgggcaggaa aggaggacac aggctctgtg gggctgcagc ccagggttgg 2580 actatgagtg tgaagccatc tcagcaagta aggccaggtc ccatgaacaa gagtgggagc 2640 acgtggcttc ctgctctgta tatggggtgg gggattccat gccccataga accagatggc 2700 cggggttcag atggagaagg agcaggacag gggatcccca ggataggagg accccagtgt 2760 ccccacccag gcaggtgact gatgaatggg catgcagggt cctcctgggc tgggctctcc 2820 ctttgtccct caggattcct tgaaggaaca tccggaagcc gaccacatct acctggtggg 2880 ttctggggag tccatgtaaa gccaggagct tgtgttgcta ggaggggtca tggcatgtgc 2940 tgggggcacc aaagagagaa acctgagggc aggcaggacc tggtctgagg aggcatggga 3000 gcccagatgg ggagatggat gtcaggaaag gctgccccat cagggagggt gatagcaatg 3060 gggggtctgt gggagtgggc acgtgggatt ccctgggctc tgccaagttc cctcccatag 3120 tcacaacctg gggacactgc ccatgaaggg gcgcctttgc ccagccagat gctgctggtt 3180 ctgcccatcc actaccctct ctgctccagc cactctgggt ctttctccag atgccctgga 3240 cagccctggc ctgggcctgt cccctgagag gtgttgggag aagctgagtc tctggggaca 3300 ctctcatcag agtctgaaag gcacatcagg aaacatccct ggtctccagg actaggcaat 3360 gaggaaaggg ccccagctcc tccctttgcc actgagaggg tcgaccctgg gtggccacag 3420 tgacttctgc gtctgtccca gtcaccctga aaccacaaca aaaccccagc cccagaccct 3480 gcaggtacaa tacatgtggg gacagtctgt acccagggga agccagttct ctcttcctag 3540 gagaccgggc ctcagggctg tgcccggggc aggcgggggc agcacgtgcc tgtccttgag 3600 aactcgggac cttaagggtc tctgctctgt gaggcacagc aaggatcctt ctgtccagag 3660 atgaaagcag ctcctgcccc tcctctgacc tcttcctcct tcccaaatct caaccaacaa 3720 ataggtgttt caaatctcat catcaaatct tcatccatcc acatgagaaa gcttaaaacc 3780 caatggattg acaacatcaa gagttggaac aagtggacat ggagatgtta cttgtggaaa 3840 tttagatgtg ttcagctatc gggcaggaga atctgtgtca aattccagca tggttcagaa 3900 gaatcaaaaa gtgtcacagt ccaaatgtgc aacagtgcag gggataaaac tgtggtgcat 3960 tcaaactgag ggatattttg gaacatgaga aaggaaggga ttgctgctgc acagaacatg 4020 gatgatctca cacatagagt tgaaagaaag gagtcaatcg cagaatagaa aatgatcact 4080 aattccacct ctataaagtt tccaagagga aaacccaatt ctgctgctag agatcagaat 4140 ggaggtgacc tgtgccttgc aatggctgtg agggtcacgg gagtgtcact tagtgcaggc 4200 aatgtgccgt atcttaatct gggcagggct ttcatgagca cataggaatg cagacattac 4260 tgctgtgttc attttacttc accggaaaag aagaataaaa tcagccgggc gcggtggctc 4320 acgcctgtaa tcccagcact ttagaaggct gaggtgggca gattacttga ggtcaggagt 4380 tcaagaccac cctggccaat atggtgaaac cccggctcta ctaaaaatac aaaaattagc 4440 tgggcatggt ggtgcgcgcc tgtaatccca gctactcggg aggctgaggc tggacaattg 4500 cttggaccca ggaagcagag gttgcagtga gccaagattg tgccactgca ctccagcttg 4560 ggcaacagag ccagactctg taaaaaaaaa aaaaaaaaaa aaaaaaagaa agaaagaaaa 4620 agaaaagaaa gtataaaatc tctttgggtt aacaaaaaaa gatccacaaa acaaacacca 4680 gctcttatca aacttacaca actctgccag agaacaggaa acacaaatac tcattaactc 4740 acttttgtgg caataaaacc ttcatgtcaa aaggagacca ggacacaatg aggaagtaaa 4800 actgcaggcc ctacttgggt gcagagaggg aaaatccaca aataaaacat taccagaagg 4860 agctaagatt tactgcattg agttcattcc ccaggtatgc aaggtgattt taacacctga 4920 aaatcaatca ttgcctttac tacatagaca gattagctag aaaaaaatta caactagcag 4980 aacagaagca atttggcctt cctaaaattc cacatcatat catcatgatg gagacagtgc 5040 agacgccaat gacaataaaa agagggacct ccgtcacccg gtaaacatgt ccacacagct 5100 ccagcaagca cccgtcttcc cagtgaatca ctgtaacctc ccctttaatc agccccaggc 5160 aaggctgcct gcgatggcca cacaggctcc aacccgtggg cctcaacctc ccgcagaggc 5220 tctcctttgg ccaccccatg gggagagcat gaggacaggg cagagccctc tgatgcccac 5280 acatggcagg agctgacgcc agagccatgg gggctggaga gcagagctgc tggggtcaga 5340 gcttcctgag gacacccagg cctaagggaa ggcagctccc tggatggggg caaccaggct 5400 ccgggctcca acctcagagc ccgcatggga ggagccagca ctctaggcct ttcctagggt 5460 gactctgagg ggaccctgac acgacaggat cgctgaatgc acccgagatg aaggggccac 5520 cacgggaccc tgctctcgtg gcagatcagg agagagtggg acaccatgcc aggcccccat 5580 ggcatggctg cgactgaccc aggccactcc cctgcatgca tcagcctcgg taagtcacat 5640 gaccaagccc aggaccaatg tggaaggaag gaaacagcat cccctttagt gatggaaccc 5700 aaggtcagtg caaagagagg ccatgagcag ttaggaaggg tggtccaacc tacagcacaa 5760 accatcatct atcataagta gaagccctgc tccatgaccc ctgcatttaa ataaacgttt 5820 gttaaatgag tcaaattccc tcaccatgag agctcacctg tgtgtaggcc catcacacac 5880 acaaacacac acacacacac acacacacac acacacacac acagggaaag tgcaggatcc 5940 tggacagcac caggcaggct tcacaggcag agcaaacagc gtgaatgacc catgcagtgc 6000 cctgggcccc atcagctcag agaccctgtg agggctgaga tggggctagg caggggagag 6060 acttagagag ggtggggcct ccagggaggg ggctgcaggg agctgggtac tgccctccag 6120 ggagggggct gcagggagct gggtactgcc ctccagggag ggggctgcag ggagctgggt 6180 actgccctcc agggaggggg ctgcagggag ctgggtactg ccctccaggg agggggctgc 6240 agggagctgg gtactgccct ccagggaggc aggagcactg ttcccaacag agagcacatc 6300 ttcctgcagc agctgcacag acacaggagc ccccatgact gccctgggcc agggtgtgga 6360
ttccaaattt cgtgccccat tgggtgggac ggaggttgac cgtgacatcc aaggggcatc 6420 tgtgattcca aacttaaact actgtgccta caaaatagga aataacccta ctttttctac 6480 tatctcaaat tccctaagca caagctagca ccctttaaat caggaagttc agtcactcct 6540 ggggtcctcc catgccccca gtctgacttg caggtgcaca gggtggctga catctgtcct 6600 tgctcctcct cttggctcaa ctgccgcccc tcctgggggt gactgatggt caggacaagg 6660 gatcctagag ctggccccat gattgacagg aaggcaggac ttggcctcca ttctgaagac 6720 taggggtgtc aagagagctg ggcatcccac agagctgcac aagatgacgc ggacagaggg 6780 tgacacaggg ctcagggctt cagacgggtc gggaggctca gctgagagtt cagggacaga 6840 cctgaggagc ctcagtggga aaagaagcac tgaagtggga agttctggaa tgttctggac 6900 aagcctgagt gctctaagga aatgctccca ccccgatgta gcctgcagca ctggacggtc 6960 tgtgtacctc cccgctgccc atcctctcac agcccccgcc tctagggaca caactcctgc 7020 cctaacatgc atctttcctg tctcattcca cacaaaaggg cctctggggt ccctgttctg 7080 cattgcaagg agtggaggtc acgttcccac agaccaccca gcaacagggt cctatggagg 7140 tgcggtcagg aggatcacac gtccccccat gcccagggga ctgactctgg gggtgatgga 7200 ttggcctgga ggccactggt cccctctgtc cctgagggga atctgcaccc tggaggctgc 7260 cacatccctc ctgattcttt cagctgaggg cccttcttga aatcccaggg aggactcaac 7320 ccccactggg aaaggcccag tgtggacggt tccacagcag cccagctaag gcccttggac 7380 acagatcctg agtgagagaa cctttaggga cacaggtgca cggccatgtc cccagtgccc 7440 acacagagca ggggcatctg gaccctgagt gtgtagctcc cgcgactgaa cccagccctt 7500 ccccaatgac gtgacccctg gggtggctcc aggtctccag tccatgccac caaaatctcc 7560 agattgaggg tcctcccttg agtccctgat gcctgtccag gagctgcccc ctgagcaaat 7620 ctagagtgca gagggctggg attgtggcag taaaagcagc cacatttgtc tcaggaagga 7680 aagggaggac atgagctcca ggaagggcga tggcgtcctc tagtgggcgc ctcctgttaa 7740 tgagcaaaaa ggggccagga gagttgagag atcagggctg gccttggact aaggctcaga 7800 tggagaggac tgaggtgcaa agagggggct gaagtagggg agtggtcggg agagatggga 7860 ggagcaggta aggggaagcc ccagggaggc cgggggaggg tacagcagag ctctccactc 7920 ctcagcattg acatttgggg tggtcgtgct agtggggttc tgtaagttgt agggtgttca 7980 gcaccatctg gggactctac ccactaaatg ccagcaggac tccctcccca agctctaaca 8040 accaacaatg tctccagact ttccaaatgt cccctggaga gcaaaattgc ttctggcaga 8100 atcactgatc tacgtcagtc tctaaaagtg actcatcagc gaaatccttc acctcttggg 8160 agaagaatca caagtgtgag aggggtagaa actgcagact tcaaaatctt tccaaaagag 8220 ttttacttaa tcagcagttt gatgtcccag gagaagatac atttagagtg tttagagttg 8280 atgccacatg gctgcctgta cctcacagca ggagcagagt gggttttcca agggcctgta 8340 accacaactg gaatgacact cactgggtta cattacaaag tggaatgtgg ggaattctgt 8400 agactttggg aagggaaatg tatgacgtga gcccacagcc taaggcagtg gacagtccac 8460 tttgaggctc tcaccatcta ggagacatct cagccatgaa catagccaca tctgtcatta 8520 gaaaacatgt tttattaaga ggaaaaatct aggctagaag tgctttatgc tcttttttct 8580 ctttatgttc aaattcatat acttttagat cattccttaa agaagaatct atccccctaa 8640 gtaaatgtta tcactgactg gatagtgttg gtgtctcact cccaacccct gtgtggtgac 8700 agtgccctgc ttccccagcc ctgggccctc tctgattcct gagagctttg ggtgctcctt 8760 cattaggagg aagagaggaa gggtgttttt aatattctca ccattcaccc atccacctct 8820 tagacactgg gaagaatcag ttgcccactc ttggatttga tcctcgaatt aatgacctct 8880 atttctgtcc cttgtccatt tcaacaatgt gacaggccta agaggtgcct tctccatgtg 8940 atttttgagg agaaggttct caagataagt tttctcacac ctctttgaat tacctccacc 9000 tgtgtcccca tcaccattac cagcagcatt tggacccttt ttctgttagt cagatgcttt 9060 ccacctcttg agggtgtata ctgtatgctc tctacacagg aatatgcaga ggaaatagaa 9120 aaagggaaat cgcattacta ttcagagaga agaagacctt tatgtgaatg aatgagagtc 9180 taaaatccta agagagccca tataaaatta ttaccagtgc taaaactaca aaagttacac 9240 taacagtaaa ctagaataat aaaacatgca tcacagttgc tggtaaagct aaatcagata 9300 tttttttctt agaaaaagca ttccatgtgt gttgcagtga tgacaggagt gcccttcagt 9360 caatatgctg cctgtaattt ttgttccctg gcagaatgta ttgtcttttc tccctttaaa 9420 tcttaaatgc aaaactaaag gcagctcctg ggccccctcc ccaaagtcag ctgcctgcaa 9480 ccagccccac gaagagcaga ggcctgagct tccctggtca aaataggggg ctagggagct 9540 taaccttgct cgataaagct gtgttcccag aatgtcgctc ctgttcccag gggcaccagc 9600 ctggagggtg gtgagcctca ctggtggcct gatgcttacc ttgtgccctc acaccagtgg 9660 tcactggaac cttgaacact tggctgtcgc ccggatctgc agatgtcaag aacttctgga 9720 agtcaaatta ctgcccactt ctccagggca gatacctgtg aacatccaaa accatgccac 9780 agaaccctgc ctggggtcta caacacatat ggactgtgag caccaagtcc agccctgaat 9840 ctgtgaccac ctgccaagat gcccctaact gggatccacc aatcactgca catggcaggc 9900 agcgaggctt ggaggtgctt cgccacaagg cagccccaat ttgctgggag tttcttggca 9960 cctggtagtg gtgaggagcc ttgggaccct caggattact ccccttaagc atagtgggga 10020 cccttctgca tccccagcag gtgccccgct cttcagagcc tctctctctg aggtttaccc 10080 agacccctgc accaatgaga ccatgctgaa gcctcagaga gagagatgga gctttgacca 10140 ggagccgctc ttccttgagg gccagggcag ggaaagcagg aggcagcacc aggagtggga 10200 acaccagtgt ctaagcccct gatgagaaca gggtggtctc tcccatatgc ccataccagg 10260 cctgtgaaca gaatcctcct tctgcagtga caatgtctga gaggacgaca tgtttcccag 10320 cctaacgtgc agccatgccc atctacccac tgcctactgc aggacagcac caacccagga 10380 gctgggaagc tgggagaaga catggaatac ccatggcttc tcaccttcct ccagtccagt 10440 gggcaccatt tatgcctagg acacccacct gccggcccca ggctcttaag agttaggtca 10500 cctaggtgcc tctgggaggc cgaggcagga gaattgcttg aacccgggag gcagaggttg 10560 cagtgagccg agatcacacc actgcactcc agcctgggtg acagaatgag actctgtctc 10620 aaaaaaaaag agaaagatag catcagtggc taccaagggc taggggcagg ggaaggtgga 10680 gagttaatga ttaatagtat gaagtttcta tgtgagatga tgaaaatgtt ctggaaaaaa 10740 aaatatagtg gtgaggatgt agaatattgt gaatataatt aacggcattt aattgtacac 10800 ttaacatgat taatgtggca tattttatct tatgtatttg actacatcca agaaacactg 10860 ggagagggaa agcccaccat gtaaaataca cccaccctaa tcagatagtc ctcattgtac 10920 ccaggtacag gcccctcatg acctgcacag gaataactaa ggatttaagg acatgaggct 10980 tcccagccaa ctgcaggtgc acaacataaa tgtatctgca aacagactga gagtaaagct 11040 gggggcacaa acctcagcac tgccaggaca cacacccttc tcgtggattc tgactttatc 11100 tgacccggcc cactgtccag atcttgttgt gggattggga caagggaggt cataaagcct 11160 gtccccaggg cactctgtgt gagcacacga gacctcccca cccccccacc gttaggtctc 11220 cacacataga tctgaccatt aggcattgtg aggaggactc tagcgcgggc tcagggatca 11280 caccagagaa tcaggtacag agaggaagac ggggctcgag gagctgatgg atgacacaga 11340 gcagggttcc tgcagtccac aggtccagct caccctggtg taggtgcccc atccccctga 11400 tccaggcatc cctgacacag ctccctcccg gagcctcctc ccaggtgaca catcagggtc 11460 cctcactcaa gctgtccaga gagggcagca ccttggacag cgcccacccc acttcactct 11520 tcctccctca cagggctcag ggctcagggc tcaagtctca gaacaaatgg cagaggccag 11580 tgagcccaga gatggtgaca gggcaatgat ccaggggcag ctgcctgaaa cgggagcagg 11640 tgaagccaca gatgggagaa gatggttcag gaagaaaaat ccaggaatgg gcaggagagg 11700 agaggaggac acaggctctg tggggctgca gcccaggatg ggactaagtg tgaagacatc 11760 tcagcaggtg aggccaggtc ccatgaacag agaagcagct cccacctccc ctgatgcacg 11820 gacacacaga gtgtgtggtg ctgtgccccc agagtcgggc tctcctgttc tggtccccag 11880 ggagtgagaa gtgaggttga cttgtccctg ctcctctctg ctaccccaac attcaccttc 11940 tcctcatgcc cctctctctc aaatatgatt tggatctatg tccccgccca aatctcatgt 12000 caaattgtaa accccaatgt tggaggtggg gccttgtgag aagtgattgg ataatgcggg 12060 tggattttct gctttgatgc tgtttctgtg atagagatct cacatgatct ggttgtttaa 12120 aagtgtgtag cacctctccc ctctctctct ctctctctta ctcatgctct gccatgtaag 12180 acgttcctgt ttccccttca ccgtccagaa tgattgtaag ttttctgagg cctccccagg 12240 agcagaagcc actatgcttc ctgtacaact gcagaatgat gagcgaatta aacctctttt 12300 ctttataaat tacccagtct caggtatttc tttatagcaa tgcgaggaca gactaataca 12360 atcttctact cccagatccc cgcacacgct tagccccaga catcactgcc cctgggagca 12420 tgcacagcgc agcctcctgc cgacaaaagc aaagtcacaa aaggtgacaa aaatctgcat 12480 ttggggacat ctgattgtga aagagggagg acagtacact tgtagccaca gagactgggg 12540 ctcaccgagc tgaaacctgg tagcactttg gcataacatg tgcatgaccc gtgttcaatg 12600 tctagagatc agtgttgagt aaaacagcct ggtctggggc cgctgctgtc cccacttccc 12660 tcctgtccac cagagggcgg cagagttcct cccaccctgg agcctcccca ggggctgctg 12720 acctccctca gccgggccca cagcccagca gggtccaccc tcacccgggt cacctcggcc 12780 cacgtcctcc tcgccctccg agctcctcac acggactctg tcagctcctc cctgcagcct 12840 atcggccgcc cacctgaggc ttgtcggccg cccacttgag gcctgtcggc tgccctctgc 12900 aggcagctcc tgtcccctac accccctcct tccccgggct cagctgaaag ggcgtctccc 12960 agggcagctc cctgtgatct ccaggacagc tcagtctctc acaggctccg acgcccccta 13020 tgctgtcacc tcacagccct gtcattacca ttaactcctc agtcccatga agttcactga 13080 gcgcctgtct cccggttaca ggaaaactct gtgacaggga ccacgtctgt cctgctctct 13140 gtggaatccc agggcccagc ccagtgcctg acacggaaca gatgctccat aaatactggt 13200 taaatgtgtg ggagatctct aaaaagaagc atatcacctc cgtgtggccc ccagcagtca 13260 gagtctgttc catgtggaca caggggcact ggcaccagca tgggaggagg ccagcaagtg 13320 cccgcggctg ccccaggaat gaggcctcaa cccccagagc ttcagaaggg aggacagagg 13380 cctgcaggga atagatcctc cggcctgacc ctgcagccta atccagagtt cagggtcagc 13440 tcacaccacg tcgaccctgg tcagcatccc tagggcagtt ccagacaagg ccggaggtct 13500 cctcttgccc tccagggggt gacattgcac acagacatca ctcaggaaac ggattcccct 13560 ggacaggaac ctggctttgc taaggaagtg gaggtggagc ctggtttcca tcccttgctc 13620 caacagaccc ttctgatctc tcccacatac ctgctctgtt cctttctggg tcctatgagg 13680 accctgttct gccaggggtc cctgtgcaac tccagactcc ctcctggtac caccatgggg 13740 aaggtggggt gatcacagga cagtcagcct cgcagagaca gagaccaccc aggactgtca 13800 gggagaacat ggacaggccc tgagccgcag ctcagccaac agacacggag agggagggtc 13860 cccctggagc cttccccaag gacagcagag cccagagtca cccacctccc tccaccacag 13920 tcctctcttt ccaggacaca caagacacct ccccctccac atgcaggatc tggggactcc 13980 tgagacctct gggcctgggt ctccatccct gggtcagtgg cggggttggt ggtactggag 14040 acagagggct ggtccctccc cagccaccac ccagtgagcc tttttctagc ccccagagcc 14100 acctctgtca ccttcctgtt gggcatcatc ccaccttccc agagccctgg agagcatggg 14160 gagacccggg accctgctgg gtttctctgt cacaaaggaa aataatcccc ctggtgtgac 14220 agacccaagg acagaacaca gcagaggtca gcactgggga agacaggttg tcctcccagg 14280 ggatgggggt ccatccacct tgccgaaaag atttgtctga ggaactgaaa atagaaggga 14340 aaaaagagga gggacaaaag aggcagaaat gagaggggag gggacagagg acacctgaat 14400 aaagaccaca cccatgaccc acgtgatgct gagaagtact cctgccctag gaagagactc 14460 agggcagagg gaggaaggac agcagaccag acagtcacag cagccttgac aaaacgttcc 14520 tggaactcaa gctcttctcc acagaggagg acagagcaga cagcagagac catggagtct 14580 ccctcggccc ctccccacag atggtgcatc ccctggcaga ggctcctgct cacaggtgaa 14640 gggaggacaa cctgggagag ggtgggagga gggagctggg gtctcctggg taggacaggg 14700 ctgtgagacg gacagagggc tcctgttgga gcctgaatag ggaagaggac atcagagagg 14760 gacaggagtc acaccagaaa aatcaaattg aactggaatt ggaaaggggc aggaaaacct 14820 caagagttct attttcctag ttaattgtca ctggccacta cgtttttaaa aatcataata 14880 actgcatcag atgacacttt aaataaaaac ataaccaggg catgaaacac tgtcctcatc 14940 cgcctaccgc ggacattgga aaataagccc caggctgtgg agggccctgg gaaccctcat 15000 gaactcatcc acaggaatct gcagcctgtc ccaggcactg gggtgcaacc aagatc 15056 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 12047 <212> TYPE: DNA <213> ORGANISM: H. sapiens <400> SEQUENCE: 4 gaattcagaa ataggggaag gttgaggaag gacactgaac tcaaagggga tacagtgatt 60 ggtttatttg tcttctcttc acaacattgg tgctggagga attcccaccc tgaggttatg 120 aagatgtctg aacacccaac acatagcact ggagatatga gctcgacaag agtttctcag 180 ccacagagat tcacagccta gggcaggagg acactgtacg ccaggcagaa tgacatggga 240 attgcgctca cgattggctt gaagaagcaa ggactgtggg aggtgggctt tgtagtaaca 300 agagggcagg gtgaactctg attcccatgg gggaatgtga tggtcctgtt acaaattttt 360 caagctggca gggaataaaa cccattacgg tgaggacctg tggagggcgg ctgccccaac 420 tgataaagga aatagccagg tgggggcctt tcccattgta ggggggacat atctggcaat 480 agaagccttt gagacccttt agggtacaag tactgaggca gcaaataaaa tgaaatctta 540 tttttcaact ttatactgca tgggtgtgaa gatatatttg tttctgtaca gggggtgagg 600 gaaaggaggg gaggaggaaa gttcctgcag gtctggtttg gtcttgtgat ccagggggtc 660 ttggaactat ttaaattaaa ttaaattaaa acaagcgact gttttaaatt aaattaaatt 720 aaattaaatt ttactttatt ttatcttaag ttctgggcta catgtgcagg acgtgcagct 780 ttgttacata ggtaaacgtg tgccatggtg gtttgctgta cctatcaacc catcacctag 840 gtattaagcc cagcatgcat tagctgtttt tcctgacgct ctccctctcc ctgactccca 900 caacaggccc cagtgtgtgt tgttcccctc cctgtgtcca tgtgttctca ttgttcagct 960 cccacttata agtgagaaca tgtggtgttt ggttttctgt ttctgtgtta gtttgctgag 1020 gataatggct tccacctcca tccatgttcc tgcaaaggac gtgatcttat tcttttttat 1080 ggttgcatag aaattgtttt tacaaatcca attgatattg tatttaatta caagttaatc 1140 taattagcat actagaagag attacagaag atattaggta cattgaatga ggaaatatat 1200 aaaataggac gaaggtgaaa tattaggtag gaaaagtata atagttgaaa gaagtaaaaa 1260 aaaatatgca tgagtagcag aatgtaaaag aggtgaagaa cgtaatagtg actttttaga 1320 ccagattgaa ggacagagac agaaaaattt taaggaattg ctaaaccatg tgagtgttag 1380 aagtacagtc aataacatta aagcctcagg aggagaaaag aataggaaag gaggaaatat 1440 gtgaataaat agtagagaca tgtttgatgg attttaaaat atttgaaaga cctcacatca 1500 aaggattcat accgtgccat tgaagaggaa gatggaaaag ccaagaagcc agatgaaagt 1560 tagaaatatt attggcaaag cttaaatgtt aaaagtccta gagagaaagg atggcagaaa 1620 tattggcggg aaagaatgca gaacctagaa tataaattca tcccaacagt ttggtagtgt 1680 gcagctgtag ccttttctag ataatacact attgtcatac atcgcttaag cgagtgtaaa 1740 atggtctcct cactttattt atttatatat ttatttagtt ttgagatgga gcctcgctct 1800 gtctcctagg ctggagtgca atagtgcgat accactcact gcaacctctg cctcctctgt 1860 tcaagtgatt ttcttacctc agcctcccga gtagctggga ttacaggtgc gtgccaccac 1920 acccggctaa tttttgtatt ttttgtagag acggggtttt gccatgttgg ccaggctggt 1980 cttgaactcc tgacatcagg tgatccacct gccttggcct cctaaagtgc tgggattaca 2040 ggcatgagcc accgtgccca accactttat ttatttttta tttttatttt taaatttcag 2100 cttctatttg aaatacaggg ggcacatata taggattgtt acatgggtat attgaactca 2160 ggtagtgatc atactaccca acaggtaggt tttcaaccca ctccccctct tttcctcccc 2220 attctagtag tgtgcagtgt ctattgttct catgtttatg tctatgtgtg ctccaggttt 2280 agctcccacc tgtaagtgag aacgtgtggt atttgatttt ctgtccctgt gttaattcac 2340 ttaggattat ggcttccagc tccattcata ttgctgtaaa ggatatgatt catttttcat 2400 ggccatgcag tattccatat tgcgtataga tcacattttc tttctttttt ttttttgaga 2460 cggagtcttg ctttgctgcc taggctggag tgcagtagca cgatctcggc tcactgcaag 2520 cttcacctcc ggggttcacg tcattcttct gtctcagctt cccaagtagc tgggactaca 2580 ggcgcccgcc accacgtccg gctaattttt ttgtgtgttt ttagtagaga tgggggtttc 2640 actgtgttag ccaggatggt cttgatctcc tgaccttgtg gtccacctgc ctcggtctcc 2700 caaagtgctg ggattacagg ggtgagccac tgcgcccggc ccatatatac cacattttct 2760 ttaaccaatc caccattgat gggcaactag gtagattcca tggattccac agttttgcta 2820 ttgtgtgcag tgtggcagta gacatatgaa tgaatgtgtc tttttggtat aatgatttgc 2880 attcctttgg gtatacagtc attaatagga gtgctgggtt gaacggtggc tctgtttaaa 2940 attctttgag aattttccaa actgtttgcc atagagagca aactaattta catttccacg 3000 aacagtatat aagcattccc ttttctccac agctttgtca tcatggtttt tttttttctt 3060 tattttaaaa aagaatatgt tgttgttttc ccagggtaca tgtgcaggat gtgcaggttt 3120 gttacatagg tagtaaacgt gagccatggt ggtttgctgc acctgtcaac ccattacctg 3180 ggtatgaagc cctgcctgca ttagctcttt tccctaatgc tctcactact gccccaccct 3240 caccctgaca gggcaaacag acaacctaca gaatgggagg aaatttttgc aatctattca 3300 tctgacaaag gtcaagaata tccagaatct acaaggaact taagcaaatt tttacttttt 3360 aataatagcc actctgactg gcgtgaaatg gtatctcatt gtggttttca tttgaatttc 3420 tctgatgatc agtgacgatg agcatttttt catatttgtt ggctgcttgt acgtcttttg 3480 agaagtgtct cttcatgcct tttggccact ttaatgggat tattttttgc tttttagttt 3540 aagttcctta tagattctgg atattagact tcttattgga tgcatagttt gtgaatactc 3600 tcttccattc tgtaggttgt ctgtttactc tattgatggc ttcttttgct gtgccgaagc 3660 atcttagttt aattagaaac cacctgccaa tttttgtttt tgttgcaatt gcttttgggg 3720 acttagtcat aaactctttg ccaaggtctg ggtcaagaag agtatttcct aggttttctt 3780 ctagaatttt gaaagtctga atgtaaacat ttgcattttt aatgcatctt gagttagttt 3840 ttgtatatgt gaaaggtcta ctctcatttt ctttccctct ttctttcttt ctttcttttc 3900 tttctttctt tctttctttc tttctttctt tctttctttc tttctttttg tccttctttc 3960 tttctttctt tctctttctt tctctctttc tttttttttt ttgatggagt attgctctgt 4020 tgcccaggct gcagtgcagc ggcacgatct cggctcactg caacctctgc ctcctgggtt 4080 caactgattc tcctgcatca gccttccaag tagctgggat tataggcgcc cgccaccacg 4140 cccgactaat ttttgtattt ttagtagaga cggggttgtg ccatgttggc caggctggtt 4200 tgaaactcct gacctcaaac gatctgcctg ccttggcctc ccaaagtgct gggattacag 4260 gtgtgagcca ctgtgcccag ccaagaatgt cattttctaa gaggtccaag aacctcaaga 4320 tattttggga ccttgagaag agaggaattc atacaggtat tacaagcaca gcctaatggc 4380 aaatctttgg catggcttgg cttcaagact ttaggctctt aaaagtcgaa tccaaaaatt 4440 tttataaaag ctccagctaa gctaccttaa aaggggcctg tatggctgat cactcttctt 4500 gctatacttt acacaaataa acaggccaaa tataatgagg ccaaaattta ttttgcaaat 4560 aaattggtcc tgctatgatt tactcttggt aagaacaggg aaaatagaga aaaatttaga 4620 ttgcatctga cctttttttc tgaattttta tatgtgccta caatttgagc taaatcctga 4680 attattttct ggttgcaaaa actctctaaa gaagaacttg gttttcattg tcttcgtgac 4740 acatttatct ggctctttac tagaacagct ttcttgtttt tggtgttcta gcttgtgtgc 4800 cttacagttc tactcttcaa attattgtta tgtgtatctc atagttttcc ttcttttgag 4860 aaaactgaag ccatggtatt ctgaggacta gagatgactc aacagagctg gtgaatctcc 4920 tcatatgcaa tccactgggc tcgatctgct tcaaattgct gatgcactgc tgctaaagct 4980 atacatttaa aaccctcact aaaggatcag ggaccatcat ggaagaggag gaaacatgaa 5040 attgtaagag ccagattcgg ggggtagagt gtggaggtca gagcaactcc accttgaata 5100 agaaggtaaa gcaacctatc ctgaaagcta acctgccatg gtggcttctg attaacctct 5160 gttctaggaa gactgacagt ttgggtctgt gtcattgccc aaatctcatg ttaaattgta 5220 atccccagtg ttcggaggtg ggacttggtg gtaggtgatt cggtcatggg agtagatttt 5280 cttctttgtg gtgttacagt gatagtgagt gagttctcgt gagatctggt catttaaaag 5340 tgtgtggccc ctcccctccc tctcttggtc ctcctactgc catgtaagat acctgctcct 5400 gctttgcctt ctaccataag taaaagcccc ctgaggcctc cccagaagca gatgccacca 5460 tgcttcctgt acagcctgca gaaccatcag ccaattaaac ctcttttctg tataaattac 5520 cagtcttgag tatctcttta cagcagtgtg agaacggact aatacaaggg tctccaaaat 5580 tccaagttta tgtattcttt cttgccaaat agcaggtatt taccataaat cctgtcctta 5640 ggtcaaacaa ccttgatggc atcgtacttc aattgtctta cacattcctt ctgaatgact 5700 cctcccctat ggcatataag ccctgggtct tgggggataa tggcagaggg gtccaccatc 5760 ttgtctggct gccacctgag acacggacat ggcttctgtt ggtaagtctc tattaaatgt 5820 ttctttctaa gaaactggat ttgtcagctt gtttctttgg cctctcagct tcctcagact 5880 ttggggtagg ttgcacaacc ctgcccacca cgaaacaaat gtttaatatg ataaatatgg 5940 atagatataa tccacataaa taaaagctct tggagggccc tcaataattg ttaagagtgt 6000
aaatgtgtcc aaagatggaa aatgtttgag aactactgtc ccagagattt tcctgagttc 6060 tagagtgtgg gaatatagaa cctggagctt ggcttcttca gcctagaatc aggagtatgg 6120 ggctgaagtc tgaagcttgg cttcagcagt ttggggttgg cttccggagc acatatttga 6180 catgttgcga ctgtgatttg gggtttggta tttgctctga atcctaatgt ctgtccttga 6240 ggcatctaga atctgaaatc tgtggtcaga attctattat cttgagtagg acatctccag 6300 tcctggttct gccttctagg gctggagtct gtagtcagtg acccggtctg gcatttcaac 6360 ttcatataca gtgggctatc ttttggtcca tgtttcaacc aaacaaccga ataaaccatt 6420 agaacctttc cccacttccc tagctgcaat gttaaaccta ggatttctgt ttaataggtt 6480 catatgaata atttcagcct gatccaactt tacattcctt ctaccgttat tctacaccca 6540 ccttaaaaat gcattcccaa tatattccct ggattctacc tatatatggt aatcctggct 6600 ttgccagttt ctagtgcatt aacatacctg atttacattc ttttacttta aagtggaaat 6660 aagagtccct ctgcagagtt caggagttct caagatggcc cttacttctg acatcaattg 6720 agatttcaag ggagtcgcca agatcatcct caggttcagt gattgctggt agccctcata 6780 taactcaatg aaagctgtta tgctcatggc tatggtttat tacagcaaaa gaatagagat 6840 gaaaatctag caagggaaga gttgcatggg gcaaagacaa ggagagctcc aagtgcagag 6900 attcctgttg ttttctccca gtggtgtcat ggaaagcagt atcttctcca tacaatgatg 6960 tgtgataata ttcagtgtat tgccaatcag ggaactcaac tgagccttga ttatattgga 7020 gcttggttgc acagacatgt cgaccacctt catggctgaa ctttagtact tagcccctcc 7080 agacgtctac agctgatagg ctgtaaccca acattgtcac cataaatcac attgttagac 7140 tatccagtgt ggcccaagct cccgtgtaaa cacaggcact ctaaacaggc aggatatttc 7200 aaaagcttag agatgacctc ccaggagctg aatgcaaaga cctggcctct ttgggcaagg 7260 agaatccttt accgcacact ctccttcaca gggttattgt gaggatcaaa tgtggtcatg 7320 tgtgtgagac accagcacat gtctggctgt ggagagtgac ttctatgtgt gctaacattg 7380 ctgagtgcta agaaagtatt aggcatggct ttcagcactc acagatgctc atctaatcct 7440 cacaacatgg ctacagggtg ggcactacta gcctcatttg acagaggaaa ggactgtgga 7500 taagaagggg gtgaccaata ggtcagagtc attctggatg caaggggctc cagaggacca 7560 tgattagaca ttgtctgcag agaaattatg gctggatgtc tctgccccgg aaagggggat 7620 gcactttcct tgacccccta tctcagatct tgactttgag gttatctcag acttcctcta 7680 tgataccagg agcccatcat aatctctctg tgtcctctcc ccttcctcag tcttactgcc 7740 cactcttccc agctccatct ccagctggcc aggtgtagcc acagtaccta actctttgca 7800 gagaactata aatgtgtatc ctacagggga gaaaaaaaaa aagaactctg aaagagctga 7860 cattttaccg acttgcaaac acataagcta acctgccagt tttgtgctgg tagaactcat 7920 gagactcctg ggtcagaggc aaaagatttt attacccaca gctaaggagg cagcatgaac 7980 tttgtgttca catttgttca ctttgccccc caattcatat gggatgatca gagcagttca 8040 ggtggatgga cacaggggtt tgtggcaaag gtgagcaacc taggcttaga aatcctcaat 8100 cttataagaa ggtactagca aacttgtcca gtctttgtat ctgacggaga tattatcttt 8160 ataattgggt tgaaagcaga cctactctgg aggaacatat tgtatttatt gtcctgaaca 8220 gtaaacaaat ctgctgtaaa atagacgtta actttattat ctaaggcagt aagcaaacct 8280 agatctgaag gcgataccat cttgcaaggc tatctgctgt acaaatatgc ttgaaaagat 8340 ggtccagaaa agaaaacggt attattgcct ttgctcagaa gacacacaga aacataagag 8400 aaccatggaa aattgtctcc caacactgtt cacccagagc cttccactct tgtctgcagg 8460 acagtcttaa catcccatca ttagtgtgtc taccacatct ggcttcaccg tgcctaacca 8520 agatttctag gtccagttcc ccaccatgtt tggcagtgcc ccactgccaa ccccagaata 8580 agggagtgct cagaattccg aggggacatg ggtggggatc agaacttctg ggcttgagtg 8640 cagagggggc ccatactcct tggttccgaa ggaggaagag gctggaggtg aatgtccttg 8700 gaggggagga atgtgggttc tgaactctta aatccccaag ggaggagact ggtaaggtcc 8760 cagcttccga ggtactgacg tgggaatggc ctgagaggtc taagaatccc gtatcctcgg 8820 gaaggagggg ctgaaattgt gaggggttga gttgcagggg tttgttagct tgagactcct 8880 tggtgggtcc ctgggaagca aggactggaa ccattggctc cagggtttgg tgtgaaggta 8940 atgggatctc ctgattctca aagggtcaga ggactgagag ttgcccatgc tttgatcttt 9000 ccatctactc cttactccac ttgagggtaa tcacctactc ttctagttcc acaagagtgc 9060 gcctgcgcga gtataatctg cacatgtgcc atgtcccgag gcctggggca tcatccactc 9120 atcattcagc atctgcgcta tgcgggcgag gccggcgcca tgacgtcatg tagctgcgac 9180 tatccctgca gcgcgcctct cccgtcacgt cccaaccatg gagctgtgga cgtgcgtccc 9240 ctggtggatg tggcctgcgt ggtgccaggc cggggcctgg tgtccgataa agatcctaga 9300 accacaggaa accaggactg aaaggtgcta gagaatggcc atatgtcgct gtccatgaaa 9360 tctcaaggac ttctgggtgg agggcacagg agcctgaact tacgggtttg ccccagtcca 9420 ctgtcctccc aagtgagtct cccagatacg aggcactgtg ccagcatcag cttcatctgt 9480 accacatctt gtaacaggga ctacccagga ccctgatgaa caccatggtg tgtgcaggaa 9540 gagggggtga aggcatggac tcctgtgtgg tcagagccca gagggggcca tgacgggtgg 9600 ggaggaggct gtggactggc tcgagaagtg ggatgtggtt gtgtttgatt tcctttggcc 9660 agataaagtg ctggatatag cattgaaaac ggagtatgaa gaccagttag aatggagggt 9720 caggttggag ttgagttaca gatggggtaa aattctgctt cggatgagtt tggggattgg 9780 caatctaaag gtggtttggg atggcatggc tttgggatgg aaataggttt gtttttatgt 9840 tggctgggaa gggtgtgggg attgaattgg ggatgaagta ggtttagttt tggagataga 9900 atacatggag ctggctattg catgcgagga tgtgcattag tttggtttga tctttaaata 9960 aaggaggcta ttagggttgt cttgaattag attaagttgt gttgggttga tgggttgggc 10020 ttgtgggtga tgtggttgga ttgggctgtg ttaaattggt ttgggtcagg ttttggttga 10080 ggttatcatg gggatgagga tatgcttggg acatggattc aggtggttct cattcaagct 10140 gaggcaaatt tcctttcaga cggtcattcc agggaacgag tggttgtgtg ggggaaatca 10200 ggccactggc tgtgaatatc cctctatcct ggtcttgaat tgtgattatc tatgtccatt 10260 ctgtctcctt cactgtactt ggaattgatc tggtcattca gctggaaatg ggggaagatt 10320 ttgtcaaatt cttgagacac agctgggtct ggatcagcgt aagccttcct tctggtttta 10380 ttgaacagat gaaatcacat tttttttttc aaaatcacag aaatcttata gagttaacag 10440 tggactctta taataagagt taacaccagg actcttattc ttgattcttt tctgagacac 10500 caaaatgaga tttctcaatg ccaccctaat tctttttttt tttttttttt tttttgagac 10560 acagtctggg tcttttgctc tgtcactcag gctggagcgc agtggtgtga tcatagctca 10620 ctgaaccctt gacctcctgg acttaaggga tcctcctgct tcagcctcct gagtagatgg 10680 ggctacaggt gcttgccacc acacctggct aattaaattt tttttttttt tttgtagaga 10740 aagggtctca ctttgttgcc ctggctgatc ttgaacttct gacttcaagt gattcttcag 10800 ccttggactc ccaaagcact gggattgctg gcatgagcca ctcaccgtgc ctggcttgca 10860 gcttaatctt ggagtgtata aacctggctc ctgatagcta gacatttcag tgagaaggag 10920 gcattggatt ttgcatgagg acaattctga cctaggaggg caggtcaaca ggaatccccg 10980 ctgtacctgt acgttgtaca ggcatggaga atgaggagtg aggaggccgt accggaaccc 11040 catattgttt agtggacatt ggattttgaa ataataggga acttggtctg ggagagtcat 11100 atttctggat tggacaatat gtggtatcac aaggttttat gatgagggag aaatgtatgt 11160 ggggaaccat tttctgagtg tggaagtgca agaatcagag agtagctgaa tgccaacgct 11220 tctatttcag gaacatggta agttggaggt ccagctctcg ggctcagacg ggtataggga 11280 ccaggaagtc tcacaatccg atcattctga tatttcaggg catattaggt ttggggtgca 11340 aaggaagtac ttgggactta ggcacatgag actttgtatt gaaaatcaat gattggggct 11400 ggccgtggtg ctcacgcctg taatctcatc actttgggag accgaagtgg gaggatggct 11460 tgatctcaag agttggacac cagcctaggc aacatggcca gaccctctct ctacaaaaaa 11520 attaaaaatt agctggatgt ggtggtgcat gcttgtggtc tcagctatcc tggaggctga 11580 gacaggagaa tcggttgagt ctgggagttc aaggctacag ggagctgcga tcacgccgct 11640 gcactccagc ctgggaaaca gagtgagact gtctcagaat ttttttaaaa aagaatcagt 11700 gatcatccca acccctgttg ctgttcatcc tgagcctgcc ttctctggct ttgttcccta 11760 gatcacatct ccatgatcca taggccctgc ccaatctgac ctcacaccgt gggaatgcct 11820 ccagactgat ctagtatgtg tggaacagca agtgctggct ctccctcccc ttccacagct 11880 ctgggtgtgg gagggggttg tccagcctcc agcagcatgg ggagggcctt ggtcagcatc 11940 taggtgccaa cagggcaagg gcggggtcct ggagaatgaa ggctttatag ggctcctcag 12000 ggaggccccc cagccccaaa ctgcaccacc tggccgtgga caccggt 12047 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5 <211> LENGTH: 858 <212> TYPE: DNA <213> ORGANISM: H. sapiens <400> SEQUENCE: 5 cgagcggccc ctcagcttcg gcgcccagcc ccgcaaggct cccggtgacc actagagggc 60 gggaggagct cctggccagt ggtggagagt ggcaaggaag gaccctaggg ttcatcggag 120 cccaggttta ctcccttaag tggaaatttc ttcccccact cctccttggc tttctccaag 180 gagggaaccc aggctgctgg aaagtccggc tggggcgggg actgtgggtt caggggagaa 240 cggggtgtgg aacgggacag ggagcggtta gaagggtggg gctattccgg gaagtggtgg 300 ggggagggag cccaaaacta gcacctagtc cactcattat ccagccctct tatttctcgg 360 ccgctctgct tcagtggacc cggggagggc ggggaagtgg agtgggagac ctaggggtgg 420 gcttcccgac cttgctgtac aggacctcga cctagctggc tttgttcccc atccccacgt 480 tagttgttgc cctgaggcta aaactagagc ccaggggccc caagttccag actgcccctc 540 ccccctcccc cggagccagg gagtggttgg tgaaaggggg aggccagctg gagaacaaac 600 gggtagtcag ggggttgagc gattagagcc cttgtaccct acccaggaat ggttggggag 660 gaggaggaag aggtaggagg taggggaggg ggcggggttt tgtcacctgt cacctgctcg 720 ctgtgcctag ggcgggcggg cggggagtgg ggggaccggt ataaagcggt aggcgcctgt 780 gcccgctcca cctctcaagc agccagcgcc tgcctgaatc tgttctgccc cctccccacc 840 catttcacca ccaccatg 858 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6 <211> LENGTH: 454 <212> TYPE: DNA <213> ORGANISM: R. rattus <400> SEQUENCE: 6 aagcttccac aagtgcattt agcctctcca gtattgctga tgaatccaca gttcaggttc 60 aatggcgttc aaaacttgat caaaaatgac cagactttat attcttacac caacatctat 120 ctgattggag gaatggataa tagtcatcat gtttaaacat ctaccattcc agttaagaaa 180 atatgatagc atcttgttct tagtcttttt cttaataggg acataaagcc cacaaataaa 240 aatatgcctg aagaatggga caggcattgg gcattgtcca tgcctagtaa agtactccaa 300 gaacctattt gtatactaga tgacacaatg tcaatgtctg tgtacaactg ccaactggga 360 tgcaagacac tgcccatgcc aatcatcctg aaaagcagct ataaaaagca ggaagctact 420 ctgcaccttg tcagtgaggt ccagatacct acag 454 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 7 <211> LENGTH: 307 <212> TYPE: DNA <213> ORGANISM: Adenovirus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (20)...(304) <400> SEQUENCE: 7 gatgaccggc tcaaccatc gcg ccc aca acg gac tat cgc aac acc act gct 52 Ala Pro Thr Thr Asp Tyr Arg Asn Thr Thr Ala 1 5 10 acc gga cta aca tct gcc cta aat tta ccc caa gtt cat gcc ttt gtc 100 Thr Gly Leu Thr Ser Ala Leu Asn Leu Pro Gln Val His Ala Phe Val 15 20 25 aat gac tgg gcg agc ttg gac atg tgg tgg ttt tcc ata gcg ctt atg 148 Asn Asp Trp Ala Ser Leu Asp Met Trp Trp Phe Ser Ile Ala Leu Met 30 35 40 ttt gtt tgc ctt att att atg tgg ctt att tgt tgc cta aag cgc aga 196 Phe Val Cys Leu Ile Ile Met Trp Leu Ile Cys Cys Leu Lys Arg Arg 45 50 55 cgc gcc aga ccc ccc atc tat agg cct atc att gtg ctc aac cca cac 244 Arg Ala Arg Pro Pro Ile Tyr Arg Pro Ile Ile Val Leu Asn Pro His 60 65 70 75 aat gaa aaa att cat aga ttg gac ggt ctg aaa cca tgt tct ctt ctt 292 Asn Glu Lys Ile His Arg Leu Asp Gly Leu Lys Pro Cys Ser Leu Leu 80 85 90 tta cag tat gat taa 307 Leu Gln Tyr Asp 95 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 8 <211> LENGTH: 95 <212> TYPE: PRT <213> ORGANISM: Adenovirus <400> SEQUENCE: 8 Ala Pro Thr Thr Asp Tyr Arg Asn Thr Thr Ala Thr Gly Leu Thr Ser 1 5 10 15 Ala Leu Asn Leu Pro Gln Val His Ala Phe Val Asn Asp Trp Ala Ser 20 25 30 Leu Asp Met Trp Trp Phe Ser Ile Ala Leu Met Phe Val Cys Leu Ile 35 40 45 Ile Met Trp Leu Ile Cys Cys Leu Lys Arg Arg Arg Ala Arg Pro Pro 50 55 60 Ile Tyr Arg Pro Ile Ile Val Leu Asn Pro His Asn Glu Lys Ile His 65 70 75 80 Arg Leu Asp Gly Leu Lys Pro Cys Ser Leu Leu Leu Gln Tyr Asp 85 90 95
* * * * *
Field of search:
No:320.1 - VECTOR, PER SE (E.G., PLASMID, HYBRID PLASMID, COSMID, VIRAL VECTOR, BACTERIOPHAGE VECTOR, ETC.)
No:325 - ANIMAL CELL, PER SE (E.G., CELL LINES, ETC.); COMPOSITION THEREOF; PROCESS OF PROPAGATING, MAINTAINING OR PRESERVING AN ANIMAL CELL OR COMPOSITION THEREOF; PROCESS OF ISOLATING OR SEPARATING AN ANIMAL CELL OR COMPOSITION THEREOF; PROCESS OF PREPARING A CO
No:93.6 - Virus or bacteriophage
No:233.1 - Adenoviridae, adeno-like virus, or Parvoviridae (e.g., adenovirus, canine parvovirus, mink enteritis virus, hemorrhagic enteritis virus, feline panleukopenia virus, egg drop syndrome virus, etc.)
No:191.1 - Disclosed amino acid sequence derived from parasitic organism (e.g., Dirofilaria, Eimeria, Trichinella, etc.)
No:23.5 - Encodes an animal polypeptide
0845537 / 1998-05-31
WO 98/39466 / 1998-08-31
WO 95/11984 / 1995-04-30
WO 95/14100 / 1995-04-30
WO 96/17053 / 1996-05-31
WO 96/34969 / 1996-10-31
WO-97/01358 / 1996-12-31
WO 98/06864 / 1998-01-31
WO 98/13508 / 1998-03-31
WO 98/35028 / 1998-07-31
Cuevas et al. Cancer Res 63:6877-6884, October 15, 2003.*
Hernandez-Alcoceba et al. Human Gene Therapy 13:1737-1750, Sep. 20, 2002.*
Li et aI abstract 445 from ASGT 71h Annual Meeting, Jun. 2004.*
Babiss et. al.; Cellular Promoters Incorporated into the Adenovirus Genome; 1987, J. Mol. Biol. 193: 643-650.*
Shi et. al.; Modulation of the Specificity and Activity of a Cellular Promoter in an Adenoviral Vector; 1997, Human Gene Therapy 8: 403-410.*
Bett et. al., packaging Capacity and Stability of Human adenovirus Type 5 Vectors, 1993, Journal of Virology, Vol. 67, No. 10: 5911-5921.*
Anderson; Human gene therapy, 1998, Nature Vol. 392: 25-30.*
Verma et. al.; Gene therapy-promises, problems and prospects, 1997, Nature, Vol. 389:239-242.*
Curiel; Strategies to Adapt Adenoviral Vectors forTargeted Delivery, 1999, Gene Therapy Strategies:158-171.*
Jam; Delivery of moleculat and cellular medicine to solid tumors, 1997, Journal of Controlled Release 53: 49-87.*
Hobbs; Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment, 1998, Proc. Natl. Acad. Sci. Vol. 95: 4607-4612.*
Walther; Targeted Vectors for Gene therapy of Cancer and Retrpvoral Infections, 1996, Molecular Biotechnology Vol. 6: 2670286.*
Dachs; Targeted Gene Therapy to Cancer: A Review. 1997, Oncolog Research Vol. 9: 313-325.*
Advani et. al.; Radiogenetic Therapy: On the Interaction of Viral therapy and Ionizing Radiation for improving Local Control of Tumors, 1997, Seminars in Oncology Vol. 24. No. 6: 633-638.*
Parr et. al..; Tumor-selective transgene expression in vivo mediated by an E2F-responsive adenoviral vector, 1997, Nature medicine Vol. 3, No. 3: 1145-1949.*
Orkin et al., “Report and Recommendations of the Panel to Assess the NIH Investment in Research on Gene Therapy.” pp. 1-20, Dec. 1995.*
Miller et al., “Targeted Vector for Gene Therapy.” FASEB, Vol. 9: 190-199, Feb. 1995.*
Ledley, F 0.,“Pharmaceutical Approach to Somatic Gene Therapy.” Pharmaceutical Research vol. 13: 1595-1613, Nov. 1996.*
Eck et al “Gene-Based Therapy-Chapter 5.”, Goodman & Gilman's The Pharmacological Basis of Therapeutics-Ninth Edition, McGraw-Hill: 77-101, 1996.*
Abe et al. (1993). “Characterization of Cis-Acting Elements Regulating Transcription of the Human DF3 Breast Carcinoma-Associated Antigen (N4UCI) Gene,” Proc. Natl. Sci. USA 90: 282-286.
Arnberg et al. (1997). “Fiber Genes of Adenoviruses with Tropism for the Eye and the Genital Tract,” Virol. 227: 239-244.
Ausubel et al. eds. (1987). Current Protocols in Molecular Bioloev Suppl. 30 Sec. 7.7.18, Table 7.7.1.
Bailey et al. (1993). “Enteric Adenovirus Type 40: Expression of EIB Proteins in Vitro and in Vivo,” Virol. 193: 631-641.
Bailey et al. (1994). “Cell Type Specific Regulation of Expression from the Ad40 E1 B Promoter in Recombinant Ad5/Ad40 Viruses,” Virol. 202: 695-706.
Behringeret al. (1988). “Dwarf Mice Produced by Genetic Ablation of Growth Hormone-Expressing Cells,” Genes Dev. 2: 453-461.
Berkner et al. (1983). “Generation of Adenovirus by Transfection of Plasmids,” Nuc. Acid Res. 11(17): 6003-6020.
Bett et al. (1993). “Packaging Capacity and Stability of Human Adenovirus Type 5 Vectors,” J. Virol. 67(10): 5911-5921.
Bett et al. (1994) “An efficient and Flexible System for Construction of Adenovirus Vectors with insertions or Deletions in Early Regions 1 and 3,” Proc. Nail. A cad. Sci. USA 91: 8802-8806.
Bridge et al. (1989). “Redundant Control of Adenovirus Late Gene Expression by Early Region 4,” J. Virol. 63(2): 631-638.
Bunn et al. (1996). “Oxygen Sensing and Molecular Adaptation to Hypoxia,” Physiol. Rev. 76(3): 839-885.
Cannio et al. (1991). “A Cell-Type Specific and Enhancer-Dependent Silencer in the Regulation of the Expression of the Human Urokinase Plasminogen Activator Gene,” Nuc. Acids Res. 19(9): 2303-2308.
Coiditz. (1993). “Epidemiology of Breast Cancer,” Cancer Suppl. 71(4): 1480-1489.
Dachs et al. (1996). “The Molecular Response of Mammalian Cells to Hypoxia and the Potential for Exploitation in Cancer Therapy,” Br. J. Cancer 74: 5 126-5132.
Dachs et al. (1997). “Targeting Gene Expression to Hypoxic Tumor Cells,” Nat. Med. 3(5): 515-520.
Feigner et al. (1989). “Cationic Liposome-Mediated Transfection,” Nature 337: 387-388.
Firth et al. (1994). “Oxygen-Related Control Elements in the Phosphoglycerate Kinase 1 and Lactate Dehydrogenase A Genes: Similarities with the Erythropoietin 3′ Enhancer,” Proc. Nati. Acad. Sci. USA 91: 6496-6500.
Flint. (1982). “Expression of Adenoviral Genetic Information in Productively infected Cells,” Biochem. Biophys. Acta 651: 175-208.
Flint. (1986). “Regulation of Adenovirus mRNA Formation,” Adv. Vir. Res. 31: 169-228.
Folkman. (1989). “What is the Evidence that Tumors are Angiogenesis Dependent?” Natl. Cancer Inst. 82(1): 4-6.
Frankel et al. (1989). “Selection and Characterization of Ricin Toxin A-Chain Mutations in Saccharomyces cerevisiae,” Mol. Cell. Biol. 9(2): 4 15-420.
Graham. (1984). “Covalently Closed Circles of Human Adenovirus DNA are Infectious,” EMBO J. 3(12): 2917-2922.
Grand. (1987). “The Structure and Functions of the Adenovirus Early Region 1 Proteins,” Biochern. J. 241: 25-38.
Grooteclaes et al. (1984). “The 6-Kilobase c-erbB2 Promoter Contains Positive and Negative Regulatory Elements Functional in Human Mammary Cell Lines,” Cancer Res. 54: 4193-4199.
Guillemin et al. (1997). “The Hypoxie Response: Huffing and HIFing,” Cell 89: 9-12.
Hallahan et al. (1995). “Spatial and Temporal Control of Gene Therapy Using Ionizing Radiation,” Nat. Med 1(8): 786-791.
Höckel et al. (1996). “Hypoxia and Radiation Response in Human Tumors,” Semin. Rad. Oncol.˜6(1): 3-9.
Hudson Ct al. (1990). “Structure and Inducible Regulation of the Human c-crb B2Ineu Promoter,” J. Biol. Chem. 265: 4389-4393.
Ido et al. (1995). “Gene Therapy for 1-lepatoma Cells Using a Retrovirus Vector Carrying Herpes Simplex Virus Thymidine Kinase Gene Under the Control of Human ci-Fetoprotein Gene Promoter,” Cancer Res. 55: 3105-3109.
Ishii et al. (1987) “Characterization of the Promoter Region of the Human C-erb B-2 Protooncogene,” Proc. Nati. Acad. Sci. USA 84: 4374-4378.
Jiang et al. (1997). “V-SRC Induces Expression of Hypoxia-inducible Factor 1(HIF-1) and Transcription of Genes Encoding Vascular Endothelial Growth Factor and Enolase 1: Involvement of HIF-1 in Tumor Progression,” Can. Res. 57: 5328-5335.
5871726 - Tissue specific and tumor growth supperssion by adenovirus comprising prostate specific antigen
Browse by classes
Automotives and Transportation
Business and Commerce
Fashion and Accessories
Hardware and Tools
Health and Medicine
Materials and Material Science
Paper and Office Materials