# Patent

## Title
Methods and compositions for screening for angiogenesis modulating compounds

## Abstract

The present invention relates to novel transcription control elements, including promoters, derived from angiogenesis-related genes, particularly the mouse VEGF gene, the mouse VEGFR-2 receptor gene, and the mouse Tie2 gene. Also disclosed are isolated polynucleotides comprising such promoters, as well as nucleic acid constructs comprising such promoters operatively linked to genes encoding a gene product, such as, a reporter, a protein, polypeptide, hormone, ribozyme, or antisense RNA, and to recombinant cells and transgenic animals comprising such nucleic acid constructs. The present invention further relates to screening methods using those recombinant cells and transgenic animals, particularly methods of screening for therapeutic compounds that modulate tumorigenesis and angiogenesis.

## Background
This application is a continuation-in-part of U.S. application Ser. No. 09/465,978, filed 16 Dec. 1999 now U.S. Pat. No. 6,867,348, from which priority is claimed under 35 USC §120, and is herein incorporated by reference in its entirety.


TECHNICAL FIELD
The present invention is in the field of molecular biology and medicine. More specifically, it relates to novel promoters, to expression cassettes, and vector constructs incorporating those promoters that are useful for generating transformed cells and transgenic animals. It further relates to methods of using those transformed cells and transgenic animals, particularly to methods of screening for compounds that modulate angiogenesis.
BACKGROUND
A requirement for cellular inflow of nutrients, outflow of waste products, and gas exchange in most tissues and organs is the establishment of a vascular supply. Several processes for blood vessel development and differentiation have been identified. One such process is termed “vasculogenesis” and takes place in the embryo. This process consists of the in situ differentiation of mesenchymal cells into hemoangioblasts, which are the precursors of both endothelial cells and blood cells. “Angiogenesis” is a second such process and involves the formation of new blood vessels from a preexisting endothelium. This process is required for (i) the development of embryonic vasculature, and (ii) a variety of post-natal processes, including, but not limited to, wound healing, tissue regeneration, and organ regeneration. Further, angiogenesis has been identified as a requirement for solid tumor growth and uncontrolled blood cell proliferation.
Vascular Endothelial Growth Factor (VEGF; also designated as vascular permeability factor (VPF) has been identified as a regulator of normal and pathological angiogenesis. VEGF is a secreted growth factor having the following properties: (i) it is an endothelial cell specific mitogen; (ii) it is angiogenic in vivo and induces vascular permeability; (iii) its expression (and expression of its receptors) has been correlated with vasculogenesis and angiogenesis during embryonic development; and (iv) it is expressed in tumor cells. The VEGF receptor appears to be expressed exclusively in adjacent small blood vessels. VEGF appears to play a crucial role in the vascularization of a wide range of tumors including, but not limited to, breast cancers, ovarian tumors, brain tumors, kidney and bladder carcinomas, adenocarcinomas and malignant gliomas. Tumors have been shown to produce ample amounts of VEGF, which stimulates the proliferation, and migration of endothelial cells (EC's). This is thought to induce tumor vascularization by a paracrine mechanism.
The angiogenic effect of VEGF appears to be mediated by its binding to high affinity cell surface VEGF receptors (VEGFR).
SUMMARY OF THE INVENTION
The present invention relates to transcription control elements derived from angiogenesis-related gene loci, in particular, transcription control elements derived from VEGFR-2, VEGF, and TIE2 gene loci. The present invention relates to methods of use of such transcription control elements, for example, in the construction of expression cassettes, vectors, recombinant cells and transgenic animals.
In one aspect, the present invention includes, but is not limited to, an isolated polynucleotide, and fragments thereof, having at least 80–85%, preferably 85–90%, more preferably 90–95%, and most preferably 98–100% sequence identity to sequence SEQ ID NO:32, SEQ ID NO:40, and/or SEQ ID NO:44. Exemplary fragments include, but are not limited to, polynucleotides having lengths of approximately 55–100, 100–250, 250–500, and 500–1,000. In particular, the present invention relates to recombinant nucleic acid molecules comprising transcription control elements derived from a mouse VEGFR-2, VEGF, and/or Tie-2 gene locus.
One embodiment of the present invention includes an isolated polynucleotide comprising, a polynucleotide having X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least about 95% identity to Y contiguous nucleotides derived from nucleotides approximately 1 through 3,564 of SEQ ID NO:32 (VEGFR-2; FIGS. 3A–3C), (ii) X equals Y, and (iii) X is greater than or equal to approximately 55. X is typically in the range of approximately 55 to 3,564 nucleotides in length including all integer values in that range. In another embodiment, the invention includes an isolated polynucleotide consisting of a polynucleotide having greater than 80% identity to the sequence presented as SEQ ID NO:32.
In another embodiment of the present invention, an isolated polynucleotide comprises, a polynucleotide having X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least about 95% identity to Y contiguous nucleotides derived from nucleotides approximately 1 through 3,762 of SEQ ID NO:44 (VEGF, FIGS. 2A–2C), (ii) X equals Y, and (iii) X is greater than or equal to approximately 50. X is typically in the range of approximately 50 to 3,762 nucleotides in length including all integer values in that range. The invention also includes an isolated polynucleotide consisting of a polynucleotide having greater than 80% identity to the sequence presented as SEQ ID NO:44.
In a further embodiment of the invention, an isolated polynucleotide comprises a polynucleotide having X contiguous nucleotides, wherein (A) (i) the X contiguous nucleotides have at least about 95% identity to Y contiguous nucleotides derived from nucleotides approximately 1 through 6,091 of SEQ ID NO:40 (TIE-2, FIGS. 6A–6D), (ii) X equals Y, and (iii) X is greater than or equal to approximately 55, or (B) (i) the X contiguous nucleotides have at least about 90% identity to Y contiguous nucleotides derived from nucleotides approximately 6,091 through 6,560 of SEQ ID NO:40, (ii) X equals Y, and (iii) X is greater than or equal to approximately 250. For polynucleotide (A) X is typically in the range of approximately 55 to 6,091 nucleotides in length including all integer values in that range, or for polynucleotide (B) X is typically in the range of approximately 250 through 469 nucleotides in length including all integer values in that range. The invention also includes an isolated polynucleotide consisting of a polynucleotide having greater than 80% identity to the sequence presented as SEQ ID NO:40.
In another embodiment the invention includes expression cassettes containing the isolated polynucleotide sequences of the present invention wherein the transcription control element(s) is operably linked with a reporter gene of interest, for example, a sequence encoding a light generating protein. Such expression cassettes may be provided in the form of a vector. Exemplary vectors include, but are not limited to procaryotic, eucaryotic, viral vectors, and shuttle vectors. Expression vectors comprise a variety of components described herein, including, but not limited to, origins of replication and selectable markers. Cells, e.g., epithelial cells or tumor cells, and transgenic animals may comprise the polynucleotides of the present invention. Further, cells comprising the polynucleotides of the present invention may be introduced into animals (e.g., wild-type, carrying a genomic modification, and/or transgenic). The transgenic animals of the present invention may, of course, comprise genomic modifications in addition to the polynucleotides of the present invention. In preferred embodiments of the present invention the transgenic animals are transgenic rodents, for example, mice, rats, or guinea pigs.
In another aspect, the present invention describes methods for monitoring expression mediated by a transcription control element, derived from an angiogenesis-related gene locus, in a living, transgenic, non-human animal, for example, a rodent. The methods comprises monitoring the expression of a reporter sequence in a living transgenic animal, where the animal comprises an expression cassette having a transcription control element derived from an angiogenesis-related gene. Such transcription control elements include, but are not limited to, a VEGFR-2-locus-derived sequence, a VEGF-locus-derived sequence, and/or a TIE2-locus-derived sequence. Some exemplary isolated polynucleotide sequences comprising transcription control elements are described above and herein. Expression of the reporter sequence may, for example, be observed to increase during monitoring typically indicating induction mediated by the selected transcription control elements. Alternately, expression of the reporter sequence may be observed to decrease during monitoring typically indicating a repression of expression mediated by the selected transcription control elements. Expression of the reporter sequence may be monitored under a variety of conditions, including, but not limited to, different stages of development of a transgenic embryo, fetus, neonate, or animal; the presence or absence of a analyte; changing environmental factors; and modified genetic backgrounds. Expression may be monitored before, during, and/or after treatment or change of conditions, as well as, such monitoring may be carried out in a single animal or multiple animals. For example, expression mediated by a transcription control element of interest may be monitored in an animal before, during, and after treatment with a selected analyte in order to observe the effect of the analyte on gene expression mediated by the selected transcription control element. Alternatively, control animals may be used for comparison where, for example, the effect of a selected analyte on expression mediated by a selected transcription control element is monitored in groups of treated and untreated animals. Monitoring may include administration of a substrate for the reporter to an animal or cell.
In preferred embodiments of the present invention the sequences encoding light generating protein are sequences encoding a luciferase.
The present invention also includes methods for monitoring expression mediated by transcription control elements, derived from an angiogenesis-related gene locus, in a cell. The methods comprises monitoring the expression of a reporter sequence in a cell, where the cell comprises an expression cassette having a transcription control element derived from an angiogenesis-related gene. Such transcription control elements include, but are not limited to, a VEGFR-2-locus-derived sequence, a VEGF-locus-derived sequence, and/or a TIE2-locus-derived sequence. Some exemplary isolated polynucleotide sequences comprising transcription control elements are described above and herein. Expression of the reporter sequence may be observed to increase during monitoring typically indicating induction mediated by the selected transcription control elements. Alternately, expression of the reporter sequence may be observed to decrease during monitoring typically indicating a repression of expression mediated by the selected transcription control elements. Expression of the reporter sequence may be monitored under a variety of conditions, including, but not limited to, cells derived from animals at different stages of development (for example from a transgenic embryo, fetus, neonate, or animal); cells derived from selected tissue types or organs; the presence or absence of a analyte; changing environmental factors; and modified genetic backgrounds. Expression may be monitored before, during, and/or after treatment or change of conditions. Such monitoring may be carried out in a high-throughput screening format. For example, expression mediated by a transcription control element of interest may be monitored employing a multi-well plate where the cells are treated with different concentrations of analyte and/or combinations of analytes. Monitoring can be carried out before, during, and after treatment with a selected analyte in order to observe the effect of the analyte on gene expression mediated by the selected transcription control element. Alternatively, control cells may be used for comparison where, for example, the effect of a selected analyte on expression mediated by a selected transcription control element is monitored in groups of treated and untreated cells. Monitoring may include administration of a substrate corresponding to the reporter that is employed. A variety of cell types may be employed in the practice of the present invention. Expression cassettes of the present invention may be introduced into a variety of cell-types homologous or heterologous with the source cell of the transcription control element. For example, transcription control elements derived from a mouse gene locus may be introduced into mouse cells, other rodent cells, human cells, avian cells, insect cells, procaryotic and/or other eucaryotic cell types. Cells may, for example, be primary cells, cultured cells, tumor cells, cell-lines, etc.
Particular analytes of interest for use in the methods of the present invention are analytes that are being screened for their effects on expression mediated by transcription control elements derived from angiogenesis-related gene loci.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts PCR conditions for genomic screening for promoters useful in exemplary expression constructs of the present invention.
FIGS. 2A–C depict the nucleotide sequence of the 6 kb promoter region of the VEGF mouse gene (SEQ ID NO:44)
FIGS. 3A–C depict the nucleotide sequence of the 4.5 kb promoter region of the VEGFR-2 mouse gene (SEQ ID NO:32).
FIG. 4 depicts the nucleotide sequence of a VEGFR-2 enhancer region (SEQ ID NO:35).
FIG. 5 is a schematic depicting engineering of the pGL3B2-KPN construct. PGL3B (Promega, Madison, Wis.) contains the yellow-green luciferase gene (Luc-YG). The construct contains a 4.5 kb fragment of the VEGFR-2 promoter and a 0.5 kb fragment of the VEGFR-2 enhancer.
FIGS. 6A–D depicts the nucleotide sequence of the 7.1 kb promoter region of the Tie2 mouse gene (SEQ ID NO:40).
FIG. 7 depicts the nucleotide sequence of a 1.7 kb enhancer region of Tie2 (SEQ ID NO:41).
FIG. 8 is a schematic depicting construction of the pGL3B2-TPN vector construct. See Example 3B below. The Tie2 promoter is cloned into the polylinkers upstream of the Luc-YG gene.
FIG. 9 is a schematic depicting construction of the pTK53 vector. Polynucleotides encoding mouse phosphoglycerate kinase 1 promoter (PGK-P), neomycin (Neo) and thymidine kinase (TK) and 5′ and 3′ linkers are introduced into a pKS backbone to produce the vector designated pTK53.
FIG. 10 is schematic depicting construction of the pTK-LucR and pTK-LucYG vectors. For pTK-LucR, a polynucleotide encoding LucR is introduced into pTK53. Thus, the pTK-LucR construct contains PGK-P, a neomycin (Neor) gene, a thymidine kinase (TK) gene and sequence encoding red luciferase (Luc-R). For pTK-LucYG, a polynucleotide encoding LucYG is introduced into pTK53. Thus, the pTK-LucYG construct contains PGK-P, a neomycin (Neor) gene, a thymidine kinase (TK) gene and a sequence encoding yellow-green luciferase (Luc-YG).
FIG. 11 is a schematic depicting the intermediate vector constructs pTKLR-Vn and pTKLG-Vn. Sequences homologous to the vitronectin gene are inserted into pTK-LucR and pTK-LucYG such that they flank the Neor gene and the Luc-R coding sequence. The pTKLR-Vn construct contains PGK-P, a neomycin (Neor) gene, a thymidine kinase (TK) gene, a sequence encoding red luciferase (Luc-R), a 1.63 kb 3′ end vitronectin gene fragment and a 2.35 kb 5′ end vitronectin gene (see Example 4B(1)(a) below). The pTKLG-Vn is identical except that it contains a sequence encoding yellow-green luciferase (Luc-YG) in place of the sequence encoding red luciferase. See Example 4B(1)(b) below.
FIG. 12 is a schematic depicting the intermediate vector construct pTKLG-FosB. FosB gene sequences (SEQ ID NO:43) are inserted into pTK-LucYG such that they flank the Neor gene and the Luc-YG coding sequence. See Example 4B(2) below.
FIG. 13 is a schematic depicting the vectors pTKLR-Vn-VP and pTKLG-Vn-VP. A 7.8 kb VEGF promoter sequence (see Example 1) was cloned into the PacI and NotI sites in the polylinker regions of the pTKLR-Vn and pTKLG-Vn intermediate vector constructs, respectively. See Example 4C(1) below.
FIG. 14 is a schematic depicting the vector constructs pTKLG-Fos-KPN and pTKLG-Vn-KPN. pTKLG-Fos-KPN was constructed using pGL3B2-KPN (FIG. 5) and pTKLG-Fos (FIG. 12). See Example 4C(2) below. The vector construct pTKLG-Vn-KPN was constructed using pGL3B2-KPN (FIG. 5) and pTKLG-Vn (FIG. 11). See Example 4C(2) below.
FIG. 15 is a schematic depicting engineering of the pTKLG-Fos-TPN construct using pGL3B2-TPN (FIG. 8) and pTKLG-Fos (FIG. 12). See Example 4C(3) below.
FIGS. 16A and 16B are graphical representations of transfection experiments performed to study the effect of neoplasticity and angiogenesis inhibitors on VEGFR2 (FIG. 16A) and Tie2 (FIG. 16B) promoter expression in endothelial cells. See Example 5 and Example 6 below.
FIGS. 17A through 17D present graphical representation of the results of bioluminescent imaging of VEGFR-2 transgenic mice from four founders over the course of 1–6 week development period (FIG. 17A, transgenic KC; FIG. 17B, transgenic KJ; FIG. 17C, transgenic KG; and FIG. 17D, transgenic KA). The figures show development dependent reduction of luciferase signal in VEGFR2-luc transgenic mice. See Example 7 below.
FIGS. 18A through 18D present graphical representation of the results of bioluminescent imaging of 2 female VEGFR-2 KA transgenic mice over the course of several estrus cycles (FIGS. 18A and 18B, mouse 1 and 2 dorsal views, respectively, and FIGS. 18C and 18D, mouse 1 and 2 ventral views, respectively). See Example 8 below.
FIGS. 19A through 19D present graphical representation of the results of bioluminescent imaging of a female VEGFR-2 KA transgenic mouse treated with estrogen antagonist ICI 182 780 over the course of several estrus cycles (FIGS. 19A and 19B, dorsal and ventral views, respectively, before treatment; FIGS. 19C and 19D, ventral and dorsal views, respectively, after treatment).). See Example 9 below.
FIG. 20 is a graphical representation of the results of bioluminescent imaging of a VEGFR-2 transgenic mouse (founder KC) implanted with growth factor-containing matrigel and a control mouse. See Example 10 below.
FIG. 21 is a graphical representation of the results of bioluminescent imaging of C57BL/6J-Tyrc-2J/+ mice implanted with LL/w cells stably transfected with the VEGF-Luc construct pTKLG-Vn-VP. See Example 11 below.



MODES FOR CARRYING OUT THE INVENTION
Throughout this application, various publications, patents, and published patent applications are referred to by an identifying citation. The disclosures of these publications, patents, and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, (F. M. Ausubel et al. eds., 1987); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR 2: A PRACTICAL APPROACH (M. J. McPherson, B. D. Hames and G. R. Taylor eds., 1995) and ANIMAL CELL CULTURE (R. I. Freshney. Ed., 1987).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “an antigen” includes a mixture of two or more such agents.
1. Definitions
As used herein, certain terms will have specific meanings.
The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably to and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, 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 is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
A “coding sequence” or a sequence which “encodes” a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in vivo when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of the coding sequence are typically determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence. Other “control elements” may also be associated with a coding sequence. A DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired polypeptide coding sequence. “Encoded by” refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed are polypeptide sequences, which are immunologically identifiable with a polypeptide encoded by the sequence.
A “transcription factor” as used herein typically refers to a protein (or polypeptide) which affects the transcription, and accordingly the expression, of a specified gene. A transcription factor may refer to a single polypeptide transcription factor, one or more polypeptides acting sequentially or in concert, or a complex of polypeptides.
Typical “control elements” include, but are not limited to, transcription promoters, transcription enhancer elements, cis-acting transcription regulating elements (transcription regulators, e.g., a cis-acting element that affects the transcription of a gene, for example, a region of a promoter with which a transcription factor interacts to induce expression of a gene), transcription initiation signals (e.g., TATA box), basal promoters, transcription termination signals, as well as polyadenylation sequences (located 3′ to the translation stop codon), sequences for optimization of initiation of translation (located 5′ to the coding sequence), translation enhancing sequences, and translation termination sequences. Transcription promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters.
“Expression enhancing sequences,” also referred to as “enhancer sequences” or “enhancers,” typically refer to control elements that improve transcription or translation of a polynucleotide relative to the expression level in the absence of such control elements (for example, promoters, promoter enhancers, enhancer elements, and translational enhancers (e.g., Shine and Delagamo sequences)).
The term “modulation” as used herein refers to both inhibition, including partial inhibition, as well as stimulation. Thus, for example, a compound that modulates expression of a reporter sequence may either inhibit that expression, either partially or completely, or stimulate expression of the sequence.
“Purified polynucleotide” refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
A “heterologous sequence” as used herein is typically refers to either (i) a nucleic acid sequence that is not normally found in the cell or organism of interest, or (ii) a nucleic acid sequence introduced at a genomic site wherein the nucleic acid sequence does not normally occur in nature at that site. For example, a DNA sequence encoding a polypeptide can be obtained from yeast and introduced into a bacterial cell. In this case the yeast DNA sequence is “heterologous” to the native DNA of the bacterial cell. Alternatively, a promoter sequence from a Tie2 gene can be introduced into the genomic location of a fosB gene. In this case the Tie2 promoter sequence is “heterologous” to the native fosB genomic sequence.
A “polypeptide” is used in it broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The subunits may be linked by peptide bonds or by other bonds, for example ester, ether, etc. As used herein, the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is typically called a polypeptide or a protein.
“Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter that is operably linked to a coding sequence (e.g., a reporter expression cassette) is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
“Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. “Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting procaryotic microorganisms or eucaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.
An “isolated polynucleotide” molecule is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith.
Techniques for determining nucleic acid and amino acid “sequence identity” also are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482–489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353–358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745–6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). A preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects “sequence identity.” Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the following internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST. When claiming sequences relative to sequences of the present invention, the range of desired degrees of sequence identity is approximately 80% to 100% and integer values therebetween. Typically the percent identities between the disclosed sequences and the claimed sequences are at least 80–85%, preferably 85–90%, more preferably 90–95%, and most preferably 98–100% sequence identity to the reference sequence (i.e., the sequences of the present invention).
Alternatively, the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two DNA, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 80–85%, preferably 85–90%, more preferably 90–95%, and most preferably 98–100% sequence identity to the reference sequence over a defined length of the molecules, as determined using the methods above. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
Two nucleic acid fragments are considered to “selectively hybridize” as described herein. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence “selectively hybridize,” or bind, to each other to form a hybrid molecule. A nucleic acid molecule that is capable of hybridizing selectively to a target sequence under “moderately stringent” typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10–14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10–14 nucleotides in length having a sequence identity of greater than about 90–95% with the sequence of the selected nucleic acid probe. Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions. The selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
A “vector” is capable of transferring gene sequences to target cells. Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors.
“Nucleic acid expression vector” or “expression cassette” refers to an assembly that is capable of directing the expression of a sequence or gene of interest. The nucleic acid expression vector includes a promoter that is operably linked to the sequences or gene(s) of interest. Other control elements may be present as well. Expression cassettes described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include a bacterial origin of replication, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a M13 origin of replication), a multiple cloning site, and a “mammalian” origin of replication (e.g., a SV40 or adenovirus origin of replication).
An “expression cassette” comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest. Such cassettes can be constructed into a “vector,” “vector construct,” “expression vector,” or “gene transfer vector,” in order to transfer the expression cassette into target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.
A variety of “reporter genes” also referred to as “reporter sequences” and “marker sequences,” i.e., genes or sequences the expression of which indicates the expression of polynucleotide sequences of interest to which the reporter gene or sequence is operably linked. Preferred are those reporter sequences that produce a protein product that is easily measured, preferably in a routine assay. Suitable reporter genes include, but are not limited to chloramphenicol acetyl transferase (CAT), light generating proteins (e.g., luciferase), and beta-galactosidase. Convenient assays include, but are not limited to calorimetric, fluorimetric and enzymatic assays. In one aspect, reporter genes may be employed that are expressed within the cell and whose extracellular products are directly measured in the intracellular medium, or in an extract of the intracellular medium of a cultured cell line. This provides advantages over using a reporter gene whose product is secreted, since the rate and efficiency of the secretion introduces additional variables that may complicate interpretation of the assay. In a preferred embodiment, the reporter gene is a light generating protein. When using the light generating reporter proteins described herein, expression can be evaluated accurately and non-invasively as described above (see, for example, Contag, P. R., et al., (1998) Nature Med. 4:245–7; Contag, C. H., et al., (1997) Photochem Photobiol. 66:523–31; Contag, C. H., et al., (1995) Mol Microbiol. 18:593–603).
“Luciferase,” unless stated otherwise, includes procaryotic and eucaryotic luciferases, as well as variants possessing varied or altered optical properties, such as luciferases that produce different colors of light (e.g., Kajiyama, N., and Nakano, E., Protein Engineering 4(6):691–693 (1991)).
“Light-generating” is defined as capable of generating light through a chemical reaction or through the absorption of radiation.
A “light generating protein” or “light-emitting protein” is a protein capable of generating light in the visible spectrum (between approximately 350 nm and 800 nm). Examples include bioluminescent proteins such as luciferases, e.g., bacterial and firefly luciferases, as well as fluorescent proteins such as green fluorescent protein (GFP).
“Light” is defined herein, unless stated otherwise, as electromagnetic radiation having a wavelength of between about 300 nm and about 1100 nm.
“Animal” as used herein typically refers to a non-human mammal, including, without limitation, farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.
A “transgenic animal” refers to a genetically engineered animal or offspring of genetically engineered animals. A transgenic animal usually contains material from at least one unrelated organism, such as from a virus, plant, or other animal. The “non-human animals” of the invention include vertebrates such as rodents, non-human primates, sheep, dogs, cows, amphibians, birds, fish, insects, reptiles, etc. The term “chimeric animal” is used to refer to animals in which the heterologous gene is found, or in which the heterologous gene is expressed in some but not all cells of the animal.
“Analyte” as used herein refers to any compound or substance whose effects (e.g., induction or repression of a specific promoter) can be evaluated using the test animals and methods of the present invention. Such analytes include, but are not limited to, chemical compounds, pharmaceutical compounds, polypeptides, peptides, polynucleotides, and polynucleotide analogs. Many organizations (e.g., the National Institutes of Health, pharmaceutical and chemical corporations) have large libraries of chemical or biological compounds from natural or synthetic processes, or fermentation broths or extracts. Such compounds/analytes can be employed in the practice of the present invention.
As used herein, the term “positive selection marker” refers to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neor) gene are resistant to the compound G418. Cells that do not carry the Neor gene marker are killed by G418. Other positive selection markers will be known to those of skill in the art. Typically, positive selection markers encode products that can be readily assayed. Thus, positive selection markers can be used to determine whether a particular DNA construct has been introduced into a cell, organ or tissue.
“Negative selection marker” refers to gene encoding a product that can be used to selectively kill and/or inhibit growth of cells under certain conditions. Non-limiting examples of negative selection inserts include a herpes simplex virus (HSV)-thymidine kinase (TK) gene. Cells containing an active HSV-TK gene are incapable of growing in the presence of gangcylovir or similar agents. Thus, depending on the substrate, some gene products can act as either positive or negative selection markers.
The term “homologous recombination” refers to the exchange of DNA fragments between two DNA molecules or chromatids at the site of essentially identical nucleotide sequences. It is understood that substantially homologous sequences can accommodate insertions, deletions, and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align (see, above).
A “knock-out” mutation refers to partial or complete loss of expression of at least a portion the target gene. Examples of knock-out mutations include, but are not limited to, gene-replacement by heterologous sequences, gene disruption by heterologous sequences, and deletion of essential elements of the gene (e.g., promoter region, portions of a coding sequence). A “knock-out” mutation is typically identified by the phenotype generated by the mutation.
A “gene” as used in the context of the present invention is a sequence of nucleotides in a genetic nucleic acid (chromosome, plasmid, etc.) with which a genetic function is associated. A gene is a hereditary unit, for example of an organism, comprising a polynucleotide sequence (e.g., a DNA sequence for mammals) that occupies a specific physical location (a “locus”, “gene locus” or “genetic locus”) within the genome of an organism. A gene can encode an expressed product, such as a polypeptide or a polynucleotide (e.g., tRNA). Alternatively, a gene may define a genomic location for a particular event/function, such as the binding of proteins and/or nucleic acids (e.g., phage attachment sites), wherein the gene does not encode an expressed product. Typically, a gene includes coding sequences, such as, polypeptide encoding sequences, and non-coding sequences, such as, transcription control elements (e.g., promoter sequences), poly-adenlyation sequences, transcriptional regulatory sequences (e.g., enhancer sequences). Many eucaryotic genes have “exons” (coding sequences) interrupted by “introns” (non-coding sequences). In certain cases, a gene may share sequences with another gene(s) (e.g., overlapping genes).
The “native sequence” or “wild-type sequence” of a gene is the polynucleotide sequence that comprises the genetic locus corresponding to the gene, e.g., all regulatory and open-reading frame coding sequences required for expression of a completely functional gene product as they are present in the wild-type genome of an organism. The native sequence of a gene can include, for example, transcriptional promoter sequences, translation enhancing sequences, introns, exons, and poly-A processing signal sites. It is noted that in the general population, wild-type genes may include multiple prevalent versions that contain alterations in sequence relative to each other and yet do not cause a discernible pathological effect. These variations are designated “polymorphisms” or “allelic variations.”
By “replacement sequence” is meant a polynucleotide sequence that is substituted for at least a portion of the native or wild-type sequence of a gene.
“Linear vector” or “linearized vector,” as used herein, is a vector having two ends. For example, circular vectors, such as plasmids, can be linearized by digestion with a restriction endonuclease that cuts at a single site in the plasmid. Preferably, the expression vectors described herein are linearized such that the ends are not within the sequences of interest.
2.0.0 Detailed Description of the Invention
Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or method parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.
2.1.0 General Overview
In one aspect, the present invention relates to novel transcription control elements derived from mouse angiogenesis-related genes, expression cassettes which include those control elements, vector constructs, cells and transgenic animals containing the expression cassettes, and methods of using the cells and transgenic animals containing the expression cassettes, for example, as modeling, screening and/or test systems. Methods of using the control elements, expression cassettes, cells, and transgenic animals of the present invention include, but are not limited to, studies involving angiogenesis, as well as tumor growth and other disease conditions, and methods for screening angiogenesis-modulating. Exemplary transcription control elements useful in the practice of the present invention include those derived from mouse VEGF locus, mouse VEGFR-2 locus and mouse TIE2 locus.
In one embodiment, the present invention relates to (1) novel transcription control elements (e.g., promoters) derived from the mouse VEGF gene locus, the mouse VEGFR-2 receptor gene locus, and the mouse TIE2 gene locus, (2) expression cassettes comprising such transcription control elements operatively linked to genes encoding a gene product, such as, a reporter, a protein, polypeptide, hormone, ribozyme, or antisense RNA, (3) recombinant cells comprising such expression cassettes, (4) methods of screening for therapeutic drugs using such cells (e.g., screening for compounds that modulate angiogenesis mediated by the novel transcription control elements of the present invention), (5) transgenic animals comprising the aforementioned novel transcription control elements, expression cassettes and vector constructs, (6) methods of monitoring angiogenesis using such animals, and (7) methods of screening for therapeutic drugs using such animals (e.g., screening for compounds that modulated angiogenesis mediated by the novel transcription control elements of the present invention).
A variety of transcription control elements are useful in the practice of the present invention, for example, transcription control elements derived from genes or gene locii associated with tumorigenesis or angiogenesis. Thus, an exemplary transcription control element can be one that is associated with proteins induced during tumorigenesis, for instance in the presence of tumor generating compounds or of tumors themselves. In this way, expression of the reporter sequence is induced in the transgenic animals of the present invention when, for example, tumors are present, and progression of the tumor can be evaluated by non-invasive imaging methods using the whole animal. Another exemplary transcription control element (e.g., promoter) is one that is derived from a gene or locus associated with angiogenesis. Because the transcription control element is linked to a reporter such as luciferase, non-invasive monitoring of the progression of angiogenesis is possible. Various forms of the different embodiments of the invention, described herein, may be combined.
Non-invasive imaging and/or detecting of light-emitting conjugates in mammalian subjects was described in U.S. Pat. No. 5,650,135, by Contag, et al., issued 22 Jul. 1997, and herein incorporated by reference. This imaging technology can be used in the practice of the present invention in view of the teachings of the present specification. In the imaging method, the conjugates contain a biocompatible entity and a light-generating moiety. Biocompatible entities include, but are not limited to, small molecules such as cyclic organic molecules; macromolecules such as proteins; microorganisms such as viruses, bacteria, yeast and fungi; eucaryotic cells; all types of pathogens and pathogenic substances; and particles such as beads and liposomes. In another aspect, biocompatible entities may be all or some of the cells that constitute the mammalian subject being imaged, for example, cells carrying the expression cassettes of the present invention expressing a reporter sequence.
Light-emitting capability is conferred on the biocompatible entities by the conjugation of a light-generating moiety. Such moieties include fluorescent molecules, fluorescent proteins, enzymatic reactions which give off photons, and luminescent substances, such as bioluminescent proteins. In the context of the present invention, light emitting capability is typically conferred on target cells by having at least one copy of a light-generating protein, e.g., a luciferase, present. In preferred embodiments, luciferase is operably linked to appropriate control elements that can facilitate expression of a polypeptide having luciferase activity. Substrates of luciferase can be endogenous to the cell or applied to the cell or system (e.g., injection into a transgenic mouse, having cells carrying a luciferase construct, of a suitable substrate for the luciferase, for example, luciferin). The conjugation may involve a chemical coupling step, genetic engineering of a fusion protein, or the transformation of a cell, microorganism or animal to express a light-generating protein.
2.2.0 Promoters
The expression cassettes, vectors, cells and transgenic animals described herein contain a sequence encoding a detectable gene product, e.g., a luciferase gene, operably linked to a transcription control element, e.g., a promoter. The promoter may be from the same species as the transgenic animal (e.g., mouse promoter used in construct to make transgenic mouse) or from a different species (e.g., human promoter used in construct to make transgenic mouse). The promoter can be derived from any gene of interest. In one embodiment of the present invention, the promoter is derived from a gene whose expression is induced during angiogenesis, for example pathogenic angiogenesis like tumor development. Thus, when a tumor begins to develop in a transgenic animal carrying a vector construct of the present invention, the promoter is induced and the animal expresses luciferase, which can then be monitored in vivo.
Exemplary transcription control elements (e.g., promoters) for use in the present invention include, but are not limited to, promoters derived from the angiogenesis-related genes and gene families described below. Those genes and gene families include, but are not limited to the following mouse genes: vascular endothelial growth factor (VEGF); VEGFR-2, also known as Flk 1; and Tie2, also known as Tek.
VEGF is a specific mitogen for EC in vitro and a potent angiogenic factor in vivo. In a tumorigenesis study, it was shown that VEGF was critical for the initial subcutaneous growth of T-47D breast carcinoma cells transplanted into nude mice, whereas other angiogenic factors, such as, bFGF can compensate for the loss of VEGF after the tumors have reached a certain size (Yoshiji, H., et al.,1997 Cancer Research 57: 3924–28). VEGF is a major mediator of aberrant EC proliferation and vascular permeability in a variety of human pathologic situation, such as, tumor angiogenesis, diabetic retinopathy and rheumatoid arthritis (Benjamin LE, et al.,1997 PNAS 94: 8761–66; Soker, S., et al.,1998 Cell 92: 735–745). VEGF is synthesized by tumor cells in vivo and accumulates in nearby blood vessels. Because leaky tumor vessels initiate a cascade of events, which include plasma extravasation and which lead ultimately to angiogenesis and tumor stroma formation, VEGF plays a pivotal role in promoting tumor growth (Dvorak, H. F., et al., 1991 J Exp Med 174:1275–8). VEGF expression was upregulated by hypoxia (Shweiki, D., et al., 1992 Nature 359:843–5). VEGF is also upregulated by overexpression of v-Src oncogene (Mukhopadhyay. D., et al.,1995 Cancer Res. 15: 6161–5), c-SRC (Mukhopadhyay, D., et al., 1995 Nature 375: 577–81), and mutant ras oncogene (Plate, K. H., et al., 1992 Nature 359: 845–8). The tumor suppressor p53 downregulates VEGF expression (Mukhopadhyay. D., et al.,1995 Cancer Res. 15: 6161–5).
A number of cytokines and growth factors, including PGF and TPA (Grugel, S., et al., 1995 J. Biological Chem. 270: 25915–9), EGF, TGF-b, IL-1, IL-6 induce VEGF mRNA expression in certain type of cells (Ferrara, N., et al., 1997 Endocr. Rev. 18: 4–25). Kaposi's sarcoma-associated herpes virus (KSHV) encoded a G-protein-coupled receptor, a homologue of IL-8 receptor, can activate JNK/SAPK and p38MAPK and increase VEGF production, thus causing cell transformation and tumorigenicity (Bais, C., et al., Nature 1998 391:86–9). VEGF overexpression in skin of transgenic mice induces angiogenesis, vascular hyperpermeability and accelerated tumor development (Larcher, F., et al., Oncogene (1998) 17:303–11).
Further angiogenesis-related genes include but are not limited to the following.
VEGF-B (cDNA sequences available on databases) is a mitogen for EC and may be involved in angiogenesis in muscle and heart (Olofsson, B., et al., 1996 Proc Natl Acad Sci USA 93:2576–81). Shown in vitro, binding of VEGF-B to its receptor VEGFR-1 leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration (Olofsson, B., et al., 1998 Proc Natl Acad Sci USA 95:11709–14).
VEGF-C (see, e.g., U.S. Pat. No. 5,916,763 and Shima et al., supra) may regulate angiogenesis of lymphatic vasculature, as suggested by the pattern of VEGF-C expression in mouse embryos (Kukk, E., et al., 1996 Development 122: 3829–37). Although VEGF-C is also a ligand for VEGFR-2, the functional significance of this potential interaction is unknown. Overexpression of VEGF-C in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement, suggesting the major function of VEGF-C is through VEGFR-3 rather than VEGFR-2 (Jeltsch M, et al., 1997 Science 276:1423–5). Shown by the CAM assay, VEGF and VEGF-C are specific angiogenic and lymphangiogenic growth factors, respectively (Oh, S. J., et al., (1997) Devel. Biol. 188: 96–109). VEGF-C overexpression in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement (Jeltsch M, et al., 1997 Science 276:1423–5).
VEGF-D (cDNA sequences available on databases) is a mitogen for EC. Given that VEGF-D can also activate VEGFR-3. it is possible that VEGF-D could be involved in the regulation of growth and/or differentiation of lymphatic endothelium (Achen, M. G., et al., 1998 Proc Natl Acad Sci USA 95: 548–53). VEGF-D is induced by transcription factor c-Fos in mouse (Orlandini, M., 1996 PNAS 93: 11675–80).
VEGFR-1 signaling pathway may regulate normal endothelial cell-cell or cell matrix interactions during vascular development, as suggested by the knockout study (Fong, G. H., et al., 1995 Nature 376: 65–69). Although VEGFR-1 has a higher affinity to VEGF than VEGFR-2, it does not transduce the mitogenic signals of VEGF in ECs (Soker, S., et al.,1998 Cell 92: 735–745). VEGFR-2 (see, e.g., Rönicke et al., Patterson et al., Kappel et al. (1999), supra) appears to be the major transducer of VEGF signals in EC that result in chemotaxis, mitogenicity and gross morphological changes in target cells (Soker, S., et al.,1998 Cell 92: 735–745). The cloning and sequencing of the 4.5 kb VEGFR-2 promoter region is described herein (see Example 2 below).
VEGFR-3 has an essential role in the development of the embryonic cardiovascular system before the emergence of lymphatic vessels, as shown by the knockout study (Dumont, D. J., et al., 1998 Science 282: 946–949). Neuropillin-1 (see, e.g., Soker et al. (1998) Cell 92:735–745) is a receptor for VEGF165. It can enhance the binding of VEGF165 to VEGFR-2 and VEGF165 mediated chemotaxis (Soker, S., et al.,1998 Cell 92: 735–745). Neuropillin1 overexpression in transgenic mice resulted in embryonic lethality. The embryos possessed excess capillaries and blood vessels. Dilated vessels and hemorrhage were also observed (Kitsukawa, T., et al., 1995 Development 121: 4309–18).
Thus, in one aspect the present invention relates to the isolation and characterization of the mouse VEFG and VEGFR-2 promoters. This section describes some information related to the VEFG and VEGFR gene families. Alternative names for some of these genes are as follows: VEGF (vascular endothelial growth factor)is also named VPF (vascular permeability factor); VEGFR-1 is also named FLT1; VEGFR-2 is also named KDR/FLK1; and VEGFR-3 is also named FLT4.
VEGF is a homodimeric 45 kDa (monomer 23 kDa) protein. VEGF has five isoforms of which VEGF165 and VEGF121 are the most abundant. Both are ligands for VEGFR-2 as well as VEGFR-1 (Soker, S., et al., JBC 271:5761–67, 1996). VEGF165 is the only VEGF isoform that binds to Neuropillin-1 (Soker, S., et al., Cell 92:735–745, 1998). VEGF is extremely unstable—its half life in circulation is only 3 minutes (Ferrara, N., et al., Nature 380:439–442, 1996; Ferrara, N., et al., Endocr Rev 18:4–25, 1997).
VEGF-B is 43% (aa) identical to VEGF and exists as a homodimer. It can also form heterodimers with VEGF (Olofsson, B., et al., Proc Natl Acad Sci USA 93:2576–81, 1996). VEGF-B is a ligand for VEGFR-1 (Olofsson, B., et al., Proc Natl Acad Sci USA 95:11709–14, 1998).
VEGF-C is 30% (aa) identical to VEGF. The mature VEGF-C is 23 kDa, the precursor protein is 35.8 kDa. VEGF-C is a ligand for VEGFR-3 as well as VEGFR-2. It induces autophosphorylation of both receptors (Joukov, V., et al., EMBO J 15:290–298, 1996).
VEGF-D is 31% (aa) identical to VEGF165 and 48% (aa) identical to VEGF-B. The mature VEGF-D is approximately 22 kDa. VEGF-D is a ligand for VEGFR-3 as well as VEGFR-2. It induces autophosphorylation of both receptors (Achen, M. G., et al., 1998 Proc Natl Acad Sci USA 95:548–53, 1998).
PIGF is 46% identical (aa) to VEGF (Maglione, D., et al., Proc Natl Acad Sci 88:9267–71, 1991) and can form heterodimers with VEGF ((Disalvo, J., et al., JBC 270:7717–23, 1995).
VEGFR-1 is an approximately 180 kDa tyrosine kinase receptor for VEGF-B (Olofsson, B., et al., Proc Natl Acad Sci USA 95:11709–14, 1998) and VEGF (de Vries, C., et al., Science 255:989–91, 1992) and PIGF (Park, J. E., et al., J Biol Chem 269:25646–54, 1994).
VEGFR-2 is an approximately 200 kDa tyrosine kinase receptor for VEGF (Termini, B I, et al., Oncogene September 6(9):1677–83, 1991), VEGF-C (Joukov, V., et al., EMBO J 15:290–298, 1996), and VEGF-D (Achen, M. G., et al., 1998 Proc Natl Acad Sci USA 95:548–53, 1998).
VEGFR-3 is a tyrosine kinase receptor (Pajusola, K., et al., Cancer Res 32:5738–43, 1992) on lymphatic EC for VEGF-C (Dumont, D. J., et al., Science 282:946–949, 1998) and VEGF-D (Achen, M. G., et al., 1998 Proc Natl Acad Sci USA 95:548–53, 1998). VEGFR-3 has a processed mature form of about 125 kDa, and an unprocessed form of about 195 kDa (Achen, M. G., et al., 1998 Proc Natl Acad Sci USA 95:548–53, 1998).
Neuropillin-1 is an approximately 130 kDa receptor tyrosine kinase. It binds VEGF165, but not VEGF121 (Soker, S., et al., Cell 92:735–745, 1998).
Expression of many of these genes has been evaluated in adults. A summary of information relating to expression follows here.
VEGF has an approximately 3.7 kb transcript. It is expressed in multiple human tissues, including heart, skeletal muscle and prostate. In mouse, VEGF is mainly expressed in heart, lung and kidney. The remaining human or mouse tissues, including brain and testis, do not express detectable or significant level of VEGF (Olofsson, B., et al., Proc Natl Acad Sci USA 93:2576–81, 1996). In another study, it was shown that VEGF is highly expressed in epithelial cells of lung alveoli, renal glomeruli and adrenal cortex and in cardiac myocytes (Berse, B., MCB 3:211–20, 1992).
VEGF-B has an approximately 1.4 kb transcript. It is expressed in a majority of human and mouse tissues. In human, VEGF-B is most prominently expressed in heart, skeletal muscle, pancreas, brain and prostate. In mouse, VEGF-B is mostly expressed heart, skeletal muscle, brain and kidney. Liver does not appear to express a significant level of VEGF-B in either humans or mice. VEGF-B and VEGF are coexpressed in many human tissues, such as heart, skeletal muscle, pancreas and prostate. In general, VEGF-B is more abundantly expressed than VEGF. VEGF-B can act as an endothelial cell growth factor (Olofsson, B., et al., Proc Natl Acad Sci USA 93:2576–81, 1996).
VEGF-C has an approximately 2.4 kb transcript that is expressed in multiple human tissues, most prominently in heart, skeletal muscle, placenta, ovary, small intestine, pancreas and prostate. Several tissues, including brain and liver, do not appear to express detectable levels of VEGF-C (Joukov, V., et al., EMBO J 15:290–298, 1996).
VEGF-D has an approximately 2.3 kb transcript that is expressed in multiple human tissues, most prominently in heart, skeletal muscle, lung, colon and small intestine. Several tissues, including brain, liver, placenta, do not appear to express detectable levels of VEGF-D (Achen, M. G., et al., 1998 Proc Natl Acad Sci USA 95:548–53, 1998).
VEGFR-1 appears to be endothelial cell specific (Peters, K. G., et al., Proc Natl Acad Sci 90:8915–19, 1993). VEGFR-1 cDNA is approximately 7.7 kb and encodes a protein of 1338 aa. It was expressed in a variety of normal tissues of adult rat (Shibuya, M., et al., Oncogene 5:519–24, 1990). In a glioma model of tumor angiogenesis, both VEGFR-1 and VEGFR-2 are specifically expressed in EC's that have penetrated into the tumor, but are absent from ECs in the normal brain tissues. VEGF expression was detectable in glioma cells along necrotic edge (Plate, K. H., et al., Cancer Research 53:5822–27, 1993).
VEGFR-2 is expressed as an approximately 7 kb transcript (Terman, B. I., et al., Oncogene September 6 (9):1677–83, 1991) that appears to be endothelial cell specific. VEGFR-2 is expressed ubiquitously in many tissues, including heart, placenta, lung and kidney. The expression levels of VEGFR-2 are relatively low in these tissues compared with neuropillin expression. Brain does not appear to express detectable levels of VEGFR-2 (Soker, S., et al., Cell 92:735–745, 1998). In situ hybridization analysis revealed a specific association of VEGFR-2 with endothelial cells at all stages of mouse development. It is abundant in proliferating endothelial cells of vascular sprouts and branching vessels of embryonic and early postnatal brain, but were drastically reduced in adult brain, where proliferation has ceased (Millauer, B., Cell 72:835–46, 1993).
VEGFR-3 is expressed as approximately 5.8 kb and 4.5 kb mRNAs. Most fetal tissues expressed VEGFR-3, with spleen, brain intermediate zone, and lung showing the highest levels. It does not appear to be expressed in the endothelial cells of blood vessels (Pajusola, K., et al., Cancer Res 32:5738–43, 1992). During embryonic development, VEGFR-3 is expressed in blood vessels but become largely restricted to the lymphatic endothelium postnatally (Kaipainen, A., et al., Proc Natl Acad Sci USA 92: 3566–3570, 1995).
Neuropillin-1 is expressed in both endothelial cells and many types of tumor cells as an approximately 7 kb transcript. Most tissues express high level of Neuropillin-1, especially in heart and placenta. Skeletal muscle, pancreas, lung and kidney also express high level of Neuropillin-1. Brain does not appear to express detectable levels of Neuropillin-1 (Soker, S., et al., Cell 92:735–745, 1998).
Some functions of these genes have been evaluated and are as follows.
VEGF is a specific mitogen for EC in vitro and a potent angiogenic factor in vivo. In vitro, VEGF binds and induces autophosphorylation of VEGFR-2 and VEGFR-1, but the mitogenic response is mediated only through VEGFR-2 (Waltenberger, J., JBC 269:26988–95, 1994). VEGF functions as a survival factor for newly formed vessels during developmental neovascularization, possibly through mediating interaction of endothelial cells with underlying matrix, but is not required for maintenance of mature vessels (Benjamin, L. E., et al., Proc Natl Acad Sci 94:8761–66, 1997). In embryogenesis, VEGF and VEGFR-2 interaction induces the birth and proliferation of endothelial cells (Hanahan, D., Science 277:48–50, 1997). Binding of VEGF to VEGFR-1 elicits endothelial cell-cell interactions and capillary tube formation, a process that follows closely proliferation and migration of endothelial cells (Hanahan, D., Science 277:48–50, 1997). In a tumorigenesis study, it was shown that VEGF is critical for the initial growth of T-47D breast carcinoma cells transplanted into nude mice, whereas other angiogenic factors such as bFGF can compensate for the loss of VEGF after the tumors have reached a certain size (Yoshiji, H., et al., Cancer Research 57:3924–28, 1997). VEGF is a major mediator of aberrant endothelial cells (EC) proliferation and vascular permeability in a variety of human pathologic situation, such as tumor angiogenesis, diabetic retinopathy and rheumatoid arthritis (Benjamin, L. E., et al., Proc Natl Acad Sci 94:8761–66, 1997, Soker, S., et al., Cell 92:735–745, 1998). VEGF induces expression of plasminogen activator (PA), PA inhibitor 1 (PAI-1), MMP, and interstitial collagenase in ECs. These findings are consistent with the proangiogenic activities of VEGF. VEGF promotes expression of VCAM-1 and ICAM-1 in EC, thus may facilitate the adhesion of activated NK cells to EC. VEGF may promote monocyte chemotaxis (Pepper, M. S., et al., BBRC 181:902–906, 1991; Ferrara, N., et al., Endocr Rev 18:4–25, 1997). Tumors are believed to be the principal source of VEGF. A correlation has been observed between VEGF expression and vessel density in human breast tumors, renal cell carcinoma and colon cancer (Fong, T. A. T., et al., Cancer Res 59:99–106, 1999). VEGF and PGF expressions were significantly upregulated in 96% and 91% of hypervascular renal carcinoma tissues compared with adjacent normal kidney tissues (Takahashi, A., et al., Cancer Res 54:4233–7, 1994).
VEGF-B is a mitogen for EC and may be involved in angiogenesis in muscle and heart (Olofsson, B., et al., Proc Natl Acad Sci USA 93:2576–81, 1996). In vitro, binding of VEGF-B to its receptor VEGFR-1 leads to increased expression and activity of urokinase-type plasminogen activator and plasminogen activator inhibitor, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration (Olofsson, B., et al., Proc Natl Acad Sci USA 95:11709–14, 1998).
VEGF-C may regulate angiogenesis of lymphatic vasculature, as suggested by the pattern of VEGF-C expression in mouse embryos (Kukk, E., et al., Development 122:3829–37, 1996). Although VEGF-C is also a ligand for VEGFR-2, the functional significance of this potential interaction is unknown. Overexpression of VEGF-C in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement, suggesting the major function of VEGF-C is through VEGFR-3 rather than VEGFR-2 (Jeltsch, M., et al., Science 276:1423–5, 1997). Using the CAM assay, VEGF and VEGF-C were shown to be specific angiogenic and lymphangiogenic growth factors, respectively (Oh, S. J., et al., Del Biol 188:96–109, 1997).
VEGF-D is a mitogen for EC. VEGF-D can also activate VEGFR-3. It is possible that VEGF-D could be involved in the regulation of growth and/or differentiation of lymphatic endothelium (Achen, M. G., et al., 1998 Proc Natl Acad Sci USA 95:548–53, 1998).
PIGF can potentiate the action of low concentrations of VEGF in vitro and in vivo (Park, J. E., et al., J Biol Chem 269:25646–54, 1994).
VEGFR-1 signaling pathway may regulate normal endothelial cell-cell or cell-matrix interactions during vascular development, as suggested by a knockout study (Fong, G. H., et al., Nature 376:65–69, 1995). Although VEGFR-1 has a higher affinity to VEGF than VEGFR-2, it does not transduce the mitogenic signals of VEGF in ECs (Soker, S., et al., Cell 92:735–745, 1998).
VEGFR-2 appears to be the major transducer of VEGF signals in EC that result in chemotaxis, mitogenicity and gross morphological changes in target cells (Soker, S., et al., Cell 92:735–745, 1998).
VEGFR-3 has an essential role in the development of the embryonic cardiovascular system before the emergence of lymphatic vessels, as shown by a knockout study (Dumont, D. J., et al., Science 282:946–949, 1998).
Neuropillin-1 is a receptor for VEGF165. It can enhance the binding of VEGF165 to VEGFR-2 and VEGF165 mediated chemotaxis (Soker, S., et al., Cell 92:735–745, 1998).
Gene regulation of some of these genes has been investigated and is discussed herein below.
In situ hybridization demonstrated VEGF mRNA was present in transplanted tumor cells but not in tumor blood vessels, indicating that immunohistochemical labeling of tumor vessels with VEGF antibodies reflects uptake of VEGF, not endogenous synthesis. VEGF protein staining was evident in adjacent preexisting venules and small veins as early as 5 hours after tumor transplant and plateaued at maximally intense levels in newly induced tumor vessels by approximately 5 days. In contrast, vessels more than approximately 0.5 mm distant from tumors were not hyperpermeable and did not exhibit immunohistochemical staining for VEGF. Vessel staining disappeared within 24–48 h of tumor rejection. These studies indicate that VEGF is synthesized by tumor cells in vivo and accumulates in nearby blood vessels. Because leaky tumor vessels initiate a cascade of events, which include plasma extravasation and which lead ultimately to angiogenesis and tumor stroma formation, VEGF plays a pivotal role in promoting tumor growth (Dvorak, H. F., et al., J Exp Med 174:1275–8, 1991). In addition, it was shown that VEGF production by stromal cells could be stimulated by transplanted tumor cells (Fukumura, D., et al., Cell, 94:715–25, 1998). Fibroblasts cultured in vitro are highly activating for VEGF promoter function compared with fibroblasts in freshly isolated tumors, indicating the culture condition did not mimic the status of normal (unactivated) tissue in vivo (Fukumura, D., et al., Cell, 94:715–25, 1998). For example, C6 tumor spheroids (C6 is a cell line derived from a rat glial tumor—C6 cells aggregate and form small spheroids in culture) implanted into nude mice became neovascularized accompanied by a gradual reduction of VEGF expression (Shweiki, D., et al., Proc Natl Acad Sci 92:768–772, 1995). The VEGF promoter region bears many of the characteristics of a house-keeping gene (Tischer, E., JBC 266:11947–11954, 1991), hence it is likely that almost any cell type could serve as a source for VEGF upon hypoxic or ischemic demand (Fukumura, D., et al., Cell, 94:715–25, 1998).
VEGF expression was upregulated by hypoxia (Shweiki, D., et al., Nature 359:843–5, 1992), due to both increased transcriptional activation and stability of its mRNA (Ikeda, E., et al., JBC 270:19761–5, 1995). In a number of in vitro studies, it was shown that hypoxia upregulates VEGF expression through the activation of PI3K/Akt pathway (Mazure, N. M., et al., Blood 90:3322–31, 1997) and HIF-1 (an enhancer induced by hypoxia and bind to VEGF promoter region) (Forsythe, J. A., MCB 16:4604–13, 1996; Mazure, N. M., et al., Blood 90:3322–31, 1997). VEGF is also upregulated by overexpression of v-Src oncogene Mukhopadhyay, D., Cancer Res. 15:6161–5, 1995), c-SRC (Mukhopadhyay, D., et al., Nature 375:577–81, 1995), and mutant ras oncogene (Plate, K. H., Nature 359:845–8, 1992). The tumor suppressor p53 downregulates VEGF expression (Mukhopadhyay, D., Cancer Res. 15:6161–5, 1995). A number of cytokines and growth factors, including PGF, TPA (Grugel, S., et al., JBC 270:25915–9,1995), EGF, TGF-b, IL-1, and IL-6 induce VEGF mRNA expression in certain type of cells (Ferrara, N., et al., Endocr Rev 18:4–25, 1997). Kaposi's sarcoma-associated herpes virus (KSHV), which encodes a G-protein-coupled receptor—a homologue of IL-8 receptor, can activate JNK/SAPK and p38MAPK and increase VEGF production, thus causing cell transformation and tumorigenicity (Bais, C., Nature 391:86–9, 1998).
The growth of androgen-dependent Shionogi carcinoma in immunodeficient mice was regressed after the mice were castrated, accompanied by decrease in VEGF expression. Two weeks after castration, a second wave of angiogenesis and tumor growth begins with a concomitant increase in VEGF expression (Jain, R. K., Proc Natl Acad Sci USA 95:10820–5, 1998).
VEGF-D is induced by transcription factor c-fos in mouse (Orlandini, M. Proc Natl Acad Sci 93:11675–80, 1996).
Overexpression of some of these genes has been evaluated using different systems.
VEGF overexpression in skin of transgenic mice induces angiogenesis, vascular hyperpermeability and accelerated tumor development (Larcher, F., et al., Oncogene 17:303–11, 1998). Retina tissue-specific VEGF overexpression in transgenic mice causes intraretinal and subretinal neovascularization (Okamoto, N., et al., Am J Pathol 151:281–91, 1997). VEGF overexpression mediated by the Tet system promotes tumorigenesis of C6 glioma cells when transplanted into nude mice. The tumors become hypervascularized with abnormally large vessels, arising from excessive fusions. The tumors were less necrotic. After VEGF expression was shut off, regression of the tumors occurred due to detachment of endothelial cells from the walls of preformed vessels and their subsequent apoptosis. Vascular collapse further leads to hemorrhages and extensive tumor necrosis (Benjamin, L. E., et al., Proc Natl Acad Sci 94:8761–66, 1997). In human-VEGF-promoter-GFP transgenic mice, implantation of solid tumor induces specific GFP expression in stromal cells. Transgenic mice were mated with T-antigen mice (able to form spontaneous mammary tumors) to generate double transgenic mice, in which spontaneous mammary tumors were formed. Strong stromal, but not tumor, expression of GFP was observed (Fukumura, D., et al., Cell, 94:715–25, 1998). A CCD camera was used to monitor GFP expression. GFP half life was shown to be between about 1.2–1.5 days (Fukumura, D., et al., Cell, 94:715–25, 1998). The transgene was integrated into the IgG locus of the chromosome through DNA recombination (Fukumura, D., et al., Cell, 94:715–25, 1998). FVB derived VEGF-GFP transgenic mice were mated with wild-type C3H mice to create hybrid mice that can be served as hosts for C3H derived tumor lines (Fukumura, D., et al., Cell, 94:715–25, 1998).
VEGF-C overexpression in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement (Jeltsch, M., et al., Science 276:1423–5, 1997).
Neuropillin-1 overexpression in transgenic mice resulted in embryonic lethality. The embryos possessed excess capillaries and blood vessels. Dilated vessels and hemorrhage were also observed (Kitsukawa, T., et al., Development 121:4309–18, 1995).
The functions of some of these genes have been evaluated in knock-out mice constructs, animal studies, and in vitro studies.
A VEGF knockout was an embryonic lethal. F1 is also embryonic lethal and angiogenesis was impaired. VEGF secretion from +/− ES cells was reduced to 50% (Carmellet, P., et al., Nature 380:435–439, 1996; Ferrara, N., et al., Nature 380:439–442, 1996).
VEGFR-1 was evaluated in a lacZ knock-in wherein a fragment of the exon that contains the ATG start codon was replaced by LacZ. Knockout mice were embryonic lethal. Blood vessels were formed, but the organization of the blood vessel was perturbed (Fong, G. H., et al., Nature 376:65–69, 1995).
VEGFR-2 was an embryonic lethal caused by defective endothelial cell development (Shalaby, F., et al., Nature 376:62–65, 1995).
VEGFR-3(LacZ Knock-in) was an embryonic lethal caused by defective blood vessel development (Dumont, D. J., et al., Science 282:946–949, 1998).
Neuropillin-1 was an embryonic lethal (Dumont, D. J., et al., Science 282:946–949, 1998).
In vitro studies showed that a mutant VEGF (a heterodimer of two mutant VEGF) (Siemeister, G., et al., Proc Natl Acad Sci 95:4625–9, 1998), as well as a GST-Exon7 (VEGF) fusion protein (Soker, S., et al., JBC 272:31582–88, 1997), was able to inhibit endothelial cell proliferation by acting as an VEGF antagonist and interfering VEGF binding to VEGFR-2 and VEGFR-1 (Siemeister, G., et al., Proc Natl Acad Sci 95:4625–9, 1998). More importantly, A VEGF neutralizing chimeric protein, containing the extracellular domain of VEGF receptor (either VEGFR-1 or VEGFR-2) fused with IgG, substantially reduced the development of retinal neovascularization when injected into mice with ischemic retinal disease (Aiello, L. P., et al., Proc Natl Acad Sci 92:10457–61, 1995).
Treatment of tumors with monoclonal antibodies directed against VEGF resulted in dramatic reduction in tumor mass due to the suppression of tumor angiogenesis (Kim, K. J., et al., Nature 362:841–44, 1993). Injection of antibodies against VEGF reduced tumor vascular permeability and vessel diameter in immunodeficient mice transplanted with human glioblastoma, colon adenocarcinoma, and melanoma (Yuan, F., et al., Proc Natl Acad Sci 93:14765–70, 1996). Retrovirus-mediated overexpression of a dominant negative form of VEGFR-2 in nude mice suppresses the growth of transplanted rat C6 glioma tumor cells (Millauer, B., et al., Nature 367:576–9, 1994) mammary, ovarian tumors and lung carcinoma (Millauer, B., et al., Cancer Res 56:1615–20, 1996).
Further promoters of interest derived from angiogenesis-related genes include, but are not limited to, the following.
Ang2 is expressed only at predominant vascular remodeling sites, such as ovary, placenta, uterus (Maisonpierre, P. C., et al., 1997 Science 277: 55–60). In glioblastoma angiogenesis, Ang2 is found to be expressed in endothelial cells of small blood vessel and capillaries while Ang1 is expressed in glioblastoma tumor cells (Stratmann, A., 1998 Am J Pathol 153: 1459–66). Ang2 is up-regulated in bovine microvascular endothelial by VEGF, bFGF, cytokines, hypoxia (Mandriota, S. J., 1998 Circ Res 83: 832–9). Ang2 transgenic overexpression disrupts angiogenesis, and is embryonic lethal (Maisonpierre, P. C., et al., 1997 Science 277: 55–60). Ang1 is widely expressed, less abundant in heart and liver (Maisonpierre, P. C., et al., 1997 Science 277: 55–60). Ang1 is expressed in mesenchymal cells and may up-regulate the expression of Tie2 in the endothelial cells (Suri, C., et al., 1996 Cell 87: 1171–1180). Ang1 overexpression in the skin of transgenic mice produces larger, more numerous, and more highly branched vessels (Suri, C., et al., Science 1998 282:468–71).
Tie2 (see, e.g., Fadel et al.; Schlaeger et al. (1995), and Schlager et al. (1997), supra) is endothelial cell specific, up-regulated during wound healing, follicle maturation (Puri, M. C., et al., 1995 EMBO J 14: 5884–91) and pathologic angiogenesis (Kaipainen, A., 1994 Cancer Research 54: 6571–77), such as, glioblastoma (Stratmann, A., 1998 Am J Pathol 153: 1459–66). Tie2 is also expressed in non-proliferating adult endothelium and endothelial cell lines (Dumont, D. J., et al. (1994) Genes & Develop. 8:1897–1909). A Tie2 activating mutation causes vascular dysmorphogenesis (Vikkula M, et al., 1996 Cell 87: 1181–1190). Tie2 mutant overexpression in transgenic mice is embryonic lethal (Dumont, D. J., et al., supra). The cloning and sequencing of the 7.1 kb promoter region of Tie2 is described herein (see Example 3 below).
Other promoters derived from angiogenesis-related genes that are useful in the practice of the present invention include, by way of example, promoters derived from the sequences encoding the following polypeptide products: PTEN (dual specificity phosphatase); BAI (brain-specific angiogenesis inhibitor); KAI1 (KANGAI 1); catenin beta-1 (cadherin-associated protein, beta); COX2 (PTGS2 cyclooxygenase 2, a.k.a. prostaglandin-endoperoxide synthase 2); MMP2 (72 kDa Type IV-A collagenase); MMP9 (92 kDa type IV-B collagenase); TIMP2 (tissue inhibitor of metalloproteinase 2); and TIMP3 (tissue inhibitor of metalloproteinase 3).
PTEN is a tumor suppressor gene and encodes a protein of 403 amino acids. (Li et al. (1997) Science 275:1943–1946; DiCristofano et al. (1998) Nature Genet. 19:348–355). Overexpression of PTEN has been shown to inhibit cell migration and it is postulated that this protein may function as a tumor suppressor by negatively regulating cell interactions with the extracellular matrix or by negatively regulating the PI3K/PKB/Akt signaling pathway. (Tamura et al. (1998) Science 280:1614–1617; Stambolic et al. (1998) Cell 95:29–29). Mutations in PTEN have been detected in cancer cell lines and in the germline of patients having Cowden disease, Lhernitte-Duclos disease and Bannayan-Zonana syndrome (diseases and syndromes which are characterized by hyperplastic/dysplastic changes in the prostate, skin and colon and which are associated with an increased risk of certain cancers, for example, breast cancer, prostate cancer and colon cancer). (Marsh et al. (1998) Hum Molec Genet. 7:507–515; Marsh et al. (1998) J Med Genet 35:881–885; Nelen et al. (1997) Hum Molec Genet 6:1383–1387).
BAI1 protein is predicted to be 1,584 amino acids in length and includes an extracellular domain, an intracellular domain and a 7-span transmembrane region similar to that of the secretin receptor (Nishimori et al. (1997) Oncogene 15:245–2150). The extracellular region of BAI1 has a single Arg-Gly-Asp (RGD) motif recognized by integrins and also has five sequences corresponding to the thrombospondin type I (accession number 188060) repeats that can inhibit angiogenesis includes by basic fibroblast growth factor (bFGF, accession number 134920). Shiratsuchi et al. (1997) Cytogenet. Cell Genet. 79:103–108, cloned 2 other brain-specific angiogenesis inhibiting genes, designated BAI2 (accession number 602683) and BAI 3 (accession number 602684). Thus, it is postulated that members of this gene family may play a role in suppression of glioblastoma.
KAI1 encodes a 267 amino acid protein that is a member of the leukocyte surface glycoprotein family. The protein has 4 hydrophobic transmembrane domains and 1 large extracellular hydrophilic domain with three potential N-glycosylation sites. (Dong et al. (1995) Science 268:884–886). Molecular analysis of KAI1 is described, for example, in Dong et al. (1997) Genomics 41:25–32. KAI1 is a tumor metastasis suppressor gene that is capable of inhibiting the metastatic process in experimental animals. Expression of KAI1 is downregulated during tumor progression of prostate, breast, lung, bladder and pancreatic cancers in humans, apparently at the transcriptional or postranscriptional level. Mashimo et al. (1998) PNAS USA 95:11307–11311, found that the tumor suppressor gene p53 can directly inactivate the KAI1 gene by interacting with the region 5′ to the coding sequence, suggesting a direct relationship between p53 and KAI1.
Catenin beta-1 is an adherens junction (AJ) protein, which are critical for establishing and maintaining epithelial cell layers, for instance during embryogenesis, wound healing and tumor cell metastasis. Molecular analysis, including description of sequence homology to plakoglobin (accession number 173325), homology to the drosophila gene “armadillo” and interactions with Lef1/Tcf DNA binding proteins, is described, for example, in Nollet et al. (1996) Genomics 32:413–424; McCrea et al. (1991) Science 254:1359–1361 and Korinek et al. (1997) Science 275:1784–1787. In addition, studies by Korinek et al., supra and Morin et al.(1997) Science 275:1787–1790, have indicated that APC (accession number 175100) negatively regulates catenin beta and that regulation of this protein is critical to the tumor suppressive effect of APC. Abnormally high levels of beta-catenin have been detected in certain human melanoma cell lines. (Rubinfeld et al. (1997) Science 275:1790–1792. Koch et al. (1999) Cancer Res. 59:269–273 report that childhood hepatoblastomas frequently carry a mutated degradation-targeting box of the beta-catenin gene. Transgenic mice that express catenin beta under the control of an epidermal promoter undergo de novo hair morphogenesis and eventually these animals develop two types of tumors—epithelioid cysts and trichofolliculomas. Gat et al. (1998) Cell 95:605–614.
COX2 encodes a cyclooxygenase and is a key regulator of prostaglandin synthesis. (Hla et al. (1992) PNAS USA 89:7384–7388; Jones et al. (1993) J. Biol. Chem. 268:9049–9054). In particular, COX2 is generally considered to be a mediator of inflammation and overexpression of COX2 in rat epithelial cells results in elevated levels of E-cadherin and Bcl2. (Tsujii & DuBois (1995) Cell 83:493–501). In co-cultures of endothelial cells and colon carcinoma cells, cells that overexpress COX2 produce prostaglandins, proangiogenic factors and stimulate both endothelial migration and tube formation. (Tsujii et al. (1998) Cell 93:705–716). Experiments conducted using APC knock-out mice have demonstrated that animals homozygous for a disrupted COX2 locus develop significantly more adenomatous polyps. (Oshima et al. (1996) Cell 87:803–809). COX-2 “knock out” mice develop severe nephropathy, are susceptible to peritonitis, exhibit reduced arachidonic acid-induced inflammation and exhibit reduced indomethacin-induced gastric ulceration. (Morham et al. (1995) Cell 83:473–482; Langenbach et al. (1995) Cell 83:483–492). Female mice that are deficient in cyclooxygenase 2 exhibit multiple reproductive failures. (Lim et al. (1997) Cell 91:197–208.
MMP2 is a metalloproteinase that specifically cleaves type IV collagen. A C-terminal fragment of MMP2, termed PEX, prevents normal biding to alpha-V/beta-3 and disrupts angiogenesis and tumor growth. (Brooks et al. (1998) Cell 92:391–400).
MMP9 is a collagenase secreted from normal skin fibroblasts. MMP9 null mice exhibit an abnormal pattern of skeletal growth plate vascularization and ossification. (Vu et al. (1998) Cell 93:411–422).
TIMP2 is a collagenase and appears to play a major role in modulating the activity of interstitial collagenase and a number of connective tissue metalloendoproteases. (Stetler-Stevenson et al. (1989) J. Biol. Chem. 264:17372–17378). Unlike TIMP1 and TIMP3, TIMP2 is not upregulated by TPA or TGF-beta. (Hammani et al. (1996) J. Biol. Chem. 271:25498–25505).
TIMP3 (Wilde et al. (1994) DNA Cell Biol. 13:711–718) is localized in the extracellular matrix in both its glycosylated and unglycosylated forms. Studies of mutant TIMP3 proteins have demonstrated that C-terminal trunctions do not bind to the extracellular matrix. (Langton et al. (1998) J. Biol. Chem. 273:16778–16781).
As one of skill in the art will appreciate in view of the teachings of the present specification, transcription control element sequences can be derived and isolated from, e.g., genomic sequences, using method known in the art in view of the teachings herein. For example, the transcription control element sequences of VEGF, VEGFR-2 and Tie2 were isolated and sequenced as described in Examples 1, 2 and 3 below. Another exemplary method of isolating promoter sequences employs a GenomeWalker® kit, commercially available from Clontech (Palo Alto, Calif.), and described on page 27 of the 1997–1998 Clontech catalog.
2.2.1 Mouse VEGF, VEGF-R2 and TIE2 Transcription Control Element Sequences
The subject nucleic acids of the present invention (e.g., as described in Examples 1, 2 and 3) find a wide variety of applications including use as hybridization probes, PCR primers, expression cassettes useful for compound screening, detecting the presence of VEGF, VEGFR-2 and/or Tie2 genes or varients thereof, detecting the presence of gene transcripts, detecting or amplifying nucleic acids encoding additional VEGF, VEGFR-2, and/or Tie2 promoter sequences or homologues thereof (as well as, structural analogs), and in a variety of screening assays.
The present invention provides efficient methods of identifying pharmacological agents or lead compounds for agents active at the level of VEGF, VEGFR-2, and Tie2 gene transcription. A wide variety of assays for transcriptional can be used based on the teaching of the present specification, including, but not limited to, cell-based transcription assays, screening in vivo in transgenic animals, and promoter-protein binding assays. For example, the disclosed luciferase reporter constructs are used to transfect cells for cell-based transcription assays. For example, primary endothelial cells are plated onto microtiter plates and used to screen libraries of candidate agents for compounds which modulate the transcriptional regulation of the VEGFR-2 and Tie2 gene promoters, as monitored by luciferase expression (See Example 5 and Example 6.).
As noted above, the present invention relates to a recombinant nucleic acid molecule comprising transcription control elements derived from a mouse VEGF gene locus. This invention provides an isolated polynucleotide comprising, a polynucleotide having X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least about 95% identity to Y contiguous nucleotides derived from nucleotides 1 through 3,762 of SEQ ID NO:44, (ii) X equals Y, and (iii) X is greater than or equal to 50. X is in the range of 50 to 3,762 nucleotides in length including all integer values in that range. The present invention may also include a nucleic acid sequence substantially complementary to said polynucleotide sequences, or fragments thereof, as well as, a nucleic acid sequence that specifically hybridizes to said polynucleotide sequences or fragments thereof. In one embodiment, the isolated polynucleotide consists of a polynucleotide having greater than 80% identity to the sequence presented as SEQ ID NO:44.
The present invention further relates to a recombinant nucleic acid molecule comprising transcription control elements derived from a mouse VEGFR-2 gene locus. This invention provides a polynucleotide having X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least about 95% identity to Y contiguous nucleotides derived from nucleotides 1 through 3,564 of SEQ ID NO:32, (ii) X equals Y, and (iii) X is greater than or equal to 55. X is in the range of 55 to 3,564 nucleotides in length including all integer values in that range. The present invention may also include a nucleic acid sequence substantially complementary to said polynucleotide sequences, or fragments thereof, as well as, a nucleic acid sequence that specifically hybridizes to said polynucleotide sequences or fragments thereof. In one embodiment, the isolated polynucleotide consists of a polynucleotide having greater than 80% identity to the sequence presented as SEQ ID NO:32.
Finally, the present invention relates to a recombinant nucleic acid molecule comprising transcription control elements derived from a mouse Tie2 gene locus. This invention provides a polynucleotide having X contiguous nucleotides, wherein in a first embodiment, (A), (i) the X contiguous nucleotides have at least about 95% identity to Y contiguous nucleotides derived from nucleotides 1 through 6,091 of SEQ ID NO:40, (ii) X equals Y, and (iii) X is greater than or equal to 55, and/or in a second embodiment, (B), (i) the X contiguous nucleotides have at least about 90% identity to Y contiguous nucleotides derived from nucleotides 6,091 through 6,560 of SEQ ID NO:40, (ii) X equals Y, and (iii) X is greater than or equal to 250. For polynucleotide (A), X is in the range of 55 to 6,091 nucleotides in length including all integer values in that range. For polynucleotide (B), X is in the range of 250 through 469 nucleotides in length including all integer values in that range. The present invention may also include a nucleic acid sequence substantially complementary to said polynucleotide sequences, or fragments thereof, as well as, a nucleic acid sequence that specifically hybridizes to said polynucleotide sequences or fragments thereof. In one embodiment, the isolated polynucleotide consists of a polynucleotide having greater than 80% identity to the sequence presented as SEQ ID NO:40.
The invention includes further transcription control element sequences (e.g., promoter sequences) identified based on the teachings of the present specification (including, but not limited to, sequence information and isolation methods, e.g., Examples 1, 2, and 3).
The nucleic acid molecules of this invention are useful for producing transfected cells and transgenic animals that are themselves useful in a variety of applications, and for screening for compounds that modulate tumorigenic and angiogenic processes (see Examples 5 through 14 below).
Those skilled in the art can practice the invention by following the guidance of the specification supplemented with standard procedures of molecular biology for the isolation and characterization of the VEGF, VEGFR-2 and Tie2 transcription control elements, their transfection into host cells, and vascular endothelial cell-specific expression of heterologous DNA operably linked to said VEGF, VEGFR-2 and Tie2 promoters. For example, DNA is commonly transferred or introduced into recipient mammal cells by calcium phosphate-mediated gene transfer, electroporation, lipofection, viral infection, and the like. General methods and vectors for gene transfer and expression may be found, for example, in M. Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press (1990). Direct gene transfer to cells in vivo can be achieved, for example, by the use of modified viral vectors, including, but not limited to, retroviruses, adenoviruses, adeno-associated viruses and herpes viruses, liposomes, and direct injection of DNA into certain cell types. In this manner, recombinant expression vectors and recombinant cells containing the novel VEGF, VEGFR-2 and Tie2 transcription control elements of the present invention operably linked to a desired heterologous gene can be delivered to specific target cells in vivo. See, e.g., Wilson, Nature, 365: 691–692 (1993); Plautz et al, Annals NY Acad. Sci., 716: 144–153 (1994); Farhood et al, Annals NY Acad. Sci., 716: 23–34 (1994) and Hyde et al Nature, 362: 250–255 (1993). Furthermore, cells may be transformed ex vivo and introduced directly at localized sites by injection, e.g., intra-articular, intracutaneous, intramuscular and the like.
Cloning and characterization of the VEGF-, VEGFR-2- and Tie2-locus-derived transcription control elements are described in Examples 1 through 3, below.
2.3.0 Expression Cassettes and Vectors
The expression cassettes described herein may typically include the following components: (1) a polynucleotide encoding a reporter gene, such as a sequence encoding a light generating protein, (2) a transcription control element operably linked to the reporter gene sequence, wherein the control element is heterologous to the coding sequences of the light generating protein (e.g., the novel VEGF, VEGF-R2 and Tie2 promoters of the present invention). Transcription control elements derived from the sequences provided herein may be associated with, for example, a basal transcription promoter to confer regulation on such a basal transcription promoter. Exemplary expression constructs are described in Examples 1 through 4.
The present invention also includes providing such expression cassettes in vectors, comprising, for example, a suitable vector backbone and optionally a sequence encoding a selection marker e.g., a positive or negative selection marker. Suitable vector backbones generally include an F1 origin of replication; a colE1 plasmid-derived origin of replication; polyadenylation sequence(s); sequences encoding antibiotic resistance (e.g., ampicillin resistance) and other regulatory or control elements. Non-limiting examples of appropriate backbones include: pBluescriptSK (Stratagene, La Jolla, Calif.); pBluescriptKS (Stratagene, La Jolla, Calif.) and other commercially available vectors.
A variety of reporter genes may be used in the practice of the present invention. Preferred are those that produce a protein product which is easily measured in a routine assay. Suitable reporter genes include, but are not limited to chloramphenicol acetyl transferase (CAT), light generating proteins (e.g., luciferase), and beta-galactosidase. Convenient assays include, but are not limited to calorimetric, fluorimetric and enzymatic assays. In one aspect, reporter genes may be employed that are expressed within the cell and whose extracellular products are directly measured in the intracellular medium, or in an extract of the intracellular medium of a cultured cell line. This provides advantages over using a reporter gene whose product is secreted, since the rate and efficiency of the secretion introduces additional variables that may complicate interpretation of the assay. In a preferred embodiment, the reporter gene is a light generating protein. When using the light generating reporter proteins described herein, expression can be evaluated accurately and non-invasively as described above (see, for example, Contag, P. R., et al., (1998) Nature Med. 4:245–7; Contag, C. H., et al., (1997) Photochem Photobiol. 66:523–31; Contag, C. H., et al., (1995) Mol Microbiol. 18:593–603).
In one aspect of the invention, the light generating protein is luciferase. Luciferase coding sequences useful in the practice of the present invention include sequences obtained from lux genes (procaryotic genes encoding a luciferase activity) and luc genes (eucaryotic genes encoding a luciferase activity). A variety of luciferase encoding genes have been identified including, but not limited to, the following: B. A. Sherf and K. V. Wood, U.S. Pat. No. 5,670,356, issued 23 Sep. 1997; Kazami, J., et al., U.S. Pat. No. 5,604,123, issued 18 Feb. 1997; S. Zenno, et al, U.S. Pat. No. 5,618,722; K. V. Wood, U.S. Pat. No. 5,650,289, issued 22 Jul. 1997; K. V. Wood, U.S. Pat. No. 5,641,641, issued 24 Jun. 1997; N. Kajiyama and E. Nakano, U.S. Pat. No. 5,229,285, issued 20 Jul. 1993; M. J. Cormier and W. W. Lorenz, U.S. Pat. No. 5,292,658, issued 8 Mar. 1994; M. J. Cormier and W. W. Lorenz, U.S. Pat. No. 5,418,155, issued 23 May 1995; de Wet, J. R., et al, Molec. Cell. Biol. 7:725–737, 1987; Tatsumi, H. N., et al, Biochim. Biophys. Acta 1131:161–165, 1992; and Wood, K. V., et al, Science 244:700–702, 1989; all herein incorporated by reference. Eucaryotic luciferase catalyzes a reaction using luciferin as a luminescent substrate to produce light, whereas procaryotic luciferase catalyzes a reaction using an aldehyde as a luminescent substrate to produce light.
Wild-type firefly luciferases typically have emission maxima at about 550 nm. Numerous variants with distinct emission maxima have also been studied. For example, Kajiyama and Nakano (Protein Eng. 4(6):691–693, 1991; U.S. Pat. No. 5,330,906, issued 19 Jul. 1994, herein incorporated by reference) teach five variant firefly luciferases generated by single amino acid changes to the Luciola cruciata luciferase coding sequence. The variants have emission peaks of 558 nm, 595 nm, 607 nm, 609 nm and 612 nm. A yellow-green luciferase with an emission peak of about 540 nm is commercially available from Promega, Madison, Wis. under the name pGL3. A red luciferase with an emission peak of about 610 nm is described, for example, in Contag et al. (1998) Nat. Med. 4:245–247 and Kajiyama et al. (1991) Port. Eng. 4:691–693.
Positive selection markers include any gene which a product that can be readily assayed. Examples include, but are not limited to, an HPRT gene (Littlefield, J. W., Science 145:709–710 (1964), herein incorporated by reference), a xanthine-guanine phosphoribosyltransferase (GPT) gene, or an adenosine phosphoribosyltransferase (APRT) gene (Sambrook et al., supra), a thymidine kinase gene (i.e. “TK”) and especially the TK gene of the herpes simplex virus (Giphart-Gassler, M. et al., Mutat. Res. 214:223–232 (1989) herein incorporated by reference), a nptII gene (Thomas, K. R. et al., Cell 51:503–512 (1987); Mansour, S. L. et al., Nature 336:348–352 (1988), both references herein incorporated by reference), or other genes which confer resistance to amino acid or nucleoside analogues, or antibiotics, etc., for example, gene sequences which encode enzymes such as dihydrofolate reductase (DHFR) enzyme, adenosine deaminase (ADA), asparagine synthetase (AS), hygromycin B phosphotransferase, or a CAD enzyme (carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase). Addition of the appropriate substrate of the positive selection marker can be used to determine if the product of the positive selection marker is expressed, for example cells which do not express the positive selection marker nptII, are killed when exposed to the substrate G418 (Gibco BRL Life Technology, Gaithersburg, Md.).
The vector typically contains insertion sites for inserting polynucleotide sequences of interest, e.g., the novel VEGF, VEGFR-2 and Tie2 promoters of the present invention. These insertion sites are preferably included such that there are two sites, one site on either side of the sequences encoding the positive selection marker, luciferase and the promoter. Insertion sites are, for example, restriction endonuclease recognition sites, and can, for example, represent unique restriction sites. In this way, the vector can be digested with the appropriate enzymes and the sequences of interest ligated into the vector.
Optionally, the vector construct can contain a polynucleotide encoding a negative selection marker. Suitable negative selection markers include, but are not limited to, HSV-tk (see, e.g., Majzoub et al. (1996) New Engl. J. Med. 334:904–907 and U.S. Pat. No. 5,464,764), as well as genes encoding various toxins including the diphtheria toxin, the tetanus toxin, the cholera toxin and the pertussis toxin. A further negative selection marker gene is the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene for negative selection in 6-thioguanine.
Exemplary promoters for use in the practice of the present invention are described above.
Vector Construction: The vectors described herein can be constructed utilizing methodologies known in the art of molecular biology (see, for example, Ausubel or Maniatis) in view of the teachings of the specification. As described above, the vector constructs containing the expression cassettes are assembled by inserting, into a suitable vector backbone, (1) polynucleotides encoding a reporter protein, such as a light-generating protein, e.g., a luciferase gene, operably linked to a transcription control element(s) of interest; (2) a sequence encoding a positive selection marker; and, optionally (3) a sequence encoding a negative selection marker. In addition, the vector construct contains insertion sites such that additional sequences of interest can be readily inserted to flank the sequence encoding positive selection marker and luciferase-encoding sequence.
A preferred method of obtaining polynucleotides, suitable regulatory sequences (e.g., promoters) is PCR. General procedures for PCR as taught in MacPherson et al., PCR: A PRACTICAL APPROACH, (IRL Press at Oxford University Press, (1991)). PCR conditions for each application reaction may be empirically determined. A number of parameters influence the success of a reaction. Among these parameters are annealing temperature and time, extension time, Mg2+ and ATP concentration, pH, and the relative concentration of primers, templates and deoxyribonucleotides. Exemplary primers are described below in the Examples. After amplification, the resulting fragments can be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.
In one embodiment, PCR can be used to amplify fragments from genomic libraries. Many genomic libraries are commercially available. Alternatively, libraries can be produced by any method known in the art. Preferably, the organism(s) from which the DNA is has no discernible disease or phenotypic effects. This isolated DNA may be obtained from any cell source or body fluid (e.g., ES cells, liver, kidney, blood cells, buccal cells, cerviovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy, urine, blood, cerebrospinal fluid (CSF), and tissue exudates at the site of infection or inflammation). DNA is extracted from the cells or body fluid using known methods of cell lysis and DNA purification. The purified DNA is then introduced into a suitable expression system, for example a lambda phage.
Another method for obtaining polynucleotides, for example, short, random nucleotide sequences, is by enzymatic digestion. As described below in the Examples, short DNA sequences generated by digestion of DNA from vectors carrying genes encoding luciferase (yellow green or red).
Polynucleotides are inserted into vector backbones using methods known in the art. For example, insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary or blunt ends on each molecule that can pair with each other and be joined with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of a polynucleotide. These synthetic linkers can contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA. Other means are known and, in view of the teachings herein, can be used.
The vector backbone may comprise components functional in more than one selected organism in order to provide a shuttle vector, for example, a bacterial origin of replication and a eucaryotic promoter.
The final constructs can be used immediately (e.g., for introduction into ES cells), or stored frozen (e.g., at −20° C.) until use. Preferably, the constructs are linearized prior to use, for example by digestion with suitable restriction endonucleases.
2.4.0 Transgenic Animals
The expression cassettes of the present invention may be introduced into the genome of an animal in order to produce transgenic animals for purposes of practicing the methods of the present invention. In a preferred embodiment of the present invention, the transgenic animal is a transgenic rodent, for example, a mouse, rat, or guinea pig. A variety of transformation techniques are well known in the art. Those methods include the following.
(i) Direct microinjection into nuclei: Expression cassettes can be microinjected directly into animal cell nuclei using micropipettes to mechanically transfer the recombinant DNA. This method has the advantage of not exposing the DNA to cellular compartments other than the nucleus and of yielding stable recombinants at high frequency. See, Capecchi, M., Cell 22:479–488 (1980).
For example, the expression cassettes of the present invention may be microinjected into the early male pronucleus of a zygote as early as possible after the formation of the male pronucleus membrane, and prior to its being processed by the zygote female pronucleus. Thus, microinjection according to this method should be undertaken when the male and female pronuclei are well separated and both are located close to the cell membrane. See U.S. Pat. No. 4,873,191 to Wagner, et al. (issued Oct. 10, 1989).
(ii) Electroporation: The DNA containing the expression cassettes of the present invention can also be introduced into the animal cells by electroporation. In this technique, animal cells are electroporated in the presence of DNA containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the DNA. The pores created during electroporation permit the uptake of macromolecules such as DNA. Procedures are described in, e.g., Potter, H., et al., Proc. Nat'l. Acad. Sci. U.S.A. 81:7161–7165 (1984); and Sambrook, ch. 16.
(iii) Calcium phosphate precipitation: The expression cassettes may also be transferred into cells by other methods of direct uptake, for example, using calcium phosphate. See, e.g., Graham, F., and A. Van der Eb, Virology 52:456–467 (1973); and Sambrook, ch.16.
(iv) Liposomes: Encapsulation of DNA within artificial membrane vesicles (liposomes) followed by fusion of the liposomes with the target cell membrane can also be used to introduce DNA into animal cells. See Mannino, R. and S. Gould-Fogerite, BioTechniques, 6:682 (1988).
(v) Viral capsids: Viruses and empty viral capsids can also be used to incorporate DNA and transfer the DNA to animal cells. For example, DNA can be incorporated into empty polyoma viral capsids and then delivered to polyoma-susceptible cells. See, e.g., Slilaty, S. and H. Aposhian, Science 220:725 (1983).
(vi) Transfection using polybrene or DEAE-dextran: These techniques are described in Sambrook, ch.16.
(vii) Protoplast fusion: Protoplast fusion typically involves the fusion of bacterial protoplasts carrying high numbers of a plasmid of interest with cultured animal cells, usually mediated by treatment with polyethylene glycol. Rassoulzadegan, M., et al., Nature, 295:257 (1982).
(viii) Ballistic penetration: Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327, 70–73, 1987.
Any technique that can be used to introduce DNA into the animal cells of choice can be employed. Electroporation has the advantage of ease and has been found to be broadly applicable, but a substantial fraction of the targeted cells may be killed during electroporation. Therefore, for sensitive cells or cells which are only obtainable in small numbers, microinjection directly into nuclei may be preferable. Also, where a high efficiency of DNA incorporation is especially important, such as transformation without the use of a selectable marker (as discussed above), direct microinjection into nuclei is an advantageous method because typically 5–25% of targeted cells will have stably incorporated the microinjected DNA. Retroviral vectors are also highly efficient but in some cases they are subject to other shortcomings, as described by Ellis, J., and A. Bernstein, Molec. Cell. Biol. 9:1621–1627 (1989). Where lower efficiency techniques are used, such as electroporation, calcium phosphate precipitation or liposome fusion, it is preferable to have a selectable marker in the expression cassette so that stable transformants can be readily selected, as discussed above.
In some situations, introduction of the heterologous DNA will itself result in a selectable phenotype, in which case the targeted cells can be screened directly for homologous recombination. For example, disrupting the gene hart results in resistance to 6-thioguanine. In many cases, however, the transformation will not result in such an easily selectable phenotype and, if a low efficiency transformation technique such as calcium phosphate precipitation is being used, it is preferable to include in the expression cassette a selectable marker such that the stable integration of the expression cassette in the genome will lead to a selectable phenotype. For example, if the introduced DNA contains a neo gene, then selection for integrants can be achieved by selecting cells able to grow on G418.
Transgenic animals prepared as above are useful for practicing the methods of the present invention. Operably linking a promoter of interest to a reporter sequence enables persons of skill in the art to monitor a wide variety of biological processes involving expression of the gene from which the promoter is derived. The transgenic animals of the present invention that comprise the expression cassettes of the present invention provide a means for skilled artisans to observe those processes as they occur in vivo, as well as to elucidate the mechanisms underlying those processes.
With respect to transgenic animals carrying expression cassettes that employ a light-generating protein as a reporter sequence, the monitoring of expression of luciferase reporter expression cassettes using non-invasive whole animal imaging has been described (Contag, C. et al, U.S. Pat. No. 5,650,135, Jul. 22, 1997, herein incorporated by reference; Contag, P., et al, Nature Medicine 4(2):245–247, 1998; Contag, C., et al, OSA TOPS on Biomedical Optical Spectroscopy and Diagnostics 3:220–224, 1996; Contag, C. H., et al, Photochemistry and Photobiology 66(4):523–531, 1997; Contag, C. H., et al, Molecular Microbiology 18(4):593–603, 1995). Such imaging typically uses at least one photo detector device element, for example, a charge-coupled device (CCD) camera.
Thus, in one exemplary embodiment, female transgenic mice carrying expression cassettes comprising the VEGFR2 promoter operably linked to a luciferase-encoding reporter sequence may be used to monitor VEGFR2-mediated angiogenesis in the female reproductive organs over the course of estrus. See Example 7 below. In a further exemplary embodiment, neonatal VEGFR-2 transgenic mice may be used to monitor angiogenesis over the course of the first eight weeks of development. See Example 8 below. The transgenic animals of the present invention that comprise the expression cassettes of the present invention also provide a means for screening analytes that may be capable of modulating such processes and thereby identifying compounds with potential pharmaceutical applications.
Methods of administration of the analyte include, but are not limited to, injection (subcutaneously, epidermally, intradermally), intramucosal (such as nasal, rectal and vaginal), intraperitoneal, intravenous, oral or intramuscular. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. For example, the analyte of interest can be administered over a range of concentration to determine a dose/response curve. The analyte may be administered to a series of test animals or to a single test animal (given that response to the analyte can be cleared from the transgenic animal).
Thus, in one exemplary embodiment, female transgenic mice carrying expression cassettes comprising the VEGFR-2 promoter operably linked to a luciferase-encoding reporter sequence may be used to monitor the effects of known endocrine disrupter compounds on VEGFR-2 angiogenesis in the female reproductive organs over the course of estrus. See Example 9 below. In another exemplary embodiment, the effects of growth factors on VEGFR-2 promoter-mediated angiogenesis may be observed using transgenic mice carrying expression cassettes comprising the VEGFR-2 promoter operably linked to a luciferase-encoding reporter gene. See Example 10 below.
In another exemplary embodiment, transgenic mice of the present invention are implanted with tumor cells, treated with compounds known to affect angiogenesis, and monitored to determine the effects of those compounds on tumor-induced angiogenesis and thereby screen for compounds which may possess angiogenesis-modulating activity. For example, VEGF and VEGFR-2 transgenic mice were first implanted with cells from several compatible tumor cell lines, then treated with a variety of putative antitumorigenic anti-angiogenic agent, and finally subjected to in vivo bioluminescent imaging to monitor the effects of those compounds on tumor growth. The results obtained were then compared with those obtained from untreated control animals. See Example 13 and Example 14 below. The results of those experiments demonstrate that the transgenic mice of the present invention may be used to screen compounds which may be effective pharmaceutical agents.
2.5.0 Monitoring Promoter Activity
Activity of the transcription control element sequences comprising the expression cassettes and vectors of the present invention may be monitored by detecting and/or quantifying the protein products encoded by the reporter sequences operably linked to those promoters. The particular method used to monitor promoter activity depends on the reporter sequence employed, and may include, for example, enzymatic assay methods, as well as, in the case of reporter sequences which encode light-generating proteins, in vitro or in vivo bioluminescent imaging.
For example, promoter activity in transgenic animals carrying the expression cassettes of the present invention may be monitored using in vivo bioluminescence imaging (see Contag et al., (see, for example, Contag, P. R., et al., (1998) Nature Med. 4:245–7; Contag, C. H., et al., (1997) Photochem Photobiol. 66:523–31; Contag, C. H., et al., (1995) Mol Microbiol. 18:593–603).
Monitoring promoter activity in turn enables one to monitor the biological processes with which that promoter is associated. It may further be employed in methods of screening analytes which modulate those processes at the promoter level (see discussion in the following section).
Thus, in one aspect of the invention, the transgenic animals carrying expression cassettes comprising promoter sequences derived from angiogenesis-related gene locii such as those described above may be used to monitor angiogenesis as it occurs during a variety of biological processes.
In one embodiment, pregnant female mice carrying VEGF, VEGFR-2 or Tie2 transgenic embryos were monitored via the in vivo bioluminescence imaging method cited above in order to model VEGF, VEGFR-2 and/or Tie2 expression in those embryos during the course of prenatal development. The pups are then monitored for 1 to 8 weeks after birth in order to study VEGF, VEGFR-2 and/or Tie2 expression during post-natal development. See Example 7 below. The results of this experiment demonstrate that the expression cassettes and transgenic animals of the present invention may be used to monitor VEGFR-2 promoter-mediated expression of bioluminescence in vivo over the course of development.
In another embodiment, VEGF, VEGFR-2 and/or Tie2 expression during the reproductive cycles of female transgenic mice is monitored via in vivo bioluminescent imaging. See, e.g., Example 8, below. The results of this experiment demonstrate that it is possible to monitor changes in VEGFR-2 promoter-mediated bioluminescence expression that occur during estrus in female mice carrying the VEGFR-2 promoter-Luc transgene cassette.
In yet another embodiment of this aspect of the invention, the effect of estrogen antagonist ICI 182 780 on estrus cycling was studied by in vivo imaging of a VEGFR2 promoter transgenic mouse that was dosed with this compound daily for 16 days. See, e.g. Example 9 below. The results of this experiment demonstrate that transgenic animals carrying the expression cassettes of the present invention may be used to investigate the effects of endocrine disrupting compounds, including but not limited the effects of estrogen antagonists such as ICI 182 789, on VEGFR-2 promoter-mediated gene expression during the estrus cycle.
The effect of growth factors on VEGFR2 expression was studied in still another embodiment of this aspect of the invention by in vivo imaging of a VEGFR2 promoter transgenic mouse that was implanted with a growth factor-containing matrigel. See, e.g. Example 10 below. The results of this experiment demonstrate that transgenic animals carrying the expression constructs of the present invention may be used to investigate the effects of growth factors on VEGFR-2 promoter-mediated gene expression during the process of angiogenesis.
In a further embodiment, VEGF expression in tumor cells was monitored by in vivo imaging of C57BL/6J-Tyrc-2J/+ mice that were implanted with LL/2, B16F1 and T241 tumor cells stably transfected with VEGF-Luc (pTKLG-Vn-VP). See, e.g. Example 11, below. The results of this experiment demonstrate that stable tumor cells generated with an expression vector comprising the VEGF promoter operably linked to a luciferase-encoding reporter sequence may be used to investigate VEGF gene expression during tumor development.
2.6.0 Screening Analytes
The methods of monitoring promoter activity discussed above may be employed for the purpose of screening analytes which modulate a variety of biological processes, the effects of which are mediated at the promoter level. Screening may be accomplished by means of in vitro assays employing transiently or stably transfected cells, and may also be conducted using the transgenic animals of the present invention discussed above, either by themselves or in conjunction with other wild-type or transformed cells or tissues that have been introduced into those animals. The particular assay method used to measure the effects of various candidate compounds on promoter activity will be determined by the particular reporter sequence present in the expression cassette carried by the cells or animals employed. As discussed above, promoter activity in transgenic animals carrying constructs employing reporter sequences encoding light-generating proteins may be measured by means of ex vivo assay methods or by means of the in vivo bioluminescent imaging technique reference previously.
Thus, one aspect of this invention is the use of the expression cassettes and vectors for use in screening for pharmacologically active agents (or compounds) that modulate VEGF, VEGFR-2 and/or Tie2 promoter activity, either by affecting signal transduction pathways that necessarily precede transcription or by directly affecting transcription of the VEGF, VEGFR-2 and/or Tie2.
For screening purposes, appropriate host cells, preferably tumor cells for monitoring VEGF promoter-mediated expression and vascular endothelial cells for monitoring VEGFR2 promoter-mediated and Tie2 promoter-mediated expression, are transformed with an expression vectors comprising a reporter gene (e.g., luciferase) operably linked to either the VEGF, VEGFR-2 or Tie2 gene promoters of this invention. The transformed cells are next exposed to various test substances and then analyzed for expression of the reporter gene. The expression exhibited by these cells can be compared to expression from cells that were not exposed to the test substance. A compound that modulates the promoter activity of the VEGF, VEGFR-2 and/or Tie2 promoter will result in modulated reporter gene expression relative to the control. See, e.g. Example 12, below.
Thus, one aspect of the invention is to screen for test compounds that regulate (i.e., stimulate or inhibit) gene expression levels mediated by the VEGF-, VEGFR-2- and/or Tie2-locus derived transcription control elements (e.g., promoters). Screening may be accomplished by, for example, (i) contacting host cells in which the VEGF, VEGFR-2 or Tie2 promoter disclosed herein is operably linked to a reporter gene with a test medium containing the test compound under conditions which allow for expression of the reporter gene; (ii) measuring the expression of the reporter gene in the presence of the test medium; (iii) contacting the host cells with a control medium which does not contain the test compound but is otherwise essentially identical to the test medium in (i), under conditions essentially identical to those used in (i); (iv) measuring the expression of reporter gene in the presence of the control medium; and (v) relating the difference in expression between (ii) and (iv) to the ability of the test compound to affect the activity of the promoter.
Alternatively, the transformed cells may be induced with a transcriptional inducer, such as IL-1 or TNF-alpha, forskolin, dibutyryl-cAMP, or a phorbol-type tumor promoter, e.g., PMA. Transcriptional activity is measured in the presence or absence of a pharmacologic agent of known activity (e.g., a standard compound) or putative activity (e.g., a test compound). A change in the level of expression of the reporter gene in the presence of the test compound is compared to that effected by the standard compound. In this way, the ability of a test compound to affect VEGF, VEGFR-2 and/or Tie2 transcription and the relative potencies of the test and standard compounds can be determined.
Thus in a further aspect, the present invention provides methods of measuring the ability of a test compound to modulate VEGF, VEGFR-2 and/or Tie2 transcription by: (i) contacting a host cell in which the VEGF, VEGFR-2 or the Tie2 promoter, disclosed herein, is operably linked to a reporter gene with an inducer of the VEGF, VEGFR-2 or the Tie2 promoter activity under conditions which allow for expression of the reporter gene; (ii) measuring the expression of the reporter gene in the absence of the test compound; (iii) exposing the host cells to the test compound either prior to, simultaneously with, or after contacting, the host cells with the inducer; (iv) measuring the expression of the reporter gene in the presence of the test compound; and (iv) relating the difference in expression between (ii) and (iv) to the ability of the test compound to modulate VEGF, VEGFR-2 or Tie2-mediated transcription.
Because different inducers are known to affect different modes of signal transduction, it is possible to identify, with greater specificity, compounds that affect a particular signal transduction pathway. Further, VEGF and VEGFR2 have been shown to be upregulated in tumor cells and proliferating endothelial cells, respectively; this upregulation appears to be necessary for tumor angiogenesis. Therefore, such assays provide a means of identifying compounds that will inhibit and/or reverse tumor growth by downregulating VEGF and VEGFR2 expression and thus preventing or reducing tumor angiogenesis.
Compounds that are especially promising as anti-tumorigenic agents are angiogenesis inhibitors. About 20 of this class of compounds are currently being tested in human trials. Most are in the early phase I or II clinical (human) studies, three are in phase II testing, and one has already yielded results as of the end of 1999. See Table 1 below.
Without being bound by any particular mechanistic explanation, the following alternatives for possible mechanisms of action of anti-angiogenic drugs are provided herein. Four general strategies are employed to investigate the action of anti-angiogenesis drugs: 1) blocking the factors that stimulate blood vessel formation; 2) making use of natural inhibitors of angiogenesis, i.e. angiostatin and endostatin; 3) blocking the action of molecules that allow newly forming blood vessels to invade surrounding tissue; and 4) incapacitating newly dividing endothelial cells.
Several distinct advantages conferred by anti-angiogenic drug therapies as compared to traditional chemotherapy approaches include the following: (1) anti-angiogenic drugs are not likely to cause bone marrow suppression, gastrointestinal symptoms, or hair loss; and (2) drug resistance may not develop as frequently happens for chemotherapeutic agents, owing to the fact that normal endothelial cells are not genetically unstable, as are most cancer cells.
Anti-angiogenic therapy may also prove useful when administered in combination with therapies directly aimed at tumor cells.
A list of anti-angiogenic agents currently in clinical trials as anti-tumorigenic agents appears below.







TABLE 1







Angiogenesis Inhibitors in Clinical Trials










Drug
Sponsor
Trial
Mechanism










Drugs that modulate matrix interaction or degradation










Marimastat
British Biotech;
Phase III against pancreas,
Synthetic MMP inhibitor



Annapolis, MD
non-small cell lung cancers,




breast cancers


Bay 12-9566
Bayer;
Phase III against lung
Synthetic inhibitor



West Haven, CT
and pancreatic cancers
of tumor growth


AG3340
Agouron;
2 trials: Phase III against
Synthetic MMP inhibitor



LaJolla, CA
non small cell lung and against




prostate cancers


CGS 27023A
Novartis;
Phase I/II
Synthetic MMP inhibitor



East Hanover, NJ


COL-3
Collagenex;
Phase I
Synthetic MMP inhibitor.



Newtown, PA/NCI

Tetracycline derivative


Neovastat
Aeterna;
Phase III against
Naturally-occurring



Sainte-Foy, Québec
non-small cell lung cancer (will
MMP inhibitor




open later in 1999)







Direct inhibition of endothelial cell function or response










TNP-470
TAP Pharmaceuticals,
Phase II against advanced cance
Synthetic analogue of



Deerfield, IL
Adults with solid tumors;
fungallin protein;




Phase I against pediatric solid
inhibits endothelial cell




tumors, tumors, lymphomas,
growth




and acute leukemias


Thalidomide
Celgene;
Phase II against



Warren, NJ
Kaposi's sarcoma,




Glioblastoma, prostate, lung,




and breast cancers


Squalamine
Magainin
Phase I
Extract from dogfish



Pharmaceuticals, Inc.;

Shark liver; inhibits



Plymouth Meeting, PA

sodium-hydrogen





exchanger, NHE3


Combretastatin
Oxigene;
Phase I;
Induction of apoptosis in



Boston, MA
Phase II to begin late 1999
Proliferating endothelial cells







Inhibition of angiogenic factor activity










Anti-VEGF
Genentech;
Phase II/III against lung, breast,
Monoclonal antibody


Antibody
S. San Francisco, CA
Prostate, colorectal,
to vascular endothelial




and renal cancers
growth factor (VEGF)


SU5416
Sugen, Inc.;
Phase I and Phase I/II against
Blocks VEGF receptor



Redwood City, CA
Kaposi's sarcoma and
signaling




solid tumors


SU6668
Sugen, Inc:
Phase I study will open in
Blocks VEGF, FGF, and



Redwood City, CA
early 1999 (London)
EGF receptor signaling


PTK787/ZK
Novartis;
Phase I against advanced cancer
Blocks VEGF receptor



East Hanover, NJ
(Germany and UK);
signaling




Phase I against glioblastoma




And Kaposi's sarcoma; and




Phase I/II against




Von Hippel Lindau disease (US)


Interferon-alfa
(commercially
Phase II/III
Inhibition of bFGF and



available)

VEGF production







Inhibition of endothelial-specific integrin survival signaling










Vitaxin
Ixsys, Inc.;
Phase II enrollment will begin
Antibody to integrin present



La Jolla, CA
in early 1999
on endothelial cell surface


EMD121974
Merck KCgaA;
Phase II/III against
Small molecule blocker of



Darmstadt, Germany
Kaposi's sarcoma, and
Integrin present on




brain tumors
endothelial cell surface




(to open later in 1999)







Non-specific mechanism of action










CAI
NCI;
Phase II/III against ovarian
Inhibitor of



Bethesda, MD
non-small cell lung, and
calcium influx




renal cell cancers


Interleukin-12
Genetics Institute;
Phase I/II against
Upregulation of



Cambridge, MA
Kaposi's sarcoma and
Interferon gamma and




solid tumors
IP-10


IM862
Cytran;
Phase III against AIDS-related
Unknown mechanism



Kirkland, WA
Kaposi's sarcoma









In another aspect of this invention, transgenic animals expressing a heterologous gene encoding a detectable product placed under the regulatory control of the VEGF-, VEGFR-2- or the Tie2-locus derived transcription control elements, as disclosed herein, may be used to determine the effect of a test compound. In particular, those animals may be used to determine the effects of a test compound on levels of gene expression mediated by a VEGF, VEGFR-2 or the Tie2 promoter in vivo. The test compound is, for example, administered to the animal and the degree of expression of the heterologous gene observed is compared to the degree of expression in the absence of administration of the test compound using, for example, whole animal luciferase-based assays as disclosed herein. Methods of generating transgenic animals are described above.
Thus, in one aspect of the invention, putative anti-tumorigenic anti-angiogenesis compounds are administered to VEGF and VEGFR-2 transgenic mice in which either growth factor-containing matrigel or tumor cells (including but not limited to B16F1 melanoma cells, LL/2 Lewis Lung carcinoma cells and T241 fibrosarcoma cells) have been introduced. Test compounds are then administered, and the expression of VEGF and VEGFR-2, as determined by in vivo bioluminescence imaging, is determined and compared with that observed for untreated controls. See Examples 13 and 14 below. Such an approach allows the visualization, via bioluminescence monitoring, of angiogenesis process which occurs in response to either growth factors or to the tumor growth; it further allows the effect of putative anti-tumorigenic anti-angiogenesis compounds on VEGF and VEGFR-2 expression during these processes to be studied.
This invention also provides transgenic animals useful as models for studying other physiological and pathological processes that involve VEGF, VEGFR-2 and/or Tie2 gene expression.
Various forms of the different embodiments of the invention, described herein, may be combined.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the devices, methods, and formulae of the present invention, and are not intended to limit the scope of what the inventor regards as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
EXPERIMENTAL EXAMPLES
Example 1
Construction of the VEGF-Luc Expression Cassette
A. Cloning of a Novel VEGF-Locus Derived Promoter Sequence
A novel VEGF promoter sequence was isolated by PCR screening of a mouse genomic library using primers based on a known partial VEGF promoter region sequence.
First, a 2240 bp mouse VEGF genomic DNA sequence containing a partial VEGF promoter region was obtained from GenBank (Accession Number: U41383). PCR primers were designed to amplify a 0.69 kb fragment (nucleotide number 958 to 1660) (VF1-VR1A; Table 2) and a 0.98 kb fragment (nucleotide number 641 to 1624) (VF2-VR2; Table 2) using that sequence.
The ability of each primer pair to amplify the predicted product was then confirmed using mouse129SvJ genomic DNA (Genome Systems) as a template. The conditions employed for PCR amplification are shown in FIG. 1.







TABLE 2







PCR Amplification Primers for VEGF, VEGFR-2 and Tie2 Promoter and


Enhancer Sequences










SEQ ID



NAME
NO:
SEQUENCE





VF1
19
ACCTCACTCTCCTGTCTCCCCTGATTCCCAA


VR1A
20
GTCTGGCGGTCACCCCCAAAAGCA


VF2
21
CCCTTTCCAAGACCCGTGCCATTTGAGC


VR2
22
ACTTTGCCCCTGTCCCTCTCTCTGTTCGC


KF1
23
GCTGCGTCCAGATTTGCTCTCAGATGCG


KR1
24
TTCTCAGGCACAGACTCCTTCTCCGTCCCT


KF2
25
CAGATGGACGAGAAAACAGTAGAGGCGTTGGC


KR2
26
GAGGACTCAGGGCAGAAAGAGAGCG


TF3
27
AGCTTAGCCTGCAAGGGTGGTCCTCATCG


TF2
28
CAAATGCACCCCAGAGAACAGCTTAGCCTGC


TR1
29
GCTTTCAACAACTCACAACTTTGCGACTTCCCG


VR2F
30
CGCTAGTGTGTAGCCGGCGCTCTC


VR2R
31
ATAAGAATGCGGCCGCCTGCACCTCGCGCTGGGCACAG


VEF
33
ACACGCCTCGAGAAATGTGCTGTCTTTAGAAGCCACTG


VER
34
ACACGCGTCGACGATCCAATAGGAAAGCCCTTCCATAAAC


VR-Not
45
ATAAGAATGCGGCCGCGGTTTCGGAGGCCGTCCGGGG


T2F
38
TATCAACACTCGGGAGGCTGAGGGAG


T2R
39
ATAAGAATGCGGCCGCACTTCCCCAGATCTCCCCATCCAGC









Finally, the primers designed and tested above were used to PCR screen a mouse 129/SvJ genomic DNA BAC (bacterial artificial chromosome) library (Genome Systems, Inc., St. Louis, Mo.) in order to isolate a novel VEGF promoter sequence. The library, on average, contained inserts of 120 kb with sizes ranging between 50 kb to 240 kb. A large genomic DNA fragment that contained VEGF promoter region was obtained.
Southern blot analysis was performed to map the location of the VEGF promoter region. A unique HindIII restriction site was mapped approximately 7.8 kb upstream of the ATG translational start codon of the VEGF gene. Over 6 kb of the 7.8 kb fragment containing the VEGF promoter region were sequenced. The sequence determined is presented in FIG. 2A–C (SEQ ID NO:44).
The present invention includes, but is not limited to, an isolated polynucleotide, and fragments thereof, having at least about 80–85%, preferably 85–90%, more preferably 90–95%, and most preferably 98–100% sequence identity to the sequence presented as SEQ ID NO:44. Exemplary fragments include, but are not limited to, polynucleotides having lengths of approximately 55–100, 100–250, 250–500, and 500–1,000. In particular, the present invention relates to a recombinant nucleic acid molecule comprising transcription control elements derived from a mouse VEGF gene locus. This invention provides an isolated polynucleotide comprising, a polynucleotide having X contiguous nucleotides, wherein (i) the X contiguous nucleotides have typically at least about 95% identity to Y contiguous nucleotides derived from nucleotides 1 through 3,762 of SEQ ID NO:44, (ii) X equals Y, and (iii) X is greater than or equal to 50. X is in the range of approximately 50 to approximately 3,762 nucleotides in length including all integer values in that range. Typically the polynucleotide sequence comprises one or more transcription control element(s). The present invention may also include a nucleic acid sequence substantially complementary to said polynucleotide sequences, or fragments thereof, as well as, a nucleic acid sequence that specifically hybridizes to said polynucleotide sequences or fragments thereof. In one embodiment, the isolated polynucleotide consists of a polynucleotide having greater than 80% identity to the sequence presented as SEQ ID NO:44.
B. Construction of the VEGF Expression Cassette
A 7.9 kb HindIII-SrfI VEGF promoter fragment was cloned into vector pSK (Stratagene, La Jolla, Calif.) that was linearized with HindIII and SmaI. The resulting construct was designated pSK-V32. A 0.3 kb 3′ end fragment was amplified by PCR (Forward primer VF1 (SEQ ID NO:19); reverse primer VR-Not (SEQ ID NO:45)) and digested with NheI and Notl. This fragment was used to replace the 0.2 kb NheI-Notl fragment of pSK-V32 and the resulting construct was designated pSK-VP, which contains a 7.8 kb VEGF promoter sequences beginning at a HindIII site 5′ to the ATG translational start codon, and ending at the ATG codon. The VEGF promoter was isolated as a HindIII-NotI fragment and cloned into the same sites of pGL3B2 (see Example 2B, below). The resulting construct was designated pGL3B2-VP.
Example 2
Construction of the VEGFR-2 Promoter-Luc YG-VEGFR-2 Enhancer Expression Cassette
A. Cloning and Re-engineering of VEGFR-2-locus Derived Promoter and Enhancer Sequences
1. Cloning and Re-engineering of a Novel VEGFR-2 Promoter Sequence
A novel VEGFR-2 promoter sequence was isolated by PCR screening of the same mouse genomic library employed in Example 1 (see above), using primers based on a known partial VEGFR-2 promoter region.
A mouse VEGFR-2 genomic DNA sequence of 1079 bp containing a partial VEGFR-2 promoter region (nucleotide position 3,561 TO 4,487) was published previously (Ronicke et al (1996) Cir. Res. 79:277–285). This sequence was used to design PCR amplification primers that were able to amplify a 0.45 kb fragment (nucleotide position 4,000 to 4,455) (KFI-KR1; Table 2) and a 0.58 kb fragment (nucleotide position 3,624 to 4,206) (KF2-KR2; Table 2).
The ability of each primer pair to amplify the predicted product was then confirmed using mouse 129SvJ genomic DNA as a template. DNA sequences for these primers are shown in Table 2 above. The PCR amplification conditions employed are shown in FIG. 1.
These primers were then used for PCR screening of a mouse 129/SvJ genomic DNA BAC library in order to isolate a novel VEGFR-2 sequence. A large genomic DNA fragment of the VEGFR-2 promoter region was obtained (the “VEGFR-2 BAC clone”).
Based on the VEGFR-2 restriction map that was published (Ronicke et al, supra), a 4.6 kb HindIII-XbaI fragment that covers the VEGFR-2 promoter region was subcloned from the VEGFR-2 BAC clone into vector pSK (Stratagene, La Jolla, Calif.) linearized with HindIII and XbaI. This construct was designated pSK-K6.
The pSK-K6 construct was then re-engineered to delete a 159 bp sequence from the ATG translational start codon to a downstream XbaI site, as follows.
A 0.3 kb 3′ end fragment was obtained by PCR amplification of pSK-K6 using the forward primer VR2F (SEQ ID NO:30) and the reverse primer VR2R (SEQ ID NO:31). This PCR product was digested with Bsu36I and NotI, and the resulting fragment was then used to replace the 0.45 kb Bsu36I-Notl fragment of pSK-K6. The resulting construct, designated pSK-KP, contains a novel 4.5kb VEGFR-2 promoter sequence beginning at a HindIII site 5′ to the ATG translational start codon, and ending at that ATG codon.
This promoter fragment was fully sequenced; and the sequence is shown in FIGS. 3A–C (SEQ ID NO:32). The present invention includes, but is not limited to, an isolated polynucleotide, and fragments thereof, having at least about 80–85%, preferably 85–90%, more preferably 90–95%, and most preferably 98–100% sequence identity to the sequence presented as SEQ ID NO:32. Exemplary fragments include, but are not limited to, polynucleotides having lengths of approximately 55–100, 100–250, 250–500, and 500–1,000. In particular, the present invention relates to a recombinant nucleic acid molecule comprising transcription control elements derived from a mouse VEGFR-2 gene locus. This invention provides a polynucleotide having X contiguous nucleotides, wherein (i) the X contiguous nucleotides have typcially at least about 95% identity to Y contiguous nucleotides derived from nucleotides 1 through 3,564 of SEQ ID NO:32, (ii) X equals Y, and (iii) X is greater than or equal to 55. X is in the range of approximately 55 to approximately 3,564 nucleotides in length including all integer values in that range. Typically the polynucleotide sequence comprises one or more transcription control element(s). The present invention may also include a nucleic acid sequence substantially complementary to said polynucleotide sequences, or fragments thereof, as well as, a nucleic acid sequence that specifically hybridizes to said polynucleotide sequences or fragments thereof. In one embodiment, the isolated polynucleotide consists of a polynucleotide having greater than 80% identity to the sequence presented as SEQ ID NO:32.
2. Cloning of a VEGFR-2 Enhancer Sequence
In a recent report, it was described that a 511 bp sequence located within the first intron of VEGFR-2 gene functions as an endothelial cell-specific expression enhancer. (Kappel et al (1999) Blood 12: 4284–4292). Accordingly, this VEGFR-2 enhancer sequence was amplified by PCR from the VEGFR-2 BAC clone DNA using the forward primer VEF (SEQ ID NO:33) and the reverse primer VER (SEQ ID NO:34). The 511 bp VEGFR-2 enhancer sequence is shown in FIG. 4 (SEQ ID NO:35).
The PCR product comprising the VEGFR-2 enhancer was then digested with XhoI and SalI and then cloned into the SalI site of the pSK vector. The resulting construct was designated pSK-KN.
B. Construction of the VEGFR-2 Promoter-luciferase-VEGFR-2 Enhancer Expression Cassette
The following scheme for constructing the VEGFR-2 Promoter-Luc YG-VEGFR-2 Enhancer expression cassette is presented in FIG. 5.
1. Re-engineering of PGL3B
The yellow-green luciferase-containing vector pGL3B (Promega, Madison, Wis.) was re-engineered as illustrated in FIG. 5. First, pGL3B was digested with NotI and then blunt ended with T4 DNA polymerase. A PmeI linker (New England Bolas) was then ligated into the vector. The resulting vector, pGL3B-Pme, was double digested with Asp718 and HindIII and ligated with a synthetic linker that resulted from annealing two complementary oligomers, GL3B-Forward (SEQ ID NO:36) and GL3B-Reverse (SEQ ID NO:37). This construct was designated pGL3B2.
2. Insertion of the VEGFR-2 Promoter and VEGFR-2 Enhancer Sequences
After re-engineering of the pGL3B2 vector was complete, the VEGFR-2 promoter was isolated from pSK-KP (see above) as a HindIII-NotI fragment and cloned into the same sites of pGL3B2. The resulting construct was designated pGL3B2-KP. Next, the VEGFR-2 enhancer was isolated from pSK-KN as a AhoI-SalI fragment and cloned into the pGL3B2-KP vector that had been linearized with SalI. The resulting construct, containing the complete VEGFR-2 promoter-LucYG-VEGFR-2 enhancer expression cassette, was designated pGL3B2-KPN.
Example 3
Construction of the Tie-2 Promoter-Luc-Tie2 Enhancer Expression Cassette
A. Cloning and Re-engineering of Tie2-locus Derived Promoter and Enhancer Sequences
1. Cloning and Re-engineering of a Novel Tie2 Promoter Sequence
A 477 bp region of the mouse Tie2 promoter has been isolated and sequenced (Fadel et al (1998) Biochem J. 330:335–343). Using this region, primers capable of amplifying a 0.45 kb (TF3-TR1; Table 2) and a 0.47 kb fragment (TF2-TR1; Table 2) of the Tie2 promoter were designed. The ability of each primer pair to amplify the predicted PCR product was confirmed using mouse 129SvJ genomic DNA as template. DNA sequences for these primers are shown in Table 2 above and PCR amplification conditions are shown in FIG. 1.
These primers were then used for PCR screening of the mouse 129/SvJ genomic DNA BAC library, and a large genomic DNA fragment containing a portion of the Tie2 promoter region was obtained. Based on the Tie2 genomic DNA restriction map previously published (Dumont et al (1994) Genes and Development 8:1897–1909), a 10.5 kb Asp718-EcoRV fragment spanning the Tie2 promoter region was subcloned from the Tie2 BAC clone which contained it into the same sites of pSK. The resulting construct was designated pSK-T67.
pSK-T67 was further engineered to delete 3.4 kb of sequence, from the ATG translational start codon to an upstream EcoRV site, as follows.
First, a 1.0 kb fragment at the 3′ end of the promoter region was PCR amplified using the forward primer T2F (SEQ ID NO:38) and the reverse primer T2R (SEQ ID NO:39). The amplification product was digested with BstAPI and NotI, and the fragment used to replace the 4.0 kb BstAPI-NotI fragment of pSK-T67. The resulting construct was designated pSK-TP.
pSK-TP thus contains a 7.1 kb fragment of the Tie2 promoter sequence, from a 5′ Asp718 site to the ATG translational start codon. This fragment was fully sequenced, and the sequence is shown in FIGS. 6A–D (SEQ ID NO:40).
The present invention includes, but is not limited to, an isolated polynucleotide, and fragments thereof, having at least about 80–85%, preferably 85–90%, more preferably 90–95%, and most preferably 98–100% sequence identity to the sequence presented as SEQ ID NO:40. Exemplary fragments include, but are not limited to, polynucleotides having lengths of approximately 55–100, 100–250, 250–500, and 500–1,000. In particular, the present invention relates to a recombinant nucleic acid molecule comprising transcription control elements derived from a mouse Tie-2 gene locus. This invention provides a polynucleotide having X contiguous nucleotides, wherein in a first embodiment, (A), (i) the X contiguous nucleotides have typically at least about 95% identity to Y contiguous nucleotides derived from nucleotides 1 through 6,091 of SEQ ID NO:40, (ii) X equals Y, and (iii) X is greater than or equal to 55, and/or in a second embodiment, (B), (i) the X contiguous nucleotides have at least about 90% identity to Y contiguous nucleotides derived from nucleotides 6,091 through 6,560 of SEQ ID NO:40, (ii) X equals Y, and (iii) X is greater than or equal to 250. For polynucleotide (A), X is in the range of approximately 55 to approximately 6,091 nucleotides in length including all integer values in that range. For polynucleotide (B), X is in the range of approximately 250 through approximately 469 nucleotides in length including all integer values in that range. Typically the polynucleotide sequence comprises one or more transcription control element(s). The present invention may also include a nucleic acid sequence substantially complementary to said polynucleotide sequences, or fragments thereof, as well as, a nucleic acid sequence that specifically hybridizes to said polynucleotide sequences or fragments thereof. In one embodiment, the isolated polynucleotide consists of a polynucleotide having greater than 80% identity to the sequence presented as SEQ ID NO:40.
2. Cloning of a Tie2 Enhancer Sequence
A 1.7 kb region within the first intron of the Tie2 gene was previously reported to function as an endothelial cell specific enhancer. (Schiaeger et al (1997) PNAS USA 94: 3058–3063). Accordingly, this 1.7 kb Tie2 enhancer region was isolated from the Tie2 Bac clone DNA by digesting with Asp718 and XhoI.
The enhancer sequence was then subcloned as a XhoI-Asp718 fragment from the Tie2 BAC clone identified above into the same sites of pSK. The Asp718 site was then converted to a SalI site using a SalI linker (New England Biolabs). The resulting construct was designated pSK-TN.
The 1.7 kb Tie2 enhancer was fully sequenced and the sequence is shown in FIG. 7 (SEQ ID NO:41).
B. Construction of the Tie2 Promoter-LucYG-Tie2 Enhancer Expression Cassette
As illustrated in FIG. 8, the Tie2 promoter was isolated from pSK-TP as a Asp718-NotI fragment and cloned into the pGL3B2 vector that was linearized with Asp718 and NotI. The resulting construct was designated pGL3B2-TP. Subsequently, the Tie2 enhancer was isolated from pSK-TN as a XhoI-SalI fragment and cloned into the pGL3B2-TP vector linearized with SalI. The resulting construct was designated pGL3B2-TPN.
Example 4
Construction of Vectors Containing the VEGF, VEGFR-2 and TIE2 Expression Cassettes
Additional vector constructs containing VEGF, VEGFR-2 and TIE2 expression constructs useful in a variety of the methods of the present invention were created as described below. All have in common one of the backbone vectors pTK-LucR or pTK-LucYG.
A. Construction of the Backbone Vectors pTK-LucR and pTK-LucYG.
1. Construction of the pTK53 Starting Vector
Initially, the vector pTK53 was constructed as illustrated in FIG. 9 by introducing sequences from the mouse phosphoglycerate kinase 1 promoter, the neomycin gene coding sequence, and the thymadine kinase gene coding sequence, as well as two synthetic polylinker sequences, into the pBluescript II KS vector (Stratagene, La Jolla, Calif.), as follows.
First, the 0.5 kb mouse phosphoglycerate kinase 1 promoter was amplified with the PGK primers PGKF (SEQ ID NO: 1) and PGK (SEQ ID NO:2) using mouse genomic DNA (Genome Systems, Inc., St. Louis, Mo.) as a template. This fragment was then double digested with EcoRI and PstI and cloned into the pBluescript II KS vector (Stratagene, La Jolla, Calif.) which was linearized with the same enzymes.
Next, the neomycin resistance gene was amplified with the PCR primers NeoF (SEQ ID NO:3) and NeoR (SEQ ID NO:4) using pNTKV1907 (Stratagene) as a template. The 1.1 kb PCR fragment obtained was double digested with PstI and BamHI and cloned into the pKS-PGK vector that was linearized with the same enzymes.
The mouse thymidine kinase gene was then cloned into the pKS-PGK-Neo vector as follows. PCR primers TKF (SEQ ID NO:5) and TKR (SEQ ID NO:6) were used to amplify the TK gene from pNTKV1907 (Stratagene). The amplified 2kb fragment obtained was next digested with BamHI and SacI and finally cloned into pKS-PGK-Neo linearized with the same enzymes. This constructed vector was designated as pTK.
Finally, synthetic linker sequences were added. A first synthetic linker, F5R5, was made by annealing two primers: a forward primer sequence F5R5F (SEQ ID NO:7) and a reverse primer sequence F5R5R (SEQ ID NO:8). This linker was inserted between the Asp718l and HindIII sites of pTK and the resulting construct was designated pTK5.
A second synthetic linker, F3R3, was made by annealing the forward primer F3R3F1 (SEQ ID NO:9) and the reverse primer F3R32 (SEQ ID NO:10). This linker was inserted between NotI and BamHI sites of pTK and the resulting construct was designated pTK53.
2. Insertion of the Luciferase Reporter Sequences LucR and LucY/G into pTK53
Schematic diagrams of the starting vector pKS and the intermediate constructs pTK and pTK53 are shown in FIG. 9.
The backbone vectors pTK-LucR and pTK-LucY/G were then constructed by introducing the luciferase reporter sequences LucR and LucY/G into pTK53.
The yellow-green luciferase gene LucY/G was isolated from the vector pGL3B (Promega) and a HindIII-SalI fragment and was then cloned into pTK53 linearized with the same enzymes. A schematic diagram of the resulting backbone vector, designated pTK-LucYG (8931 bp), is shown in FIG. 10.
Similarly, the red luciferase gene LucR was isolated from the vector pGL3-red (obtained from Dr. Christopher Contag, Stanford University, Stanford, Calif.; Eames, B. F., et al., 1998, J. Invest. Med. 46 (1):94A) as a HindIII-SalI fragment and was then cloned into pTK53 linearized with the same enzymes. The resulting backbone vector, designated pTK-LucR (8931 bp), is also shown schematically in FIG. 10.
B. Insertion of Vitronectin and FosB Sequences
1. Insertion of Vitronectin Sequences into the pTK-LucYG and pTK-LucR Backbone Vectors to Produce the Intermediate Constructs pTKLG-Vn and pTKLR-Vn
Vitronectin (Vn) is an abundant glycoprotein present in plasma and the extracellular matrix of most tissues. In a previous study, it was shown that heterozygous mice carrying one normal and one null VN allele and homozygous null mice completely deficient in vitronectin demonstrate normal development, fertility, and survival. These results suggest that VN is not essential for cell adhesion and migration during normal mouse development (Zheng, X., et al., Proc Natl Acad Sci USA 1995 92:12426–30). The sequence of the vitronectin gene is available in the GenBank database (Accession No. X72091; SEQ ID NO:42).
a. Insertion of the Vitronectin Sequence into the pTK-LucR Backbone Vector to Produce the Intermediate Construct pTKLR Vn
The intermediate vector construct pTKLR-Vn was generated by inserting vitronectin (Vn) DNA sequences into the pTK-LucR vector constructed in Example 4A (see above).
A mouse vitronectin genomic DNA sequence of 5004 bp was obtained from the GenBank database (Accession No. X72091). The sequence served as the basis for designing the forward primer VN1F (SEQ ID NO:12) and the reverse primer VN1R (SEQ ID NO:11) which were used to PCR amplify a 1.63 kb 3′ end vitronectin fragment using mouse C57BL/6 genomic DNA as a template (Genome Systems, Inc., St. Louis, Mo.). The fragment obtained was digested with SwaI and NsiI and cloned into pTK-LucR (linearized with SwaI and SbfI). This intermediate construct was designated as pTK-LucR3.
Subsequently, a 2.35 kb 5′ end vitronectin fragment was PCR amplified from the same template DNA using the reverse primer VN2R (SEQ ID NO:13) and the forward primer VN2F (SEQ ID NO:14). This fragment was digested with SalI and AscI and cloned into pTK-LucR linearized with SalI and AscI. This construct was designated as pTKLR-Vn. FIG. 11 shows the restriction map of pTKLR-Vn.
The polylinker between the neomycin gene and the luciferase gene may be used to insert the VEGF promoter (see, e.g., Example 4C1 below) or other promoters or expression cassettes of interest.
b. Insertion of the Vitronectin Sequence into the pTK-LG Backbone Vector to Produce the Intermediate Construct pTKLG-Vn
pTKLG-Vn was made by replacing the red luciferase gene (excised as a NotI-SalI fragment) in pTKLR-Vn with the yellow green luciferase gene (isolated as a NotI-SalI fragment) from pGL3B2 (see above). The restriction map of pTKLG-Vn is shown in FIG. 11.
The polylinker between the luciferase gene and the neomycin gene may be used to insert promoters or expression cassettes of interest.
2. Insertion of FosB Sequences into the pTK-LucYG Backbone Vector to Produce the Intermediate Construct pTKLG-Fos
FosB is one of the members of the Fos family. It plays a functional role in transcriptional regulation. It has been shown that FosB mice are born at a normal frequency, are fertile and present no obvious phenotypic or histologic abnormalities (Gruda et al (1996) Oncogene 12:2177–2185).
A 28.8 kb genomic region that contains mouse FosB DNA was obtained from the GenBank database sequence (Accession No. AF093624). The sequence served as the basis for designing the PCR amplification primers FosB1F and FosB1R. The forward primer FosB1F (SEQ ID NO:15) and the reverse primer FosB1R (SEQ ID NO:16) were used to obtain a 1.71 kb 5′ end FosB fragment using mouse C57BL/6 genomic DNA as a template. This fragment was digested with SwaI and SbfI and cloned into pTK-LucYG linearized with SwaI and SbfI. This construct was designated as pTK-LucYG3.
Subsequently, a 1.58 kb 3′ end FosB fragment was amplified from the same template DNA with the forward primer FosB2F (SEQ ID NO:17) and the reverse primer FosB2R (SEQ ID NO:18). This fragment was digested with SalI and AscI and cloned into pTK-LucYG linearized with SalI and AscI. The construct was designated as pTKLG-Fos, and is depicted in FIG. 12.
The polylinker between the luciferase gene and the neomycin gene may be used to insert promoters and expression cassettes of interest.
C. Insertion of Expression Cassettes
1. Introduction of the VEGF Promoter Sequence into the Intermediate Vector Constructs pTKLR-Vn and pTKLG-Vn to Create the Expression Cassette Vectors pTKLR-Vn-VP and pTKLG-Vn-VP
pTKLR-Vn-VP and pTKLG-Vn-VP, depicted schematically in FIG. 13, were made by cloning the 7.8 kb PacI-NotI VEGF promoter fragment from pGL3B2-VP described above into the PacI and NotI sites in the polylinker regions of pTKLR-Vn and pTKLG-Vn, respectively.
2. Insertion of the VEGFR-2 Promoter-LucYG-VEGFR-2 Enhancer Cassette into the Intermediate Vector Constructs pTKLG-Vn and pTKLG-Fos to Create the Expression Cassette Vectors pTKLG-Vn-KPN and pTKLG-Fos-KPN
The VEGFR-2 promoter-luciferase-enhancer cassette was isolated from pGL3B2-KPN as a PacI-SalI fragment and cloned into the same sites of pTKLG-Vn. The resulting construct was designated pTKLG-Vn-KPN. See FIG. 14.
Similarly, the VEGFR-2 promoter-luciferase-enhancer cassette was isolated from pGL3B2-KPN (see above) as a PacI-SalI fragment and cloned into the same sites of pTKLG-Fos. See FIG. 12. The resulting construct was designated pTKLG-Fos-KPN. See FIG. 14.
3. Insertion of the Tie2 Promoter-LucYG-Tie2 Enhancer Cassette into the Intermediate Vector Construct pTKLG-Fos to Create the Expression Cassette Vector pTKLG-Fos-TPN.
The Tie2 promoter-Luciferase-enhancer cassette was isolated from pGL3B2-TPN as a PacI-SalI fragment and cloned into the pTKLG-Fos vector linearized with PacI and SalI. The resulting construct was designated pTKLG-Fos-TPN. See FIG. 15.
Example 5
High Through-Put Screening of Putative Angiogenesis Modulating Compounds via the VEGFR-2 Promoter Sequence
Putative angiogenesis-affecting compounds can be screened by monitoring their ability to modulate VEGFR-2 promoter-mediated bioluminescence in transfected cells. Vascular endothelial cells that are transfected with VEGFR2-Luc constructs, such as pGL3B2-KPN or pTKLG-Vn-KPN, are useful for high-through-put screening of compounds that can inhibit VEGFR2 promoter-mediated gene expression. This approach provides a means for discovering compounds capable of modulating angiogenesis and neoplasticity.
Briefly, transfected vascular endothelial cells prepared in the preceding example are plated into 96 well plates and used for high-through-put screening of a compound library. The compounds that reduce luciferase activity are selected for secondary screening. Upon confirmation by the secondary screening procedure, the candidate compounds are further tested in the VEGFR2-LucYG transgenic mouse models as described in Example 14 below.
As an example, the expression construct pGL3B2-KPN (see Example 2 above) was used to transiently transfect primary bovine endothelial cells (Clonetics) using lipofectamine (Promega). The cells were seeded onto 96-well plastic culturing plates (Nunc) prior to transfection. The transfection was carried out according to the manufacture's instructions (Promega). Plasmid pRL-TK (Promega), containing Renilla luciferase driven by the thymidine kinase promoter, was used as an internal control in all transfection experiments. The primary bovine endothelial cells were cultured in EGM-2 MV medium (Clonetics) at 37 C in 5% CO2, 95% air. After transfection, the cells were lysed with passive lysis buffer (Promega) and assayed with the Dual-Luciferase Reporter Assay System (Promega) for luciferase activity.
Several angiogenesis and neoplasticity inhibitors (Sigma) were then tested for their effects on VEGFR-2 promoter-mediated luciferase expression in the transfected primary bovine endothelial cells described above. The tested compounds included the neoplasticity inhibitor mithramycin, and angiogenesis inhibitors 2-methoxyestradiol, thalidomide, and fumagillin.
Briefly, 24 hours after transfection with pGL3B2-KPN, the cells were treated with selected angiogenesis and neoplasticity inhibitors for 36 hours and assayed for luciferase activity. Cells treated with mithramycin (1 μM), fumagillin (10 μM) and 2-methoxyestradiol (10 μM) all showed significant decrease of luciferase activity, indicating that these compounds can inhibit VEGFR2 expression mediated by the 4.5 kb VEGFR-2 promoter. See FIG. 16A.
These results suggest that sequences derived from the 4.5 kb VEGFR-2 promoter are useful for high-through-put screening for compounds capable of modulating VEGFR-2-mediated angiogenesis.
Example 6
High Through-Put Screening of Putative Angiogenesis Modulating Compounds Via the Tie2 Promoter Sequence
Putative angiogenesis-affecting compounds can be screened by monitoring their ability to modulate Tie-2 promoter-mediated bioluminescence in transfected cells. Vascular endothelial cells that are transfected with Tie2-Luc constructs (e.g., pGL3B2-TP) are useful for high-through-put screening of compounds that can inhibit Tie2 promoter-mediated gene expression. These approaches provide a means for discovering compounds capable of modulating angiogenesis and neoplasticity.
Briefly, transfected vascular endothelial cells prepared in the preceding example are plated into 96 well plates and used for high-through-put screening of a compound library. The compounds that reduce luciferase activity are selected for secondary screening.
As an example, the expression construct pGL3B2-TP prepared above (see Example 3B above) was used to transiently transfect primary bovine endothelial cells (Clonetics) using lipofectamine (Promega). The cells were seeded onto 96-well plastic culturing plates (Nunc) prior to transfection. The transfection was carried out according to the manufacture's instructions (Promega). Plasmid pRL-TK (Promega), containing Renilla luciferase driven by the thymidine kinase promoter, was used as an internal control in all transfection experiments. The primary bovine endothelial cells were cultured in EGM-2 MV medium (Clonetics) at 37 C in 5% CO2, 95% air. After transfection, the cells were lysed with passive lysis buffer (Promega) and assayed with the Dual-Luciferase Reporter Assay System (Promega) for luciferase activity.
Several angiogenesis and neoplasticity inhibitors (Sigma) were then tested for their effects on the expression of Tie-2 expression in the transfected primary bovine endothelial cells described above.
Briefly, 24 hours after transfection with pGL3B2-TP, the cells were treated with selected angiogenesis and neoplasticity inhibitors for 36 hours and assayed for luciferase activity. The tested compounds included the neoplasticity and angiogenesis inhibitors mithramycin, 2-methoxyestradiol, thalidomide and fumagillin. Cells treated with mithramycin (1 μM) and fumagillin (10 μM) showed significant decrease of luciferase activity, indicating that these compounds can inhibit Tie2 expression mediated by the 7.1 kb Tie2 promoter. See FIG. 16B.
These results suggest that sequences derived from the 7.1 kb Tie2 promoter are useful for screening compounds that may be capable of modulating Tie2-mediated angiogenesis.
Example 7
In vivo Monitoring of VEGFR-2-Mediated Angiogenesis During Development
Four lines of transgenic mice carrying the VEGFR-2 promoter-LucYG-VEGFR-2 enhancer expression cassette were obtained using pGL3B2-KPN combined with known methods for generating transgenic mice (see the discussion above). These animals (“founders”) were bred to non-transgenic mates to produce litters of from 8 to 10 pups (“F1 animals”).
Two F1 animals from each of the four VEGFR2 founders (designated KA, KC, KG and KJ) were continuously imaged from the age of one to six weeks according to the methods described in U.S. Pat. No. 5,650,135 to Contag et al. All of the mice imaged exhibited rapid reduction in VEGFR-2-mediated expression of bioluminescence over the course of development. All mice showed high level of signal when imaged at the age of 1 week. The signal decreased dramatically in all the mice when they were imaged at the age of 2 weeks. Further decrease of the signal was observed when the mice were imaged at 4 weeks and the signals decreased to basal levels when the mice reached 6 weeks. The observed signal intensities were quantified. As shown in FIGS. 17A, 17B, 17C, and 17D, the KC, KJ, KG and KA individuals, respectively, showed 7,000-, 7,000-, 1300-, and 20–1000 fold reductions, respectively, in the luciferase signals observed during the monitored development period.
These experiments demonstrate that the expression cassettes and transgenic animals of the present invention may be used to monitor VEGFR-2 promoter-mediated expression of bioluminescence in vivo over the course of development.
Example 8
In vivo Monitoring of VEGFR-2-Mediated Angiogenesis During Estrus
Two female VEGFR2 transgenic F1 mice from founder KA (See Example 7 above) were subjected to in vivo imaging of bioluminescence both dorsally and ventrally over a 16 day period according to the methods described in U.S. Pat. No. 5,650,135 to Contag et al. The results are presented graphically in FIGS. 18A–18D (FIGS. 18A and 18B, mouse 1 and 2 dorsal views, respectively, and FIGS. 18C and 18D, mouse 1 and 2 ventral views, respectively). Both mice exhibited cyclic variation in the levels of bioluminescence detected over periods of from 3 to 6 days. In general, higher levels of luminescence were detectable ventrally as compared to dorsally. The signals appeared to be localized in the ovary (dorsal view) and uterus (ventral view). Both ovaries were clearly visible in most of the dorsal views. The uterus, connecting tubes and ovaries were visible in some of the ventral views. Quantification of the signals showed more than a 100 fold difference in signal intensity over the course of the estrus cycle.
These results demonstrate that it is possible to monitor changes in VEGFR-2 promoter-mediated bioluminescence expression that occur during estrus in female mice carrying the VEGFR-2-Luc transgene cassette. They may be applied in a number of ways. For example, the transgenic mice may be used with the in vivo imaging protocol described to test the efficacy of estrogen antagonists (see the following example), to screen for endocrine disrupters, and to test the efficacy of compounds targeted to estrogen-dependent tumors.
Example 9
Examining the Effect of Endocrine Disrupters on VEGFR-2 Promoter-Mediated Angiogenesis During Estrus
The effect of the known estrogen antagonist ICI 182 780 (Tocris Cookson Ltd. Bristol, UK) on gene expression mediated by the VEGFR-2 promoter was investigated using the methods described in the preceding example.
A mature female VEGFR2 transgenic F1 mouse from founder KA (See Example 7 above) was imaged daily for 16 days according to the methods described in U.S. Pat. No. 5,650,135 to Contag et al. The bioluminescence signal remained high for a maximum of from 3–5 days, dropping back to a basal level for the interim periods.
The same mouse was subsequently treated with ICI 182 780 by subcutaneous injection at a dosage of 30mg/kg/day for another 16 day period, and was imaged daily for the duration of the treatment. After an initial lag of several days following commencement of treatment, the uterine signal remained high for 9 days before dropping back to basal level. See FIGS. 19A–19D (FIGS. 19A and 19B, dorsal and ventral views, respectively, before treatment; FIGS. 19C and 19D, ventral and dorsal views, respectively, after treatment).
These results suggest that transgenic animals carrying the expression constructs of the present invention may be used to investigate the effects of endocrine disrupting compounds, including but not limited to estrogen antagonists such as ICI 182 789, on VEGFR-2-mediated gene expression during the estrus cycle.
Example 10
Examining the Effect of Growth Factors on VEGFR-2 Promoter-Mediated Angiogenesis
The effect of the growth factors on gene expression mediated by the VEGFR-2 promoter was investigated.
Transgenic mice carrying the VEGFR-2 promoter-LucYG-VEGFR-2 enhancer expression cassette were obtained using pGL3B2-KPN by known methods for generating transgenic mice (see the discussion above). These founder animals were bred to non-transgenic mates to produce litters of from 8 to 10 F1 animals.
A mature F1 mouse carrying the VEGFR-2 promoter-LucYG-VEGFR-2 enhancer expression cassette was injected into the center of the abdominal midline with 500 μl of matrigel containing a growth factor cocktail (2 fig VEGF, 0.5 μg bFGF, 0.5 μg aFGF). A control F1 mouse carrying the VEGFR-2 promoter-LucYG-VEGFR-2 enhancer expression cassette was injected with matrigel alone. Both mice were imaged for 7 days after the matrigel injection. In the mouse that were injected with the growth factor containing matrigel, an induction of VEGFR2 mediated luciferase signal was detected 1 day after the matrigel injection. This signal went up at day 2 and reached a peak at day 4. By day 7, the signal dropped back to the basal level. This growth factor-mediated luciferase induction was absent in the mouse that was injected with matrigel alone. See FIG. 20.
This experiment demonstrates that transgenic animals carrying the expression constructs of the present invention may be used to investigate the effects of growth factors on VEGFR-2 gene expression during the angiogenesis process.
Example 11
Monitoring of VEGF Promoter-Mediated Bioluminescence in C57BL/6J-Tyrc-2J/+ Mice During Tumor Development Using Tumor Cell Lines Stably Transfected with pTKLG-Vn-VP
The pTKLG-Vn-VP vector construct described in Example 4 above was used to stably transfect several tumor cell lines including LL/2 (Lewis lung carcinoma), B 16-F1 (mouse melanoma) and T241 (mouse fibrosarcoma) to generate tumor cell lines expressing VEGF-mediated bioluminescence. The transfection was accomplished using lipofectamine as described in the manufacturer's instructions (Promega).
All tumor cells were cultured in DMEM medium plus 10% fetal bovine serum (Gibco BRL) at 37° C. in 5% CO2 and 95% air. After transfection, cells were selected with G418 (Gibco BRL) in a concentration range of 600–800 μg/ml. After 2–3 weeks of selection, stable clones were recovered and tested for luciferase expression.
A luciferase-expressing LL/2 clone was selected, and 1×106 cells from this clone were injected into C57BL/6J-Tyrc-2J/+ mice and monitored for luciferase activity for a period of 26 days. In three separate injection sites (FIG. 21, Mouse 1, solid diamonds; Mouse 2, solid squares; and Mouse 3, solid triangles), luciferase activities were detected 1 day after the injection. The signal reached a peak at day 10, followed by a decrease of the signal at day 11. The signal went up again from day 14 and reached a second peak at day 23. By day 26, the signal started to drop again and the mice died a few days after the last image. See FIG. 21.
This experiment demonstrates that stable tumor cells generated with the VEGF expression vector pTKLG-Vn-VP of the present invention can be used to investigate VEGF gene expression during tumor development.
Example 12
High Through-Put Screening of Putative Angiogenesis and Neoplasticity-Modulating Compounds Via the VEGF Promoter Sequence
The tumor cells stably transfected with pTKLG-Vn-VP that were generated in the preceding example are useful for high-through-put screening of compounds that can inhibit VEGF promoter-mediated gene expression. This approach provides a means for discovering compounds capable of modulating angiogenesis and neoplasticity.
Briefly, the stably transfected tumor cells or transfected vascular endothelial cells prepared in the preceding example are plated into 96 well plates and used for primary screening of a compound library. The compounds that reduce luciferase activity are selected for secondary screening. Upon confirmation by the secondary screening, the candidate compounds are further tested in the VEGF-Luc transgenic mice model or mice carrying VEGF-luc stable tumor cells, as described in Example 13 below.
Example 13
In vivo Monitoring of the Effects of Angiogenesis-Affecting Compounds on Tumor-Induced Angiogensis Mediated by VEGF
The effects of anti-angiogenesis compounds on tumor-induced VEGF expression may be tested in mice carrying the VEGF promoter expression cassettes of the present invention.
Transgenic mice carrying the VEGF promoter expression cassette are obtained by direct injection of the DNA into nuclei of zygotes derived from FVB mice. The FVB/VEGF transgenic mice are then bred with C57BL/6J-Tyrc-2J/+ mice to generate hybrid transgenic mice. The hybrid transgenic animals carry the VEGF-Luc transgene and also allow the growth of C57BL/6-derived tumor cells. C57BL/6-derived tumor cells, including but not limited to B16F1 (melanoma), LL/2 (Lewis Lung carcinoma), and T241 (mouse fibrosarcoma) cells are then introduced into the hybrid transgenic mice.
The effects of a variety of putative anti-tumorigenic anti-angiogenic agents, including SU5416, PD173034, TNP-470, 2-methoxyestradiol, paclitaxel and PTK787, are then tested by administering 2 doses per compound to each of 3 tumor-carrying hybrid VEGF transgenic mice. The mice are imaged according to the methods described in U.S. Pat. No. 5,650,135 to Contag et al., and the results compared with those obtained for untreated controls.
Alternatively, non-transgenic mice, such as C57BL/6J-Tyrc-2J/+ mice, can be implanted with tumor cells that have been stably transfected with the VEGF-Luc construct pTKLG-Vn-VP, including but not limited to, stably transfected B16F1 (melanoma) LL/2 (Lewis Lung carcinoma) and T241 (mouse fibrosarcoma) cells. The effects of the putative anti-tumorigenic anti-angiogenic agents listed above on VEGF expression are then tested by administering 2 doses per compound to each of 3 tumor-carrying mice. The mice are imaged according to the methods described in U.S. Pat. No. 5,650,135 to Contag et al., and the results are compared with those obtained for untreated controls.
Example 14
In vivo Monitoring of the Effects of Angiogenesis-Modulating Compounds on Tumor Induced Angiogenesis Mediated by VEGFR-2
The effects of anti-angiogenesis compounds on VEGFR-2 expression induced by implanted growth factor-containing matrigel and by implanted tumor cells may be tested in mice carrying the VEGFR-2 promoter expression cassettes of the present invention.
Transgenic mice carrying the VEGFR2 promoter expression cassette are obtained by direct injection of the DNA into nuclei of zygote derived from FVB mice. The FVB/VEGFR2 transgenic mice are then bred with C577BL/6J-Tyrc-2J/+ mice to generate hybrid mice that carry the VEGFR2-luc transgene and that allow the growth of C57BL/6 derived tumor cells. C57BL/6 derived tumor cells, including but not limited to B16F1 (melanoma) LL/2 (Lewis Lung carcinoma) and T241 (mouse fibrosarcoma) cells, are then introduced into the transgenic mice as discussed in Example 10 above.
The effects of a variety of putative anti-tumorigenic anti-angiogenic agents, including SU5416, PD173034, TNP-470, 2-methoxyestradiol, paclitaxel and PTK787, are then tested by administering 2 doses per compound to each of 3 tumor-carrying hybrid VEGFR2 transgenic mice. The mice are imaged according to the methods described in U.S. Pat. No. 5,650,135 to Contag et al., and the results compared with those obtained for untreated controls.
Alternatively, FVB/VEGFR2 transgenic mice, are implanted with growth factor-containing matrigel as discussed in Example 10. The effects of the putative anti-tumorigenic anti-angiogenic agents listed above on VEGFR2 expression during growth factor-induced angiogenesis process are then tested by administering 2 doses per compound to the matrigel-carrying mice. The mice are imaged according to the methods described in U.S. Pat. No. 5,650,135 to Contag et al., and the results are compared with those obtained for untreated controls.
As is apparent to one of skill in the art, various modification and variations of the above embodiments can be made without departing from the spirit and scope of this invention. These modifications and variations are within the scope of this invention.

## Claims

1. An isolated polynucleotide comprising either nucleotides 1 through 3,762 of SEQ ID NO:44 or a nucleic acid sequence that is at least 95% identical to the nucleotide sequence 1 through 3,762 of SEQ ID NO:44, wherein said isolated polynucleotide controls transcription of a gene operably linked thereto.

2. An isolated polynucleotide consisting of either SEQ ID NO:44 or a nucleic acid sequence that is at least 95% identical to the sequence of SEQ ID NO:44, wherein said isolated polynucleotide controls transcription of a gene operably linked thereto.

3. An expression cassette comprising:
(a) the isolated polynucleotide of claim 1 comprising a transcription control element; and
(b) a reporter sequence, operably linked to said transcription control element.

4. The expression cassette of claim 3, wherein said reporter sequence encodes a light-generating protein.

5. An expression cassette comprising:
(a) the isolated polynucleotide of claim 2 comprising a transcription control element; and
(b) a reporter sequence, operably linked to said transcription control element.

6. The expression cassette of claim 5, wherein said reporter sequence encodes a light-generating protein.

7. A vector comprising
(a) the isolated polynucleotide of claim 1 comprising a transcription control element;
(b) a reporter sequence, operably linked to said transcription control element; and
(c) a vector backbone.

8. The vector of claim 7, wherein said reporter sequence encodes a light-generating protein.

9. The vector of claim 7, wherein said vector backbone comprises an origin of replication.

10. The vector of claim 7, wherein said vector is a shuttle vector.

11. The vector of claim 7, wherein said vector backbone further comprises a selectable marker.

12. An isolated cell comprising the expression cassette of claim 3.