WO2003096986A2 - Compositions and methods for treatment of wounds - Google Patents

Abstract

The present invention provides for compositions of proteins, peptides, fragments and derivatives thereof, of p27, in modulating cell motility for prevention of metastases in tumors and to promote wound healing. In particular, the ability of p27 to induce cell migration was mapped to a C-terminal scatter domain (residues 118-158) that was independent of previously described cell cycle arrest functions of p27 that reside in the N-terminus.

Description

COMPOSITIONS AND METHODS FOR TREATMENT OF WOUNDS

The present application claims the benefit of U.S. provisional application number

60/381,310 filed May 17, 2002, and which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention.

The present invention provides for compositions of p27 for modulating cell migration and cytoskeletal rearrangement, including filopodia and lamellipodia. These compositions are important for treatment of cancer by preventing metastasis ofthe tumor, thereby allowing for treatment ofthe localized tumor. Further, by modulating cell motility, the present invention provides for accelerated wound healing, for example in the treatment of burn victims, thereby, decreasing the risk of infection, hemorrhaging and disfigurement.

2. Background. The presence of tumor metastases is one ofthe most significant factors affecting survival of cancer patients. Metastatic tumor growth is characterized by aberrant cell cycle regulation, loss of cell-cell contact, increased motility, adherence to and subsequent invasion of extracellular matrix at a secondary site, followed by uncontrolled proliferation and angiogenesis. However, molecular events that contribute to metastatic progression are currently poorly understood. Several lines of evidence suggest that hepatocyte growth factor (HGF) (also known as Scatter Factor) signaling contributes to metastasis via Met receptor signaling.

HGF was independently identified as a growth factor for hepatocytes and an effector of epithelial cell motility. In normal tissues, HGF is secreted by cells of mesenchymal origin to affect epithelial cells expressing the Met receptor tyrosine kinase. Met receptor activation initiates recruitment of adaptor proteins and phosphorylation of a variety of downstream signal transduction molecules. Homozygous deletion ofthe HGF gene results in embryonic lethality in mice, due to improper formation ofthe placenta, suggesting a critical role for HGF in development. However, deregulated and/or constitutive HGF/Met signaling can contribute to increased cell motility, invasion and proliferation in the context of tumorigenesis and metastasis. Indeed, increased expression of HGF and/or Met has been observed in numerous tumor types, including hepatocellular carcinomas.

Previously we reported that HGF signaling in human hepatocellular carcinoma cells results in increased cell migration, actin cytoskeletal rearrangement, including filopodia formation, and elevated levels ofthe p27 cyclinxdk inhibitor tumor suppressor protein. High levels of p27 protein have also been reported in several other human malignancies. p27, a member ofthe Cip/Kip family of cell cycle inhibitors, is a nuclear protein that negatively regulates Gi cell cycle progression by sequestering and inactivating cyclin E/A:Cdk2 complexes.

Although p27 is characterized as a tumor suppressor, inactivating point mutations with loss of heterozygosity are rarely observed in human cancer; rather, alteration ofthe machinery regulating p27 protein stability has been observed in numerous tumor cells. Indeed, p27 mRNA levels remain unchanged during the cell cycle, whereas p27 protein levels drop as cells transit from early G] phase into late Gi/S phase. Processes that regulate p27 protein degradation involve Thr- 187 phosphorylation by cyclin E:Cdk2 complexes followed by sCF^Skp2/Cksl complex mediated ubiquitination and degradation by the 26S proteosome. p27 degradation has now been reported to occur in both the cytoplasm and nucleus of cells.

HGF treatment of hepatocellular carcinoma cells results in increased cell motility, actin cytoskeletal rearrangements, and, paradoxically, stabilization ofthe p27 tumor suppressor protein. Although several studies have correlated a poor patient outcome with loss or low levels of p27 expression in multiple human malignancies, high levels of p27 tumor suppressor protein have been previously reported in several human malignancies, including: esophageal, colon, breast and small-cell lung carcinoma. Consistent with this observation, cytoplasmic localization of p27 has been correlated with anchorage-independent growth in culture, aggressive tumor progression and poor survival in vivo. In mammalian cells, migration is dependent upon rearrangement ofthe actin cytoskeleton by the Rho family of small GTPases, including Rac, Rho and Cdc42. Filopodia formation is a cdc42-dependent event that produces actin microspikes at the cell membrane that aid the motility of some cell types. Transduced p27 protein induces filopodia formation, suggesting that activation of cdc42 is a downstream event in a p27- dependent pathway.

It would be advantageous to provide therapeutic treatment of various diseases states whereby either the cell growth cycle is arrested or motility of cells is inhibited (such as metastatic tumor cells), or alternatively, provide for increased motility cells for wound healing. Especially advantageous would be the use of such treatment whereby cell cycle growth arrest and motility of cells are independently treated using similar molecules.

SUMMARY OF THE INVENTION

The invention provides for compositions of p27 proteins, peptides, derivatives and fragments thereof for the prevention of metastasis of tumor cells; cell growth arrest in diseases characterized by cell cycle abnormalities; and for expedited wound healing, especially in diseases where wound healing is impeded by disease such as diabetes.

In particular, the invention defines a novel role for p27 in cell motility in response to HGF signaling that is independent ofthe G_\ cell cycle arrest functions of p27. This is of importance in the treatment of wounds, especially in diseases which result in the slow healing of wounds such as, for example, diabetes. For example, hepatocyte growth factor (HGF) signaling resulted in p27 phosphorylation on Ser- 10 that was coupled with nuclear export of p27 into the cytoplasm. Cytosolic p27 localization was required for p27-mediated cell motility and actin cytoskeletal rearrangements, independent of cell cycle arrest.

In a preferred embodiment, the invention provides for a p27 fusion protein comprising a covalently linked protein transduction domain and a p27 domain, wherein the p27 domain further comprises full length p27 or fragments thereof. The transducing domain ofthe fusion protein ofthe invention is preferably comprises TAT protein or fragments thereof.

In a preferred embodiment, the invention provides for a p27 fusion protein comprising a covalently linked protein transduction domain and a p27 domain, wherein the p27 domain comprises full length p27 or comprises at least about 50%, 60%, 70%, 80%, 90%) or 95% ofthe amino acid sequence ofthe wild type full length p27 domain, but need not contain the entire amino acid sequence ofthe wild type full length p27 domain, i.e. the p27 domain ofthe fusion protein may contain less than 100%, 95%, 90%, 80%, 70%, 60% or 55% ofthe amino acid sequence ofthe wild type full length p27 domain. The percentage of amino acids, as compared to the full length p27 (100%) comprising the desired length ofthe p27 domain can be, for example, identical in sequence to wild type p27 protein or may contain mutations as discussed herein. The p27 fusion protein preferably comprises a transducing domain comprising a TAT protein; and, comprises one or more His and/or HA-epitope tags. As used herein,

"percentage" of a protein or peptide refers to the number of consecutive amino acids that comprise a desired peptide as compared to the full length wild type protein. By way of example if the wild type full length protein is 100 amino acids long from the carboxy 1 to the amino terminal end, then a peptide that comprises at least about 50% ofthe amino acid sequence ofthe wild type full length protein will comprise about 50 amino acids. The 50 amino acids do not have to correspond to amino acid positions in the wild type protein but can comprise mutations, deletions and the like. For example, the 50 amino acids can comprise any number of amino acids selected from any portion ofthe wild type protein. If the desired protein is the p27 domain and the desired fragment comprises about 50%) ofthe p27 domain then about 50% ofthe amino acids that comprise the full length p27 domain can be selected from the N-terminal end, the C-terminal end or any position thereof. The peptide may also comprise only N-terminal or C-terminal amino acid sequences.

In one embodiment ofthe invention, the p27 fusion protein further comprises 6x-

His and HA-epitope tags useful for intracellular detection ofthe fusion protein as described in the examples which follow. In another embodiment ofthe invention, mutants of wild type p27 are generated in the various domains of p27 such that, transduction of said mutants result in increased cell motility as defined by cell motility assays, and allow for the locomotion of cells to wounded areas ofthe patient thereby allowing for wound healing.

Preferred mutants are described in the Examples which follow, but any mutations which modulate the function of p27 are preferred. For example, p27 mutations which cause the arrest ofthe cell cycle in the Gl phase, thereby inhibiting tumor growth; p27 mutations which inhibit motility of cells such that metastatic tumor cells are inhibited from metastasizing to different locations in patient. In this case, these mutants are important for treatment of, for example, malignancies.

In another aspect ofthe invention, the fusion proteins are transduced in epithelial and/or non epithelial cells thereby allowing for treatment of skin wound healing, skin disorders such as melanomas; wound healing of tissues and organs due to, for example, surgery; treatment of epithelial and underlying tissues, such as for example, in burn victims; inhibition ofthe growth cycle in, for example, diseases associated with abnormal cell growth such as cancer; inhibition of metastatic tumor cells by inhibiting the locomotion of tumor cells.

In general, the fusion proteins ofthe invention are used therapeutically where cell functions, such as cell motility, filopodia formation, and growth, can be modulated. Examples include but are not limited to regeneration of blood vessel linings after treatment with, for example, catheters for atherosclerotic patients; hemophiliacs; diseases associated with slow wound healing; healing of surgical wounds or accident victims and the like.

Treatment of patients is not limited to mammals such as humans, and livestock but can be used for treating any species which express p27.

Preferred fusion proteins ofthe invention comprise a TAT domain covalently linked to p27 proteins, peptides, fragments, or derivatives thereof. One example of a preferred p27 mutant is one which retains the at least about 20 carboxy terminal amino acid residues, more preferably at least about 50 carboxy terminal, most preferably at least about 100 carboxy terminal amino acid residues. In a preferred embodiment the p27 protein domain is comprised amino acid residues 105 to 198 or fragments thereof. In another aspect ofthe invention, the p27 carboxy terminus comprises amino acid residues 118 to 158 or fragments thereof.

In another aspect ofthe invention, the carboxy terminal portion ofthe p27 protein may comprise mutations that modulate the motility of cells when the fusion proteins ofthe invention are transduced into said cells.

In another preferred embodiment the p27 domain ofthe fusion proteins comprises a serine to alanine substitution at amino acid position 10. In another aspect ofthe invention the p27 domain ofthe fusion protein ofthe invention comprises a serine amino acid at position 10 of the amino terminal portion ofthe p27 protein. In another aspect ofthe invention the p27 fusion protein which comprises a serine at amino acid position 10 is phosphorylated by Hepatocyte growth factor activated kinase resulting in nuclear export of said fusion protein into the cytoplasm and results in the generation of filopodia. Filopodia formation is independent of

cell cycle arrest as described infra, and is therefore of significance in modulating one aspect of a cell function such as cell motility, while leaving another cell function unhindered such as, a normal cell growth cycle. This underscores the importance for using the fusion proteins ofthe invention for treatment of wounds without interfering with the growth cycle ofthe cell.

In another preferred embodiment, the p27 protein ofthe fusion proteins ofthe invention comprise the amino terminal portion ofthe p27 protein. As described infra, cell cycle arrest functions map to the amino terminal portion ofthe p27 protein. This again underscores the importance of being able to treat another disease condition such as tumor cell growth or inhibition of metastasis of tumor cells without affecting other cell functions.

In preferred embodiments, the p27 protein may comprise amino terminal and carboxy terminal portions ofthe p27 protein. Either of these domains may comprise one or more mutations, or be of varying length. The choice ofthe domain will be dictated by the disease which is sought to be treated.

In another aspect ofthe invention, a cocktail of fusion proteins, comprises various mutations, amino acid residues, fragments, and the like may be administered to a patient in need of such therapy.

In another aspect ofthe invention, antisense oligonucleotides are used for the treatment of patients suffering from or susceptible to diseases, such as those characterized by abnormal cell growth, metastasis of tumor cells, wound healing and the like.

In another aspect, the invention provides for a method for selectively transducing pathologic hyperproliferative mammalian cells comprising retroviral-mediated transduction of pathologic cells with a nucleic acid encoding a gene product ofthe fusion proteins ofthe invention. The methodology provided involves the introduction of a stably expressed gene which produces the fusion proteins ofthe invention into a heterogeneous cell preparation, for example, a heterogeneous cell preparation comprising both normal and pathologic hyperproliferative cells; and, under suitable conditions, selectively transducing phenotypically pathologic hyperproliferative cells; wherein, such treatment results in suppressing the pathologic phenotype; and, reinfusing the treated cell preparation into the patient.

Also provided by this invention is a method for treating a pathology in a subject patient caused by the absence of, or the presence of a pathologically mutated p27 gene. For example, using antisense nucleic acids which suppress the activity ofthe pathologically mutated p27 and/or delivering wild type p27 into the subject patient to restore normal functions ofthe p27.

Other aspects ofthe invention are disclosed infra.

BRIEF DESCRIPTION OF THE FIGURES Figure IA and IB shows that HGF signaling induces nuclear export of p27 to the cytoplasm. Figure 1 A is a photograph showing an analysis of endogenous cellular p27 localization using immunocytochemistry. Figure IB is a graph showing that nuclear export of p27 occurs in a Crm-1 dependent fashion. HepG2 cells were treated with HGF or PBS control for 20 hr, followed by treatment with leptomycin B (LMB; hatched bars) or mock treatment (solid bars) for an additional 3 hr. Cells were fixed and stained for p27 (dark blue) or cyclin B (light blue). Data are plotted as percentage of cells having a significantly greater extent of nuclear versus cytoplasmic immuno-detectable protein.

Figures 2 A and 2B shows photographs of immunocytochemically stained HGF treated cells. Cyclin A:Cdk2 complexes remain in the nucleus and cytoplasmic p27 localizes with actin in HGF treated cells. Figure 2 A is a photograph of results obtained with HepG2 cells treated with control PBS or HGF for 24 hr and analyzed by immunocytochemistry for cyclin A (FITC; green) and Cdk2 (TRITC; red) localization. Corresponding nuclei were counterstained with DAPI (blue). Figure 2B is a photograph showing that p27 co-localizes with F-actin. HepG2 cells treated with HGF for 24 hr were analyzed by immunocytochemistry for endogenous p27 (FITC; green) and F-actin (TRITC; red) localization. Corresponding nuclei were counterstained with DAPI (blue). Arrows indicate areas in merged image with co-localized (yellow) cytoplasmic p27 and F-actin.

Figure 3A -3B are immunoblots showing that endogenous p27 is phosphorylated on Ser- 10 in response to HGF. Figure 3 A shows an immunoblot of HepG2 cells treated with HGF. HepG2 cells treated with HGF for 20 hr were metabolically labeled with [³²P]-orthophosphate followed by anti-p27 immunoprecipitation (top panel). Total p27 protein levels were determined by anti-p27 immunoblotting (bottom panel). Figure 3B is an immunoblot showing HGF- dependent Ser- 10 phosphorylation. p27 was immunoprecipitated from lysates of control and HGF treated HepG2 cells with pan anti-p27 antibodies followed by immunoblot analysis with anti-phospho-Thr-187 (top panel), anti-phospho-Ser-10 (middle panel) and pan anti-p27 (bottom panel) antibodies. Figure 4A is a schematic representation ofthe generation of transducible TATp27 fusion proteins. The N-terminal leader contains the 11 amino acid TAT protein transduction domain, hemaglutinin (HA) epitope tag and six-histidine purification tag. (CBD is the cyclinxdk binding domain of p27). TATp27-WT represents full-length wild type ρ27 protein, whereas TATρ27- 158 and TATp27-l 18 designate terminal truncations at respective residues. TATp27-QM contains four single point mutations in actin interacting domain of Farlp-like motif. TATp27- KK contains two inactivating point mutations that disrupt p27 binding to cyclinxdk complexes. TATρ27-S10A contains an Alanine substitution for Serine at residue 10.

Figure 4B is a photograph of results obtained with immunocytochemistry showing the intracellular localization of TATp27 proteins. HepG2 cells were treated with control PBS or transducible TATp27 proteins and visualized by immunocytochemistry using anti-HA antibody and FITC-conjugated secondary antibody.

Figure 4C is a graph showing the cell cycle analysis of transduced primary cells. Contact inhibited, G] arrested and released human SiFT diploid fibroblasts were treated with control PBS and transducible TATp27 proteins as indicated for 24 hr followed by cell cycle analysis. Data represent the mean of three independent observations.

Figure 5 A is a photograph showing that transducible TATp27 proteins induce cell migration. HepG2 cells were treated for 24 hr with control PBS, HGF or TATp27 fusion proteins as indicated and observed for cell scattering. The scattering phenotype is characterized by loss of cell-cell contact and increased distance between neighboring cells.

Figure 5B is a graph showing the quantification of cell migration. Cells treated in figure 5 A above were stained with hematoxylin and recorded by digital microscopy at 0, 8 and 24 hr post-treatment. Distance between colony nuclei measured at 0 hr was subtracted from distance at 8 hr or 24 hr to calculate average distance of migration. Data represents a minimum of 50 random fields and 300 measurements per treatment for each of three separate experiments. Figure 5C is a photograph using immunostaining to show TATp27 protein localization in response to HGF. HepG2 cells were treated with control PBS (left panels) or HGF (right panels) for 20 hr, followed by treatment with control PBS, TATρ27-WT protein or TATp27-S10A protein for an additional 3 hr. TATp27 proteins were visuahzed by immunostaining with anti- HA antibodies (HA epitope tag is present in N-terminal leader of all TAT-fusion proteins) and FITC-anti-mouse IgG (green). Corresponding nuclei were counterstained with DAPI (blue).

Figure 6 A is a photograph showing wound-migration analysis of wild type and p27- deficient murine embryonic fibroblasts (MEFs). A wound area was mechanically induced by a single passage of a microtome blade across culture plate surface to confluent MEF monolayers. After 24 hr, cells were fixed and stained with hematoxylin. The wound edge is noted as a digitally-drawn line over the image.

Figure 6B is a photograph showing confluent p27-deficient 3T3 cells which were subjected to the wound-migration assay described in (A) by single passage of a rubber policeman followed by treatment with transducible TATp27 proteins. p27-WT and p27-158 proteins rescued wound-migration defect, whereas mock treatment (PBS) and TATp27-l 18 protein (lacking the p27 scatter domain) failed to induce cell migration. The wound edge is noted as a digitally-drawn line over the image.

Figure 7 is a schematic representation showing a model of HGF signaling via p27 to mediate cell migration. HGF binding to the Met receptor activates HAcK (HGF-Activated Kinase), that phosphorylates Ser-10 on p27. Phosphorylation of Ser-10 is required for export of p27 from the nucleus to the cytoplasm in a Crml -dependent fashion. In the cytoplasm, p27 interacts directly/indirectly with actin remodeling proteins, thereby inducing cytoskeletal rearrangement accompanied by cell migration. p27-dependent cell migration requires Ser-10 phosphorylation and the p27 scatter domain (residues 118-158), but not a functional cyclinxdk binding domain. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for TAT p27 fusion proteins, peptides, derivatives and fragments thereof, for treatment of diseases such as metastatic cancer by inhibiting cell cycle and motility of cells. The invention also provides for the promotion of wound healing by increasing the cell migration of both epithelial and non-epithelial cells, for example, fibroblasts.

In particular, the role of ρ27 protein was investigated in response to HGF activation of the Met receptor in human hepatocellular carcinoma cells. It was found that HGF signaling resulted in p27 phosphorylation on Ser-10 that was coupled with nuclear export of p27 into the cytoplasm. Cytosohc p27 localization was required for p27-mediated cell motility and actin cytoskeletal rearrangements, independent of cell cycle arrest. Taken together, these observations define a novel cytosolic-dependent role for p27 in cell motility.

In a preferred embodiment, the invention provides for a p27 fusion protein comprising a covalently linked protein transduction domain and a p27 domain, wherein the p27 domain comprises full length p27 or comprises at least about 50%, 60%, 70%, 80%, 90% or 95% ofthe amino acid sequence ofthe full length p27 domain. The percentage of amino acids, as compared to the full length p27 (100%) comprising the desired length ofthe p27 domain can be, for example, identical in sequence to wild type p27 protein or may contain mutations as discussed herein. The p27 fusion protein preferably comprises a transducing domain comprising a TAT protein; and, comprises one or more His and/or HA-epitope tags.

Preferred p27 fusion proteins comprises p27 carboxy terminal amino acids, wherein the carboxy terminal can comprise amino acid residues 105 to 198.

In accordance with the invention, the fusion protein is transduced into mammalian cells. The transduced fusion protein induces cell migration for the promotion of wound healing, as determined by cell motility assays. Preferred mutants include but are not limited to p27 fusion proteins wherein the carboxy terminal amino acid residues 118 to 158 are deleted. Preferably, the p27 fusion protein induces formation of filopodia and lamellipodia. In another preferred embodiment, the p27 fusion protein is transduced into tumor cells to inhibit metastasis. Preferred mutants, include but are not limited to p27 fusion proteins comprising, for example, a serine to alanine mutation at amino acid residue number 10 of ρ27.

hi another preferred embodiment, the invention provides for a method of treating a patient suffering from or susceptible to tumor cell growth, the method comprising administering to the patient a therapeutically effective amount of a p27 fusion protein. The p27 fusion protein preferably comprises a covalently linked protein transducing domain and a p27 domain, wherein the protein is lacking the functional carboxy terminus scatter domain, as measured by cell migration assays. The scattering domain comprises carboxy terminal amino acid residues 118- 158.

In another preferred embodiment, the invention provides for a method of treating a patient suffering from, or susceptible to, metastatic tumor cells, the method comprising admimstering to the patient a therapeutically effective amount of p27 fusion protein which lacks the serine amino acid at position 10. The various methods of treating the patient are discussed below. For example, antisense oligonucleotides are administered to a patient to inhibit the motility of cells and for treating metastasis of tumor cells.

In another preferred embodiment, the invention provides for a method for treatment of wounds, the method comprising topical or systemic administering to a patient with p27 fusion proteins which increase cell motility; wherein, cells migrate into the wound area and accelerate healing. The transduced cells can be epithelial or non-epithelial cells. Especially preferred are fibroblasts. The fusion proteins are preferably comprises full length p27 protein, or for example, the proteins comprise of p27-158 ( amino acid positions 1 to amino acid position 158).

In accordance with the invention, the novel role for p27 in cell migration and wound healing that is independent of cell cycle arrest functions, is important for the therapeutic use of p27 proteins, peptides, fragments and derivatives thereof, in diseases with cell cycle abnormalities or diseases associated with hyperproliferative cell disorders, wound healing, disease whereby wound healing is slow such as in diabetes, and burn victims.

As an illustrative example, which is not meant to limit or construe the invention in any way, the fusion proteins and/or oligonucleotides ofthe invention are used in promoting wound healing. Migration of cells into a wounded area to drive closure remains a rate-limiting step in wound healing. Therefore, application of agents that enhance cell migration by either topical, local or systemic delivery, pending the extent ofthe wounded area and/or individual, would serve to expedite the wound healing process. In this illustrative example, a wounded area is treated with transducible p27 proteins and/or derivative protein domains and peptides to enable and enhance cell migration. By applying the above transducible p27 proteins, peptides or derivatives thereof, serve to recruit cells into the wounded spaces and result in dramatic decreases in time to heal the wounded area. The proteins and/or the oligonucleotides ofthe invention recruit epithelial and non-epithelial cells, especially fibroblasts, to the wounded area. Preferred proteins or peptides include, but are not limited to, p27-WT and p27-158 proteins (see figure 6 and the examples which follow. Decreasing the healing time has the advantage that such a treatment will result in a dramatic reduction in patient exposure to pathogenic microorganisms, decreased rates of dehydration, and decreases costs of hospital visits. Administration ofthe therapeutic molecules ofthe invention can be either topically or systemically. Methods are described infra.

In accordance with the invention, the oligonucleotides, proteins, peptides, mutants and fragments thereof are important in the therapy of cancer. The ability of these molecules to prevent anti-metastases of tumor cells by use ofthe molecules ofthe invention, lacking or comprising mutations in the terminal scattering domain which result in a non-functional terminal scattering protein, is an important function ofthe molecules. For example, tumors treated with such molecules arrest cell cycle growth and inhibit the metastasis ofthe tumor cells. Preferred molecules are described, but are not restricted to the Examples which follow. As used herein, the term "hyperproliferative cells" includes but is not limited to cells having the capacity for autonomous growth, i.e., existing and reproducing independently of normal regulatory mechanisms. Hyperproliferative diseases may be categorized as pathologic, i.e., deviating from normal cells, characterizing or constituting disease, or may be categorized as non-pathologic, i.e., deviation from normal but not associated with a disease state. Pathologic hyperproliferative cells are characteristic ofthe following disease states, thyroid hyperplasia— Grave's Disease, psoriasis, benign prostatic hypertrophy, Li-Fraumeni syndrome including breast cancer, sarcomas and other neoplasms, bladder cancer, colon cancer, lung cancer, various leukemias and lymphomas. Examples of non-pathologic hyperproliferative cells are found, for instance, in mammary ductal epithelial cells during development of lactation and also in cells associated with wound repair. Pathologic hyperproliferative cells characteristically exhibit loss of contact inhibition and a decline in their ability to selectively adhere which implies a change in the surface properties ofthe cell and a further breakdown in intercellular communication. These changes include stimulation to divide and the ability to secrete proteolytic enzymes. The present invention will allow for high dose chemotherapy and/or radiation therapy, followed by autologous bone marrow reconstitution with hematopoietic cell preparations in which phenotypically pathologic cells have been reconstituted with genes encoding the fusion proteins ofthe invention. Application ofthe present invention will result in diminished patient relapses which occur as a result of reinfusion of pathologic hyperproliferative cells contaminating autologous hematopoietic cell preparations.

The p27 scatter domain is required for induction of both cell migration and actin cytoskeletal rearrangements, specifically, filopodia formation. Transduction or microinj ection of constitutively active cdc42 protein into cells results in filopodia formation in less than 10 min. In contrast, the first signs of filopodia formation and cell migration are not detectable until >4 hr post-treatment with TATp27 protein. Importantly, TATp27 reaches an intracellular maximum concentration in <15 min. Therefore, it is unlikely that unmodified p27 alone is capable of activating cdc42 directly and suggests that p27 either requires post-translational modification and/or assembly into macromolecular complexes prior to becoming competent to activate Rho GTPases. Rescue ofthe cell migration defect in p27-deficient murine embryonic fibroblasts (MEFs) and 3T3 cells by p27 reconstitution genetically places p27 in a cell motility pathway, independent of its cell cycle arrest functions. Whereas hepatocytes are epithelial cells and express the Met receptor, fibroblasts are non-epithelial cells that do not express Met. This broadens the involvement of p27 in cell motility to non-epithelial cells and p27 can be important in non-Met receptor signaling pathways. Indeed, development and tissue regeneration following injury are biological processes that require tight coupling of cell migration and cell cycle regulation.

Metastatic tumor growth is one ofthe most important factors in predicting the survival of cancer patients. HGF/c-Met signaling mediates phenotypes characteristic of tumor progression and enhanced metastatic potential. HGF treatment of hepatocellular carcinoma cells results in increased cell motility, actin cytoskeletal rearrangements, and, paradoxically, stabilization ofthe p27 tumor suppressor protein. Although several studies have correlated a poor patient outcome with loss or low levels of p27 expression in multiple human malignancies, high levels of p27 tumor suppressor protein have been previously reported in several human malignancies, including: esophageal, colon, breast and small-cell lung carcinoma. Consistent with this observation, cytoplasmic localization of p27 has been correlated with anchorage-independent growth in culture, aggressive tumor progression and poor survival in vivo.

Here, a novel role for p27 in cell motility is defined, in response to HGF signaling that is independent ofthe Gi cell cycle arrest functions of p27. The data presented here support a model in which an HGF- Activated Kinase (HAcK) phosphorylates p27 on Ser-10 resulting in nuclear export of p27 to the cytoplasm (Figure 7). These events then position p27 for its function in cell migration and cytoskeletal rearrangement, particularly filopodia formation. Consistent with these observations, we detected cytoplasmic p27 localized to areas of active cytoskeletal rearrangement. Importantly, the ability of p27 to induce cell migration mapped to a C-terminal scatter domain (residues 118-158) that was independent of previously described cell cycle arrest functions of p27 that reside in the N-terminus and mapped to a C-terminal scatter domain (residues 118-158). Loss or disruption ofthe C-terminal p27 scatter domain abolishes both actin rearrangement and cell scattering. However, the scatter domain is not sufficient for cell migration as HAcK phosphorylation of p27 on Ser-10 and export to the cytoplasm are also required for p27-mediated cell migration. Indeed, p27 harboring an Ala substitution at position 10 failed to be exported from the nucleus in an HGF-dependent manner and also failed to induce cell migration.

Subcellular localization of p27 is a regulated process that has been the subject of intense investigation. Here, it is demonstrated that HGF-induced nuclear export of p27 occurs in a Crm- 1 dependent manner that was dependent on HAcK phosphorylation of Ser- 10 on ρ27. However, p27 has no intrinsic nuclear export sequence (NES) motif, suggesting that cytoplasmic translocation occurs in conjunction with nuclear export proteins. Although Jabl, a nuclear export protein, has been identified as a p27 binding protein via yeast 2-hybrid assays that mediates nuclear export of p27, direct interaction between endogenous proteins has not yet been detected. Thus, biochemical identification of both the HAcK and the nuclear export protein(s) that recognize phospho-Ser-10 on p27 is important to determine this mechanism.

The p27 scatter domain is required for induction of both cell migration and actin cytoskeletal rearrangements, specifically, filopodia formation. These observations are evolutionarily reminiscent of alpha factor pheromone signaling in yeast. Farlp, the yeast cell cycle inhibitor, mediates both a G_\ cell cycle arrest and schmoo formation that orients the actin cytoskeleton toward opposite mating partner. In response to alpha factor, Farlp translocates from the nucleus to the cytoplasm and interacts with cdc42p. Consistent with this functional analogy, mutation of a Farlp-like sequence motif present in the p27 scatter domain inactivates both cell migration and induction of filopodia formation functions of p27, but preserves the cell cycle arrest functions.

In mammalian cells, migration is dependent upon rearrangement ofthe actin cytoskeleton by the Rho family of small GTPases, including Rac, Rho and Cdc42. Filopodia formation is a cdc42-dependent event that produces actin microspikes at the cell membrane that aid the motility of some cell types. Transduced p27 protein induces filopodia formation, suggesting that activation of cdc42 is a downstream event in a p27-dependent pathway. Transduction or microinjection of constitutively active cdc42 protein into cells results in filopodia formation in less than 10 min. In contrast, the first signs of filopodia formation and cell migration are not detectable until >4 hr post-treatment with TATp27 protein. Importantly, TATp27 reaches an intracellular maximum concentration in <15 min. Therefore, it is unlikely that unmodified p27 alone is capable of activating cdc42 directly and suggests that p27 either requires post- translational modification and/or assembly into macromolecular complexes prior to becoming competent to activate Rho GTPases.

As used herein, "p27 transducing proteins" or "TATp27 fusion proteins" will be used interchangeably. These terms refer to p27 wild type, peptides, fragments, derivatives or mutants thereof, fused to a transducing protein, preferably an HIN TAT protein or fragment thereof. The TAT proteins useful in the invention have been described in US Patent No: 6,221,355 to Dowdy, which is incoφorated herein by reference.

As used herein, a cell has been "transformed", "transduced", by exogenous or heterologous nucleic acids and/or amino acids, proteins and the like, when such nucleic and/or amino acids, proteins and the like, have been introduced inside the cell.

In general, the transduction domain ofthe fusion molecule can be nearly any synthetic or naturally-occurring amino acid sequence that can transduce or assist in the transduction ofthe fusion molecule. For example, transduction can be achieved in accord with the invention by use of a protein sequence and particularly an HIV TAT protein or fragment thereof that is covalently linked to the fusion molecule. Alternatively, the transducing protein can be the Antennapedia homeodomain or the HSV VP22 sequence, or suitable transducing fragments thereof such as those known in the field.

The type and size ofthe transducing amino acid sequence will be guided by several parameters including the extent of transduction desired. Preferred sequences will be capable of transducing at least about 20%, 25%, 50%, 75%, 80% or 90% ofthe cells of interest, more preferably at least about 95%, 98%% and up to about 100% ofthe cells. Transduction efficiency, typically expressed as the percentage of transduced cells as compared to the total number of cells, can be determined by several conventional methods such as those specific microscopical methods discussed below (e.g., flow cytornetric analysis).

Additionally preferred transducing sequences will manifest cell entry and exit rates (sometimes referred to as ki and k₂, respectively) that favor at least picomolar amounts ofthe fusion molecule in the cell. The entry and exit rates ofthe amino acid sequence can be readily determined or at least approximated by standard kinetic analysis using detectably-labeled fusion molecules. Typically, the ratio ofthe entry rate to the exit rate will be in the range of from between about 5 to about 100 up to about 1000.

Particularly preferred, are transducing amino acid sequences that include at least a peptide featuring substantial alpha-helicity. It has been discovered that transduction is optimized when the transducing amino acid sequence exhibits significant alpha-helicity. Also preferred are those sequences having basic amino acid residues that are substantially aligned alone at least one face ofthe peptide. Typically such preferred transduction sequences are synthetic protein or peptide sequences.

Additional transducing sequences in accord with this invention include a TAT fragment that comprises at least amino acids 49 to 56 of TAT up to about the full-length TAT sequence. A preferred TAT fragment includes one or more amino acid changes sufficient to increase the alpha-helicity of that fragment. In most instances, the amino acid changes introduced will involve adding a recognized alpha-helix enhancing amino acid. Alternatively, the amino acid changes will involve removing one or more amino acids from the TAT fragment that impede alpha helix formation or stability. In more specific embodiments, the TAT fragment will include at least one amino acid substitution with an alpha-helix enhancing amino acid. Preferably the TAT fragment is made, for example, by standard peptide synthesis techniques although recombinant DNA approaches may be preferred in some cases. Additional transduction proteins of this invention include the TAT fragment in which the TAT 49-56 sequence has been modified so that at least two basic amino acids in the sequence are substantially aligned along at least one face ofthe TAT fragment and preferably the TAT 49-56 sequence. In one embodiment, that alignment is achieved by making at least one specified amino acid addition or substitution to the TAT 49-56 sequence. Illustrative TAT fragments include at least one specified amino acid substitution in at least amino acids 49-56 of TAT which substitution aligns the basic amino acid residues ofthe 49-56 sequence along at least one face of the segment and preferably the TAT 49-56 sequence.

Additional transduction proteins in accord with this invention include the TAT fragment in which the TAT 49-56 sequence includes at least one substitution with an alpha-helix enhancing amino acid. In one embodiment, the substitution is selected so that at least two basic amino acid residues in the TAT fragment are substantially aligned along at least one face of that TAT fragment. In a more specific embodiment, the substitution is chosen so that at least two basic amino acid residues in the TAT 49-56 sequence are substantially aligned along at least one face of that sequence.

Additionally provided are chimeric transducing proteins that include parts of at least two different transducing proteins. For example, chimeric transducing proteins can be formed by fusing two different TAT fragments, e.g., one from HIV-1 and the other from HIV-2. Alternatively, other transducing proteins can be formed by fusing a desired transducing protein to heterologous amino acid sequences such as 6xHis, (sometimes referred to as "HIS"), EE, HA or Myc.

As mentioned above, the fusion molecule ofthe present invention also includes a fused p27 domain. p27, a member ofthe Cip/Kip family of cell cycle inhibitors, is a nuclear protein that negatively regulates G_\ cell cycle progression by sequestering and inactivating cyclin E/A:Cdk2 complexes. p27-deficient mice present multiple organ hyperplasia, suggesting that p27 is an important regulator of cell differentiation and proliferation in vivo. Although p27 is characterized as a tumor suppressor, inactivating point mutations with loss of heterozygosity are rarely observed in human cancer; rather, alteration ofthe machinery regulating p27 protein stability has been observed in numerous tumor cells. Indeed, p27 mRNA levels remain unchanged during the cell cycle, whereas p27 protein levels drop as cells transit from early Gi phase into late Gi/S phase. Processes that regulate p27 protein degradation involve Thr-187 phosphorylation by cyclin E:Cdk2 complexes followed by scF^slφ2/cksl complex mediated ubiquitination and degradation by the 26S proteosome. p27 degradation has now been reported to occur in both the cytoplasm and nucleus of cells.

In a preferred embodiment, p27 proteins, fragments and derivatives thereof are generated by PCR based strategies. These strategies are described in detail in the examples which follow. However, a variety of amplification approaches can be utilized, e.g. a standard polymerase chain reaction, a ligase chain reaction, reverse transcriptase polymerase chain reaction, Rolling Circle polymerase chain reaction, multiplex polymerase chain reaction and the like.

As used herein, "derivatives" refers to modified p27 either by mutation of wild type p27 or by the addition of various groups such as carbohydrates, chemical moieties, localization sequences and the likes. Mutations can be introduced by insertions, deletions, transversions and the like.

In a preferred embodiment, recombinant p27 proteins were generated as follows. This is an illustrative example and is not meant to limit or construe the invention in any way. PCR- based strategies were used to generate truncation mutants ofthe human p27 cDNA from the pTAT-HA-p27 expression vector at amino acid residues p27-158 and p27-l 18. Similarly, a Ser- 10-Ala point mutant (p27-S10A) and a quadruple point mutant, p27-QM (S140A, Q141 A,

A149E, II 5 IE), were generated. The coding region of each plasmid was PCR amplified, with Ncol and EcoRI restriction endonuclease recognition sites added at the N- and C-terminus, respectively, and were cloned into pTAT-HA expression vector. All constructs were verified by DNA sequencing. All TAT-fusion proteins contain an N-terminal 6x-His purification tag, 11 residue (YGRKKRRQRRR) TAT protein transduction domain and HA-epitope tag. Fusion proteins were purified as previously described (Becker-Hapak, M., McAllister, S. S., and Dowdy, S. F. (2001). Methods 24, 247-56; Nagahara, H., Vocero-Akbani, A. M., Snyder, E. L., Ho, A., Latham, D. G., Lissy, N. A., Becker-Hapak, M., Ezhevsky, S. A., and Dowdy, S. F. (1998). Nature Medicine 4, 1449-52). Briefly, bacterial lysates containing recombinant TAT- fusion proteins were sonicated in 8 M urea, passed over a Ni-NTA resin (Qiagen), eluted with immidazole, loaded in 4 M urea onto a Mono S column attached to an FPLC (Amersham- Pharmacia), eluted with 1 M NaCl, and desalted into PBS on Sephadex G-25 exchange column (Amersham-Pharmacia). All TAT fusion proteins were sterile filtered and stored in 10% glycerol at -80 °C.

Particularly preferred fusion proteins ofthe invention, include, but are not limited to TAT wild type p27; nuclear exporting fusion proteins such as TATp27 Ser 10; TATp27-N-terminal (amino acid residues 1-103); TAT p27 C-terminal truncation proteins; TATp27 mutants that delete or inactivate the cyclinxdk binding domain; TATp27fusion proteins comprises SQ_xAxl- motif; and any other mutants which enhance cell motility, arrest the metastases of cells, and promote wound healing. Preferred mutants are fully described in the examples which follow.

In another preferred embodiment, the nucleotide sequences ofthe invention may be used for their ability to selectively form duplex molecules with complementary stretches of genes or RNAs or to provide primers for amplification of DNA or RNA from tissues. For example, from patient tumor cells, from cells in disease states characterized by abnormal cell cycles or motility. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.

"Stringency" is meant the combination of conditions to which nucleic acids are subject that cause the duplex to dissociate, such as temperature, ionic strength, and concentration of additives such as formamide. Conditions that are more likely to cause the duplex to dissociate are called "higher stringency", e.g. higher temperature, lower ionic strength and higher concentration of formamide. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50° C. to about 70° C. Such high stringency conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating p27 genes or detecting p27 mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

For certain applications, for example, substitution of amino acids by site-directed mutagenesis, it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Thus, hybridization conditions can be readily manipulated depending on the desired results.

The phrase "hybridizing conditions" and its grammatical equivalents, when used with a maintenance time period, indicates subjecting the hybridization reaction admixture, in context of the concentration ofthe reactants and accompanying reagents in the admixture, to time, temperature, pH conditions sufficient to allow the polynucleotide probe to anneal with the target sequence, typically to form the nucleic acid duplex. Such time, temperature and pH conditions required to accomplish the hybridization depend, as is well known in the art on the length ofthe polynucleotide probe to be hybridized, the degree of complementarity between the polynucleotide probe and the target, the guanidine and cytosine content ofthe polynucleotide, the stringency ofthe hybridization desired, and the presence of salts or additional reagents in the hybridization reaction admixture as may affect the kinetics of hybridization. Methods for optimizing hybridization conditions for a given hybridization reaction admixture are well known in the art. As mentioned, any amplification procedure can be used, for example, multiplex PCR, LCR, RT-PCR, RCA and the like. "Amplification", as used herein, refers to any in vitro process for increasing the number of copies of a nucleotide sequence or sequences, i.e., creating an amplification product which may include, by way of example additional target molecules, or target-like molecules or molecules complementary to the target molecule, which molecules are created by virtue ofthe presence ofthe target molecule in the sample. These amplification processes include but are not limited to multiplex PCR, Rolling Circle PCR, ligase chain reaction (LCR) and the like. In a situation where the target is a nucleic acid, an amplification product can be made enzymatically with DNA or RNA polymerases or transcriptases. Nucleic acid amplification results in the incorporation of nucleotides into DNA or RNA. As used herein, one amplification reaction may consist of many rounds of DNA replication. PCR is an example of a suitable method for DNA amplification. For example, one PCR reaction may consist of 30-100 "cycles" of denaturation and replication.

The earliest method for DNA amplification was the polymerase chain reaction (PCR) which operated only on linear segments of DNA and produced linear segments using specific primer sequences for the 5'- and 3'-ends of a segment of DNA whose amplification was desired. As an improvement on this method, linear rolling circle amplification (LRCA) uses a target DNA sequence that hybridizes to an open circle probe to form a complex that is then ligated to yield an amplification target circle and a primer sequence and DNA polymerase is added. The amplification target circle (ATC) forms a template on which new DNA is made, thereby extending the primer sequence as a continuous sequence of repeated sequences complementary to the ATC but generating only about several thousand copies per hour. An improvement on LRCA is use of exponential RCA (ERCA) with additional priming sequences that bind to the replicated ATC-complement sequences to provide new centers of amplification, thereby providing exponential kinetics and greatly increased amplification. Exponential rolling circle amplification (ERCA) employs a cascade of strand displacement reactions but is limited to use of the initial single stranded RCA product as a template for further DNA synthesis using individual single stranded primers that attach to said product but without additional rolling circle amplification.

Each of these methods makes use of one or more oligonucleotide primers or splice templates able to hybridize to or near a given nucleotide sequence of interest. After hybridization ofthe primer, the target-complementary nucleic acid strand is enzymatically synthesized, either by extension ofthe 3' end ofthe primer or by transcription, using a promoter- primer or a splice template. In some amplification methods, such as PCR, rounds of primer extension by a nucleic acid polymerizing enzyme is alternated with thermal denaturation of complementary nucleic acid strands. Other methods, such as those of WO91/02818, Kacian and Fultz, U.S. Pat. No. 5,480,783; McDonough, et al., WO 94/03472; and Kacian, et al., WO 93/22461, are isothermal transcription-based amplification methods.

In each amplification method, however, side reactions caused by hybridization ofthe primer to non-target sequences can reduce the sensitivity ofthe target-specific reaction. These competing "mismatches" may be reduced by raising the temperature ofthe reaction. However, raising the temperature may also lower the amount of target-specific primer binding as well.

Thus, according to this aspect ofthe invention, primers having high target affinity, and comprising modified nucleotides in the target binding region, may be used in nucleic acid amplification methods to more sensitively detect and amplify small amounts of a target nucleic acid sequence, by virtue ofthe increased temperature, and thus the increased rate of hybridization to target molecules, while reducing the degree of competing side-reactions (cross- reactivity) due to non-specific primer binding. Preferred oligonucleotides contain at least one cluster of modified bases, but less than all nucleotides are modified in preferred oligonucleotides.

In another preferred embodiment, modified oligonucleotide primers are used in a nucleic acid amplification reaction in which a target nucleic acid is RNA. See, e.g., Kacian and Fultz, supra. The target may be the initially present nucleic acid in the sample, or may be an intermediate in the nucleic acid amplification reaction. In this embodiment, the use of preferred 2'-modified primers, such as oligonucleotides containing 2'-O-methyl nucleotides, permits their use at a higher hybridization temperature due to the relatively higher melting temperature (T_m ) conferred to the hybrid, as compared to the deoxyoligonucleotide ofthe same sequence. Also, due to the preference of such 2'-modified oligonucleotides for RNA over DNA, competition for primer molecules by non-target DNA sequences in a test sample may also be reduced. Further, in applications wherein specific RNA sequences are sought to be detected amid a population of DNA

"Amplification products", "amplified products" "PCR products" or "amplicons" comprise copies of the target sequence and are generated by hybridization and extension of an amplification primer. This term refers to both single stranded and double stranded amplification primer extension products which contain a copy ofthe original target sequence, including intermediates ofthe amplification reaction.

"Target" or "target sequence" refers to nucleic acid sequences to be amplified. These include the original nucleic acid sequence to be amplified, its complementary second strand and either strand of a copy ofthe original sequence which is produced in the amplification reaction. The target sequence may also be referred to as the template for extension of hybridized amplification primers.

"Nucleotide" as used herein, is a term of art that refers to a base-sugar-phosphate combination. Nucleotides are the monomeric units of nucleic acid polymers, i.e. of DNA and RNA. The term includes ribonucleoside triphosphates, such as rATP, rCTP, rGTP, or rUTP, and deoxyribonucleotide triphosphates, such as dATP, dCTP, dUTP, dGTP, or dTTP. A "nucleoside" is a base-sugar combination, i.e. a nucleotide lacking phosphate. It is recognized in the art that there is a certain interchangability in usage ofthe terms nucleoside and nucleotide. For example, the nucleotide deoxyuridine triphosphate, dUTP, is a deoxyribonucleoside triphosphate. After incorporation into DNA, it serves as a DNA monomer, formally being deoxyuridylate, i.e. dUMP or deoxyuridine monophosphate. One may say that one incorporates dUTP into DNA even though there is no dUTP moiety in the resultant DNA. Similarly, one may say that one incorporates deoxyuridine into DNA even though that is only a part ofthe substrate molecule.

The term "nucleic acid" is defined to include DNA and RNA, and their analogs, and is preferably DNA. Further, the methods ofthe present invention are not limited to the detection of mRNAs. Other RNAs that may be of interest include tRNAs, rRNAs, and snRNAs.

"Incorporating" as used herein, means becoming part of a nucleic acid polymer.

"Terminating" as used herein, means causing a treatment to stop. The term includes means for both permanent and conditional stoppages. For example, if the treatment is enzymatic, a permanent stoppage would be heat denaturation; a conditional stoppage would be, for example, use of a temperature outside the enzyme's active range. Preferred methods of termination include the use of abasic regions. It is also expedient to use deoxyribonucleoside triphosphates as chain termination molecules which are modified at the 3' position ofthe deoxyribose in such a way that they have no free -OH group but are nevertheless accepted as a substrate by the polymerase. Examples of such chain termination molecules are 3' fluoro, 3'-O- alkyl and 3Η-modified deoxyribonucleosides. 3'-H-modifϊed deoxyribonucleotides are preferably used as chain termination molecules i.e. dideoxyribonucleoside triphosphates (ddNTP). It is preferable to use unlabeled chain termination molecules in the method according to the invention but it is also possible to use labeled chain termination molecules as known to a person skilled in the art. Any type of termination procedures are intended to fall within the scope of this term.

"Oligonucleotide" as used herein refers collectively and interchangeably to two terms of art, "oligonucleotide" and "polynucleotide". Note that although oligonucleotide and polynucleotide are distinct terms of art, there is no exact dividing line between them and they are used interchangeably herein. An oligonucleotide is said to be either an adapter, adapter/linker or installation oligonucleotide (the terms are synonymous) if it is capable of installing a desired sequence onto a predetermined oligonucleotide. An oligonucleotide may serve as a primer unless it is "blocked". An oligonucleotide is said to be "blocked," if its 3' terminus is incapable of serving as a primer.

The term "probe" refers to a strand of nucleic acids having abase sequence substantially complementary to a target base sequence. Typically, the probe is associated with a label to identify a target base sequence to which the probe binds, or the probe is associated with a support to bind to and capture a target base sequence.

"Oligonucleotide-dependent amplification" as used herein refers to amplification using an oligonucleotide or polynucleotide or probe to amplify a nucleic acid sequence. An oligonucleotide-dependent amplification is any amplification that requires the presence of one or more oligonucleotides or polynucleotides or probes that are two or more mononucleotide subunits in length and that end up as part ofthe newly-formed, amplified nucleic acid molecule.

"Primer" as used herein refers to a single-stranded oligonucleotide or a single-stranded polynucleotide that is extended by covalent addition of nucleotide monomers during amplification. Nucleic acid amplification often is based on nucleic acid synthesis by a nucleic acid polymerase. Many such polymerases require the presence of a primer that can be extended to initiate such nucleic acid synthesis. Here through the selection of primers, modified or otherwise, which determine the average molecular weight ofthe DNA segments (or size), the result can be achieved that the variations of size or molecular weights for the DNA segments formed by the various primer pairs only prevents superimposition or overlap.

The term "label" refers to a molecular moiety capable of detection including, by way of example, without limitation, radioactive isotopes, enzymes, luminescent agents, dyes, and detectable intercalating agents. Any suitable means of detection may be employed, thus, the label maybe an enzyme label, a fluorescent label, a radioisotopic label, a chemiluminescent label, etc. Examples of suitable enzyme labels include alkaline phosphatase, acetylcholine esterase, α- glycerol phosphate dehydrogenase, alkaline phosphatase, asparaginase, β-galactosidase, catalase, δ-5-steroid isomerase, glucose oxidase, glucose-6-phosphate dehydrogenase, luciferase, malate dehydrogenase, peroxidase, ribonuclease, staphylococcal nuclease, triose phosphate isomerase, urease, and yeast alcohol dehydrogenase. Examples of suitable fluorescent labels include fluorescein label, an isothiocyanate label, arhoda ine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, a fluorescamine label, 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, and rhodamine. Preferred fluorescent labels are fluorescein (5- carboxyfluorescein-N-hydroxysuccinimide ester) and rhodamine (5,6-tetramethyl rhodamine), etc. Examples of suitable chemiluminescent labels include luminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate label, a luciferin label an aequorin label. Alternatively, the sample may be labeled with non-radioactive label such as biotin. The biotin labeled probe is detected via avidin or streptavidin through a variety of signal generating systems known in the art. Labeled nucleotides are preferred form of detection label since they can be directly incorporated into the products of PCR during synthesis. Examples of detection labels that can be incorporated into amplified DNA include nucleotide analogs such as BrdUrd (Hoy and Schimke, Mutation Research, 290:217-230 (1993)), BrUTP (Wansick et al., J. Cell Biology, 122:283-293 (1993)) and nucleotides modified with biotin (Langer et al., Proc. Natl. Acad. Sci. USA, 78:6633 (1981)) or with suitable haptens such as digoxygenin (Kerkhof, Anal. Biochem., 205:359-364 (1992)). Suitable fluorescence-labeled nucleotides are Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yu et al., Nucleic Acids Res., 22:3226-3232 (1994)). A preferred nucleotide analog detection label for DNA is Cyanine-5-dUTP or BrdUrd (BUDR triphosphate, Sigma), and a preferred nucleotide analog detection label is Biotin- 16-uridine-5 '-triphosphate (Biotin- 16-dUTP, Boehringher Mannheim).

The term "agent" is used in a broad sense, in reference to labels, and includes any molecular moiety which participates in reactions which lead to a detectable response.

Typically, the hybridization product to be amplified, functions in PCR as a primed template comprises polynucleotide as a primer hybridized to a target nucleic acid as a template. In PCR, the primed template is extended to produce a strand of nucleic acid having a nucleotide sequence complementary to the template, i.e., template complement. Through a series of primer extension reactions, an amplified nucleic acid product is formed that contains the specific nucleic acid sequence complementary to the hybridization product.

If the template whose complement is to be produced is in the form of a double stranded nucleic acid, it is typically first denatured, usually by melting into single strands, such as single stranded DNA. The nucleic acid is then subjected to a first primer extension reaction by treating or contacting nucleic acid with a first polynucleotide synthesis primer having as a portion of its nucleotide sequence, a sequence selected to be substantially complementary to a portion ofthe sequence ofthe template. The primer is capable of initiating a primer extension reaction by hybridizing to a specific nucleotide sequence. Typically, for PCR applications, suitable primers are at least about 10 nucleotides in length, more typically at least about 15, 20, 25 or 30 nucleotides in length.

The primer extension reaction is accomplished by mixing an effective amount ofthe primer with the template nucleic acid, and an effective amount of nucleic acid synthesis inducing agent to form the primer extension reaction admixture. The admixture is maintained under polynucleotide synthesizing conditions for a time period, which is typically predetermined, sufficient for the formation of a primer extension reaction product.

The primer extension reaction is performed using any suitable method. Generally, it occurs in a buffered aqueous solution, preferably at a pH of about 7 to 9, most preferably, about 8. Preferably, a molar excess (for genomic nucleic acid, usually 10⁶ :1 primeπtemplate) ofthe primer is admixed to the buffer containing the template strand. A large molar excess is preferred to improve the efficiency ofthe process. For polynucleotide primers of about 10 to 30 nucleotides in length, a typical ratio is in the range of about 50 ng to 1 μg, preferably about 250 ng of primer per 100 ng to about 500 ng of mammalian genomic DNA or per 10 to 50 ng of plasmid DNA. As little as 50 ng of genomic DNA can be used.

The deoxyribonuclotide triphosphates (dNTPs), dATP, dCTP, dGTP and dTTP are also admixed to the primer extension reaction admixture to support the synthesis of primer extension products and depends on the size and number of products to be synthesized. One of skill in the art can determine the number of cycles and temperatures for performing PCR. Typically, the primer extension reaction admixture solution is heated to about 95°C for 5 min followed by 35 cycles of 95°C for 45 sees, 55°C for 45 sees, and 72°C for 1 min followed by 72°C for 10 min. After heating, the solution is allowed to cool to room temperature which is preferable for primer hybridization. To the cooled mixture is added an appropriate agent for inducing or catalyzing the primer extension reaction and the reaction is allowed to occur under conditions known in the art. The synthesis reaction may occur at from room temperature up to a temperature above which the inducing agent no longer functions efficiently. Thus, for example, if DNA polymerase is used as the inducing agent, the temperature is generally no greater than about 40°C unless the polymerase is heat stable.

The inducing agent may be any compound or system which will function to accomplish the synthesis ofthe primer extension products, including enzymes. Suitable enzymes for this purpose include for example E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, recombinant modified T7 DNA polymerase, other available DNA polymerase, reverse transcriptase and other enzymes including heat stable enzymes which will facilitate the combination of nucleotides in the proper manner to form the primer extension products which are complementary to each nucleic acid strand. Heat stable DNA polymerase is used in the most preferred embodiment by which PCR is conducted in a single solution in which the temperature is cycled. Representative heat stable polymerases are DNA polymerases isolated from Bacillus stearothermophilus (BioRad), Thermus Thermophilus (FINZYMΕ, ATCC#27634), Thermus species (ATCC #31674), Thermus aquaticus strain TV1151B (ATCC 25105), Sulfolobus acidocaldarius described by Bukrashuili et al. Biochem. Biophys. Acta 1008:102-7 (1989) and Εlie et al. Biochem. Biophys. Acta 951:261-7 (1988) and Thermus filiformis (ATCC #43280). Particularly, the preferred polymerase is Taq DNA polymerase available from a variety of sources including Taq Gold (Applied Biosystems) Perkin Elmer Cetus (Norwalk, Conn.), Promega (Madison, Wis.) and Stratagene (La Jolla, Calif.) and AmpliTaq.TM. DNA polymerase, a recombinant Taq DNA polymerase available from Perkin- Elmer Cetus. Generally, the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand until the synthesis terminates, producing molecules of different lengths. There may be inducing agents, however, which initiate synthesis at the 5' end and proceed in the above direction using the same process.

The primer extension reaction product is subjected to a second primer extension reaction by treating it with a second polynucleotide synthesis primer having a preselected nucleotide sequence. The second primer is capable of initiating the second reaction by hybridizing to a nucleotide sequence, preferably at least about 20 nucleotides in length and more preferably a predetermined amount thereof with the first product preferably, a predetermined amount thereof to form a second primer extension reaction admixture. The admixture is maintained under polynucleotide synthesizing conditions for a time period, sufficient for the formation of a second primer extension reaction product.

PCR is carried out simultaneously by cycling, i.e., performing in one admixture, the above described first and second primer extension reactions, each cycle comprising polynucleotide synthesis followed by denaturation ofthe double stranded polynucleotides formed. Methods and systems for amplifying a specific nucleic acid sequence are described in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, to Mullis et al; and the teachings in PCR

Technology, Ehrlich, ed. Stockton press (1989); Faloona et al., Methods in Enzymol. 155:335-50 (1987): Polymerase Chain Reaction, Ehrlich, eds. Cold Spring Harbor Laboratories Press (1989), the contents of which are hereby incorporated by reference.

The hybridization reaction mixture is maintained in the contemplated method under hybridizing conditions for a time period sufficient for the polynucleotide probe to hybridize to complementary nucleic acid sequences present in the sample to form a hybridization product, i.e., a complex containing probe and target nucleic acid. Typical hybridizing conditions include the use of solutions buffered to pH values between 4 and 9, and are carried out at temperatures from 18 °C to 75 °C, preferably at least about 22 °C to at least about 37 °C, more preferably at least about 37 °C and for time periods from at least 0.5 seconds to at least 24 hours, preferably 30 min, although specific hybridization conditions will be dependent on the particular primer used.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization, as in PCR, for detection of expression of corresponding genes, such as p27 or variations thereof, as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing ofthe hybridized surface to remove non- specifically bound probe molecules, hybridization is detected, or even quantified, by means of the label.

In another preferred embodiment, p27 oligonucleotides, proteins, peptides, or derivatives thereof are generated using nucleic acid sequences of p27 chosen by the user. For applications in which the nucleic acid segments ofthe present invention are incoφorated into vectors, such as plasmids, cosmids or viruses, these segments may be combined with other DNA sequences, such as promoters, polyadenylation signals, restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. The term "DNA construct" and "vector" are used herein to mean a purified or isolated polynucleotide that has been artificially designed and which comprises at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their natural environment.

Once the coding sequence of p27 wild type, mutants or fragments thereof, are selected, the gene can be inserted into an appropriate expression system. The gene can be expressed in any number of different recombinant DNA expression systems.

Examples of expression systems known to the skilled practitioner in the art include bacteria such as E. Coli, yeast such as Saccharomyces cerevisia and Pichia pastoris, baculovirus, and mammalian expression systems such as in Cos or CHO cells. In one embodiment, polypeptides are expressed in E. coli and in baculovirus expression systems. A complete gene can be expressed or, alternatively, fragments ofthe gene encoding portions of polypeptide can be produced.

A most preferred vector is the pTAT-HA expression vector (Nagahara, H., Vocero- Akbani, A. M., Snyder, E. L., Ho, A., Latham, D. G., Lissy, N. A., Becker-Hapak, M., Ezhevsky, S. A., and Dowdy, S. F. (1998). Nature Medicine 4, 1449-52).

As used herein, the term "administering a molecule to a cell" (e.g., an expression vector, nucleic acid, amino acid, fusion proteins, a delivery vehicle, agent, and the like) refers to transducing, transfecting, microinjecting, electroporating, or shooting, the cell with the molecule. In some aspects, molecules are introduced into a target cell by contacting the target cell with a delivery cell (e.g., by cell fusion or by lysing the delivery cell when it is in proximity to the target cell).

Deletion of sequences from the wild type p27 gene used for expression can be achieved by standard techniques. For example, fortuitously-placed restriction enzyme sites can be used to excise the desired gene fragment, or PCR-type amplification can be used to amplify only the desired part ofthe gene. The skilled practitioner will realize that such changes must be designed to not change the translational reading frame for downstream portions ofthe protein-encoding sequence. Minigenes or gene fusions encoding the desired polypeptide can be constructed and inserted into expression vectors by standard methods, for example, using PCR methodology.

The gene or gene fragment encoding a p27 polypeptide can be inserted into an expression vector by standard subcloning techniques. In one embodiment, an E. coli expression vector is used that produces the recombinant polypeptide as a fusion protein, allowing rapid affinity purification ofthe protein. Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, NJ.), the maltose binding protein system (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6x-His system (Qiagen, Chatsworth, Calif.).

In a most preferred embodiment, the fusion protein comprises an N terminal 6x-His purification tag, a TAT protein transduction domain of about 11 amino acid residues and an HA- epitope tag.

Some of these systems produce recombinant polypeptides bearing only a small number of additional amino acids, which are unlikely to affect the physical and chemical properties ofthe recombinant polypeptide. For example, both the FLAG system and the 6x-His system add only short sequences, both of that are known to be poorly antigenic and which do not adversely affect folding ofthe polypeptide to its native conformation. Other fusion systems produce polypeptide where it is desirable to excise the fusion partner from the desired polypeptide. In one embodiment, the fusion partner is linked to the recombinant polypeptide by a peptide sequence containing a specific recognition sequence for a protease.

In another embodiment, the expression system used is one driven by the baculovirus polyhedron promoter. The gene encoding the polypeptide can be manipulated by standard techniques in order to facilitate cloning into the baculovirus vector. One baculovirus vector is the pBlueBac vector (Invitrogen, Sorrento, Calif.). The vector carrying the gene for the polypeptide is transfected into Spodopterafrugiperda (Sf9) cells by standard protocols, and the cells are cultured and processed to produce the recombinant antigen. See Summers et al., A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station.

As an alternative to recombinant polypeptides, synthetic p27 peptides can be prepared.

Such peptides are at least six amino acid residues long, and may contain up to approximately 35 residues, which is the approximate upper length limit of automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, Calif.).

In one embodiment, amino acid sequence variants of the polypeptide can be prepared.

These may, for instance, be minor sequence variants ofthe polypeptide that arise due to natural variation within the population, variations in tumor cells, abnormal cells, cells associated with diseases in characterized by abnormal cell cycle or motility. Sequence variants can be prepared by standard methods of site-directed mutagenesis such as those described below.

Amino acid sequence variants ofthe polypeptide can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues ofthe native protein which are not essential for function. Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell. Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties ofthe polypeptide such as stability against proteolytic cleavage. Substitutions preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Insertional variants include fusion proteins such as those used to allow rapid purification ofthe polypeptide and also can include hybrid proteins containing sequences from other proteins and polypeptides which are homologues ofthe polypeptide. For example, an insertional variant could include portions ofthe amino acid sequence ofthe polypeptide from one species, together with portions ofthe homologous polypeptide from another species. Preferably, variants include p27 polypeptides from tumors, abnormal cells, and the like. Other insertional variants can include those in which additional amino acids are introduced within the coding sequence ofthe polypeptide.

Preferred mutants include, but are not limited to: a Ser 10-Ala point mutant (p27-S10A) or a quadruple point mutant such as p27-QM (S140A, Q141 A, A149E, 115 IE), described in the examples which follow. In particular, any mutant of p27 that may arrest the metastatic potential of tumor cells or promotes wound healing, are useful for the generation ofthe therapeutic fusion proteins ofthe invention.

In a preferred embodiment, PCR-based strategies are used to generate truncation mutants ofthe human p27 cDNA from the pTAT-HA-p27 expression vector. Preferred truncation mutants are at amino acid residues 158 and 118. Other techniques well known in the art may be used to generate mutants useful in the therapeutic applications, in accordance with the invention.

Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis ofthe underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, incoφorating one or more ofthe foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence ofthe desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides ofthe deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction ofthe sequence being altered. In general, the technique of site-specific mutagenesis is well known in the art. As will be appreciated, the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.

In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single- stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis ofthe mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.

The preparation of sequence variants ofthe selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes maybe obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

Further aspects ofthe present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide. The term "purified protein or peptide" as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally- obtainable state, i.e., in this case, relative to its purity within a cell expressing p27. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.

Generally, "purified" will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component ofthe composition, such as constituting about 50% or more ofthe proteins in the composition.

Various methods for quantifying the degree of purification ofthe protein or peptide will be known to those of skill in the art in light ofthe present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity ofthe fraction, to compare it to the specific activity ofthe initial extract, and to thus calculate the degree of purity, herein assessed by a "- fold purification number". The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

A preferred method of purification is described in the examples which follow. Briefly, bacterial lysates containing recombinant TAT-fusion proteins were sonicated in 8 M urea, passed over a Ni-NTA resin (Qiagen), eluted with immidazole, loaded in 4 M urea onto a Mono S column attached to an FPLC (Amersham-Pharmacia), eluted with 1 M NaCl, and desalted into PBS on Sephadex G-25 exchange column (Amersham-Pharmacia). All TAT fusion proteins were sterile filtered and stored in 10% glycerol at -80 °C. Other techniques may also be used.

The fusion proteins ofthe present invention can be separated and purified by appropriate combinations of known techniques. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

Methods for purification ofthe fusion proteins utilize several chemical and physical properties ofthe fusion proteins. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electrical charge such as ion-exchange column chromatography, methods utilizing specific affinity such as affinity chromatograph, methods utilizing a difference in hydrophobicity such as reverse-phase high performance liquid chromatograph and methods utilizing a difference in isoelectric point, such as isoelectric focusing electrophoresis, metal affinity columns such as Ni-NTA. See generally Sambrook et al. and Ausubel et al. for disclosure relating to these methods.

It is preferred that the fusion proteins ofthe present invention be substantially pure. That is, the fusion proteins have been isolated from cell substituents that naturally accompany it so that the fusion proteins are present preferably in at least 80% or 90% to 95% homogeneity (w/w). Fusion proteins having at least 98 to 99% homogeneity (w/w) are most preferred for many pharmaceutical, clinical and research applications. Once substantially purified the fusion protein should be substantially free of contaminants for therapeutic applications. Once purified partially or to substantial purity, the soluble fusion proteins can be used therapeutically, or in performing in vitro or in vivo assays as disclosed herein. Substantial purity can be determined by a variety of standard techniques such as chromatography and gel electrophoresis.

There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater-fold purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., Biochem. Biophys. Res. Comm., 76:425, 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.

Purified fusion proteins ofthe invention are preferably transduced into cells by the methods described the in examples which follow. In general, Human HepG2 hepatocellular carcinoma cells and primary human SiFT diploid fibroblasts are maintained in DME plus 5% or 10% FBS, respectively, by methods well known in the art. p27-deficient and strain matched wild type MEFs are immortalized using a 3T3 protocol and maintained in DME plus 10% FBS as described (Groth et al., 2000). Cells are transduced by addition of purified TAT-fusion proteins directly to cell culture media. Transduction efficiency is verified by immunoblotting for the HA epitope contained in all p27 fusion proteins. HepG2 cells are treated with about 100 nM TATp27 proteins for about 6 hr and trypsinized for about 15 min to degrade extracellular and external membrane-bound protein. Cell pellets are washed about 3x with PBS and immunoblotted with anti-HA antibodies (1 :1000; BabCo). Functionality of TATp27 fusion proteins is determined by cell cycle analysis of synchronized primary diploid human fibroblasts, SiFT, as described ( Ezhevsky, S. A., Nagahara, H., Vocero-Akbani, A. M., Gius, D. R., Wei, M. C, and Dowdy, S. F. (1997). Proc Natl Acad Sci U S A 94, 10699-704). Briefly, 48 hr high density, contact-inhibited Gi arrested SiFTs are replated at low density, treated with about 200 nM TATp27 fusion proteins and analyzed for cell cycle position by propidium iodide staining and flow cytometry. Nucleic acid encoding a desired fusion protein can be introduced into a host cell by standard techniques for transfecting cells. The term "transfecting" or "transfection" is intended to encompass all conventional techniques for introducing nucleic acid into host cells, including calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinj ection, viral transduction and/or integration. Suitable methods for transfecting host cells can be found in Sambrook et al, and other laboratory textbooks.

Cells transduced by the fusion molecules ofthe present invention can be assayed for viability by standard methods. In one approach, cell viability can be readily assayed by measuring DNA replication following or during transduction. For example, a preferred assay involves cell uptake of one or more detectably-labeled nucleosides such as radiolabelled thymidine. The uptake can be conveniently measured by several conventional approaches including trichloroacetic acid (TCA) precipitation followed by scintillation counting. Other cell viability methods include well know trypan blue exclusion techniques.

As noted, fusion molecules ofthe present invention are efficiently transduced into target cells or groups of such cells. Transduction efficiency can be monitored and quantified if desired by one or a combination of different strategies.

For example, one approach involves an in vitro assay that measures uptake ofthe fusion protein by the cell. The assay includes detectably-labeling the fusion protein with, e.g., a radioactive atom, fluorescent, phosphorescent, or luminescent tag (e.g., fluorescein, rhodamine or FITC) and then measuring uptake ofthe labeled fusion protein. Alternatively, the fusion protein can be labeled with an enzyme capable of forming a detectable label such as horseradish peroxidase, β-galactosidase, chloramphenicol acetyl transferase or luciferase. In a preferred approach, it is possible to genetically fuse a desired fusion protein to the well-known green fluorescent protein (GFP) and then assaying the fusion protein. Uptake can be measured by several conventional methods such as by quantifying labeled cells in a standard cell sorter (e.g., FACS), by fluorescence microscopy or by autoradiography. See generally Sambrook et al. and Ausubel et al. infra for disclosure relating to the assays.

Biologically, GFP acts to shift the color of bioluminescence from blue to green in luminous coelenterates (jellyfish, hydroids, sea pansies, and sea pens) and to increase the quantum yield of light emission. This fluorescence can be visualized directly on culture plates upon illumination with either blue- or long- wave ultraviolet (UN) light. Any ofthe vectors designed for protein expression can be used to make constructs to express GFP in different cells or organisms, either alone or as a fusion protein.

Preferred fusion proteins ofthe invention are capable of transducing at least about 20%, to 80%, and more preferably at least about 90%, 95%, 99% up to 100% ofthe total number of target cells as determined by any conventional methods for monitoring protein uptake by cells and particularly the FACS or related microscopical techniques. The total number of target cells can be estimated by standard techniques.

The fusion proteins ofthe invention can be administered to cells in vivo or in vitro by one or a combination of strategies.

For example, the fusion proteins can be administered to primary or immortalized cells growing in culture in vitro by conventional cell culture techniques that generally include contacting the cells with the fusion protein and allowing the fusion protein to transduce through the cells for a specified period of time. Typically, cell media will be removed from the cells prior to the contact to increase fusion protein concentration.

In addition, the fusion proteins can be administered to cells in vivo, for example, by using a specified delivery mechanism suitable for introduction of fusion proteins into those cells. In general, the type of delivery mechanism selected will be guided by several considerations including the location ofthe cells, the degree of transduction needed to induce motility ofthe cells, and the general health ofthe cells. Preferred methods for determining intracellular localization ofthe fusion proteins, include but are not limited to immunofluorescence, leptomycin B assays and other techniques well known to one of skill in the art.

In certain embodiments, the invention relates to TAT p27 fusion proteins which include the p27 intrinsic nuclear localization sequence and nuclear exporting signals. (For a general discussion of nuclear protein import, see, e.g., Gorlich, D. Curr. Opin. Cell. Biol. (1997) 9:412- 419). Thus, the effective nuclear concentration of a fusion protein having a TAT domain, a p27 domain and a nuclear import and/or nuclear export domain. The data presented here support a model in which an HGF- Activated Kinase (HAcK) phosphorylates p27 on Ser-10 resulting in nuclear export of p27 to the cytoplasm (Figure 7). Thus, a desirable TATp27 fusion protein will include a p27 comprising Ser-10 as described in the examples which follow.

In particular, we demonstrate that HGF-induced nuclear export of p27 occurs in a Crm-1 dependent manner that is dependent on HAcK phosphorylation of Ser-10 on p27. However, p27 has no intrinsic nuclear export sequence (NES) motif, suggesting that cytoplasmic translocation occurs in conjunction with nuclear export proteins. Although Jabl, a nuclear export protein, has been identified as a p27 binding protein via yeast 2-hybrid assays that mediates nuclear export of p27, direct interaction between endogenous proteins has not yet been detected.

Another example of a modulator of nuclear export is leptomycin B. Thus, fusion proteins which comprise leptomycin analogs or derivatives, can be prepared. Leptomycin B has a carboxylate group which can be conveniently coupled to fusion proteins ofthe invention.

Figure 7 is an illustrative model of HGF signaling through p27 to mediate cell migration. HGF binding to the Met receptor activates HAcK (HGF-Activated Kinase), that phosphorylates Ser-10 on p27. Phosphorylation of Ser-10 is required for export of p27 from the nucleus to the cytoplasm in a Crml -dependent fashion. In the cytoplasm, p27 interacts directly/indirectly with actin remodeling proteins, thereby inducing cytoskeletal rearrangement accompanied by cell migration. p27-dependent cell migration requires Ser-10 phosphorylation and the p27 scatter domain (residues 118-158), but not a functional cyclinxdk binding domain.

The invention also provides methods of treatment, using the fusion proteins ofthe invention, which methods in general will comprise administration of a therapeutically effective amount of one or more ofthe fusion proteins discussed above to a mammal, particularly a human, suffering from or susceptible to diseases associated with abnormal cell cycles, metastatic tumors. In another embodiment, the fusion proteins ofthe invention are administered to patients for the promotion of wound healing, especially in those patients suffering from burns, diabetes and the like.

As used herein, the terms "cancer," "neoplasm," and "tumor," are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that normally manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and/or which is detectable because ofthe expression of one or more cancer-specific antigens in a sample obtainable from a patient.

As used herein, "patient" refers to any animal or mammal, especially a human.

The fusion proteins ofthe invention may be administered by a variety of suitable routes including oral, topical (including transdermal, buccal or sublingual), nasal and parenteral (including intraperitoneal, subcutaneous, intravenous, intradermal or intramuscular injection. See generally Reminington's Pharmaceutical Sciences, Mack Pub. Co., Easton, Pa., 1980. Nasal or oral routes leading significant contact believe one or more ofthe fusion proteins and with airway epithelia, lung tissue being generally preferred.

The fusion proteins ofthe present invention can be administered as a sole active agent, in combination with one or more other fusion proteins as provided herein or in combination with other medicaments. Administration of two or more medicaments, including the fusion proteins of the invention is illustrative of a "cocktail" or "cocktail" therapy.

While one or more fusion proteins ofthe invention may be administered alone, they also may be present as part of a pharmaceutical composition in mixture with conventional excipient, preferably a pharmaceutically acceptable organic or inorganic carrier substances that is generally suitable for oral or nasal delivery as mentioned previously. However, in some cases, other modes of administration may be indicated in which case the fusion protein can be combined with a vehicle suitable for parenteral, oral or other desired administration and which do not deleteriously react with the fusion proteins and are not deleterious to the recipient thereof.

Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the fusion proteins.

As used herein, the term "pharmaceutically acceptable carrier" encompasses any ofthe standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton (1975)). Pharmaceutically acceptable carriers are sterile, and pyrogen free. For parenteral application, particularly suitable are solutions, preferably oily or aqueous solutions as well as, suspensions, emulsions, or implants, including suppositories. Ampules are convenient unit dosages.

For enteral application, particularly suitable are tablets, dragees or capsules having talc and/or carbohydrate carrier binder or the like, the carrier preferably being lactose and/or corn starch and/or potato starch. A syrup, elixir or the like can be used wherein a sweetened vehicle, is employed. Sustained release compositions can be formulated including those wherein the active component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.

Therapeutic fusion proteins ofthe invention also may be incoφorated into liposomes. The incoφoration can be carried out according to known liposome preparation procedures, e.g. sonication and extrusion.

It will be appreciated that the actual preferred amounts of active fusion proteins used in a given therapy will vary according to the specific fusion protein being utilized, the particular anti- pathogen system formulated, the mode of application, the particular site of administration, etc. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art using conventional dosage determination tests conducted with regard to the foregoing guidelines.

In another preferred embodiment, antisense therapy is used. Antisense therapy involves the administration of exogenous oligonucleotides that bind to a target nucleic acid, typically an RNA molecule, located within cells. The term antisense is so given because the oligonucleotides are typically complementary to mRNA molecules ("sense strands") which encode a cellular product. The ability to use anti-sense oligonucleotides to inhibit expression of mRNAs, and thereby to inhibit protein expression in vivo, is well documented. Anti-sense agents typically need to continuously bind all target RNA molecules so as to inactivate them or alternatively provide a substrate for endogenous ribonuclease H (Rnase H) activity. Sensitivity of RNA/oligonucleotide complexes, to Rnase H digestion can be evaluated by standard methods (see, e.g., Donia, B. P., et al., J. Biol. Chem. 268 (19):14514-14522 (1993); Kawasaki, A. M., et al., J. Med. Chem. 6(7):831-841 (1993)).

The present invention employs oligonucleotides 8 to 50 nucleotides in length which are specifically hybridizable with portions ofthe p27 which are involved in cell motility (for the prevention of tumor metastasis) or the portions which of p27 which are involved in abnormal cell growth and are capable of inhibiting cell cycle growth abnormalities. Li preferred embodiments, oligonucleotides are targeted to the translation initiation site of: the carboxy terminal scatter domain (translated into amino acid residues 118-158) for prevention of cell motility, targeting of the amino terminal regions which are responsible for abnormal cell growth or any derivatives or fragments thereof. Details of p27 mutants are described in the Examples which follow.

This relationship between an oligonucleotide and the nucleic acid sequence to which it is targeted is commonly referred to as "antisense." "Targeting" an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a foreign nucleic acid (RNA or DNA) from an infectious agent such as oncogenic viruses that may disrupt p27 functioning. The targeting process also includes , determination of a site or sites within the nucleic acid sequence for the oligonucleotide interaction to occur such that the desired effect, i.e., modulation of gene expression, will result. Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.

In antisense therapies, administered oligonucleotides ofthe present invention, contacts (interacts with) the targeted gene or mRNA from the gene, whereby expression ofthe gene is modulated, and frequently expression is inhibited rather than increased. Such modulation of expression suitably can be a difference of at least about 10% or 20% relative to a control, more preferably at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% difference in expression relative to a control. It will be particularly preferred where interaction or contact with an oligonucleotide ofthe invention results in complete or essentially complete modulation of expression relative to a control, e.g., at least about a 95%, 97%, 98%, 99% or 100% inhibition of or increase in expression relative to control. A control sample for determination of such modulation can be comparable cells (in vitro or in vivo) that have not been contacted with the desired oligonucleotides.

In the context of this invention "modulation" means either inhibition or stimulation.

Inhibition of target gene expression is presently the preferred form of modulation. This modulation can be measured, in samples derived from either in vitro or in vivo (animal) systems, in ways which are routine in the art, for example by PCR, Southern blot, Northern blot or Western blot or ELISA assay of protein expression as taught in the examples ofthe instant application. "Hybridization," in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them. "Specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding ofthe oligonucleotide to the target interferes with the normal function ofthe target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non- target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted. fri the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term "oligonucleotide" also includes oligomers or polymers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake, increased stability in the presence of nucleases, or enhanced target affinity. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incoφorated more resistant to nuclease digestion than the native oligodeoxynucleotide. Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides. A number of modifications have also been shown to increase binding (affinity) ofthe oligonucleotide to its target. Affinity of an oligonucleotide for its target is routinely determined by measuring the T_m of an oligonucleotide/target pair, which is the temperature at which the oligonucleotide and target dissociate. Dissociation is detected spectrophotometrically. The higher the T_m, the greater the affinity ofthe oligonucleotide for the target. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance.

Specific examples of some preferred oligonucleotides envisioned for this invention may contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar ("backbone") linkages. Most preferred are phosphorothioates and those with CH₂ -NH-O-CH₂, CH₂-N(CH₃)-O-CH₂, CH₂-O-N(CH₃)-CH₂, CH₂-N(CH₃) -N(CH₃) -CH₂and O-N(CH₃) -CH₂-CH₂ backbones (where phosphodiester is O-P-O-CH₂). Also preferred are oligonucleotides having moφholino backbone structures.

In other preferred embodiments, such as the protein-nucleic acid or peptide-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms ofthe polyamide backbone. P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science 1991, 254, 1497. Other preferred oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one ofthe following at the 2' position: OH, SH, SCH , F, OCN, OCH₃OCH₃, OCH₃O(CH₂) _nCH₃, O(CH₂) _nNH₂ or O(CH₂) _nCH₃ where n is from 1 to about 10; d to do lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-, S-, orN-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂ CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyallcylamino; substituted silyl; an RNA cleaving group; a cholesteryl group; a folate group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Folate, cholesterol or other groups which facilitate oligonucleotide uptake, such as lipid analogs, may be conjugated directly or via a linker at the 2' position of any nucleoside or at the 3' or 5' position ofthe 3'-terminal or 5'-terminal nucleoside, respectively. One or more such conjugates may be used. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place ofthe pentofuranosyl group. Other preferred embodiments may include at least one modified base form or "universal base" such as inosine.

The oligonucleotides in accordance with this invention preferably are from about 8 to about 50 nucleotides in length. In the context of this invention it is understood that this encompasses non-naturally occurring oligomers as hereinbefore described, having 8 to 50 monomers.

The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis ofthe oligonucleotides is well known by one skilled in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as those available from Glen Research, Sterling NA, to synthesize modified oligonucleotides such as cholesterol-modified oligonucleotides.

Methods of inhibiting, for example, metastasis or cell growth are provided, in which the cells or tissues are contacted with an oligonucleotide ofthe invention. In the context of this invention, to "contact" means to add the oligonucleotide to cells, or vice versa, or to add the oligonucleotide to a biological sample, or vice versa, or to add the oligonucleotide to cells tissues in situ, i.e., in an animal. In the context of this invention a "biological sample" is a preparation or isolate of cells or tissues (such as a biopsy sample).

For prophylactics and therapeutics, methods of preventing tumor cell metastasis, and of treating diseases associated with abnormal cell growth or hypeφroliferation, and wound healing, are provided. The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill in the art. Antisense oligonucleotides may be formulated in a pharmaceutical composition, which may include carriers, thickeners, diluents, buffers, preservatives, surface active agents, liposomes or lipid formulations and the like in addition to the oligonucleotide. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, liposomes, diluents and other suitable additives.

The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal), oral, by inhalation, or parenteral, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.

Dosing is dependent on severity and responsiveness ofthe condition to be treated, with course of treatment lasting from several days to several months or until a reduction in cell growth or motility (routinely measured by assays described in the examples which follow) is effected or a diminution of disease state is achieved. Optimal dosing schedules are routinely calculated from measurements of drug accumulation in the body. Persons of ordinary skill can easily and routinely determine optimum dosages, dosing methodologies and repetition rates. Therapeutically or prophylactically effective amounts (dosages) may vary depending on the relative potency of individual compositions, and can generally be routinely calculated based on molecular weight and EC₅₀ in in vitro and/or animal studies. For example, given the molecular weight of compound (derived from oligonucleotide sequence and chemical structure) and an effective dose such as an ICso, for example (derived experimentally), a dose in mg/kg is routinely calculated. In general, dosage is from 0.001 μg to 100 g and may be admimstered once or several times daily, weekly, monthly or yearly, or even every 2 to 20 years.

The invention also provides for the treatment of a wide variety of diseases using gene therapy methods. For example, there are approximately 5,000 bone marrow transplantations (BMT) each year (The BBI Newsletter, 156 (1991)). Most of these are performed on leukemia and lymphoma patients. A growing number of BMT are being done to support more intensive therapeutic approaches to breast and lung cancers, as well as for other indications (Droz, J. P. Eur. J. Can., 27:831-35 (1991); Menichella, G. Br. J. Haem., 79:444-50 (1991); Osbourne, C. K. Breast Can. Res. Trtmt., 20:511-14 (1991)). Many of these patients are candidates for inhibition of abnormal cell growth or inhibition of metastasis using oligonucleotides coding for the fusion proteins ofthe invention. In preferred embodiments a retrovirus encoding the fusion proteins of the invention can be efficacious, in reversing the malignant phenotype of several leukemia and lymphoma cell lines as measured by abrogation or substantial inhibition of colony formation in soft agar assays, and as determined, for example, by reversing/inhibiting the ability of tumor cells to grow in nude mice following introduction ofthe genes encoding for the fusion proteins ofthe invention.

The present invention is useful for use in gene therapy for "negative purging" of pathologic hypeφroliferative cells that contaminate preparations of autologous hematopoietic cells used for bone marrow reconstitution. More specifically, the present invention relates to a method for depleting a suitable sample of pathologic mammalian hypeφroliferative cells contaminating hematopoietic precursors during bone marrow reconstitution via the introduction of a stably-expressed genes encoding the fusion proteins ofthe invention, into the cell preparation (whether derived from autologous peripheral blood or bone marrow). As used herein, a "suitable sample" is defined as a heterogeneous cell preparation obtained from a patient, e.g., a mixed population of cells containing both phenotypically normal and pathogenic cells.

The preferred delivery system for the genes encoding the fusion proteins ofthe invention, is a replication-incompetent retroviral vector. As used herein, the term "retroviral" includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the coding sequence into dividing cells. As used herein, the terms "replication-incompetent" is defined as the inability to produce viral proteins, precluding spread ofthe vector in the infected host cell. An example of such vector is RN, which has been described in detail by Chen et al., Science, 250:1576-80 (1990). Another example of a replication-incompetent retroviral vector is LΝL6 (Miller, A. D. et al., BioTechniques 7:980-990 (1989)).

The methodology of using replication-incompetent retroviruses for retro viral-mediated gene transfer of gene markers is well established (Correll, P. H. et al., N4SUSA, 86:8912 (1989); Bordignon, C. et al., EN4SUSA, 86:8912-52 (1989); Culver, K. et al., EN4SUSA, 88:3155 (1991); Rill, D. R. et al., Blood, 79(10):2694-700 (1992)). Clinical investigations have shown that there are few or no adverse effects associated with the viral vectors (43: Anderson, Science, 256:808-13 (1992)). However, these methods have been limited to transfers of "gene markers" such as the neomycin gene that merely function as "tracking agents" for marking malignant cells before, and locating malignant cells after, reinfusion of bone marrow, however, the transduction of gene markers confers little clinical benefit to the affected patient who does not receive protection against subsequent relapse (Rill, D. R. et al. Blood, supra). The subject invention eliminates the necessity ofthe time consuming procedure of transducing cell samples with a selectable marker gene, such as neomycin, to identify pathologic cells to facilitate subsequent attempts to remove those cells before reinfusion into the patient. Other vectors are suitable for use in this invention and will be selected for efficient delivery ofthe nucleic acid encoding the desired gene, in accordance with the invention. The nucleic acid can be DNA, cDNA or RNA.

The subject invention provides a "shotgun" procedure whereby the cell sample is contacted with a retroviral vector in the absence of selective medium that does not necessarily contain a selectable marker gene, but notwithstanding, possesses the ability to simultaneously selectively target and transduce only the pathologic cell population in the heterogeneous cell preparation. Other methods of efficient delivery or insertion of a gene of interest into a cell are well known to those of skill in the art and comprise various molecular cloning techniques. As used herein, the terms "insertion or delivery" encompass methods of introducing an exogenous nucleic acid molecule into a cell.

A variety of techniques have been employed in an attempt to deplete marrow of pathologic hypeφroliferative cells before reinfusion, utilizing "purging" methods, e.g., monoclonal antibodies or chemotoxins (Kaizer H. et al., Blood, 65:1504 (1985); Gorin, N. C. et al., Blood, 67:1367 (1986); De Fabritiis, P. et al., Bone Marrow Transplant, 4:669 (1989)). As used herein, the term "pathologic" includes abnormalities and malignancies induced by mutations and failures in the genetic regulatory mechanisms that govern normal differentiation that are not the result of gene loss or mutation.

The invention confers related advantages as well. These advantages include: (a) the use of a recombinant retroviral vector that does not require a selectable marker gene in combination with a short-term infection in the absence of selective medium eliminating the time consuming procedure traditionally employed to "selectively mark" the target cells before any "purging" of such cells is attempted, thereby dramatically reducing the time traditionally required for preparing hematopoietic cells for transplants; and (b) the retroviral mediated delivery methodology ofthe subject invention offers selective targeting of pathologic hypeφroliferative cells in resting cultures of hematopoietic cells as a result ofthe higher infection frequency by the retroviral delivery system into actively dividing tumor cells (Miller et al., Mol. Cell. Biol., 10(8):4239-42 (1990)).

The ex-vivo introduction of a gene encoding the fusion proteins ofthe invention, via an efficient delivery system into pathologic hypeφroliferative cells contaminating peripheral blood- or marrow-derived autologous hematopoietic cells will facilitate suppression ofthe hypeφroliferative phenotype, by, for example, inducing transformation ofthe cell to a mature or benign phenotype or, alternatively, by inducing apoptosis or programmed cell death, thereby allowing patients receiving ABMT to have a longer, relapse-free survival. As used herein, the term "mature or benign cell" refers to the phenotypic characteristic of inability to invade locally or metastasize.

This invention further provides a method for transducing a pathologic hypeφroliferative mammalian cell by contacting the cell with a suitable retroviral vector containing a nucleic acid encoding a gene product having the functions ofthe fusion proteins discussed infra, under suitable conditions such that the cell is transduced. Additionally, the nucleic acid is DNA, cDNA or RNA.

The suitable conditions for contacting can be by infecting the sample cells in the absence of selective medium. "Suitable retroviral vector" has been defined above. This method is particularly useful when the pathological cells being contacted are prostate cells, psoriatic cells, thyroid cells, breast cells, colon cellsj lung cells, sarcoma cells, leukemic cells or lymphoma cells.

The suitable time period for contacting can be less than about ten hours, or more specifically, about four hours. Transduction can be known to be complete, for example, when the hypeφroliferative phenotype is characterized by the transduced cell expressing a mature or benign phenotype or by apoptosis or death ofthe transduced cell. This method has been shown to reduce tumor formation or tumorigenicity in a subject. This method can be practiced ex vivo or in vivo. The practice ofthe ex vivo method is described above. When the method is practiced in vivo, the retroviral vector can be added to a pharmaceutically acceptable carrier and systemically administered to the subject. In one embodiment, the subject is a mammal, such as a human patient. Acceptable "pharmaceutical carriers" are well known to those of skill in the art and have been discussed above.

As used herein, the term "administering" for in vivo puφoses means providing the subject with an effective amount ofthe vector, effective to inhibit hypeφroliferation ofthe target cell. Methods of administering pharmaceutical compositions are well known to those of skill in the art and include, but are not limited to, microinj ection, intravenous or parenteral administration. As discussed above, administration can be effected continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of admimstration are well known to those of skill in the art and will vary with the vector used for therapy, the puφose ofthe therapy, the cell or tumor being treated, and the subject being treated.

All documents mentioned herein are incoφorated herein by reference.

The present invention is further illustrated by the following Examples. These Examples are provided to aid in the understanding ofthe invention and are not construed as a limitation thereof.

EXAMPLES

Materials and Methods

Generation of Recombinant TATp27Transducing Proteins PCR-based strategies were used to generate truncation mutants ofthe human p27 cDNA

(Polyak et al., 1994) from the pTAT-HA-p27 expression vector at amino acid residues p27-158 and p27-l 18. Similarly, a Ser- 10- Ala point mutant (p27-S10A) and a quadruple point mutant, ρ27-QM (S140A, Q141 A, A149E, I151E) were generated. The coding region of each plasmid was PCR amplified, with Ncol and EcoRI restriction endonuclease recognition sites added at the N- and C-terminus, respectively, and were cloned into pTAT-HA expression vector. All constructs were verified by DNA sequencing. All TAT-fusion proteins contained an N-terminal 6x-His purification tag, 11 residue (YGRKKRPvQRRR) TAT protein transduction domain and HA-epitope tag. Fusion proteins were purified as previously described (Becker-Hapak et al., 2001; Nagahara et al., 1998). Briefly, bacterial lysates containing recombinant TAT-fusion proteins were sonicated in 8 M urea, passed over a Ni-NTA resin (Qiagen), eluted with immidazole, loaded in 4 M urea onto a Mono S column attached to an FPLC (Amersham- Pharmacia), eluted with 1 M NaCl, and desalted into PBS on Sephadex G-25 exchange column (Amersham-Pharmacia). All TAT fusion proteins were sterile filtered and stored in 10% glycerol at -80 °C.

Cell Culture, Protein Transduction and Cell Cycle Analysis

Human HepG2 hepatocellular carcinoma cells and primary human SiFT diploid fibroblasts were maintained in DME plus 5% or 10% FBS, respectively, as described (Ezhevsky et al., 1997; Nagahara et al., 1998). p27-deficient and strain matched wild type MEFs were immortalized using a 3T3 protocol and maintained in DME plus 10% FBS as described ( Groth, A., Weber, J. D., Willumsen, B. M., Sherr, C. J., and Roussel, M. F. (2000). JBiol Chem 275, 27473-80). Cells were transduced by addition of purified TAT-fusion proteins directly to cell culture media. Transduction efficiency was verified by immunoblotting for the HA epitope contained in all p27 fusion proteins. HepG2 cells were treated with 100 nM TATp27 proteins for 6 hr and trypsinized 15 min to degrade extracellular and external membrane-bound protein. Cell pellets were washed 3x with PBS and immunoblotted with anti-HA antibodies (1:1000; BabCo) as described (Ezhevsky et al., 1997). Functionality of TATp27 fusion proteins was determined by cell cycle analysis of synchronized primary diploid human fibroblasts, SiFT, as described (Ezhevsky et al., 1997). Briefly, 48 hr high density, contact-inhibited Gi arrested SiFTs were replated at low density, treated with 200 nM TATp27 fusion proteins and analyzed for cell cycle position by propidium iodide staining and flow cytometry.

Immunocytochemistry and Leptomycin B Assays

Intracellular localization of TATp27 fusion proteins was determined by immunostaining for the HA epitope (present in the N-terminal leader on all TAT fusion proteins); endogenous proteins (p27, cyclin A, cyclin B and Cdk2) were visualized by direct immunofluorescence. HepG2 cells were plated into eight-well chamber slides (Nunc Nalgene), allowed to adhere overnight, then treated with PBS, 20 ng/ml HGF (Sigma) or 100 nM TAT fusion proteins in fresh media. At given time points, cells were washed extensively with PBS, fixed in 3.7% paraformaldehyde, permeabilized with ice cold 100% ethanol (EtOH) and blocked with 1% BSA for 10 min. Cells were incubated for 30 min at 42 ^°C with anti-HA (1:1000, BabCo), anti-p27 (1:1000, Transduction Labs), anti-Cyclin A (1:500, C-19 Santa Cruz) monoclonal antibodies or rabbit polyclonal anti-Cdk2 antibody (1:500, kindly provided by Dr. Helen Piwnica- Worms) (Xu et al., 1994). To control for antibody specificity, cells were incubated with non-immune serum or PBS. Cells were then washed and incubated with either FITC-conjugated anti-mouse IgG or TRITC-conjugated anti-rabbit IgG (Sigma) for 20 min at 42°C and mounted in the presence of Slowfade reagent (Molecular Probes). Where indicated, actin filaments were visualized by staining with TRITC-conjugated phalloidin (100 ng/ml; Sigma) for 30 min to 1 hr at room temperature. In noted cases, cell nuclei were counterstained with 0.2 μg/ml 4,6-diamidino-2- phenylindole (DAPI; Sigma).

For leptomycin B assays, HepG2 cells were plated into 8-well chamber slides and treated with either PBS or 20 ng/ml HGF for 20 hr. Culture media was exchanged and 10 ng/ml leptomycin B (LMB) was added with or without HGF for 3 hr at 37 °C followed by immunocytochemistry as described above using monoclonal antibodies to p27 (1 :500) or cyclin B (1 :200; Santa Cruz). Randomly selected fields were analyzed and individual cells were scored for nuclear, cytoplasmic, or, nuclear and cytoplasmic staining.

Analysis of p27 Phosphorylation Status HepG2 cells were treated with either PBS or 10 ng/ml HGF for 16 hr, starved of phosphate for 45 min then labeled with 3.75 mCi PO₄ for 4 hr in the presence of either HGF or PBS. Cells were lysed in IP buffer (250 mM NaCl, 50 mM HEPES, 2mM EDTA, 0.5% NP-40, including DTT, aprotinin, PMSF and leupeptin), precleared with zysorbin (Zymed) and immunoprecipitated overnight with anti-p27 monoclonal antibody plus protein G Sepharose. Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose. The same membrane was used to detect both ³²P-labeled p27 by phosphorimage analysis and total p27 protein levels by immunoblotting using polyclonal anti-p27 (1 :1000). Phospho-specific antibodies to Thr-187 and Ser-10 of p27 were used to detect phosphorylation status of respective residues. Lysates from HepG2 cells that were treated with HGF or PBS control for 24 hr were immunoprecipitated using monoclonal antibody to p27, followed by immunoblotting using anti- p27 (1:1000), anti-phospho-Thr-187 (1:200) and anti-ρhosρho-Ser-10 (1:500) polyclonal antibodies.

Cell Migration HepG2 cells were plated at low density to form discrete 5-10 cell colonies and allowed to adhere overnight. PBS, 5 ng/ml HGF or 100 nM TAT fusion proteins were added to fresh cell culture media for indicated time periods. Colonies were considered positive for scattering if cell- cell contact was lost and the distance between cells increased upon microscopic observation. Cells were fixed and stained with hematoxylin and digital images were used to quantify cell distances. Distance between colony cell nuclei was measured at 0 hr and subtracted from that at 8 hr or 24 hr to yield the average total distance of cell migration.

MEF and 3T3 Wound-Migration Assay

. Cell migration of p27^"A MEFs was compared to that of MEFs from matched wild type embryos using a migration assay on confluent monolayer. Briefly, sub-confluent MEFs in 6- well plates were incubated overnight with DME plus 0.1%-0.5% FCS. The confluent monolayers were scraped with either a rubber policeman or the edge of a sterile microtome blade (model 818, Leica Instruments GmbH) in an area measuring approximately 1.5 x 0.3 cm. To measure cell migration, the cultures were incubated at 37° C for 24 hr, washed with PBS, fixed with absolute methanol, and stained with hematoxylin. Cells that had migrated into the denuded area were observed using an Axiovert 25 microscope (Carl Zeiss, Inc.) (lOOx magnification). Random fields were observed along the wound edge in duplicate culture wells. Reconstitution of p27 was performed by addition of lOOnM TATp27 proteins at the time of denuding. Cells were fixed, stained with hematoxylin and photographed 24 hr post-treatment. Example 1

HGF Signals Nuclear Export of Endogenous p27 to the Cytoplasm

HGF treatment of HepG2 human hepatocellular carcinoma cells results in elevated p27 protein levels. The majority of p27 regulation occurs via post-translational mechanisms including phosphorylation and subcellular localization. The effects of Met receptor activation by HGF treatment on the subcellular distribution of p27 protein over time, were investigated by immunocytochemistry. Human HepG2 hepatocellular carcinoma cells treated with PBS control (Figure 1 A, left panels) or HGF (20 ng/ml; right panels) were analyzed for endogenous p27 localization by immunocytochemistry. At indicated time points, cells were stained with anti-p27 antibodies and FITC-conjugated anti-mouse IgG (green). Corresponding cell nuclei were visualized by DAPI (blue). Results represent a minimum of four independent observations. p27 remained nuclear in control PBS treated cells throughout the time course as determined by counterstaining with DAPI (Figure 1 A, left panels).

In contrast, HGF treatment of HepG2 cells resulted in redistribution of p27 from the nucleus to the cytoplasm in a time-dependent fashion (Figure 1 A, right panels). Nuclear export of p27 occurs in a Crm-1 dependent fashion. HepG2 cells were treated with HGF or PBS control for 20 hr, followed by treatment with leptomycin B (LMB; hatched bars) or mock treatment (solid bars) for an additional 3 hr. Cells were fixed and stained for p27 (dark blue) or cyclin B (light blue). Data are plotted as percentage of cells having a significantly greater extent of nuclear versus cytoplasmic immuno-detectable protein. p27 was first detected in the cytoplasm of HGF treated cells at 12 hr, and by 24 hr, the majority of p27 was present in the cytoplasm (Figure 1 A, right panels).

p27 has an intrinsic nuclear localization sequence (NLS), but lacks a nuclear export

(NES) motif. To determine whether HGF inhibits nuclear import or induces nuclear export of p27, HepG2 cells were treated with leptomycin B (LMB), a specific inhibitor of Crml -dependent nuclear export (Nishi et al., 1994) and analyzed by immunocytochemistry. To control for LMB specificity in HepG2 cells, cyclin B localization, which is exported from the nucleus in a Crml- dependent manner, was examined. Cyclin B was retained in the nucleus of cells treated with LMB (Figure IB, left panel). Importantly, HGF treatment did not alter cyclin B localization compared to control treated cells in either the presence or absence of LMB (Figure IB, right panel). In control PBS treated cells, p27 remained nuclear both in the presence and absence of LMB (Figure IB, left panel). In contrast, LMB treatment prevented the cytoplasmic accumulation of endogenous p27 in HGF treated cells (Figure IB, right panel). These observations indicate that HGF signaling results in Crml -dependent nuclear export of p27 to the cytoplasm.

p27 is thought to associate with and inhibit the activity of cyclin A:Cdk2 complexes. Therefore, to determine whether nuclear export of p27 involved concomitant export of cyclinxdk2 complexes from the nucleus, HGF and control treated HepG2 cells were analyzed by immunocytochemistry for endogenous cyclin A and Cdk2 localization. HepG2 cells treated with control PBS or HGF for 24 hr were analyzed by immunocytochemistry for cyclin A (FITC; green) and Cdk2 (TRITC; red) localization. Corresponding nuclei were counterstained with DAPI (blue). Both cyclin A and Cdk2 remained localized to the nucleus after 24 hr of HGF or control treatment with little or no detectable difference between either group (Figure 2A). These observations demonstrate that HGF-dependent nuclear export of p27 to the cytoplasm does not involve accompanying export of cyclin A:Cdk2 complexes.

Cellular responses to HGF treatment include increased motility associated with extensive rearrangement ofthe actin cytoskeleton. Given the HGF induced cytoskeletal rearrangements and export of endogenous p27 to the cytoplasm, p27 localization with respect to F-actin, was investigated. HepG2 cells were treated for 24 hr with HGF and triply stained for endogenous p27 (FITC; detected as a green coloration), F-actin (TRITC; detected as a red coloration), and nuclei/DNA (DAPI; detected as a blue coloration) (Figure 2B). Arrows indicate areas in merged image with co-localized (detected as a yellow coloration) cytoplasmic p27 and F-actin. HGF treated cells contained a substantial amount of endogenous p27 co-localized with F-actin at both the membrane and in areas of organized actin filaments (Figure 2B, bottom right panel). Taken together, these observations demonstrate that HGF signaling results in the specific export of endogenous nuclear p27 to the cytoplasm, exclusive of cyclinxdk complexes, where it co- localizes with F- actin.

Example 2 HGF Induces Ser-10 Phosphorylation on p27

It is thought that phosphorylation of Thr- 187 is required for p27 degradation and phosphopeptide mapping has potentially identified Ser-10 as a second predominant phosphorylation site on p27 that both increases protein stability and mediates nuclear export of p27 to the cytoplasm. The phosphorylation status of p27 in HGF treated cells, was examined. HepG2 cells treated with HGF for 20 hr were metabohcally labeled with [³²P]-orthophosphate followed by anti-p27 immunoprecipitation (Figure 3A, top panel). Total p27 protein levels were determined by anti-p27 immunoblotting (Figure 3 A, bottom panel). HGF treated cells, containing predominantly cytoplasmic p27, showed a significant level of endogenous p27 phosphorylation compared to p27 from control cells (Figure 3A).

To determine the phosphorylation status of specific p27 residues in response to HGF signaling, p27 was immunoprecipitated from lysates of control and HGF treated HepG2 cells with pan anti-p27 antibodies followed by immunoblot analysis with anti-phospho-Thr-187 (top panel), anti-phospho-Ser-10 (middle panel) and pan anti-p27 (bottom panel) antibodies. Total p27 was immunoprecipitated from lysates of HepG2 cells grown in 5% serum plus or minus HGF for 24 hr, followed by immunoblotting. Low levels of Thr- 187 phosphorylation were equally detected in HGF and control treated cells (Figure 3B, top panel). In contrast, Ser-10 phosphorylation was dramatically increased in HGF treated cells compared to control cells (Figure 3B, middle panel). These observations demonstrate that HGF/Met signaling specifically regulates phosphorylation of p27 on Ser-10.

Example 3 p27 C-terminal Domain Mediates Cell Migration

The observation that endogenous p27 was specifically exported from the nucleus to the cytoplasm in HGF treated cells suggested that p27 may be positioned for a function in cell migration. Previously, we established that transduction of full length TATp27-WT (wild type) protein into HepG2 cells was sufficient for both cell migration and filopodia formation, whereas transduction of TATp27-N-terminal protein (residues 1-103) that retained the cyclinxdk binding domain was insufficient to induce either phenotype (Nagahara et al., 1998). Although the C- terminal portion of p27 outside ofthe cyclinxdk binding domain is evolutionarily conserved, little is known about its structure or function. To determine the C-terminal p27 structural elements required for cell migration, a series of transducible TATp27 C-terminal truncation proteins as well as several proteins containing point mutations in full length TATp27 protein, were generated (Figure 4A). In addition to the 11 amino acid TAT protein transduction domain (PTD) in the N-terminal leader, all TAT-fusion proteins contained 6x-His purification and HA- epitope tags. All TATp27 fusion proteins were expressed in bacteria and purified under similar conditions. Figure 4A shows a schematic representation of TATp27 fusion proteins. N-terminal leader contains the 11 amino acid TAT protein transduction domain, hemagglutinin (HA) epitope tag and six-histidine purification tag. CBD, cyclinxdk binding domain of p27. TATp27-WT represents full-length wild type p27 protein, whereas TATp27- 158 and TATp27- 118 designate terminal truncations at respective residues. TATp27-QM contains four single point mutations in actin interacting domain of Farlp-like motif. TATp27-KK contains two inactivating point mutations that disrupt p27 binding to cyclinxdk complexes. TATp27-S10A contains an Alanine substitution for Serine at residue 10.

To determine the transduction efficiency of TATp27 fusion proteins, HepG2 cells were treated with equal concentrations of each protein for 1 hr, washed extensively, fixed and analyzed by anti-HA immunocytochemistry. Figure 4B shows the intracellular localization of TATp27 proteins. HepG2 cells were treated with control PBS or transducible TATp27 proteins and visualized by immunocytochemistry using anti-HA antibody and FITC-conjugated secondary antibody. Consistent with a growing body of transducible TAT-fusion proteins (Wadia and Dowdy, 2002), each TATp27 fusion proteins transduced into cells and were visualized equally in the cytoplasm and nucleus of cells 1 hr after addition to the media (Figure 4B). In addition, immunoblotting for the HA epitope in lysates from HepG2 cells treated with TATp27 fusion proteins and trypsinized to remove any extracellular TATp27 protein, independently confirmed transduction of TATp27 proteins into cells.

To ascertain if TATp27 fusion proteins were refolded and biologically active intracellularly, contact-inhibited and released primary human fibroblasts (SiFT) were transduced with TATp27 proteins and analyzed for Gi cell cycle arrest 24 hr post-treatment. Control PBS treated cells progressed from G_\ into S/G₂/M phases as did cells treated with either negative control TATp27-C-terminal protein (residues 105-198; deleted for the N-terminal cyclinxdk binding domain) or TATp27-KK protein, which contains an inactivating mutation in the cyclinxdk binding domain (Figure 4A, C). In contrast, treatment with TATp27-WT, TATρ27- 158, TATp27-l 18 and TATp27-QM fusion proteins that retained the cyclinxdk binding domain induced a Gi cell cycle arrest (Figure 4C). Taken together, these data confirm that TATp27 proteins efficiently transduce into cells and maintain intracellular biological activity to induce Gi cell cycle arrest.

To investigate the involvement of p27 in cell migration, small colonies of 5-10 HepG2 cells were treated with PBS control, HGF or TATp27-WT and observed over a 24 hr period. Cell migration was defined by loss of cell-cell contact and increased distance between cells in a colony. Figure 5B shows the results of quantification of cell migration. Cells treated as described above, were stained with hematoxylin and recorded by digital microscopy at 0, 8 and 24 hr post-treatment. Distance between colony nuclei measured at 0 hr was subtracted from distance at 8 hr or 24 hr to calculate average distance of migration. Data represents a minimum of 50 random fields and 300 measurements per treatment for each of three separate experiments. Control PBS treated cells retained cell-cell contacts and maintained discrete colonies with average distances between cell nuclei of less than 3 μm (Figure 5 A, B). As expected, HGF treatment resulted in disruption of cell-cell contacts and accumulation of significant distances between colony cell nuclei in excess of 15 μm (Figure 5 A, B). Treatment of cells with TATp27- WT full length protein induced cell migration to near identical distances as HGF treatment (Figure 5 A, B). In addition, TATp27-WT protein treatment induced a greater extent of cell scattering compared to HGF treatment at 8 hr, while the two treatments resulted in similar migration distances by 24 hr (Figure 5B, right panel). These data suggest that direct transduction of TATp27-WT accelerates the rate of cell migration.

p27 was originally identified as an inhibitor of G] cell cycle progression (Hengst and Reed, 1998; Sherr and Roberts, 1999). To address whether p27 must retain its cell cycle arrest function to induce cell migration, HepG2 cells were treated with TATp27-KK full-length protein that contains a mutation in the cyclin binding domain and is unable to mediate Gi cell cycle arrest (Figure 4C). Surprisingly, TATp27-KK protein induced cell scattering to an extent comparable to HGF and TATρ27-WT protein treatments (Figure 5A, B). Thus, the ability of p27 to mediate cell cycle arrest is not a requirement for p27 induction of cell migration.

The N-terminus of p27 contains the functional cyclinxdk binding domain, but fails to induce cell migration. Suφrisingly, the C-terminal region of ρ27 (residues 108-198) was also insufficient to induce cell scattering. Therefore, to delineate the region of p27 required to induce cell migration, HepG2 colony cells were treated with TATp27-158 and TATp27-l 18 C-terminal truncation proteins. TATp27-158 protein induced cell migration to similar distances as HGF and TATp27-WT protein treatments (Figure 5 A, B). In contrast, TATp27-l 18 C-terminal truncation protein failed to disrupt cell-cell contacts and maintained cell-cell distances of less than 3 μm (Figure 5 A, B). These data suggest that the p27 "scatter domain" required for induction of actin rearrangement and cell motility resides between residues 118-158.

Example 4 p27 and Farlp Similarities

Analogous to HGF treatment of mammalian cells, alpha factor treatment of yeast cells results in nuclear export of Farlp, a cyclinxdk inhibitor, to the cytoplasm followed by reorientation ofthe actin cytoskeleton into a schmoo structure. The sequence ofthe p27 "scatter domain" (residues 118-158) was compared to the C-terminus of Farlp involved in schmoo formation. Although there is limited sequence identity between the two distantly related and functionally analogous proteins, a SQx₇AxI-motif present within the p27 scatter domain (residues 140-151) was found in the Farlp C-terminal Cdc42p/Bemlp/Cdc24p-binding region (Figure 4A).

To test the requirement of this motif for p27-mediated cell migration, a transducible full length p27 protein that contained a mutation of each residue, termed TATp27-QM (quadruple mutant) was generated (Figure 4A). TATp27-QM protein efficiently transduced into cells and induced a Gi cell cycle arrest (Figure 4B, C). However, TATp27-QM protein treatment failed to induce migration of HepG2 cells (Figure 5A, B). These observations indicate that the SQx₇AxI- motif present in the p27 scatter domain is important for the ability of p27 to induce cell migration. In addition to cell migration, HGF, T ATp27- WT, and TATρ27- 158 proteins induced filopodia (actin microspikes) formation, whereas TATp27-118, TATp27-QM and TATp27-N' proteins failed to induce filopodia (data not shown). Taken together, these observations define a novel C-terminal scatter domain in p27 that resides between residues 118-158.

Example 5

HGF-Induced Phosphorylation on Ser-10 Mediates p27 Nuclear Export and is Required for Cell Migration

The requirement for Ser-10 phosphorylation during induction of cell migration was examined due to the phosphorylation of endogenous p27 on Ser-10 and concomitant nuclear export to the cytoplasm of HGF treated cells. A transducible full length p27 protein that contained a Ser to Ala mutation at position 10, termed TATp27-S10A (Figure 4A), was generated. TATp27-S10A protein transduced into cells and induced a Gi cell cycle arrest (Figure 5C; data not shown). However, TATp27-S10A protein failed to induce migration of treated HepG2 cells (Figure 5A, B).

Next, the intracellular localization of TATp27 proteins in response to HGF treatment by immunocytochemistry, was examined. Figure 5C shows the results obtained for TATp27 protein localization in response to HGF. HepG2 cells were treated with control PBS (left panels) or HGF (right panels) for 20 hr, followed by treatment with control PBS, TATp27-WT protein or TATp27-S 10A protein for an additional 3 hr. TATp27 proteins were visualized by immunostaining with anti-HA antibodies (HA epitope tag is present in N-terminal leader of all TAT-fusion proteins) and FITC-anti-mouse IgG (green). Corresponding nuclei were counterstained with DAPI (blue). Both TATp27-WT and TATp27-S10A proteins were present in the nucleus of control PBS treated cells (Figure 5C, left panels). Consistent with the translocation of endogenous p27 (Figure IA), TATp27-WT protein was exported from the nucleus into the cytoplasm of HGF treated cells (Figure 5C, right panels). In addition, TATp27- 158, TATp27-l 18 and TATp27-QM proteins were also exported from the nucleus in an HGF- dependent fashion. In contrast, TATp27-S10A protein remained in the nucleus of HGF treated cells (Figure 5C, right panels). Taken together, these observations establish that p27-mediated cell migration requires S er- 10 phosphorylation-dependent cytoplasmic localization of p27 bearing an intact C-terminal scatter domain (118-158), but not a functional cyclinxdk binding domain.

Example 6 p2 T'^~ Cells Fail to Migrate

To confirm the placement of p27 in a cell motility pathway, the ability of wild type and p27-deficient primary murine embryonic fibroblasts (MEFs) to migrate into and repopulate a denuded region of confluent cells was examined using a standard migration assay. MEFs isolated from strain-matched wild type mice efficiently migrated into the denuded region in 24 hr (Figure 6A, left panel). In contrast, p27-deficient MEFs failed to migrate into the denuded region (Figure 6A, right panel). Consistent with this observation, p27-deficient 3T3 derivates (immortalized cells) also failed to migrate into and repopulate the denuded area (Figure 6B, top left panel), demonstrating that p27 was also essential for cell migration after immortalization. p27-deficient 3T3 cells were next assayed for migration potential by reconstitution of p27 with transducible TATp27-WT protein. Both TATp27-WT and TATp27-l 58 proteins complemented the migration defect in p27-deficient 3T3 cells and rescued the ability to induce cell migration (Figure 6B). In contrast, TATp27-l 18 protein failed to complement the migration defect in p27- deficient 3T3 cells (Figure 6B). Taken together, these observations demonstrate a role for p27 in a cell motility pathway that functions in both epithelial and non-epithelial derived cells that is independent of Gi cell cycle arrest functions of p27. Example 7 Wound Healing

Migration of cells into a wounded area to drive closure remains a rate-limiting step in wound healing. Therefore, application of agents that enhance cell migration by either topical, local or systemic delivery, pending the extent ofthe wounded area and/or individual, would serve to expedite the wound healing process. A wounded area is treated with transducible p27 proteins and/or derivative protein domains and peptides to enable and enhance cell migration. By applying the above transducible p27 proteins, peptides or derivatives thereof, would serve to recruit cells into the wounded spaces and result in dramatic decreases in time to heal the wounded area.

Figure 6 A shows the wound-migration analysis of wild type and p27-deficient murine embryonic fibroblasts (MEFs). A wound area was mechanically induced by a single passage of a microtome blade across culture plate surface to confluent MEF monolayers. After 24 hr, cells were fixed and stained with hematoxylin. The experiment was repeated three times with indistinguishable results. The wound edge is noted as a digitally-drawn line over the image.

Figure 6B shows that p27 protein rescues cell migration defect. Confluent p27-deficient 3T3 cells were subjected to the wound-migration assay described in (A) by single passage of a rubber policeman followed by treatment with transducible TATp27 proteins. p27-WT and p27- 158 proteins rescued wound-migration defect, whereas mock treatment (PBS) and TATp27-l 18 protein (lacking the p27 scatter domain) failed to induce cell migration. The wound edge is noted as a digitally-drawn line over the image.

Decreasing the healing time has the advantage that such a treatment will result in a dramatic reduction in patient exposure to pathogenic microorganisms, decreased rates of dehydration, and decreases costs of hospital visits.

Claims

What is claimed is:

1. A p27 fusion protein comprising a covalently linked protein transduction domain and a p27 domain, wherein the p27 domain comprises at least about 50% of the amino acid sequence ofthe full length p27 domain.

2. The p27 fusion protein of claim 1 , wherein the p27 domain comprises at least about 60% of the amino acid sequence ofthe full length ρ27 domain.

3. The p27 fusion protein of claim 1 , wherein the p27 domain comprises at least about 70% of the amino acid sequence ofthe full length p27 domain.

4. The p27 fusion protein of claim 1, wherein the p27 domain comprises at least about 80% of the amino acid sequence ofthe full length p27 domain.

5. The p27 fusion protein of claim 1 , wherein the p27 domain comprises at least about 90% of the amino acid sequence ofthe full length p27 domain.

6. The p27 fusion protein of claim 1 , wherein the p27 domain comprises at least about 95 % of the amino acid sequence ofthe full length p27 domain.

7. The p27 fusion protein of claim 1 , wherein the p27 domain comprises the amino acid sequence ofthe full length p27 domain.

8. The p27 fusion protein of claim 1 , wherein the transducing domain comprises TAT protein.

9. The p27 fusion protein of claim 2, wherein the fusion protein comprises one or more His and or HA-epitope tags.

10. The p27 fusion protein of claim 1 , wherein the ρ27 domain comprises p27 carboxy terminal amino acids.

11. The p27 fusion protem of claim 10, wherein the carboxy terminal comprises amino acid residues 105 to 198.

12. The p27 fusion protein of claim 10, wherein the fusion protein is transduced into mammalian cells.

13. The p27 fusion protein of claim 12, wherein the transduced fusion protein induces cell migration for the promotion of wound healing, as determined by cell motility assays.

14. The p27 fusion protein of claiml 0, wherein the carboxy terminal amino acid residues 118 to 158 are deleted.

15. The p27 fusion protein of claim 12, wherein the protein induces formation of filopodia and lamellipodia.

16. The p27 fusion protein of claim 15, wherein the protein is transduced into tumor cells to inhibit metastasis.

17. The p27 fusion protein of claim 1 , wherein the protein comprises a serine to alanine mutation at amino acid residue number 10 of p27.

18. The p27 fusion protein of claim 17, wherein the protein is transduced into mammalian cells and induces the cells' growth cycle arrest.

19. The p27 fusion protem of claim 18, wherein the protein aπests the cell cycle at the Gi position.

20. The p27 fusion protein of claims 12 or 18, wherein the mammalian cells are epithelial cells.

21. The p27 fusion protein of claims 12 or 18, wherein the mammalian cells are non- epithelial cells.

22. The p27 fusion protein of claim 1 , wherein the fusion protein comprises a serine amino acid at position 10 of p27 protein.

23. The p27 fusion protein of claim 22, wherein the serine at amino acid position 10 is phosphorylated by Hepatocyte growth factor activated kinase and results in nuclear export of the fusion protein into the cytoplasm.

24. The p27 fusion protein of claim 23, wherein localization ofthe fusion protein in the cytoplasm induces filopodia formation.

25. The p27 fusion protein of claim 24, wherein the filopodia formation is independent of Gi cell cycle aπest.

26. The p27 fusion protein of claim 24, wherein cell cycle arrest functions map to the amino terminal portion ofthe protein.

27. The p27 fusion protein of claim 24, wherein cell migration induction by said fusion protein maps to the carboxy terminus portion ofthe p27 protein and induces cell migration independently ofthe cell cycle aπest functions ofthe amino terminal portion ofthe p27 protein.

28. A method of treating a patient suffering from or susceptible to tumor cell growth, the method comprising: aclmimstering to the patient a therapeutically effective amount of a p27 fusion protein comprises a covalently linked protein transducing domain and a p27 domain, wherein the protein is lacking the functional carboxy terminus scatter domain, as measured by cell migration assays.

29. The method of claim 28, wherein the fusion protein is co-administered with a one or more other p27 fusion proteins.

30. The method of claim 28, wherein the fusion protein is co-administered with a chemotherapeutic agent.

31. A method of treating a patient suffering from, or susceptible to, metastatic tumor cells, the method comprising: administering to the patient a therapeutically effective amount of p27 fusion protein which lacks the serine amino acid at position 10.

32. The method of claim 31, wherein antisense oligonucleotides are administered to a patient to inhibit the motility of cells and for treating metastasis of tumor cells.

33. The method of claim 32, wherein antisense oligonucleotides aπest the cell cycle of tumor cells.

34. A method for treatment of wounds, the method comprising: topical or systemic administering to a patient p27 fusion proteins which increase cell motility; wherein, cells migrate into the wound area and accelerate healing.

35. The method of claim 34, wherein the cells are epithelial or non-epithelial cells.

36. The method of claim 35, wherein the cells are fibroblasts.

37. The method of claim 34, wherein cells are transduced with p27 fusion proteins, peptides, or mutants.

38. The method of claim 34, wherein the fusion proteins comprise full length p27 protein.

39. The method of claim 34, wherein the fusion proteins comprise p27-l 58.