WO1999058156A1

WO1999058156A1 - Cancer prevention by selective delivery methods

Info

Publication number: WO1999058156A1
Application number: PCT/US1999/009848
Authority: WO
Inventors: Orla M. Conneely; Daniel Medina; Lakashmi Sivaraman; David Shine; Bert W. O'malley
Original assignee: Baylor College Of Medicine
Priority date: 1998-05-12
Filing date: 1999-05-06
Publication date: 1999-11-18
Also published as: AU3883299A

Abstract

The present invention is directed to compositions and methods that utilize injection delivery to introduce genes selectively to cancerous cells. Compositions preferably comprise an adenoviral gene delivery system containing a gene encoding a toxin. Methods of the invention comprise delivery of the recombinant vector into the mammary epithelium by intraductal injection. Delivery is highly efficient throughout the mammary epithelium. Compositions and methods of the invention are useful for treatment, and in particular prevention, of cancers including breast, prostate and lung cancers.

Description

CANCER PREVENTION BY SELECTIVE DELIVERY METHODS

Background of the Invention

1. Field of the Invention The present invention relates to compositions and methods for the treatment and, in particular, the prevention, of cancer. Compositions comprise novel vector constructs for delivering genetic information to target cells and to therapeutic agents that work in conjunction with products expressed from the genetic information delivered. Methods of the invention comprise novel techniques for selectively delivering genetic information to target cells and, in particular, to epithelial cells. Both compositions and methods of the invention are useful for prevention of many forms of cancer including breast cancer, prostate cancer and lung cancer.

2. Description of the Background

Structure and Development of the Mammary Gland: The mammary gland develops postnatally as an arborized structure consisting of epithelial cells that give rise initially to a large main duct that proliferates and branches into secondary ducts that end in small club-shaped terminal end buds (TEB). Further sprouting of ducts results in an increased density of TEB, increased branching of secondary ducts and formation of alveolar buds (AB). Upon exposure to estrogen and progesterone during the ovarian cycle, some alveolar buds begin to differentiate into smaller units, the alveoli. During pregnancy, rapid epithelial cell proliferation begins again, but this time is associated with extensive differentiation of alveolar buds into alveolar lobules that will serve as the functional unit of milk production. The tree-like mammary epithelium resides in the mammary fat pad surrounded by stromal fibroblast cells and adipose tissue. The ovarian steroid hormones, estrogen and progesterone are major regulators of mammary epithelial cell proliferation and differentiation. Estrogen is a major stimulus of ductal growth and TEB sprouting and proliferation. Progesterone also stimulates epithelial cell proliferation by increasing ductal side branching, but also is the primary stimulus of differentiation to produce mature alveolar lobules. The effects of estrogen and progesterone are mediated by specific nuclear receptors for both hormones that act as hormone activated transcription factors to regulate the expression of genes associated with mammary epithelial cell proliferation and differentiation. Etiology of Breast Cancer: In the United States, a woman living to age 85 has about an 1 1% lifetime risk of developing breast cancer. Age, reproductive history, genetic background and premalignant lesions are the major risk factors. Early menarche, late pregnancy and nulliparity are associated with increased risk. Genetic predisposition accounts for approximately 10% of cases. The identification of several genetic mutations including BRCA1 and BRCA2 associated with hereditary breast cancer has greatly improved diagnosis of susceptibility to the disease. The only current preventative approach for women who are diagnosed with a genetic predisposition to breast cancer is radical mastectomy. Comparative analysis of human and rodent mammary tumorigenesis suggests that rapidly proliferating cells that are concentrated in the terminal end buds of rodents and the interlobular terminal duct in humans are the most susceptible to tumorigenesis. Thus, these proliferating cells as well as those in the mammary epithelium throughout the gland may provide suitable targets for gene delivery and alternative approaches aimed at decreasing tumor susceptibility by inhibiting proliferation of susceptible cells.

Role of Steroid hormones in Mammary Tumorigenesis: In the case of mammary gland tumorigenesis, the effects of progesterone and estrogen can be either stimulatory or inhibitory or both and such effects are dose and stage dependent. The hormonal effects are known to be mediated by specific high affinity intracellular receptor proteins that are members of a superfamily of related transcription factors. Studies on the ontogeny of mouse mammary gland responsiveness to ovarian steroid hormones have indicated that receptors for estrogen and progesterone (ER and PR respectively) are present in both stromal and epithelial cells. The estrogen and progesterone receptors in epithelial cells are responsive to their ligands at four and seven weeks of age, respectively. The essential role of these receptors in mediating mammary developmental responses to estrogen and progesterone has been confirmed recently by the generation of null mutant mice lacking functional receptors for both hormones. These mice display grossly impaired ductal epithelial proliferation and branching in the case of the estrogen receptor null mutants and significant ductal development but decreased arborization and an absence of alveolar differentiation in the case of the progesterone receptor null mutants.

Estrogen and progesterone also play an important role in the development of mammary tumors. Estrogen receptors are expressed only in a subset of scattered epithelial cells. However, most mammary tumors in humans and the rat are initially estrogen dependent and can be inhibited in the early stages by the anti-estrogen, tamoxifen, supporting the conclusion that these tumors arise in estrogen responsive cells. These observations indicate that progesterone receptors, whose expression is known to be induced by estrogen in the mammary gland, may mediate the tumorigenic effects of estrogen. The observation that 60-70% of human mammary tumors and 90% of rat mammary tumors are initially estrogen dependent and can be blocked by tamoxifen, together with the high degree of susceptibility of proliferating cells within the terminal end buds to tumorigenesis. suggest that estrogen may influence the activity of proliferating cells either directly or indirectly to promote neoplastic transformation. This suggests that estrogen responsive cells also may be a suitable target for prophylactic approaches to inhibit proliferation of tumor susceptible cells and prevent later development of mammary tumors.

The mechanism by which steroid hormone receptors mediate hormone induced signal transduction has been studied extensively in tissue culture and cell-free systems. Binding of steroids to their cognate receptors results in the formation of activated receptor dimers that bind to specific enhancer DNA elements located in the promoter regions of hormone-responsive genes. Ligand dependent activation is accompanied by a removal of receptor-bound corepressor proteins that inhibit transcriptional activation by steroid receptors and an induction of binding of coactivator proteins that facilitate functional interaction of steroid receptors with the general transcription machinery. Activation or repression of specific genes by steroid receptors represents the manifestation of the hormonal response. Prostate Cancer:

Prostate cancer like breast cancer, is derived from epithelial cells whose growth is initially androgen hormone dependent, then progresses to hormone independence. Prostate tumors are initially responsive to antiandrogen therapy but later become non- responsive. Tumors appear to arise in androgen responsive epithelial cells and in cells with high proliferative index, therefore toxic gene delivery that ablates either the whole epithelium, proliferative epithelial cells or androgen responsive epithelial cells should be effective in preventing tumor development in susceptible individuals. Summary of the Invention

The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides new compositions and methods for the prevention of cancer.

One embodiment of the invention is directed to compositions useful for selective delivery of anti -cancer agents to target cells and, in particular, mammary epithelial cells to prevent breast cancer. Agents which can be utilized include effective chemicals, toxins and genetic sequences which encode proteins effective in the prevention of cancer.

Another embodiment of the invention is directed to methods for the selective targeting of therapeutically-effective agents to epithelial cells for the prevention of cancer and other cell proliferative disorders. Selective delivery comprises intraductal administration and, preferably, direct intraductal injection of effective agents.

Another embodiment of the invention is directed to methods for prevention of breast, prostate or lung cancer in a patient comprising administering an agent for selective ablation of proliferating tumor-susceptible epithelial cells.

Another embodiment of the invention is directed to methods for prevention of cancer in a patient comprising administering an agent for selective ablation of steroid-responsive tumor-susceptible cells. Agents comprise toxin-encoding genes which permit selective expression of the toxins in steroid-responsive cells.

Another embodiment of the invention is directed to methods for the treatment of prostate cancer in a patient comprising administering an agent, such as an agent that selectively targets epithelial cells, by intraurethral injection. Another embodiment of the invention is directed to methods for selective delivery of a gene to mammary epithelial cells comprising administering an agent containing exogenous DNA to said cells by intraductal injection.

Another embodiment of the invention is directed to compositions for the prevention or other treatment of an epithelial cell derived cancer comprising an adenoviral vector containing a cytotoxic gene.

Another embodiment of the invention is directed to methods for the selective destruction of rapidly proliferating epithelial cells comprising the steps of administering an agent comprising an adenoviral vector comprising a constitutive promoter coupled to the coding region of the tk gene and administering an effective amount of gancyclovir.

Another embodiment of the invention is directed to methods for the selective destruction of tumor-susceptible epithelial cells comprising administering an adenoviral vector comprising a cytotoxic gene. Another embodiment of the invention is directed to methods for preventing an epithelial cell derived cancer disposed in a duct and having an accessible route of ductal delivery comprising administering into said duct an agent that selectively targets epithelial cells.

Other embodiments and advantages of the invention are set forth, in part, in the description which follows and, in part, will be obvious from this description or may be learned from the practice of the invention. Description of the Drawings

Figure 1(A) Rat mammary gland 48 hours after injection with tracking dye. Figure 1(B) β-galactosidase expression pattern in rat mammary gland 2 days after injection with the Ad-CMV-βgal reporter gene construct.

Figure 1(C) β-galactosidase expression pattern in rat mammary gland 2 days after injection with the Ad-CMV-βgal reporter gene construct. Figure 1(D) High magnification of β-galactosidase expression patterns in rat mammary gland 2 days after injection with the Ad-CMV-βgal reporter gene construct. Figure 1(E) β-galactosidase expression patterns in rat mammary gland 4 days after injection with the Ad-CMV-βgal reporter gene construct. Figure 1 (F) β-galactosidase expression patterns in rat mammary gland 9 days after injection with the Ad-CMV-βgal reporter gene construct. Figure 2(A) Expression pattern of the estrogen-dependent Ad-ERE-tk-βgal reporter gene construct in the rat mammary gland in the absence of estradiol benzoate. Figure 2(B) Expression pattern of the estrogen-dependent Ad-ERE-tk-βgal reporter gene construct in the rat mammary gland following injection of estradiol benzoate.

Figure 2(C) Expression of the Ad-CMV-βgal reporter gene construct in the rat mammary gland in the absence of estradiol benzoate. Figure 2(D) Expression pattern of the estrogen-dependent Ad-ERE-tk-βgal reporter gene construct in the region of the rat mammary gland close to the nipples in the absence of estradiol benzoate.

Figure 2(E) Expression pattern of the estrogen-dependent Ad-ERE-tk-βgal reporter gene construct in the region of the rat mammary gland close to the nipples following injection of estradiol benzoate. Figure 2(F) Expression of the CMV-βgal reporter gene construct in the region of the rat mammary gland close to the nipples in the absence of estradiol benzoate. Figure 2(G) Expression pattern of the estrogen-dependent Ad-ERE-tk-βgal reporter gene construct in the small duct region of the rat mammary gland in the absence of estradiol benzoate. Figure 2(H) Expression pattern of the estrogen-dependent Ad-ERE-tk-βgal reporter gene construct in the small duct region of the rat mammary gland following injection of estradiol benzoate. Figure 2(1) Expression of the CMV-βgal reporter gene construct in the small duct region of the rat mammary gland in the absence of estradiol benzoate. Figure 3(A) Expression pattern of the estrogen-dependent Ad-ERE-tk-βgal reporter gene construct in the region of the rat mammary gland close to the nipples in the absence of estradiol benzoate. Figure 3(B) Expression pattern of the estrogen-dependent Ad-ERE-tk-βgal reporter gene construct in the small ducts of the rat mammary gland in the absence of estradiol benzoate. Figure 3(C) Expression pattern of the estrogen-dependent Ad-ERE-tk-βgal reporter gene construct in the region of the rat mammary gland close to the nipples following injection of estradiol benzoate. Figure 3(D) Expression pattern of the estrogen-dependent Ad-ERE-tk-βgal reporter gene construct in the small ducts of the rat mammary gland following injection of estradiol benzoate. Figure 4(A) Construction of pADL.l/RSV. Figure 4(B) Construction of pADL.1 /RSV-tk. Figure 4(C) Generation of the Ad-RSV-tk recombinant adenovirus.

Figure 5(A) BrdU staining of the ductal region of rat mammary gland infected with

Ad-RSV-tk recombinant adenovirus without gancyclovir treatment.

Figure 5(B) BrdU staining of the alveolar lobules of rat mammary gland infected with Ad-RSV-tk recombinant adenovirus without gancyclovir treatment. Figure 5(C) BrdU staining of the terminal end buds of rat mammary gland infected with Ad-RSV-tk recombinant adenovirus without gancyclovir treatment.

Figure 5(D) Ductal region of rat mammary gland infected with Ad-RSV-tk recombinant adenovirus after treatment with gancyclovir. Figure 5(E) Alveolar lobules of rat mammary gland infected with Ad-RSV-tk recombinant adenovirus after treatment with gancyclovir.

Figure 5(F) Terminal end buds of rat mammary gland infected with Ad-RSV-tk recombinant adenovirus after treatment with gancyclovir. Figure 6(A) Whole mount analysis of Ad-RSV-tk recombinant adenovirus infected mammary tissue without gancylcovir treatment and labeled with BrdU. Figure 6(B) Whole mount analysis of Ad-RSV-tk recombinant adenovirus infected mammary tissue without gancylcovir treatment and labeled with BrdU. Figure 6(C) Whole mount analysis of Ad-RSV-tk recombinant adenovirus infected mammary tissue without gancylcovir treatment and labeled with BrdU. Figure 6(D) Whole mount analysis of Ad-RSV-tk recombinant adenovirus infected mammary tissue BrdU labeled after treatment with gancylcovir. Figure 6(E) Whole mount analysis of Ad-RSV-tk recombinant adenovirus infected mammary tissue BrdU labeled after treatment with gancylcovir. Figure 6(F) Whole mount analysis of Ad-RSV-tk recombinant adenovirus infected mammary tissue BrdU labeled after treatment with gancylcovir.

Description of the Invention

As embodied and broadly described herein, the present invention is directed to compositions and methods for the prevention and treatment of cancer.

Conventional approaches to the treatment of cancer involve the selective destruction of cancerous cells. Anti-cancer agents, effective at therapeutic doses, are often rendered ineffective at the doses required to prevent undesirable side effects.

Current approaches to prevent the development of breast cancer are extremely limited and involve either radical mastectomy or treatment with the antiestrogen, tamoxifen. Alternative preventative strategies that will eliminate the necessity for surgical mastectomy are needed. While tamoxifen preventative therapy has recently shown to significantly reduce the risk of development of breast cancer in women by 45%, potential side effects of tamoxifen, including increased risk of uterine cancer, are well documented. Further, the current treatment period is lengthy at five years. Finally, uncertainty exists as to whether tumor cells may regrow after cessation of treatment if tamoxifen is cytostatic rather than cytotoxic.

It has been discovered that cells and, in particular, breast epithelial cells, can be selectively targeted for destruction by intraductal administration of cytotoxins and, preferably, intraductal injection. Accordingly, agents such as recombinant vectors that express tissue or cell-specific anti-cancer agents, can be used in such delivery systems to prevent various forms of cancer. The compositions and methods disclosed herein may be broadly applied to the prevention of all epithelial cell derived cancers in which there is an accessible route of ductal delivery.

It has been discovered that the mammary epithelium can be selectively targeted by intraductal injection of an adenoviral vector. A foreign gene may be introduced into the mammary epithelium by injection into the main duct of the mammary gland an adenoviral vector carrying the foreign gene under the control of a promoter. For example, in one embodiment, the foreign gene may be a gene encoding a toxin under the control of a constitutive promoter, such as a cytomegalovirus (CMV) promoter. Using a CMV-βgal construct, it has been demonstrated that CMV promoter expression so administered is limited to the epithelium and substantially absent from the stromal compartment of the mammary gland. Specifically, an adenoviral vector carrying a gene encoding β-galactosidase under the control of the CMV promoter (CMV βgal) was injected into the main duct of the mammary gland. Expression from the promoter was limited to the mammary epithelium. The epithelium of the mammary gland is the region from which mammary tumors arise.

In another embodiment, a foreign gene is introduced into a specific region of the mammary epithelium, namely, the subset of epithelial cells that respond to estrogen by induction of expression of estrogen dependent target genes. To demonstrate and obtain this cell specific expression, an adenoviral vector containing the lacZ reporter gene, which encodes the β-galactosidase enzyme, under the transcriptional control of a promoter that contains at least one copy of a cis-acting estrogen responsive element (ERE) and the thymidine kinase (tk) promoter may be used. Since mammary tumors are known to arise initially in cells that are responsive to estrogen and progress from an estrogen dependent state to an estrogen independent state, targeting of foreign genes to these cells provides a gene therapy approach to modify activity of cells from which mammary tumors arise. For example, an adenoviral vector comprising a steroid- responsive promoter, such as an estrogen-responsive promoter, coupled to a cytotoxic gene, such as the tk gene, may be used to selectively target steroid-responsive cells. Treatment of cells carrying the steroid-responsive cytotoxic gene with gancyclovir would trigger destruction of the cells expressing the tk gene.

These methods demonstrate the ability to deliver exogenous genes to the epithelial cells of the mammary gland by intraductal injection into the cannulated main duct of the mammary gland, β-galactosidase expression occurs throughout the mammary epithelium, but is absent from stromal cells and adipose cells within the gland. These methods also demonstrate that delivery of an estrogen receptor responsive target gene (ERE-tk-βgal) to the mammary epithelium permits expression of β- galactosidase in a subset of epithelial cells in an estrogen dependent manner. In addition, using the novel compositions and methods of the present invention, it has been discovered that proliferating mammary epithelial cells may be selectively ablated.

For example, an alternative adenoviral vector which contains the gene encoding thymidine kinase (tk) under the control of the constitutive Rous sarcoma virus (RSV) promoter was used to examine the feasibility of selectively ablating subsets of epithelial cells by the gene delivery approach. Expression of the thymidine kinase gene in mammary epithelial cells of rats administered the drug gancyclovir results in phosphorylation of gancyclovir and incorporation of the phosphorylated nucleotide into replicating strands of DNA in rapidly proliferating cells. The nucleotide acts as a terminator of DNA polymerization, resulting in cessation of replication and ultimate death of replicating cells. While most mammary epithelial cells are efficiently transduced by the virus, proliferating rather than differentiated epithelial cells are selectively ablated by the virally expressed thymidine kinase. This approach allows for selectively ablating proliferating cells that are susceptible to neoplastic transformation by toxin activation using adenoviral gene delivery to the mammary epithelium. The approach leaves the non-proliferating epithelium and general mammary gland architecture intact, but drastically lowers susceptibility of the gland to tumorigenesis. This procedure provides an alternative relatively non-invasive approach to mastectomy to significantly decrease or eliminate tumor susceptibility in women predisposed to breast cancer disease. Further, the procedure also provides a suitable approach to prevent tumor reoccurrence in women who have been treated for breast cancer by hormonal therapy or lumpectomy.

Accordingly, one embodiment of the invention is directed to compositions and methods for selectively targeting intraductal epithelial cells of the mammalian mammary gland with anti-cancer agents for the treatment of localized non- invasive proliferative disorders and their progression to invasive cancer. Compositions may comprise agents that selectively or generally eliminate populations of proliferative cells. Preferably, compositions comprise a recombinantly engineered viral vector containing genetic information to be expressed in the target cell. Another embodiment of the invention is directed to methods comprising intraductal infection of anti-cancer agents and, preferably, recombinant viral vectors. These methods are especially useful for the prevention of cancer.

Another embodiment of the invention is directed to a composition for the prevention of breast cancer comprising an agent that selectively targets mammary epithelial cells. In a preferred embodiment, the agent is an adenoviral vector containing a cytotoxic gene.

Another embodiment is directed to a composition for the prevention or other treatment of an epithelial cell derived cancer comprising an adenoviral vector containing a cytotoxic gene. Preferably, the cancer is glandular tissue and is accessible via ductal delivery of the composition. Cancers which may be prevented or treated using the compositions and methods of the present invention include breast cancer, prostate cancer, lung cancer, and any other cancer of epithelial origin, particularly those accessible by retrograde injection into a duct.

The adenoviral vector may further comprise a constitutive promoter coupled to the cytotoxic gene. Constitutive promoters such as a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, a herpes simplex virus (HSV) promoter, an SV40 promoter or any suitable promoter may be used. The cytotoxic gene may be any suitable directly cytotoxic gene, for example, the gene encoding diphtheria toxin or the gene encoding ricin or the gene encoding caspase. Caspase is a gene product that promotes cell death by apoptosis. Alternately, the cytotoxic gene may be a suicide gene, for example the thymidine kinase (tk) gene. Suicide genes can make targeted cells susceptible to specific drugs. Administering the drug to cells carrying such suicide genes results in cell death. For example, cells expressing the tk gene are killed following treatment with the drug gancylcovir, whereas cells not expressing the tk gene are unharmed by gancylcovir treatment. Other suitable cytotoxic genes may also be used.

The adenoviral vector may alternately comprise a steroid-responsive promoter coupled to the cytotoxic gene in which the promoter contains an enhancer element. The enhancer element may be an estrogen-responsive, an androgen-responsive or a progesterone-responsive enhancer element. In a preferred embodiment, the cancer to be prevented by the composition of the present invention is breast cancer and the promoter is estrogen-responsive and contains a cis-acting estrogen-responsive enhancer element. In another preferred embodiment, the cancer to be prevented is prostate cancer and the promoter is responsive to the steroid hormone androgen. Another embodiment of the invention is directed to a method for the selective destruction of tumor-susceptible epithelial cells comprising administering an adenoviral vector comprising a cytotoxic gene. The cytotoxic gene may be a directly cytotoxic gene or a suicide gene.

In a preferred embodiment, the tumor-susceptible epithelial cells are accessible via ductal delivery and the vector is administered intraductally. For example, when the tumor-susceptible epithelial cells are mammary cells, the anti-cancer agent is preferably administered intraductally. When the tumor-susceptible epithelial cells are prostate cells, the anti-cancer agent is preferably administered intraurethrally.

Another embodiment of the present invention is directed to compositions and methods for the prevention or other treatment of steroid-responsive cancers, by administering to a patient an adenoviral vector comprising a cytotoxic gene coupled to a steroid-responsive promoter. This method may optionally comprise the step of administering the steroid to which the promoter is responsive.

Another embodiment of the invention is directed to methods for preventing or otherwise treating an epithelial cell derived cancer disposed in a duct and having an accessible route of ductal delivery comprising administering into said duct an agent that selectively targets epithelial cells. Preferably, the agent comprises an adenoviral vector. More preferably, the agent comprises an adenoviral vector coupled or functionally linked to a cytotoxic gene. Suitable types of cytotoxic genes include, but are not limited to, directly cytotoxic genes and suicide genes. In a preferred embodiment, the cytotoxic gene is coupled to a constitutive promoter. In another preferred embodiment, the cytotoxic gene is coupled to a steroid-responsive promoter. Another embodiment is directed to a method for the selective destruction of rapidly proliferating epithelial cells comprising the steps of administering an agent comprising an adenoviral vector comprising a constitutive promoter coupled or functionally linked to the coding region of the tk gene, and administering an effective amount of gancyclovir. The agent may be administered intraductally or intraurethrally. The types of rapidly proliferating epithelial cells which may be targeted by this method include prostate cancer and breast cancer cells. The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention. Examples Example 1 Experimental Animals

Female Wistar Furth rats (28 days old) were obtained from Harlan Sprague Dawley, Inc., Indianapolis, IN. They were anesthetized and either sham- operated or ovariectomized prior to experimentation. All animal studies were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals. Example 2 Recombinant Adenovirus Construction and Large-scale Production Replication defective recombinant adenoviruses expressing β- galactosidase (βgal) under the control of the cis-acting estrogen response element (ERE) and either the Elb or thymidine kinase (tk) minimal promoters were constructed. For pERE-tk-βgal shuttle vector construction, the 191 bp Xba I - Bgl II ERE-tk fragment containing a single copy of the ERE sequence upstream of the tk promoter was isolated from plasmid pEREl 5 and ligated upstream of a 3.4 Kb Hind III - Dra I β-galactosidase fragment from pCHl 10 (Pharmacia Biotech Inc., NJ), and the 153 bp poly A' fragment from SV40 DNA in the pXCJL Ad vector. For the pERE4-Elb-βgal shuttle vector construction, a synthetic oligonucleotide containing four copies of the ERE sequence located upstream of the Elb minimal promoter was subcloned into the pqEl spl adenoviral shuttle vector. Both adenoviral shuttle vectors were CsCl₂-purified and were then cotransfected with pJM107 into 293 cells using N-(l -(2,3-dioleoyloxyl)propyl)- N,N,N-trimethylammoniummethyl sulfate mediated transfection method according to the manufacturer's instructions to allow homologous recombination to occur (Boehringer Mannheim Biochemicals, Indianapolis, IN). The 293 cell line is a transformed human kidney cell line. Individual plaques were isolated and amplified in 293 cells. Viral DNAs were prepared and the recombinant adenovirus (Ad) was identified by polymerase chain reaction (PCR) and Southern analysis. Selected clones of Ad-ERE-tk-βgal and Ad-ERE4-Elb-βgal were obtained by plaque purification and propagated in 293 cells. Cells were harvested 36 to 48 hours after infection. Cell pellets were then resuspended in phosphate buffered saline (50 mM Na phosphate, 100 mM NaCl, pH 7.4; PBS), lysed by three freeze/thaw cycles, centrifuged at 1000 x g for 5 minutes to remove cell debris, and the virus was purified by CsCl₂ gradient centrifugation. Concentrated virus was immediately dialyzed, aliquoted, and stored at -80°C. Viral titers were determined by O.D.₂₆₀ measurement or plaque assay. The control virus Ad-CMV-βgal where βgal is under the control of the constitutive cytomegalovirus (CMV) promoter used in this study was constructed in a similar manner.

Example 3 In Vivo Estrogen-Induced Transactivation of ERE-Reporter Activity in Rat Mammary Gland via Adenoviral Vector Infection Twenty-eight day old female Wistar Furth rats were anesthetized and ovariectomized to reduce the circulating estrogen and progesterone. Ten days later, rats receiving the adenovirus were first anesthetized and infused with 10 μl adenovirus in conjunction with a vital tracking dye (indigo carmine, 50 μg/10 μl) through intraductal injection with a blunt-ended 20-26 gauge needle. At the same time, rats receiving hormonal treatment were given estrogen benzoate (EB) suspension in sesame oil (100 μg/0.2 ml) s.c. Twenty-four hours later, the animals were then anesthetized and the mammary fat pad was removed for X-gal staining. Rats were then euthanized with CO₂.

Example 4 β-galactosidase Assay and X-gal Staining in Tissues For X-gal staining in the mammary gland, rats were anesthetized and fat pads containing the mammary gland were removed. Fat pads were fixed in fresh cold 2% paraformaldehyde solution containing 0.1 M PIPES, pH 6.9, 2 mM MgCl₂, 1.25 mM EGTA for 1 -2 hours, washed with PBS three times, and permeabilized with 0.02% NP40, 0.01% Na deoxycholate, and 2 mM MgCl₂ in PBS for 1 hour. Fat pads were stained immediately with staining solution containing 25 M K₃Fe(CN₆), 25 mM K₄Fe(CN₆), 2 mM MgCl₂, 0.02% NP40. 0.01% Na deoxycholate, 0.5 mg/ml X-gal in PBS, pH 8.1 at 37°C for 12-16 hours. After staining and photography, the glands were subsequently dehydrated, embedded in paraffin, and sectioned serially for microscope examination and photography. Example 5 Introduction of foreign genes into the mammary gland by adenoviral delivery

Indigo carmine was injected into cells as a vital tracking dye to examine the physical penetration of the dye throughout the mammary epithelial compartments to determine the feasibility of introducing foreign genes into the mammary gland. Rats were anesthetized and the main ducts were cannulated with a blunted 21 -26 gauge needle. A single gland can accommodate injections of 2-40 μl of dye suspension depending on the age of the animal. Using this procedure, the success of each injection could be monitored and the complete glandular structure visualized within seconds after the injection. The tracking dye diffused out of the mammary ducts completely within 20 hours and became invisible. This dye was therefore used in combination with the adenovirus preparations to monitor the success of injection. In the first experiments, an adenoviral construct that contains the gene encoding β-galactosidase under the control of the constitutive CMV promoter (Ad-CMV-βgal) was injected in combination with tracking dye into the mammary gland to examine its degree of infectivity in the mammary epithelium. To determine the conditions under which infection by the adenovirus is optimal, studies were performed to determine the appropriate titer of adenovirus to deliver to the gland and the appropriate length of time between administration of the virus and measurement of gene expression. Mammary glands were infused with varying amounts of the virus in conjunction with tracking dye and the mammary fat pads were dissected, fixed, and stained with X-gal at various time points after the injection. A multiplicity of infection of 10: 1 was found to be sufficient to infect the epithelial cells with the assumption that the number of mammary epithelial cells per gland is approximately 3 x 10⁷. In the CMV-βgal experiment shown in Figure 1 , rats were anesthetized and the mammary glands infused with 10 μl of tracking dye either alone (Figure 1 A), or in combination with 4 x 10⁷ pfu Ad-CMV-βgal (Figures IB- IF), an adenoviral construct that contains the gene encoding β-galactosidase, the lacZ gene, under the control of the constitutive CMV promoter. The mammary fat pads were dissected, fixed, and stained with X-gal solution at various time points post intraductal injection of the recombinant virus. Blue cells resulting from X-gal treatment are an indication of β-galactosidase activity.

As shown in Figures IB and IC, strong β-gal expression was observed 2 days after injection with Ad-CMV-β-gal and persisted throughout regions of the gland. Figure IE shows β-gal expression was diminished four days after injection with Ad-CMV-βgal. Figure I F reveals β-gal expression was undetectable nine days after injection with Ad-CMV-βgal. βgal staining was localized to the luminal epithelial compartment (LE) of the mammary gland and not the stromal compartment (S), as shown in Figure ID, which indicates that the virus was preferentially taken up by luminal epithelial cells.

Example 6 In situ localization of estrogen receptor dependent gene expression

The estrogen responsiveness of the Ad-ERE-tk-βgal adenoviral reporter gene construct was tested to reconstitute estrogen-dependent reporter gene expression in situ in the mammary gland. The responses obtained for the Ad-ERE-tk-βgal reporter construct are shown in Figure 2 and Figure 3. Briefly, ovariectomized rats were anesthetized and the mammary glands infused with 1.6 x 10⁸ pfu Ad-ERE-tk-βgal (Figures 2A, 2B, 2D, 2E, 2G, 2H, 3A-3D) or 4 x 10⁷ pfu Ad-CMV-βgal (Figures 2C, 2F, and 21), both in conjunction with tracking dye at a final volume of 10 μl. Half of the Ad-ERE-tk-βgal injected glands were subsequently injected with either control vehicle sesame oil (Figures 2A, 2D, 2G, 3 A and 3B) or 100 μg of estrodiaol benzoate (Figures 2B, 2E 2H, 3C and 3D) at a final volume of 0.2 ml. 24 hours later, the mammary fat pads were dissected, fixed, and stained with X-gal solution to assay for βgal activity, β-galactosidase positive cells are the cells that stain blue. Sections were also counter stained with nuclei fast red. Figures 2D and 2G are high power magnifications of Figure 2A, Figures 2E and 2H are high power magnifications of Figure 2B, and Figures 2F and 21 are high power magnifications of Figure 2C.

In the absence of estrogen (Figures 2A, 2D, 2G, 3 A and 3B), basal expression of β-galactosidase was observed in the rat mammary glands. However, in the mammary glands treated for one day with estradiol benzoate. a strong localized βgal expression signal was detected (Figures 2B, 2C, 2E, 2F, 2H, 21, 3C and 3D). The expression pattern of the estrogen-responsive Ad-ERE-tk-βgal construct was strikingly different from that observed with the constitutive βgal expression construct, Ad-CMV- βgal (compare Figures 2B and 2C, or 2E and 2F, or 2H and 21). Thus, despite the ability of the virus to penetrate epithelial cells located throughout the mammary ductal system as demonstrated by the Ad-CMV-βgal construct (Figures 2C, 2F, and 21), estrogen-dependent reporter gene expression was concentrated in the small ducts (Figures 2H and 3D) as compared to regions close to the nipples (Figures 2E and 3C). The arrows in Figures 2A-2F indicate the location of nipples that connect to the mammary main ducts and subsequently the lobulo-alveolar structures. High power magnification (Figures 2D-2I) more clearly illustrates the expression of β-galactosidase at regions close to the nipples (Figures 2E-2F) and also at distal regions of the glands (Figures 2H-2I). These data indicate that estrogen responsiveness is localized to the small ducts of the gland and is minimal in the large ductal epithelia of these glands. Example 7 Selective delivery of adenoviral gene toxin to the mammary epithelium. Construction of recombinant Ad-RSV-tk virus

The strategy behind the generation of the recombinant adenovirus ADV/RSV-tk is shown in Figure 4. First, the 2.8kb Bglll/BamHI fragment containing the HSV-tk gene and poly (A) tail was inserted into the BamHI site of plasmid pADL.l/RSV (Figure 4B), which was obtained by insertion of the RSV-LTR promoter into the Xbal and Clal sites of the shuttle vector pXCJL.l (Figure 4A). In the resulting plasmid, pADL.l/RSV-tk, the HSV-tk gene is under the control of the RSV-LTR. To generate the desired recombinant adenovirus, pADL.l/RSV-tk and pJM17, a plasmid containing the complete adenovirus genome, were co-transfected into 293 cells by calcium phosphate precipitation (Figure 4C). Recombinant Ad-RSV-tk adenovirus (ADV/RSV-tk in Figure 4C) was isolated from a single plaque, expanded in 293 cells, and purified by double cesium chloride ultracentrifugation. Virus titer was determined by optical absorbance at 260 nm. Injection of adenoviral gene toxin

Forty-five day old rats were divided into two groups. The #4 abdominal mammary glands of both groups were administered 10⁸ pfu of Ad-RSV-tk by intraductal injection into the cannulated main duct. Twelve hours after the injection of virus, one group of rats was injected intraperitoneally with gancyclovir (10 mg/kg body weight) and injections were repeated twice daily thereafter for 4 days. On day 5, all animals were injected with bromodeoxyuridine (BrdU) at a concentration of 50 mg/kg body weight and sacrificed two hours later. Because BrdU is incorporated into replicating DNA, the measurement of the incorporation of BrdU provides an index of the number of proliferating cells in the mammary gland following each treatment condition. Figure 5 shows the results of one experiment.

Briefly, following the treatment described above, the #4 mammary glands were excised, dissected in two and prepared for whole mount staining to examine overall mammary gland morphology or fixed in Methacarn and sectioned (5 μm sections) for immunohistochemical analysis of BrdU labeled cells using a commercially available cell proliferation assay kit (Amersham Inc.). Figures 5A-5C show the number of BrdU positive cells in the ducts (5 A), alveolar lobules (5B) and terminal end buds (5C) in adenoviral transduced epithelium in the absence of gancyclovir treatment. Figures 5D-5F reveal a striking loss of BrdU-positive proliferating epithelial cell in the ducts (5D), alveolar lobules (5E) and terminal end buds (5F) after treatment of viral transduced rats with gancyclovir. BrdU labeled cells were visualized using a Zeiss axioscope. Comparison of the number of BrdU labeled cells in gancyclovir treated versus untreated glands provides a direct measure of the number of proliferating cells remaining in the mammary glands following delivery of the tk gene and treatment with gancyclovir. Figure 6 shows the overall morphology of the mammary gland after injection of the Ad-RSV-tk in the absence and presence of gancyclovir. Figures 6A-6C show BrdU labeled mammary tissue infected with Ad- RSV-tk without gancyclovir treatment. Figures 6D-6F show BrdU labeled mammary tissue infected with Ad-RSV-tk after gancyclovir treatment. These results show that after ablation of proliferating epithelial cells, no changes in the overall morphology of the mammary epithelium are observed indicating that this cytotoxic approach does not adversely affect the morphology of the mammary gland. Example 8 Prostate Cancer

The above approach is also useful as a prophylactic treatment for prostate cancer. Briefly, the experimental approach comprises intraurethrally injecting vectors carrying (1 ) direct cytotoxins such as ricin or diphtheria toxin under the control of constitutively active promoters such as RSV or CMV to ablate all epithelial cells, (2) the thymidine kinase gene under control of constitutively active promoters to selectively ablate proliferating cells, or (3) toxin genes under the control of an androgen responsive promoter to direct expression of the toxin to androgen responsive cells. These vectors are administered to patients who are at high risk for prostate cancer to prevent occurrence or to patients who have responded to antiandrogen therapy or are in remission to prevent reoccurrence.

These relatively non-invasive approaches to cancer prevention constitute a major advancement over conventional procedures such as mastectomy and orchiectomy. Because of the selective expression of the exogenous gene in epithelial cells, the intraductal injection technique should be applicable for administration of pharmaceutical agents for prevention of tumor development in cases where tumors are of epithelial cell origin and in which there is an accessible duct or tract space for delivery. Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All U.S. patents and patent applications, including provisional applications, and all other documents referenced herein, for whatever reason, are specifically incorporated by reference. It is intended that the specification and examples be considered exemplary only, with the true scope and spirit of the invention indicated by the following claims.

Claims

We Claim:

1. A composition for the prevention of breast cancer comprising an agent that selectively targets mammary epithelial cells.

2. The composition of claim 1 wherein the agent is an adenoviral vector containing a cytotoxic gene.

3. A method for prevention of breast, prostate or lung cancer in a patient comprising administering an agent for selective ablation of proliferating tumor- susceptible epithelial cells.

4. The method of claim 3 wherein the agent is an adenoviral vector containing a cytotoxic gene.

5. A method for prevention of cancer in a patient comprising administering an agent for selective ablation of steroid-responsive tumor-susceptible cells.

6. The method of claim 5 wherein the cancer is breast cancer or prostate cancer.

7. The method of claim 5 wherein the agent is a toxin gene which permits selective expression of said toxin in steroid-responsive cells.

8. The method of claim 5 wherein the agent is a toxin gene which is administered by intraductal injection into the mammary glands.

9. A method for the treatment of prostate cancer in a patient comprising administering an agent by intraurethral injection.

10. A method for delivery of a gene to mammary epithelial cells comprising administering an agent containing exogenous DNA to said cells by intraductal injection.

1 1. The method of claim 10 wherein the agent selectively ablates gene expression of said cells.

12. The method of claim 10 wherein the agent selectively ablates gene expression of proliferating epithelial cells.

13. The method of claim 10 wherein the agent selectively ablates gene expression of steroid responsive epithelial cells.

14. A method for the selective destruction of rapidly proliferating epithelial cells comprising the steps of: administering an agent comprising an adenoviral vector comprising a constitutive promoter coupled to the coding region of the thymidine kinase gene; and administering an effective amount of gancyclovir.

15. The method of claim 14 wherein the agent is administered intraductally or intraurethrally.

16. The method of claim 14 wherein the rapidly proliferating epithelial cells are prostate cancer or breast cancer.

17. A method for the selective destruction of a tumor-susceptible epithelial cell comprising administering an adenoviral vector comprising a cytotoxic gene.

18. The method of claim 17 wherein the tumor-susceptible epithelial cell is accessible via ductal delivery and the vector is administered intraductally.

19. The method of claim 17 wherein the tumor-susceptible epithelial cell is a mammary epithelial cell and the agent is administered intraductally.

20. The method of claim 17 wherein the tumor-susceptible epithelial cell is a prostatic epithelial cell and the agent is administered intraurethrally.

21. The method of claim 17 wherein the cytotoxic gene is a directly cytotoxic gene or a suicide gene.

22. The method of claim 17 wherein the tumor-susceptible epithelial cell is steroid- responsive and the adenoviral vector comprises a cytotoxic gene coupled to a steroid- responsive promoter.

23. A method for preventing an epithelial cell derived cancer disposed in a duct and having an accessible route of ductal delivery comprising administering into said duct an agent that selectively targets epithelial cells.

24. The method of claim 23 wherein the agent comprises an adenoviral vector.

25. The method of claim 24 wherein the adenoviral vector is coupled to a cytotoxic gene.

26. The method of claim 25 wherein the cytotoxic gene is a directly cytotoxic gene or a suicide gene.

27. The method of claim 25 wherein the cytotoxic gene is coupled to a constitutive promoter.

28. The method of claim 25 wherein the cytotoxic gene is coupled to a steroid- responsive promoter.

29. The method of claim 23 wherein the cancer is breast cancer and the agent is administered intraductally.

30. The method of claim 23 wherein the cancer is prostate cancer and the agent is administered intraurethrally.

31. A composition for the prevention of an epithelial cell derived cancer comprising an adenoviral vector containing a cytotoxic gene.

32. The composition of claim 31 wherein the cancer is accessible via ductal delivery.

33. The composition of claim 31 wherein the cytotoxic gene is a directly cytotoxic gene selected from the group consisting of a gene encoding diphtheria toxin, a gene encoding ricin and a gene encoding caspase.

34. The composition of claim 31 wherein the cytotoxic gene is a suicide gene.

35. The method of claim 34 wherein the suicide gene is thymidine kinase.

36. The composition of claim 31 wherein the vector further comprises a constitutive promoter coupled to the cytotoxic gene.

37. The composition of claim 36 wherein the constitutive promoter is selected from the group consisting of a cytomegalovirus promoter or a Rous sarcoma virus promoter.

38. The composition of claim 31 wherein the vector further comprises a steroid- responsive promoter coupled to the cytotoxic gene.

39. The composition of claim 38 wherein the steroid is estrogen and the cancer is breast cancer.

40. The composition of claim 38 wherein the steroid is androgen the cancer is prostate cancer.

41. The composition of claim 31 wherein the cancer is breast cancer, prostate cancer or lung cancer.