EP0973902A1

EP0973902A1 - Radioisotope concentrator methods and compositions

Info

Publication number: EP0973902A1
Application number: EP98920832A
Authority: EP
Inventors: Robert B. Mandell; Charles J. Link, Jr.
Original assignee: Human Gene Therapy Research Institute
Current assignee: Human Gene Therapy Research Institute
Priority date: 1997-04-10
Filing date: 1998-04-09
Publication date: 2000-01-26
Also published as: AU7358098A; CA2286860A1; JP2001523955A; WO1998045443A1

Abstract

The present invention pertains to combination nuclear medicine involving radiopharmaceuticals for diagnostic imaging and for therapy of tumors. More specifically the invention includes pharmaceutical compositions and methods of treatment, diagnosis, and radioimaging by genetic engineering designed to accumulate radioisotopes in cells. The combination of genetic engineering to confer radioisotope accumulation by cells with introduction of radioisotopes has many uses including radioimaging in vitro or in vivo to locate or identify gene expression, radiation therapy for killing neoplastic and non-neoplastic cells, and diagnostic applications. Genetic constructs, vectors, transformed cells and methods are disclosed.

Description

TITLE: RADIOISOTOPE CONCENTRATOR METHODS AND

COMPOSITIONS

FIELD OF THE INVENTION The present invention pertains to combination radiotherapy for imaging a destruction of cells and more specifically to pharmaceutical compositions, and methods of treatment by genetic engineering designed to concentrate radioisotopes in tumor cell destruction, non-neoplastic tissue destruction such as keloid scar tissue, adipose tissue, cardiac hypertrophy, etc., or for radioimaging of transduced cells either ex vivo or in vivo, as well as those additional cells subject to a bystander effect.

BACKGROUND OF THE INVENTION

The limited ability of anti-neoplastic therapy to distinguish neoplastic from normal cells on the basis of proliferative behavior has inspired a search for biochemical characteristics of neoplastic cells that are tumor specific rather than proliferation specific. Unfortunately current molecular genetic studies have failed to support the expectation that such characteristics are a consistent feature of neoplastic cells. Rather these studies suggest that the neoplastic state can be explained without postulating tumor specific functions, but merely the operation of normal proliferation-specific functions at abnormal levels, as a result of changes (sometimes minimal) in the structure of growth- regulatory genes or changes in their number or chromosomal environment. This conclusion suggests that continued search for highly specific attributes of neoplastic cells cannot be relied upon for a general solution to the problems of cancer therapy. Major reductions in the lethality of cancer will require alternative approaches that do not depend on the natural occurrence of such attributes.

One alternative strategy entails the artificial creation of differences between normal and neoplastic cells through prophylactic use of gene insertion techniques. In other words, manufacturing biochemical differences which can be exploited to systematically and specifically target neoplastic cells for destruction. This invention involves combination genetic engineering of cells and radiation treatment to inhibit proliferation and kill neoplastic cells for ex vivo protocols such as bone marrow purging or in vivo for treatment of tumors.

Gene insertion protocols are used to artificially manufacture biochemical differences in target tumor cells which are then exploited to specifically render the cells susceptible to the effects of radiation, by causing the cells to take- up and accumulate radioisotopes leading to tumor cell death.

Thus an object of the present invention is to provide therapeutic materials and procedures for visualizing or treating tumors and for non neoplastic cell destruction as well, using, for example, radioisotopes such as

¹³¹1, ^{1 5}I, ¹⁸⁶RE, ⁶ Cu, ⁶⁷Cu, ^etc-

Another object of the invention is to kill tumor using gene therapy to engineer biochemical differences in tumor cells which are exploited to confer radiation therapy sensitivity. Another object of the invention is to genetically engineer tumor cells to contain a gene which causes radioisotope concentration in that cell, which upon exposure to radioisotopes causes accumulation of the same within the cell and targeted cell death.

Another object of the invention is to genetically engineer cells for radioisotope concentration to provide for radioimaging of tumors or of transformed cells in vivo.

SUMMARY OF THE INVENTION

In accordance with an aspect of the present invention, there is provided a method of concentrating radioisotopes in cells for diagnostic as well as therapeutic methods using radiopharmaceuticals. These methods include radioimaging of transduced cells and radiation therapy for destruction of tumor and non-neoplastic cells. The method comprises transducing cells in vivo, ex vivo, or in vitro with a nucleic acid (DNA or RNA) sequence encoding an agent which is capable of providing for the uptake by transformed cells of radionuclides causing their accumulation in transformed cells as well as those cells subject to a bystander effect, upon expression of the nucleic acid sequence encoding the agent and subsequent radiation therapy.

In a preferred embodiment the agent encoded by the nucleic acid is a radioisotope concentrator or transporter which targets and enhances the effects as well as lethality of administered radioactive elements whether for diagnostic radioimaging or for radiation therapy.

DESCRIPTION OF THE FIGURES

Figure 1 is a graph depicting an iodine uptake experiment for several murine and human tumor cell lines as described in the Examples section. As can be seen, the cells which contain the sodium iodide symporter experienced considerably higher iodine uptake than control cells which were the FRTL cells with ice or cells with no vector.

Figures 2(a)-2(d) are graphs depicting saturation kinetics of ¹²⁵I accumulation. Saturation kinetics of ¹²⁵I uptake is linear as a function of the external [r] as determined by Lineweaver-Burk plot. (Lineweaver H et al, "The determination of enzyme dissociation constants", J Am Chem Soc, 1934; 56:658-660). The calculated kM was 31.3 μM and 34.7 μM iodide in 2(A) A375 melanoma and 2(C) IGROV ovarian cancer cells, respectively. These values are similar to that previously reported for rat thyroid FRTL-5 cells. (Weiss SJ et al., "Iodide transport in a continuous line of cultured cells from rat thyroid", Endocrinology, 1984; 114:1090-1098) The results in graphs 2(B) and 2(D) show the NIS activity in 2(B) A375 melanoma and 2(D) IGROV ovarian carcinoma cells that contain no retroviral vector or were transduced with the LNISN vector. Time expressed in minutes after exposure to ¹²⁵I (0.345 nM) in a total concentration of 30 μM Nal. Note the rapid and sustained ¹²⁵I uptake.

Figure 3 is a graph depicting in vitro survival of transduced and non- transduced tumor cells upon exposure to lOμCi/ml ¹³¹I after 6 hours. Percent survival is based on counts of cell colonies still proliferating after treatment. This demonstrates that tumors expressing the NIS can be selectively killed using standard, well-established protocols for ¹³¹I-mediated thyroid ablation. Figure 4 is a graph depicting tumor cell lines after 12 hours of exposure to 133nM Na ¹³¹I (lOμCi/ml) 30μM Nal.

Figure 5 is a graph depicting percent survival of BNL.1ME cells to 6 hour ¹³¹I 8 days post exposure. Figure 6 is a Plasmid map of the Herpes Simplex Viral pHE700 vector.

Amp^R, ampicillin resistant; "a", HSV-1 packaging signal; HSV-tk promoter, HSV-1 thymidine kinase promoter; hyg+, hygromycin resistance; MCS, multi- cloning site; ΔEBNA-1, modified EBV nuclear antigen gene; ori P, EBV unique latent replication origin; ori S, HSV-1 replication origin. Figure 7 is a diagram depicting the proposed mechanism of the effects of photochemical modification of pHE-tk vector containing tandem repeats of the Hstk gene expression unit. In permissive cells transduced with unmodified vector, both DNA replication and gene expression occur (top). After PUVA treatment, interstrand DNA crosslinks inhibit viral replication (Hanson CV, "Photochemical inactivation of viruses with psoralens: an overview", Blood Cells, 1992; 18:7-25), but permit transgene expression from unaffected transcription units.

Figure 8 is a graph depicting Hstk gene activity expressed from PUVA modified vectors in IGROV ovarian carcinoma cells. The graph depicts IGROV cell proliferation after exposure to pHE-tk vector, measured by ³H-thymidine incorporation. Vectors were treated with TMP (0 or 1 μg/ml) and differing lengths of UVA exposure (0 to 7.5 KJ/m²). Tumor cells were plated at IO⁴ cells/well and proliferation assays were done as previously described. (Link, CJ, et al., "Preliminary in vitro efficacy and toxicities studies of the herpes simplex thymidine kinase gene system for the treatment of breast cancer", Hybridoma. 1995; 14:143-147; Section 1: Escherichia coli, plasmids, and bacteriophages. In: Ausubel FM et al (eds). Current protocols in molecular biology. Vol. 1. John Wiley and Sons: New York, 1989, pp. 1,4.2-1.4.3) Each sample is shown with GCV (0 or 5.o μg/ml) illustrating the decrease in vector cytotoxicity while still expressing functional Hstk activity. Incorporated ³H- thymidine is measured as counts per min (cpm) and expressed as percent of control (no GCV).

Figure 9 is a graph depicting the theoretical effect of increasing tumor mass diameter on dose transmission. Increasing the radius of the tumor cell mass results in less of the β-particles being transmitted from the mass.

Figure 10 is a plasmid map of the Herpes Simplex Virus pHE850 vector.

Amp^R, ampicillin resistant; "a", packaging signal; HSV-tk promoter, HSV-1 thymidine kinase promoter; hyg+, hygromycin resistance; MCS, multi-cloning site; ΔEBNA-1, modified EBV nuclear antigen gene; ori P, EBV unique latent replication origin; ori S, replication origin; GFP, green fluorescent protein.

DETAILED DESCRIPTION OF THE INVENTION

The field of nuclear medicine has long used radiopharmaceuticals for diagnostic (radioimaging) as well as therapeutic (radiation therapy for destruction of both neoplastic and non neoplastic cells).

Radionucleics used in nuclear medicine are all synthetic and are either generator produced, reactor produced, or cyclotron produced. Several important parameters affect the ultimate use of a radionucleic for diagnostic or therapeutic protocols. The half life of the radionuclide is critical as it must be long enough for imaging or for destruction of target tissue yet must also have an acceptable clearance pattern so that effective life is long enough for imaging. Ideally a radiopharmaceutical's effective half life should equal 1.5 times the duration of the diagnostic procedure. Iodine 121, 131, and 125 all have acceptable half lives compared to their clearance pattern so that the effective life is long enough for imaging. Availability is also a critical parameter as several of the radionuclides are rare. 133^Xe molybdenum 99, and ¹³¹I all of which are by-products of the fission of uranium-235. These isotopes are produced in great quantity in nuclear reactors and are regarded by the nuclear power industry as waste products. Target to nontarget ratio is also essential. An additional critical aspect of treatment by radiopharmaceuticals includes target to non-target ratio. If a ratio is not high enough, 5:1 minimum per planar imaging, and 2:1 for single photon emission computed tomography (SPECT) imaging, a non- diagnostic scan can result making it difficult or impossible to distinguish pathology from background. The results of a very low target to non-target ratio can be a non-diagnostic scan resulting in unnecessary radiation dose, a delay in the diagnosis and the ultimate need to repeat the procedure. The target to non-target ratio where important in diagnostic radiopharmaceutical use, it is absolutely essential for therapeutic procedures. A low target to non-target ratio can result in inadequate treatment of the primary disease and delivery of a potentially lethal radiation dose to bone marrow or other radiosensitive tissues. Several groups of nuclides are currently used as radiopharmaceuticals. The first group is comprised of the positive emitting isotopes carbon-11, nitrogen-13, oxygen-15 and fluorine- 18 all of which are cyclotron produced. These have very short half lives and the first three limit their use to facility at or near the cyclotron site. While ¹⁸F is possible to transport, practicalities make it virtually impossible.

The second group includes the gamma emitters cobalt-57⁶⁷Ga, ^mIn, ¹²³I, and ²⁰¹T1. These are also cyclotron produced, have long half lives and are easily transportable. ²⁰¹TI is of particular interest. The majority of photons collected by the camera for image formation are the low energy mercury-201 x- rays since the percent abundance of the 135 and 167-keV gamma rays is low.

The third group includes gallium-68, krypton-81m, rubidium-82, "mTc and nsmjN _an ₀f which are generator produced radionuclides. Of particular note is "mTc because of its ideal imaging energy and physical half life as well as its ability to bind so many compounds.

The final group includes 133^Xe molybdenum 99, and ¹³¹I all of which are by-products of the fission of uranium-235. These isotopes are produced in great quantity in nuclear reactors and are regarded by the nuclear power industry as waste products. Traditionally the limiting factor in design of appropriate radiopharmaceuticals is simply the physiological function of the target organ and is wholly dependent on organ processes such as antigen antibody reactions, physical trapping, receptor cite binding, removal of intentionally damaged cells from circulation, and transport of a chemical species across the cell membrane and into a cell by a normally operative metabolic process. According to the invention however the organ may be modified to actively transport the radiopharmaceutical of the invention thereby allowing radionuclides with attractive features for both radioimaging and radionuclide therapy such as ¹³¹I, ²⁰¹T1, and ^99mTC, to be used with any organ by active transport through the thyroid iodide symporter gene. Radioiodine has an excellent half life profile, ¹²⁵I has a half Ufe of sixty days, ¹³¹I has a half life of 8.08 days. It is abundantly available and has a good target to nontarget ratio. It's use, however, has been limited to thyroid carcinoma due to its active transport in the thyroid. Thyroid uptake of radioiodide is by active transport. The first step involves trapping of the iodide, it then undergoes intermediate synthesis involving a thyroglobuUn intermediate (organification) and is ultimately converted into T₃ and T₄ by the process of coupUng. Initial locaUzation following intravenous injection is in the thyroid stomach teratoids and chloroidplexus. Ultimately the iodide is stored in the thyroid as thyroxines with a Tbiol of approximately 3 weeks or cleared through the kidneys.

Another radionuclide with good parameters is ²⁰¹T1 which is also actively taken in through the Na⁺/K⁺ pump. Myocardial perfusion imaging is routinely formed with ²o^ιTl in the form of ThalUum (Tl¹⁺). This involves utilization of a normally operative metabolic pathway for handUng potassium since the Tl¹⁺ is a potassium analogue and is therefore handled efficiently by the well documented ATPase driven sodium potassium pump mechanism. Initial locaUzation of the Tl¹⁺ following intravenous injection is in the heart, liver and muscle. Ultimately it is recycled so that very little is cleared to the kidneys. The whole body Tbiol is approximately ten days. Imaging tumors of neuroendocrine origin also falls under the category of active transport although metabolic incorporation is perhaps a better description. The ¹²³I or ¹³¹ImBG injected is similar in structure to guanethidine. The precursor of epinephrin so that these tumors which include pheochromocytomas, neuroblastomas, paragangliomas, carcinoid type tumors medullary hyperplasia attempt to use it as a substrate for synthesis of hormones. The material thus accumulates within them and the accumulated tracer activity simply increases in the tumor as a function of time.

Detailed description of radioiodine uptake studies are disclosed in Hee- Myung Park, Chapter 59, "The Thyroid Gland", Nuclear Medicine, Vol. 1; Robert E. Henkin, 1996, Mosby-Year Book Inc., the disclosure of which is incorporated herein by reference. Technetium-99 Pertechnetate is also trapped by the same mechanism as iodide and can be used to estimate thyroid functional status as well. Advantages of use of this radiopharmaceutical include much lower radiation absorbed doses and shorter duration for the test. Applicants invention solves the problem of normal tissue toxicity due to nonspecific action. The invention, comprising gene-activated radionuclide accumulation, reduces normal tissue compUcation to negligible levels. To date there have been few attempts to combine radiopharmaceuticals with genetic engineering protocols, Wechselbaum et al demonstrated the feasibility of upregulating tumor necrosis factor expression following exposure to large doses of radiation. Wechselbaum et al., Int. J. Radiation Oncology, Biol. Phys., 24: 565, 567 (1992). Wechselbaum and colleagues proposed the possibility of regulating transcription of genes encoding cytotoxic proteins by placing radiation response elements in front of genetic constructs which may activate or amplify radiation induced signals or genes that encode proteins which increase radiation tolerance of dose Umiting normal tissues. See Wechselbaum et al. "Gene Therapy Targeted by Ionizing Radiation", Supra, the disclosure of which is herein incorporated by reference.

Another combination therapy comprises use of compounds which are selectively metaboUzed in Herpes transformed cells to radiation sensitizers. It involves use of Herpes Simplex Virus thymidine kinase gene expression^" to phosphorylate radiation sensitizers that are otherwise inactive in mammalian cells. Upon administration of pyrimidine nucleoside analogs, tumor cells which have been transformed with the HSV-tk gene metabolize these compounds to an active radiation sensitizer and render those cells more susceptible to radiation therapy. This protocol is described at length in PCT Publication No. WO 96/26743, published September 6, 1996 to Link et al., the disclosure of which is expressly incorporated by reference.

The invention herein describes a methodology whereby radioisotopes are selectively accumulated in transformed cells for use in radioimaging or radiotherapy. According to the invention target cells are transformed with a nucleic acid which is a radioisotope concentrator and upon expression of the nucleic acid sequence encoding the agent and subsequent radiation therapy the radioisotopes are accumulated in target cells.

In a preferred embodiment the agent is a radioisotope concentrator which causes uptake by transformed cells of radioisotopes to direct ionizing radiation to these cells and to thereby enhance the effects of radiation in these ceUs.

As used herein the term "radioisotope concentrator gene" shall be interpreted to include any nucleic acid sequence the expression of which causes the cell to import, either actively or passively, a radiopharmaceutical including precursors or conjugated derivatives thereof.

One example of such a radioisotope concentrator gene useful for the present invention includes the sodium-iodide symporter gene, which causes uptake of iodine and upon exposure to radioactive iodine by the thyroid.

Another radioisotope concentrator gene useful for the present invention includes the (Na⁺-K⁺) activated ATPase which is responsible for uptake of the radioisotope ThalUum. See, Henkin et al., "Nuclear Medicine", 1996 by Mosby Year Book, Inc. Vol. II, Chapter 93 "Thallium-201 Chloride and Technetium- 99m Sestamibi in Tumor Imaging" incorporated herein by reference. ThaUium-201, (²⁰¹T1) binds 2 sites on the ATPase enzyme and is accumulated by ceUs. Its uptake is inhibited by ouabain, digitalis, and the diuretic furosemide because the agents block the sodium potasium pump. ²⁰¹ /1 has been used in radioimaging of tumor cells and accumulates mainly in the area of viable tumor cells. See Waxman AD, "Thallium-201 in nuclear oncology", In Freeman LM, editor: Nuclear Medicine annual, New York, 1991, 193-209, Raven Press; Waxman A, "Thallium scintigraphy in the differentiation of malignant from benign mass abnormalities of the breast", J Nucl Med, 31:767, 1990 (abstract); Waxman AD, "Thallium scintigraphy in the evaluation of mass abnormahties of the breast", J. Nucl Med, 34(1): 18-23, 1993, the disclosure of which are hereby incorporated by reference. 99m Tc-sestamibi is another agent taken up by cells through the Na+-K+ pump and can be used in radioimaging of transformed ceUs expressing the Na⁺-K⁺ ATPase. Any such radioisotope concentrator gene can be used in the methods of the invention and one of skill in the art can identify numerous variations of gene/radioisotope combinations which are intended to be within the scope of this invention. The gene encoding Na⁺-K⁺ ATPase can easily be located by one of skill in the art by searching data bases such as Genbank, or by developing primers and using PCR amplification using analogous sequences from other species according to methods known in the art, and as disclosed in, for example, Ausubel FM et al, (eds.) "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, New York, 1989. Recently, the cloning and characterization of the thyroid iodide transporter gene from the rat has been reported and is herein incorporated by reference. Dai, G., Levy, O. & Carrasco, N., "Cloning and Characterization of the Thyroid Iodide Transporter", Nature, 379:458-460 (1996). The Na+/1- symporter (NIS) gene was isolated by using an iodide accumulation assay in the Xenopus laevis oocytes after injection with RNA transcripts. When NIS transcripts were introduced this permitted the accumulation of 800 pmol per cell of iodide. The primary sequence analysis demonstrated a gene of 2,839 bp in length with a predicted open reading frame of 1854 bp that encodes a protein of 618 amino acids. This small cDNA size will easily permit cloning into either RV or HSV based vectors. The NIS protein has 12 putative membrane spanning domains. This molecular pump functions as a NaH-/l- symporter and allows for the initial step of thyroid hormone synthesis. This protein therefore allows for the detection of l¹²⁵ uptake in thyroid tissue and for the ability to ablate hyperactive thyroid tissue or to treat well differentiated thyroid cancers with ¹³¹I. This interesting and unique property of thyroid tissue allows for very focused radiotherapy with minimal, if any, systemic side effects from therapy except the potential need for thyroid supplements. Additional information on NIS gene and its activity, as well as methods for detecting iodine accumulation are disclosed in "Iodide Transport in a Continuous Line of Cultured Cells From Rat Thyroid". Weiss, S.J. et al., Endocrinology, Vol. 114(4):1090-1098 herein incorporated by reference.

Since the main function of the thyroid is to trap and organify iodide, the logical choice for thyroid scanning agent is an iodine tracer, namely, radioiodine. There are several radioiodines available: ¹²³I, ¹³¹I, and ¹²⁵I. However, ¹²⁵I is no longer used for in vivo thyroid studies because of its low energy (28 keV) and very long half-Ufe (60 days). For imaging the thyroid morphology, ⁹°-^mTc pertechnetate is also satisfactory in most cases. Other agents include thalUum-201, galhum-67, ¹³¹I or ¹²³I metaiodobenzylguanidine (MIBG), "^mTc(V) dimercaptosuccinic acid (DMSA), fluorine-18 fluorodeoxyglucose (FDG), and "^mTc sestamibi. These secondary imaging agents are used mainly to image thyroid neoplasms and their metastases.

Radioimaging procedures using radioiodine have long been known in the art and are disclosed in detail in Nuclear Medicine, Vol. I and II, Mosby Press, 1996. The disclosure of which is incorporated by reference. Iodine in its various isotopic forms has also been used to radiolabel monoclonal antibodies for imaging and for therapy. Classic radioiodination methods involve covalent attachment of radioiodine to tyrosine residues in proteins. The process involves mild oxidation of iodine at slightly alkaUne pH using oxidizing agents such as chloramine-T, idogen, or lactoperoxidase. Indirect iodination procedures via the Bolton-hunter reagent involves radioiodinating a secondary molecule, typically an active ester which then reacts with N-terminal and lysine amino groups of monoclonal antibodies. Disclosure of radioiodination of monoclonal antibodies is disclosed at A. Michael Zimmer, "New approaches to radiolabeUng monoclonal antibodies", Chapter 40, Nuclear Medicine, Vol. 1, pp. 511-515, Henkin, Robert E (ed.) 1996, Mosby-Year Book Inc., supra.

The NIS gene has made radioactive iodine therapy a standard treatment for thyroid cancer. A majority of adenocarcinomas are removed surgically and radioiodine has been used as an injunctive therapy for management of thyroid adenocarcinoma for more than twenty years. The efficacy of radioiodine therapy is directly related to tumor uptake and retention due to the presence of the sodium iodide symporter gene which is expressed in thyroid cells. Efficient uptake and response to radioiodine is observed in tumors that are differentiated in cell type such as papillary or follicular whereas undifferentiated tumors and medullary carcinomas rarely concentrate radioiodine. Effected tumor uptake is approximately 0.5% of the radioiodine dose per gram with a biologic half life of approximately 4 days. From administration of 5.6 Gbq (equivalent to 150 mCi of ¹³¹I) a tumor may receive as much as 25 cGy or five times the absorbed dose that can be dehvered by a course of external radiation therapy. Moreover the dose is delivered to every functional metastases regardless of size or location in the body and tumor tissue will receive several hundred times the radiation exposure received by the rest of the body. Doses of radioiodine effective for treatment for uptake and killing of tumor cells are accomplished by the administration of 1.9 to 5.6 Gbq (50-150 mCi of ¹³¹I. Radio imaging procedures are used to identify the presence of iodine and to track its accumulation in tumor ceUs. These bovine TSH (thyroid stimulating hormone) is often given to patients to increase the accumulation of radioiodine in thyroid cancer metastases in which endogenous thyroid stimulating hormone is suppressed. Once adequate tumor uptake can be assured by radioiodine imaging studies therapeutic doses of radioiodine are administered. Traditionally the dose of radioiodine varies between 3.7 to 7.4 Gbq (100-200 mCi). It has been suggested that uniformly good response is possible with minimal tumor dose of 8,000 to 10,000 cGY if dosimetry is based upon estimation of the mass of post- surgical residual cancer tissue percent uptake in cancerous lesions and effective half life of radioiodine in the lesions. Limiting factors on the amount of radioiodine that can be safely administered include the possibility of complications from damaging effects of radioiodine on normal or vital tissues. Whole body radiation from usual therapeutic doses of radioiodine is estimated to be 20-40 cGy. Doses of iodine range between as low as 30 mCi up to as much as 300 mCi. The standard imperical dose is 150-200 mCi and methods of administration of radioiodine tracking its accumulation and managing side effects are known in the art and are described in the following publications which are incorporated in their entirety by reference. Cancer Treatment, 4th Ed., Charles M. Haskel, MD, FACP, W.B. Saunders Co., 1995. "Radiation to the thyroid can induce thyroid neoplasms both benign and malignant"; Nuclear Medicine Communications. (1993) 14:736-735. "Radionuclides and Therapy of Thyroid Cancer", O'Doherty, M.J. et al; Flower, M.A., et al, "Radiation Dose Assessment in Radioiodine Therapy to Practical Implementation Use in Quantitative Scanning and PET with Initial Results on Thyroid Carcinoma", Radiother. Oncol.. 1989, 15:345-57; O'Connel, M.E.A., et al., "Radiation Dose Assessment in Radioiodine Therapy Dose Response Relationships in Differentiated Thyroid Carcinoma Using Quantitative Scanning and PET", Radiother. Oncol., (in press); Leper, R., "Controversies in the Treatment of Thyroid Cancer, the New York Memorial Hospital Approach", Thyroid Today, (1982) 1-4. Mouse models of thyroid cancer and treatment protocols for administration of radioiodine are also standard in the art and are disclosed in Yoshio, Kasuga "The Effect of Xenotransplantation of Human Thyroid Tissue Following Radioactive Iodine-induced Thyroid Ablation on Thyroid Function in the Nude Mouse". Clin. Invest. Med., 14(4):277-281, (1981) which discloses a mouse model mimicking human thyroid cancer based upon transplantation of human thyroid tissues and expression of these cells in mice and concomitant treatment with radioactive iodine at doses of 0.2 ml. See also Active Radiologica Ther. Physics, Biol. 10(6) December 1971 "Determination of the ¹³¹I Dose to the Mouse Thyroid" by G. WaUnder. Any peptide or protein which causes the accumulation of radionuclides, whether radioisotope or beam therapy can be used in the method of invention. The methods of the invention can be used for radioimaging of transformed cells. This can include diagnostic protocols such as radioimaging of tumors or for ex-vivo protocols as a marker to track the expression by transformed ceUs of incorporated genes, one of which includes a radioisotope concentrator gene. The methods can also be used to target various compounds to transformed cells via conjugation of pharmaceuticals or other agents to a radioisotope which is selectively taken up by transformed cells. In a preferred embodiment the methods include radiotherapy for destruction of neoplastic cells such as tumor cells as well as non neoplastic cells to accumulate radiopharmaceuticals and destroy transformed cells.

The radiotherapy of the invention may also be combined with other traditional radio-sensitizers to further concentrate the effects. Examples of traditional drugs which have been reported to sensitize cells to therapeutic radiation include those in U.S. Patent No. 4,628,047 reports use of diltiazem (chemical name: d-3-aceotxy-cis-2, 3-dihydro-5-[2-(dimethylamino) ethyl[-2-(p- methoxyphenyl]-l,5-benzo-thiazepin-4(5H)-l) to enhance the sensitivity of a variety of types of cancer cells toward cytotoxic agents such as doxorubicin. For such administration the radiation sensitizer precursor or conjugate thereof can be combined with a pharmaceutically acceptable carrier such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives. The liquid vehicles and excipients are conventional and are commerciaUy available. Illustrative thereof are distilled water, physiological saUne, aqueous solutions of dextrose and the like. Traditionally IV therapy is preferred.

In general, in addition to the active compounds, the pharmaceutical compositions of this invention may contain suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. In addition to administration with conventional carriers, active ingredients may be administered by a variety of specialized delivery drug techniques which are known to those of skill in the art, such as portable infusion pumps. In accordance with an aspect of the present invention, there is provided a method of accumulating radionucUdes in cells for diagnostic or therapeutic purposes. The method comprises transducing cells in vivo or in vitro with a nucleic acid (DNA or RNA) sequence encoding a radioisotope concentrates gene which is capable of providing for the accumulation of administered radionuclides upon expression of the nucleic acid sequence encoding the agent. Radioisotopes useful for the invention include any which can be paired with a concentrator gene for cellular uptake. Potential radioisotopes include but are not limited to the radio nuclide metals ¹⁸<⁵RE, ¹⁸⁸RE, ^Cu, <"Cu, "yttrium, κ>⁹Pd, ²¹²Bi, ²⁰³PB, ²¹²Pb, ²¹¹At, ⁹⁷Ru, weRh, ¹⁹⁸Au, ¹⁹ Ag, and ⁱ"I. Any radioisotope concentrator gene the nucleotide sequence of which is known or is ascertainable by one of skiU in the art can be used. One of skill in the art can search for such genes in publically accessible databases such as Genbank to identify other such genes which act similarly.

In a preferred embodiment, the radioisotope concentrator gene is an iodide symporter gene, Na/k+ ATPase, or calcium transporter.

Next according to the invention radiotherapy is administered to the cells. The radioisotope is administered according to known protocols disclosed and incorporated herein in an amount effective to visuaUze transformed cells or to destroy the growth of the transduced tumor cells. These dosages are standard and are known in the art as described supra. The invention may allow for lower dosages of the radio element with equal effectiveness due to the concentration of the elements in transformed cells. The radioisotope may be administered to the host or to the in vivo cells in an amount known in the art as radiotherapy has long been employed and tolerance as well as toxicity data is well known. The radioisotopes are administered systemically, such as, for example, by intravenous administration, by parenteral administration, by intraperitoneal administration, or by intramuscular administration, or any other administration protocol acceptable for the in vivo or ex vivo protocol.

According to the invention, when producer cells or other expression media including the radioisotope concentrator gene are administered to cells, metabolic cooperation, a "bystander effect", will result, i.e., tumor cells which are not transduced with the nucleic acid sequence encoding the radioisotope concentrator gene may also be selectively visualized or killed upon administration of radiation therapy.

The methods of the invention should have a potent bystander effect since ¹³¹I energy can travel 7 mm from the point of particle decay in tissues. This fact has been repeatedly viewed as strong supportive evidence for using anti-tumor antibodies radiolabeled with ¹³¹I as the therapeutic agents. Divgi, C.R., "Status of Monoclonal Antibodies for Diagnosis and Therapy of Cancer", Oncology 10:939-953 (1996). Bystander killing is a key element of this novel approach because it implies that gene delivery does not have to target 100% of the tumor cells in a recurrent lesion. This is essential because current gene delivery methods that are being tested in the Phase I or II trials are fairly inefficient in humans. The use of ¹³¹I in humans is well characterized in terms of dose and monitoring. The administration of 5.6 Gbq of ¹³¹I usually results in a radiation dose equivalent to 25,000 cGy to a thyroid tumor. Hershman, J.M., Blahd, W.H. & Gordon, H.E. in Cancer Treatment (ed. Haskell, CM.) 743-752 (W.B. Saunders, Philadelphia, 1995).

According to the invention a method for gene delivery must be employed to overcome the complex problem of gene delivery and expression in target tissues. The invention in its simplest embodiment includes a polynucleotide sequence which provides for the expression of a radioisotope concentrator gene in transformed ceUs. In a preferred embodiment the polynucleotide sequence is an expression construct which includes a gene encoding a radioisotope concentrator element and certain regulatory elements operatively linked to the gene. One such element is a promoter which when operatively linked to the gene can provide for the transcription and ultimate expression of the gene in cells. The promoter can be constitutive or inducible. A multitude of promoters which are active in mammalian cells are known in the art and the selection of a suitable promoter is an expedient easily optimized by those of skill in the art and contemplated herein. Several suitable promoters are disclosed in Maniantis, "Molecular Cloning", Cold Spring Harbor Press, 1989. In a preferred embodiment the promoter is a viral promoter that is located within a viral vector for gene delivery which is operatively linked to a multiple cloning site for insertion of the radioisotope concentrator gene. The expression construct also typically includes a termination or polyadenylation sequence operatively linked to the radioisotope concentrator gene. The construct of the invention may be further comprised within a vector or gene transfer vehicle as described hereinafter.

Inducible promoters can be used with the methods of the invention in radioimaging to visuaUze activity of the promoter in activating the radioisotope concentrator gene and any other genes operatively linked to the same promoter as in, for example, a diagnostic turbidoscan to identify whether a particular promoter is active in a particular cell. For example tyrosine hydroxylase promoter could be used to define tyrosine hydroxylase activity in part of the brain. This could provide valuable prognostic or diagnostic information for Parkinson's disease.

In yet another embodiment the promoter is a tumor specific promoter which can provide for selective expression of the gene in not only transformed cells, but transformed tumor cells. In this embodiment the polynucleotide sequences of the invention can be used diagnostically or therapeutically. Transformation of tumor cells with a NIS gene or its equivalent operatively Unked to a tumor specific promoter such as those disclosed in Example 7 can be used to image tumors in which regulated expression of the NIS gene occurs and ¹²⁵I is taken up.

Thus the vectors of the invention can be tied to cell specific promoters or inducible promoters and with radioimaging based upon radioiodine uptake can give very specific information about the state of a cell. Two methods of gene delivery have been shown successful in other experiments are preferred. The first method is retroviral (RV) delivery, Moolten, F.L., "Tumor Chemosensitivity Conferred by Inserted Herpes Thymidine Kinase Genes: Paradigm for a Prospective Cancer Control Strategy", Cancer Res. 46:5276-5281 (1986); Link, C.J., Kolb, E. & Muldoon, R., "Preliminary In Vitro Efficacy and Toxicities Studies of the Herpes Simplex Thymidine Kinase Gene System for the Treatment of Breast Cancer", Hybridoma 14:143-147 (1995). This gene delivery method is based on work initially performed by Xandra Breakfield's group using the direct implantation of RV vector producing cells (VPC). Short, M.P., et al., "Gene Delivery to Glioma Cells in Rat Brain by Grafting of a Retrovirus Packaging Cell Line", j Neurosci. Res., 27:427-439 (1990). Using this gene delivery method, a Phase 1 clinical trial for patients with recurrent ovarian cancer that employs intraperitoneal infusion of xenogeneic VPC to introduce the Herpes simplex thymidine kinase (HS-tk) gene has been employed by applicants. Link, C.J., Moorman, D., Seregina, T., Levy, J.P. & Schabold, K.J., "A Phase I Trial of In Vivo Gene Therapy With the Herpes Simplex Thymidine Kinase/Ganciclόvir System for the Treatment of Refractory or Recurrent Ovarian Cancer", Human Gene Ther., Vol. 7, In Press (1996). As such the invention comprises in one embodiment a novel retroviral vector comprising a nucleic acid sequence which encodes a radioisotope concentrator protein such as NIS.

The second and more novel method of gene delivery is an amplicon system based on HSV-1. This gene delivery vector was developed by applicants as described herein. It has been found that at an MOI of 3-10 plaque forming units (pfu/ml) of applicants pHE vector transduces >95% of tumor cells in vitro. HSV vectors will also allow for high efficiency gene transfer in vivo into rodent tumors. Boviatsis, E.J., et al., "Long-Term Survival of Rats Harboring Brain Neoplasms Treated With Ganciclovir and a Herpes Simplex Virus Vector That Retains an Intact Thymidine Kinase Gene", Cancer Res., 54:5745-5751 (1994). In these preferred experimental models, gene transfer was efficient enough to induce complete remission and long term survival in some animals.

In a preferred embodiment, a packaging cell line is transduced with a retroviral vector, such as those hereinabove described, which includes the Na⁺/I- symporter gene. The transduced packaging cells are administered in vivo or ex vivo to the tumor in an acceptable pharmaceutical carrier and in an amount effective to inhibit, prevent, or destroy the growth of the tumor. Upon administration of the producer cells to the tumor, the producer cells generate viral particles including a gene encoding the Na+/I- symporter. Such viral particles transduce the adjacent tumor cells.

The human or animal host organism is then given the appropriate corresponding radioisotope. In the case of Na⁺/L symporter gene the radioisotope is ¹³¹I. Upon expression of the gene the radioisotopes are selectively concentrated in transformed cells. As hereinabove mentioned, a "bystander effect" may also occur, whereby non- transduced tumor cells also may be killed as well.

The method of the present invention for direct in vivo therapy utilizing retroviruses is particularly useful when the targeted tumor is in or surrounded by a tissue made up of cells which are relatively quiescent mitotically, such as liver, skin, bone, muscle, bladder, prostate, kidney, adrenal, pancreas, heart, blood vessel and thyroid tissue, among others. The inventive approach also should be useful against tumors located in the subarachnoid space, in the peritoneum, and in the pleural cavity. In addition, tumors in organs the loss of which, in whole or part, is generally well-tolerated are preferred targets of a treatment according to the present invention such as the liver, for example.

Targeting of the vectors to tumor sites for the methods of the invention can be accomplished through a number of protocols. The vectors can be mechanically injected directly at the target site. This can be accomplished with plasmid DNA, retroviral vectors (MMKV or HIV based) Herpes Simplex Viral Vectors (either amplicons or near whole virus vectors), adenoviral vectors, Sandai virus vectors, or DNA polylysine antibody complexes. Direction can also occur through selective uptake of foreign material (phagocytosis or macrophage). Another method includes viral targets on dividing tumor cells such as the epo receptor or use an antibody fused to viral envelopes. As discussed earlier tissue or tumor specific promoters could also be used as disclosed in Example 7.

Direct injection of the producer cells minimizes undesirable propagation of the virus in the body, especially when replication-competent retroviral vectors are used. Because most cells of the body express receptors for amphotropic retroviral vectors, any vector particle which escapes from the local environment of the tumor should immediately bind to another cell. Most cells are not in cycle, however, and therefore will not integrate the genes carried by the vector and will not express any genes which it contains. Thus, the proportion of potential target cells which are in cycle at the time of exposure will be small, and systemic toxic effects on normal tissues will be minimized.

Tumors which may be treated in accordance with the present invention include malignant and non-malignant tumors. Malignant (including primary and metastatic) tumors which may be treated include, but are not Umited to, those occurring in the adrenal glands; bladder; bone; breast; cervix; endocrine glands (including thyroid glands, the pituitary gland, and the pancreas); colon; rectum; heart; hematopoietic tissue; kidney; liver; lung; muscle; nervous system; brain; eye; oral cavity; pharynx; larynx; ovaries; penis; prostate; skin (including melanoma); testicles; thymus; and uterus. Examples of such tumors include apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), plasmacytoma, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, throphoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, leydig cell tumor, papilloma, sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paragangUoma, paraganglioma nonchromaffin, antiokeratoma, angiolymphoid hyperplasia with eosinophilia, angioma scle rosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (e.g., Ewing's experimental, Kaposi's, and mast-cell), neoplasms and for other such cells.

GENETIC ENGINEERING METHODS

The nucleic acid sequence which encodes the radioisotope concentrator agent is contained in an appropriate expression vehicle which transduces the tumor cells. Certain proprietary expression vehicles have been discussed supra, however, and appropriate gene transfer vehicle may be employed by the method of the invention. The invention also includes these novel expression vehicles as well as their use in radiotherapy. Such expression vectors include, but are not Umited to, eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial vectors), and viral vectors. In one embodiment, the expression vector is a viral vector. Viral vectors which may be employed include, but are not Umited to, retroviral vectors, adenovirus vectors, and adeno-associated virus vectors.

In a preferred embodiment, a packaging cell line is transduced with a viral vector containing the nucleic acid sequence encoding the agent or factor which provides for the inhibition, prevention, or destruction of the tumor cells upon expression of the nucleic acid sequence encoding the agent to form a producer cell line including the viral vector. The producer cells then are administered to the tumor, whereby the producer cells generate viral particles capable of transducing the tumor cells.

In a preferred embodiment, the viral vector is a retroviral or adenoviral vector. Examples of retroviral vectors which may be employed include, but are not Umited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.

Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic ceUs. Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.

These new genes have been incorporated into the proviral backbone in several general ways. The most straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences within the long terminal repeat (LTR). Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.

Efforts have been directed at minimizing the viral component of the viral backbone, largely in an effort to reduce the chance for recombination between the vector and the packaging-defective helper virus within packaging cells. A packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.

In one embodiment, the retroviral vector may be one of a series of vectors described in Bender, et al., J. Virol. 61:1639-1649 (1987), based on the N2 vector (Armentano, et al., J. Virol., 61:1647-1650) containing a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems. These changes have also reduced the likelihood that viral proteins would be expressed. In the first of these vectors, LNL-XHC, there was altered, by site-directed mutagenesis, the natural ATG start codon of gag to TAG, thereby eliminating unintended protein synthesis from that point.

In Moloney murine leukemia virus (MoMuLV), 5' to the authentic gag start, an open reading frame exists which permits expression of another glycosylated protein (pPr80S^aS). Moloney murine sarcoma virus (MoMuSV) has alterations in this 5' region, including a frameshift and loss of glycosylation sites, which obviate potential expression of the amino terminus of pPr80S^ag. Therefore, the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5' portion of MoMuSV. The 5' structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells. In a final alteration to reduce overlap with packaging-defective helper virus, Miller has eliminated extra env sequences immediately preceding the 3' LTR in the LN vector (Miner, et al., Biotechniαues, 7:980-990, 1989). The paramount need that must be satisfied by any gene transfer system for its application to gene therapy is safety. Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector. Miller, et al. have developed the combination of the pPAM3 plasmid (the packaging-defective helper genome) for expression of retroviral structural proteins together with the LN vector series to make a vector packaging system where the generation of recombinant wild-type retrovirus is reduced to a minimum through the elimination of nearly all sites of recombination between the vector genome and the packaging-defective helper genome (i.e. LN with pPAM3).

In one embodiment, the retroviral vector may be a Moloney Murine Leukemia Virus of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al. (1987) and MiUer, et al. (1989). Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon. The term "mutated" as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragment or truncations thereof, are not expressed.

In another embodiment, the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs. Preferred cloning sites are selected from the group consisting of NotI, SnaBI, Sail, and Xhol. In a preferred embodiment, the retroviral vector includes each of these cloning sites.

When a retroviral vector including such cloning sites is employed, there may also be provided a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, Sail, and Xhol located on the retroviral vector. The shuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.

The shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art. The shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems. The shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria. Thus, for example, the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc. The vector includes one or more promoters. Suitable promoters which may be employed include, but are not Umited to, the retroviral LTR; the SV40 promoter; and the human cy tome galo virus (CMV) promoter described in Miller, et al., Biotechniαues, 7:(9):980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not Umited to, the histone, pol III, and β-actin promoters). Other viral promoters which may be employed include, but are not Umited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein. The vector then is employed to transduce a packaging cell line to form a producer cell Une. Examples of packaging cells which may be transfected include, but are not Umited to the PE501, PA317, Ψ2, Ψ-AM, PA12, T19-14X, VT-19-17-H2, ΨCRE, ΨCRIP, GP+E-86, GP+envAM12, and DAN cell Unes. The vector containing the nucleic acid sequence encoding the agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells upon expression of the nucleic acid sequence encoding the agent may transduce the packaging cells through any means known in the art. Such means include, but are not Umited to, electroporation, the use. of liposomes, and CaPO-j precipitation. The producer cells then are administered directly to or adjacent to the tumor in an amount effective to inhibit, prevent, or destroy the growth of the tumor upon subsequent radiation therapy. In general, the producer cells are administered in an amount tolerated by the patient, it is desirable to inject as many producer cells as possible. The exact amount of producer cells to be administered is dependent upon various factors, including but not Umited to, the type of the tumor and the size of the tumor.

In general, the producer ceUs are administered directly to or adjacent to the tumor by injection. The producer cells are administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient. The carrier may be a liquid carrier such as, for example, a saline solution.

Upon administration of the producer cells to the tumor, the producer cells generate viral particles. The viral particles then transduce the surrounding tumor cells. Because tumor ceUs, and in particular cancerous tumor cells, in general are actively replicating ceUs, the retroviral particle would be integrated into and expressed preferentially or exclusively in the tumor cells as opposed to normal cells.

In a preferred embodiment the invention comprises a viral vector which commonly infects humans and packaging cell Une which is human based. For example vectors derived from viruses which commonly infect humans such as

Herpes Virus, Epstein Barr Virus, may be used which do not express an active α-galactosyl envelope.

In a most preferred embodiment the vector comprises a Herpes Simplex

Virus plasmid vector. Herpes simplex virus type-1 (HSV-1) has been demonstrated as a potential useful gene delivery vector system for gene therapy, Glorioso, J.C., "Development of Herpes Simplex Virus Vectors for Gene Transfer to the Central Nervous System. Gene Therapeutics: Methods and Applications of Direct Gene Transfer", Jon A. Wolff, Editor, 1994 Birkhauser Boston, 281-302; Kennedy, P.G., "The Use of Herpes Simplex Virus Vectors for Gene Therapy in Neurological Diseases", Q J Med, Nov. 1993, 86(ll):697-702; Latchman, D.S., "Herpes Simplex Virus Vectors for Gene Therapy", Mol Biotechnol, Oct. 1994, 2(2):179-95.

HSV-1 vectors have been used for transfer of genes to muscle. Huard, J., "Herpes Simplex Virus Type 1 Vector Mediated Gene Transfer to Muscle", Gene Therapy, 1995, 2, 385-392; and brain, KapUtt, M.G., "Preproenkephalin Promoter Yields Re ion- Specific and Long- Term Expression in Adult Brain After Direct In Vivo Gene Transfer Via a Defective Herpes Simplex Viral Vector", Proc Natl Acad Sci USA, Sep 13, 1994, 91(19):8979-83, and have been used for murine brain tumor treatment, Boviatsis, E.J., "Long-Term Survival of Rats Harboring Brain Neoplasms Treated With Ganciclovir and a Herpes Simplex Virus Vector That Retains an Intact Thymidine Kinase Gene", Cancer Res, Nov 15, 1994, 54(22):5745-51; Mineta, T., "Treatment of Malignant Gliomas Using Ganciclovir-Hypersensitive, Ribonucleotide Reductase-Deficient Herpes Simplex Viral Mutant", Cancer Res, Aug 1, 1994, 54(15):3963-6.

Helper virus dependent mini-viral vectors have been developed for easier operation and their capacity for larger insertion (up to 140 kb), Geller,

Al, "An Efficient Deletion Mutant Packaging System for Defective Herpes

Simplex Virus Vectors: Potential AppUcations to Human Gene Therapy and

Neuronal Physiology", Proc Natl Acad Sci USA, Nov 1990, 87(22):8950-4;

Frenkel, N., "The Herpes Simplex Virus Amplicon: A Versatile Defective Virus Vector", Gene Therapy. 1. Supplement 1, 1994. Replication incompetent HSV amplicons have been constructed in the art, one example is the pHSVlac vector by Geller et al, Science, 241, Sept. 1988, incorporated herein by reference.

These HSV amplicons contain large deletions of the HSV genome to provide space for insertion of exogenous DNA. Typically they comprise the HSV-1 packaging site, the HSV-1 "ori S" repUcation site and the IE 4/5 promoter sequence. These virions are dependent on a helper virus for propagation. Primarily two types of mutant helper viruses have been developed to minimize recombination. Other complementary HSV helper virus systems are contemplated herein and are within the scope of those of skill in the art. One such system which has been developed is a temperature-sensitive mutant. An HSV temperature-sensitive (TS) mutant has been developed with a TS mutation in the IE3 gene. Davison et al, 1984, J. Gen. Virol., 65:859-863. Consequently this virus has an IE phenotype, does not replicate DNA, does not significantly alter cellular physiology, and does not produce progeny virus at 37°C. Virus is grown at the permissive temperature of 37°C. TS mutants however have had a tendency to revert to wild type.

In contrast a second helper virus system is a deletion mutant with the majority of the IE3 gene simply deleted. These do not revert to wild type. Therefore HSV-1 vectors packaged using a deletion mutant as helper virus is the most preferred helper virus of the invention. See for example Patterson et al., 1990, J. Gen. Virol., 71:1775-1783. Other replication incompetent helper viruses can be used and one of skiU in the art will appreciate that other mutations in the IE genes or other genes which result in a replication incompetent helper virus which will provide the appropriate replication and expression functions and which are coordinated with the helper cell Une and vector are contemplated within this invention. Any ceU Une can be used for this step so long as it is capable of expressing the IE3 or replication dependent gene, or obtaining a helper cell line which has already been transformed and is commercially available. Any cell line can be used by introducing pHE and the plasmid containing the IE3 gene simultaneously. Next, the vector is delivered to the helper cell line by electroporation, calcium phosphate DNA transfection or any other suitable method. Any cell Une can be used by introducing pHE and the plasmid containing the IE3 gene simultaneously. The cells are next infected with a helper virus IE3 deletion mutant or other corresponding deletion mutant which is replication incompetent. The IE3 gene or other such gene in the helper cell Une complements the helper virus resulting in a productive HSV-1 infection and the resulting virus stock consists of HSV-1 particles containing either vector DNA or helper virus DNA, all of which are replication incompetent. Further information about helper cell lines and the methodology is disclosed in Geller et al., PNAS, 87:8950-8954, November 1990, "An Efficient Deletion Mutant Packaging System for Defective Herpes Simplex Virus Vectors: Potential Applications to Human Gene Therapy and Neuronal Physiology". The invention comprises a HSV mini vector which combines a replication incompetent HSV amplicon with other viral sequences such as those from Epstein-Barr virus, human papillomavirus, or bovine papillomavirus type 1 which aUow the vector to be maintained in the cell in episomal form achieving a 10 times greater titer, and a very large DNA insert capacity.

One embodiment of the present invention involves a helper virus- dependent mini-viral vector comprising: (a) the HSV-1 "a" sequence for the package/cleavage signal and an "ori S" repUcation origin for the replication packaging of the plasmid (in response to signals to replicate and package from the helper virus); (b) an Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) gene and an EBV latent origin of replication (ori P) which allow the vector to be maintained in episomal form within the nucleus for replication without integration to the host genome and for even replication into each of two dividing cells; preferably (c) genes from prokaryotic cells for propagation of the vector in E. coli (a selectable marker gene such as the ampicillin resistance or tetracycline resistance gene and the col. El ori) and (d) a sequence encoding a radioisotope concentrator protein such as NIS. Optionally the vector may also comprise prokaryotic genes that provide for a second selectable marker such as the genes for positive Hygromycin selection.

In this particular embodiment the packaging function of mini-vector DNA into Herpes simplex viral capsids is provided by a helper virus and a helper cell Une.

In yet another embodiment the HSV vector can be engineered to produce a helper free viral vector as in Mann et al., "Construction of a Retro- Virus Packaging Mutant and its Use to Produce Helper-Free Defective Retrovirus", 33 Sal., p. 153-159, May 1983, Journal of Virology, September 1989, pp. 3822-3829, September 1989; Samulski "Helper Free Stocks of Recombinant Adeno-Associated Viruses: Normal Integration Does Not Require Viral Gene Expression"; and Kohn et al., "High Efficiency Gene Transfer Into Mammalian Cells: Generation of Helper-Free Recombinant Retrovirus With Broad Mammalian Host Range", PNAS, 81:6349-6353, October 1984. See also Okasinki, U.S. Patent No. 4,970,155 "HSV HELPER VIRUS INDEPENDENT VECTOR", incorporated herein by reference.

All references cited herein are hereby expressly incorporated in their entirety. The invention will now be described with respect to the following examples; however, the scope of the present invention is not intended to be limited thereby.

EXAMPLE 1 RETROVIRAL TRANSDUCTION Breast cancer that locally recurs after radiation therapy and that is refractory to chemotherapy represents a difficult clinical challenge. Patients often cannot tolerate further external beam radiotherapy without being subjected to prohibitive toxicity. Likewise, further chemotherapy can provide significant toxicity without great benefit, especially after combined modality therapy. In this setting, an ideal therapy would provide more local and effective treatment for better palliation of these significant lesions without substantial or Ufe threatening side effects. Towards this end, a number of laboratories are investigating molecular approaches that are based on targeted gene transfer, Han, X., Kasahara, N. & Kan, Y.W., "Ligand-Directed Retroviral Targeting of Human Breast Cancer Cells", Proc. Natl. Acad. Sci U.S.A., 92:9747-9751 (1995) or promoter specificity, Harris, J.D., Gutierrez, A.A., Hurst, H.C., Sikora, K. & Lemoine, N.R., "Gene Therapy for Cancer Using Tumor-Specific Prodrug Activation", Gene Ther., 1:170-175 (1994). These experiments are in the early stages of development, but have shown some promise. Our proposal focuses on a new idea of how to concentrate tumor killing radiation in a well circumscribed area. A gene will be transferred to breast cancer cells that will make them mimic the iodide uptake of normal thyroid follicular cells. In our project, we wiU compare traditional retroviral (RV) vector delivery to our novel Herpes simplex virus (HSV) vector delivery of the thyroid iodide transporter gene. Retroviral or Herpes Viral vectors for effective transfer a functional thyroid symporter gene to breast cancer cells in vitro.

Cloning of the thyroid iodide transporter gene and subcloning into the LXSN retroviral vector. The rat FRTL-5 thyroid-derived cell line was used as a source for RNA that was purified using the Rneasy kit (Qiagen) to isolate the thyroid iodide transporter gene as described in Dai et al., Supra. The RNA was then treated by a RT-PCR method to generate cDNA (CLONETECH). These were then used as templates for PCR reactions to amplify the transporter. The primers for PCR amplification were a 5' 31mer that started at position #90 of the reported sequence and 3' 31mer starting at position #1950. PCR amplification with these primers added a Kpn I site to the 5' end and a Xho I site to the 3' end of the gene. A DNA fragment of the appropriate size (1.8 kb) was obtained, restriction digested with Xho I and Kpn I, and cloned into the appropriate sites in pREP7 (Invitrogen). Subsequent sequence analysis of one of the clones revealed a sequence that was identical to the published report, Supra.

The NIS gene was then cloned into the LXSN retroviral backbone that that contains a multi-cloning site and the neo^r gene under SV40 promoter control: Link C.J., Kolb E, Muldoon R., "PreUminary in vitro efficacy and toxicities studies of the herpes simplex thymidine kinase gene system for the treatment of breast cancer", Hybridoma, 1995, 14:143-147; Link CJ et al., "A phase I trial of in vivo gene therapy with the Herpes simplex thymidine kinase/ganciclovir system for the treatment of refractory or recurrent ovarian cancer", Human Gene Ther, 1996, 7:1161-1179. The vector also contains modifications to minimize breakouts of replication competent retrovirus. Miller, A.D. & Buttimore, C, "Redesign of Retrovirus Packaging Cell Lines to Avoid Recombination Leading to Helper Virus Production", Molec. Cell Biol, 6:2895-2902 (1986). The vector has been well described elsewhere and the inventors have previously used LXSN for in vitro transfer and killing of breast cancer cells using the HS-tk gene and ganciclovir approach. Link C.J., Kolb E, Muldoon R., "Preliminary in vitro efficacy and toxicities studies of the herpes simplex thymidine kinase gene system for the treatment of breast cancer", Hybridoma, 1995, 14:143-147. The resulting vector was named LNISN.

In vitro analysis for NIS activity. The previously published protocol by Weiss et al. was used to determine the uptake of ¹²⁵I into LNISN vector transduced EMT-6 breast cancer cells. Weiss, S.J., Philp, N.J. & Grollman, E.F., "Iodide Transport in a Continuous Line of Cultured Cells From Rat Thyroid", Endocrinology, 114:1090-1098 (1984). Briefly, iodide uptake was initiated by adding 500 μl of 0.345-.345 μM carrier free Nal¹²⁵ and 5-300 μM Nal. After no longer than 120 minute incubation reactions were terminated by washing cells with ice cold Hank's Balanced Salt Solution (HBSS) pH 7.3. 1% Triton X-100 was added and samples were counted in a scintillation counter. To evaluate this assay, we first analyzed ¹²⁵I uptake in FRTL-5 rat thyroid cells. These results are shown in Figure 1 and demonstrate rapid and effective ^{1 5}I uptake. Control cells were incubated on ice to block symporter function and as a result do not show ¹²⁵I uptake. Direct comparisons were performed between cells with no vector and cells previously transduced with LNISN vector supernates and selected in G418 for 14 days. Comparison of ¹²⁵I intracellular levels in the NIS transduced cancer cells with nontransduced (negative control) cells demonstrates direct evidence of NIS gene function. This level of concentration is comparable to that obtained in normal thyroid tissue in vivo. Furthermore the uptake is rapid and sustained (Figures 1 and 2(a-d)). Comparison of ¹²⁵I intracellular levels in the test cells compared to the negative control cells will provide direct evidence of NIS gene function. Direct injection of NIS RNA transcripts into Xenopus oocytes resulted in a greater than 30-fold increase of intracellular iodide concentration. This level of concentration is comparable to that obtained in normal thyroid tissue in vivo. Retroviral transduction of the NIS gene to selectively radiosensitize cancer cells to ¹³¹I. The LNISN vector was used to transduce human tumor cell lines followed by incubation with ¹³¹I for in vitro clonogenic assays to determine efficacy of this approach. Briefly, iodide uptake was initiated by adding 30 μCi/ml of Nal¹³¹ and 30 μM Nal. The survival rate of tumor cells transduced with LNISN to cells with no vector (no NIS gene) was compared. Cells were exposed for 6 hours to the solution containing ¹³¹I. (Figure 3). Next, cells were trypsin digested and then plated at cell numbers between 1.25xl0² and lxlO⁴ cells per plate in quadruplicate for each data point. After 7 to 10 days the cells were fixed and stained. Macroscopic colonies containing <50 cells were counted. Survival measurements were corrected for plating efficiency. Results are shown in Figures 3-5. These results show selective radiation killing of the cells engineered to express the NIS gene when compared to control cells. Under these initial conditions some killing of control cells does occur, but greater kilUng effect is consistently observed in the NIS expressing cells. These results demonstrate that the NIS gene functions as predicted by ¹²⁵I uptake experiments. As can be seen from the figures, kilUng was observed in A375, BNL.1ME, CT26 and IGROV cells which were transduced with the NIS gene (Figures 4 and 5).

In vivo gene delivery with vector producing cells. Implantation of retroviral VPC was used as the first method to deliver the NIS gene into tumor cell targets. The implantation of VPC to deliver genes into tumor cells effectively was first demonstrated in a brain tumor model by engrafting lacZ VPC into rodents with C6 glioma tumors. Staining demonstrated efficient gene transfer into the glioma. (Short MP et al., "Gene delivery to glioma cells in rat brain by grafting of a retrovirus packaging cell line", J Neurosci Res, 1990; 27:427-439) The anti-tumor effect in Herpes Simplex type I thymidine kinase (Hstk) VPC implantation has been evaluated in several in vivo tumor models. Growing 9L gliosarcoma in the rat brain has been used as a model for human glioblastoma multiforme. (Culver KW et al., "In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors", Science, 1992; 256:1550-1552). More recent work suggests that in part this 9L tumor destruction by Hstk VPC is mediated by the immune system in vivo and does not require drug selection for tumor regression. (Tapscott SJ et al., "Gene therapy of rat 9L gliosarcoma tumors by transduction with selectable genes does not require drug selection", Proc Natl Acad Sci, USA, 1994; 91:8185- 8189). Another group has demonstrated anti- tumor efficacy in a rat brain tumor model as weU, using the implantation of C6 glioma cells, but with the addition of replication competent helper virus. (Takamiya Y et al., 'An experimental model of retrovirus gene therapy for malignant brain tumors", J Neurosurg, 1993; 79:104-110). Evidence for anti-tumor efficacy of the Hstk system has also been demonstrated in a rat model of colorectal metastasis to the liver. (Caruso M et al., "Regression of established macroscopic liver metastases after in situ transduction of a suicide gene", Proc Natl Acad Sci (USA) 1993; 90:7024-7028) in this model, BDIX rats were injected under the liver capsule with a syngeneic colon carcinoma cell line. Necropsy at the end of GCV treatment revealed that approximately one third of the animals were pathologically tumor free. The following two experiments demonstrate the feasibility of VPC implantation.

Antitumor efficacy of LTKOSN VPC against MC38 intraperitoneal colonic adenocarcinoma. C57B1/6 mice were injected with an intraperitoneal inoculation of MC38 colonic adenocarcinoma (kindly provided by S. Rosenberg, NCI). An experiment was performed in an attempt to determine the most effective LTKOSN clone for clinical trial (Table 1). The vector LTKOSN contains the Hstk gene transcribed from the viral long terminal repeat (LTR) and a bacterial neomycin resistance (neo^r) gene transcribed from an internal SV40 (simian virus 40) early promoter (LTR- HStk-SV-neo^r-LTR) in the LXSN backbone. Animals received injections of 2xl0⁴ MC38 tumor cells suspended in 1 ml Hank's balanced salt solution (HBSS) into the peritoneal cavity on day 1. On day 6 the animals received the treatment cells suspended in 1 ml HBSS. Five days later the animals were treated with a 14 day course of GCV. All cones showed some degree of activity compared to animals injected with tumor alone. LTKOSN.2 (clone 2), however, showed better activity than clones 9, 10, or 12. No evidence of toxicity was observed in the surviving animals. Animals that died during the experiment had death caused by progressive intraperitoneal carcinomatosis. This experiment establishes the efficacy of treating intraperitoneal adenocarcinoma with injections of LTKOSN.2 VPC followed by GCV treatment.

TABLE 1. EFFICACY OF LTKOSN VPC CLONES ON INTRAPERITONEAL MC38

COLONIC ADENOCARCINOMA

MC38 LTKOSN LTKOSN Animals Free of Disease

Group no vector Clone # VPC

(# cells) (# cells) +GCV -GCV

A 2.5xl0⁴ NA None 05 0/5

B 2.5xl0⁴ 2 lxlO⁷ 5/10 0/3

C 2.5xl0⁴ 9 lxlO⁷ 1/10 0/3

D 2.5xl0⁴ 10 lxlO⁷ 2/10 0/3

E 2.5xl0⁴ 12 lxlO⁷ 2/10 0/3

Estimation of in vivo transduction efficiency of LTKOSN.2 VPC into MC 38 intraperitoneal adenocarcinoma. C57B1/6 mice were injected with an intraperitoneal inoculation of 2xl0⁴ MC 38 colonic adenocarcinoma ceUs suspended in 1 ml HBSS IP (day 1). On day 7 the animals received lxlO⁷ LTKOSN.2 VPC cells suspended in 1 ml HBSS. On day 27 the mice were sacrificed. Tumors were digested and placed in culture with RPMI + 10% FBS with penicillin, streptomycin, and fungizone. The cells recovered in culture were of MC 38 morphology. VPC have not been detected in these mice after 14 days. Two days later, the cells were used in a clonogenic assay to determine the percentage of MC 38 cells which had been transduced in vivo by the LTKOSN.2 vector. MC 38 NV cells (negative control) and MC 38 cells from the LTKOSN/2 VPC treated mice were seeded in triplicate in tissue culture dishes at selected densities. The next day 1 mg/ml G418 was added to 3 of 6 dishes in each set. Nine days later the cells were fixed with methanol: acetic acid and stained with crystal violet. Three separate clonogenic assays were set up from 2 animals (Table 2). All colonies with more than 50 cells were counted, and a ratio was calculated: % Resistant = G418 resistant colonies/total colonies in absence of drug. These results demonstrate in vivo transduction by the LTK)SN.2 VPC. Although, only 47% of these cells are transduced, 50% of the animals remain tumor free (Tables 1 and 2). These experiments establish the utility of this approach for efficient intraperitoneal gene delivery.

TABLE 2

IN VTVO LTKOSN.2 VPC TRANSDUCTION FREQUENCIES INTO

INTRAPERITONEAL MC 38 ADENOCARCINOMA

Assay Control: No Vector LTKOSN.2 VPC Treated

G418 G418

Plating Resistant Plating Resistant

Efficiency Colonies Efficiency Colonies

(%) (%) (%) (%)

1 4.4 0 3.5 43

2 8.8 1.4 4.0 45

3 7.1 0 3.6 54

Mean±S.D. 6.8±2.2 0.5+0.8 3.7+0.3 47±5.9

EXAMPLE 2 Anti-tumor therapy with Herpes simplex viral vectors. One of two broad categories of HSV-based vectors are amplicons. (Spaete RR et al., "The herpes simplex virus amplicon: A new eucaryotic defective-virus cloning- amplifying vector", Cell, 1982; 30:295-304) Plasmids with HSV-1 lytic replication origins (ori S) and HSV-1 terminal packaging signal sequences can be amplified and packaged into infectious HSV-1 virions in the presence of helper virus. (Spaete RR et al., "The herpes simplex virus amplicon: A new eucaryotic defective-virus cloning-amplifying vector", Cell, 1982; 30:295-304; Kwong AD et al, "Herpes simplex virus amplicon: Effect of size on replication of constructed defective genomes containing eukaryotic DNA sequences", J Virol, 1984; 51:595-603; Geller Al et al, "A defective HSV-1 vector expresses Escherichia coli β-galactosidase in cultured peripheral neurons", Science, 1988; 241:1667-1669; Geller Al et al, "Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli beta-galactosidase", Proc Natl Acad Sci USA, 1990; 87:1149-1153). This plasmid system permits easier cloning and carries genomic information between prokaryotic and eukaryotic cells as a shuttle vector. (Kwong AD et al., "The Herpes simplex virus amplicon. Efficient expression of a chimeric chicken ovalbumin gene amplified within defective virus genomes", Virology, 1985; 142:421-425). The amplicon systems retain the merits of HSV-1 vectors, but viral stocks tend to have lower titers and production is time consuming. (Spaete RR et al., "The herpes simplex virus amplicon: A new eucaryotic defective-virus cloning-amplifying vector", Cell, 1982; 30:295-304). Previously reported amplicon systems were unable to package and maintain large size DNA fragments (>15 kb) efficiently. (Kwong AD et al, "Herpes simplex virus amplicon: Effect of size on replication of constructed defective genomes containing eukaryotic DNA sequences", J Virol. 1984; 51:595-603). HsV vectors have demonstrated efficient gene transfer in vivo. (PaleUa TD et al., "Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex type 1 vector", Gene, 1989; 80:137- 144; Chiocca EA et al., "Transfer and expression of the lacZ gene in rat brain neurons by herpes simplex virus mutants", New Biol, 1990; 2:739-746; Anderson JK et al, "Gene Transfer into mammalian central nervous system using herpesvirus vectors: extended expression of bacterial lacZ gene in neurons using the neuron-specific enolase promoter", Human Gene Ther, 1992; 3:487-499; Ho D et al., "Altering central nervous system physiology with a defective herpes simplex virus vector expressing the glucose transporter gene", Proc Natl Acad Sci USA, 1993; 90:3655-3659; During MJ et al, "Long-term behavioral recovery in Parkinsonian rats by an HSV vector expressing tyrosine hydroxylase", Science, 1994; 266:1399-1403; Glorioso JC et al., "Development and application of herpes simplex virus vectors for human gene therapy", Annu Rev Microbiol, 1995; 49:675-710). Several groups are developing HSV vectors as anti-cancer therapeutics. The cytopathic effects of modified HSV viruses and vectors can be used to excellent advantage against tumor cells. Martuza and coworkers first used thymidine kinase (Hstk) negative HSV to treat human gliomas in nude mice. (Martuza RL et al., "Experimental therapy of human glioma by means of a genetically engineered virus mutant", Science, 1991; 252:854-856). Some mice treated with only IO⁵ pfu of defective virus had prolonged survival. These initial modified HSV vectors were replication competent. Because of concerns about infection (especially viral encephalitis) other groups developed HSV vectors defective in other genes such as ribonucleotide reductase transcription factors ICP4 or ICPO, _γι34.5 gene. (Boviatsis EJ et al., "Antitumor activity and reporter gene transfer into rat brain neoplasms inoculated with herpes simplex virus vectors defective^' in thymidine kinase or ribonucleotide reductase", Gene Ther. 1994; 1:323-331; Takamiya Y et al., "Gene therapy of maUgnant brain tumors: a rat glioma line bearing the herpes simplex virus type 1-thymidine kinase gene and wild type retrovirus kills other tumor ceUs", J Neurosci Res, 1992; 33:493-503; Mineta T et al., "Treatment of malignant gliomas using ganciclovir-hypersensitive, ribonucleotide reductase-deficient herpes simplex viral mutant", Cancer Res, 1994; 54:3963-3966; Chambers R et al., "Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma", Proc Natl Acad Sci USA, 1995; 92:1411- 1415). The addition of GCV treatment to defective HSV containing an intact Hstk gene also enhances tumor kilUng. (Boviatsis EJ et al., "Long-term survival of rats harboring brain neoplasms treated with ganciclovir and a herpes simplex virus vector that retains an intact thymidine kinase gene", Cancer Res, 1994; 54:5745-5751). Recently efforts have focused on HSV mutants with multiple specific gene deletions to enhance their therapeutic index. (Glorioso JC et al., "Development and application of herpes simplex virus vectors for human gene therapy", Annu Rev Microbiol, 1995; 49:675-710; Mineta T et al., "Attenuated multi-mutated herpes simplex virus- 1 for the treatment of malignant gliomas", Nat Med, 1995; 1:938-943). These data suggest that our HSV amplicon vector will be useful to transfer the NIS gene into tumors in vivo.

Gene delivery with the pHE700 Herpes simplex amplicon vector. Plasmids containing a HSV-1 lytic replication origin (ori S) and a HSV-1 terminal packaging signal sequences, can be amplified and packaged into infectious HSV-1 virions in the presence of transacting helper virus. (Spaete RR et al., "The herpes simplex virus amplicon: A new eucaryotic defective- virus cloning-amplifying vector", Cell, 1982; 30:295-304; Kwong AD et al, "Herpes simplex virus amplicon: Effect of size on replication of constructed defective genomes containing eukaryotic DNA sequences", J Virol, 1984; 51:595-603; Geller Al et al, "A defective HSV-1 vector expresses Escherichia coli β-galactosidase in cultured peripheral neurons", Science, 1988; 241:1667- 1669; Geller Al et al, "Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli beta-galactosidase", Proc Natl Acad Sci USA, 1990; 87:1149- 1153). Our novel amplicon vector, pHE, contains HSV-1 ori S and packaging sequences that permit vector replication and packaging into HSV-1 virions. We have constructed HSV amplicons that also contain Epstein-Barr virus (EBV) sequences to maintain the plasmid as an episome in the transfected cell nucleus. (Westphal EM et al., "A novel infectious mini-HSV for high efficiency gene transfer into human cancer cells", Cancer Gene Ther, 1995; 2:324). EBV has been demonstrated to contain a unique latent replication origin (ori. P) which directs viral self-replication and maintenance in cells without entering the lytic cycle. (Yates JL et al., "Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells", Nature, 1985; 313:812-815; Reisman D et al., "A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components", Mol Cell Biol, 1985; 5:1822-1832). The Epstein-Barr virus nuclear antigen 1 (EBNA-1) encodes a DNA binding transactivator for ori P. (Yates JL et al., "Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells", Nature, 1985; 313:812-815; Reisman D et al., "A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis- acting components", Mol Cell Biol, 1985; 5:1822-1832; Rawlins DR et al., "Sequence-specific interactions of cellular nuclear factor I and Epstein-Barr virus nuclear antigen with herpes virus DNAs", Cancer Cells, 1986; 4:525-542; Goldsmith K et al., "Identification of EBNA1 amino acid sequences required for the interaction of the functional elements of the Epstein-Barr virus latent origin of DNA replication", J Virol. 1993; 67:3418-3426). Investigators previously demonstrated that plasmid vectors containing ori P and expressing the EBNA-1 gene were more effective eukaryotic expression vectors. (Yates JL et al., "Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells", Nature, 1985; 313:812-815). Various groups have used such EBNA-1 based vectors for expression in human tumors with therapeutic intent. (Judde JG et al., "Use of Epstein-Barr virus nuclear antigen- 1 in targeted therapy of EBV- associated neoplasia", Human Gene Ther, 1996; 7:647-653). The combination of the HSV amplicon with the EBV sequences improves the ease of use of the HSV amplicon system. Our replication incompetent pHE vectors maintain wide tropism for delivering transgene(s) into both dividing and quiescent cells with high efficiency both in vitro and in vivo. This improved vector could be produced at high titer and could package and carry a vector with a 21 kb DNA insert.

Episomal maintenance and amplicon vector packaging. The maintenance of pHE vector as an episome was demonstrated by transfection of pHE700-lac into E5 cells and selection with hygromycin. The pHE700-lac vector contains the bacterial LacZ gene cloned into the multi-cloning site of pHE700 (Figure 6). By day 16 of drug selection, almost all cells expressed β- galactosidase. To generate viral stocks, these selected E5 cells containing pHE700-lac plasmid were infected with dl20 helper virus (kindly provided by N. DeLuca, University of Pittsburgh). The resulting supernatants contain both the pHE700-lac vector and helper virus. The multiplicity of infection (MOI) of the helper virus added was between 0.01 to 0.1 to induce viral vector production within 24-36 hours. The average titer obtained was 2xl0⁶ bfu/ml with a ratio of pHE700-lac vector (bfu) to dl20 helper virus (pfu) of 1:10.

Transduction and expression of the pHE700-lac vector in vitro. The pHE700-lac containing supernatants were used to transduce human target cells in vitro. The β-galactosidase gene expression was evaluated after infection with pHE700-lac vector (3-10 MOI) in various cultured human cells, including VA13 normal fibroblasts. All cells were fixed and stained with X-gal two days after infection. The expression continued for approximately 2 weeks with a peak expression occurring 48-72 hours after transduction.

In vivo expression of the pHE700-lac vector in the rats and mice. The efficiency of pHE700-lac vector for gene delivery into the central nervous system was evaluated. The rat caudate nucleus or hippocampus were injected with pHE700-lac viral vector supernatant. Viral stock (2 x 10⁵ bfu) was stereotaxically injected unilaterally into the rat brain. High β-galactosidase expression was found around the injection site within the caudate and in the dentate gyrus following hippocampal injection as determined by X-gal staining 48 hours post injection. Gene expression in these brain areas was detected between 2 and 14 days after injection and was most prominent between 2 to 7 days. We have also demonstrated effective gene transfer of the amplicon vector into subcutaneous A375 human melanoma xenografts in athymic nude mice (data not shown).

HSV amplicon vector cytotoxicity. The dl20 HSV helper virus is necessary to package this HSV amphcon vector (DeLuca NA et al., "Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate -early regulatory protein ICP4", J Virol, 1985; 56:558-570; DeLuca NA et al., "Activities of herpes simplex virus type 1(HSV-

1) ICP4 genes specifying nonsense peptides", Nucleic Acids Res, 1987; 15:4491-

4511). Helper virus dl20 has deletions of both IE3 gene loci to prevent viral replication in normal cells, but permits replication in the E5 helper cell line expressing the IE3 gene (DeLuca NA et al., "Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4", J Virol, 1985; 56:558-570; DeLuca NA et al., "Activities of herpes simplex virus type l(HSV-l) ICP4 genes specifying nonsense peptides", Nucleic Acids Res. 1987; 15:4491-4511). This helper virus causes substantial cytotoxicity to infected normal cells in vitro (data not shown). Unfortunately, there is no published method currently available to separate HSV amplicon vectors from helper virus and still maintain high amplicon titers. Because of this cytotoxicity concern, we developed a novel method that uses psoralen and UVA light (PUVA) to reduce the cytotoxicity of HSV vectors but retain high level gene expression (Figure 7). Psoralens are polycyclic planar molecules that form covalent, cyclobutane- type linkages (Hanson CV et al., "Photochemical inactivation of DNA and RNA viruses by psoralen derivatives", J Gen Virol, 1978; 40:345-358). Previous studies applying crosslinking methods with psoralen and UVA completely inactivated virus by blocking DNA replication and viral gene expression to inactivate viruses. (Hanson CV, "Photochemical inactivation of viruses with psoralens: an overview", Blood Cells, 1992; 18:7-25; Redfield DC et al., "Psoralen inactivation of influenza and herpes simplex viruses of virus- infected cells", Infect Immun, 1981; 32:1216-1226; Swanstrom R et al., "Interaction of psoralen derivatives with the RNA genome of Rous Sarcoma Virus", Virology, 1981; 113:613-622; Alter HJ et al., "Photochemical decontamination of blood components containing hepatitis B and Non-A, non-B virus", Lancet, 1988; 2:1446-1450; Lin L et al., "Photochemical inactivation of cell-associated human immunodeficiency virus in platelet concentrates", Blood, 1993; 82:292-297; Cotten M et al, "Psoralen treatment of adenovirus particles eliminates virus replication and transcription while maintaining the endosomolytic activity of the virus capsid", Virology, 1994; 205:254-261). In our experiments, the appropriate PUVA dose induces DNA crosslinks in the vector that result in differential inactivation of viral replication and transgene expression. Prior PUVA treatment inhibits viral replication in the E5 helper cells while retaining gene expression of a reporter gene product.

To evaluate the selective elimination of cytotoxicity of a HSV amplicon vector while maintaining the transgene activity, a cell proliferation assay that measured cellular DNA synthesis was used as previously described. (Link, CJ, et al., "Preliminary in vitro efficacy and toxicities studies of the herpes simplex thymidine kinase gene system for the treatment of breast cancer", Hybridoma, 1995; 14:143-147). The pHE-tk vector was generated by inserting the Hstk gene into the pHE vector multi-cloning site under CMV promoter control (Figure 6). CeUs expressing Hstk can phosphorylate ganciclovir (GCV) to a toxic form that induces cell death. (Moolten, FL, "Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: Paradigm for a prospective cancer control strategy", Cancer Res, 1986, 46:5276-5281). The tests were performed with IGROV ovarian carcinoma cells transduced with pHE-tk vector packaged with helper virus dl20. Untreated pHE-tk vector stocks significantly reduced cellular proliferation indicating the cytotoxic effect of the helper virus and pHE-tk vector on the infected cells. (Figure 8). However, cells infected with the PUVA treated vector substantially maintained their proliferation rate (Figure 8). Importantly, while the cytotoxicity from PUVA treated pHE-tk vector was reduced to near that of cells not exposed to vector, we found that the vector retained functional transgene expression. GCV reduced ceUular proliferation under conditions where only small inhibitory effects from the modified vector were found. This anti- proliferative effect of GCV on cells transduced by PUVA treated vector indicates Hstk enzyme function (Figure 8). Thus, PUVA treatment prevented vector mediated cytotoxicity, but permitted Hstk gene expression. Our results demonstrate that the combination of TMP and UVA irradiation results in the differential inactivation of HSV vectors. We postulate that interstrand DNA crosslinks block viral DNA replication and randomly inactivate different regions of the vector genome. Genes located near the crosslinks are likely inactivated, but gene expression can be complemented by transcription from other vectors that have different inactivated regions or from other transcription units in amplicon vectors (Figure 7). Thus, the crosslinked DNA retains measurable transgene expression while prohibiting replication of any single vector. Thus photochemical modification should prevent any lytic viral 5 replication that might occur due to the rescue of recombinant wild-type virus both in vitro or in vivo. These PUVA treated vectors are ideal candidates for in vivo anti-tumor strategies using therapeutic genes such as the NIS gene.

EXAMPLE 3 l o DEMONSTRATION OF RETROVIRAL AND HERPES VIRAL

VECTORS GENE TRANSFER TO OVARIAN CANCER CELLS IN VITRO AND INCREASED CELL DESTRUCTION BY ¹³*I

RADIOTHERAPY. In vitro efficacy of retroviral vector transduction to transfer the NIS

15 gene into tumor spheroids. Our initial data indicated significant kilUng induced by ¹³¹I in NIS transduced melanoma and ovarian cancer cells. However, these results may be better studied under conditions where greater bystander kilUng effects can be appreciated. For this purpose, we will use spheroid tumors grown in soft agar. (Courtenay VD et al., "Growth of human

20 tumor cell colonies from biopsies using two soft-agar techniques", Br J Cancer, 1978; 38:77-81; Tveit KM et al., "Colony-forming abiUty of human ovarian carcinomas in the Courtenay soft agar assay", Anticancer Res, 1989; 9:1577- 1582). The increased radius of the tumor mass in this system will allow greater overall deposition of energy into the nearby cells in a manner

25 analogous to that of in vivo tumor cell masses. The dose from accumulated ¹³¹I is affected by cell density, radius and the stopping power of the beta energy from the decay of ¹³¹I. The fraction of internalized dose from accumulated iodide transmitted out of (lost by) cells can be simpUfied to the following equation: Transmitted Fraction = e ^μt; where μ = stopping power for beta

30 energy (3.009 m²/kg) x cell density in kg/m³ (approx. 1.3 x 10³ kg/m³) and t = the cell radius in meters. (Troulfanidis N (1995) "Measurement and detection of radiation", Vol. 2nd edition. Taylor and Francis, Washington, D.C.; (1992) The health physics and radiological handbook. In: Schleien B(Ed). Scinta, Inc., Silver Spring, MD). Small changes in radius exponentially affect the absorbed dose. For example, a round cell with an 8.0 μm radius will absorb only 3.1% of the energy (theoretical maximum dose) from internalized ¹³¹I, while cell mass with a 580 μm radius will absorb over 90% of the possible dose. This relationship is illustrated by Figure 9. Therefore small tumor-like cell aggregates will receive a significantly higher dose from accumulated ¹³¹I than cells in a monolayer. This model system more closely mimics the geometry of a tumor in vivo and thus should more accurately demonstrate the efficacy of the NIS for gene-directed radiotherapy for ovarian cancer.

We will use established protocols to grow small cell aggregates of our tumor cell Unes (with and without NIS) in soft agar. (Courtenay VD et al., "Growth of human tumor cell colonies from biopsies using two soft-agar techniques", Br J Cancer, 1978; 38:77-81; Tveit KM et al., "Colony-forming ability of human ovarian carcinomas in the Courtenay soft agar assay", Anticancer Res, 1989; 9:1577-1582). Individual "spheroids" from 0.5 to 4 mm in diameter will be exposed to ¹³¹I under similar conditions described previously for monolayer cells. Efficacy will be determined by measuring spheroid growth after exposure. Bystander effect will be assessed using mixed-population spheroids containing various percentages of NIS transduced and no vector cells. The next step in the project is to test a HSV amplicon vector to transfer a functional NIS gene to tumor spheroids in culture.

Clone a HSV vector containing both the Green Fluorescent Protein and NIS gene. A key part of evaluating and developing any gene therapy strategy is to accurately measure gene transfer efficiency. This data is essential in order to determine the level of vector transduction required_, to obtain a therapeutic level of the transgene in vivo. The second series of HSV vectors that will be cloned will not contain G418 drug selectable markers (neo^r)- Our vectors will express a variant of the green fluorescent protein (GFP). This codon modified GFP gene also contains a serine 65 to threonine mutation to greatly enhance the fluorophore activity. (Chalfie M et al., "Green Fluorescent Protein as a marker for gene expression", Science, 1994; 263:802- 805; Heim R et al., "Improved green fluorescence", Nature, 1995; 373:663-664). We have recently demonstrated the use of a codon optimized, red shifted mutant GFP gene to demonstrate retroviral gene delivery to cells without the need for any fixation techniques (Muldoon RR et al., "Tracing and quantitation of retroviral mediated transfer using a humanized, red shifted green fluorescent protein gene", Bio Techniques, 1997; 22:162-167; Levy JP et al., "Retroviral transfer and expression of humanized, red shifted green fluorescent protein into human tumor cells", Nature Biotechnol, 1996; 14:610- 614). Single copy gene expression is visualized in tumor cells within 24 hours after exposure to retroviral supernates under a fluorescence microscope or by FACS analysis using a standard FITC filter set. This marker allows the detection of in vitro and in vivo gene transfer by the direct observation of living tissues or frozen sections (without the need for fixation). The marker allows gene transfer to be observed on an individual cell basis. Excellent GFP gene expression and translation is visible in living tumor cell, transduced by a pHE700 vector. Recently a new HSV amplicon vector has been constructed, pHE850, that contains a red-shifted, codon optimized GFP gene and a separate multicloning site (Figure 10). This new vector will be used for transfer of the NIS gene into tumor cells, since it also permits rapid determinations of gene transfer efficiency by observing GFP co-expression

Cloning of the NIS gene into the pHE8NIS amplicon vector The NIS gene open reading frame will be cloned into the multi-cloning site of the pHE850 vector backbone (Figure 10). This vector contains a large multicloning site that aUows for direct subcloning from pREP eukaryotic expression plasmids. The NIS gene was previously subcloned into pREP7 flanked by Kpn I and Xho I sites and these will be used to subclone into the multi-cloning site of pHE850. The amplicon undergoes rolling circle replication in E5 cells containing an IE 3 gene that permits trans- complementation of dl20 (IE3 gene defective) helper virus. In the presence of helper virus, the amplicon replicates until a viral DNA of 152 kb long is obtained and then packaged. Thus, for the pHE8NIS plasmid of approximately 14.6 kb, at least 10 expression cassettes will be present as tandem repeats in the resulting viral vector. This should provide strong levels of NIS gene expression in targeted cancer cells. pHE8NIS viral vector production. The closed pHE8NIS plasmid will be transfected into E5 cells expressing the repUcation permissive IE3 gene with Lipofection per the manufacture's protocol (GibcoBRL). One day later, transfected cells will be placed in selection with Hygromycin B (ICN Biomedical Inc., Aurora, Ohio). Hygromycin resistant colonies will be trypsin digested and cells plated onto dishes. After stable selection for two weeks in hygromycin, the population of transfected cells will be examined for GFP expression. If positive for fluorescence, the cells will then be transduced with dl20 helper virus. To generate vector stocks, 3 x IO⁶ hygromycin resistant cells containing pHE8NIS will be plated on a 10-cm dish. After cells grow to confluence, 0.01 to 0.1 MOI of dl20 virus in 1 ml of Opti-MEM (Gibco, BRL) will be added for 2 hours at 37°C to allow infection. The virus solutions are then removed and fresh medium is added for an additional 24 to 36 hours. After 48-72 hours cells wiU be lysed. Supernates obtained, after centrifugation to remove debris, will be tested for green forming units (gfu/ml) to define supernate vector titer.

PUVA treatment of pHE8NIS vector supernates. For PUVA treatment, TMP (Trioxsalen, Sigma) in various concentrations (0 to 5 μg/ml) will be added to virus stocks for 30 minutes at 37°C with differing lengths of UVA exposure (0-7.5 kJ/m²). The UVA source will be a Spectroline Model XX-15A bulb (Westbury, New York). One ml of viral stock will be placed into one well of a 6-well plate and irradiated for various times (1 min = 0.5 kJ/m² @ 365 nm). The virus replication ability will be measured 3 days after incubation by staining permissive E5 cells with crystal violet. GFP will be visualized by fluorescence microscopy.

In vitro efficiency and efficacy with the pHE8NIS vector. An experimental design will be employed in a similar fashion to that previously demonstrated in examples herein. Three to ten MOI of pHE8NIS vector will be apphed to accompUsh gene transfer and expression. GFP expression will be observed by fluorescent microscopy of living cells in culture at 6, 12, 18, 24 and 36 hours after transduction. The number of GFP positive cells and GFP negative cells will be recorded per high powered field to calculate the vector transduction efficiency for the MOI used. Uptake of ¹²⁵I will be determined as previously demonstrated using an optimal MOI as revealed by the GFP analysis. After the completion of tests in tumor cell clonogenic assays for uptake of ¹²⁵I and killing with ¹³¹I, studies will be conducted with spheroid tumors in soft agar under conditions developed for the retroviral vector efficacy experiments. EXAMPLE 4

EVALUATION OF INVIVO GENE TRANSFER USINGVPC TO TRANSFERARVVECTOR

For the in vivo testing animals will first be administered low amounts of ¹³¹I (0.2 mCi) to ablate the thyroid gland and will then be placed on Synthroid supplements as described previously. (Kasuga Y et al., "The effect of xenotransplantation of human thyroid tissue following radioactive iodine - induced thyroid ablation on thyroid function in the nude mouse", Clin Invest Med, 1991; 14:277-281). Standardized retroviral stocks will be generated in PA317 packaging cells as previously described. (Link, CJ, et al., "Preliminary in vitro efficacy and toxicities studies of the herpes simplex thymidine kinase gene system for the treatment of breast cancer", Hybridoma, 1995; 14:143- 147). The vector will first be transiently transfected into GP+E86 ecotropic packaging cells and then the resulting supernates wiU be used to transduce PA317 cells growing in log phase. The cells will be cloned by Umiting dilution and 20 clones will be titered for activity. Several of the highest titer LNISN VPC will be grown to large scale and used for in vivo tests.

Test efficacy of pretransduced tumor model for response with ¹³¹I. In order to insure that the planned ¹³¹I treatment dose of 0.3 mCi is effective, an initial small experiment will be conducted with ¹³¹I. SK-OV-3 tumor cells will be transduced with the LNISN vector and an individual clone with stable transgene expression will be obtained. Athymic nude mice (nu/nu) will be injected subcutaneously with lOxlO⁶ SK-OV-3 tumor cells into the anterior abdominal wall. This cell number usually results in a solid 3-4 mm tumor mass within 5-7 days (data not shown). The cells administered will be transduced ex vivo before implantation according to Table 3. Group D will determine if tumors cells expressing the NIS gene combined with cells not expressing the NIS gene (one to one ratio) can be destroyed by bystander radiation killing after ¹³¹I administration. Fourteen days later, animals will receive a single intravenous injection of 0.3 mCi of ¹³¹I (Kasuga Y et al., "The effect of xenotransplantation of human thyroid tissue following radioactive iodine-induced thyroid ablation on thyroid function in the nude mouse", Clin Invest Med, 1991; 14:277-281; Walinder G, "Determination of the 131 1 dose to the mouse thyroid", Acta Radio Ther Phys Biol, 1971; 558-578). Responses will be determined by tumor measurements recorded biweekly (mm3).

TABLE 3

IN VIVO ANTI-TUMOR ACTIVITY OF "ⁱi AGAINST

PRETRANSDUCED TUMORS

Group Injected SK OV-3 cells transduced by: Number of mice in group

#cells Vector 131J No isotope

A lOxlO⁶ None 5 0

B lOxlO⁶ LXSN 5 0

C lOxlO⁶ LNISN 5 3

D 5xl0⁶ LNISN 5 3 5xl0⁶

Test efficacy after in vivo gene transfer with vector producer cell injections followed by ¹³¹I administration. Previous experiments have demonstrated effective gene transfer into target tumor cells by the direct implantation of retroviral VPC (Short MP et al., "Gene delivery to glioma cells in rat brain by grafting of a retrovirus packaging cell line", J Neurosci Res, 1990; 27:427-439; Link CJ et al, "A phase I trial of in vivo gene therapy with the Herpes simplex thymidine kinase/ganciclovir system for the treatment of refractory or recurrent ovarian cancer", Human Gene Ther, 1996; 7:1161-1179; Caruso M et al., "Regression of established macroscopic liver metastases after in situ transduction of a suicide gene", Proc Natl Acad Sci (USA) 1993; 90:7024-7028). This approach will be performed with the SK-OV-3 tumor model (Table 4). Five positive control mice (group A) will be injected with lOxlO⁶ SK-OV-3 cells pretransduced with LNISN vector. Eight mice each in groups B, C and D will be injected into the abdominal cavity with lOxlO⁶ SK- OV-3 tumor cells followed by treatment with either (B) LNChRG VPC (transfers GFP gene, but no NIS gene), (C) HBSS only or (D) LNISN VPC. Three days after the injection of treatment cells, 5 mice in each group will be injected intravenously of 0.3 mCi of ¹³¹I. Responses will be determined by biweekly observations of the animals for cachexia of ascites. Animals from group B through D not treated with ¹³¹I will be evaluated for neo^r expression by G418 selection by the same method used for Table 2. This aim should establish in vivo efficacy of the isotope concentrator concept.

Table 4.

EFFICACY AFTER IN VIVO GENE TRANSFER WITH MURINE VECTOR PRODUCER CELLS FOLLOWED BY ¹ *I

Number of mice

VPC No

Group Treatment injected 131J isotope

A LNISN transduced None 5 0 SK-OV-3 ceUs

B LNChRG VPC lOxlO⁶ 5 3

C HBSS only None 5 3

D LNISN VPC 10x10⁶ 5 3

EXAMPLE 5

IN VIVO GENE TRANSFER EFFICIENCY USING HERPES SIMPLEX VIRUS (HSV) AMPLICON VECTORS EXPRESSING THE GREEN FLUORESCENT PROTEIN (GFP) AND EFFICACY BY TRANSFER OF THE NIS GENE FOLLOWED BY ^{1 1}I ADMINISTRATION.

Anti-tumor gene therapy requires highly efficient gene transfer and expression of therapeutic genes. HSV vectors offer both of these properties and will be the gene transfer vehicle for our anti-tumor approach with NIS gene therapy. These vectors are extremely attractive since they efficiently transduce nondividing cells (Go stage) and have large carrying capacity.

Most human epithelial neoplasms have a significant percentage of their cells in the GO stage of the cell cycle, for example, breast cancers have only approximately 9% of their cells in S phase. (Witzig TE et al., "DNA ploidy and percent S-phase as prognostic factors in node-positive breast cancer: results from patients enrolled in two prospective randomized trials", J Clin Oncol, 1993; 11:351-359). Murine retroviral vectors do not transduce these Go cells, since mitosis is required to transduce cells. (Takamiya Y et al., "Gene therapy of malignant brain tumors: a rat glioma Une bearing the herpes simplex virus type 1-thymidine kinase gene and wild type retrovirus kills other tumor cells", J Neurosci Res, 1992; 33:493-503). Plasmids containing a HSV-1 lytic replication origin (ori S) and a HSV-1 terminal packaging signal sequences, can be amplified and packaged into infectious HSV-1 virions in the presence of transacting helper virus. (Spaete RR et al., "The herpes simplex virus amplicon: A new eucaryotic defective-virus cloning-amplifying vector", Cell, 1982; 30:295-304; Kwong AD et al, "Herpes simplex virus amplicon: Effect of size on replication of constructed defective genomes containing eukaryotic DNA sequences", J Virol. 1984; 51:595-603; Geller Al et al, "A defective HSV-1 vector expresses Escherichia coli β-galactosidase in cultured peripheral neurons", Science, 1988; 241:1667-1669; Geller Al et al, "Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli beta-galactosidase", Proc Natl Acad Sci USA, 1990; 87:1149-1153). This approach uses an HSV amplicon system because of the Umited ability to transduce tumors with retroviral approaches. Again this aim has a dual purpose of basic science investigation of the delivery system and kilUng method and of developing preclinical data for a human clinical trial.

Evaluate gene transfer and expression of GFP in SK-OV-3 murine model. Athymic nude mice (nu/nu) mice that are permissive for the growth of SK-Ov-3 human ovarian carcinoma cells will be used. The injection of lOxlO⁶ cells on day 1 will result in a 3-4 mm tumor by day 5-7. Five animals that will serve as positive controls for GFP expression will be injected with lOxlO⁶ SK- OV-3 cells previously transduced with LNChRG vector containing a modified GFP gene. Three remaining groups of 5 animals each will receive various doses (5 x IO⁸, 1 x IO⁹, or 5 x IO⁹ gfu/ml) of pHE8NIS vector (suspended in 100 μl of HBSS) injected directly into the subcutaneous tumor. Animals will then be sacrificed and tumors will be examined by fluorescent microscopy of frozen sections or FACS analysis of tumor cell suspension after trypsin digestion. We have previously demonstrated the direct visualization of GFP expression from frozen tissue sections after injection of eukaryotic expression plasmids containing a modified GFP gene (data not shown). Next, a series of similar experiments will be conducted with intraperitoneal tumors to determine gene transfer efficiency. The injection of 10 x IO⁶ cells on day 1 will result in multiple intraperitoneal tumors in all animals by day 7-10 after inoculation (Link, unpubhshed observations). Three groups of 5 mice each will receive direct intraperitoneal injections of 1 ml of HBSS containing 1 x IO⁷, 1 x IO⁸ or 1 x IO⁹ gfu/ml of pHE8NIS vector. Analysis for GFP expression will be performed 2 to 5 days after injection.

Evaluate for anti-tumor efficacy using the pHE8NIS vector followed by ¹³¹I treatment in a murine xenograft model. Athymic nude (nu/nu) mice will be obtained (Harpan-Sprague). Animals will be fed sterile mouse food (Teklad) and H2O ad libitum. Sani-chip and microisolator mouse cages (Lab products) will be used. Cages will be kept in our fill service rodent faciUty. Mice in groups A through E (Table 5) will be injected intraperitoneally with 10xl0^G SK-OV-3 cells on day 1. This will result in multiple intraperitoneal tumors in the animals by day 7-10 after innoculation and animal death by approximately 30 days after tumor injection (Link, unpublished observations). Control groups will be (A) HBSS only and (B) pHE-tk vector (control for HSV vector cytotoxicity). Groups (C), (D) and (E) will be injected with three dose levels of pHE8NIS vector (Table 5). Mice in group F will receive SK-OV-3 cells pretransduced with LNISN retroviral vector and will serve as positive controls for tumor killing by ¹³¹I. Three days after the injection of treatment cells, five animals from each group will receive an intravenous injection of 0.3 mCi of ¹³¹I. Animals will be observed daily for evidence of toxicity or infection after vector administration. Responses will be determined by biweekly observations of the animals for cachexia or ascites. Complete responses will be defined as animals that have complete tumor destruction (no visible disease at necropsy). This will be determined when the animals are sacrificed on day 60 (this time frame should permit death to occur in all untreated or control animals). Remaining tumor deposits in mice surviving at 60 days will be harvested. A separate portion of this tumor will be sent for histopathology staining for cellular infiltrates present in the tumors.

TABLE 5

EFFICACY AFTER IN VTVO GENE TRANSFER WITH pHE8NIS

VECTOR INJECTIONS FOLLOWED BY «ⁱl

Number mice

Group Treatment Amount ¹³¹I none

A HBSS only None 5 0

B pHE-tk 5xl0⁹ pfu/ml 5 3

C pHE8NIS 5xl0⁸ gfu/ml 5 3

D pHE8NIS 2xl0⁹ gfu/ml 5 3

E pHE8NIS 5xl0⁹ gfu/ml 5 3

F LNISN transduced None 5 3

SK-OV-3 ceUs Day 0: Tumor implantation into the peritoneal cavity Day 5: Vector administered into the peritoneal cavity Day 8: ¹³¹I administered (0.3 mCi)

EXAMPLE 6

EX VIVO PROTOCOLS FOR PURGING OF

CELLS USING RADIOISOTOPE CONCENTRATOR THERAPY.

Adoptive transfer of lymphocytes modified to treat graft versus host disease (GvHD) after allogeneic bone marrow transplantation.

Allogeneic BMT (bone marrow transplant) cures leukemia by means of myeloablation induced by the preparative regimen and by transfer in the bone marrow aUograft of immunocompetent donor cells that exert an anti-leukemic effect called Graft-versus-Leukemia (GvL). Horowitz MM et al. "Graft- Versus- Leukemia Reaction After Bone Marrow Transplantation", Blood 1990; 75:555- 562; Weiden, P.L., Horowitz M.M., "Graft-vs-Leukemia Effects in Clinical Bone Marrow Transplant", Hematology 1990; 12:691-708. Recently direct evidence for this anti-leukemic effect was demonstrated by the infusion of donor peripheral blood leukocytes into patients with relapse after allogeneic BMT (Table I). The International Bone Marrow Transplant Registry analyzed data from 2,254 patients who underwent HLA-identical sibling BMT for early leukemia and found a significant reduction in the relapse risk for patients who developed GvHD. Horowitz MM et al. "Graft-Versus-Leukemia Reaction After Bone Marrow Transplantation", Blood 1990; 75:555-562; Weiden, P.L., Horowitz M.M., "Graft-vs-Leukemia Effects in Clinical Bone Marrow Transplant", Hematology 1990; 12:691-708. Sadly, GvHD is not always treatable and causes substantial patient morbidity and mortality. The removal of mature T-cells from the graft results in effective prevention of acute and chronic GvHD. This benefit of T-cell depletion is offset by increased graft failure and leukemia relapse so that overall survival is not improved. Marmont, A.M. et al., "T-Cell Depletion of HLA-identical Transplants in Leukemia", Blood. 1991; 78:2120-2130. T-cell depletion increases leukemia relapse in AML and ALL because of the loss of GvHD. In the case of cell CML there is also a GvL effect (independent of GvHD) which may be lost during the process of T-cell depletion. Kolb and colleagues first reported that for patients who relapse with CML after aUogeneic BMT, leukocyte infusions from the original donor can induce remission. Kolb, H.J. et al., "Donor Leukocyte Transfusion for Treatment of Recurrent Chronic Myelogenous Leukemia in Marrow Transplant Recipients", Blood, 1990; 76:2462-2465. Existing data finds that patients with CML who have cytogenetic relapse only or chronic phase respond better to this treatment than patients with more advanced disease. In conclusion, allogeneic BMT is associated with a GvL effect that has a GvHD-dependent and a GvHD-independent component. Current drug therapy for GvHD is only partially effective and progressive GvHD destruction of recipient tissues is often fatal. Therefore, it is highly desirable to be able to destroy adoptively transferred lymphocytes only if they cause GvHD.

NR-not reported; AL-acute leukemia

EXAMPLE 7

TUMOR SPECIFIC PROMOTERS USEFUL FOR THE INVENTION

The following is a list of tumor specific promoters which may be useful for the invention, many other such promoters are known and available to those of skill in the art in such resources as genbank. Tumor Specific Promoters Useful for the Invention include but are not hmited to the following:

0gb:HSU24128 Human prohormone convertase (PCl/3) gene, promoter and 5' flanking

R73368519-73377389-/gopherlib/data/db/.genbank-92/gbpri.seq gopher.nih.gov 70

0gb:HUMPRDAlA Homo sapiens PRADl/cyclin Dl proto-oncogene, promoter region and

R131139159-131144243-/gopherHb/data/db/.genbank-92/gbpri.seq gopher.nih.gov 70 0gb:MMTIMP3MI M.musculus (Balb/C) TIMP-3 gene for metalloproteinase-3 tissue R21667172-21676635-/gopherUb/data/db/.genbank-92/gbrod.seq gopher.nih.gov 70

0gb:S76735 HrMA4 alpha=muscle-specific actin {promoter} [Halocynthia

R91434337-91436077-/gopherlib/data/db/.genbank-92/gbinv.seq gopher.nih.gov 70

0gb:SPU16263 Strongylocentrotus purpuratus cytoplasmic actin I (SpCyl) gene, R94170781-94174495-/gopherhb/data/db/.genbank-92/gbinv.seq gopher.nih.gov 70

0gb:SUSMSP130A S.purpuratus cell surface glycoprotein (mspl30) gene, 5' flank and

R95059129-95062574-/gopherhb/data/db/.genbank-92/gbinv.seq gopher.nih.gov 70

0gb:SUSMSP130B S.purpuratus cell surface glycoprotein (mspl30) mRNA, 5' end. R95062574-95068440-/gopherlib/data/db/.genbank-92/gbinv.seq gopher.nih.gov 70

0gb:TBVSGll8A T. brucei promoter region for variant-specific surface glycoprotein

R96463813-96467678-/gopherlib/data/db/.genbank-92/gbinv.seq gopher.nih.gov 70

0gb:BTU15731 Bos taurus somatotropin receptor gene, exon 1 and liver-specific

R8476848-8482291-/gopherlib/data/db/.genbank-92/gbmam.seq gopher.nih.gov

70

0gb:DOGCAMII Dog gene for calmodulin, exon 1.

R10631174-10634811-/gopherUb/data/db/.genbank-92/gbmam.seq gopher.nih.gov 70

0gb:LC15LOPRO L.cuniculus 15-lipoxygenase gene, promoter region.

R12667022-12669806-/gopherlib/data/db/.genbank-92/gbmam.seq gopher.nih.gov 70

0gb:MDU32208 Monodelphis domestica ubiquitin C-terminal hydrolase (PGP9.5) gene,

R12823493-12828679-/gopherUb/data/db/.genbank-92/gbmam.seq gopher.nih.gov 70

0gb:OALGB Ovis aries beta-lactoglobulin gene.

R14266321-14279312-/gopherUb/data/db/.genbank-92/gbmam.seq gopher.nih.gov 70

0gb:OCKK3 O.cuniculus keratin K3 gene.

R15952615-15964713-/gopherhb/data/db/.genbank-92/gbmam.seq gopher.nih.gov 70

0gb:RAB15LOX Rabbit erythroid cell-specific 15-hpoxygenase (15-lox) gene, R19673279-19689638-/gopherUb/data/db/.genbank-92/gbmam.seq gopher.nih.gov 70

Ogb:RABSURFA Rabbit lung surfactant protein A related gene, complete gene and

R22419294-22432460-/gopherhb/data/db/.genbank-92/gbmam.seq gopher.nih.gov 70

0gb:S55298 LINE/c-MYC (junction sequence} [dogs, transmissible venereal tumor, R22998775-23000354-/gopherlib/data/db/.genbank-92/gbmam.seq gopher.nih.gov 70

0gb:S64695 luteinizing hormone beta-subunit [sheep, Genomic, 1779 nt]. R23183638-23187681-/gopherUb/data/db/.genbank-92/gbmam.seq gopher.nih.gov 70

0gb:S65740 K3 keratin [rabbits, Genomic, 6045 nt].

R23217050-23227115-/gopherhb/data/db/.genbank-92/gbmam.seq gopher.nih.gov 70 Ogb:SSIKBAG S.scrofa IkBa gene (promoter region.

R25337255-25341446-/gopherlib/data/db/.genbank-92/gbmam.seq gopher.nih.gov 70

0gb:A08215 Patatin gene and promoter sequence.

R4651664-4655250-/gopherlib/data/db/.genbank-92/gbpat.seq gopher.nih.gov 70

0gb:A15840 Promoter region proteinase gene from pSKIII.

Claims

What is claimed is:

1. A method of accumulating radioisotopes in cells and those cells subject to a bystander effect comprising: transforming said cells with a polynucleotide sequence which encodes a radioisotope concentrator; and thereafter contacting said cells with a radioisotope so that said radioisotope is imported to said transformed ceUs.

2. The method of claim 1 wherein said radioisotope concentrator is a sodium iodide symporter.

3. The method of claim 1 wherein said radioisotope concentrator is Na⁺/K⁺ ATPase.

4. The method of claim 2 wherein said radioisotope is selected from the group consisting of ^{1 1}I, ¹²⁵I, ¹³¹I-MIBG, ¹²³I and ⁹⁹T pertechnetate.

5. The method of claim 1 wherein said radioisotope is ¹³¹I.

6. The method of claim 3 wherein said radioisotope is ²⁰¹T1.

7. The method of claim 3 wherein said radioisotope is ⁹⁹T.

8. The method of claim 1 wherein said radioisotope concentrator is a bone Ca transporter.

9. The method of claim 8 wherein said radioisotope is ⁹⁹Tc.

10. The method of claim 1 further comprising the step of: imaging said cells to identify expression of said radioisotope concentrator gene.

11. The method of claim 1 wherein said radioisotope is administered in an amount effective for kiUing said transformed cell.

12. The method of claim 10 wherein said radioisotope is administered in an amount affective for radioimaging said transformed cell.

13. The method of claim 1 wherein said transformation comprises: delivering to said group of cells a producer cell line of a replication deficient vector for expressing viral a radioisotope concentrator.

14. The method of claim 4 wherein said delivery of said vector comprises direct injection of said producer cell line.

15. The method of claim 14 wherein said vector is a herpes simplex virus amplicon vector.

16. The method of claim 14 wherein said vector is a retroviral vector.

17. A method for killing tumor cells in an organism comprising: delivering into said tumor a vector for expressing in replicating cells a polynucleotide encoding a radioisotope concentrator gene; expressing said factor in replicating cells in said tumor; administering vector to said organism by a route and in an amount effective for said vector to concentrate radioisotopes in said tumor cells which express said vector and by said bystander effect, sensitize other cells in said tumor which do not express said concentrator gene, and exposing said tumor cells to radionuclide therapy.

18. The method of claim 17 wherein said delivery of said polynucleotide comprises: delivering to said tumor a producer cell Une of a replication deficient vector for expressing a radioisotope concentrator.

19. The method of claim 18 wherein said delivery of said vector comprises direct injection of said producer cell line.

20. An expression construct for accumulation of radionuclides in transformed ceUs comprising: a nucleotide sequence which encodes a radioisotope concentrator gene; a promoter operable linked to said gene; and a polyadenylation signal.

21. The construct of claim 20 wherein said promoter is a tumor specific promoter.

22. The construct of claim 20 wherein said radioisotope concentrator gene is selected from the group consisting of: a sodium iodide symporter gene, a sodium potassium adenosine triphosphatase gene, and a bone calcium transporter gene.

23. The construct of claim 22 wherein said radioisotope concentrator gene is a sodium iodide symporter gene.

24. The construct of claim 23 wherein said sodium iodide symporter gene is isolated from FRTL cells.

25. A viral vector comprising a radioisotope concentrator gene.

26. The vector of claim 25 wherein said vector is a Herpes Simplex Viral vector.

27. The vector of claim 25 wherein said vector is a retroviral vector.

28. The vector of claim 25 wherein said radioisotope concentrator gene is selected from the group consisting of a sodium iodide symporter gene, a sodium potassium adenosine triphosphatase gene, and a bone calcium transporter gene.

29. The vector of claim 28 wherein said radioisotope concentrator gene is a sodium iodide symporter gene.

30. The vector of claim 29 wherein said sodium iodide symporter gene is isolated from FRTL cells.

31. A cell transformed with the viral vector of claim 28.

32. A vector producer cell Une transformed with the vector of claim 25.