Oncolytic virus-induced expression of PTEN-Long

Abstract

Novel modified viruses containing recombinant PTEN-Long and methods of using the viruses to treat cancer are disclosed.

Description

Detailed Description of the Invention

[0001] (Statement of Government Support) This work was supported by NIH 1R01NS064607 and NIH P01 CA163205 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 368,822, filed July 29, 2016, and U.S. Provisional Patent Application No. 62 / 479,671, filed March 31, 2017, each of which is expressly incorporated by reference in its entirety.

[0003] [Background technology] Glioblastoma (GBM) is a World Health Organization grade IV astrocytoma with poor patient survival rates, characterized by extreme heterogeneity and invasiveness, as well as resistance to radiation and chemotherapy drugs. Brain metastases are 2-3 times more frequent than GBM and, together with breast cancer, are among the top three brain metastatic tumor types. The median survival rate for patients receiving current standard of care is less than 18 months for GBM and 2-25 months for breast cancer brain metastases (BCBM). Novel therapies for brain tumors and cancer in general are needed.

[0004] Glioblastoma (GBM) is a World Health Organization grade IV astrocytoma with poor patient survival rates, characterized by extreme heterogeneity and invasiveness, as well as resistance to radiation and chemotherapy drugs. Brain metastases are two to three times more frequent than GBM and, together with breast cancer, are among the top three brain metastatic tumor types. The average survival rate for patients receiving current standard of care is less than 18 months for GBM and 2-25 months for breast cancer brain metastases (BCBM). Novel therapies for brain tumors and cancer in general are needed.

[0005] [Summary of the invention] Methods and compositions relating to recombinant viruses expressing a mammalian PTEN-Long gene are disclosed. In one aspect, disclosed herein is a recombinant virus of any preceding aspect, wherein the recombinant virus is a recombinant HSV-1 virus comprising a PTEN-Long gene.

[0006] In one aspect, disclosed herein is a recombinant virus of any preceding aspect, where the virus has been modified to target tumor cells or to place a gene important for viral replication under the control of a tumor-specific promoter.

[0007] In one aspect, disclosed herein is a recombinant virus of any preceding aspect, wherein the PTEN-Long gene is operatively linked to a tissue-specific promoter, an inducible promoter, or a constitutive promoter.

[0008] Also disclosed herein is a method of treating cancer comprising administering to a subject the recombinant virus of any of the preceding aspects. In one aspect, disclosed herein is a method of inhibiting cancer and / or inhibiting metastasis comprising administering to a subject the recombinant virus of any preceding aspect.

[0009] Also disclosed herein is a method of inhibiting PD-L1 in cancer cells, comprising administering to a subject a virus of any of the preceding embodiments. In one aspect, disclosed herein is a method of increasing the number of infiltrating CD8 T cells at a site of cancer in a subject comprising administering to the subject a virus of any preceding aspect.

[0010] Also disclosed is a method of increasing the number of infiltrating natural killer cells at a site of cancer in a subject comprising administering to the subject a virus of any preceding embodiment. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and, together with the description, illustrate the disclosed compositions and methods. [Brief description of the drawings]

[0011] [Figure 1A] FIG. 1 shows the PTEN-Long transgene expression vector in the HSVQuick backbone. [Figure 1B] FIG. 1 shows that transgene expression is detectable within 3 hours of infection in U87ΔEGFR glioma cells (WB). [Figure 1C] FIG. 1 shows that HSV-P10 infection dramatically affects Akt phosphorylation in infected DB7 cells compared to HSVQ control (WB). [Diagram 2] Representative hematoxylin and eosin (H&E) and PTEN staining from sections of human breast cancer brain metastases (top, 40x) and FVB / N mice with intracranial DB7 (bottom, 20x). [Diagram 3] FIG. 1 shows that HSV containing a PTEN-Long expression vector (HSV-P10) can replicate and spread in glioma cells in vitro similarly to the control HSVQ virus. [Figure 4A] Figure 1 shows that PTEN-Long expression does not inhibit HSV-P10 cell killing in multiple cell lines. Figure 2 shows the results of MTT assays (DB7, mouse breast cancer, MDA-MB-468, human breast cancer, LN229 and U87ΔEGFR, human glioma). [Figure 4B] Figure 1 shows that PTEN-Long expression does not inhibit HSV-P10 cell killing in multiple cell lines. Figure 2 shows the results of MTT assays (DB7, mouse breast cancer, MDA-MB-468, human breast cancer, LN229 and U87ΔEGFR, human glioma). [Figure 4C] Figure 1 shows that PTEN-Long expression does not inhibit HSV-P10 cell killing in multiple cell lines. Figure 2 shows the results of MTT assays (DB7, mouse breast cancer, MDA-MB-468, human breast cancer, LN229 and U87ΔEGFR, human glioma). [Figure 4D]Figure 1 shows that PTEN-Long expression does not inhibit HSV-P10 cell killing in multiple cell lines. Figure 2 shows the results of MTT assays (DB7, mouse breast cancer, MDA-MB-468, human breast cancer, LN229 and U87ΔEGFR, human glioma). [Figure 5A] Figure 1 shows the survival rate of FVB / N mice bearing DB7-dsRed tumors.Virus was injected intratumorally 7 days after tumor implantation. [Figure 5B] Figure 1 shows the survival rate of nude mice bearing DB7 tumors.Virus was injected intratumorally 7 days after tumor implantation. [Figure 6A] Figure 1 shows brain immune system infiltration 7 days after virus treatment.Figure 2 shows the ratio of CD11b+ F4 / 80+ microglia (CD45lo-int) to macrophages (CD45hi). [Figure 6B] FIG. 1 shows brain immune system infiltration 7 days after virus treatment. Percentage of MHC-II+ microglia (red) and macrophages (green). [Figure 6C] Figure 1 shows brain immune system infiltration 7 days after viral treatment. Figure 2 shows infiltrating CD49b+ CD335+ NK cells. [Figure 6D] Figure 1 shows brain immune system infiltration 7 days after virus treatment. Figure 2 shows infiltrating cytotoxic CD3+ CD8+ T cells. [Figure 7] Figure 1 shows that HSV-P10 inhibits virus-induced PD-L1 expression on the surface of infected and adjacent uninfected DB7 breast cancer cells 24 hpi, as measured by flow cytometry. [Figure 8] Sera from tumor-bearing FVB / N mice collected 7 days after treatment were analyzed for antibodies against DB7 cell lysates, n=3 per group, ELISA measurements. [Figure 9] FIG. 1 illustrates a depiction of the pathway by which PTEN-Long-expressing recombinant viruses (such as HSV-P10, also known as RAPTOR) can attack cancer cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] As shown in Figure 9, anti-tumor antibody responses are increased and AKT phosphorylation is decreased, thus decreasing PD-L1 expression and preventing the inhibitory signals that reduce T cell proliferation and NK cell proliferation, thus increasing the infiltration of NK cells and T cells and allowing them to exert anti-tumor activity.

[0013] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that, unless otherwise specified, they are not limited to particular synthetic methods, or to particular recombinant biotechnology methods, or to particular reagents, unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0014] A.Definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0015] Ranges may be expressed as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that each of the endpoints of a range is significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that a number of values ​​are disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. It is also understood that when a value is disclosed, "less than or equal to," "greater than or equal to," and possible ranges between values ​​are also disclosed, as would be well understood by one of ordinary skill in the art. For example, if the value "10" is disclosed, "greater than or equal to 10" is also disclosed, as well as "less than or equal to 10." It is also understood that data are provided in many different formats throughout the application, and that the data represent endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it is understood that data points greater than 10 and 15, equal to or greater than 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15 are considered to be disclosed, as well as between 10 and 15. It is also understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0016] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes examples when the event or circumstance occurs and examples when it does not occur.

[0017] "Reduction" can refer to any change that results in a lesser amount of a symptom, composition, or activity. A substance is also understood to reduce the gene output of a gene when the gene output of a gene product containing the substance is less relative to the output of a gene product without the substance. A reduction can also be, for example, a change in a disease symptom such that the symptom is less than previously observed.

[0018] "Inhibit," "inhibiting," and "inhibition" refer to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, or any amount in between, compared to native or control levels.

[0019] Here, "treatment", "treat" or "treating" can refer to a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms. Treatment can be any reduction from the native level, including but not limited to the complete removal of the disease, condition, or symptoms of the disease or condition. Thus, in the disclosed methods, "treatment" can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in the severity of the disease or disease progression suffered. For example, the disclosed methods for reducing or inhibiting the effects of cancer (such as inhibiting or reducing metastasis) are considered to be treatments if there is a 10% reduction in one or more symptoms of the disease in a subject with the disease compared to the native level in the same subject or a control subject. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction or any amount in between compared to the native level or control level. It is understood and contemplated herein that "treatment" does not necessarily mean curing a disease or condition, but refers to improving the appearance of a disease or condition.

[0020] Throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosure pertains. The references disclosed are also individually and specifically incorporated by reference into this specification for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0021] B. Composition Disclosed are the components used to prepare the disclosed compositions, and the compositions themselves used within the methods disclosed herein. Where these and other substances are disclosed herein, and where combinations, subsets, interactions, groups, etc. of these substances are disclosed, it is understood that specific references to the various individual and collective combinations and permutations of each of these compounds may not be expressly disclosed, but each is specifically contemplated and described herein. For example, where a particular PTEN-Long expressing recombinant virus is disclosed and discussed, and where a number of modifications that can be made to a number of molecules including PTEN-Long and / or the viral backbone are discussed, unless specifically indicated to the contrary, all combinations and permutations of PTEN-Long and any viral backbone with possible modifications are specifically contemplated. Thus, where types of molecules A, B, and C are disclosed, as well as examples of types of molecules D, E, and F and combinations of molecules A-D, each is individually and collectively contemplated, and combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed, even if each is not individually listed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the subgroups AE, BF and CE will be considered as disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in the disclosed methods of making and using the compositions. Thus, when there are multiple additional steps that can be performed, it is understood that each of these additional steps can be performed by any particular embodiment or combination of embodiments of the disclosed method.

[0022] Tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN) is a phosphatase that inhibits the AKT signaling pathway in cells. PTEN is mutated or deleted in approximately 50% of all high-grade gliomas and is lost in 33-49% of all breast cancers. PTEN-Long is a recently discovered alternative start site mutant of PTEN that has 173 additional amino acids at the N-terminus. This longer form of PTEN is secreted, membrane permeable, and maintains its phosphatase activity after secretion and reintroduction into cells. Disclosed herein are novel tumor treatments (e.g., brain tumor treatments) using modified viruses (e.g., modified herpes simplex virus (HSV-P10)) that secrete PTEN-Long from infected cells.

[0023] Delivery of compositions to cells In one aspect, it is understood and contemplated herein that the functional and therapeutic effects disclosed herein are the result of expression of PTEN-Long in cells, such as, for example, cancer cells. There are numerous compositions and methods that can be used to deliver nucleic acids (such as PTEN-Long expressing nucleic acids) to cells, either in vitro or in vivo. These methods and compositions can be primarily categorized into two types: viral delivery systems and non-viral delivery systems. For example, nucleic acids can be delivered via numerous direct delivery systems, such as electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material into cells or carriers such as cationic liposomes. Thus, in one aspect, viral vectors, plasmids, phages, cosmids that contain PTEN-Long. Also disclosed herein is a method of treating cancer in a subject, comprising administering to the subject PTEN-Long, which is delivered to the cancer cells via lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via carriers such as transfer of genetic material into cells or cationic liposomes. Suitable means for transfection, including viral vectors, chemical transfectants, or physical-mechanical methods, such as electroporation and direct diffusion of DNA, are described, for example, by Wolff, JA, et al., Science, 247, 1465-1468 (1990) and Wolff, JANature, 352, 815-818, (1991). Such methods are known in the art and can be readily adapted for use with the compositions and methods described herein. In certain cases, the methods will be modified to function specifically with large DNA molecules. Additionally, these methods can be used to target specific diseases and cell populations by using the targeting properties of the carriers.

[0024] a) Nucleic Acid-Based Delivery Systems A transcription vector can be any nucleotide construct (e.g., a plasmid) used to deliver genes into cells or used as part of a general strategy for delivering genes, e.g., as part of a recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).

[0025] As used herein, a plasmid or viral vector is a vehicle that transports the disclosed nucleic acid, such as PTEN-Long, into cells without degradation and contains a promoter that results in the expression of genes in the cells to which the nucleic acid is delivered. Viral vectors are, for example, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, polioviruses, AIDS viruses, neurotrophic viruses, Sindbis viruses, and other RNA viruses, including those viruses with HIV backbones. Any virus family that shares the properties of these viruses that make them suitable for use as vectors is also preferred. Retroviruses include mouse Maloney leukemia virus, MMLV, and retroviruses that express the preferred properties of MMLV as vectors. Retroviral vectors can carry larger gene payloads, i.e., transgenes or marker genes, than other viral vectors, and for this reason are commonly used vectors. However, they are not useful in non-proliferating cells. Adenoviral vectors are relatively stable and easy to handle, have high titers, can be delivered in aerosol formulations, and can transfect non-dividing cells. Pox viral vectors are large, have several sites for inserting genes, are heat stable and can be stored at room temperature. A preferred embodiment is a viral vector that has been genetically engineered to suppress the immune response of the host organism elicited by viral antigens. A preferred vector of this type will carry the coding region for interleukin 8 or 10.

[0026] Viral vectors can have a higher processivity (gene transfer ability) than chemical or physical methods for introducing genes into cells. In general, viral vectors contain nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and a promoter for controlling transcription and replication of the viral genome. When engineered as a vector, the virus generally has one or more of the early genes removed, and a gene or gene / promoter cassette is inserted into the viral genome in place of the removed viral DNA. This type of construct can carry up to about 8 kb of foreign genetic material. The essential functions of the removed early genes are generally provided by cell lines engineered to express the gene products of the early genes in trans.

[0027] (1) Oncolytic vector In one aspect, the viral vector can be oncolytic. Oncolytic viruses selectively infect and kill cancer cells, either through direct lysis of infected cells, secretion of toxins and / or stimulation of host anti-tumor immune responses. Oncolytic viruses can be any type of viral vector, including but not limited to adenovirus, HSV, reovirus, vaccinia virus, VSV and measles virus. For example, HSV-based oncolytic viruses can contain a null mutation or deletion of ICP34.5 (also referred to herein as neurovirulence factor gamma-34.5). This mutation results in an HSV vector that can no longer replicate in terminally differentiated, non-dividing cells, but retains the ability to infect and lyse rapidly dividing cells, such as cancer. In one aspect, the viral vector backbone of the disclosed modified viruses expressing PTEN-Long can be an oncolytic HSV vector, such as, for example, HSVQuick (HSVQ), HSV1716, HF10 (lacking UL56 protein expression) and OncoVex GM-CSF (lacking functional expression of ICP34.5 and GM-CSF). The oncolytic virus can also be an oncolytic adenovirus, including but not limited to, ONYX-015, H101 and KH901, an oncolytic reovirus, including but not limited to, Reolysin and RT3D, an oncolytic vaccinia virus, including but not limited to, GL-ONC1 and JX-594, and an oncolytic measles virus, including but not limited to, MV-NIS. The choice of viral vector may depend on the tropism of the viral backbone, the transgene size or availability to the scientist. Thus, disclosed herein are PTEN-Long expressing HSV viral vectors, in which the viral backbone is an oncolytic HSV-1 backbone that has been modified to target tumor cells and / or to place genes important for viral replication under the control of a tumor-specific promoter, such as, for example, HSVQuick (HSVQ).

[0028] (2) Herpes virus vector Herpesvirus is an animal virus belonging to the Herpesviridae family. Herpesvirus has a wide host range and can accept large transgene payloads. In addition, herpesvirus has the ability to establish and persist indefinitely latent infection in a subject.

[0029] Herpes virus vectors, such as herpes simplex virus-1 vectors (e.g., HSVQuick), can be attenuated to remove genes that are not essential for replication but affect host cell range. For example, the virus can be modified to contain deletions in the neurovirulence factor gamma-34.5 and / or the viral ribonucleotide reductase ICP6. Further modifications can include deletions or null mutations in ICP47 and / or GM-CSF expression.

[0030] The disclosed herpes virus vectors can also be replication-deficient. HSV encodes more than 80 viral gene products, about half of which are essential for viral replication. It is relatively easy to create replication-deficient mutants by deleting one of the essential genes and providing the gene product in trans by a complementing cell line. Although such mutant viruses are unable to replicate in non-complementing cells, they can retain cytotoxicity due to the fact that they still express many viral gene products. Viral replication is also easily disrupted by null mutations in immediate early genes that can be complemented in vitro in trans, allowing for the direct production of high-titer pure preparations of non-pathogenic vectors. Transduction with replication-deficient vectors results in latent infection in both neuronal and non-neuronal tissues, and the vectors are non-pathogenic, unable to reactivate, and long-lasting. For example, the HSVQuick viral vector (HSVQ) has been modified to restrict infection to non-neuronal tissues. In one aspect, a recombinant virus comprising a mammalian PTEN-Long gene is disclosed herein, where the recombinant virus is a herpes virus, such as, for example, a herpes simplex virus. In some aspects, a PTEN-Long expressing HSV viral vector is disclosed herein, where the viral backbone is an HSV-1 backbone modified to target tumor cells and / or place genes important for viral replication under the control of a tumor-specific promoter, such as, for example, HSVQuick (HSVQ). HSVQ viruses expressing the PTEN-Long transgene are referred to herein as HSV P10 or RAPTOR viruses (see, for example, FIG. 1A).

[0031] (3) Retroviral vector A retrovirus is an animal virus that belongs to the family Retroviridae and includes any type, subfamily, genus or tropism. Retroviral vectors are generally described by Verma, IM "Retroviral vectors for gene transfer".

[0032] Retroviruses are essentially packages that encase a nucleic acid cargo inside. The nucleic acid cargo carries with it a packaging signal to ensure that the replicated daughter molecules are efficiently packaged within the package coat. In addition to the packaging signal, there are a number of molecules required in cis for replication and packaging of the replicated virus. In general, retroviral genomes contain the gag, pol, and env genes, which are involved in making the protein membrane. The gag, pol, and env genes are generally replaced by the foreign DNA to be introduced into the target cell. Retroviral vectors typically contain a packaging signal for incorporation into the package coat, a sequence that signals the start of the gag transcription unit, elements required for reverse transcription including a primer binding site for binding to the tRNA primer for reverse transcription, terminal repeat sequences that induce RNA strand switching during DNA synthesis, a purine-rich sequence 5' to 3' LTR that acts as a priming site for synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTR that allow the insertion of the retroviral DNA state into the host genome. Removal of the gag, pol and env genes allows approximately 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed and packaged into new retroviral particles during replication. This amount of nucleic acid is sufficient for delivery of one to many genes depending on the size of each transcript. It is preferred to include either a positive or negative selectable marker in the insert along with other genes.

[0033] Because the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line that has been transfected or transformed with a retrovirus that contains the replication and packaging machinery but lacks a packaging signal. When a vector carrying a selection DNA is transfected into this cell line, the vector containing the gene of interest is replicated and packaged into new retroviral particles by the machinery provided in cis by the helper cells. The genome for the machinery is not packaged because it lacks the essential signals.

[0034] (4) Adenovirus Vector The construction of replication-deficient adenoviruses has been described (Berkner et al., J. Virology 61:1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987); Zhang, "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis," BioTechniques 15:868-872 (1993)). The advantage of using these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, because they can replicate inside the initially infected cell but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve highly efficient gene transfer following direct in vivo delivery to airway epithelia, hepatocytes, vascular endothelium, CNS tumors, and numerous other tissue sites. Recombinant adenoviruses achieve gene transfer by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, similar to wild-type or replication-deficient adenoviruses.

[0035] The viral vector can be an adenovirus-based viral vector that has had the E1 gene removed, and these virions are produced in a cell line, such as the human 293 cell line. In another preferred embodiment, both the E1 and E3 genes are removed from the adenoviral genome.

[0036] (5) Adeno-associated virus vector Another type of viral vector is based on the adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is non-pathogenic to humans. AAV-type vectors can transport approximately 4-5 kb, and wild-type AAV is known to stably insert into chromosome 19. Vectors that contain this site-specific integration property are preferred. A particularly preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen (San Francisco, CA), which can contain marker genes such as the herpes simplex virus thymidine kinase gene, HSV-tk and / or the gene encoding the green fluorescent protein GFP.

[0037] In another type of AAV virus, the AAV comprises a pair of inverted terminal repeats (ITRs) flanking at least one cassette containing a promoter that directs cell-specific expression operably linked to a heterologous gene, heterologous in this context refers to any nucleotide sequence or gene not derived from AAV or B19 parvovirus.

[0038] Generally, the coding regions of AAV and B19 are deleted, resulting in a safe, non-cytotoxic vector. The AAV ITRs or variants thereof confer infectivity and site-specific integration but not cytotoxicity, and the promoter drives cell-specific expression. U.S. Patent No. 6,261,834 is incorporated herein by reference for material related to AAV vectors.

[0039] Thus, the disclosed vectors provide DNA molecules that are capable of integrating into mammalian chromosomes without substantial toxicity. Genes inserted into viruses or retroviruses usually contain promoters and / or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence of DNA that functions when in a relatively fixed location with respect to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and can contain upstream elements and response elements.

[0040] (6) Large payload viral vectors Molecular genetic experiments with giant human herpesviruses have provided the means by which large heterologous DNA fragments can be cloned, propagated, and established in cells that can be infected by herpesviruses (Sun et al., Nature genetics 8:33-41, 1994; Cotter and Robertson, Curr Opin Mol Ther 5:633-644, 1999). These giant DNA viruses (Herpes Simplex Virus (HSV) and Epstein-Barr Virus (EBV)) have the ability to deliver human heterologous DNA fragments of >150kb to specific cells. EBV recombinants can maintain large fragments of DNA in infected B cells as episomal DNA. Individual clones of up to 330kb carrying human genomic inserts have been shown to be genetically stable. Maintenance of these episomes requires a specific EBV nuclear protein, EBNA1, which is constitutively expressed upon EBV infection. In addition, these vectors can be used for transfection and large amounts of protein can be produced transiently in vitro. The herpesvirus amplicon system has also been used to infect cells that are able to package DNA fragments of >220kb and stably maintain the DNA as episomes.

[0041] Other useful systems include, for example, replicating vaccinia virus vectors and host-restricted non-replicating vaccinia virus vectors. b) Non-nucleic acid based systems As disclosed herein, PTEN-Long can be delivered to target cells in a variety of ways. For example, the composition can be delivered via electroporation, or via lipofection, or via calcium phosphate precipitation. The delivery mechanism selected will depend in part on the type of cell targeted, and on whether the delivery is occurring, for example, in vivo or in vitro.

[0042] Thus, in addition to the disclosed PTEN-Long and HSVQ vectors containing the PTEN-Long transgene, the compositions can include lipids such as liposomes, for example, cationic liposomes (e.g., DOTMA, DOPE, DC cholesterol) or anionic liposomes. The liposomes can further include proteins to facilitate targeting of specific cells, if desired. Administration of the composition containing the compound and cationic liposomes can be administered to the bloodstream to the target organ or inhaled into the airways to target cells of the airways. For liposomes, see, for example, Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Felgner et al. Proc. Natl. Acad. Sci USA, 84:7413-7417 (1987); U.S. Patent No. 4,897,355. Additionally, the compound can be administered as a component of a microcapsule that can be targeted to a particular cell type, such as a macrophage, or at a site where diffusion of the compound from the microcapsule or delivery of the compound is designed for a particular rate or dosage.

[0043] In the above-described methods involving administration and uptake of exogenous DNA into cells of a subject (i.e., gene transduction or transfection), delivery of the composition to the cells can be via a variety of mechanisms. As an example, delivery can be via liposomes, using commercially available liposome formulations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Quagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to standard procedures in the art. In addition, the disclosed nucleic acids or vectors can be delivered in vivo by electroporation, technology for delivery available from Genetronics, Inc. (San Diego, CA), as well as by SONOPORATION machines (ImaRx Pharmaceutical Corp., Tuscon, AZ).

[0044] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al. al., Biochem. Pharmacol, 42:2062-2065, (1991)). These techniques can be used for a variety of other specific cell types. Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid-mediated drug targeting to colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-tropic tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutively or ligand-induced. These receptor clusters in clathrin-coated pits enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted and then either recycled to the cell surface, stored intracellularly or degraded in liposomes.Internalization pathways serve diverse functions such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and receptor level regulation. Many receptors follow more than one intracellular pathway depending on cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0045] The nucleic acid that is delivered to cells and is to be integrated into the host cell genome generally contains integration sequences.These sequences are often virus-related sequences, particularly when a virus-based system is used.These viral integration systems can also be integrated into the nucleic acid that is to be delivered using a non-nucleic acid-based delivery system, such as liposome, so that the nucleic acid that is included in the delivery system is integrated into the host genome.

[0046] Other common techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems generally rely on sequences flanking the nucleic acid to be expressed that have sufficient homology with a target sequence in the host cell genome for recombination to occur between the vector nucleic acid and the target nucleic acid, allowing the delivered nucleic acid to be integrated into the host genome. These systems and the methods required to promote homologous recombination are known to those skilled in the art.

[0047] c) In vivo / ex vivo As described above, the compositions can be provided in a pharma- ceutically acceptable carrier and delivered to the subject's cells in vivo and / or ex vivo by a variety of mechanisms known in the art (e.g., naked DNA uptake, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis, etc.).

[0048] If ex vivo methods are used, the cells or tissues can be removed and maintained outside the body according to standard protocols known in the art. The composition can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection, or proteoliposomes. The transduced cells can then be injected (e.g., in a pharma- ceutically acceptable carrier) or transplanted homogeneously back into the subject through standard methods for the cell or tissue type. Standard methods are known for transplanting or injecting a variety of cells into a subject.

[0049] Expression system The nucleic acid delivered to the cell generally includes an expression control system. For example, genes inserted into viral or retroviral systems usually include a promoter and / or enhancer to help control the expression of the desired gene product. A promoter is generally a sequence of DNA that functions when in a relatively fixed position relative to the transcription start site. A promoter includes core elements required for basic interaction of RNA polymerase and transcription factors, and can include upstream elements and response elements.

[0050] a) Viral promoters and enhancers Preferred promoters controlling transcription from vectors in mammalian host cells may be derived from a variety of sources, such as the genomes of viruses, such as polyoma, Simian Virus 40 (SV40), adenovirus, retrovirus, Hepatitis B virus, and most preferably, cytomegalovirus, or from heterologous mammalian promoters, such as the β-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273:113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenway, PJ et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or related species are also useful herein.

[0051] Enhancers generally refer to sequences of DNA that function at variable distances from the transcription start site and can be located either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78:993 (1981)) or 3' (Lusky, ML, et al., Mol. Cell Bio. 3:1108 (1983)) to the transcription unit. In addition, enhancers can be located within introns (Banerji, J Let al., Cell 33:729 (1983)) and within the coding sequence itself (Osborne, TF, et al., Mol. Cell Bio. 4:1293 (1984)). Enhancers are usually 10-300 bp in length and function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain responsive elements that mediate the regulation of transcription. Promoters may also contain responsive elements that mediate the regulation of transcription. Enhancers often determine the regulation of gene expression. Currently, many enhancer sequences are known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), but generally enhancers from eukaryotic cell viruses will be used for general expression. Preferred examples are the SV40 enhancer at the rear of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer at the rear of the replication origin and adenovirus enhancers.

[0052] Promoters and / or enhancers may be specifically activated, either by light or by specific chemical events that trigger their function. The system can be controlled by agents such as tetracycline and dexamethasone. There are also methods to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0053] In some embodiments, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In some constructs, the promoter and / or enhancer region is active in all types of eukaryotic cells, even if it is only expressed in a certain type of cell at a certain time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are the SV40 promoter, the cytomegalovirus (full-length promoter) and the retroviral vector LTR. In some aspects, the constitutive promoter is a promoter expressed in a viral backbone, such as the immediate early gene promoter for HSV. Thus, in one aspect, a recombinant virus is disclosed herein that includes a mammalian PTEN-Long gene, which is operably linked to a constitutive promoter, such as, for example, the HSV IE4 / 5 promoter.

[0054] In some embodiments, the promoter can be a tissue-specific or inducible promoter to restrict expression to a particular tissue when certain conditions are met or when the supervising physician wishes to start and / or stop expression. Inducible promoters can include any inducible promoter known in the art, including but not limited to tetracycline-inducible transgenic systems (tetracycline transactivator (tTA) or "Tet-Off" and reverse tetracycline transactivator (rtTA) or "Tet-On") and Cre-lox. Tissue-specific promoters are promoters whose expression is restricted to a particular tissue / cell type. Tissue-specific promoters are known in the art and can include any tissue-specific promoter known or commercially available. The selection of the tissue-specific promoter is based on the cell type in which expression is desired. Thus, in one aspect, a recombinant virus is disclosed herein that includes a mammalian PTEN-Long gene, the PTEN-Long gene operably linked to a tissue-specific or inducible promoter.

[0055] Any specific control element can be cloned into a construct expression vector and used that has been shown to be selectively expressed in a particular cell type, such as melanoma cells. The promoter of the glial fibrillary acetic protein (GFAP) has been used to selectively express genes in cells of glial origin.

[0056] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription, which affects the expression of the mRNA. These regions are transcribed as polyadenylation segments in the untranslated portion of the mRNA encoding the tissue factor protein. The 3' untranslated region also contains a transcription termination site. It is preferred that the transcription unit also contains a polyadenylation region. One advantage of this region is that it increases the likelihood that the transcription unit will be processed and transported like an mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that a homologous polyadenylation signal is used in the transgene construct. In some transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcription unit contains other standard sequences, either alone or in combination with the above sequences, to improve expression from the construct or its stability.

[0057] b) Marker Viral vectors may contain nucleic acid sequences encoding marker products that are used to determine whether the gene has been delivered to a cell and is expressed after delivery. Preferred marker genes are the E. coli lacZ gene, which encodes β-galactosidase, and green fluorescent protein.

[0058] In some embodiments, the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully introduced into mammalian host cells, the transformed mammalian host cells can survive when placed under selective pressure. There are two different categories of selection regimes that are widely used. The first category is based on the metabolism of the cells and the use of mutant cell lines that lack the ability to grow independent of supplemented media. Two examples are CHO DHFR cells and mouse LTK cells. These cells lack the ability to grow without the addition of nutrients such as thymidine or hypoxanthine. These cells lack certain genes necessary for a complete nucleotide synthesis pathway and therefore cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the corresponding genes, thereby altering their growth requirements. Individual cells that have not been transformed with the DHFR or TK genes cannot survive in unsupplemented medium.

[0059] The second category is dominant selection, which refers to selection schemes used with any cell type and does not require the use of mutant cell lines. These schemes generally use a drug to stop the growth of the host cells. Those cells with the new gene express a protein that confers drug resistance and can survive the selection. Examples of such dominant selection use the drugs neomycin (Southern P. and Berg, P., J. Molec. Appl. Genet. 1:327 (1982)), mycophenolic acid (Mulligan, RC and Berg, P. Science 209:1422 (1980)) or hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5:410-413 (1985)). Those three examples use bacterial genes under eukaryotic control to confer resistance to the appropriate drugs G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

[0060] Sequence similarity In the present specification, the use of the words homology and identity is understood to mean the same thing as similarity.Thus, for example, when the use of the word homology is used between two non-natural sequences, it is understood that this does not necessarily indicate the evolutionary relationship between these two sequences, but rather focuses on the similarity or relatedness between their nucleic acid sequences.Many of the methods for determining the homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity, regardless of whether they are evolutionarily related or not.

[0061] It is understood that one way to determine any known variants and derivatives or possible variants of the genes and proteins disclosed herein is by defining the variants and derivatives in terms of homology to a particular known sequence. For example, SEQ ID NO: 1 specifies the particular sequence of the PTEN-Long gene. Specifically disclosed are variants of these and other genes and proteins disclosed herein that have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent homology to the described sequence. Those skilled in the art will readily understand how to determine the homology of two nucleic acids, such as two proteins or genes. For example, homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0062] In general, it is understood that one way to determine any known variants and derivatives or possible variants of the genes and proteins disclosed herein is by defining the variants and derivatives in terms of their homology to a particular known sequence. The identity of the particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of the genes and proteins disclosed herein typically have at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent homology to the described or natural sequence. Those skilled in the art will readily understand how to determine the homology of two nucleic acids, such as two proteins or genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0063] Other methods for calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0064] It is understood that any of the methods may generally be used, and that in certain cases the results of these various methods may differ, but one of skill in the art will understand that if identity is found by at least one of these methods, the sequence will be said to have the stated identity, and will be disclosed herein.

[0065] For example, as used herein, a sequence described as having a particular percent homology to another sequence refers to a sequence having the described homology as calculated by any one or more of the above-mentioned methods. For example, if a first sequence is calculated to have 80 percent homology to a second sequence using the Zuker method, then the first sequence has 80 percent homology to the second sequence as defined herein, even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other methods. As another example, if a first sequence is calculated to have 80 percent homology to a second sequence using both the Zuker and Pearson-Lipman algorithms, the first sequence has 80 percent homology to the second sequence as defined herein, even if the first sequence does not have 80 percent homology to the second sequence as calculated by either the Smith-Waterman, Needleman-Wunsch, Jaeger, or other algorithms. As yet another example, if a first sequence is calculated to have 80 percent homology to a second sequence using each of the algorithms, the first sequence has 80 percent homology to the second sequence as defined herein (although in practice, different algorithms will often result in different calculated homology percentages).

[0066] Hybridization / Selective Hybridization The term hybridization generally refers to a sequence-driven interaction between at least two nucleic acid molecules, such as a primer or probe and a gene. A sequence-driven interaction refers to an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide-specific manner. For example, G interacting with C or A interacting with T is a sequence-driven interaction. Generally, a sequence-driven interaction occurs on the Watson-Crick or Hoogsteen face of a nucleotide. The hybridization of two nucleic acids is influenced by many conditions and parameters known to those skilled in the art. For example, the salt concentration, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.

[0067] Parameters for selective hybridization between two nucleic acid molecules are known to those of skill in the art. For example, in some embodiments, selective hybridization conditions can be defined as stringent hybridization conditions. For example, the stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps. For example, hybridization conditions that achieve selective hybridization may include hybridization in a high ionic strength solution (6X SSC or 6X SSPE) at a temperature of about 12-25°C below Tm (the melting temperature at which half of the molecules dissociate from their hybridization partner), followed by washing at a combination of temperature and salt concentration selected such that the washing temperature is about 5°C to about 20°C below Tm. Temperature and salt conditions are easily determined empirically in preliminary experiments in which samples of reference DNA immobilized on a filter were hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringency. Hybridization temperatures are generally higher for DNA-RNA and RNA-RNA hybridizations. Conditions can be used to achieve stringency as described above and as known in the art. Preferred stringent hybridization conditions for DNA. DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE, followed by washing at 68°C. Hybridization and washing stringency can be reduced, if desired, as the desired degree of complementarity decreases, and also depending on the GC or AT richness of any region where variability is sought. Similarly, hybridization and washing stringency can be increased, if desired, as the desired homology increases, and also depending on the GC or AT richness of any region where high homology is desired, all as known in the art.

[0068] Another way to define selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to another nucleic acid. For example, in some embodiments, selective hybridization conditions will be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non-limiting primer is in excess, for example, 10 or 100 or 1000 times. This type of assay is used when both the limiting primer and the non-limiting primer are in excess, for example, at their k d or only one of the nucleic acid molecules is 10-fold, 100-fold, or 1000-fold less than their k d The process can be carried out under conditions exceeding 1000 .mu.m.

[0069] Another way to define selective hybridization is to look at the percentage of primers that undergo enzymatic manipulation under conditions where hybridization is necessary to promote the desired enzymatic manipulation. For example, in some embodiments, selective hybridization conditions will be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primers undergo enzymatic manipulation under conditions that promote enzymatic manipulation. For example, if the enzymatic manipulation is DNA extension, selective hybridization conditions would be those where at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended. Preferred conditions also include those conditions suggested by the manufacturer or indicated in the art as being suitable for the enzyme to perform the manipulation.

[0070] It is understood that, like homology, there are various methods disclosed herein for determining the level of hybridization between two nucleic acid molecules.It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, and unless otherwise indicated, it will be sufficient to meet the parameters of any of the methods.For example, 80% hybridization is required, and only if hybridization occurs within the required parameters in any one of these methods will it be considered disclosed herein.

[0071] It is understood that one of skill in the art would understand that if a composition or method, either collectively or individually, meets any one of these criteria for determining hybridization, it is a composition or method disclosed herein.

[0072] nucleic acid There are various molecules disclosed herein that are nucleic acid-based, including, for example, the nucleic acid encoding PTEN-Long or fragments thereof and various functional nucleic acids. The disclosed nucleic acids are, for example, composed of nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. For example, when a vector is expressed in a cell, it is understood that the expressed mRNA generally consists of A, C, G, and U. Similarly, for example, when an antisense molecule is introduced into a cell or cellular environment, for example, through exogenous delivery, it is understood that it is beneficial for the antisense molecule to consist of nucleotide analogs that reduce degradation of the antisense molecule in the cellular environment.

[0073] a) Nucleotides and related molecules A nucleotide is a molecule that includes a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked through their phosphate and sugar moieties forming an internucleoside linkage. The base moiety of a nucleotide can be adenine-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is ribose or deoxyribose. The phosphate moiety of a nucleotide is a pentavalent phosphate. Non-limiting examples of nucleotides would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). There are many varieties of these types of molecules available in the art and can be used herein.

[0074] A nucleotide analog is a nucleotide that contains some type of modification in either the base, sugar, or phosphate moiety. Modifications to nucleotides are known in the art and include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-aminoadenine, as well as modifications in the sugar or phosphate moieties. There are many different types of these molecules available in the art and can be used herein.

[0075] Nucleotide substitutes are molecules that have similar functionality to nucleotides, but do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but bind together through a moiety other than the phosphate moiety. Nucleotide substitutes can conform to a double helix type structure when interacting with an appropriate target nucleic acid. There are many different of these types of molecules available in the art and are available herein.

[0076] Other types of molecules (conjugates) can be attached to nucleotides or nucleotide analogs, for example to enhance cellular uptake. Conjugates can be chemically attached to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties, such as cholesterol moieties. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556). There are many different types of these molecules available in the art and are available herein.

[0077] A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, N1, and C6 positions of a purine base nucleotide, nucleotide analog, or nucleotide substitute, and the C2, N3, and C4 positions of a pyrimidine base nucleotide, nucleotide analog, or nucleotide substitute.

[0078] A Hoogsteen interaction is an interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analogue exposed in the major groove of double-stranded DNA. The Hoogsteen face contains the reactive groups (NH2 or O) at the N7 and C6 positions of purine nucleotides.

[0079] b) Array There are various sequences related to the protein molecules involved in the signal transduction pathways disclosed herein, such as PTEN-Long or any of the nucleic acids disclosed herein to make PTEN-Long, all of which are encoded by or are nucleic acids. Sequences for human and other analogs of these genes, and alleles, and splice variants, and other types of variants of these genes, are available in various protein and gene databases, including Genbank. Those skilled in the art will understand how to resolve sequence discrepancies and differences, and how to adapt compositions and methods for a particular sequence for other related sequences. Primers and / or probes can be designed for any sequence given the information disclosed herein and are known in the art.

[0080] Pharmaceutical Carriers / Drug Delivery As mentioned above, the composition can also be administered in vivo in a pharma- ceutically acceptable carrier. By "pharmaceutical acceptable" is meant a biologically or otherwise desirable substance, i.e., the substance may be administered to a subject together with the nucleic acid or vector without causing any undesired biological effects or interacting in a detrimental manner with any of the other components of the pharmaceutical composition in which the substance is included. The carrier would naturally be selected to minimize degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be known to one of skill in the art.

[0081] The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, externally, topically, etc., including local intranasal administration or administration by inhalation. As used herein, "local intranasal administration" refers to delivery of the composition to the nose and nasal cavity through one or both nostrils, and can include delivery by spray or droplet mechanism or delivery via aerosolization of the nucleic acid or vector. Administration of the composition by inhalation can be through the nose or mouth via delivery by spray or droplet mechanism. Delivery can also be directly to any area of ​​the respiratory system (e.g., lungs) via intubation. The exact amount of the composition required will vary from subject to subject, depending on the subject's race, age, weight, and general condition, the severity of the allergic disease being treated, the particular nucleic acid or vector used, the mode of administration of the composition, etc. Thus, it is not possible to specify exact amounts for all compositions. However, appropriate amounts can be determined by one of ordinary skill in the art using only routine experimentation in light of the teachings herein.

[0082] Parenteral administration of compositions, when used, is generally characterized by injection.Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, solids suitable for dissolving or suspending in liquid before injection, or emulsions.The most recent improved method for parenteral administration involves the use of slow-release or sustained-release systems to maintain a constant dosage.See, for example, U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0083] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al. al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drug targeting to colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-tropic tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutively or ligand-induced. These receptor clusters in clathrin-coated pits enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted and then either recycled to the cell surface, stored intracellularly or degraded in liposomes.Internalization pathways serve diverse functions such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and receptor level regulation. Many receptors follow more than one intracellular pathway depending on cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0084] a) Pharmaceutically acceptable carrier The compositions containing the antibodies can be used therapeutically in combination with a pharma- ceutically acceptable carrier.

[0085] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995. In general, an appropriate amount of a pharma- ceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharma- ceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Further, carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing an antibody, the matrices being in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to one skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the composition to be administered.

[0086] Pharmaceutical carriers are known to those skilled in the art. These are most commonly considered to be standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0087] Pharmaceutical compositions may include, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. Pharmaceutical compositions may also include one or more active ingredients, such as antibacterial agents, anti-inflammatory agents, anesthetics, etc.

[0088] The pharmaceutical compositions may be administered in a number of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example, by intravenous drip, subcutaneous injection, intraperitoneal injection, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0089] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solutions are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases.

[0090] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0091] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets.Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0092] Some of the compositions may potentially be administered as pharma- ceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric, hydrobromic, perchloric, nitric, thiocyanic, sulfuric, and phosphoric acid, and organic acids such as formic, acetic, propionic, glycolic, lactic, pyruvic, oxalic, malonic, succinic, maleic, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkylamines, and arylamines, and substituted ethanolamines.

[0093] b) Therapeutic use The effective amount and schedule for administering the composition may be determined empirically, and making such a determination is within the skill of one of ordinary skill in the art. The dosage range of administration of the composition is a dosage large enough to produce the desired effect of affecting the symptoms of the disease. The dosage should not be so large as to cause adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, etc. In general, the dosage will vary according to the age, condition, sex, and extent of the disease of the patient, the route of administration, or whether other drugs are included in the regimen, and can be determined by one of ordinary skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. The dosage can be varied and can be administered in one or more doses administered daily, for one or several days. Guidance can be found in the literature regarding the appropriate dosage for a given class of pharmaceutical product. For example, guidance in selecting an appropriate dose for an antibody can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. Typical daily doses of antibodies for single use can range from about 1 μg up to 100 mg or more per kg of body weight per day, based on the factors listed above.

[0094] C. Methods of Treating Cancer It is understood and contemplated herein that the disclosed PTEN-Long expressing recombinant virus is effective for the treatment of cancer.Therefore, in one aspect, disclosed herein is a method for treating cancer in a subject, comprising administering to the subject any of the PTEN-Long expressing viruses disclosed herein.For example, disclosed herein is a method for treating cancer in a subject, comprising administering to the subject a recombinant virus comprising mammalian PTEN-Long gene (such as an oncolytic HSV virus vector comprising PTEN-Long transgene, for example, HSV-P10).

[0095] The PTEN-Long expressing viruses disclosed herein can be used to treat any disease in which uncontrolled cell proliferation occurs, such as cancer. A non-limiting list of different types of cancer includes: lymphoma (Hodgkin's and non-Hodgkin's), leukemia, carcinoma, solid tissue cancer, squamous cell carcinoma, adenocarcinoma, sarcoma, glioma, high-grade glioma, blastoma, neuroblastoma, plasmacytoma, histiocytoma, melanoma, adenoma, anoxic tumor, myeloma, AIDS-related lymphoma or sarcoma, metastatic cancer, or cancer in general.

[0096] A representative, non-limiting list of cancers for which the disclosed PTEN-Long expressing viruses can be used to treat includes lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer including small cell lung cancer and non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the oral cavity, throat, larynx and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer and epithelial cancer, renal cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck carcinoma, colon cancer, hematopoietic cancer, testicular cancer, colon cancer, and rectal cancer.

[0097] The recombinant viruses expressing mammalian PTEN-Long disclosed herein may also be used to treat precancerous conditions such as cervical dysplasia and anal dysplasia, other dysplasias, highly dysplastic epithelia, hyperplasias, atypical hyperplasias and neoplasias.

[0098] In one aspect, the disclosed PTEN-Long expressing viruses can be administered therapeutically to inhibit, reduce or treat cancer already present in a subject. However, it is also contemplated herein that the PTEN-Long expressing viruses can be administered prophylactically to inhibit or reduce metastasis. Thus, in one aspect, disclosed herein is a method of inhibiting or predicting metastasis of cancer in a subject, comprising administering to the subject a recombinant virus expressing a mammalian PTEN-Long gene.

[0099] As disclosed herein, the ability to treat, inhibit, and / or reduce cancer is due, in part, to the activation of NK cells and CD8 + This may be a result of increased infiltration of immune cells such as T cells. Generally, CD8 T cells and NK cells will inhibit the site of infection and / or cancer from infiltrating. Cancer cells often use PD1-PD-L1 interaction as a checkpoint to inhibit the infiltration of NK cells and CD8 T cells. PD1 is expressed on T cells, and upon binding to its ligand PD-L1, a signaling pathway is triggered to shut down PD1, which inhibits proliferation. Cancer cells increase PD-L1 to avoid T cell and NK cell mediated killing. The disclosed PTEN-Long expressing recombinant virus inhibits the expression of PD-L1. By inhibiting PD-L1, the PD1-PD-L1 checkpoint is inhibited, allowing NK cells and CD8 T cells to infiltrate and kill the cancer. Thus, in one aspect, disclosed herein is a method of increasing the number of infiltrating CD8 T cells and / or infiltrating NK cells at the site of cancer in a subject, comprising administering to the subject any of the PTEN-Long expressing recombinant viruses disclosed herein. For example, in one aspect, disclosed herein is a method of increasing the number of infiltrating CD8 T cells and / or infiltrating NK cells at a site of cancer in a subject comprising administering to the subject a recombinant virus comprising a mammalian PTEN-Long gene (such as an oncolytic HSV viral vector, e.g., HSV-P10, comprising a PTEN-Long transgene).

[0100] [Example] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and evaluate the compounds, compositions, articles, devices and / or methods claimed herein, are intended to be purely illustrative, and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in °C or is ambient temperature, and pressure is at or near atmospheric pressure.

[0101] Example 1 Oncolytic HSV-1 vectors were generated using the HSVQuik system. This system uses the G207 viral backbone, which is an F strain HSV-1 doubly deleted for the neurovirulence factor gamma-34.5 and the viral ribonucleotide reductase ICP6 (Figure 1A). The virus conditionally replicates in tumor cells of non-neuronal origin, is defective in protein kinase R signaling, and produces its own ribonucleotide reductase during division. The PTEN-L transgene contains an ATG start site 519 bp upstream of the canonical PTEN start site (SEQ ID NO:1). This second start site is mutated in the transgene, eliminating the canonical PTEN by mutating its second ATG start site to ATA using site-directed mutagenesis. This mutation promotes expression of the entire PTEN-L gene and prevents expression of the short intracellular form of PTEN. The mutated PTEN-L transgene was cloned into the G207 viral backbone according to the HSVquik protocol.

[0102] HSV-P10 was amplified and purified using a standard sucrose purification protocol. Ten to twenty 150 mm plates of 70% confluent Vero cells were infected at a low MOI in a minimal volume of 2%-FBS DMEM. The infected plates were transferred to 34C in a humidified incubator containing 5% CO2 + air. When the majority of the cells on the plate began to round (usually on day 3-4), the cells and medium were collected by scraping. The cells were pelleted by centrifugation and the supernatant was transferred to a 50 ml conical tube for separation. All cells were resuspended together in 20-30 ml of supernatant, and the cells and medium were flash frozen in liquid nitrogen and stored at -80C. The frozen cells were thawed to 4C in a 37C water bath and sonicated to release the viral particles from the intact cells. The cell debris was pelleted again by centrifugation and the medium containing the viral particles was combined with the separated medium without the cells. Viral particles were pelleted by ultracentrifugation and resuspended in sterile saline. The resuspended virus was layered on top of a 30% sucrose layer and the separated layers were ultracentrifuged again to purify only the viral particles. The pelleted virus was resuspended in a minimal volume of 20% glycerol in sterile saline and stored at -80C in 20 μl aliquots.

[0103] To test the in vitro and in vivo efficacy of PTEN-L expressing virus (HSV-P10 also called RAPTOR), U87 glioblastoma cells were infected with HSV-P10 (i.e., RAPTOR) at a multiplicity of infection of 1, and the expression of PTEN-Long was measured at various time points (Figure 1B). PTEN-Long expression is observed within 4 hours after infection. Next, to compare the effect of PTEN-Long on the phosphorylation of AKT, DB7 cells were infected with cell lysates from uninfected cells (NV) or cells infected with HSVQ vector only (Q) or cells infected with HSV-P10 (i.e., PTEN-Long expressing HSVQ) (R). Here, DB7 cells infected with HSVQ-P10 expressed PTEN-Long and had reduced AKT phosphorylation compared to HSVQ infected cells.

[0104] PTEN is commonly lost in breast cancer brain metastases (BCBM). To confirm this, representative hematoxylin and eosin (H&E) and PTEN staining from sections of human breast cancer brain metastases (top, 40x) and FVB / N mice bearing intracranial DB7 (mouse breast cancer cells) (bottom, 20x) are shown. The staining confirms the marked loss of PTEN in BCBM cells.

[0105] To demonstrate that the RAPTOR virus (i.e., HSV-P10) has the same cell tropism and growth ability as the HSVQ virus, LN229 cells (a glioblastoma cell line), U251-T3 cells (glioma cells), and U87ΔEGFR cells (glioma cells) were infected with either HSVQ or HSV-P10 at a multiplicity of infection (MOI) of 0.1. Cells were observed for viability and infection. As shown in Figure 3, both HSVQ and HSV-P10 were able to infect all three cell types and completely lyse LN229 and U251-T3 cells.

[0106] Next, MTT assays were performed in DB7 cells (Figure 4A), MDA-MB-468 cells (human breast cancer) (Figure 4B), LN229 cells (Figure 4C), and U87ΔEGFR cells (Figure 4D), and it was observed that PTEN-Long expression did not inhibit HSV-P10 cell killing in multiple cell lines. In each cellular scenario, the rate of lysis of infected cells was identical. The differences in magnitude were determined to be due to variations in the viral load administered to the cells, rather than true differences in viral infection, as evidenced by any differences that were fully maintained over the 96-hour culture.

[0107] To determine how expression of PTEN-Long affected in vivo tumor burden, immunocompetent FVB / N mice bearing DB7-dsRed tumors and nude mice bearing DB7 were infected with saline control or HSVQ viral backbone or PTEN-Long-expressing HSV-P10 (i.e., RAPTOR) (Figure 5). By day 40 post-infection, 90% of mock-infected animals and 50% of HSVQ-infected animals had died. In contrast, at day 100 post-infection, more than 90% of HSV-P10 animals were alive. In immunocompromised nude mice, all tumor-bearing mice died by day 15 in the HSVQ and saline groups and by day 19 in the RAPTOR-infected group. This indicates that HSV-P10 promotes survival in mice bearing breast cancer brain metastases.

[0108] To determine the mechanism of protection in HSV-P10-infected mice, infected tumor cells were removed and assayed for the presence of infiltrating macrophages, NK cells, and CD8 T cells (Figure 6). Staining for CD11b+ and F4 / 80+ revealed that macrophages (CD45 hi ) against CD11b + F4 / 80 + Microglia (CD45 lo-int It was observed that the ratio of IL-1, IL-2, and IL-3 decreased in HSQV and HSV-P10 cells, with relatively more macrophages present in HSV-P10-infected cells (Figure 6A). When measured to determine the activation state of microglia and macrophages, it was further observed that the percentage of activated cells was also increased in HSVQ and ISV-P10-infected cells, with more pronounced activation in the HSV-P10 population (Figure 6B). Measurements of infiltrating NK cells (Figure 6C) and infiltrating CD8 T cells (Figure 6D) showed that while HSVQ had no statistical increase in the percentage of infiltrating NK cells or infiltrating CD8 T cells, RAPTOR-infected cells (i.e., HSV-P10-infected cells) showed a significant increase in the percentage of NK cells and CD8 T cells compared to HSVQ and mock infection.

[0109] Hypothesizing that the increase in infiltrating NK cells and CD8 T cells could be due to a decrease in PD-L1 expression, which may normally function to signal through PD1 to inhibit proliferation of NK cells and CD8 T cells, cells were tested for PD-L1 expression (Figure 7). Mock-infected DB7 cells showed barely detectable levels of PD-L1, as would be expected. Also as expected, HSVQ-infected cells showed virus-induced PD-L1 expression 24 hours post-infection. However, cells infected with PTEN-Long-expressing HSV-P10 (i.e., RAPTOR) showed a significant reduction in PD-L1 expression. This reduction in PD-L1 is a direct result of PTEN-Long expression, as the only difference between the HSVQ backbone and HSV-P10 is the PTEN-Long transgene.

[0110] In addition, HSV-P10-infected animals were able to produce anti-DB-7 antibodies (Fig. 8 ). conclusion HSV-P10 virus is a herpes simplex virus type 1 with a double deleted gamma-34.5 gene and an insertion mutation in viral ICP6 containing GFP and PTEN-Long expression vectors. HSV-P10: expresses PTEN-L in infected cells, secretes PTEN-L from infected cells, and also reduces Akt phosphorylation in infected cells. HSV-P10 shows equivalent replication in glioma cells compared to HSVQ, and kills glioma and breast cancer cell lines with the same efficacy as HSVQ.

[0111] However, HSV-P10 promotes survival in immune-competent mice bearing DB7 breast cancer brain metastases. This is in part a consequence of increased recruitment of activated immune effector cells in the DB7 tumor-bearing brain hemisphere that are no longer suppressed by expression of PD-L1, as shown herein that HSV-P10 reduces PD-L1 expression in infected and adjacent uninfected cells. In addition, HSV-P10 induces robust antigen spreading against DB7 cancer antigens.

[0112] HSV-P10 acts in three major ways to induce tumor cell death. First, the virus can selectively replicate in and destroy infected tumor cells. Second, HSV-P10 induces secretion of the tumor suppressor PTEN-Long, which can act to locally slow the growth of uninfected tumor cells and also act as a damage-associated molecular pattern molecule to recruit the innate immune system to the site of infection. Third, HSV-P10 acts as a "cancer vaccine" that induces an adaptive immune response against tumor cells by exposing non-self tumor peptides to the immune system via tumor cell lysis and immune cell recruitment to the site of infection (Figure 9).

[0113] E.References 1 Cerami, E., Demir, E., Schultz, N., Taylor, B. S., & Sander, C. (2010). “Automated network analysis identifies core pathways in glioblastoma.” Plos One, 5(2), e8918.

[0114] 2 Hopkins, B. D., Fine, B., Steinbach, N., Dendy, M., Rapp, Z., Shaw, J., Parsons, R. (2013). “A secreted PTEN phosphatase that enters cells to alter signaling and survival.” Science (New York, NY), 341(6144), 399–402.

[0115] 3 Mineharu Y. Kamran N, Lowenstein PR, Castro MG. "Blockade of mTOR signaling via rapamycin combined with immunotherapy augments antiglioma cytotoxic and memory T-cell functions." Mol Cancer Ther. 2014;13(12):3024~3036.

[0116] 4 Terada, K., Wakimoto, H., Tyminski, E., Chiocca, E. Ak, & Saeki, Y. (2006). "Development of a rapid method to generate multiple oncolytic HSV vectors and their in vivo evaluation using syngeneic mouse tumor models." Gene Therapy, 13(8), 705~14.

[0117] 5 Zhang L, Zhang S, Yao J, et al. "Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth." Nature. 2015;527(7576):100~104.

[0118] F. Sequence Accession No.: 1 PTEN-Long coding region

Claims

1. A recombinant oncolytic herpes simplex virus (HSV) comprising a herpes simplex virus-1 (HSV-1) backbone and a mammalian PTEN-Long gene.

2. The recombinant virus of claim 1 , wherein the virus targets tumor cells.

3. 3. The recombinant virus of claim 1 or 2, wherein the virus has been modified to place a gene important for viral replication under the control of a tumor-specific promoter.

4. The recombinant virus of any one of claims 1 to 3, wherein the HSV-1 backbone is HSVQuik (HSVQ).

5. The recombinant virus of any one of claims 1 to 4, wherein the PTEN-Long gene is operably linked to a tissue-specific promoter.

6. The recombinant virus of any one of claims 1 to 4, wherein the PTEN-Long gene is operably linked to an inducible promoter.

7. The recombinant virus of any one of claims 1 to 4, wherein the PTEN-Long gene is operably linked to a constitutive promoter.

8. 8. The recombinant virus of claim 1 for use in a method of treating cancer in a subject comprising administering the recombinant virus to the subject.

9. The recombinant virus of any one of claims 1 to 7, for use in a method for inhibiting PD-L1 in cancer cells, comprising administering the recombinant virus to a subject.

10. 8. The recombinant virus of any one of claims 1 to 7, for use in a method of increasing the number of infiltrating CD8 T cells or natural killer cells at a site of cancer in a subject, comprising administering the recombinant virus to the subject.

11. The recombinant virus described in claim 8, wherein the cancer is a metastatic cancer.

12. 10. Use of a recombinant virus according to any one of claims 1 to 7 for the manufacture of a medicament for the treatment of cancer, wherein said treatment of cancer comprises administering to a subject a recombinant virus comprising a mammalian PTEN-Long gene.

13. 10. An ex vivo method for inhibiting PD-L1 in cancer cells, comprising contacting the cancer cells with a recombinant virus of any one of claims 1 to 7.

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