ONCOLYTIC VIRUS AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority benefit of U.S. Provisional Application No.

63/154,118, filed February 26, 2021, the disclosure of which is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A

TEXT FILE

[0002] A sequence listing is provided herewith as a text file, “AMUN-

N001WO_SEQ_LIST_ST25.txt,” created on February 11, 2022 and having a size of 66000 bytes. The contents of the text file are incorporated by reference herein in their entirety.

BACKGROUND

[0003] Recently there has been a tremendous interest in developing oncolytic viruses for cancer therapy and for improving immunotherapies. However, to date only one oncolytic virus, Herpes virus expressing GM-CSF, has been approved for clinical use in treating melanomas. Therefore, there is a great need for oncolytic viruses that can be used for cancer therapy and for improving immunotherapies.

SUMMARY

[0004] The present disclosure provides an oncolytic virus comprising a nucleic acid encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) and a soluble form of the TGF-B receptor-II (sTGF-BRII). The present disclosure also provides compositions comprising the oncolytic virus and treatment methods using the oncolytic virus. The treatment methods include local and/or systemic administration of the oncolytic virus for treating cancers, such as solid tumors.

[0005] In one aspect, the disclosure provides an oncolytic virus comprising a nucleic acid encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) and a soluble form of the TGF-B receptor-II (sTGF-BRII). In some embodiments, the oncolytic virus is an adenovirus, herpes virus, lentivirus, vaccinia virus, Reo virus, maraba virus, Newcastle disease virus, sendai virus, pox virus, poliovirus, myxoma virus, or retrovirus. In some embodiments, the oncolytic virus is an adenovirus. Some embodiments provide an oncolytic virus that preferentially replicates in cancer cells. In some embodiments, the expression of genes required for replication of the virus is under the control of a promoter active in the cancer cells, such as a telomerase reverse transcriptase (TERT) promoter. In some embodiments, the expression of sTGF-BRII is under the control of a cytomegalovirus (CMV) promoter. In some embodiments, the expression of GM-CSF is under the control of an adenoviral E1B promoter. In some embodiments, the virus comprises a capsid protein that specifically binds to a cancer cell that overexpresses an adenovirus receptor. In some embodiments, the cancer cell is a breast cancer cell, skin cancer cell, lung cancer cell, renal cancer cell, ovarian cancer cell, prostate cancer cell, pancreatic cancer cell, brain cancer cell, musculoskeletal cancer, astrocytoma cancer cell, cervical cancer cell, testicular cancer cell, hepatic cancer cell, lymphoma cancer cell, colon cancer cell, or bladder cancer cell. In some embodiments, the cancer cell is a breast cancer cell, such as a triple negative breast cancer cell. In some embodiments, the cancer cell is a skin cancer cell, such as a melanocyte. In some embodiments, the nucleic acid encoding the GM-CSF is codon-optimized to increase expression of the GM-CSF in a human subject. In some embodiments, the GM-CSF is a human GM-CSF. In some embodiments, the sTGF-BRII is fused to a heterologous protein. In some embodiments, the heterologous protein comprises a half-life-extending moiety, such as an immunoglobulin Fc region.

[0006] Another aspect of the disclosure is drawn to a pharmaceutical composition comprising: a therapeutically effective amount of the oncolytic virus described above and a pharmaceutically acceptable excipient, diluent, or carrier.

[0007] Still another aspect of the disclosure is directed to a method of treating cancer in a subject, the method comprising: administering a therapeutically effective amount of the oncolytic virus described above or the composition described above to the subject. In some embodiments of the method, the cancer is breast cancer, skin cancer, lung cancer, bladder cancer, renal cancer, astrocytoma, hepatic cancer, lymphoma, colon cancer, or head/neck cancer. In some embodiments, the administering comprises an intratumoral or intravenous administration. In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the method further comprises administering to the subject one or more of an immunotherapy, a chemotherapy, a surgical treatment, a radiation therapy, a hormone therapy, an anti -cancer small molecule, a growth factor inhibitor therapy, CAR-T therapy, or a cytokine therapy. In some embodiments, an immunotherapy is administered to the subject. In some embodiments, a chemotherapy is administered to the subject. In some embodiments, an immune checkpoint inhibitor is administered to the subject. In some embodiments, the immune checkpoint inhibitor comprises an inhibitor of PD-1, PD-L1, CTLA-4, LAG-3, BCH-3, BCH-4, or TIM-3. In some embodiments, the inhibitor is an antibody. [0008] Other features and advantages of the disclosure will be apparent from the following detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS [0009] Included in the drawings are the following figures: [0010] FIGS. 1A-1B. Schematics of adenoviral vectors based on Ad5 vector.

[0011] FIG. 1C. Virus replication assay to measure virus production using the listed adenoviral vectors.

[0012] FIGS. 2A-2B. Effect of adenovirus comprising the vectors depicted in FIG. 1 A on tumor volume and tumor weight in a mouse model for mammary tumor. [0013] FIGS. 3A-3C. Cytotoxicity of replicating viruses rAd.sT.GM, rAd.GM, rAd.sT, rAd.Null and replication-deficient Ad(El-) in mouse breast cancer cells 4T1 and in human breast cancer cells (MDA-MB-231 and MCF-7).

[0014] FIGS. 4A-4B. Concentration of TGFbRIIFc and hGM-CSF in virus infected cell cuture supernatant. [0015] FIGS. 5A-5B. sTGFbRIIFc expression in 4T1 tumors in mice on day 10 and day

25 post-infection.

[0016] FIGS. 5C-5D. GM-CSF expression in 4T1 tumors in mice on day 10 and day 25 post-infection.

DETAILED DESCRIPTION [0017] The present disclosure provides an oncolytic virus comprising a nucleic acid encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) and a soluble form of the TGF-B receptor-II (sTGF-BRII). The present disclosure provides compositions comprising the oncolytic virus and treatment methods using the oncolytic virus. The treatment methods include local and/or systemic administration of the oncolytic virus for treating cancers, such as solid tumors.

[0018] Before exemplary embodiments of the present invention are described, it is to be understood that this invention is not limited to particular embodiments described, 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, since the scope of the present invention will be limited only by the appended claims.

[0019] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials may now be described. Any and all publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0021] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an adenovirus virion” includes a plurality of such virions and reference to “the vector” includes reference to one or more vectors, “a mutation” refers to one or more mutations, and so forth.

[0022] It is further noted that the claims may be drafted to exclude any element which may be optional. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.

[0023] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent such publications may set out definitions of a term that conflicts with the explicit or implicit definition of the present disclosure, the definition of the present disclosure controls. [0024] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

DEFINITIONS

[0025] A "recombinant adenovirus vector" as used herein refers to an adenovirus vector comprising a polynucleotide sequence not of adenoviral origin ( i.e ., a polynucleotide heterologous to the adenovirus), typically a sequence of interest for expression in a cell. In general, the heterologous polynucleotide is flanked by at least one, and generally by two inverted terminal repeat sequences (ITRs). The term recombinant adenovirus vector encompasses both adenoviral vector particles and adenoviral vector plasmids.

[0026] An "adenovirus" or " adenoviral particle" or " adenovirus vector particle" refers to a viral particle composed of at least one adenovirus capsid protein (typically of all of the capsid proteins of a wild-type adenovirus) and an encapsidated polynucleotide adenovirus vector. If the particle comprises a heterologous polynucleotide {i.e., a polynucleotide other than a wild-type adenovirus genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as a "recombinant adenovirus vector particle" or simply a "rAd vector". Thus, production of rAd particle necessarily includes production of rAd vector, as such a vector contained within a rAd particle.

[0027] An "infectious" virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus. As used herein, an “infectious” virus or viral particle is one that can access a target cell, can infect a target cell, and can express a heterologous nucleic acid in a target cell. Thus, “infectivity” refers to the ability of a viral particle to access a target cell, infect a target cell, and express a heterologous nucleic acid in a target cell. Infectivity can refer to in vitro infectivity or in vivo infectivity. Assays for counting infectious viral particles are described in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles can be expressed as the number of viral genome (vg) copies. The ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as “transduction.” The ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay ( e.g ., where the virus comprises a nucleotide sequence encoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and/or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (ELISA). Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art.

[0028] The term "polynucleotide" or “nucleic acid” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single- stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a nucleic acid encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0029] A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.

[0030] "Recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0031] A "control element" or "control sequence" is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter. Reference to a gene promoter refers to both the promoter for the expression of the gene as found in nature as well as modified versions of the natural promoter, e.g, a shortened promoter, chimeric promoter, a mutated promoter, etc.

[0032] "Operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

[0033] An "expression vector" is a vector comprising a region which encodes a polypeptide of interest, and is used for effecting the expression of the protein in an intended target cell. An expression vector also comprises control elements operatively linked to the coding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[0034] "Heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, a recombinant adenovirus that includes a heterologous nucleic acid encoding a heterologous gene product is an adenovirus that includes a nucleic acid not normally included in a naturally occurring, wild-type adenovirus, and the encoded heterologous gene product is a gene product not normally encoded by a naturally occurring, wild-type adenovirus.

[0035] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: a) inhibiting the disease, i.e., arresting or slowing its development; and (b) relieving the disease, i.e., causing regression of the disease. One form of treatment includes prevention of occurrence of a disease or delay in onset of the disease and/or reduced severity of the disease after onset of the disease in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it. [0036] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals ( e.g ., horses, camels, etc.); mammalian farm animals (e.g., sheep, goats, cows); mammalian pets (dogs, cats, etc.); and rodents (e.g, mice, rats, etc.). In some cases, the individual is a human.

[0037] The term "pharmaceutically acceptable" refers to a non-toxic material which preferably does not interfere with the action of the active ingredient of the pharmaceutical composition. In particular, the term "pharmaceutically acceptable" means that the subject substance has been approved by a governmental regulatory agency for use in animals, and particularly humans, or in U.S. Pat. Pharmacopoeia, European Pharmacopoeia or other recognized pharmacopoeias for use in animals and in particular humans.

[0038] The term "cancer" as used herein refers to a cancer of any kind and origin including tumor-forming cells, blood cancers and/or transformed cells.

[0039] The term "cancer cell" includes cancer or tumor-forming cells, transformed cells or a cell that is susceptible to becoming a cancer or tumor-forming cell.

[0040] The term "recombinant oncolytic virus" refers to an engineered oncolytic virus, such as an adenovirus, generated in vitro using recombinant DNA technology and/or an oncolytic virus derived from such a recombined oncolytic virus (e.g, progeny virus).

[0041] The term "oncolytic" as used herein refers to a tumor-selective replicating virus that induces cell death in the infected cell, and/or tissue. Although normal or non-tumor cells may be infected, tumor cells are infected and lysed preferentially in comparison to the normal or non-tumor cells. For example, an oncolytic virus induces at least 5-fold, at least 6-fold, at least 10-fold, at least 15-fold, or at least 20-fold more cell death in a population of cancer cells compared to normal cells.

[0042] The term "normal tissue" as used herein refers to non-cancer tissue and/or tissue derived from a subject that is free of cancer of the particular tissue (e.g, when the tissue is pancreas, "normal tissue" can be derived from a subject that does not have pancreatic cancer). The term "normal cell of the same tissue type" as used herein refers to a cell or cells derived from such normal tissue.

[0043] The term "resistant cancer" or "chemotherapeutic resistant cancer" refers to a cancer that has decreased sensitivity to one or more chemotherapeutic drugs, for example by amplifying a gene that allows it to persist in the presence of the drug, for example by increasing expression of pumps that decrease amount of the drug inside a cancer cell, such as, MDR1. [0044] The term "antibody" as used herein is intended to include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals. The term "antibody fragment" as used herein is intended to include Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof and bi-specific antibody fragments. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bi-specific antibody fragments and other fragments can also be synthesized by recombinant techniques. Methods for making antibodies are well known in the art.

[0045] The term "nucleic acid" includes DNA and RNA and can be either double- stranded or single-stranded.

[0046] The term "sequence identity" as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e. g. , gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i. e., % identity=number of identical overlapping positions/total number of positions times). The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc Natl Acad Sci USA 872264-2268, modified as in Karlin and Altschul, 1993, Proc Natl Acad Sci U S A 90 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs. [0047] As used herein, "contemporaneous administration" and "administered contemporaneously" mean that two substances are administered to a subject such that they are both biologically active in the subject at the same time. The exact details of the administration will depend on the pharmacokinetics of the two substances in the presence of each other, and can include administering one substance within 24 hours of administration of the other, if the pharmacokinetics are suitable. In particular embodiments, two substances will be administered substantially simultaneously, i.e. within minutes of each other, or in a single composition that comprises both substances.

[0048] As used herein, the phrase "effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, in the context or treating a cancer, an effective amount is an amount that for example induces remission, reduces tumor burden, and/or prevents tumor spread or growth compared to the response obtained without administration of the oncolytic virus provided herein. Effective amounts may vary according to factors such as the disease state, age, sex, weight of the subject. The amount of an oncolytic virus herein that will correspond to such an amount will vary depending upon various factors, such as the pharmaceutical formulation, the route of administration, the type of cancer, the severity of the cancer, and the like, but can nevertheless be routinely determined by one skilled in the art.

[0049] The term "replication competent" refers to any viral vector that is not deficient in any gene function required for viral replication in specific cells or tissues. The vector must be capable of replicating and being packaged, but might replicate only conditionally in specific cells or tissues. Replication competent adenoviral vectors disclosed herein may be engineered to reduce or eliminate their ability to replicate in normal cells while retaining their ability to replicate efficiently in cancer cells.

RECOMBINANT ONCOLYTIC VIRUS

[0050] The present disclosure provides an oncolytic virus comprising a nucleic acid encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) and a soluble form of the transforming growth factor-beta (TGF-B) receptor-II (sTGF-BRII). This oncolytic virus may be used for treating cancer, such as solid tumors as well as metastatic cancer, in part by providing GM-CSF and sTGF-BRII in the blood stream of a subject receiving the oncolytic virus. In addition, the oncolytic virus may inhibit tumor metastasis.

[0051] The oncolytic virus may be an adenovirus, herpes virus, lentivirus (e.g., an integration deficient lentivirus), vaccinia virus, Reo virus, maraba virus, New Castle disease virus, sendai virus, pox virus, poliovirus, myxoma virus, or retrovirus. In certain aspects, the oncolytic virus may be a replication-competent oncolytic virus.

[0052] In certain aspects, the oncolytic virus is a vaccinia virus, such as an orthopox virus, non-limiting examples of which include Western Reserve, Wyeth, and Copenhagen strains, optionally modified to increase cancer selectivity. Such modifications include, but are not limited to non-functional thymidine kinase gene, non-functional vaccinia growth factor gene, and non-functional type 1 interferon-binding gene.

[0053] In another aspect, the oncolytic virus is a herpes simplex virus (HSV), such as

HSV1, HSV2 or HSV1716 strain. The HSV may include mutations that allow the virus to replicate in actively dividing cells, such as in cancer cells but which prevent significant replication in normal cells. Such mutations include disruption of the genes encoding ICP34.5 (i.e., g34.5), ICP6 and thymidine kinase.

[0054] In certain aspects, the oncolytic virus may be an adenovirus. The adenovirus may be a replication-competent adenovirus that can replicate in a host cell, such as, a mammalian cancer cell. The adenovirus may be of any of the 57 human adenovirus serotypes (HAdV-1 to 57). In one embodiment, the adenovirus is a subgroup B, for example Ad3, Ad5, Ad7, Adi 1, Adl4, Adl6, Ad21, Ad34 or Ad51. In some examples, the oncolytic adenovirus is a replication competent Ad5 serotype adenovirus or a hybrid serotype adenovirus comprising an Ad5 component. The adenovirus may be a wild-type strain or may be genetically modified to enhance tumor selectivity, for example by attenuating the ability of the virus to replicate within normal quiescent cells without affecting the ability of the virus to replicate in tumor cells or by increasing replication of the virus in cancer cells as compared to normal cells. Non-limiting examples of replication competent oncolytic adenoviruses that can be used to provide sTGF- BRII and GM-CSF expression in a subject, such as a human, include Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, ONYX-015, ColoAdl, H101 and AD5/3. Onyx-015 is a hybrid of virus serotype Ad2 and Ad5 with deletions in the E1B-55K and E3B regions to enhance cancer selectivity. HI 01 is a modified version of Onyx-015. ICOVIR-5 and ICOVIR-7 comprise an Rb- binding site deletion of El A and a replacement of the El A promoter by an E2F promoter. ColoAdl is a chimeric Addllp/Ad3 serotype. AD5/3 is a serotype 5/3 capsid-modified adenovirus (the Ad5 capsid protein knob is replaced with a knob domain from serotype 3). In some cases, the oncolytic adenovirus may be an adenoviral dl01/07 mutant that can replicate in a variety of cancer cells regardless of their genetic defects. The dl01/07 mutant adenovirus includes a mutant El A protein in which amino acids from position 4-25 and 111-123 are deleted. The mutant El A protein cannot bind with p300 or Rb proteins. Thus, dl01/07 is ineffective for S-phase progression in normal cells and cannot replicate in normal cells.

However, cancer cells are able to progress to S-phase, permitting virus replication in cancer cells. In other cases, the adenovirus may include a wild type El A protein that binds with p300 or Rb proteins. The expression of the El A gene may be placed under the control of human TERT promoter to provide for selective replication of the virus in human cancer cells. The recombinant adenovirus may include an active El A gene and an inactivated E1B 19 gene, an inactivated E1B 55 gene, or an inactivated E1B 19/E1B 55 gene. As used herein the term “inactivation” in the context of a gene means that the transcription and/or translation of the gene is reduced or absent, and thus the function of the protein encoded by the gene is reduced or undetectable. For example, the inactivated E1B 19 gene is a gene that cannot produce an active E1B 19 kDa protein due to mutation (substitution, addition, partial deletion or total deletion) of the gene. When E1B 19 is deleted, cell apoptosis can be increased, and when the E1B 55 gene is deleted, tumor cell specificity can be increased. The term deletion, used in connection with the viral genome sequence in the present disclosure, means complete deletion or partial deletion of a gene. The adenovirus of the present disclosure may include an active El A gene, an inactivated E1B 19/E1B 55 gene, and an inactivated E3 gene.

[0055] The cancer cell that is targeted by the disclosed oncolytic virus may be a breast cancer cell, skin cancer cell, lung cancer cell, renal cancer cell, ovarian cancer cell, prostate cancer cell, pancreatic cancer cell, brain cancer cell, astrocytoma cancer cell, hepatic cancer cell, lymphoma cancer cell, colon cancer cell, or bladder cancer cell. In certain embodiments, the cancer cell may be a human cancer cell. In particular embodiments, the breast cancer cell may be a triple negative breast cancer cell, the skin cancer cell may be a melanocyte.

[0056] The nucleic acid(s), encoding the sTGF-BRII and GM-CSF proteins, present in the oncolytic virus may be a DNA or RNA which may be single- or double-stranded depending upon the type of oncolytic virus. In certain aspects, the nucleic acid may be double-stranded DNA. In certain aspects, the nucleic acid(s) encoding the human sTGF-BRII and human GM- CSF may be a codon-optimized sequence(s), where the codons are codons from highly expressed human genes. The nucleic acids may be present in a single vector, such as a viral vector, that includes, at a minimum, a nucleic acid encoding sTGF-BRII, a nucleic acid encoding GM-CSF, and one or more promoters controlling expression of sTGF-BRII and GM-CSF, and 5' and 3' inverted terminal repeats (ITRs) flanking the promoter(s), the sTGF-BRII-encoding polynucleotide, and the GM-CSF-encoding polynucleotide. The vector may also include polynucleotides encoding proteins required for replication of the oncolytic virus. In certain embodiments, the viral vector only provides expression of the two therapeutic proteins, sTGF- BRII and GM-CSF, while in other embodiments, the viral vector can include additional nucleic acids encoding other therapeutic proteins or polynucleotides, such as RNA.

[0057] Nucleic acids encoding sTGF-BRII and GM-CSF can be inserted, e.g ., at any nonessential location in the oncolytic virus genome so long as the oncolytic virus remains replication-competent. In one embodiment, the oncolytic virus is an adenovirus with a heterologous nucleic acid comprising a sequence encoding sTGF-BRII inserted downstream of a heterologous promoter. In another embodiment, a heterologous nucleic acid comprising a sequence encoding GM-CSF is inserted in the E3 region of a replication-competent adenovirus backbone. The E3 region is nonessential for viral replication; however, the E3 proteins play a role in regulating host immune response. The replication-competent adenovirus can comprise a full or partial E3 deletion. For example, the replication-competent adenovirus can comprise deletions of one, two, three or more open reading frames (ORFs) in the E3 region, with the heterologous nucleic acid encoding GM-CSF inserted in its place. In one embodiment, the gpl9k and 6.7K genes are deleted and the heterologous nucleic acid encoding GM-CSF is inserted into a gpl9k/6.7K-deleted E3 region. In related aspects, the full E3 region is deleted from the replication-competent adenovirus backbone and the heterologous nucleic acid GM-CSF is inserted in place of the full E3 region.

[0058] The recombinant adenovirus of the present disclosure includes a promoter functional in animal cells, e.g ., mammalian cells, such as, human cells. Suitable promoters include promoters derived from mammalian virus and promoters derived from the genome of mammalian cells, such as CMV (Cytomegalovirus) promoter, a chicken beta actin promoter, human b-actin / CMV hybrid promoter, chicken b-actin / CMV hybrid promoter, CMV-actin- globin (CAG) hybrid promoter, Math Promoter, VGLUT3 promoter, parvalbumin promoter, calretinin promoter, calbindin 28k promoter, prestin promoter, a liver-specific or liver- preferential promoter, e.g., albumin promoter, U6 promoter, HI promoter, MLV (Murine Leukemia Virus) LTR (Long terminal repeat) promoter, adenovirus early promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothionine promoter, beta-actin promoter, human IL -2 promoter of gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human GM-CSF gene, inducible promoter, cancer cell-specific promoter (e.g, TERT promoter, modified TERT Promoter, PSA promoter, PSMA promoter, CEA promoter, Survivin promoter, E2F promoter, modified E2F promoter, AFP promoter, modified AFP promoter, E2F-AFP hybrid promoter, and E2F-TERT hybrid promoter) and tissue-specific promoters (e.g, albumin promoter), human phosphogly cerate kinase (PGK) promoter, mouse phosphoglycerate kinase (PGK) promoter, etc. In certain aspects, two different promoters for controlling expression of GM-CSF and sTGF-B-RII are used. For example, a CMV promoter may be used to drive sTGF-B-RII expression and a E1B promoter may drive expression of GM-CSF. A polyadenylation sequence may be linked downstream of a nucleic acid encoding GM-CSF and/or sTGF-B-RII. The polyadenylation sequence may be a bovine growth hormone terminator, a polyadenylation sequence derived from SV40, HIV-1 poly A, b- globin poly A, HSV TK poly A, or polyomavirus poly A, etc.

[0059] In certain instances, the recombinant vector may be an Ad5 vector or Ad2 vector.

Adenovirus produced using Ad5 vector can transduce cell expressing Coxsackie-Adenovirus Receptor (CAR). Ad5 vector may include Ad5 ITRs. The absence or the presence of low levels of the coxsackievirus and adenovirus receptor (CAR) on several tumor types can limit the efficacy of the oncolytic adenovirus. Various peptide motifs may be added to the fiber knob, for instance an RGD motif (RGD sequences mimic the normal ligands of cell surface integrins), Tat motif, polylysine motif, NGR motif, CTT motif, CNGRL motif, CPRECES motif or a strept-tag motif. A motif can be inserted into the HI loop of the adenovirus fiber protein. Modifying the capsid allows CAR-independent target cell infection. Specific receptors found exclusively or preferentially on the surface of cancer cells may be used as a target for adenoviral binding and infection, such as EGFRvIII.

[0060] In certain aspects, one or both of sTGF-BRII and GM-CSF may be a fusion protein where a heterologous protein is fused to the amino acid sequence of sTGF-BRII and/or GM-CSF at either the N-terminus or the C-terminus. For example, sTGF-BRII and/or GM-CSF may be fused to a heterologous protein that increases its serum half-life and/or increases solubility. Such heterologous proteins that increase serum half-life include the Fc region of immunoglobulins, e.g ., human IgGl, human serum albumin, etc. Fusion to maltose binding protein may be used to increase solubility of sTGF-BRII and/or GM-CSF. In other examples, only sTGF-BRII may be expressed as a fusion protein, where the heterologous protein comprises a half-life-extending moiety. The half-life-extending moiety may be a Fc region of a human IgGl. As used herein, Fc region refers to the Fc region found in immunoglobulins and variants thereof. Variants can include sequences that have deletions, insertions, or substitutions or other modifications, such as modified glycosylation that retain the half-life-extending effect associated with the naturally occurring Fc region of an immunoglobulin.

[0061] The sTGF-BRII protein may be encoded by a nucleic acid comprising a polynucleotide sequence from any source, e.g. , a cDNA or a synthetic sequence. The sTGF- BRII protein may include only the extracellular region of the TGF-BRII such that the receptor is secreted from cells expressing the receptor. The sTGF-BRII binds to the ligands for TGF-BRII but do not activate TGF-B signaling, thus decreasing TGF-B signaling. In certain aspects, the subject to whom the oncolytic virus is administered is a mammal and the polynucleotide sequence encoding sTGF-BRII protein is the TGF-BRII cDNA sequence from the mammal. In certain aspects, the subject may be human and the sTGF-BRII may be a human sTGF-BRII. The human sTGF-BRII may have an amino acid sequence that is at least 80%, 85%, 90%, 95%,

96%, 97%, 98%, or 99% or a 100% identical to the amino acid sequence set forth in SEQ ID NO:l. In some instances, the human sTGF-BRII may include amino acid differences from SEQ ID NO:l, such as deletions, insertions, or substitutions. The amino acid differences may be such that they do not significantly affect binding to TGF-BRII ligands, such as, TGF-B1, TGF-B2, and/or TGF-B3. The sequence of SEQ ID NO:l is as follows:

[0062] MGRGLLRGLWPLHI VLWTRI AS TIPPH V QK S VNNDMI VTDNN GA VKFPQ

LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PY HDFILED AASPKCIMKEKKKPGETFFMC SC S SDECNDNIIF S EEYNT SNPD .

[0063] The nucleic acid encoding GM-CSF may be from any source and may encode a functionally active variant ( e.g ., fragment or mutant) of GM-CSF. In certain examples, the GM- CSF is secreted from cells infected with the oncolytic virus. In certain aspects, the subject to whom the oncolytic virus is administered is a mammal and the GM-CSF protein sequence matches (e.g. is identical to) the GM-CSF protein sequence from the mammal. In certain aspects, the subject may be a human and the GM-CSF may be a human GM-CSF. The human GM-CSF may have an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or a 100% identical to the amino acid sequence set forth in SEQ ID NO:2. In some instances, the human GM-CSF may include amino acid differences from SEQ ID NO:2, such as deletions, insertions, or substitutions. The amino acid differences may be such that they do not significantly affect GM-CSF activity.

[0064] In certain aspects, the Fc region fused to the N-terminus and/or the C-terminus of

GM-CSF or sTGF-BRII may include an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or a 100% identical to the amino acid sequence set forth in SEQ ID NO:3. In some instances, the Fc region may include amino acid differences from SEQ ID NO:3, such as deletions, insertions, or substitutions. The amino acid differences may be such that they do not significantly affect the half-life-extending property of the Fc region. The sequence of SEQ ID NO:3 is as follows:

[0065] MDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR

TPEVTC VVVD V SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV S V LTVLHQDWLNGKEYKCKVSTKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. [0066] In certain instances, the oncolytic virus comprises a nucleic acid encoding a human GM-CSF and a sTGF-BRIIFc. sTGF-BRIIFc may have an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or a 100% identical to the amino acid sequence set forth in SEQ ID NO:6.

Compositions comprising subject oncolytic virus [0067] Also provided herein are compositions, such as pharmaceutical compositions comprising a therapeutically effective amount of the oncolytic virus disclosed herein and a pharmaceutically acceptable excipient, diluent, or carrier.

[0068] The compositions of the invention may comprise an oncolytic virus alone, or in combination with one or more other viruses ( e.g ., a second oncolytic virus comprising a nucleic acid encoding one or more different therapeutic genes). In some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different oncolytic viruses, each having one or more different therapeutic genes. In certain aspects, the composition may include an additional therapeutic agent for treatment of cancer.

[0069] Suitable carriers may be readily selected by one of skill in the art in view of the type of cancer for which the oncolytic virus is administered. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate- buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. Optionally, the compositions of the present disclosure may contain, in addition to the virus and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0070] In some embodiments, the oncolytic virus compositions are formulated to reduce aggregation of viral particles in the composition, particularly where high virus concentrations are present (e.g, about 10 ¹³ genome copies “GC’Vml or more). Methods for reducing aggregation of viral particles are well known in the art and include, for example, addition of surfactants, pH adjustment, and salt concentration adjustment.

[0071] Formulation of pharmaceutically acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. [0072] Typically, these formulations may contain at least about 0.1% of the oncolytic virus or more, although the percentage of the oncolytic virus may, of course, be varied and may conveniently be between about 1 about 80% (e.g, between about 2-70%) or more of the weight or volume of the total formulation. Naturally, the amount of oncolytic virus in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the oncolytic virus. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0073] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

[0074] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the recipient.

[0075] Sterile injectable solutions are prepared by incorporating the active virions in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. The viral compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, and the like.

[0076] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into target cells.

TREATMENT METHODS

[0077] Treatment methods comprise administering to a subject having cancer a therapeutically effective amount of the oncolytic virus described in the present application. The administering may include a single administration, or alternatively comprises a series of administrations. For example, the oncolytic virus described herein may be administered at least once daily, once weekly, or once monthly over a period of two or more months. As another example, the oncolytic virus is administered once every 2, 3, or 4 weeks for 4 cycles. The length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the patient, the concentration, the activity of the oncolytic virus described herein, and/or a combination thereof. It will also be appreciated that the effective dosage used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regimen. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. In addition, the treatment regimen may change when another cancer therapy is administered.

[0078] The dosage administered will vary depending on the use and known factors such as the pharmacodynamic characteristics of the particular substance, and its mode and route of administration, age, health, and weight of the individual recipient, nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. Dosage regimen may be adjusted to provide the optimum therapeutic response.

[0079] A "therapeutically effective amount" will fall in a relatively broad range that can be determined through experimentation and/or clinical trials. For example, a therapeutically effective dose can be on the order of from about 10 ⁶ to about 10 ¹⁵ of the virus particles, e.g, from about 10 ⁸ to 10 ¹² viral genomes (vg), from about 10 ⁶ vg to about 10 ¹⁵ vg of the oncolytic virus, e.g. , from about 10 ⁸ vg to 10 ¹² vg. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose-response curves.

[0080] In some embodiments, more than one administration (e.g, two, three, four or more administrations) may be employed to achieve the desired level of gene expression. In some cases, the more than one administration is administered at various intervals, e.g, daily, weekly, twice monthly, monthly, every 3 months, every 6 months, yearly, etc. In some cases, multiple administrations are administered over a period of time from 1 month to 2 months, from 2 months to 4 months, from 4 months to 8 months, from 8 months to 12 months, from 1 year to 2 years, from 2 years to 5 years, or more than 5 years.

[0081] In certain aspects, the oncolytic virus provided herein may be administered sequentially or contemporaneously with at least one other anti-cancer therapy. The anti-cancer therapy may be an immunotherapy, a chemotherapy, a surgical treatment, a radiation therapy, a hormone therapy, an anti-cancer small molecule, a growth factor inhibitor therapy, or a cytokine therapy.

[0082] The type of cancer that can be treated in a subject by administering the oncolytic virus of the present disclosure may be a primary cancer or a metastasized tumor. In certain embodiment, the cancer is breast cancer (e.g, triple negative breast cancer), skin cancer (e.g, melanoma), head or neck cancer, astrocytoma, hepatic cancer, lymphoma (e.g, lymphocytic lymphoma, primary central nervous system lymphoma, or pediatric lymphoma), colon cancer, gastric cancer, lung cancer, non-small cell lung cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, cervical cancer, bone cancer, non-small cell cancer, bone cancer, blood cancer, uterine cancer, rectal cancer, cancer near the anus, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, Thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, salivary gland cancer, sarcoma cancer, pseudomyxoma, hepatoblastoma, testicular cancer, glioblastoma, cleft lip cancer, ovarian germ cell tumor, basal cell carcinoma, multiple myeloma, gallbladder cancer, choroidal melanoma, barter bulge cancer, peritoneal cancer, tongue cancer, small cell carcinoma, neuroblastoma, duodenal cancer, ureter cancer, meningioma, renal pelvis cancer, vulvar cancer, thymic cancer, central nervous system (CNS) tumor, spinal cord tumor, brain stem glioma, and pituitary adenoma. The cancer may be a recurrent cancer.

Recombinant Oncolytic Virus Administration Methods [0083] The oncolytic virus provided herein may be delivered to a subject according to any appropriate methods known in the art. The virus, may be suspended in a physiologically compatible carrier or a pharmaceutically acceptable excipient or diluent ( i.e ., in a composition), may be administered to a subject, such as, for example, a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate ( e.g ., Macaque).

[0084] The virus is administered in sufficient amounts to transfect the target cells, e.g., solid tumor cells, and to provide sufficient levels of gene transfer and expression without undue off-target effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, intravenous, direct delivery (e.g, by injection) into a tumor, direct delivery to the selected organ (e.g, intraportal or intrahepatic delivery to the liver, delivery to brain via a cannula), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intramuscular, subcutaneous, intradermal, intrathecal, and other parenteral routes of administration. In certain aspects the administration may be into the brain, e.g, intraparenchymal, intracerebroventricular, intra cistema magna or subpial. Routes of administration may be combined, if desired.

[0085] Moreover, in certain instances, it may be desirable to deliver the virions to the

CNS of a subject. By "CNS" is meant all cells and tissue of the brain and spinal cord of a vertebrate. Thus, the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage and the like. Recombinant oncolytic virus may be delivered directly to the CNS or brain by injection into, e.g, the ventricular region, as well as to the striatum (e.g, the caudate nucleus or putamen of the striatum), spinal cord and neuromuscular junction, or cerebellar lobule, with a needle, catheter or related device, using neurosurgical techniques known in the art, such as by stereotactic injection.

[0086] In a further aspect, the present invention provides a virion of the present invention or a pharmaceutical composition of the present invention for use as a medicament. [0087] In a further aspect, the present invention provides an oncolytic virus of the present invention or a pharmaceutical composition of the present invention for use in a method of treating cancer in a subject in need thereof.

[0088] The dose of an oncolytic virus required to achieve a particular "therapeutic effect," e.g, the units of dose in genome copies/per kilogram of body weight (GC/kg), will vary based on several factors including, but not limited to: the route of administration, the level of gene expression required to achieve a therapeutic effect, the type of cancer being treated, and the like.

[0089] An effective amount of an oncolytic virus is an amount sufficient to infect a desired number of cancer cells. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the size/location of the tumor, and may thus vary among animal and tissue. For example, an effective amount of an oncolytic virus is generally in the range of from about 1 ml to about 100 ml of solution containing from about 10 ⁹ to 10 ¹⁶ genome copies. In some cases, a dosage between about 10 ¹¹ to 10 ¹² genome copies is appropriate. In certain embodiments, 10 ¹² viral genome copies is effective to target brain, liver, and pancreatic tissues. In certain embodiments, the dosage of the oncolytic virus is 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , or 10 ¹⁴ genome copies per kg. In certain embodiments, the dosage of the oncolytic virus is 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , or 10 ¹⁵ genome copies per subject.

[0090] In certain aspects, a therapeutically effective dose of an oncolytic virus of the present disclosure provides a level and/or activity of GM-CSF in the subject that is at least 10% higher than the normal GM-CSF level and/or activity, respectively ( e.g ., at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, or higher than the normal GM-CSF level). The GM-CSF level and/or activity may be determined using a blood sample or part thereof, e.g., serum or plasma from a subject who has been administered the virus. In certain aspects, a therapeutically effective dose of an oncolytic virus of the present disclosure provides a reduction in TGF-B activity that is at least 10% less (e.g., at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, or even lower than the normal TGF-B activity). The TGF-B activity may be determined using a blood sample or part thereof, e.g, serum or plasma from a subject who has been administered the virus.

Combination Therapy

[0091] In certain aspects, the treatment of a subject having cancer with an oncolytic virus comprising a nucleic acid encoding a sTGF-BRII and a GM-CSF as disclosed herein may or may not involve another anti-cancer therapy, such as, an immunotherapy, a chemotherapy, a surgical treatment, a radiation therapy, a hormone therapy, an anti-cancer small molecule, a growth factor inhibitor therapy, CAR-T therapy, or a cytokine therapy.

[0092] In some instances, a subject may be treated systemically, including with the subject oncolytic virus, with or without one or more additional reagents. By “systemic treatment”, as used herein, is meant a treatment that is not directed solely to target a specific tumor (such as, e.g, a primary tumor or a defined secondary tumor) or a specific cancer containing tissue (such as, e.g. , the liver in the case of liver cancer, the blood in the case of a blood cancer). Systemic treatments will generally be directed to the subject’s body as a whole and may include, but are not limited to, e.g. , systemic radiation therapy, systemic chemotherapy, systemic immunotherapy, combinations thereof and the like.

[0093] In some instances, a subject may be treated locally, including with the subject oncolytic virus, with or without one or more additional reagents. By “local treatment”, as used herein, is meant a treatment that is specifically directed to the location of a tumor (such as, e.g. , a primary tumor or a defined secondary tumor) or specifically directed to a cancer containing tissue (such as, e.g. , the liver in the case of liver cancer, the blood in the case of a blood cancer). In some instances, local treatment may also be administered in such a way as to affect the environment surrounding a tumor, such as tissue surrounding the tumor, such as tissue immediately adjacent to the tumor. Local treatment will generally not affect or not be targeted to tissues distant from the site of cancer including the site of a tumor, such as a primary tumor. Useful local treatments that may be administered in addition to, or in combination with, a subject oncolytic virus, e.g. , include but are not limited to surgery, local radiation therapy, local cryotherapy, local laser therapy, local topical therapy, combinations thereof, and the like.

[0094] Radiation therapy includes, but is not limited to, x-rays or gamma rays that are delivered from either an externally applied source such as a beam, or by implantation of small radioactive sources.

[0095] Suitable antibodies for use in cancer treatment include, but are not limited to, naked antibodies, e.g. , trastuzumab (Herceptin) , bevacizumab (Avastin™), cetuximab (Erbitux™), panitumumab (Vectibix™), Ipilimumab (Yervoy™), rituximab (Rituxan), alemtuzumab (Lemtrada™), Ofatumumab (Arzerra™), Oregovomab (OvaRex™), Lambrolizumab (MK-3475), pertuzumab (Peijeta™), ranibizumab (Lucentis™), etc., and conjugated antibodies, e.g. , gemtuzumab ozogamicin (Mylortarg™), Brentuximab vedotin (Adcetris™), 90Y-labelled ibritumomab tiuxetan (Zevalin™), 13 ll-labelled tositumoma (Bexxar™), etc.

[0096] Suitable antibodies for use in cancer treatment also include, but are not limited to, antibodies raised against tumor-associated antigens. Such antigens include, but are not limited to, CD20, CD30, CD33, CD52, EpCAM, CEA, gpA33, Mucins, TAG-72, CAIX, PSMA, Folate-binding protein, Gangliosides (e.g, GD2, GD3, GM2), Ley, VEGF, VEGFR, Integrin alpha- V-beta-3, Integrin alpha-5-beta-l, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILRl, TRAILR2, RANKL, FAP, Tenascin, Programmed Death-Ligand 1 (PD-L1), androgen receptor (AR), Bruton's Tyrosine Kinase (BTK), BCR-Abl, c-kit, PIK3CA, EML4- ALK, KRAS, ALK, ROS1, AKTl, BRAF, MEKJ, MEK2, NRAS, RAC1, ESR1, etc.

[0097] Biological response modifiers suitable for use in connection with the methods of the present disclosure include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine/threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; ( 4) apoptosis receptor agonists; (5) interleukin-2; (6) interferon-a.; (7) interferon-g; (8) inhibitors of angiogenesis; and (9) antagonists of tumor necrosis factor.

[0098] Chemotherapeutic agents or antineoplastic agents are non-peptidic (i.e., non- proteinaceous) compounds that reduce proliferation of cancer cells and/or kill cancer cells, and encompass cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents ( e.g ., nitrosoureas), antimetabolites (e.g, methotrexate), antitumor antibiotics (e.g, anthracyclins), plant alkaloids (e.g, vinca alkaloids, taxanes), toposiomerase inhibitors, and steroid hormones.

[0099] Agents that act to reduce cellular proliferation are known in the art and widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[00100] Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6- mercaptopurine (6-MP), pentostatin, 5 -fluorouracil (5-FU), methotrexate, 10-propargyl-5,8- dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatine, and gemcitabine.

[00101] Hormone modulators and steroids (including synthetic analogs) that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g, prednisone, dexamethasone; estrogens and pregestins, e.g. , hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, and adrenocortical suppressants, e.g, aminoglutethimide; 17a-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex. Estrogens stimulate proliferation and differentiation, therefore compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids may inhibit T cell proliferation.

[00102] Immune checkpoint inhibitor suitable for use include a PD-1 (programmed cell death-1) antagonist, a PD-Ll (programmed cell death-ligand 1) antagonist, a PD-L2 (programmed cell death-ligand 2) antagonist, a CD27 (cluster of differentiation 27) antagonist, CD28 (cluster of differentiation 28) antagonist, CD70 (cluster of differentiation 70) antagonist, CD80 (cluster of differentiation 80, also known as B7-1) antagonist, CD86 (cluster of differentiation 86, also known as B7-2) antagonist, CD137 (cluster of differentiation 137) antagonist, CD276 (cluster of differentiation 276) antagonist, KIRs (killer-cell immunoglobulin like receptors) antagonist, LAG3 (lymphocyte-activation gene 3) antagonist, TNFRSF4 (tumor necrosis factor receptor superfamily, member 4, also known as CD134) antagonist, GITR (glucocorticoid-induced TNFR-related protein) antagonist, GITRL (glucocorticoid-induced TNFR-related protein ligand) antagonist, 4-1BBL (4- IBB ligand) antagonist, CTLA-4 ( cytolytic T lymphocyte associated antign-4) antagonist, A2AR (Adenosine A2A receptor) antagonist, VTCN1 (V-set domain-containing T-cell activation inhibitor 1) antagonist, BTLA (B- and T-lymphocyte attenuator) antagonist, IDO (Indoleamine 2,3 -di oxygenase) antagonist, TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3) antagonist, VISTA (V-domain Ig suppressor of T cell activation) antagonist, KLRA (killer cell lectin- like receptor subfamily A) antagonists, and the like.

[00103] The immune checkpoint inhibitor may be an antibody. Examples of antibodies to checkpoint inhibitor molecules include Nivolumab (BMS-936558, MDX- 1 106, ONO-4538), a fully human Immunoglobulin G4 (lgG4) monoclonal PD-1 antibody; Lambrolizumab (MK- 3475), a humanized monoclonal IgG4 PD-1 antibody, BMS-936559, a fully human IgG4 PD-L1 antibody, LAG-3 antagonistic antibodies such as relatlimab (BMS-986016; Bristol-Myers Squibb), IMP701 (Immutep), TSR-033 (anti-LAG-3 mAb; TESARO, Inc.).

[00104] In certain aspects, a subject suitable for treatment using a method of the present disclosure include an individual having a cancer; an individual diagnosed as having a cancer; an individual being treated for a cancer with chemotherapy, radiation therapy, antibody therapy, surgery, etc.; an individual who has been treated for a cancer ( e.g ., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery), and who has failed to respond to the treatment; an individual who has been treated for a cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery), and who initially responded to the treatment but who subsequently relapsed, i.e., the cancer recurred.

[00105] The methods of the present disclosure may be employed to target and treat a variety of cancers, including, e.g ., primary cancer, secondary cancers, re-growing cancers, recurrent cancers, refractory cancers and the like. For example, in some instances, the methods of the present disclosure may be employed as an initial treatment of a primary cancer identified in a subject. In some instances, the methods of the present disclosure may be employed as a non primary (e.g, secondary or later) treatment, e.g, in a subject with a cancer that is refractory to a prior treatment, in a subject with a cancer that is re-growing following a prior treatment, in a subject with a mixed response to a prior treatment (e.g, a positive response to at least one tumor in the subject and a negative or neutral response to at least a second tumor in the subject), and the like.

[00106] The methods of treating described herein may, in some instances, be performed in a subject that has previously undergone one or more conventional treatments. For example, the methods described herein may, in some instances, be performed following a conventional cancer therapy including, but not limited to, e.g, conventional chemotherapy, conventional radiation therapy, conventional immunotherapy, or surgery. In some instances, the methods described herein may be used when a subject has not responded to, or is refractory to, a conventional therapy. In some instances, the methods described herein may be used when a subject has responded to a conventional therapy.

[00107] Two, three, or more of the treatments described herein may be combined in any order for treating cancer in a subject, which treatments may be administered simultaneously or contemporaneously. Such treatments include a dual-therapy and a triple-therapy.

KITS

[00108] Aspects of the present disclosure also include kits. The kits may include, e.g, any combination of the oncolytic virus, reagents, compositions, formulations, cells, nucleic acids, viral vectors, or the like, described herein. A subject kit can include one or more of: an oncolytic virus, an oncolytic viral vector, a cell line for producing the oncolytic virus. Kits may be configured for various purposes, including, e.g, treatment kits (e.g, where a kit may include a dose of an oncolytic virus), kits for the oncolytic virus, kits comprising antibodies for detecting expression of GM-CSF and/or sTGF-BRII, and the like.

[00109] Optional components of the kit will vary and may include, e.g, a buffer; or a bacterial growth inhibitor. Where a subject kit comprises a subject nucleic acid, the nucleic acid may also have restriction sites, multiple cloning sites, primer sites, etc. and the kit may include restriction enzymes, primers, etc.

[00110] The various components of the kit may be present in separate containers or certain compatible components may be pre-combined into a single container, as desired.

[00111] In addition to the above-mentioned components, a subject kit can include instructions for using the components of the kit to practice a subject method. The instructions for practicing a subject method are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, e.g ., compact disc-read only memory (CD-ROM), digital versatile disk (DVD), diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. , via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

EXEMPLARY NON-LIMITING ASPECTS OF THE DISCLOSURE [00112] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of ordinary skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below. It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

[00113] Such aspects may include:

1. An oncolytic virus comprising a nucleic acid encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF) and a soluble form of the TGF-B receptor-II (sTGF-BRII).

2. The oncolytic virus of aspect 1, wherein the oncolytic virus is an adenovirus, herpes virus, lentivirus, vaccinia virus, Reo virus, maraba virus, New Castle disease virus, sendai virus, pox virus, poliovirus, myxoma virus, or retrovirus. 3. The oncolytic virus of aspect 1 or 2, wherein the oncolytic virus is an adenovirus.

4. The oncolytic virus of any one of aspects 1-3, wherein the oncolytic virus preferentially replicates in cancer cells.

5. The oncolytic virus of any one of aspects 1-4, wherein the expression of genes required for replication of virus is under the control of a promoter active in the cancer cells.

6. The oncolytic virus of aspect 5, wherein the promoter active in cancer cells comprises a telomerase reverse transcriptase (TERT) promoter, wherein optionally the TERT promoter is a human TERT promoter.

7. The oncolytic virus of any one of aspects 1-6, wherein the expression of sTGF- BRII is under the control of a cytomegalovirus (CMV) promoter.

8. The oncolytic virus of any one of aspects 1-7, wherein the expression of GM- CSF is under the control of an adenoviral E1B promoter.

9. The oncolytic virus of any one of aspects 1-8, wherein the virus comprises a capsid protein that specifically binds to a cancer cell which overexpress an adenovirus receptor.

10. The oncolytic virus of aspect 9, wherein the cancer cell is a breast cancer cell, skin cancer cell, lung cancer cell, renal cancer cell, ovarian cancer cell, prostate cancer cell, pancreatic cancer cell, brain cancer cell, musculoskeletal cancer, astrocytoma cancer cell, cervical cancer cells, testicular cancer cells, hepatic cancer cell, lymphoma cancer cell, colon cancer cell, or bladder cancer cell.

11. The oncolytic virus of aspect 10, wherein the cancer cell is a breast cancer cell.

12. The oncolytic virus of aspect 11, wherein the breast cancer cell is a triple negative breast cancer cell.

13. The oncolytic virus of aspect 10, wherein the cancer cell is a skin cancer cell.

14. The oncolytic virus of aspect 13, wherein the skin cancer cell is a melanocyte.

15. The oncolytic virus of any one of aspects 1-14, wherein the nucleic acid encoding the GM-CSF is codon-optimized to increase expression of the GM-CSF in a human subject.

16. The oncolytic virus of any one of aspects 1-15, wherein the GM-CSF is a human GM-CSF.

17. The oncolytic virus of any one of aspects 1-16, wherein the sTGF-BRII is fused to a heterologous protein.

18. The oncolytic virus of aspect 17, wherein the heterologous protein comprises a half-life-extending moiety. 19. The oncolytic virus of aspect 18, wherein the half-life-extending moiety comprises an immunoglobulin Fc region.

20. A pharmaceutical composition comprising: a therapeutically effective amount of the oncolytic virus of any one of aspects 1-19 and a pharmaceutically acceptable excipient, diluent, or carrier.

21. A method of treating cancer in a subject, the method comprising: administering a therapeutically effective amount of the oncolytic virus of any one of aspects 1-19 or the composition of aspect 20 to the subject.

22. The method of aspect 21, wherein the cancer is breast cancer, skin cancer, lung cancer, bladder cancer, renal cancer, astrocytoma, hepatic cancer, lymphoma, colon cancer, or head/neck cancer.

23. The method of aspect 21 or 22, wherein the administering comprises an intratumoral or intravenous administration.

24. The method of any one of aspects 21-23, wherein the cancer is a primary cancer.

25. The method of any one of aspects 21-23, wherein the cancer is metastatic cancer.

26. The method of any one of aspects 21-25, comprising administering to the subject one or more of an immunotherapy, a chemotherapy, a surgical treatment, a radiation therapy, a hormone therapy, an anti-cancer small molecule, a growth factor inhibitor therapy, CAR-T therapy, or a cytokine therapy.

27. The method of any one of aspects 21-26, comprising administering to the subject an immunotherapy.

28. The method of any one of aspects 21-27, comprising administering to the subject a chemotherapy. 29. The method of any one of aspects 21-28, comprising administering to the subject an immune checkpoint inhibitor.

30. The method of aspect 29, wherein the immune checkpoint inhibitor comprises an inhibitor of PD-1, PD-L1, CTLA-4, LAG-3, BCH-3, BCH-4, or TIM-3.

31. The method of aspect 30, wherein the inhibitor is an antibody.

EXAMPLES

[00114] As can be appreciated from the disclosure provided above, the present disclosure has a wide variety of applications. Accordingly, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. Thus, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used ( e.g ., amounts, dimensions) but some experimental errors and deviations should be accounted for.

EXAMPLE 1: RAD.ST.GM TNHTBTTS 4T1 TUMOR GROWTH IN IMMUNE COMPETENT BALB/C

MICE

[00115] Methods. To establish a mouse mammary tumor syngeneic mouse model, 2 x 10 ⁶ 4T1 cells per mouse were injected subcutaneously (day 0) into the dorsal right flank of female BALB/c mice (4-6 weeks old). Mice were monitored every day. On day 6, tumor dimensions were measured (in mm) using a caliper. Then, tumor-bearing mice were divided into seven groups without statistical differences between each group. The adenoviral vectors diagrammed in Fig. 1 were tested. rAD.sT.GM vector expressed both soluble TGFbRIIFc and GM-CSF proteins, with CMV promoter (CMVp) controlling TGFbRIIFc expression while expression of GM-CSF is under the control of adenoviral E1B promoter (ElBp). rAd.GM vector only expresses GM-CSF, under the control of adenoviral E1B promoter. rAd.sT vector only expresses soluble TGFbRIIFc, under the control of CMV promoter. rAd.null vector served as control. In each of the vectors, the expression of the El A gene is under the control of the human TERT promoter to provide for selective replication of the virus in cancer cells. Adenovirus were produced that included rAD.sT.GM vector, rAd.GM vector, rAd.sT vector, or rAd.null vector. The adenoviruses were injected directly into the tumors (5xl0 ¹⁰ viral particles/per injection, each injection IOOmI volume). Two injections were administered on each of days 7 and 9.

Tumor volumes were measured over time and calculated by the following formula: tumor volume = (width ² x length)/2.

[00116] The tumor volumes were monitored on various days shown in Fig. 2. Animals were euthanized on day 25, tumors were removed, and tumor weights were measured.

[00117] Results. The results indicated that while the tumors treated with buffer grew over time, the intratumoral injection of all the adenoviruses inhibited tumor growth. Among the adenoviruses tested, rAd.sT. GM produced the strongest inhibition of the tumor growth. (Fig.

2A). Importantly, rAd.sT. GM produced inhibition of tumor growth which was statistically superior to all other viruses tested (Fig. 2A). Most notable is the superior response of rAd.sT. GM compared to both the rAd.GM (p<0.01) and rAd.sT (p<0.05), indicating a synergism between TGFbRIIFc and GM-CSF. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001.

[00118] On day 25, mice were euthanized, and tumors were removed and weighed.

Again, among all the treatment groups, rAd.sT. GM was most effective in reducing the tumor weights (Fig. 2B). * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. [00119] These results show that the direct injection of rAd.sT. GM virus into mammary tumors produces strong inhibition of tumor growth.

[00120] rAd.LIGHT adenovirus results in expression of LIGHT (also known as tumor- necrosis factor (TNF) superfamily member 14 (TNFSF14)). rAd.LIGHT adenovirus inhibits tumor growth via activation of anti -turn or immune responses (Dai et al., Cancer Gene Therapy 27, 923-933 (2020)). rAd.LIGHT reduced 4T1 mouse mammary tumor growth and tumor weight. However, the adenovirus expressing both LIGHT and sTGFbRIIFc, /. e. , rAd.sT. LIGHT, was less effective in inhibiting tumor growth and volume compared to rAd. LIGHT and rAd.sT alone. This result shows the unpredictability of effect of combining two molecules, LIGHT and sTGFbRIIFc, that are each individually effective in treating cancer. This unpredictability makes the discovery of the combined synergistic effect of sTGFbRIIFc with GM-CSF for treating cancers even more important, since it provides for a significant improvement in treatment of cancer.

[00121] Cell lines and adenoviruses. The mouse mammary tumor cell line, 4T1, was purchased from ATCC. 4T1 cells were cultured in Eagle’s Minimal Essential Medium (EMEM) plus 10% fetal calf serum (FCS). Human embryonic kidney cells, HEK293 (ATCC) were cultured in Dulbecco’s Minimal Essential Medium (DMEM) supplement with 10% FCS. All adenoviruses were grown in HEK293 cells and purified by double cesium chloride gradients. [00122] Animal studies. All procedures for animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) atNorthShore University Health System.

[00123] Statistical analysis. Data are presented as mean±s.e.m. All statistical analyses were performed using GraphPad Prism software version 5 (GraphPad software, San Diego, CA, USA). Longitudinal data were analyzed by using a two-way repeated measure ANOVA followed by Bonferroni post hoc tests for the tumor growth data obtained over-time. One-way ANOVA followed by Bonferroni post hoc tests were performed to analyze other data. Differences were considered significant at two-sided p<0.05.

[00124] rAd.sT.GM was constructed by using a modified system that have three gene expression units: CMVp-controlled sTGFpRIIFc, TERTp-controlled El A and ElBp-controlled GM-CSF that also harbors an IRES-linked E1B55K. Three control replicating adenoviruses: rAd.GM, rAd.sT and rAd.Null are devoid of either sTGFpRIIFc or GM-CSF, or both. Nonreplicating adenoviruse Ad(El-).Null was constructed by the Ad-easy System.

[00125] Vector Construction.

[00126] Construction of Ad-GM-RE:

[00127] 1. Enzyme digestion: pShuttle-cmv was digested by Mfel and dephosphorylated.

Then, the larger fragment was recovered. TE-TP-GM (dIRES) was digested with Mfel, and then the larger fragment was also obtained.

[00128] 2. The fragments from step 1 were ligated with T4 DNA Ligase, transformed into

E. coli DH5a, and selected by Kanamycin.

[00129] 3. Picked up more than 24 clones, and grown in LB overnight (37 ^° C, 250rpm).

[00130] 4. Plasmids were extracted using conventional techniques.

[00131] 5. Identification by enzyme digestion. Using pshuttle-cmv-TGFpRIIFc as control.

Notl (GC'GGCC_GC) [CciNI] cuts 2 times at positions 966, 1257 generating fragment sizes 291, 10693. Control, Notl (GC'GGCC GC) [CciNI] cuts 1 time at position 966. One clone identified as containing correctly inserted GM-CSF, named “pShuttle-cmv-GM-RE (dIRES)”. [00132] 6. Grew the correct clone from the second batch, and obtained more plasmids.

[00133] 7. pShuttle-cmv-GM-RE (dIRES) was digested with Pmel and

Dephosphorylated.

[00134] 8. Linear plasmid was obtained by using ethanol precipitation.

[00135] 9. IOmI linear plasmid was transformed into BJ5183-Adeasy competent cells by using Electroporation (2.5KV).

[00136] 10. 12 clones were picked from the plates and grown overnight (37 ^° C, 250rpm).

[00137] 11. Extraction of the plasmids and identification by using Pad digestion. 7 clones were correctly inserted and were named “Ad -GM-RE (dIRES)”.

[00138] 12. Transformed the plasmids from #2 clone into E. coli DH5a and then extracted the plasmids for transfection.

[00139] 13. Identification of the plasmids by using Pad, again.

[00140] 14. 173pl plasmid Ad -GM-RE (dIRES) was cut by Pad (7m1), and dephosphorylated. Then, the Pad linear Ad -GM-RE (dIRES) was recovered by using ethanol precipitation. Total volume was 35m1.

[00141] 15. Transfection:

[00142] DayO: HEK293 cells were seeded into 6-well plate at a density of 5xl0 ⁶ cells/well (one 10cm plate could be seeded into 15 wells).

[00143] Day 1 : Transfection- the Pad lined Ad -GM-RE (dIRES) was transfected into HEK293 cells according to the protocol. (Two wells, 5m1 and 10m1 respectively.) The culture medium was replaced by 5% FBS DMEM (2ml) 6 hours after transfection.

[00144] Day4: The culture medium was replaced by fresh 5% FBS DMEM (2ml).

[00145] Day7: 2ml fresh 5% FBS DMEM (2ml) were added to each well.

[00146] Day8-10: Plaques were observed.

[00147] DaylO: 2ml fresh 5% FBS DMEM (2ml) were added to each well.

[00148] Dayl2-Dayl6: Adenovirus (rAd.GM) was collected and purified.

[00149] Construction of Ad-TGFfRIIFc-RE:

[00150] 1. Enzyme digestion:

[00151] pShuttle-cmv-TGFpRIIFc-GM-RE (dIRES) was digested by Spel and

Dephosphorylated. Then, the larger fragment was recovered. TE-TP-E1 A was digested with Mfel, and then the larger fragment was obtained.

[00152] 2. The fragments from stepl were recombined in BJ5183 bacteria.

[00153] 3. 12 clones were picked and grown in LB overnight (37°C, 250rpm).

[00154] 4. Plasmids were extracted using conventional techniques. [00155] 5. Identification by enzyme digestion. pshuttle-cmv-TGFpRIIFc-GM-RE dIRES was used as a control. Kpnl (G GTAC'C) [neoschizomers Acc65I,Asp718I] cuts 2 times at positions 2203 and 4186, generating fragment sizes 1983 and 9810 bp. Control, Kpnl (G GTAC'C) [neoschizomers Acc65I,Asp718I] cuts 2 times, at positions 2203 and 4623, generating fragment sizes 24209810 bp. Sail (G'TCGA C) cuts 3 times, at positions 2193,

2205, and 4129, generating fragment sizes of 12, 1924, and 9857 bp. Control, Sail (G'TCGA_C) cuts 3 times, at positions 2193, 2205, and 4566, generating fragment sizes of 12, 2361, and 9857 bp. All clones were correctly inserted and named “pshuttle-cmv-TGFbetaRIIFc-RE”.

[00156] 6. Grew the correct clone #11, and obtained more plasmids.

[00157] 7. pShuttle-cmv-TGFbetaRIIFc-RE was digested with Pmel and dephosphory 1 ated .

[00158] 8. Linear plasmid was obtained by using ethanol precipitation.

[00159] 9. IOmI linear plasmid was transformed into BJ5183-Adeasy competent cells by using Electroporation (2.5KV).

[00160] 10. 12 clones were picked from the plates and grown overnight (37 ^° C, 250rpm).

[00161] 11. The plasmids were extracted and identified using Pad digestion. One clone was correct and was named “Ad-TGFbetaRIIFc-RE”.

[00162] 12. Transformed the plasmids from #6 clone into E. coli DH5a and then extracted the plasmids for transfection.

[00163] 13. Plasmids were again identified using Pad, and also by PCR amplification.

[00164] 14. 173pl plasmid Ad-TGFbetaRIIFc-RE was cut by Pad (7m1), and dephosphorylated. Then, the Pad linearized Ad-TGFbetaRIIFc-RE was recovered using ethanol precipitation. Total volume was 35m1.

[00165] 15. Transfection:

[00166] DayO: HEK293 cells were seeded into 6-well plates at a density of 5xl0 ⁶ cells/well (one 10cm plate could be seeded into 15 wells).

[00167] Day 1 : Transfection the Pad linearized Ad-TGFbetaRIIFc-RE into HEK293 cells according to the protocol. (Two wells, 5m1 and 10m1 respectively.) The culture medium was replaced by 5% FBS DMEM (2ml) 6 hours after transfection.

[00168] Day4: The culture medium was replaced by fresh 5% FBS DMEM (2ml).

[00169] Day7: 2ml fresh 5% FBS DMEM (2ml) were added to each well.

[00170] Day8-10: Plaques were observed.

[00171] DaylO: 2ml fresh 5% FBS DMEM (2ml) were added to each well.

[00172] Dayl2-Dayl6: Adenovirus (rAd.sT) was collected and purified. [00173] Construction of Ad-Null-RE:

[00174] Method 1:

[00175] 1. Enzyme digestion:

[00176] pShuttle-cmv-TGFpRIIFc-RE was digested by Xhol, and the larger fragment was recovered.

[00177] 2. The fragment was self-ligated by using T4DNA Ligase.

[00178] Method2:

[00179] 1. Enzyme digestion:

[00180] pShuttle-cmv-GM-RE (dIRES) was digested by Spel and dephosphorylated.

Then, the fragment was recovered. TE-TP-E1 A was digested by Mfel, and dephosphorylated. Then, the larger fragment was recovered.

[00181] 2. The fragments from stepl were recombined in BJ5183 bacteria.

[00182] 3. 12 clones were picked from each method, and grown in LB overnight (37 ^° C,

250rpm).

[00183] 4. Plasmids were extracted (#1-6 clones were chosen from a total of 12 clones).

[00184] 5. Plasmids were identified by enzyme digestionusing pshuttle-cmv-ElBp-Light-

RE and pshuttle-cmv-TGFpRIIFc-RE as controls. Xhol cuts 1 time. One band for both methods, control (pshuttle-cmv-TGFpRIIFc-RE). Xhol cuts 2 times producing 2 bands; one band is 1267 bp. Control (pshuttle-cmv-ElBp-Light-RE), Xhol cuts 1 time producing 1 band. Kpnl (G GTAC'C) [neoschizomers Acc65I,Asp718I] for recombination methods cuts 2 times, at positions 957 and 2940, generating fragment sizes of 1983 and 8564 bp. For Xhol digestion and self-ligation method, there is only one band. Control, pshuttle-cmv-TGFpRIIFc-RE, Kpnl (G GTAC'C) [neoschizomers Acc65I,Asp718I] cuts 2 times, at positions 2203 and 4186, generating fragment sizes of 1983 and 9810 bp. pshuttle-cmv-ElBp-Light-RE, Kpnl (G GTAC'C) [neoschizomers Acc65I,Asp718I] cuts 3 times, at positions 957, 4367, and 4564, generating fragment sizes of 3410, 197, and 8564 bp. Correct cloanes were named “pshuttle- cmv-Null-RE”.

[00185] 6. Grew the correct clone #6 and obtained more plasmid.

[00186] 7. pShuttle-cmv-Null-RE was digested with Pmel and dephosphorylated.

[00187] 8. Linear plasmid was obtained using ethanol precipitation.

[00188] 9. IOmI linearized plasmid was transformed into BJ5183-Adeasy competent cells by using Electroporation (2.5KV).

[00189] 10. 12 clones were picked from the plates and grown overnight (37 ^° C, 250rpm). [00190] 11. Plasmids were extracted and identified using Pad digestion and PCR amplification. Correct clones were named “Ad-Null-RE”.

[00191] 12. Transformed the plasmids from #6 clone into E. coli DH5a and then plasmids were extracted for transfection.

[00192] 13. Plasmids were identified using Pad, again.

[00193]

[00194] 14. 173pl plasmid Ad-Null-RE was cut by Pad (7m1), and dephosphorylated.

Then, the Pad linearized Ad-Null-RE was recovered by using ethanol precipitation. Total volume was 35m1.

[00195] 15. Transfection:

[00196] DayO: HEK293 cells were seeded into 6-well plates at a density of 5xl0 ⁶ cells/well.

[00197] (One 10cm plate could be seeded into 15 wells.)

[00198] Day 1 : ThePad-linearized Ad-Null-RE was transfected into HEK293 cells according to the protocol. (Two wells, 5m1 and 10m1 respectively.) The culture medium was replaced by 5% FBS DMEM (2ml) 6 hours after transfection.

[00199] Day4: The culture medium was replaced by fresh 5% FBS DMEM (2ml).

[00200] Day7: 2ml fresh 5% FBS DMEM (2ml) was added to each well.

[00201] Day8-10: Plaques were observed.

[00202] Day 10: Fresh 5% FBS DMEM (2ml) was added to each well.

[00203] Day 12-Day 16: Adenovirus (rAd.Null) was collected and purified.

[00204] Construction of Ad-TGFfRIIFc-GM-RE

[00205] 1. Enzyme digestion:

[00206] pShuttle-cmv-TGFpRIIFc-RE was digested by Spel and dephosphorylated. Then, the larger fragment was recovered. TE-PPT-GM was digested with Mfel and EcoRI to get the 4.5kb fragment.

[00207] 2. The fragments from stepl were recombined in BJ5183 bacteria.

[00208] 3. 12 clones were picked and grown in LB overnight (37 ^° C, 250rpm).

[00209] 4. Plasmid extraction.

[00210] 5. Identification by enzyme digestion. pshuttle-cmv-TGFpRIIFc-RE was used as a control. Bglll (A'GATC T) cuts 3 times, at positions 947, 4782, and 6813, generating fragment sizes of 3835, 2031, and 7278 bp. Control, Bglll (A'GATC T) cuts 2 times, at positions 947 and 5462, generating fragment sizes of 4515 and 7278 bp. Scal+Spel double digests yielded fragments of 488 bp and 12656 bp. Control, Scal+Spel yielded a fragment of 11793 bp. Correct clones were named “pshuttle-cmv-TGFbetaRIIFc-RE”.

[00211] 6. Correct clone #1 was grown and more plasmid was obtained.

[00212] 7. pShuttle-cmv-TGFbetaRIIFc-GM-RE was digested with Pmel and dephosphory 1 ated .

[00213] 8. Linearized plasmid was obtained by using ethanol precipitation.

[00214] 9. IOmI linearized plasmid was transformed into BJ5183-Adeasy competent cells using Electroporation (2.5KV).

[00215] 10. 12 clones were picked from the plates and grown overnight (37°C, 250rpm).

[00216] 11. Plasmids were extracted and identified using Pad digestion. Correct clones were named “Ad-TGFbetaRIIFc-GM-RE”.

Construction of rAd T GM

[00217] 12. Plasmids from #12 Ad-TGFbetaRIIFc-GM-RE clone were amplified in E. coli DH5a and then the plasmids were extracted for transfection.

[00218] 13. Plasmids were identified by using Pad again, and by PCR amplification.

[00219] 14. 173pl plasmid Ad-TGFbetaRIIFc-GM-RE was cut by Pad (7m1), and dephosphorylated. Then, the Pad-linearized Ad-TGFbetaRIIFc-GM-RE was recovered by ethanol precipitation. Total volume was 35m1.

[00220] 15. Transfection:

[00221] DayO: HEK293 cells were seeded into 6-well plate at a density of 5xl0 ⁶ cells/well.

[00222] (One 10cm plate could be seeded into 15 wells.)

[00223] Day 1 : Pad-linearized Ad-TGFbetaRIIFc-GM-RE was transfected into HEK293 cells according to the protocol. (Two wells, 5m1 and 10m1 respectively.) The culture medium was replaced by 5% FBS DMEM (2ml) 6 hours after transfection.

[00224] Day4: The culture medium was replaced by fresh 5% FBS DMEM (2ml).

[00225] Day7: Fresh 5% FBS DMEM (2ml) was added to each well.

[00226] Day8-10: The plaques were observed.

[00227] Day 10: Fresh 5% FBS DMEM (2ml) was added to each well.

[00228] Dayl2-Dayl6: Adenovirus (rAd.sT.GM) was collected and purified.

Sequences:

[00229] Complete genome sequence of rAd.sT.GM:

1 TCCCTTCCAG CTCTCTGCCC CTTTTGGATT GAAGCCAATA TGATAATGAG 51 GGGGTGGAGT TTGTGACGTG GCGCGGGGCG TGGGAACGGG GCGGGTGACG

101 TAGTAGTGTG GCGGAAGTGT GATGTTGCAA GTGTGGCGGA ACACATGTAA

151 GCGACGGATG TGGCAAAAGT GACGTTTTTG GTGTGCGCCG G TG TACACAG

201 GAAGTGACAA TTTTCGCGCG GTTTTAGGCG GATGTTGTAG TAAATTTGGG

251 CGTAACCGAG TAAGATTTGG CCATTTTCGC GGGAAAAC TG AATAAGAGGA

301 AGTGAAATCT GAATAATTTT GTGTTACTCA TAGCGCGTAA NNNNTAATAG

351 TAATCAATTA CGGGGTCATT AGTTCATAGC CCATATATGG AGTTCCGCGT

401 T AC AT AAC T T ACGGTAAATG GCCCGCCTGG CTGACCGCCC AACGACCCCC

451 GCCCATTGAC GTCAATAATG ACGTATGTTC CCATAGTAAC GCCAATAGGG

501 ACTTTCCATT GACGTCAATG GGTGGAGTAT TTACGGTAAA CTGCCCACTT

551 GGCAGTACAT CAAGTGTATC ATATGCCAAG TACGCCCCCT ATTGACGTCA

601 ATGACGGTAA ATGGCCCGCC TGGCATTATG CCCAGTACAT GACCTTATGG

651 GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG CTATTACCAT

701 GGTGATGCGG TTTTGGCAGT ACATCAATGG GCGTGGATAG CGGTTTGACT

751 CACGGGGATT TCCAAGTCTC CACCCCATTG ACGTCAATGG GAGTTTGTTT

801 TGGCACCAAA ATCAACGGGA CTTTCCAAAA TGTCGTAACA ACTCCGCCCC

851 ATTGACGCAA ATGGGCGGTA GGCGTGTACG GTGGGAGGTC TATATAAGCA

901 GAGCTGGTTT AGTGAACCGT CAGATCCGCT AGAGATCTCG AGCTCAAGCT

951 TACCTGCCAT GGGTCGGGGG CTGCTCAGGG GCCTGTGGCC GCTGCACATC

1001 GTCCTGTGGA CGCGTATCGC CAGCACGATC CCACCGCACG TTCAGAAGTC

1051 GGTTAATAAC GACATGATAG TCACTGACAA CAACGGTGCA GTCAAGTTTC

1101 CACAACTGTG TAAATTTTGT GATGTGAGAT TTTCCACCTG TGACAACCAG

1151 AAATCCTGCA TGAGCAAC TG CAGCATCACC TC CATC TG TG AGAAGCCACA

1201 GGAAGTCTGT GTGGCTGTAT GGAGAAAGAA TGAC GAGAAC ATAACACTAG

1251 AGACAGTTTG CCATGACCCC AAGCTCCCCT ACCATGACTT TATTCTGGAA

1301 GATGCTGCTT CTCCAAAGTG CATTATGAAG GAAAAAAAAA AGCCTGGTGA

1351 GACTTTCTTC ATGTGTTCCT GTAGCTCTGA TGAGTGCAAT GACAACATCA

1401 TCTTCTCAGA AGAATATAAC ACCAGCAATC CTGACATGGA TCCCAAATCT

1451 TGTGACAAAA CTCACACATG CCCACCGTGC CCAGCACC TG AACTCCTGGG

1501 GGGACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA

1551 TCTCCCGGAC CCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAA

1601 GACCCTGAGG TCAAGTTCAA CTGGTACGTG GACGGCGTGG AGGTGCATAA

1651 TGCCAAGACA AAGCCGCGGG AGGAGCAGTA CAACAGCACG TACCGTGTGG

1701 TCAGCGTCCT CACCGTCCTG CACCAGGACT GGCTGAATGG CAAGGAGTAC

1751 AAGTGCAAGG TATCCACCAA AGCCCTCCCA GCCCCCATCG AGAAAAC CAT

1801 CTCCAAAGCC AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC

1851 CATCCCGGGA TGAGCTGACC AAGAACCAGG TCAGCC TGAC CTGCCTAGTC

1901 AAAGGCTTCT ATCCCAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA 1951 GCCGGAGAAC AACTACAAGG CCACGCCTCC CGTGCTGGAC TCCGACGGCT

2001 CCTTCTTCCT CTACAGCAAG CTCACCGTGG ACAAGAGCAG GTGGCAGCAG

2051 GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGC ACAACCACTA

2101 CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAATAAAGG CGCGCCAGTA

2151 TTGGATACTA GAGTCGAGCA TGCATCTAGT CGACGGTACC GTCGACGCGG

2201 CCGCTCGAGC CTAAGCTTCT AGATAAGATA TCCGATCCAC CGGATCTAGA

2251 TAACTGATCA TAATCAGCCA TACCACATTT GTAGAGGTTT TACTTGCTTT

2301 AAAAAACCTC CCACACCTCC CCCTGAACCT GAAACATAAA ATGAATGCAA

2351 TTGTTGTTGT TAACTTGTTT ATTGCAGCTT ATAATGGTTA CAAATAAAGC

2401 AATAGCATCA CAAATTTCAC AAATAAAGCA TTTTTTTCAC TGCATTCTAG

2451 TTGTGGTTTG TCCAAACTCA TCAATGTATC TTAACGCGCG GCCGCTGGCC

2501 CCTCCCTCGG GTTACCCCAC AGCTTAGGCC GATTCGACCT CTCTCCGCTG

2551 GGGCCCTCGC TGGCGTCCCT GCACCCTGGG AGCGCGAGCG GCGCGCGGGC

2601 GGGGAAGCGC GGCCCAGACC CCCGGGTCCG CCCGGAGCAG CTGCGCTGTC

2651 GGGGCCAGGC CGGGCTCCCA GTGGATTCGC GGGCACAGAC GCCCAGGACC

2701 GCGCTTCCCA CGTGGCGGAG GGACTGGGGA CCCGGGCACC CGTCCTGCCC

2751 CTTCACCTTC CAGCTCCGCC TCCTCCGCGC GGACCCCGCC CCGTCCCGAC

2801 CCCTCCCGGG TCCCCGGCCC AGCCCCCTCC GGGCCCTCCC AGCCCCTCCC

2851 CTTCCTTTCC GCGGCCCCGC CCTCTCCTCG CGGCGCGAGT TTCAGGCAGC

2901 GCTGCGTCCT GCTGCGCACG TGGGAAGCCC TGGCCCCGGC CACCCCCGCG

2951 GAATTCATGA GACATATTAT CTGCCACGGA GGTGTTATTA CCGAAGAAAT

3001 GGCCGCCAGT CTTTTGGACC AGCTGATCGA AGAGGTACTG GCTGATAATC

3051 TTCCACCTCC TAGCCATTTT GAACCACCTA CCCTTCACGA ACTGTATGAT

3101 TTAGACGTGA CGGCCCCCGA AGATCCCAAC GAGGAGGCGG TTTCGCAGAT

3151 TTTTCCCGAC TCTGTAATGT TGGCGGTGCA GGAAGGGATT GACTTACTCA

3201 CTTTTCCGCC GGCGCCCGGT TCTCCGGAGC CGCCTCACCT TTCCCGGCAG

3251 CCCGAGCAGC CGGAGCAGAG AGCCTTGGGT CCGGTTTCTA TGCCAAACCT

3301 TGTACCGGAG GTGATCGATC TTACCTGCCA CGAGGCTGGC TTTCCACCCA

3351 GTGACGACGA GGATGAAGAG GGTGAGGAGT TTGTGTTAGA TTATGTGGAG

3401 CACCCCGGGC ACGGTTGCAG GTCTTGTCAT TATCACCGGA GGAATACGGG

3451 GGACCCAGAT ATTATGTGTT CGCTTTGCTA TATGAGGACC TGTGGCATGT

3501 TTGTCTACAG TAAGTGAAAA TTATGGGCAG TGGGTGATAG AGTGGTGGGT

3551 TTGGTGTGGT AATTTTTTTT TTAATTTTTA CAGTTTTGTG GTTTAAAGAA

3601 TTTTGTATTG TGATTTTTTT AAAAGGTCCT GTGTCTGAAC CTGAGCCTGA

3651 GCCCGAGCCA GAACCGGAGC CTGCAAGACC TACCCGCCGT CCTAAAATGG

3701 CGCCTGCTAT CCTGAGACGC CCGACATCAC CTGTGTCTAG AGAATGCAAT

3751 AGTAGTACGG ATAGCTGTGA CTCCGGTCCT TCTAACACAC CTCCTGAGAT

3801 ACACCCGGTG GTCCCGCTGT GCCCCATTAA ACCAGTTGCC GTGAGAGTTG 3851 GTGGGCGTCG CCAGGCTGTG GAATGTATCG AGGACTTGCT TAACGAGCCT

3901 GGGCAACCTT TGGACTTGAG CTGTAAACGC CCCAGGCCAT AAGGTGTAAA

3951 CCTGTGATTG CGTGTGTGGT TAACGCCTTT GTTTGCTGAA TGAGTTGATG

4001 TAAGTTTAAT AAAGGGTGAG ATAATGTTTA ACTTGCATGG CGTGTTAAAT

4051 GGGGCGGGGC TTAAAGGGTA TATAATGCGC CGTGGGCTAA TCTTGGTTAC

4101 ATCTGACCTC ACTAGTATGT GGCTGCAGAG CCTGCTGCTC TTGGGCACTG

4151 TGGCCTGCAG CATCTCTGCA CCCGCCCGCT CGCCCAGCCC CAGCACGCAG

4201 CCCTGGGAGC ATGTGAATGC CATCCAGGAG GCCCGGCGTC TCCTGAACCT

4251 GAGTAGAGAC ACTGCTGCTG AGATGAATGA AACAGTAGAA GTCATCTCAG

4301 AAATGTTTGA CCTCCAGGAG CCGACCTGCC TACAGACCCG CCTGGAGCTG

4351 TACAAGCAGG GCCTGCGGGG CAGCCTCACC AAGCTCAAGG GCCCCTTGAC

4401 CATGATGGCC AGCCACTACA AGCAGCACTG CCCTCCAACC CCGGAAACTT

4451 CCTGTGCAAC CCAGATTATC ACCTTTGAAA GTTTCAAAGA GAACCTGAAG

4501 GACTTTCTGC TTGTCATCCC CTTTGACTGC TGGGAGCCAG TCCAGGAGTG

4551 AGTCGACGAA TTAATTCGCT GTCTGCGAGG GCCAGCTGTT GGGGTGAGTA

4601 CTCCCTCTCA AAAGCGGGCA TGACTTCTGC GCTAAGATTG TCAGTTTCCA

4651 AAAACGAGGA GGATTTGATA TTCACCTGGC CCGCGGTGAT GCCTTTGAGG

4701 GTGGCCGCGT CCATCTGGTC AGAAAAGACA ATCTTTTTGT TGTCAAGCTT

4751 GAGGTGTGGC AGGCTTGAGA TCTGGCCATA CACTTGAGTG ACAATGACAT

4801 CCACTTTGCC TTTCTCTCCA CAGGTGTCCA CTCCCAGGTC CAACTGCAGG

4851 TCGAGCATGC ATCTAGGGCG GCCAATTCCG CCCCTCTCCC TCCCCCCCCC

4901 CTAACGTTAC TGGCCGAAGC CGCTTGGAAT AAGGCCGGTG TGCGTTTGTC

4951 TATATGTGAT TTTCCACCAT ATTGCCGTCT TTTGGCAATG TGAGGGCCCG

5001 GAAACCTGGC CCTGTCTTCT TGACGAGCAT TCCTAGGGGT CTTTCCCCTC

5051 TCGCCAAAGG AATGCAAGGT CTGTTGAATG TCGTGAAGGA AGCAGTTCCT

5101 CTGGAAGCTT CTTGAAGACA AACAACGTCT GTAGCGACCC TTTGCAGGCA

5151 GCGGAACCCC CCACCTGGCG ACAGGTGCCT CTGCGGCCAA AAGCCACGTG

5201 TATAAGATAC ACCTGCAAAG GCGGCACAAC CCCAGTGCCA CGTTGTGAGT

5251 TGGATAGTTG TGGAAAGAGT CAAATGGCTC TCCTCAAGCG TATTCAACAA

5301 GGGGCTGAAG GATGCCCAGA AGGTACCCCA TTGTATGGGA TCTGATCTGG

5351 GGCCTCGGTG CACATGCTTT ACATGTGTTT AGTCGAGGTT AAAAAAACGT

5401 CTAGGCCCCC CGAACCACGG GGACGTGGTT TTCCTTTGAA AAACACGATG

5451 ATAAGCTTGC CACAACTCGA CGAGTTTTAT AAAGGATAAA TGGAGCGAAG

5501 AAACCCATCT GAGCGGGGGG TACCTGCTGG ATTTTCTGGC CATGCATCTG

5551 TGGAGAGCGG TTGTGAGACA CAAGAATCGC CTGCTACTGT TGTCTTCCGT

5601 CCGCCCGGCG ATAATACCGA CGGAGGAGCA GCAGCAGCAG CAGGAGGAAG

5651 CCAGGCGGCG GCGGCAGGAG CAGAGCCCAT GGAACCCGAG AGCCGGCCTG

5701 GACCCTCGGG AATGAATGTT GTACAGGTGG CTGAACTGTA TCCAGAACTG 5751 AGACGCATTT TGACAATTAC AGAGGATGGG CAGGGGCTAA AGGGGGTAAA

5801 GAGGGAGCGG GGGGCTTGTG AGGCTACAGA GGAGGC TAGG AATCTAGCTT

5851 TTAGCTTAAT GACCAGACAC CGTCCTGAGT GTATTACTTT TCAACAGATC

5901 AAGGATAATT GCGCTAATGA GCTTGATCTG CTGGCGCAGA AGTATTCCAT

5951 AGAGCAGCTG ACCACTTACT GGCTGCAGCC AGGGGATGAT TTTGAGGAGG

6001 CTATTAGGGT ATATGCAAAG GTGGCACTTA GGCCAGATTG CAAGTACAAG

6051 ATCAGCAAAC TTGTAAATAT CAGGAATTGT TGCTACATTT CTGGGAACGG

6101 GGCCGAGGTG GAGATAGATA CGGAGGATAG GGTGGCCTTT AGATG TAGC A

6151 TGATAAATAT GTGGCCGGGG GTGCTTGGCA TGGACGGGGT GGTTATTATG

6201 AATGTAAGGT TTACTGGCCC CAATTTTAGC GGTACGGTTT TCCTGGCCAA

6251 TACCAACCTT ATCC TACACG GTGTAAGCTT CTATGGGTTT AACAATACC T

6301 GTGTGGAAGC CTGGACCGAT GTAAGGGTTC GGGGCTGTGC CTTTTACTGC

6351 TGCTGGAAGG GGGTGGTGTG TCGCCCCAAA AGCAGGGCTT CAATTAAGAA

6401 ATGCCTCTTT GAAAGGTGTA CCTTGGGTAT CCTGTCTGAG GGTAACTCCA

6451 GGGTGCGCCA CAATGTGGCC TCCGACTGTG GTTGCTTCAT GCTAGTGAAA

6501 AGCGTGGCTG TGATTAAGCA TAACATGGTA TGTGGCAACT GCGAGGACAG

6551 GGCCTCTCAG ATGCTGACCT GCTCGGACGG CAACTGTCAC CTGCTGAAGA

6601 CCATTCACGT AGCCAGCCAC TCTCGCAAGG CCTGGCCAGT GTTTGAGCAT

6651 AACATACTGA CCCGCTGTTC CTTGCATTTG GGTAACAGGA GGGGGGTGTT

6701 CCTACCTTAC CAATGCAATT TGAGTCACAC TAAGATAT TG CTTGAGCCCG

6751 AGAGCATGTC CAAGGTGAAC CTGAACGGGG TGTTTGACAT GACCATGAAG

6801 ATCTGGAAGG TGCTGAGGTA C GATGAGAC C CGCACCAGGT GCAGACCCTG

6851 CGAGTGTGGC GG TAAACATA TTAGGAACCA GCCTGTGATG CTGGATGTGA

6901 CCGAGGAGCT GAGGCCCGAT CACTTGGTGC TGGCCTGCAC CCGCGCTGAG

6951 TTTGGCTCTA GC GATGAAGA TACAGATTGA GGTACTGAAA TGTGTGGGCG

7001 TGGCTTAAGG GTGGGAAAGA ATATATAAGG TGGGGGTCTT ATGTAGTTTT

7051 GTATCTGTTT TGCAGCAGCC GCCGCCGCCA TGAGCACCAA CTCGTTTGAT

7101 GGAAGCATTG TGAGCTCATA TTTGACAACG CGCATGCCCC CATGGGCCGG

7151 GGTGCGTCAG AATGTGATGG GCTCCAGCAT TGATGGTCGC CCCGTCCTGC

7201 CCGCAAACTC TACTACCTTG ACC TACGAGA CCGTGTCTGG AACGCCGTTG

7251 GAGAC TGCAG CCTCCGCCGC CGCTTCAGCC GCTGCAGCCA CCGCCCGCGG

7301 GATTGTGACT GACTTTGCTT TCCTGAGCCC GC TTGCAAGC AGTGCAGCTT

7351 CCCGTTCATC CGCCCGCGAT GACAAGTTGA CGGCTCTTTT GGCACAATTG

7401 GATTCTTTGA CCCGGGAACT TAATGTCGTT TC TCAGCAGC TGTTGGATCT

7451 GCGCCAGCAG GTTTCTGCCC TGAAGGCTTC CTCCCCTCCC AATGCGGTTT

7501 AAAACATAAA TAAAAAACCA GACTCTGTTT GGATTTGGAT CAAGCAAGTG

7551 TCTTGCTGTC TTTATTTAGG GGTTTTGCGC GC GC GG TAGG CCCGGGACCA

7601 GCGGTCTCGG TCGTTGAGGG TCCTGTGTAT TTTTTCCAGG ACGTGGTAAA 7651 GGTGACTCTG GATGTTCAGA TACATGGGCA TAAGCCCGTC TCTGGGGTGG

7701 AGGTAGCACC AC TGCAGAGC TTCATGCTGC GGGGTGGTGT TGTAGATGAT

7751 CCAGTCGTAG CAGGAGCGCT GGGCGTGGTG CCTAAAAATG TCTTTCAGTA

7801 GCAAGCTGAT TGCCAGGGGC AGGCCCTTGG TGTAAGTGTT TACAAAGCGG

7851 TTAAGCTGGG ATGGGTGCAT ACGTGGGGAT ATGAGATGCA TCTTGGACTG

7901 TATTTTTAGG TTGGCTATGT TCCCAGCCAT ATCCCTCCGG GGATTCATGT

7951 TGTGCAGAAC CACCAGCACA GTGTATCCGG TGCACTTGGG AAATTTGTCA

8001 TGTAGCTTAG AAGGAAATGC GTGGAAGAAC TTGGAGACGC CCTTGTGACC

8051 TCCAAGATTT TCCATGCATT CGTCCATAAT GATGGCAATG GGCCCACGGG

8101 CGGCGGCCTG GGCGAAGATA TTTCTGGGAT CACTAACGTC ATAGTTGTGT

8151 TCCAGGATGA GATCGTCATA GGCCATTTTT ACAAAGCGCG GGCGGAGGGT

8201 GCCAGAC TGC GGTATAATGG TTCCATCCGG CCCAGGGGCG TAGTTACCCT

8251 CACAGATTTG CATTTCCCAC GCTTTGAGTT CAGATGGGGG GATCATGTCT

8301 ACC TGCGGGG CGATGAAGAA AACGGTTTCC GGGGTAGGGG AGATCAGCTG

8351 GGAAGAAAGC AGGTTCCTGA GCAGCTGCGA CTTACCGCAG CCGGTGGGCC

8401 CGTAAATCAC ACCTATTACC GGGTGCAACT GGTAGTTAAG AGAGC TGCAG

8451 CTGCCGTCAT CCCTGAGCAG GGGGGCCACT TCGTTAAGCA TGTCCCTGAC

8501 TCGCATGTTT TCCCTGACCA AATCCGCCAG AAGGCGCTCG CCGCCCAGCG

8551 ATAGCAGTTC TTGCAAGGAA GCAAAGTTTT TCAACGGTTT GAGACCGTCC

8601 GCCGTAGGCA TGCTTTTGAG CGTTTGACCA AGCAGTTCCA GGCGGTCCCA

8651 CAGCTCGGTC ACCTGCTCTA CGGCATCTCG ATCCAGCATA TCTCCTCGTT

8701 TCGCGGGTTG GGGCGGCTTT CGCTGTACGG CAGTAGTCGG TGCTCGTCCA

8751 GACGGGCCAG GGTCATGTCT TTCCACGGGC GCAGGGTCCT CGTCAGCGTA

8801 GTCTGGGTCA CGGTGAAGGG GTGCGCTCCG GGCTGCGCGC TGGCCAGGGT

8851 GCGCTTGAGG CTGGTCCTGC TGGTGCTGAA GCGCTGCCGG TCTTCGCCCT

8901 GCGCGTCGGC CAGGTAGCAT TTGACCATGG TGTCATAGTC CAGCCCCTCC

8951 GCGGCGTGGC CCTTGGCGCG CAGCTTGCCC TTGGAGGAGG CGCCGCACGA

9001 GGGGCAGTGC AGACTTTTGA GGGCGTAGAG CTTGGGCGCG AGAAATACCG

9051 ATTCCGGGGA GTAGGCATCC GCGCCGCAGG CCCCGCAGAC GGTCTCGCAT

9101 TCCACGAGCC AGGTGAGCTC TGGCCGTTCG GGGTCAAAAA CCAGGTTTCC

9151 CCCATGCTTT TTGATGCGTT TCTTACCTCT GGTTTCCATG AGCCGGTGTC

9201 CACGCTCGGT GACGAAAAGG CTGTCCGTGT CCCCGTATAC AGACTTGAGA

9251 GGCCTGTCCT CGAGCGGTGT TCCGCGGTCC TCCTCGTATA GAAACTCGGA

9301 CCACTCTGAG ACAAAGGC TC GCGTCCAGGC CAGCACGAAG GAGGCTAAGT

9351 GGGAGGGGTA GCGGTCGTTG TCCAC TAGGG GGTCCACTCG CTCCAGGGTG

9401 TGAAGACACA TGTCGCCCTC TTCGGCATCA AGGAAGGTGA TTGGTTTGTA

9451 GGTGTAGGCC ACGTGACCGG GTGTTCCTGA AGGGGGGCTA TAAAAGGGGG

9501 TGGGGGCGCG TTCGTCCTCA CTCTCTTCCG CATCGCTGTC TGCGAGGGCC 9551 AGCTGTTGGG GTGAGTACTC CCTCTGAAAA GCGGGCATGA CTTCTGCGCT

9601 AAGATTGTCA GTTTCCAAAA ACGAGGAGGA TTTGATATTC ACCTGGCCCG

9651 CGGTGATGCC TTTGAGGGTG GCCGCATCCA TCTGGTCAGA AAAGACAATC

9701 TTTTTGTTGT CAAGCTTGGT GGCAAACGAC CCGTAGAGGG CGTTGGACAG

9751 CAACTTGGCG ATGGAGCGCA GGGTTTGGTT TTTGTCGCGA TCGGCGCGCT

9801 CCTTGGCCGC GATGTTTAGC TGCACGTATT CGCGCGCAAC GCACCGCCAT

9851 TCGGGAAAGA CGGTGGTGCG CTCGTCGGGC ACCAGGTGCA CGCGCCAACC

9901 GCGGTTGTGC AGGGTGACAA GGTCAACGCT GGTGGCTACC TCTCCGCGTA

9951 GGCGCTCGTT GGTCCAGCAG AGGCGGCCGC CCTTGCGCGA GCAGAATGGC

10001 GGTAGGGGGT CTAGCTGCGT CTCGTCCGGG GGGTCTGCGT CCACGGTAAA

10051 GACCCCGGGC AGCAGGCGCG CGTCGAAGTA GTCTATCTTG CATCCTTGCA

10101 AGTCTAGCGC CTGCTGCCAT GCGCGGGCGG CAAGCGCGCG CTCGTATGGG

10151 TTGAGTGGGG GACCCCATGG CATGGGGTGG GTGAGCGCGG AGGCGTACAT

10201 GCCGCAAATG TCGTAAACGT AGAGGGGCTC TCTGAGTATT CCAAGATATG

10251 TAGGGTAGCA TCTTCCACCG CGGATGCTGG CGCGCACGTA ATCGTATAGT

10301 TCGTGCGAGG GAGCGAGGAG GTCGGGACCG AGGTTGCTAC GGGCGGGCTG

10351 CTCTGCTCGG AAGACTATCT GCCTGAAGAT GGCATGTGAG TTGGATGATA

10401 TGGTTGGACG CTGGAAGACG TTGAAGCTGG CGTCTGTGAG ACCTACCGCG

10451 TCACGCACGA AGGAGGCGTA GGAGTCGCGC AGCTTGTTGA CCAGCTCGGC

10501 GGTGACCTGC ACGTCTAGGG CGCAGTAGTC CAGGGTTTCC TTGATGATGT

10551 CATACTTATC CTGTCCCTTT TTTTTCCACA GCTCGCGGTT GAGGACAAAC

10601 TCTTCGCGGT CTTTCCAGTA CTCTTGGATC GGAAACCCGT CGGCCTCCGA

10651 ACGGTAAGAG CCTAGCATGT AGAACTGGTT GACGGCCTGG TAGGCGCAGC

10701 ATCCCTTTTC TACGGGTAGC GCGTATGCCT GCGCGGCCTT CCGGAGCGAG

10751 GTGTGGGTGA GCGCAAAGGT GTCCCTGACC ATGACTTTGA GGTACTGGTA

10801 TTTGAAGTCA GTGTCGTCGC ATCCGCCCTG CTCCCAGAGC AAAAAGTCCG

10851 TGCGCTTTTT GGAACGCGGA TTTGGCAGGG CGAAGGTGAC ATCGTTGAAG

10901 AGTATCTTTC CCGCGCGAGG CATAAAGTTG CGTGTGATGC GGAAGGGTCC

10951 CGGCACCTCG GAACGGTTGT TAATTACCTG GGCGGCGAGC ACGATCTCGT

11001 CAAAGCCGTT GATGTTGTGG CCCACAATGT AAAGTTCCAA GAAGCGCGGG

11051 ATGCCCTTGA TGGAAGGCAA TTTTTTAAGT TCCTCGTAGG TGAGCTCTTC

11101 AGGGGAGCTG AGCCCGTGCT CTGAAAGGGC CCAGTCTGCA AGATGAGGGT

11151 TGGAAGCGAC GAATGAGCTC CACAGGTCAC GGGCCATTAG CATTTGCAGG

11201 TGGTCGCGAA AGGTCCTAAA CTGGCGACCT ATGGCCATTT TTTCTGGGGT

11251 GATGCAGTAG AAGGTAAGCG GGTCTTGTTC CCAGCGGTCC CATCCAAGGT

11301 TCGCGGCTAG GTCTCGCGCG GCAGTCACTA GAGGCTCATC TCCGCCGAAC

11351 TTCATGACCA GCATGAAGGG CACGAGCTGC TTCCCAAAGG CCCCCATCCA

11401 AGTATAGGTC TCTACATCGT AGGTGACAAA GAGACGCTCG GTGCGAGGAT 11451 GCGAGCCGAT CGGGAAGAAC TGGATCTCCC GCCACCAATT GGAGGAGTGG

11501 CTATTGATGT GGTGAAAGTA GAAGTCCCTG CGACGGGCCG AACACTCGTG

11551 CTGGCTTTTG TAAAAACGTG CGCAGTACTG GCAGCGGTGC ACGGGCTGTA

11601 CATCCTGCAC GAGGTTGACC TGACGACCGC GCACAAGGAA GCAGAGTGGG

11651 AATTTGAGCC CCTCGCCTGG CGGGTTTGGC TGGTGGTCTT CTACTTCGGC

11701 TGCTTGTCCT TGACCGTCTG GCTGCTCGAG GGGAGTTACG GTGGATCGGA

11751 CCACCACGCC GCGCGAGCCC AAAGTCCAGA TGTCCGCGCG CGGCGGTCGG

11801 AGCTTGATGA CAACATCGCG CAGATGGGAG CTGTCCATGG TCTGGAGCTC

11851 CCGCGGCGTC AGGTCAGGCG GGAGCTCCTG CAGGTTTACC TCGCATAGAC

11901 GGGTCAGGGC GCGGGCTAGA TCCAGGTGAT ACCTAATTTC CAGGGGCTGG

11951 TTGGTGGCGG CGTCGATGGC TTGCAAGAGG CCGCATCCCC GCGGCGCGAC

12001 TACGGTACCG CGCGGCGGGC GGTGGGCCGC GGGGGTGTCC TTGGATGATG

12051 CATCTAAAAG CGGTGACGCG GGCGAGCCCC CGGAGGTAGG GGGGGCTCCG

12101 GACCCGCCGG GAGAGGGGGC AGGGGCACGT CGGCGCCGCG CGCGGGCAGG

12151 AGCTGGTGCT GCGCGCGTAG GTTGCTGGCG AACGCGACGA CGCGGCGGTT

12201 GATCTCCTGA ATCTGGCGCC TCTGCGTGAA GACGACGGGC CCGGTGAGCT

12251 TGAGCCTGAA AGAGAGTTCG ACAGAATCAA TTTCGGTGTC GTTGACGGCG

12301 GCCTGGCGCA AAATCTCCTG CACGTCTCCT GAGTTGTCTT GATAGGCGAT

12351 CTCGGCCATG AACTGCTCGA TCTCTTCCTC CTGGAGATCT CCGCGTCCGG

12401 CTCGCTCCAC GGTGGCGGCG AGGTCGTTGG AAATGCGGGC CATGAGCTGC

12451 GAGAAGGCGT TGAGGCCTCC CTCGTTCCAG ACGCGGCTGT AGACCACGCC

12501 CCCTTCGGCA TCGCGGGCGC GCATGACCAC CTGCGCGAGA TTGAGCTCCA

12551 CGTGCCGGGC GAAGACGGCG TAGTTTCGCA GGCGCTGAAA GAGGTAGTTG

12601 AGGGTGGTGG CGGTGTGTTC TGCCACGAAG AAGTACATAA CCCAGCGTCG

12651 CAACGTGGAT TCGTTGATAT CCCCCAAGGC CTCAAGGCGC TCCATGGCCT

12701 CGTAGAAGTC CACGGCGAAG TTGAAAAACT GGGAGTTGCG CGCCGACACG

12751 GTTAACTCCT CCTCCAGAAG ACGGATGAGC TCGGCGACAG TGTCGCGCAC

12801 CTCGCGCTCA AAGGCTACAG GGGCCTCTTC TTCTTCTTCA ATCTCCTCTT

12851 CCATAAGGGC CTCCCCTTCT TCTTCTTCTG GCGGCGGTGG GGGAGGGGGG

12901 ACACGGCGGC GACGACGGCG CACCGGGAGG CGGTCGACAA AGCGCTCGAT

12951 CATCTCCCCG CGGCGACGGC GCATGGTCTC GGTGACGGCG CGGCCGTTCT

13001 CGCGGGGGCG CAGTTGGAAG ACGCCGCCCG TCATGTCCCG GTTATGGGTT

13051 GGCGGGGGGC TGCCATGCGG CAGGGATACG GCGCTAACGA TGCATCTCAA

13101 CAATTGTTGT GTAGGTACTC CGCCGCCGAG GGACCTGAGC GAGTCCGCAT

13151 CGACCGGATC GGAAAACCTC TCGAGAAAGG CGTCTAACCA GTCACAGTCG

13201 CAAGGTAGGC TGAGCACCGT GGCGGGCGGC AGCGGGCGGC GGTCGGGGTT

13251 GTTTCTGGCG GAGGTGCTGC TGATGATGTA ATTAAAGTAG GCGGTCTTGA

13301 GACGGCGGAT GGTCGACAGA AGCACCATGT CCTTGGGTCC GGCCTGCTGA 13351 ATGCGCAGGC GGTCGGCCAT GCCCCAGGCT TCGTTTTGAC ATCGGCGCAG

13401 GTCTTTGTAG TAGTCTTGCA TGAGCCTTTC TACCGGCACT TCTTCTTCTC

13451 CTTCCTCTTG TCCTGCATCT CTTGCATCTA TCGCTGCGGC GGCGGCGGAG

13501 TTTGGCCGTA GGTGGCGCCC TCTTCCTCCC ATGCGTGTGA CCCCGAAGCC

13551 CCTCATCGGC TGAAGCAGGG CTAGGTCGGC GACAACGCGC TCGGCTAATA

13601 TGGCCTGCTG CACCTGCGTG AGGGTAGACT GGAAGTCATC CATGTCCACA

13651 AAGCGGTGGT ATGCGCCCGT GTTGATGGTG TAAGTGCAGT TGGCCATAAC

13701 GGACCAGTTA ACGGTCTGGT GACCCGGCTG CGAGAGCTCG GTGTACCTGA

13751 GACGCGAGTA AGCCCTCGAG TCAAATACGT AGTCGTTGCA AGTCCGCACC

13801 AGGTACTGGT ATCCCACCAA AAAGTGCGGC GGCGGCTGGC GGTAGAGGGG

13851 CCAGCGTAGG GTGGCCGGGG CTCCGGGGGC GAGATCTTCC AACATAAGGC

13901 GATGATATCC GTAGATGTAC CTGGACATCC AGGTGATGCC GGCGGCGGTG

13951 GTGGAGGCGC GCGGAAAGTC GCGGACGCGG TTCCAGATGT TGCGCAGCGG

14001 CAAAAAGTGC TCCATGGTCG GGACGCTCTG GCCGGTCAGG CGCGCGCAAT

14051 CGTTGACGCT CTAGACCGTG CAAAAGGAGA GCCTGTAAGC GGGCACTCTT

14101 CCGTGGTCTG GTGGATAAAT TCGCAAGGGT ATCATGGCGG ACGACCGGGG

14151 TTCGAGCCCC GTATCCGGCC GTCCGCCGTG ATCCATGCGG TTACCGCCCG

14201 CGTGTCGAAC CCAGGTGTGC GACGTCAGAC AACGGGGGAG TGCTCCTTTT

14251 GGCTTCCTTC CAGGCGCGGC GGCTGCTGCG CTAGCTTTTT TGGCCACTGG

14301 CCGCGCGCAG CGTAAGCGGT TAGGCTGGAA AGCGAAAGCA TTAAGTGGCT

14351 CGCTCCCTGT AGCCGGAGGG TTATTTTCCA AGGGTTGAGT CGCGGGACCC

14401 CCGGTTCGAG TCTCGGACCG GCCGGACTGC GGCGAACGGG GGTTTGCCTC

14451 CCCGTCATGC AAGACCCCGC TTGCAAATTC CTCCGGAAAC AGGGACGAGC

14501 CCCTTTTTTG CTTTTCCCAG ATGCATCCGG TGCTGCGGCA GATGCGCCCC

14551 CCTCCTCAGC AGCGGCAAGA GCAAGAGCAG CGGCAGACAT GCAGGGCACC

14601 CTCCCCTCCT CCTACCGCGT CAGGAGGGGC GACATCCGCG GTTGACGCGG

14651 CAGCAGATGG TGATTACGAA CCCCCGCGGC GCCGGGCCCG GCACTACCTG

14701 GACTTGGAGG AGGGCGAGGG CCTGGCGCGG CTAGGAGCGC CCTCTCCTGA

14751 GCGGTACCCA AGGGTGCAGC TGAAGCGTGA TACGCGTGAG GCGTACGTGC

14801 CGCGGCAGAA CCTGTTTCGC GACCGCGAGG GAGAGGAGCC CGAGGAGATG

14851 CGGGATCGAA AGTTCCACGC AGGGCGCGAG CTGCGGCATG GCCTGAATCG

14901 CGAGCGGTTG CTGCGCGAGG AGGACTTTGA GCCCGACGCG CGAACCGGGA

14951 TTAGTCCCGC GCGCGCACAC GTGGCGGCCG CCGACCTGGT AACCGCATAC

15001 GAGCAGACGG TGAACCAGGA GATTAACTTT CAAAAAAGCT TTAACAACCA

15051 CGTGCGTACG CTTGTGGCGC GCGAGGAGGT GGCTATAGGA CTGATGCATC

15101 TGTGGGACTT TGTAAGCGCG CTGGAGCAAA ACCCAAATAG CAAGCCGCTC

15151 ATGGCGCAGC TGTTCCTTAT AGTGCAGCAC AGCAGGGACA ACGAGGCATT

15201 CAGGGATGCG CTGCTAAACA TAGTAGAGCC CGAGGGCCGC TGGCTGCTCG 15251 ATTTGATAAA CATCCTGCAG AGCATAGTGG TGCAGGAGCG CAGCTTGAGC

15301 CTGGCTGACA AGGTGGCCGC CATCAACTAT TCCATGCTTA GCCTGGGCAA

15351 GTTTTACGCC CGCAAGATAT ACCATACCCC TTACGTTCCC ATAGACAAGG

15401 AGGTAAAGAT CGAGGGGTTC TACATGCGCA TGGCGCTGAA GGTGCTTACC

15451 TTGAGCGACG ACCTGGGCGT TTATCGCAAC GAGCGCATCC ACAAGGCCGT

15501 GAGCGTGAGC CGGCGGCGCG AGCTCAGCGA CCGCGAGCTG ATGCACAGCC

15551 TGCAAAGGGC CCTGGCTGGC ACGGGCAGCG GCGATAGAGA GGCCGAGTCC

15601 TACTTTGACG CGGGCGCTGA CCTGCGCTGG GCCCCAAGCC GACGCGCCCT

15651 GGAGGCAGCT GGGGCCGGAC CTGGGCTGGC GGTGGCACCC GCGCGCGCTG

15701 GCAACGTCGG CGGCGTGGAG GAATATGACG AGGACGATGA GTACGAGCCA

15751 GAGGACGGCG AGTACTAAGC GGTGATGTTT CTGATCAGAT GATGCAAGAC

15801 GCAACGGACC CGGCGGTGCG GGCGGCGCTG CAGAGCCAGC CGTCCGGCCT

15851 TAACTCCACG GACGACTGGC GCCAGGTCAT GGACCGCATC ATGTCGCTGA

15901 CTGCGCGCAA TCCTGACGCG TTCCGGCAGC AGCCGCAGGC CAACCGGCTC

15951 TCCGCAATTC TGGAAGCGGT GGTCCCGGCG CGCGCAAACC CCACGCACGA

16001 GAAGGTGCTG GCGATCGTAA ACGCGCTGGC CGAAAACAGG GCCATCCGGC

16051 CCGACGAGGC CGGCCTGGTC TACGACGCGC TGCTTCAGCG CGTGGCTCGT

16101 TACAACAGCG GCAACGTGCA GACCAACCTG GACCGGCTGG TGGGGGATGT

16151 GCGCGAGGCC GTGGCGCAGC GTGAGCGCGC GCAGCAGCAG GGCAACCTGG

16201 GCTCCATGGT TGCACTAAAC GCCTTCCTGA GTACACAGCC CGCCAACGTG

16251 CCGCGGGGAC AGGAGGACTA CACCAACTTT GTGAGCGCAC TGCGGCTAAT

16301 GGTGACTGAG ACACCGCAAA GTGAGGTGTA CCAGTCTGGG CCAGACTATT

16351 TTTTCCAGAC CAGTAGACAA GGCCTGCAGA CCGTAAACCT GAGCCAGGCT

16401 TTCAAAAACT TGCAGGGGCT GTGGGGGGTG CGGGCTCCCA CAGGCGACCG

16451 CGCGACCGTG TCTAGCTTGC TGACGCCCAA CTCGCGCCTG TTGCTGCTGC

16501 TAATAGCGCC CTTCACGGAC AGTGGCAGCG TGTCCCGGGA CACATACCTA

16551 GGTCACTTGC TGACACTGTA CCGCGAGGCC ATAGGTCAGG CGCATGTGGA

16601 CGAGCATACT TTCCAGGAGA TTACAAGTGT CAGCCGCGCG CTGGGGCAGG

16651 AGGACACGGG CAGCCTGGAG GCAACCCTAA ACTACCTGCT GACCAACCGG

16701 CGGCAGAAGA TCCCCTCGTT GCACAGTTTA AACAGCGAGG AGGAGCGCAT

16751 TTTGCGCTAC GTGCAGCAGA GCGTGAGCCT TAACCTGATG CGCGACGGGG

16801 TAACGCCCAG CGTGGCGCTG GACATGACCG CGCGCAACAT GGAACCGGGC

16851 ATGTATGCCT CAAACCGGCC GTTTATCAAC CGCCTAATGG ACTACTTGCA

16901 TCGCGCGGCC GCCGTGAACC CCGAGTATTT CACCAATGCC ATCTTGAACC

16951 CGCACTGGCT ACCGCCCCCT GGTTTCTACA CCGGGGGATT CGAGGTGCCC

17001 GAGGGTAACG ATGGATTCCT CTGGGACGAC ATAGACGACA GCGTGTTTTC

17051 CCCGCAACCG CAGACCCTGC TAGAGTTGCA ACAGCGCGAG CAGGCAGAGG

17101 CGGCGCTGCG AAAGGAAAGC TTCCGCAGGC CAAGCAGCTT GTCCGATCTA 17151 GGCGCTGCGG CCCCGCGGTC AGATGCTAGT AGCCCATTTC CAAGCTTGAT

17201 AGGGTCTCTT ACCAGCACTC GCACCACCCG CCCGCGCCTG CTGGGCGAGG

17251 AGGAGTACCT AAACAACTCG CTGCTGCAGC CGCAGCGCGA AAAAAACCTG

17301 CCTCCGGCAT TTCCCAACAA CGGGATAGAG AGCCTAGTGG ACAAGATGAG

17351 TAGATGGAAG ACGTACGCGC AGGAGCACAG GGACGTGCCA GGCCCGCGCC

17401 CGCCCACCCG TCGTCAAAGG CACGACCGTC AGCGGGGTCT GGTGTGGGAG

17451 GACGATGACT CGGCAGACGA CAGCAGCGTC CTGGATTTGG GAGGGAGTGG

17501 CAACCCGTTT GCGCACCTTC GCCCCAGGCT GGGGAGAATG TTTTAAAAAA

17551 AAAAAAGCAT GATGCAAAAT AAAAAACTCA CCAAGGCCAT GGCACCGAGC

17601 GTTGGTTTTC TTGTATTCCC CTTAGTATGC GGCGCGCGGC GATGTATGAG

17651 GAAGGTCCTC CTCCCTCCTA CGAGAGTGTG GTGAGCGCGG CGCCAGTGGC

17701 GGCGGCGCTG GGTTCTCCCT TCGATGCTCC CCTGGACCCG CCGTTTGTGC

17751 CTCCGCGGTA CCTGCGGCCT ACCGGGGGGA GAAACAGCAT CCGTTACTCT

17801 GAGTTGGCAC CCCTATTCGA CACCACCCGT GTGTACCTGG TGGACAACAA

17851 GTCAACGGAT GTGGCATCCC TGAACTACCA GAACGACCAC AGCAACTTTC

17901 TGACCACGGT CATTCAAAAC AATGACTACA GCCCGGGGGA GGCAAGCACA

17951 CAGACCATCA ATCTTGACGA CCGGTCGCAC TGGGGCGGCG ACCTGAAAAC

18001 CATCCTGCAT ACCAACATGC CAAATGTGAA CGAGTTCATG TTTACCAATA

18051 AGTTTAAGGC GCGGGTGATG GTGTCGCGCT TGCCTACTAA GGACAATCAG

18101 GTGGAGCTGA AATACGAGTG GGTGGAGTTC ACGCTGCCCG AGGGCAACTA

18151 CTCCGAGACC ATGACCATAG ACCTTATGAA CAACGCGATC GTGGAGCACT

18201 ACTTGAAAGT GGGCAGACAG AACGGGGTTC TGGAAAGCGA CATCGGGGTA

18251 AAGTTTGACA CCCGCAACTT CAGACTGGGG TTTGACCCCG TCACTGGTCT

18301 TGTCATGCCT GGGGTATATA CAAACGAAGC CTTCCATCCA GACATCATTT

18351 TGCTGCCAGG ATGCGGGGTG GACTTCACCC ACAGCCGCCT GAGCAACTTG

18401 TTGGGCATCC GCAAGCGGCA ACCCTTCCAG GAGGGCTTTA GGATCACCTA

18451 CGATGATCTG GAGGGTGGTA ACATTCCCGC ACTGTTGGAT GTGGACGCCT

18501 ACCAGGCGAG CTTGAAAGAT GACACCGAAC AGGGCGGGGG TGGCGCAGGC

18551 GGCAGCAACA GCAGTGGCAG CGGCGCGGAA GAGAACTCCA ACGCGGCAGC

18601 CGCGGCAATG CAGCCGGTGG AGGACATGAA CGATCATGCC ATTCGCGGCG

18651 ACACCTTTGC CACACGGGCT GAGGAGAAGC GCGCTGAGGC CGAAGCAGCG

18701 GCCGAAGCTG CCGCCCCCGC TGCGCAACCC GAGGTCGAGA AGCCTCAGAA

18751 GAAACCGGTG ATCAAACCCC TGACAGAGGA CAGCAAGAAA CGCAGTTACA

18801 ACCTAATAAG CAATGACAGC ACCTTCACCC AGTACCGCAG CTGGTACCTT

18851 GCATACAACT ACGGCGACCC TCAGACCGGA ATCCGCTCAT GGACCCTGCT

18901 TTGCACTCCT GACGTAACCT GCGGCTCGGA GCAGGTCTAC TGGTCGTTGC

18951 CAGACATGAT GCAAGACCCC GTGACCTTCC GCTCCACGCG CCAGATCAGC

19001 AACTTTCCGG TGGTGGGCGC CGAGCTGTTG CCCGTGCACT CCAAGAGCTT 19051 CTACAACGAC CAGGCCGTCT ACTCCCAACT CATCCGCCAG TTTACCTCTC

19101 TGACCCACGT GTTCAATCGC TTTCCCGAGA ACCAGATTTT GGCGCGCCCG

19151 CCAGCCCCCA CCATCACCAC CGTCAGTGAA AACGTTCCTG CTCTCACAGA

19201 TCACGGGACG CTACCGCTGC GCAACAGCAT CGGAGGAGTC CAGCGAGTGA

19251 CCATTACTGA CGCCAGACGC CGCACCTGCC CCTACGTTTA CAAGGCCCTG

19301 GGCATAGTCT CGCCGCGCGT CCTATCGAGC CGCACTTTTT GAGCAAGCAT

19351 GTCCATCCTT ATATCGCCCA GCAATAACAC AGGCTGGGGC CTGCGCTTCC

19401 CAAGCAAGAT GTTTGGCGGG GCCAAGAAGC GCTCCGACCA ACACCCAGTG

19451 CGCGTGCGCG GGCACTACCG CGCGCCCTGG GGCGCGCACA AACGCGGCCG

19501 CACTGGGCGC ACCACCGTCG ATGACGCCAT CGACGCGGTG GTGGAGGAGG

19551 CGCGCAACTA CACGCCCACG CCGCCACCAG TGTCCACAGT GGACGCGGCC

19601 ATTCAGACCG TGGTGCGCGG AGCCCGGCGC TATGCTAAAA TGAAGAGACG

19651 GCGGAGGCGC GTAGCACGTC GCCACCGCCG CCGACCCGGC ACTGCCGCCC

19701 AACGCGCGGC GGCGGCCCTG CTTAACCGCG CACGTCGCAC CGGCCGACGG

19751 GCGGCCATGC GGGCCGCTCG AAGGCTGGCC GCGGGTATTG TCACTGTGCC

19801 CCCCAGGTCC AGGCGACGAG CGGCCGCCGC AGCAGCCGCG GCCATTAGTG

19851 CTATGACTCA GGGTCGCAGG GGCAACGTGT ATTGGGTGCG CGACTCGGTT

19901 AGCGGCCTGC GCGTGCCCGT GCGCACCCGC CCCCCGCGCA ACTAGATTGC

19951 AAGAAAAAAC TACTTAGACT CGTACTGTTG TATGTATCCA GCGGCGGCGG

20001 CGCGCAACGA AGCTATGTCC AAGCGCAAAA TCAAAGAAGA GATGCTCCAG

20051 GTCATCGCGC CGGAGATCTA TGGCCCCCCG AAGAAGGAAG AGCAGGATTA

20101 CAAGCCCCGA AAGCTAAAGC GGGTCAAAAA GAAAAAGAAA GATGATGATG

20151 ATGAACTTGA CGACGAGGTG GAACTGCTGC ACGCTACCGC GCCCAGGCGA

20201 CGGGTACAGT GGAAAGGTCG ACGCGTAAAA CGTGTTTTGC GACCCGGCAC

20251 CACCGTAGTC TTTACGCCCG GTGAGCGCTC CACCCGCACC TACAAGCGCG

20301 TGTATGATGA GGTGTACGGC GACGAGGACC TGCTTGAGCA GGCCAACGAG

20351 CGCCTCGGGG AGTTTGCCTA CGGAAAGCGG CATAAGGACA TGCTGGCGTT

20401 GCCGCTGGAC GAGGGCAACC CAACACCTAG CCTAAAGCCC GTAACACTGC

20451 AGCAGGTGCT GCCCGCGCTT GCACCGTCCG AAGAAAAGCG CGGCCTAAAG

20501 CGCGAGTCTG GTGACTTGGC ACCCACCGTG CAGCTGATGG TACCCAAGCG

20551 CCAGCGACTG GAAGATGTCT TGGAAAAAAT GACCGTGGAA CCTGGGCTGG

20601 AGCCCGAGGT CCGCGTGCGG CCAATCAAGC AGGTGGCGCC GGGACTGGGC

20651 GTGCAGACCG TGGACGTTCA GATACCCACT ACCAGTAGCA CCAGTATTGC

20701 CACCGCCACA GAGGGCATGG AGACACAAAC GTCCCCGGTT GCCTCAGCGG

20751 TGGCGGATGC CGCGGTGCAG GCGGTCGCTG CGGCCGCGTC CAAGACCTCT

20801 ACGGAGGTGC AAACGGACCC GTGGATGTTT CGCGTTTCAG CCCCCCGGCG

20851 CCCGCGCGGT TCGAGGAAGT ACGGCGCCGC CAGCGCGCTA CTGCCCGAAT

20901 ATGCCCTACA TCCTTCCATT GCGCCTACCC CCGGCTATCG TGGCTACACC 20951 TACCGCCCCA GAAGACGAGC AACTACCCGA CGCCGAACCA CCACTGGAAC

21001 CCGCCGCCGC CGTCGCCGTC GCCAGCCCGT GCTGGCCCCG ATTTCCGTGC

21051 GCAGGGTGGC TCGCGAAGGA GGCAGGACCC TGGTGCTGCC AACAGCGCGC

21101 TACCACCCCA GCATCGTTTA AAAGCCGGTC TTTGTGGTTC TTGCAGATAT

21151 GGCCCTCACC TGCCGCCTCC GTTTCCCGGT GCCGGGATTC CGAGGAAGAA

21201 TGCACCGTAG GAGGGGCATG GCCGGCCACG GCCTGACGGG CGGCATGCGT

21251 CGTGCGCACC ACCGGCGGCG GCGCGCGTCG CACCGTCGCA TGCGCGGCGG

21301 TATCCTGCCC CTCCTTATTC CACTGATCGC CGCGGCGATT GGCGCCGTGC

21351 CCGGAATTGC ATCCGTGGCC TTGCAGGCGC AGAGACACTG ATTAAAAACA

21401 AGTTGCATGT GGAAAAATCA AAATAAAAAG TCTGGACTCT CACGCTCGCT

21451 TGGTCCTGTA ACTATTTTGT AGAATGGAAG ACATCAACTT TGCGTCTCTG

21501 GCCCCGCGAC ACGGCTCGCG CCCGTTCATG GGAAACTGGC AAGATATCGG

21551 CACCAGCAAT ATGAGCGGTG GCGCCTTCAG CTGGGGCTCG CTGTGGAGCG

21601 GCATTAAAAA TTTCGGTTCC ACCGTTAAGA ACTATGGCAG CAAGGCCTGG

21651 AACAGCAGCA CAGGCCAGAT GCTGAGGGAT AAGTTGAAAG AGCAAAATTT

21701 CCAACAAAAG GTGGTAGATG GCCTGGCCTC TGGCATTAGC GGGGTGGTGG

21751 ACCTGGCCAA CCAGGCAGTG CAAAATAAGA TTAACAGTAA GCTTGATCCC

21801 CGCCCTCCCG TAGAGGAGCC TCCACCGGCC GTGGAGACAG TGTCTCCAGA

21851 GGGGCGTGGC GAAAAGCGTC CGCGCCCCGA CAGGGAAGAA ACTCTGGTGA

21901 CGCAAATAGA CGAGCCTCCC TCGTACGAGG AGGCACTAAA GCAAGGCCTG

21951 CCCACCACCC GTCCCATCGC GCCCATGGCT ACCGGAGTGC TGGGCCAGCA

22001 CACACCCGTA ACGCTGGACC TGCCTCCCCC CGCCGACACC CAGCAGAAAC

22051 CTGTGCTGCC AGGCCCGACC GCCGTTGTTG TAACCCGTCC TAGCCGCGCG

22101 TCCCTGCGCC GCGCCGCCAG CGGTCCGCGA TCGTTGCGGC CCGTAGCCAG

22151 TGGCAACTGG CAAAGCACAC TGAACAGCAT CGTGGGTCTG GGGGTGCAAT

22201 CCCTGAAGCG CCGACGATGC TTCTGAATAG CTAACGTGTC GTATGTGTGT

22251 CATGTATGCG TCCATGTCGC CGCCAGAGGA GCTGCTGAGC CGCCGCGCGC

22301 CCGCTTTCCA AGATGGCTAC CCCTTCGATG ATGCCGCAGT GGTCTTACAT

22351 GCACATCTCG GGCCAGGACG CCTCGGAGTA CCTGAGCCCC GGGCTGGTGC

22401 AGTTTGCCCG CGCCACCGAG ACGTACTTCA GCCTGAATAA CAAGTTTAGA

22451 AACCCCACGG TGGCGCCTAC GCACGACGTG ACCACAGACC GGTCCCAGCG

22501 TTTGACGCTG CGGTTCATCC CTGTGGACCG TGAGGATACT GCGTACTCGT

22551 ACAAGGCGCG GTTCACCCTA GCTGTGGGTG ATAACCGTGT GCTGGACATG

22601 GCTTCCACGT ACTTTGACAT CCGCGGCGTG CTGGACAGGG GCCCTACTTT

22651 TAAGCCCTAC TCTGGCACTG CCTACAACGC CCTGGCTCCC AAGGGTGCCC

22701 CAAATCCTTG CGAATGGGAT GAAGCTGCTA CTGCTCTTGA AATAAACCTA

22751 GAAGAAGAGG ACGATGACAA CGAAGACGAA GTAGACGAGC AAGCTGAGCA

22801 GCAAAAAACT CACGTATTTG GGCAGGCGCC TTATTCTGGT ATAAATATTA 22851 CAAAGGAGGG TATTCAAATA GGTGTCGAAG GTCAAACACC TAAATATGCC

22901 GATAAAACAT TTCAACCTGA ACC TCAAATA GGAGAATC TC AGTGGTACGA

22951 AACTGAAATT AATCATGCAG CTGGGAGAGT CC TTAAAAAG ACTACCCCAA

23001 TGAAACCATG TTACGGTTCA TATGCAAAAC CCACAAATGA AAATGGAGGG

23051 CAAGGCATTC TTGTAAAGCA ACAAAATGGA AAGC TAGAAA GTCAAGTGGA

23101 AATGCAATTT TTCTCAACTA CTGAGGCGAC CGCAGGCAAT GGTGATAAC T

23151 TGACTCCTAA AGTGGTATTG TACAGTGAAG ATGTAGATAT AGAAACCCCA

23201 GACACTCATA TTTCTTACAT GCCCACTATT AAGGAAGG TA ACTCACGAGA

23251 ACTAATGGGC CAACAATCTA TGCCCAACAG GCCTAATTAC ATTGCTTTTA

23301 GGGACAATTT TATTGGTCTA ATGTATTACA ACAGCACGGG TAATATGGGT

23351 GTTCTGGCGG GCCAAGCATC GCAGTTGAAT GCTGTTGTAG ATTTGCAAGA

23401 CAGAAACACA GAGCTTTCAT ACCAGCTTTT GC TTGATTCC ATTGGTGATA

23451 GAACCAGGTA CTTTTCTATG TGGAATCAGG CTGTTGACAG C TATGATCCA

23501 GATGTTAGAA TTATTGAAAA TCATGGAACT GAAGATGAAC TTCCAAATTA

23551 CTGCTTTCCA CTGGGAGGTG TGATTAATAC AGAGACTCTT ACCAAGGTAA

23601 AACCTAAAAC AGGTCAGGAA AATGGATGGG AAAAAGATGC TACAGAATTT

23651 TCAGATAAAA ATGAAATAAG AGTTGGAAAT AATTTTGCCA TGGAAATCAA

23701 TCTAAATGCC AACCTGTGGA GAAATTTCCT GTACTCCAAC ATAGCGCTGT

23751 ATTTGCCCGA CAAGCTAAAG TACAGTCCTT CCAACGTAAA AATTTCTGAT

23801 AACCCAAACA CCTACGACTA CATGAACAAG CGAGTGGTGG CTCCCGGGTT

23851 AGTGGACTGC TACATTAACC TTGGAGCACG CTGGTCCCTT GAC TATATGG

23901 ACAACGTCAA CCCATTTAAC CACCACCGCA ATGCTGGCCT GCGCTACCGC

23951 TCAATGTTGC TGGGCAATGG TCGCTATGTG CCCTTCCACA TCCAGGTGCC

24001 TCAGAAGTTC TTTGCCATTA AAAACCTCCT TCTCCTGCCG GGC TCATACA

24051 CCTACGAGTG GAAC TTCAGG AAGGATGTTA ACATGGTTCT GCAGAGCTCC

24101 C TAGGAAATG ACCTAAGGGT TGACGGAGCC AGCATTAAGT TTGATAGCAT

24151 TTGCCTTTAC GCCACCTTCT TCCCCATGGC CCACAACACC GCCTCCACGC

24201 TTGAGGCCAT GC TTAGAAAC G AC AC C AAC G ACCAGTCCTT TAACGAC TAT

24251 CTCTCCGCCG CCAACATGCT CTACCCTATA CCCGCCAACG CTACCAACGT

24301 GCCCATATCC ATCCCCTCCC GCAACTGGGC GGCTTTCCGC GGCTGGGCCT

24351 TCACGCGCCT TAAGACTAAG GAAACCCCAT CACTGGGCTC GGGCTACGAC

24401 CCTTATTACA CCTACTCTGG CTCTATACCC TACCTAGATG GAACCTTTTA

24451 CCTCAACCAC ACCTTTAAGA AGGTGGCCAT TACCTTTGAC TCTTCTGTCA

24501 GCTGGCCTGG CAATGACCGC CTGCTTACCC CCAACGAGTT TGAAATTAAG

24551 CGCTCAGTTG ACGGGGAGGG TTACAACGTT GCCCAGTGTA ACATGAC CAA

24601 AGACTGGTTC CTGGTACAAA TGCTAGCTAA CTACAACATT GGC TACCAGG

24651 GCTTCTATAT CCCAGAGAGC TAC AAGGAC C GCATGTAC TC CTTCTTTAGA

24701 AACTTCCAGC CCATGAGCCG TCAGGTGGTG GATGATACTA AATACAAGGA 24751 CTACCAACAG GTGGGCATCC TACACCAACA CAACAACTCT GGATTTGTTG

24801 GCTACCTTGC CCCCACCATG CGCGAAGGAC AGGCCTACCC TGCTAACTTC

24851 CCCTATCCGC TTATAGGCAA GACCGCAGTT GACAGC AT TA CCCAGAAAAA

24901 GTTTCTTTGC GATCGCACCC TTTGGCGCAT CCCATTCTCC AGTAACTTTA

24951 TGTCCATGGG CGCACTCACA GACCTGGGCC AAAACCTTCT CTACGCCAAC

25001 TCCGCCCACG CGCTAGACAT GACTTTTGAG GTGGATCCCA TGGACGAGCC

25051 CACCCTTCTT TATGTTTTGT TTGAAGTCTT TGACGTGGTC CGTGTGCACC

25101 GGCCGCACCG CGGCGTCATC GAAACCGTGT ACCTGCGCAC GCCCTTCTCG

25151 GCCGGCAACG CCACAACATA AAGAAGCAAG CAACATCAAC AACAGCTGCC

25201 GCCATGGGCT CCAGTGAGCA GGAAC TGAAA GCCATTGTCA AAGATCTTGG

25251 TTGTGGGCCA TATTTTTTGG GCACC TATGA CAAGCGCTTT CCAGGCTTTG

25301 TTTCTCCACA CAAGCTCGCC TGCGCCATAG TCAATACGGC CGGTCGCGAG

25351 ACTGGGGGCG TACACTGGAT GGCCTTTGCC TGGAACCCGC ACTCAAAAAC

25401 ATGCTACCTC TTTGAGCCCT TTGGCTTTTC TGACCAGCGA C TCAAGCAGG

25451 TTTACCAGTT TGAGTACGAG TCACTCCTGC GCCGTAGCGC CATTGCTTCT

25501 TCCCCCGACC GCTGTATAAC GCTGGAAAAG TCCACCCAAA GCGTACAGGG

25551 GCCCAACTCG GCCGCCTGTG GACTATTCTG CTGCATGTTT CTCCACGCCT

25601 TTGCCAACTG GCCCCAAACT CCCATGGATC ACAACCCCAC CATGAACCTT

25651 ATTACCGGGG TACCCAACTC CATGC TCAAC AGTCCCCAGG TACAGCCCAC

25701 CCTGCGTCGC AACCAGGAAC AGCTCTACAG CTTCCTGGAG CGCCACTCGC

25751 CCTACTTCCG CAGCCACAGT GCGCAGATTA GGAGCGCCAC TTCTTTTTGT

25801 CAC TTGAAAA ACATGTAAAA ATAATGTACT AGAGACAC TT TCAATAAAGG

25851 CAAATGCTTT TATTTGTACA CTCTCGGGTG ATTATTTACC CCCACCCTTG

25901 CCGTCTGCGC CGTTTAAAAA TCAAAGGGGT TCTGCCGCGC ATCGCTATGC

25951 GCCACTGGCA GGGACACGTT GCGATAC TGG TGTTTAGTGC TCCAC TTAAA

26001 CTCAGGCACA ACCATCCGCG GCAGCTCGGT GAAGTTTTCA CTCCACAGGC

26051 TGCGCACCAT CACCAACGCG TTTAGCAGGT CGGGCGCCGA TATCTTGAAG

26101 TCGCAGTTGG GGCCTCCGCC CTGCGCGCGC GAGTTGCGAT ACACAGGGTT

26151 GCAGCAC TGG AACACTATCA GCGCCGGGTG GTGCACGCTG GCCAGCACGC

26201 TCTTGTCGGA GATCAGATCC GCGTCCAGGT CCTCCGCGTT GCTCAGGGCG

26251 AACGGAGTCA ACTTTGGTAG CTGCCTTCCC AAAAAGGGCG CGTGCCCAGG

26301 CTTTGAGTTG CACTCGCACC GTAGTGGCAT CAAAAGGTGA CCGTGCCCGG

26351 TCTGGGCGTT AGGATACAGC GCCTGCATAA AAGCCTTGAT CTGCTTAAAA

26401 GCCACCTGAG CCTTTGCGCC T TC AGAGAAG AACATGCCGC AAGACTTGCC

26451 GGAAAAC TGA TTGGCCGGAC AGGCCGCGTC GTGCACGCAG CACCTTGCGT

26501 CGGTGTTGGA GATC TGCACC ACATTTCGGC CCCACCGGTT CTTCACGATC

26551 TTGGCCTTGC TAGACTGCTC CTTCAGCGCG CGCTGCCCGT TTTCGCTCGT

26601 CACATCCATT TCAATCACGT GCTCCTTATT TATCATAATG CTTCCGTGTA 26651 GACACTTAAG CTCGCCTTCG ATCTCAGCGC AGCGGTGCAG CCACAACGCG

26701 CAGCCCGTGG GCTCGTGATG CTTGTAGGTC ACCTCTGCAA ACGAC TGCAG

26751 GTACGCCTGC AGGAATCGCC CCATCATCGT CACAAAGG TC TTGTTGCTGG

26801 TGAAGGTCAG CTGCAACCCG CGGTGCTCCT CGTTCAGCCA GGTCTTGCAT

26851 ACGGCCGCCA GAGC TTCCAC TTGGTCAGGC AGTAGTTTGA AGTTCGCCTT

26901 TAGATCGTTA TCCACGTGGT ACTTGTCCAT CAGCGCGCGC GCAGCCTCCA

26951 TGCCCTTCTC CCACGCAGAC ACGATCGGCA CACTCAGCGG GTTCATCACC

27001 GTAATTTCAC TTTCCGCTTC GCTGGGCTCT TCCTCTTCCT CTTGCGTCCG

27051 CATACCACGC GCCACTGGGT CGTCTTCATT CAGCCGCCGC ACTGTGCGCT

27101 TACCTCCTTT GCCATGCTTG ATTAGCACCG GTGGGTTGCT GAAACCCACC

27151 ATTTGTAGCG CCACATCTTC TCTTTCTTCC TCGCTGTCCA CGATTACCTC

27201 TGGTGATGGC GGGCGCTCGG GCTTGGGAGA AGGGCGCTTC TTTTTCTTCT

27251 TGGGCGCAAT GGCCAAATCC GCCGCCGAGG TCGATGGCCG CGGGCTGGGT

27301 GTGCGCGGCA CCAGCGCGTC TTGTGATGAG TCTTCCTCGT CCTCGGACTC

27351 GATACGCCGC CTCATCCGCT TTTTTGGGGG CGCCCGGGGA GGCGGCGGCG

27401 ACGGGGACGG GGACGACACG TCCTCCATGG TTGGGGGACG TCGCGCCGCA

27451 CCGCGTCCGC GCTCGGGGGT GGTTTCGCGC TGCTCCTCTT CCCGACTGGC

27501 CATTTCCTTC TCCTATAGGC AGAAAAAGAT CATGGAGTCA G TC GAGAAGA

27551 AGGACAGCC T AACCGCCCCC TCTGAGTTCG CCACCACCGC CTCCACCGAT

27601 GCCGCCAACG CGCCTACCAC CTTCCCCGTC GAGGCACCCC CGCTTGAGGA

27651 GGAGGAAGTG AT TATC GAGC AGGACCCAGG TTTTGTAAGC GAAGACGACG

27701 AGGACCGCTC AGTACCAACA GAGGATAAAA AGCAAGACCA GGACAACGCA

27751 GAGGCAAACG AGGAACAAGT CGGGCGGGGG GACGAAAGGC ATGGCGACTA

27801 CCTAGATGTG GGAGACGACG TGCTGTTGAA GCATCTGCAG CGCCAGTGCG

27851 CCATTATCTG CGACGCGTTG C AAGAGC GCA GCGATGTGCC CCTCGCCATA

27901 GCGGATGTCA GCCTTGCCTA CGAACGCCAC C TAT TC TC AC CGCGCGTACC

27951 CCCCAAACGC CAAGAAAACG GCACATGCGA GCCCAACCCG CGCCTCAACT

28001 TCTACCCCGT ATTTGCCGTG CCAGAGGTGC TTGCCACCTA TCACATCTTT

28051 TTCCAAAACT GCAAGATACC CCTATCCTGC CGTGCCAACC GCAGCCGAGC

28101 GGACAAGCAG CTGGCCTTGC GGCAGGGCGC TGTCATACCT GATATCGCCT

28151 CGCTCAACGA AGTGCCAAAA ATCTTTGAGG GTCTTGGACG CGACGAGAAG

28201 CGCGCGGCAA ACGCTCTGCA ACAGGAAAAC AGCGAAAATG AAAGTCACTC

28251 TGGAGTGTTG GTGGAACTCG AGGGTGACAA CGCGCGCCTA GCCGTACTAA

28301 AACGCAGCAT CGAGGTCACC CACTTTGCCT ACCCGGCACT TAACCTACCC

28351 CCCAAGGTCA TGAGCACAGT CATGAGTGAG CTGATCGTGC GCCGTGCGCA

28401 GCCCCTGGAG AGGGATGCAA ATTTGCAAGA ACAAACAGAG GAGGGCC TAC

28451 CCGCAGTTGG CGACGAGCAG CTAGCGCGCT GGCTTCAAAC GCGCGAGCCT

28501 GCCGACTTGG AGGAGCGACG CAAAC TAATG ATGGCCGCAG TGCTCGTTAC 28551 CGTGGAGCTT GAGTGCATGC AGCGGTTCTT TGCTGACCCG GAGATGCAGC

28601 GCAAGCTAGA GGAAAC AT TG CACTACACCT TTCGACAGGG CTACGTACGC

28651 CAGGCCTGCA AGATCTCCAA CGTGGAGCTC TGCAACCTGG TCTCCTACCT

28701 TGGAATTTTG CACGAAAACC GCC TTGGGCA AAACGTGCTT CATTCCACGC

28751 TCAAGGGCGA GGCGCGCCGC GACTACGTCC GCGACTGCGT TTACTTATTT

28801 CTATGCTACA CCTGGCAGAC GGCCATGGGC GTTTGGCAGC AGTGCTTGGA

28851 GGAGTGCAAC CTCAAGGAGC TGCAGAAAC T GCTAAAGCAA AAC TTGAAGG

28901 ACC TATGGAC GGCCTTCAAC GAGCGCTCCG TGGCCGCGCA CCTGGCGGAC

28951 ATCATTTTCC CCGAACGCCT GCTTAAAACC CTGCAACAGG GTCTGCCAGA

29001 CTTCACCAGT CAAAGCATGT TGCAGAACTT TAGGAACTTT ATCCTAGAGC

29051 GCTCAGGAAT CTTGCCCGCC ACCTGCTGTG CACTTCCTAG CGACTTTGTG

29101 CCCATTAAGT ACCGCGAATG CCCTCCGCCG CTTTGGGGCC ACTGCTACCT

29151 TCTGCAGCTA GCCAACTACC TTGCCTACCA CTCTGACATA ATGGAAGACG

29201 TGAGCGGTGA CGGTCTACTG GAG TG TC AC T GTCGCTGCAA CCTATGCACC

29251 CCGCACCGCT CCCTGGTTTG CAATTCGCAG CTGCTTAACG AAAGTCAAAT

29301 TATCGGTACC TTTGAGCTGC AGGGTCCCTC GCCTGACGAA AAGTCCGCGG

29351 CTCCGGGGTT GAAACTCACT CCGGGGCTGT GGACGTCGGC TTACCTTCGC

29401 AAATTTGTAC CTGAGGACTA CCACGCCCAC GAGATTAGGT TCTACGAAGA

29451 CCAATCCCGC CCGCCAAATG CGGAGCTTAC CGCCTGCGTC ATTACCCAGG

29501 GCCACATTCT TGGCCAATTG C AAGC CATC A ACAAAGCCCG CCAAGAGTTT

29551 CTGCTACGAA AGGGACGGGG GGTTTACTTG GACCCCCAGT CCGGCGAGGA

29601 GCTCAACCCA ATCCCCCCGC CGCCGCAGCC CTATCAGCAG CAGCCGCGGG

29651 CCCTTGCTTC CCAGGATGGC ACCCAAAAAG AAGC TGCAGC TGCCGCCGCC

29701 ACCCACGGAC GAGGAGGAAT ACTGGGACAG TCAGGCAGAG GAGGTTTTGG

29751 ACGAGGAGGA GGAGGACATG ATGGAAGAC T GGGAGAGCCT AGACGAGGAA

29801 GCTTCCGAGG TCGAAGAGGT GTCAGACGAA ACACCGTCAC CCTCGGTCGC

29851 ATTCCCCTCG CCGGCGCCCC AGAAATCGGC AACCGGTTCC AGCATGGCTA

29901 CAACCTCCGC TCCTCAGGCG CCGCCGGCAC TGCCCGTTCG CCGACCCAAC

29951 CGTAGATGGG ACACCACTGG AACCAGGGCC GGTAAGTCCA AGCAGCCGCC

30001 GCCGTTAGCC CAAGAGCAAC AACAGCGCCA AGGCTACCGC TCATGGCGCG

30051 GGCACAAGAA CGCCATAGTT GCTTGCTTGC AAGACTGTGG GGGCAACATC

30101 TCCTTCGCCC GCCGCTTTCT TCTCTACCAT CACGGCGTGG CCTTCCCCCG

30151 TAACATCCTG CATTAC TACC GTCATCTCTA CAGCCCATAC TGCACCGGCG

30201 GCAGCGGCAG CGGCAGCAAC AGCAGCGGCC ACACAGAAGC AAAGGCGACC

30251 GGATAGCAAG ACTCTGACAA AGCCCAAGAA ATCCACAGCG GCGGCAGCAG

30301 CAGGAGGAGG AGCGCTGCGT CTGGCGCCCA ACGAACCCGT ATCGACCCGC

30351 GAGCTTAGAA ACAGGATTTT TCCCACTCTG TATGCTATAT T TC AACAGAG

30401 CAGGGGCCAA GAACAAGAGC TGAAAATAAA AAACAGGTCT CTGCGATCCC 30451 TCACCCGCAG CTGCCTGTAT CACAAAAGCG AAGATCAGCT TCGGCGCACG

30501 CTGGAAGACG CGGAGGCTCT CTTCAGTAAA TACTGCGCGC TGACTCTTAA

30551 GGACTAGTTT CGCGCCCTTT CTCAAATTTA AGCGCGAAAA CTACGTCATC

30601 TCCAGCGGCC ACACCCGGCG CCAGCACCTG TCGTCAGCGC CATTATGAGC

30651 AAGGAAATTC CCACGCCCTA CATGTGGAGT TACCAGCCAC AAATGGGACT

30701 TGCGGCTGGA GCTGCCCAAG ACTACTCAAC CCGAATAAAC TACATGAGCG

30751 CGGGACCCCA CATGATATCC CGGGTCAACG GAATCCGCGC CCACCGAAAC

30801 CGAATTCTCT TGGAACAGGC GGCTATTACC ACCACACCTC GTAATAACCT

30851 TAATCCCCGT AGTTGGCCCG CTGCCCTGGT GTACCAGGAA AGTCCCGCTC

30901 CCACCACTGT GGTACTTCCC AGAGACGCCC AGGCCGAAGT TCAGATGACT

30951 AACTCAGGGG CGCAGCTTGC GGGCGGCTTT CGTCACAGGG TGCGGTCGCC

31001 CGGGCAGGGT ATAACTCACC TGACAATCAG AGGGCGAGGT ATTCAGCTCA

31051 ACGACGAGTC GGTGAGCTCC TCGCTTGGTC TCCGTCCGGA CGGGACATTT

31101 CAGATCGGCG GCGCCGGCCG TCCTTCATTC ACGCCTCGTC AGGCAATCCT

31151 AACTCTGCAG ACCTCGTCCT CTGAGCCGCG CTCTGGAGGC ATTGGAACTC

31201 TGCAATTTAT TGAGGAGTTT GTGCCATCGG TCTACTTTAA CCCCTTCTCG

31251 GGACCTCCCG GCCACTATCC GGATCAATTT ATTCCTAACT TTGACGCGGT

31301 AAAGGACTCG GCGGACGGCT ACGACTGAAT GTTAAGTGGA GAGGCAGAGC

31351 AACTGCGCCT GAAACACCTG GTCCACTGTC GCCGCCACAA GTGCTTTGCC

31401 CGCGACTCCG GTGAGTTTTG CTACTTTGAA TTGCCCGAGG ATCATATCGA

31451 GGGCCCGGCG CACGGCGTCC GGCTTACCGC CCAGGGAGAG CTTGCCCGTA

31501 GCCTGATTCG GGAGTTTACC CAGCGCCCCC TGCTAGTTGA GCGGGACAGG

31551 GGACCCTGTG TTCTCACTGT GATTTGCAAC TGTCCTAACC TTGGATTACA

31601 TCAAGATCTT TGTTGCCATC TCTGTGCTGA GTATAATAAA TACAGAAATT

31651 AAAATATACT GGGGCTCCTA TCGCCATCCT GTAAACGCCA CCGTCTTCAC

31701 CCGCCCAAGC AAACCAAGGC GAACCTTACC TGGTACTTTT AACATCTCTC

31751 CCTCTGTGAT TTACAACAGT TTCAACCCAG ACGGAGTGAG TCTACGAGAG

31801 AACCTCTCCG AGCTCAGCTA CTCCATCAGA AAAAACACCA CCCTCCTTAC

31851 CTGCCGGGAA CGTACGAGTG CGTCACCGGC CGCTGCACCA CACCTACCGC

31901 CTGACCGTAA ACCAGACTTT TTCCGGACAG ACCTCAATAA CTCTGTTTAC

31951 CAGAACAGGA GGTGAGCTTA GAAAACCCTT AGGGTATTAG GCCAAAGGCG

32001 CAGCTACTGT GGGGTTTATG AACAATTCAA GCAACTCTAC GGGCTATTCT

32051 AATTCAGGTT TCTCTAGAAT CGGGGTTGGG GTTATTCTCT GTCTTGTGAT

32101 TCTCTTTATT CTTATACTAA CGCTTCTCTG CCTAAGGCTC GCCGCCTGCT

32151 GTGTGCACAT TTGCATTTAT TGTCAGCTTT TTAAACGCTG GGGTCGCCAC

32201 CCAAGATGAT TAGGTACATA ATCCTAGGTT TACTCACCCT TGCGTCAGCC

32251 CACGGTACCA CCCAAAAGGT GGATTTTAAG GAGCCAGCCT GTAATGTTAC

32301 ATTCGCAGCT GAAGCTAATG AGTGCACCAC TCTTATAAAA TGCACCACAG 32351 AACATGAAAA GCTGCTTATT C GC CACAAAA ACAAAATTGG CAAGTATGC T

32401 GTTTATGCTA TTTGGCAGCC AGGTGACACT ACAGAGTATA ATGTTACAGT

32451 TTTCCAGGGT AAAAGTCATA AAACTTTTAT GTATACTTTT CCATTTTATG

32501 AAATGTGCGA CATTACCATG TACATGAGCA AACAGTATAA GTTGTGGCCC

32551 CCACAAAATT GTGTGGAAAA CACTGGCACT TTCTGCTGCA CTGCTATGCT

32601 AATTACAGTG CTCGCTTTGG TCTGTACCCT ACTCTATATT AAATACAAAA

32651 GCAGACGCAG CTTTATTGAG GAAAAGAAAA TGCCTTAATT TACTAAGTTA

32701 CAAAGCTAAT GTCACCAC TA ACTGCTTTAC TCGCTGCTTG CAAAACAAAT

32751 TCAAAAAGTT AGCATTATAA TTAGAATAGG ATTTAAACCC CCCGGTCATT

32801 TCCTGCTCAA TACCATTCCC C TGAACAAT T GACTCTATGT GGGATATGC T

32851 CCAGCGCTAC AACC TTGAAG TCAGGCTTCC TGGATGTCAG CATCTGACTT

32901 TGGCCAGCAC CTGTCCCGCG GATTTGTTCC AGTCCAACTA CAGCGACCCA

32951 CCCTAACAGA GATGACCAAC ACAACCAACG CGGCCGCCGC TACCGGACTT

33001 ACATC TACCA CAAATACACC CCAAGTTTCT GCCTTTGTCA ATAAC TGGGA

33051 TAACTTGGGC ATGTGGTGGT TCTCCATAGC GCTTATGTTT GTATGCCTTA

33101 TTATTATGTG GCTCATCTGC TGCCTAAAGC GCAAACGCGC CCGACCACCC

33151 ATC TATAGTC CCATCATTGT GCTACACCCA AACAATGATG GAATC CATAG

33201 ATTGGACGGA CTGAAACACA TGTTCTTTTC TCTTACAGTA TGATTAAATG

33251 AGACATGATT CCTCGAGTTT TTATATTACT GACCCTTGTT GCGCTTTTTT

33301 GTGCGTGCTC CACATTGGCT GCGGTTTCTC ACATCGAAGT AGACTGCATT

33351 CCAGCCTTCA CAGTCTATTT GCTTTACGGA TTTGTCACCC TCACGCTCAT

33401 CTGCAGCCTC ATCACTGTGG TCATCGCCTT TATCCAGTGC ATTGACTGGG

33451 TCTGTGTGCG CTTTGCATAT CTCAGACACC ATCCCCAGTA CAGGGACAGG

33501 ACTATAGCTG AGCTTCTTAG AATTCTTTAA TTATGAAATT TACTGTGACT

33551 TTTCTGCTGA TTATTTGCAC CCTATCTGCG TTTTGTTCCC CGACCTCCAA

33601 GCC TCAAAGA CATATATCAT GCAGATTCAC TCGTATATGG AATATTCCAA

33651 GTTGCTACAA TGAAAAAAGC GATCTTTCCG AAGCCTGGTT ATATGCAATC

33701 ATCTCTGTTA TGGTGTTCTG CAGTACCATC TTAGCCCTAG C TATATATC C

33751 CTACCTTGAC ATTGGCTGGA AACGAATAGA TGCCATGAAC CACCCAACTT

33801 TCCCCGCGCC CGCTATGCTT CCACTGCAAC AAGTTGTTGC CGGCGGCTTT

33851 GTCCCAGCCA ATCAGCCTCG CCCCACTTCT CCCACCCCCA C TGAAATCAG

33901 CTACTTTAAT C TAACAGGAG GAGATGACTG ACACCCTAGA TCTAGAAATG

33951 GACGGAATTA TTACAGAGCA GCGCCTGCTA GAAAGACGCA GGGCAGCGGC

34001 CGAGCAACAG CGCATGAATC AAGAGCTCCA AGACATGGTT AACTTGCACC

34051 AGTGCAAAAG GGGTATCTTT TGTCTGGTAA AGCAGGCCAA AGTCACC TAC

34101 GACAGTAATA CCACCGGACA CCGCCTTAGC TACAAGTTGC CAACCAAGCG

34151 TCAGAAATTG GTGGTCATGG TGGGAGAAAA GCCCATTACC ATAAC TCAGC

34201 ACTCGGTAGA AACCGAAGGC TGCATTCACT CACCTTGTCA AGGACCTGAG 34251 GATCTCTGCA CCCTTATTAA GACCCTGTGC GGTCTCAAAG ATCTTATTCC

34301 CTTTAACTAA TAAAAAAAAA TAATAAAGCA TCACTTACTT AAAATCAGTT

34351 AGCAAATTTC TGTCCAGTTT ATTCAGCAGC ACCTCCTTGC CCTCCTCCCA

34401 GCTCTGGTAT TGCAGCTTCC TCCTGGCTGC AAACTTTCTC CACAATCTAA

34451 ATGGAATGTC AGTTTCCTCC TGTTCCTGTC CATCCGCACC CACTATCTTC

34501 ATGTTGTTGC AGATGAAGCG CGCAAGACCG TC TGAAGATA CCTTCAACCC

34551 CGTGTATCCA TATGACACGG AAACCGGTCC TCCAACTGTG CCTTTTCTTA

34601 CTCCTCCCTT TGTATCCCCC AATGGGTTTC AAGAGAGTCC CCCTGGGGTA

34651 CTCTCTTTGC GCCTATCCGA ACCTCTAGTT ACCTCCAATG GCATGCTTGC

34701 GCTCAAAATG GGCAACGGCC TCTCTCTGGA CGAGGCCGGC AACCTTACCT

34751 CCCAAAATGT AACCACTGTG AGCCCACCTC TCAAAAAAAC CAAGTCAAAC

34801 ATAAACCTGG AAATATCTGC ACCCCTCACA GTTACCTCAG AAGCCCTAAC

34851 TGTGGCTGCC GCCGCACCTC TAATGGTCGC GGGCAACACA CTCACCATGC

34901 AATCACAGGC CCCGCTAACC GTGCACGACT CCAAAC TTAG CATTGCCACC

34951 CAAGGACCCC TC AC AG TG TC AGAAGGAAAG CTAGCCCTGC AAACATCAGG

35001 CCCCCTCACC ACCACCGATA GCAGTACCCT TACTATCACT GCCTCACCCC

35051 CTCTAACTAC TGCCACTGGT AGC TTGGGCA TTGACTTGAA AGAGCCCATT

35101 TATACACAAA ATGGAAAACT AGGAC TAAAG TACGGGGCTC CTTTGCATGT

35151 AAC AG AC GAC CTAAACACTT TGACCGTAGC AACTGGTCCA GGTGTGACTA

35201 T TAATAATAC TTCCTTGCAA ACTAAAGTTA CTGGAGCCTT GGGTTTTGAT

35251 TCACAAGGCA ATATGCAACT TAATG TAGC A GGAGGACTAA GGATTGATTC

35301 TCAAAACAGA CGCC TTATAC TTGATGTTAG TTATCCGTTT GATGC TCAAA

35351 ACCAACTAAA TCTAAGACTA GGACAGGGCC CTCTTTTTAT AAACTCAGCC

35401 CACAACTTGG ATATTAACTA CAACAAAGGC CTTTACTTGT TTACAGCTTC

35451 AAACAAT TC C AAAAAGCTTG AGGTTAACCT AAGCACTGCC AAGGGGTTGA

35501 TGTTTGACGC TACAGC CATA GCCATTAATG CAGGAGATGG GCTTGAATTT

35551 GGTTCACCTA ATGCACCAAA CACAAATCCC CTCAAAACAA AAATTGGCCA

35601 TGGCCTAGAA TTTGATTCAA ACAAGGC TAT GGTTCCTAAA CTAGGAACTG

35651 GCCTTAGTTT TGACAGCACA GGTGCCATTA CAGTAGGAAA CAAAAATAAT

35701 GATAAGC TAA CTTTGTGGAC CACACCAGCT CCATCTCCTA ACTGTAGACT

35751 AAATGCAGAG AAAGATGC TA AACTCACTTT GGTCTTAACA AAATGTGGCA

35801 GTCAAATACT TGCTACAGTT TCAGTTTTGG CTGTTAAAGG CAGTTTGGCT

35851 CCAATATCTG GAACAGTTCA AAGTGCTCAT CTTATTATAA GATTTGACGA

35901 AAATGGAGTG CTACTAAACA ATTCCTTCCT GGACCCAGAA TATTGGAACT

35951 TTAGAAATGG AGATCTTACT GAAGGCACAG CC TATACAAA CGCTGTTGGA

36001 TTTATGCCTA ACCTATCAGC TTATCCAAAA TC TC AC GG TA AAACTGCCAA

36051 AAGTAACATT GTCAGTCAAG TTTACTTAAA CGGAGACAAA ACTAAACCTG

36101 T AAC AC T AAC CATTACACTA AACGGTACAC AGGAAACAGG AGACACAACT 36151 CCAAGTGCAT ACTCTATGTC ATTTTCATGG GACTGGTCTG GCCACAACTA

36201 CATTAATGAA ATATTTGCCA CATCCTCTTA CACTTTTTCA TACATTGCCC

36251 AAGAATAAAG AATCGTTTGT GTTATGTTTC AACGTGTTTA TTTTTCAATT

36301 GCAGAAAATT TCAAGTCATT TTTCATTCAG TAGTATAGCC CCACCACCAC

36351 ATAGC TTATA CAGATCACCG TACCTTAATC AAACTCACAG AACCCTAGTA

36401 TTCAACCTGC CACCTCCCTC CCAACACACA GAGTACACAG TCCTTTCTCC

36451 CCGGCTGGCC TTAAAAAGCA TCATATCATG GGTAACAGAC ATATTCTTAG

36501 GTGTTATATT CCACACGGTT TCCTGTCGAG CCAAACGCTC ATCAGTGATA

36551 TTAATAAACT CCCCGGGCAG CTCACTTAAG TTCATGTCGC TGTCCAGCTG

36601 CTGAGCCACA GGCTGCTGTC CAACTTGCGG TTGCTTAACG GGCGGCGAAG

36651 GAGAAGTCCA CGCCTACATG GGGGTAGAGT CATAATCGTG CATCAGGATA

36701 GGGCGGTGGT GC TGCAGCAG CGCGCGAATA AACTGCTGCC GCCGCCGCTC

36751 CGTCCTGCAG GAATACAACA TGGCAGTGGT CTCCTCAGCG ATGATTCGCA

36801 CCGCCCGCAG CATAAGGCGC CTTGTCCTCC GGGCACAGCA GCGCACCCTG

36851 ATCTCACTTA AATCAGCACA GTAACTGCAG CACAGCACCA CAATATTGTT

36901 CAAAATCCCA CAGTGCAAGG CGCTGTATCC AAAGCTCATG GCGGGGACCA

36951 CAGAACCCAC GTGGCCATCA TACCACAAGC GCAGGTAGAT TAAGTGGCGA

37001 CCCCTCATAA ACACGCTGGA CATAAACATT ACCTCTTTTG GCATGTTGTA

37051 ATTCACCACC TCCCGGTACC ATATAAACC T CTGATTAAAC ATGGCGCCAT

37101 CCACCACCAT CC TAAACCAG CTGGCCAAAA CCTGCCCGCC GGC TATACAC

37151 TGCAGGGAAC CGGGAC TGGA ACAATGACAG TGGAGAGCCC AGGACTCGTA

37201 ACCATGGATC ATCATGCTCG TCATGATATC AATGTTGGCA CAACACAGGC

37251 ACACGTGCAT ACACTTCCTC AGGAT TACAA GCTCCTCCCG CGTTAGAACC

37301 ATATCCCAGG GAACAACCCA TTCCTGAATC AGCGTAAATC CCACACTGCA

37351 GGGAAGACC T CGCACGTAAC TCACGTTGTG CATTGTCAAA GTGTTACATT

37401 CGGGCAGCAG CGGATGATCC TCCAGTATGG TAGCGCGGGT TTCTGTCTCA

37451 AAAGGAGGTA GACGATCCCT ACTGTACGGA GTGCGCCGAG ACAACCGAGA

37501 TCGTGTTGGT CGTAGTGTCA TGCCAAATGG AACGCCGGAC GTAGTCATAT

37551 TTCCTGAAGC AAAACCAGGT GCGGGCGTGA CAAACAGATC TGCGTCTCCG

37601 GTCTCGCCGC TTAGATCGCT CTGTGTAGTA GTTGTAGTAT ATCCACTCTC

37651 TCAAAGCATC CAGGCGCCCC CTGGCTTCGG GTTCTATGTA AACTCCTTCA

37701 TGCGCCGCTG CCCTGATAAC ATCCACCACC GCAGAATAAG CCACACCCAG

37751 CCAACCTACA CATTCGTTCT GCGAGTCACA CACGGGAGGA GCGGGAAGAG

37801 C TGGAAGAAC CATGTTTTTT TTTTTATTCC AAAAGATTAT CCAAAACCTC

37851 AAAATGAAGA TCTATTAAGT GAACGCGCTC CCCTCCGGTG GCGTGGTCAA

37901 ACTCTACAGC CAAAGAACAG ATAATGGCAT TTGTAAGATG TTGCACAATG

37951 GCTTCCAAAA GGCAAACGGC CCTCACGTCC AAGTGGACGT AAAGGCTAAA

38001 CCCTTCAGGG TGAATCTCCT C TATAAACAT TCCAGCACCT TCAACCATGC 38051 CCAAATAATT CTCATCTCGC CACCTTCTCA ATATATCTCT AAGCAAATCC 38101 CGAATATTAA GTCCGGCCAT TGTAAAAATC TGCTCCAGAG CGCCCTCCAC 38151 CTTCAGCCTC AAGCAGCGAA TCATGATTGC AAAAATTCAG GTTCCTCACA 38201 GACCTGTATA AGATTCAAAA GCGGAACATT AACAAAAATA CCGCGATCCC 38251 GTAGGTCCCT TCGCAGGGCC AGC TGAACAT AATCGTGCAG GTCTGCACGG 38301 ACCAGCGCGG CCACTTCCCC GCCAGGAACC ATGACAAAAG AACCCACACT 38351 GATTATGACA CGCATACTCG GAGCTATGCT AACCAGCGTA GCCCCGATGT 38401 AAGCTTGTTG CATGGGCGGC GATATAAAAT GCAAGGTGCT GCTCAAAAAA 38451 TCAGGCAAAG CCTCGCGCAA AAAAGAAAGC ACATCGTAGT CATGC TCATG 38501 CAGATAAAGG CAGGTAAGCT CCGGAACCAC CACAGAAAAA GACACCATTT 38551 TTCTCTCAAA CATGTCTGCG GGTTTCTGCA TAAACACAAA ATAAAATAAC 38601 AAAAAAACAT TTAAACATTA GAAGCCTGTC TTACAACAGG AAAAACAACC 38651 C TTATAAGCA TAAGAC GGAC TACGGCCATG CCGGCGTGAC CGTAAAAAAA 38701 CTGGTCACCG TGAT TAAAAA GCACCACCGA CAGCTCCTCG GTCATGTCCG 38751 GAG TC ATAAT GTAAGACTCG G TAAACACAT CAGGTTGATT CATCGGTCAG 38801 TGC TAAAAAG CGACCGAAAT AGCCCGGGGG AATACATACC CGCAGGCGTA 38851 GAGACAACAT TACAGCCCCC ATAGGAGGTA TAACAAAATT AATAGGAGAG 38901 AAAAACACAT AAACACCTGA AAAACCCTCC TGCCTAGGCA AAATAGC AC C 38951 CTCCCGCTCC AGAACAACAT ACAGCGCTTC ACAGCGGCAG CCTAACAGTC 39001 AGCCTTACCA GTAAAAAAGA AAACC TATTA AAAAAACACC ACTCGACACG 39051 GCACCAGCTC AATCAGTCAC AGTGTAAAAA AGGGCCAAGT GCAGAGCGAG 39101 TATATATAGG ACTAAAAAAT GACGTAACGG TTAAAGTCCA CAAAAAACAC 39151 C C AGAAAAC C GCACGCGAAC CTACGCCCAG AAAC GAAAGC CAAAAAACCC 39201 ACAACTTCCT CAAATCGTCA CTTCCGTTTT CCCACGTTAC GTAACTTCCC 39251 ATTTTAAGAA AACTACAATT CCCAACACAT ACAAGTTACT CCGCCCTAAA 39301 ACCTACGTCA CCCGCCCCGT TCCCACGCCC CGCGCCACGT CACAAACTCC 39351 ACCCCCTCAT TATCATATTG GCTTCAATCC AAAATAAGGT ATATTATTGA 39401 TGAT (SEQ ID NO : 4)

[00230] humanTERT promoter sequence:

[00231] GGCCCCTCCC TCGGGTTACC CCACAGCTTA GGCCGATTCG

ACCTCTCTCC GCTGGGGCCC TCGCTGGCGT CCCTGCACCC TGGGAGCGCG AGCGGCGCGC GGGCGGGGAA GCGCGGCCCA GACCCCCGGG TCCGCCCGGA GCAGCTGCGC TGTCGGGGCC AGGCCGGGCT CCCAGTGGAT TCGCGGGCAC AGACGCCCAG GACCGCGCTT CCCACGTGGC GGAGGGACTG GGGACCCGGG CACCCGTCCT GCCCCTTCAC CTTCCAGCTC CGCCTCCTCC GCGCGGACCC CGCCCCGTCC CGACCCCTCC CGGGTCCCCG GCCCAGCCCC CTCCGGGCCC TCCCAGCCCC TCCCCTTCCT TTCCGCGGCC CCGCCCTCTC CTCGCGGCGC GAGTTTCAGG CAGCGCTGCG TCCTGCTGCG CACGTGGGAA GCCCTGGCCC CGGCCACCCC CGCG (SEQ ID NO: 5)

[00232] Human sTGFpRIIFc sequence:

[00233] MGRGLLRGLWPLHI VLWTRI AS TIPPH V QK S VNNDMI VTDNN GA VKFPQ L CKF CD VRF STCDN QK S CM SN C SIT SICEKPQE V C V A VWRKNDENITLET V CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS EEYNTSNPDMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTC VVVD V SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS V LTVLHQDWLNGKEYKCKVSTKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLDSDGSFF

LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:6)

[00234] E1A sequence:

[00235] MRHIICHGGVITEEMAASLLDQLIEEVLADNLPPPSHFEPPTLHELYDLD

VTAPEDPNEEAVSQIFPDSVMLAVQEGIDLLTFPPAPGSPEPPHLSRQPE QPEQRALGPVSMPNLVPEVIDLTCHEAGFPPSDDEDEEGEEFVLDYVEHP GHGCRSCHYHRRNTGDPDIMC SLC YMRTCGMF VY SK (SEQ ID NO: 7)

[00236] GM-CSF sequence:

[00237] MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTA

AEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASH YKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE (SEQ ID NO:2)

[00238] E1B55K sequence:

[00239] MERRNP SERGVP AGF S GH AS VE S GCET QE SP AT VVFRPPGDNTD GGA A A

A AGGSQAAAAGAEPMEPESRPGPSGMNVVQVAELYPELRRILTITEDGQGL KGVKRERGACEATEEARNLAF SLMTRHRPECITF QQIKDNC ANELDLLAQ KYSIEQLTTYWLQPGDDFEEAIRVYAKVALRPDCKYKISKLVNIRNCCYI SGNGAEVEIDTEDRVAFRCSMINMWPGVLGMDGVVIMNVRFTGPNFSGTV FL ANTNLILHGV SF Y GFNNT C VE AWTD VRVRGC AF YCCWKGVV CRPKSRA SIKKCLFERC TLGIL SEGN SRVRHN V ASDC GCFML VK S V A VIKHNM V C GN CEDRASQMLTC SDGNCHLLKTIHVASHSRK AWPVFEHNILTRC SLHLGNR RGVFLP Y QCNL SHTKILLEPE SMSK VNLN GVFDMTMKIWK VLR YDETRTR

CRPCECGGKHIRNQPVMLDVTEELRPDHLVLACTRAEFGSSDEDTD (SEQ ID NO: 8) PROPHETIC EXAMPLES 2-6:

EXAMPLE 2: CHARACTERIZATION OF EXPRESSION FROM RAD.ST.GM, RAD.ST, AND RAD.ST.GM VECTORS

[00240] Cell lines and adenoviruses. Human mammary tumor cell lines, MCF-7 (ATCC, Manassas, VA), MDA-MB-231 (ATCC, Manassas, VA), and the mouse mammary tumor cell line, 4T1 (ATCC, Manassas, VA) are maintained as described earlier (Katayose, D., et al.,

(1995) Clin Cancer Res 1, 889-897; Craig, C., et al. (1997) Oncogene 14, 2283-2289; Zhang, Z., et al. (2012) Cancer Gene Ther 19, 630-636). All media components are purchased from Thermo Fisher Scientific (Waltham, MA). sTGFfiRIIFc and GM-CSF expression in breast cancer cells infected with rAd.sT.GM [00241] Experiments are conducted to examine if the infection of breast cancer cells can produce high levels of sTGFBRIIFc. Breast tumor cells (0.2xl0 ⁶ cells per well in 6-well plates) are plated in DMEM containing 10% FBS and incubated at 37°C overnight. The next morning, cells are infected with 100 plaque-forming units (pfu)/cell of rAd.sT.GM, rAd.sT, rAd.GM, or rAd.Null adenovirus for 24 hours. Cells are washed and incubated with DMEM without FBS for 24 hours. Media and cells are subjected to Western blot analysis as previously described (Katayose, D., et al., (. upra ); Craig, C., {supra)). Blots are probed with antibody reactive against TGFBRII (H-567; Santa Cruz Biotechnology, Santa Cruz, CA), or actin protein (1-19; Santa Cruz Biotechnology, Santa Cruz, CA) as control.

[00242] Expected Results. Infection of MDA-MB-231 and MCF-7 cells with rAd.sT.GM and rAd.sT show a 60-80 Kd protein band of sTGFBRIIFc. in the extracellular media. However, sTGFBRIIFc protein is not detectable in the media of the tumor cells infected with rAd.GM or rAd.Null. sTGFfiRIIFc and GMCSF expression in human breast cancer cells infected with rAd.sT.GM

[00243] Methods. Supernatants obtained from the rAd.sT.GM, rAd.sT, rAd.GM, or rAd.Null infected cells are also analyzed for sTGFBRIIFc and GM-CSF expression by ELISA, using a published method (Yang, Y. A., et al., (2002 ) J Clin Invest 109, 1607-1615). In brief, 96-well plates (Nunc, USA) are coated with the anti -human IgG-Fc specific capture antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), incubated with various dilutions of the samples, followed by detection with biotinylated anti-human TGFBRII antibody (R&D systems, Minneapolis, MN). The detection is carried out with streptavidin-conjugated peroxidase using the TMB/HRP substrate (BioFX Laboratories, Owing Mills, MD). After stopping the reaction with IN HC1, the absorbance is measured at 450 nm using a SPECTRA max Plus ELISA plate reader (Molecular Devices, Sunnyvale, CA). Standard curves of sTGFBRIIFc are used to calculate the sTGFBRIIFc concentrations in the test samples. Human GM-CSF levels are determined using a commercially available ELISA kit from Invitrogen.

[00244] Expected Results. Based on the results reported in Example 1, incorporation of an additional GMCSF gene in rAd.sT.GM, does not seem to adversely affect the expression of sTGFBRIIFc. Thus, infection of breast tumor cells with rAd.sT.GM and rAd.sT is expected to produce sTGFBRIIFc that is subsequently released into the media and breast tumor cells infected with rAd.sT.GM or rAd.GM are expected to produce GMCSF.

EXAMPLE 3: ADENOVIRUS CYTOTOXICITY AND REPLICATION

[00245] Adenovirus cytotoxicity. Tumor cells are seeded in 96-well plates at a density of lxlO ³ cells/well, and the following day they are infected with various doses of oncolytic adenoviruses (rAd.sT.GM, rAd.sT, rAd.GM, or rAd.Null) and a non-replicating virus Ad(El-).Null, ranging from 2 VPs/cell to 1.25 x 10 ⁵ VPs/cell. Incubations are continued for seven days. Cell survival is determined by the sulforhodamine B staining assay (Katayose, D., et al., {supra)).

[00246] Expected Results. All oncolytic viruses including rAd.sT.GM are expected to produce dose-dependent cytotoxicity in human breast tumor cells, as well as mouse mammary tumor cells. Based on the IC50 values (viral dose required to kill 50% of the cells), oncolytic viruses are usually about 100 times more cytotoxic than a non-replicating virus such as Ad(El-).Null.

[00247] Viral replication assay. Cells are plated in 6-well plates at about 70% confluence and the viral titers are examined using a published method (Seth, P., et al. (2006) Hum Gene Ther 17, 1152-1160; Wang, Z. G., et al. (2006) Mol Cancer Ther 5, 367-373) with some modifications. Cells are infected with rAd.sT.GM, rAd.sT, rAd.GM, rAd.Null or Ad(El ).Null for 3 hours at a multiplicity of infection (MOI) of 100. Cells are washed with DMEM and incubated in 1 ml DMEM for additional one hour at 37°C. At the end of the incubation, cells are washed and either collected in 0.5 ml growth media and frozen at -70°C or are maintained in 2 ml of growth media for an additional 48 hours. Media and cells are collected, and cells are subjected to three cycles of freezing and thawing to release the viruses. The viral titers in 3 -hour or 48-hour crude viral lysates are determined using the Adeno-X Rapid titer kit (Clutch, Mountain view, CA). Viral burst size (an increase in hexon-expressing positive cells from 3 hours to 48 hours), is used as an indicator of viral replication, as described (Hu, Z., et al. (2012) Hum Gene Ther 23, 871-882). [00248] Expected Results. rAd.sT.GM is expected to replicate in human breast cancer cells, MDA-MB-231 and MCF-7. At 3 hours, viral titers may be quite low, which should significantly increase at 48 hours (increase in viral titers from 3 hours to 48 hours is described as the “viral burst size”). On the other hand, infection of MDA-MB-231 or MCF-7 cells with Ad(El ).Null is expected to result in less than a 2-fold increase in viral burst size, since Ad(El ).Null is expected to be replication-deficient in the breast cancer cells.

EXAMPLE 4: EXPRESSION OF STGFBRTTFC IN VIVO AND ORTHOTOPIC XENOGRAFT MODEL [00249] Methods. 4Tlluc2 mouse tumors are established in BALB/c mice. Various viruses (Ad.sT, rAd.sT.GM, rAd.GM and rAd.Null) and buffer are administered intratumorally into 4Tlluc2 tumors (5.0 x 10 ¹⁰ VPs/mouse). After 48 hours, the sTGFpRIIFc levels in the blood are measured by ELISA, as described herein. To detect sTGFpRIIFc expression in the tumors, tumor sections are also subjected to immunohistochemical (IHC) staining for sTGFpRIIFc.

[00250] Expected Results. The intratumoral injection of rAd.sT.GM and rAd.sT are expected to result in secretion of sTGFpRIIFc and its detection in the blood. Intratumoral injection of rAd.sT.GM and rAd.GM is expected to result in secretion of GMCSF and its detection in the blood. sTGFpRIIFc expression is expected to be readily detected in the subcutaneous tumors of mice injected with rA.sT.GM and rAd.sT.

[00251] To establish an orthotopic xenograft model of breast cancer, 6.5x 10 ⁵ 4Tl-luc cells (IOOmI) are injected into number 3 and 4 mammary fat pads of BALB/c mice (4-6 weeks old). When tumors are visible (on day 7 after cell injection), tumor volumes are measured by caliper and calculated using the following formula: tumor volume = (Width ² Length)/2 The tumor burden is also analyzed by real-time bioluminescence imaging (BLI).

EXAMPLE 5: SAFETY/TOXICITY STUDIES FOLLOWING INTRATUMORAL DELIVERY OF ONCOLYTIC VIRUSES IN BALB/C MICE BEARING 4TlLUC2 TUMORS

[00252] Methods. To establish subcutaneous tumors, 2 x 10 ⁶ 4Tlluc2 cells per mouse

(day 0) are injected into the dorsal right flank of female BALB/c mice (6-8 weeks old). Mice are monitored every day. When the solid subcutaneous tumors are established (day 12), rAd.sT.GM, rAd.sT, rAd.GM and the vehicle control (PBS buffer) are administered intratumorally (5.0 x 10 ¹⁰ VPs in 100 pi per mouse). Two days (48 hours) after the viral injection (on day 14), whole- mouse blood is withdrawn via cardiac puncture under anesthesia before animals are euthanized. Subcutaneous tumors are removed. Mouse serum is prepared from the blood and kept frozen. Tumor samples are fixed in 10% NBF solution for histology sample preparation and analyses are performed as described (Xu, W., et al. (2014) Mol Ther 22, 1504-1517).

[00253] Hepatic and systemic toxicity following the intratumoral administration of the test oncolytic viral vectors in BALB/c mice are evaluated. Serum samples obtained from the above experiment are analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels. ALT is generally considered a good marker for hepatic damage and LDH is a good indicator of systemic toxicity (Xu, W., et al. {supra)). Commercially available kits are used to detect enzyme levels.

[00254] Expected Results. Subclinical hepatic injury is possible, but the serum values of ALT, AST and LDH are expected to be within normal limits and not to exceed 2.5 times the upper limit of normal in most individual test subjects.

EXAMPLE 6: COMBINATION THERAPY

[00255] The effectiveness of the combination of rAd.sT.GM with immune checkpoint inhibitors anti -PD- 1 and anti-CTLA-4 in inhibiting tumor growth and metastases is tested using 4T1 tumors established in BALB/c mice.

[00256] Methods. As described above, 4T1 cells are injected (day 0) into the right flank of 4-6-week-old BALB/c mice (5xl0 ⁶ cells/mouse). On day 7 following tumor cell injections, tumor volumes are calculated by the following formula: tumor volume = (Width ² xLength)/2. Tumor bearing mice are divided into these groups: (i) rAd.sT.GM, (ii) anti-PD-1, (iii) anti- CTLA-4, (iv) anti-PDland anti-CTLA-4, (v) rAd.sT.GM, anti-PD-1, (vi) rAd.sT.GM and anti- CTLA-4, (vii) rAd.sT.GM, anti-PD-1 and anti-CTLA-4, and (viii) buffer groups, without statistical differences among the groups. On day 7, rAd.sT,GM (2 5 10 ¹⁰ VPs in IOOmI) or PBS is administrated directly into the tumors. A second injection (2.5* 10 ¹⁰ VPs or PBS) is administered on day 10. On days 8, 10, 13, and 15, anti-PD-1 and anti-CTLA-4 antibodies are administered individually or together intraperitoneally (0.2 mg per mouse/each antibody) in the groups indicated as receiving the listed agent. The tumor volumes in the injected areas are monitored on day 10, 14, 17, 21, and 25. Mice are euthanized on day 25. Whole lungs are excised and photographed. Lung-surface tumor lesions are counted for each group. Parts of the lungs are processed for H&E staining according to a published protocol (Yang, Y., et al. (2019) Hum Gene Ther 30, 1117-1132). Pulmonary metastatic burdens are quantified by using Image J software (NIH, Bethesda, MD) for each lung section. [00257] Expected Results. The triple treatment is expected to be more effective in reducing tumor volume and metastasis than dual treatments and single treatment.

[00258] Statistical analysis. All data are analyzed using GraphPad Prism software version 5 (GraphPad software, San Diego, CA). For all statistical analyses, data are plotted as the mean ± standard error of the mean (SEM). Longitudinal data are analyzed using a two-way repeated measure ANOVA followed by Bonferroni post hoc pair wise tests for the data obtained over the time course. For multiple group analyses, one-way ANOVA followed by Bonferroni post hoc tests adjusting for multiplicity are performed. Student’s /-tests are performed to compare two sets of data. Differences are considered significant at two-sided p < 0.05.

EXAMPLE 7: ADENOVIRAL-MEDIATED REPLICATION AND CYTOTOXICITY ASSAYS [00259] All viral vectors were produced and purified to Good Laboratory Practice standards in Vector Development Lab/ Gene Vector Core of Baylor College of Medicine, Houston, TX. They also determined the concentration and titers of these viruses and performed sterility and endotoxin assays for the quality control and verification. All were maintained in 20mM Hepes, pH 7.8, 150mM NaCl + 10% glycerol buffer with the concentration of 5.0x1012 viral particles (VPs)/ml and stored in -80°C deep freezer with 500pl/aliquot. The total viral particles for each virus are ranging from 3.0-4.9xl0 ¹³ . Their titers are ranging from 1.6xlO ^u - l.OxlO ¹² pfu/ml. All viruses are tested negative for sterility. Their endotoxin levels are < 0.1 EU/ml except for rAd.Null (0.15 EU/ml). They all are below the endotoxin limits set by FDA for an investigational drug in the early clinical development.

[00260] For characterization, adenoviral-mediated replication and cytotoxicity assays were performed and adenoviral-mediated sTGFpRIIFc and GM-CSF expression was examined in both human and mouse breast cancer cell lines.

[00261] Briefly, for replication assay, human breast cancer cells (MDA-MB-231 and MCF-7) and mouse breast cancer cells (4Tl-luc2) were plated into a 6-well plates (2xl0 ⁵ cells/well). The next day, cells were infected with 2.5xl0 ⁴ VPs/cell of adenoviruses. Three hours after infection, cells were washed with phosphate-buffered saline buffer (PBS), and crude viral lysates were either collected immediately (3 h samples) or 48 hours after infection with completed cell culture media. The viral titers in 3- or 48-h crude viral lysates were determined using the Adeno-X Rapid Titer Kit (Clutch, Mountain view, CA). Viral burst size (an increase in hexon-expressing positive cells from 3 to 48 h) was used as an indicator of viral replication. [00262] For cytotoxicity assay, breast cancer cells were seeded into 96-well plates (l.OxlO ³ cells/well). The next day, cells were infected with various doses of adenoviruses (ranging from 80 to 1.25xl0 ⁶ VPs/cell and continued to culture for 7 days. Uninfected cells were used as the control. Cell survival was determined by sulforhodamine B staining and the lab’s standard protocol described in previous publications.

[00263] For Adenoviral-mediated sTGFpRIIFc and GM-CSF expression, breast cancer cells were plated into a 6-well plates (2xl0 ⁵ cells/well). The next day, cells were infected with 2.5xl0 ⁴ VPs/cell of adenoviruses. Twenty-four hours after infection, cells were washed with PBS, and the media were changed to serum-free media. Then, the incubations continued for another 24 hours and media were collected. To quantify sTGFpRIIFc protein in media, enzyme- linked immunosorbent assay (ELISA) was conducted using anti-human IgG, Fey Fragment Specific antibody and biotinylated anti-TGFp RII (BAF241) antibody. To quantify GM-CSF protein in media, anti-human GM-CSF monoclonal antibody (Clone 6804, ELISA Capture) and biotinylated anti- human GM-CSF monoclonal antibody (Clone 3209, ELISA Detection) were used in ELISA.

[00264] Results

[00265] All replicating adenoviruses: rAd.sT.GM, rAd.GM, rAd.sT and rAd.Null produced much higher levels of viral replication in human breast cancer cells (MDA-MB-231 and MCF-7) (with an average ratio of 1506 for 231 and 2699 for MCF-7) than those in mouse breast cancer cells (4Tl-luc2) (Average is 2.45). It is consistent with the nature of human adenovirus and our previous studies (Zhang, et al, 2021, doi:10.1038/cgt.2012.41; Xu, et al, 2020, DOI: 10.1089/hum.2020.078). However, when comparing to the control replication- deficient Ad(El-), all replicating viruses have significantly higher replication levels in all cells. [00266] Cytotoxicity assay. All adenoviruses: rAd.sT.GM, rAd.GM, rAd.sT, rAd.Null and replication-deficient Ad(El-). Null produced dose-dependent cytotoxicity in both human and mouse breast cancer cells. Cytotoxicity of replicating adenoviruses (50% survival rate at 1.0-5.0 xlO ⁴ VPs/cell in human cells and 1.0 xlO ⁵ VPs/cell in mouse cells) is much greater than replication-deficient Ad(El-). Null (50% survival rate is at around l.OxlO ⁶ VPs/cell in all cells). Mouse breast cancer cells 4T1, even with the much lower replication potentials for all replicating viruses, is susceptible to the killing ability of adenoviruses. See Figs. 3A-3C. This suggests, in addition to viral replication, there might be additional mechanisms and factors that contribute toward viral-induced cytotoxicity in the mouse cells. For example, in this experiment, the prolonged incubation with adenoviruses would increase viral-induced cytotoxicity in the mouse cells. Interestingly, for rAd.sT.GM, even has the lowest levels of replication when comparing to other replicating viruses, can kill 4T1 cells as effectively as the other replicating viruses (50% survival rates are around 1.0 xlO ⁵ VPs/cell for all replicating viruses).

EXAMPLE 8: ADEN O VIRAL-MEDIATED STGFBRTTFC ANP GM-CSF EXPRESSION [00267] Infection of the human and mouse breast cancer cells with rAd.sT.GM and rAd.sT produced high levels of sTGFpRIIFc protein, which was secreted into the cell culture media and detected by sTGFpRIIFc ELISA. sTGFpRIIFc protein concentration in those media are ranging from 3.9-5.3 pg/ml with an average amount of 4.5 pg/ml by our ELISA method. No sTGFpRIIFc expression was detected in cells infected with rAd.GM, rAd.Null and Ad(El-). Null. There was no significant difference of sTGFpRIIFc expression levels among all cell types and between cell infected with rAd.sT.GM and those with rAd.sT.

[00268] For GM-CSF expression, the high levels of GM-CSF were detected only in cells infected with adenoviruses with GM-CSF gene: rAd.sT.GM and rAd.GM. GM-CSF concentration are ranging from 159-1398 ng/ml with an average amount of 547.5 ng/ml by ELISA. Human breast cancer cells 231 have higher levels of GM-CSF expression (Avg: 995.5 ng/ml) when compared to another human cell MCF7 (Avg: 338.5 ng/ml) and mouse cell 4T1 (Avg: 308.5 ng/ml). It can be due to high replication levels of adenovirus and endogenous expression of human GM-CSF in 231 cells, since a low levels of GM-CSF expression has been detected from the mock and Ad(El-). Null infection in 231 cells (mock: 78 ng/ml; Ad(El-).

Null: 71 ng/ml). Importantly, 4T1 cells can express high levels of sTGFpRIIFc and/or human GM-CSF when infected with corresponding viruses. Also, the new adenoviruses: rAd.sT.GM elicited high levels of sTGFpRIIFc protein and human GM-CSF in all cells types including mouse breast cancer cell line: 4T1. See FIGS. 4A-4B.

The expression of sTGFpRIIFc and human GM-CSF in 4T1 tumors from all groups was also quantified by qRT-PCR. sTGFpRIIFc expression in tumors (day 10) from rAd.sT or rAd.sT.GM treated mice is 115717.69 or 118065.27 times (average value of relative quantification (RQ)) higher than that of the buffer group. By using non-parametric Kruskal- Wallis one-way analysis of variance to compare all groups, rAd.sT vs the buffer group is P < 0.05 and rAd.sT.GM vs the buffer group is P < 0.01. This statistical difference is due to the rAd.sT. GM-treated group has less variation in sTGFpRIIFc expression which can be visually assessed by the sizes of error bars on the graph. We also analyzed sTGFpRIIFc expression in day 25 tumor samples. sTGFpRIIFc mRNA level is 4.338 times (average) higher than that of the buffer group in rAd.sT-treated samples and 11.940 times (average) higher in rAd.sT. GM-treated samples. No significant difference has been detected among all groups by using Kruskal-Wallis test. Next, viral-induced human GM-CSF expression in tumors was determined by qRT-PCR.

On day 10 (viruses were injected intratum orally on day 7 and day 9), the average expression levels by RQ are 74526.09 times higher than that of the buffer group in rAd.GM treated tumors, and 79320.71 times higher in rAd.sT.GM treated tumors. By non-parametric Kruskal-Wallis one-way analysis, rAd.GM vs the buffer group is P < 0.05 and rAd.sT.GM vs the buffer group is P < 0.01. The variation of GM-CSF expression in rAd.sT.GM treated group is smaller than that in rAd.GM group, and as a result, it is more significant by Kruskal-Wallis test. For day 25 samples, the average expression levels of human GM-CSF are 4.34 times higher than that of the buffer group in rAd.GM treated tumors, and 11.94 times higher in rAd.sT.GM treated tumors, but no significant difference has been detected among all day 25 groups by using Kruskal- Wallis test. When taken together with relative quantification data of sTGFpRIIFc, not only expression of either sTGFpRIIFc or human GM-CSF elicited by rAd.sT.GM didn’t interfere with each other, but also rAd.sT.GM seems to have a more stable expression profile and a more prolonged presence of both sTGFpRIIFc and human GM-CSF in tumors. See Figs. 5A-5D. [00269] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. 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, since the scope of the present invention will be limited only by the appended claims.

[00270] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.