Modified oncolytic virus

Abstract

To provide an oncolytic agent and a cancer treatment method using the oncolytic agent.SOLUTION: An oncolytic virus is provided, the oncolytic virus comprising (i) a gene encoding a fusogenic protein and (ii) an immune stimulatory molecule or a gene encoding an immune stimulatory molecule.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to oncolytic immunotherapeutic agents and the use of oncolytic immunotherapeutic agents in the treatment of cancer. [Background technology]

[0002] Viruses have the inherent ability to invade cells with high efficiency. After cell entry, viral genes are expressed and the virus replicates. This usually results in the death of the infected cell and the release of antigenic components of the cell as the cell ruptures upon death. As a result, virus-mediated cell death tends to result in an immune response against these cellular components, including both those derived from the host cell and those encoded by or incorporated into the virus itself. The immune response is also amplified due to the host's recognition of so-called damage-associated molecular patterns (DAMPs), which aid in the activation of the immune response.

[0003] Viruses also engage various mediators of the innate immune response as part of the host response to recognition of viral infection, for example, through Toll-like receptors, cGAS / STING signaling, and recognition of pathogen-associated molecular patterns (PAMPs), which result in inflammation and activation of the interferon response, which are also immunogenic signals to the host. These immune responses can provide immunogenic benefits to cancer patients, such that immune responses to tumor antigens provide a systemic overall benefit resulting in the treatment of non-virally infected tumors, including micrometastatic disease, and provide vaccination against recurrence.

[0004] The combined direct ("oncolytic") effect of the virus and the immune response to tumor antigens (including non-self "neo-antigens," i.e., derived from specific mutated genes in individual tumors) is called "oncolytic immunotherapy."

[0005] Viruses can also be used as delivery vehicles ("vectors") to express heterologous genes inserted into the viral genome in infected cells. These properties make viruses useful in a variety of biotechnological and medical applications. For example, viruses expressing heterologous therapeutic genes can be used in gene therapy. In the context of oncolytic immunotherapy, the delivered genes may include genes encoding specific tumor antigens, genes intended to induce an immune response or increase the immunogenicity of released antigens after viral replication and cell death, genes intended to shape the immune response generated, genes that increase the general immune activation state of the tumor, or genes that increase the direct oncolytic properties (i.e., cytotoxic effect) of the virus. Importantly, viruses have the ability to deliver encoded molecules intended to help initiate, augment, or shape a systemic anti-tumor immune response directly and selectively to tumors. This may have the benefit of, for example, reduced toxicity or concentrating beneficial effects in tumors (including those not infected with the virus) rather than off-target effects in normal (i.e., non-cancerous) tissues compared to systemic administration of these same molecules or other molecules targeting the same pathway.

[0006] Numerous viruses, such as herpes simplex virus (HSV), have demonstrated utility in oncolytic treatment of cancer. For use in oncolytic treatment of cancer, HSV must be rendered ineffective so that it is no longer pathogenic but can invade and kill tumor cells. Numerous null mutations in HSV have been identified, including disruption of the genes encoding ICP34.5, ICP6, and / or thymidine kinase, which do not prevent the virus from replicating in tumor tissue in culture or in vivo, but do prevent significant replication in normal tissues. HSV with only the ICP34.5 gene disrupted replicates in many tumor cell types in vitro and selectively replicates in tumor tissue but not in surrounding tissues in mouse tumor models. Clinical trials of HSV with deleted ICP34.5, or deleted ICP34.5 and ICP6, have also demonstrated safety and selective replication in human tumor tissue.

[0007] As discussed above, oncolytic viruses such as HSV can also be used to deliver therapeutic genes in the treatment of cancer. This type of ICP34.5-deleted virus, further deleted for ICP47 and encoding a heterologous gene for GM-CSF, has also been tested in clinical trials, including a Phase 3 trial in melanoma, where safety and efficacy in humans have been demonstrated. Data demonstrated tumor responses in injected tumors and, to a lesser extent, in uninjected tumors. Responses tended to be durable (months to years), and responding patients appeared to achieve a survival benefit. Each of these demonstrated the involvement of the immune system in cancer treatment in addition to a direct oncolytic effect. However, this and other data on oncolytic viruses have generally shown that not all tumors respond to treatment, and not all patients achieve a survival benefit. As a result, improvements to this technology of oncolytic therapy are clearly needed. These may serve to increase the direct oncolytic effect of the therapy, the antitumor immunostimulatory effect of the therapy, or both of these effects together.

[0008] Recently, it has been shown that oncolytic immunotherapy can provide additive or synergistic therapeutic effects in combination with immune checkpoint blockade (i.e., inhibition or "antagonism" of immune checkpoint pathways), also known as immune co-inhibitory pathway blockade. Checkpoint (immune co-inhibitory pathway) blockade is typically intended to block host immune inhibitory mechanisms that serve to prevent the development of autoimmunity. However, in cancer patients, these mechanisms may also serve to inhibit or block the induction of potentially beneficial effects of any tumor-induced immune response. Alternatively, the immune response may not be fully enhanced due to a lack of activation or a lack of complete activation of immune-enhancing pathways. Therefore, agents that alleviate these blockades (inhibit "immune co-inhibitory pathways") or stimulate immune-enhancing pathways (i.e., activate or "agonize" "immune co-stimulatory pathways") are attractive for testing and development as cancer treatments. Targets of such approved or experimental drugs include CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, VISTA, CSF1R, IDO, CEACAM1, GITR, 4-1-BB, KIR, SLAMF7, OX40, CD40, ICOS, or CD47.

[0009] For many of these approaches targeting immune co-inhibitory pathways or co-inhibitory pathways to be successful, a pre-existing immune response against the tumor is required, i.e., it can enhance the pre-existing immune response or alleviate the blockade of the anti-tumor immune response. The presence of an inflamed tumor microenvironment, which indicates such an ongoing response, is also required. A pre-existing immune response against tumor neoantigens appears to be particularly important for the activity of immune co-inhibitory pathway blockade and related drugs. Only some patients may have an ongoing immune response against tumor antigens, including neoantigens and / or an inflamed tumor microenvironment, both of which are required for optimal activity of these drugs. Therefore, oncolytic agents that can induce immune responses against tumor antigens, including neoantigens, and / or induce an inflamed tumor microenvironment are attractive for use in combination with immune co-inhibitory pathway blockade and immune-enhancing agents. This may explain the promising combined anti-tumor effects of oncolytic agents and immune co-inhibitory pathway blockade observed to date in mice and humans.

[0010] The indoleamine 2,3-dioxygenase (IDO) pathway contributes to tumor-induced tolerance by creating a tolerogenic environment in tumors and tumor-draining lymph nodes through both direct suppression of T cells and enhanced local regulatory T cell (Treg)-mediated immunosuppression. IDO catalyzes the rate-limiting step in tryptophan degradation along the kynurenine pathway, and both the reduction in local tryptophan concentrations and the generation of immunomodulatory tryptophan metabolites contribute to IDO's immunosuppressive effects. IDO is chronically activated in many cancer patients, with IDO activation correlating with a broader range of disease. It can also function as an antagonist to other activators of antitumor immunity. Therefore, inhibitors of the IDO pathway are being developed as anticancer drugs, particularly in combination with checkpoint blockade agents targeting CTLA-4, PD-1, or PDL-1.

[0011] The above discussion demonstrates that there remains significant room for improvement in oncolytic agents and cancer treatments that use oncolytic agents. Summary of the Invention

[0012] The present invention provides oncolytic viruses expressing a fusogenic protein and at least one immunostimulatory molecule. The oncolytic viruses of the present invention provide improved cancer treatment through improved direct oncolytic effects, viral replication, and tumor spread mediated by the fusogenic protein, which (i) increases the amount of tumor antigens, including neoantigens, released for the induction of anti-tumor immune responses, and (ii) enhances the expression of virally encoded immunostimulatory molecules. Expression of immunostimulatory molecules further increases and enhances the anti-tumor immune effect. Anti-tumor efficacy is improved when the oncolytic viruses of the present invention are used as a single agent and when the viruses are used in combination with other anti-cancer modalities, such as chemotherapy, targeted drug treatment, radiation, immune checkpoint blockade, and / or immune-enhancing agents.

[0013] Thus, the present invention provides an oncolytic virus comprising (i) a gene encoding a fusogenic protein and (ii) a gene encoding an immunostimulatory molecule. The virus may encode more than one fusogenic protein and / or more than one immunostimulatory molecule.

[0014] The fusogenic protein is preferably a glycoprotein derived from gibbon ape leukemia virus (GALV) in which the R transmembrane peptide has been mutated or deleted (GALV-R-). The immunostimulatory molecule is preferably GM-CSF and / or an agonist of an immune costimulatory pathway, such as GITRL, 4-1-BBL, OX40L, ICOSL, or CD40L, or a modified version of any of them. Examples of modified versions include agonists of costimulatory pathways that are secreted rather than membrane-bound, and / or agonists modified to form protein multimers. The immunostimulatory molecule can be a protein capable of blocking CTLA-4-mediated signaling, such as an antibody or fragment thereof that binds to CTLA-4.

[0015] The virus can be a modified clinical isolate, such as a modified clinical isolate of a virus, which kills two or more tumor cell lines more quickly and / or at lower doses in vitro than one or more reference clinical isolates of the same virus.

[0016] The virus is preferably a herpes simplex virus (HSV), such as HSV 1. HSV typically does not express functional ICP34.5 and / or functional ICP47, and / or expresses the US11 gene as an immediate early gene.

[0017] The present invention also provides: - a pharmaceutical composition comprising the virus of the invention and a pharmaceutically acceptable carrier or diluent, - a virus of the invention for use in a method for treating the human or animal body by therapy, - a virus of the invention for use in a method for treating cancer, the method optionally comprising administering a further anti-cancer agent. - a product containing the virus of the invention in a sterile vial, ampoule or syringe, - a method for treating cancer comprising administering a therapeutically effective amount of the virus or pharmaceutical composition of the present invention to a patient in need thereof, the method optionally comprising administering a further anti-cancer agent, which anti-cancer agent is optionally an antagonist of an immune co-inhibitory pathway or an agonist of an immune co-stimulatory pathway, - use of a virus of the invention in the manufacture of a medicament for use in a method for treating cancer, the method optionally comprising administering a further anti-cancer agent, which anti-cancer agent is optionally an antagonist of an immune co-inhibitory pathway or an agonist of an immune co-stimulatory pathway; - A method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of an oncolytic virus, an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway and an additional antagonist of an immune co-inhibitory pathway or an agonist of an immune co-stimulatory pathway. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows the structure of an exemplary virus of the invention, comprising a GALV-R-encoding gene and a GM-CSF-encoding gene inserted into the ICP34.5 locus, with the ICP47 gene deleted so that the US11 gene is under the control of the ICP47 immediate-early promoter. Figure 1 also shows similar viruses expressing only the GALV-R-encoding gene (second panel) or only the GM-CSF-encoding gene (third panel). Also shown are viruses in which the ICP34.5 and ICP47 genes are deleted but which do not contain the inserted genes. [Figure 2] FIG. 1 shows the structure of an exemplary virus of the invention, including a GALV-R-encoding gene, a GM-CSF-encoding gene, and a CD40L-encoding gene. [Figure 3]Figure 3 shows the differential ability of eight top-ranked HSV1 clinical isolates to kill Fadu, SK-mel-28, A549, HT1080, MIA-PA-CA-2, HT29, and MDA-MB-231 human tumor cell lines, as assessed by crystal violet staining 24 or 48 hours after infection at an MOI of 0.1, 0.01, or 0.001, as indicated. The first and second ranked virus strains for each cell line are shown. Virus RH018A was ranked first for each of the Fadu, HT1080, MIA-PA-CA-2, and HT29 cell lines, and second for each of the SK-mel-28, A549, and MDA-MB-231 cell lines. RH004A was ranked first in both RH018A and RH015A for the HT29 cell line, first in the SK-mel-28 and A549 cell lines, and second in the Fadu cell line. RH023A was ranked first in the MDA-MB-231 cell line and second in the HT1080 cell line. RH031A was ranked second in each of the MIA-PA-CA-2 and HT29 cell lines. RH040A was ranked second in both the HT29 cell line. [Figure 4] Figure 1 shows a comparison of strain RH018A, the highest ranked strain of all strains tested, with the "average" strain from the screen (i.e., strain RH065A). Approximately 10-fold less strain RH018A was required to kill an equal percentage of cells than strain RH065A, as shown by crystal violet staining 24 or 48 hours post-infection at MOIs of 0.1, 0.01, and 0.001 in SK-mel-28, HT1080, MDA-MB-231, Fadu, MIA-PA-CA-2, and A549 cell lines. [Figure 5-1]1 shows the structure of HSV1 viruses modified by deletion of ICP34.5 and ICP47 so that the US11 gene is under the control of the ICP457 immediate-early promoter and contains a heterologous gene at the ICP34.5 locus. Viruses were constructed using the RH018A strain unless otherwise noted in the figure. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-1. [Figure 5-4] This is a continuation of Figure 5-1. [Figure 5-5] This is a continuation of Figure 5-1. [Figure 5-6] This is a continuation of Figure 5-1. [Figure 5-7] This is a continuation of Figure 5-1. [Figure 5-8] This is a continuation of Figure 5-1. [Figure 5-9] This is a continuation of Figure 5-1. [Figure 5-10] This is a continuation of Figure 5-1. [Figure 5-11] This is a continuation of Figure 5-1. [Figure 6] FIG. 1 shows the results of an ELISA to detect expression of human or mouse GM-CSF in the supernatants from BHK cells infected with virus 16 (mGM-CSF and GALVR-), virus 17 (hGM-CSF and GALVR-), and virus 19 (mGM-CSF). [Figure 7-1] FIG. 1 shows a comparison of the cell killing ability of the ICP34.5 deleted, GALVR- and GFP-expressing RH018A strain (virus 10) with a virus expressing GFP only (virus 12) as determined by crystal violet staining in three cell lines at low magnification. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 8-1] FIG. 1 shows a comparison of the cell killing ability of the ICP34.5 and ICP47 deleted, GALVR- and GM-CSF expressing RH018A strain (virus 17) with a conventional strain with the same modifications, as determined by crystal violet staining in four cell lines. [Figure 8-2] This is a continuation of Figure 8-1. [Figure 9] Figure 1 shows the efficacy of virus 16 (ICP34.5 and ICP47 deleted, GALVR- and mGM-CSF-expressing) in treating mice bearing bilateral A20 lymphoma tumors. Right flank tumors were injected with virus or vehicle, and the effect on tumor size was observed for 30 days. The virus was effective against both injected and uninjected tumors. [Figure 10]

[0023] Figure 1 demonstrates the effects of virus 15 (ICP34.5 and ICP47 deleted, GALVR- and GFP-expressing) and virus 24 (ICP34.5 and ICP47 deleted, GFP-expressing) on ​​rat 9L cells in vitro as assessed by crystal violet staining. The virus expressing GALV (virus 15) showed increased killing of rat 9L cells in vitro compared to the virus not expressing GALV (virus 24). [Figure 11-1]Figure 1 shows the antitumor effect of virus 16 in Balb / c mice bearing mouse CT26 tumors on both the left and right flanks. Groups of 10 mice were then treated with the following: vehicle (three injections into the right flank tumor every other day), 5 x 10 pfu of virus 16 (mRP1) injected into the right flank tumor every other day, anti-mouse PD1 alone (10 mg / kg, intraperitoneally, every 3 days, BioXCell clone RMP1-14), anti-mouse CTLA-4 (3 mg / kg, intraperitoneally, every 3 days, BioXCell clone 9D9), virus 16 with anti-mouse PD1, virus 16 with anti-mouse CTLA-4, 1-methyltryptophan (I-MT, an IDO inhibitor (5 mg / ml in drinking water)), anti-mouse PD1 with 1-methyltryptophan, or anti-mouse PD1 with 1-methyltryptophan and virus 16. The effect on tumor size was observed for an additional 30 days. Greater tumor reduction was observed in animals treated with the combination of virus and checkpoint blockade than in the single-treatment groups. Figure 11A shows that combining virus 16 and anti-PD1 has a superior anti-tumor effect than either anti-PD1 or virus alone. Figure 11B shows that the anti-tumor effect of virus 16 in combination with anti-CTLA-4 is better than the anti-tumor effect of either virus 16 or anti-CTLA-4 alone. Figure 11C shows that enhanced tumor reduction was observed using both anti-PD1 and IDO inhibition with virus 16 compared to anti-PD1 and I-MT inhibition in the absence of virus. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 11-3] This is a continuation of Figure 11-1. [Figure 12-1] FIG. 1 shows the enhanced antitumor activity of virus 16 in combination with immune checkpoint blockade in bilateral murine A20 tumors in Balb / c mice compared to either virus alone or checkpoint blockade alone (anti-PD1). [Figure 12-2] This is a continuation of Figure 12-1. [Figure 12-3] This is a continuation of Figure 12-1. [Figure 12-4] This is a continuation of Figure 12-1. [Figure 13] Figure 1 shows the structures of ICP34.5 and ICP47 deletion viruses expressing GALVR-, GM-CSF-, and codon-optimized anti-mouse or anti-human CTLA-4 antibody constructs (secreted scFv molecules linked to human or mouse IgG1 Fc regions). The scFvs contain linked ([G4S]3) light and heavy variable chains from antibody 9D9 (US2011044953: murine version) and ipilimumab (US20150283234, human version). The structures of the resulting CTLA-4 inhibitors are also shown. [Figure 14] Figure 1 shows the antitumor effects of virus 16 and virus 19 in a human xenograft model (A549). Virus 16, virus 19, or vehicle was injected three times over a one-week period at three different dose levels (N=10 / group). The doses of virus used are indicated. The antitumor effect of virus 16 expressing GALV was better than that of virus 19, which does not express GALV. [Figure 15]

[0033] Figure 1 shows the effect of a virus of the present invention expressing GALVR- on 9L cells in the flanks of Fischer 344 rats. The following treatments were administered to groups of rats (10 per group) in one flank of each rat, three times a week for three weeks: 50 μl of vehicle, 50 μl of 10 pfu / ml of virus 19 (expressing mGM-CSF but not GALV R-), or 50 μl of 10 pfu / ml of virus 16 (expressing both murine GM-CSF and GALV R-). The effect on tumor growth was then observed for an additional 30 days. Superior tumor control and regression was observed with the virus expressing GM-CSF and GALV R- compared to the virus expressing GM-CSF alone. [Figure 16] Figure 1 shows the antitumor effects of viruses expressing anti-mCTLA-4 (virus 27), mCD40L (virus 32), mOX4OL (virus 35), and m4-2BBL (virus 33), each of which also has mGM-CSF and GALV-R, compared to virus 16 (expressing GALV and mGM-CSF). DETAILED DESCRIPTION OF THE INVENTION

[0019] Brief Description of Sequence Listing SEQ ID NO: 1 is the nucleotide sequence of mouse GM-CSF. SEQ ID NO: 2 is the nucleotide sequence of a codon-optimized version of mouse GM-CSF. SEQ ID NO: 3 is the nucleotide sequence of human GM-CSF. SEQ ID NO: 4 is the nucleotide sequence of a codon-optimized version of human GM-CSF. SEQ ID NO: 5 is the amino acid sequence of mouse GM-CSF. SEQ ID NO: 6 is the amino acid sequence of human GM-CSF. SEQ ID NO: 7 is the nucleotide sequence of GALV-R-. SEQ ID NO: 8 is the nucleotide sequence of a codon-optimized version of GALV-R- (the first three nucleotides are arbitrary). SEQ ID NO: 9 is the amino acid sequence of GALV-R-. SEQ ID NO: 10 is the nucleotide sequence of a codon-optimized version of the human membrane-bound version of CD40L. SEQ ID NO: 11 is the amino acid sequence of the human membrane-bound version of CD40L. SEQ ID NO: 12 is the nucleotide sequence of a codon-optimized version of the multimeric secreted version of human CD40L. SEQ ID NO: 13 is the amino acid sequence of the multimeric secreted version of human CD40L. SEQ ID NO: 14 is the nucleotide sequence of a codon-optimized version of the multimeric secreted version of mouse CD40L. SEQ ID NO: 15 is the amino acid sequence of the multimeric secreted version of mouse CD40L. SEQ ID NO: 16 is a codon-optimized version of the nucleotide sequence of wild-type human CD40L. SEQ ID NO: 17 is the amino acid sequence of wild-type human CD40L. SEQ ID NO: 18 is a codon-optimized version of the nucleotide sequence of wild-type mouse CD40L. SEQ ID NO: 19 is the amino acid sequence of wild-type mouse CD40L. SEQ ID NO:20 is the nucleotide sequence of a codon-optimized version of mouse 4-1BBL. SEQ ID NO: 21 is the nucleotide sequence of a codon-optimized version of human 4-1BBL. SEQ ID NO: 22 is the nucleotide sequence of a codon-optimized version of secretory murine 4-1BBL. SEQ ID NO: 23 is the nucleotide sequence of a codon-optimized version of human secreted 4-1BBL. SEQ ID NO: 24 is the nucleotide sequence of a codon-optimized version of mouse GITRL. SEQ ID NO: 25 is the nucleotide sequence of a codon-optimized version of human GITRL. SEQ ID NO: 26 is the nucleotide sequence of a codon-optimized version of secreted mouse GITRL. SEQ ID NO: 27 is the nucleotide sequence of a codon-optimized version of secreted human GITRL. SEQ ID NO: 28 is the nucleotide sequence of a codon-optimized version of mouse OX40L. SEQ ID NO: 29 is the nucleotide sequence of a codon-optimized version of human OX40L. SEQ ID NO: 30 is the nucleotide sequence of a codon-optimized version of secreted mouse OX40L. SEQ ID NO: 31 is the nucleotide sequence of a codon-optimized version of secreted human OX40L. SEQ ID NO: 32 is the nucleotide sequence of a codon-optimized version of mouse ICOSL. SEQ ID NO: 33 is the nucleotide sequence of a codon-optimized version of human ICOSL. SEQ ID NO: 34 is the nucleotide sequence of a murine scFv CTLA-4 antibody. The first 6 nucleotides and the last 8 nucleotides are restriction sites added for cloning purposes. SEQ ID NO: 35 is the nucleotide sequence of a murine scFv CTLA-4 antibody. The first 6 nucleotides and the last 8 nucleotides are restriction sites added for cloning purposes. SEQ ID NO: 36 is the nucleotide sequence of the CMV promoter. SEQ ID NO:37 is the nucleotide sequence of the RSV promoter. SEQ ID NO: 38 is the nucleotide sequence of BGH polyA. SEQ ID NO:39 is the nucleotide sequence of SV40 late polyA. SEQ ID NO:40 is the nucleotide sequence of the SV40 enhancer promoter. SEQ ID NO: 41 is the nucleotide sequence of rabbit beta-globulin (RBG) polyA. SEQ ID NO: 42 is the nucleotide sequence of GFP. SEQ ID NO: 43 is the nucleotide sequence of the MoMuLV LTR promoter. SEQ ID NO: 44 is the nucleotide sequence of the EF1a promoter. SEQ ID NO: 45 is the nucleotide sequence of HGH polyA.

[0020] Detailed Description of the Invention Oncolytic viruses The virus of the present invention is oncolytic. An oncolytic virus is a virus that infects and replicates in tumor cells, thereby killing the tumor cells. Therefore, the virus of the present invention is replicable. Preferably, the virus is selectively replicable in tumor tissue. A virus is selectively replicable in tumor tissue if it replicates more effectively in tumor tissue than in non-tumor tissue. The ability of a virus to replicate in different tissue types can be determined using standard techniques in the art.

[0021] The oncolytic effect depends on the virus replicating in and killing the initially infected cell, and on the progeny virions continuing to infect and kill other tumor cells, resulting in intratumoral proliferation. Thus, the ability of the virus of the present invention to effectively kill tumor cells and replicate within tumors results in optimal direct antitumor effects. Furthermore, tumor cell lysis associated with efficient proliferation and viral replication maximizes the amount of tumor antigens released, and therefore the efficacy of the induced antitumor immune response.

[0022] The virus of the present invention can be any virus with these properties, such as herpesvirus, poxvirus, adenovirus, retrovirus, rhabdovirus, paramyxovirus, or reovirus, or any species or strain within these larger groups.The virus of the present invention can be wild-type (i.e., not modified from the parent virus species), or can have gene disruption or gene addition.Which of these depends on the virus species used.Preferably, the virus is a herpesvirus species, more preferably a HSV strain, including HSV1 and HSV2 strains, and most preferably a HSV1 strain.In a particularly preferred embodiment, the virus of the present invention is based on a clinical isolate of the virus species used.Clinical isolates may be selected based on those with particularly advantageous properties for cancer treatment.

[0023] A clinical isolate may have a surprisingly good antitumor effect compared to other strains of the same virus isolated from other patients, where the patient is an individual carrying the virus species being tested. The virus strains used for comparison to identify viruses useful in the present invention may be isolated from patients or otherwise healthy volunteers (i.e., other than those carrying the virus species being tested), preferably from otherwise healthy volunteers. The HSV1 strains used to identify viruses of the present invention are typically isolated from cold sores of individuals carrying HSV1, typically by swabbing using, for example, Virocult (Sigma) brand wipes / containers containing transport media, and then transported to a facility for further testing.

[0024] After isolating the viruses to be compared from individuals, virus stocks are typically prepared by propagating the isolated viruses, for example, on BHK or Vero cells. Preferably, this is done after no more than three cycles of freeze-thawing between collecting the sample and propagating it, for example, on BHK or Vero cells, to prepare the virus stock for further use. More preferably, the virus sample has undergone two or fewer cycles of freeze-thawing, more preferably one cycle of freeze-thawing, and most preferably no cycles of freeze-thawing, before preparing the stock for further use. Lysates from virus-infected cell lines prepared in this manner after isolation are typically compared by testing the virus's ability to kill tumor cell lines in vitro. Alternatively, virus stocks can be stored under appropriate conditions, for example, by freezing, before testing. The viruses of the present invention may have surprisingly good antitumor effects compared to other strains of the same virus isolated from other individuals, preferably compared to those isolated from more than five, more preferably more than ten, and most preferably more than 20 other individuals.

[0025] Stocks of clinical isolates identified as viruses for modification to produce the viruses of the present invention (i.e., having surprisingly good properties for killing tumor cells compared to other viral strains to which they are compared) can be stored under appropriate conditions, before and after modification, and used to generate further stocks as needed.

[0026] A clinical isolate is a strain of a virus species that has been isolated from its natural host. Clinical isolates are preferably isolated for the purpose of testing and comparing the clinical isolate with other clinical isolates of that virus species for desired properties, particularly the ability to kill human tumor cells. Clinical isolates that can be used for comparison also include those present in clinical repositories, i.e., those derived from clinical samples previously collected for clinical diagnostic or other purposes. In either case, clinical isolates used for comparison and identification of the viruses of the present invention preferably undergo minimal in vitro culture before being tested for the desired properties, preferably only sufficient culture to generate a sufficient stock for comparative testing purposes. Thus, viruses used for comparison to identify the viruses of the present invention can also include deposited strains, where the deposited strain is an HSV1 strain isolated from a patient, preferably an HSV1 strain isolated from a patient's herpes labialis.

[0027] The virus can be a modified clinical isolate, where the clinical isolate kills two or more tumor cell lines in vitro more rapidly and / or at lower doses than one or more reference clinical isolates of the same species of virus. Typically, the clinical isolate kills two or more tumor cell lines within 72 hours, preferably within 48 hours, and more preferably within 24 hours of infection at a multiplicity of infection (MOI) of 0.1 or less, preferably at an MOI of 0.01 or less, and more preferably at an MOI of 0.001 or less. Preferably, the clinical isolate kills a broad range of tumor cell lines, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or for example all, of the following human tumor cell lines: U87MG (glioma), HT29 (colorectal), LNCaP (prostate), MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu (squamous cell carcinoma), MCF7 (breast), A549 (lung), MIAPACA-2 (pancreatic), CAPAN-1 (pancreatic), HT1080 (fibrosarcoma).

[0028] Thus, the viruses of the invention can kill cells from two or more, e.g., 3, 4, 5, 6, 7 or more different types of tumors, e.g., from two or more, e.g., 3, 4, 5, 6, 7 or more solid tumors, including, but not limited to, colorectal tumor cells, prostate tumor cells, breast tumor cells, ovarian tumor cells, melanoma cells, squamous cell carcinoma cells, lung tumor cells, pancreatic tumor cells, sarcoma cells and / or fibrosarcoma cells.

[0029] Tumor cell line kill can be determined by any suitable method. Typically, a sample is first isolated from a patient, preferably from a cold sore in the case of HSV1, and used to infect BHK cells or another suitable cell line, such as Vero cells. Positive samples are typically identified by the presence of cytopathic effect (CPE) 24-72 hours post-infection, e.g., 48 hours post-infection, and confirmed to be the target virus species by, e.g., immunohistochemistry or PCR. A virus stock is then generated from the positive sample. Samples from the virus stock are typically tested and compared with other samples similarly generated using swabs from different patients. Testing can be performed by determining the level of CPE achieved over a range of multiplicities of infection (MOI) and at various time points post-infection.

[0030] For example, 80% confluent cell lines can be infected with virus samples at MOIs of 1, 0.1, 0.01, and 0.001, and duplicate plates can be incubated for 24 and 48 hours at 37°C and 5% CO2, after which the extent of viral cell killing can be determined. This can be determined, for example, by fixing the cells with glutaraldehyde and staining with crystal violet using standard methods. The level of cell lysis can then be assessed by standard methods, such as visual observation, microscopy (cell counting), and photography. This method can be repeated with cells incubated for shorter periods, such as 8, 12, or 16 hours, or for longer periods, such as 72 hours, or at additional MOIs, such as 0.0001 or less, before cell killing is determined.

[0031] Growth curve experiments can also be performed to evaluate the ability of different clinical isolates to replicate in tumor cell lines in vitro. For example, 80% confluent cell lines are infected with virus samples at MOIs of 1, 0.1, 0.01, and 0.001, incubated at 37°C and 5% CO2, and typically lysed by freeze / thawing at 0, 8, 16, 24, and 48 hours post-infection, after which the degree of viral cell death is determined. This can be determined, for example, by assessing viral titer using a standard plaque assay.

[0032] A clinical isolate of the present invention can kill infected tumor cell lines more quickly and / or at a lower MOI than other clinical isolates to which it is compared, preferably 2, 3, 4, 5, or 10 or more other clinical isolates of the same virus species. A clinical isolate of the present invention typically kills a 10%, 25%, or 50% greater percentage of tumor cells present at a particular MOI and time point than at least one, preferably 2, 3, 4, 5, or 10 or more other clinical isolates of the same virus type compared at the same MOI and time point. A clinical isolate of the present invention typically kills the same or a greater percentage of tumor cells at an MOI that is half or less than half the MOI at which one or more, preferably 2, 3, 4, 5, 10, or 15 or more other clinical isolates of the same virus species used for comparison at the same time point, typically 12, 24, and / or 48 hours, kill the same percentage of tumor cells. Preferably, a clinical isolate of the present invention kills the same or a greater percentage of tumor cells at an MOI 5 or 10 times lower than the MOI at which one or more, preferably 2, 3, 4, 5, 10, or 15 or more, other clinical isolates of the same virus species used for comparison kill the same percentage of tumor cells at the same time point, typically 12, 24, and / or 48 hours. The improved tumor cell killing ability of the virus of the present invention is typically achieved compared to at least 50%, 75%, or 90% of the other clinical isolates of the same virus species used for comparison. The virus is preferably compared to at least four other virus strains of the same species, for example, 7, 9, 19, 39, or 49 other virus strains.

[0033] The isolated strains can be tested, for example, in batches of 4-8 virus strains at a time, against 4-8 tumor cell lines at a time. For each batch of experiments, the degree of killing achieved is ranked for each cell line from best (i.e., fewest surviving cells / MOI at each time point) to worst (i.e., most surviving cells / MOI at each time point) for the viruses compared in that experiment. The virus strain from each experiment that shows the best results across the range of tumor cell lines tested (i.e., consistently ranks as one of the best at killing the cell line) can then be directly compared in further experiments with other clinical isolates and / or other tumor cell lines to identify the best virus strain overall, for example, among the 20 or more virus strains sampled. The virus ranked as best overall is the virus of the present invention.

[0034] In a preferred embodiment, the virus of the invention is a strain selected from: strain RH018A, which has provisional accession number ECCAC16121904; strain RH004A, which has provisional accession number ECCAC16121902; strain RH031A, which has provisional accession number ECCAC16121907; strain RH040B, which has provisional accession number ECCAC16121908; strain RH015A, which has provisional accession number ECCAC16121903; strain RH021A, which has provisional accession number ECCAC16121905; strain RH023A, which has provisional accession number ECCAC16121906; and strain RH047A, which has provisional accession number ECCAC16121909.

[0035] More preferably, the virus of the invention is a strain selected from: strain RH018A, which has provisional accession number ECCAC16121904; strain RH004A, which has provisional accession number ECCAC16121902; strain RH031A, which has provisional accession number ECCAC16121907; strain RH040B, which has provisional accession number ECCAC16121908; and strain RH015A, which has provisional accession number ECCAC16121903.

[0036] Most preferably, the virus of the present invention is strain RH018A having accession number EACC16121904.

[0037] The HSV of the present invention can selectively replicate in tumors, such as human tumors. Typically, HSV replicates efficiently in target tumors but not in non-tumor tissues. The HSV may contain one or more mutations in one or more viral genes that inhibit replication in normal tissues but still allow replication in tumors. The mutations may be, for example, mutations that prevent the expression of functional ICP34.5, ICP6, and / or thymidine kinase by HSV.

[0038] In one preferred embodiment, the gene encoding ICP34.5 is mutated to confer selective oncolytic activity to HSV. Mutations in the gene encoding ICP34.5 that prevent the expression of functional ICP34.5 are described in Chou et al. (1990) Science 250:1262-1266, Maclean et al. (1991) J. Gen. Virol. 72:631-639, and Liu et al. (2003) Gene Therapy 10:292-303, which are incorporated herein by reference. Additionally, the gene encoding ICP6 and / or the gene encoding thymidine kinase can be inactivated, as can other genes, as long as such inactivation does not prevent viral infection or replication within tumors.

[0039] HSV may contain additional mutations that enhance HSV replication in tumors. The resulting enhancement of viral replication in tumors not only leads to direct "oncolytic" tumor cell killing by the virus, but also enhances the level of heterologous (i.e., genes inserted into the virus, in the case of the virus of the present invention, genes encoding fusogenic proteins and immunomodulatory molecules) gene expression, increasing the amount of tumor antigens released when tumor cells die. Both of these may also improve the immunogenic properties of therapeutics for cancer treatment. For example, in a preferred embodiment of the present invention, deletion of the gene encoding ICP47 in a manner that places the US11 gene under the control of the immediate-early promoter that normally regulates the expression of the gene encoding ICP47 results in enhanced replication in tumors (see Liu et al., 2003, incorporated herein by reference).

[0040] Other mutations can also be introduced into the virus of the present invention, placing the sequence encoding HSV late gene US11 under the control of a promoter that is not dependent on viral replication.This mutation allows US11 to be expressed before HSV replication occurs, enhancing viral replication in tumors.In particular, this mutation enhances the replication of HSV that lacks the gene encoding functional ICP34.5.

[0041] Thus, in one embodiment, the HSV of the present invention comprises a US11 gene operably linked to a promoter, the activity of which is independent of viral replication. The promoter can be an immediate-early (IE) promoter or a non-HSV promoter active in mammalian, preferably human, tumor cells. The promoter can be, for example, a eukaryotic promoter, such as a promoter derived from a mammalian, preferably human, genome. The promoter can be a ubiquitous promoter (such as a β-actin or tubulin promoter) or a cell-specific promoter, such as a tumor-specific promoter. The promoter can be a viral promoter, such as the Moloney murine leukemia virus long terminal repeat (MMLV LTR) promoter or a human or murine cytomegalovirus (CMV) IE promoter. HSV immediate-early (IE) promoters are well known in the art. The HSV IE promoter can be a promoter driving expression of ICP0, ICP4, ICP22, ICP27, or ICP47.

[0042] The above-mentioned genes, whose functional inactivation confers tumor-selective properties to the virus, can be rendered functionally inactive by any suitable method, for example, by deleting or replacing all or part of the gene and / or its regulatory sequences, or by inserting one or more nucleic acids into or in place of the gene and / or its regulatory sequences. For example, the viruses of the invention can be generated using homologous recombination methods, which are standard in the art. Alternatively, bacterial artificial chromosome (BAC)-based approaches can be used.

[0043] As used herein, the term "gene" is intended to mean a nucleotide sequence that encodes a protein, i.e., the coding sequence of a gene. The various genes mentioned above can be rendered non-functional by mutating the gene itself or the regulatory sequences adjacent to the gene, such as promoter sequences. Deletions can remove one or more portions of a gene, the entire gene, or the entire gene and all or part of the regulatory sequences. For example, deletion of only a single nucleotide within a gene resulting in a frameshift can occur. However, larger deletions can occur, such as deletion of at least about 25%, more preferably at least about 50%, of the entire coding and / or non-coding sequence. In one preferred embodiment, a gene that is functionally inactive is deleted. For example, the entire gene and, optionally, part of the flanking sequences can be removed from the virus. If two or more copies of a gene are present in the viral genome, both copies of the gene are rendered functionally inactive.

[0044] A gene can be inactivated by replacing it with another sequence, for example, by replacing all or part of the endogenous gene with a heterologous gene and, optionally, a promoter sequence. If the promoter sequence is not replaced, the heterologous gene can be inserted so that it is regulated by the promoter of the non-functionalized gene. In the HSV of the present invention, the gene encoding ICP34.5 is preferably rendered non-functional by inserting a heterologous gene, and a promoter sequence operably linked thereto, and optionally other control elements, such as a polyadenylation sequence, into each of the loci encoding ICP34.5.

[0045] The viruses of the present invention are used to express fusogenic proteins and immunostimulatory proteins in tumors. This is typically achieved by inserting heterologous genes encoding the fusogenic protein and the immunostimulatory protein into the genome of a selectively replication-competent virus, each of which is under the control of a promoter sequence. Because replication of such viruses occurs selectively in tumor tissue, the expression of the fusogenic protein and the immunostimulatory protein by the virus is also enhanced in tumor tissue compared to non-tumor tissues of the body. Enhanced expression occurs when expression is greater in tumors than in other tissues of the body. Thus, the present invention provides the benefit of selectively expressing both fusogenic proteins and immunostimulatory proteins in tumors, combined with the anti-tumor effect provided by oncolytic virus replication.

[0046] The viruses of the invention may contain, in addition to the fusogenic and immunostimulatory proteins, one or more further heterologous genes, such as further fusogenic or immunostimulatory proteins.

[0047] fusogenic proteins The viruses of the present invention contain a gene encoding a fusogenic protein. The fusogenic protein can be any heterologous protein capable of promoting the fusion of a cell infected with a virus of the present invention to another cell. A fusogenic protein, preferably a wild-type or modified viral glycoprotein (i.e., one modified to enhance its fusogenic properties), is a protein capable of inducing cell-cell fusion (syncitia formation) of cells in which it is expressed. Examples of fusogenic glycoproteins include VSV-G, syncytin-1 (derived from human endogenous retrovirus-W (HERV-W)), or syncytin-2 (derived from HERVFRDE1), paramyxovirus SV5-F, measles virus-H, measles virus-F, RSV-F, and glycoproteins from retroviruses or lentiviruses, such as gibbon ape leukemia virus (GALV), murine leukemia virus (MLV), Mason-Pfizer simian virus (MPMV), and equine infectious anemia virus (EIAV), from which the R transmembrane peptide has been deleted (R-version). In a preferred embodiment, the fusogenic protein is derived from GALV and the R peptide has been deleted (GALV-R-).

[0048] The viruses of the invention can contain multiple copies of a gene encoding a fusogenic protein, preferably 1 or 2 copies. The viruses can contain two or more different fusogenic proteins, including any of the fusogenic proteins listed above.

[0049] The fusogenic proteins expressed by the viruses of the invention may be identical to naturally occurring proteins or may be modified proteins.

[0050] The gene encoding the fusogenic protein (fusogenic gene) can have a naturally occurring nucleic acid sequence or a modified sequence. The sequence of the fusogenic gene can be modified, for example, to enhance the fusogenic properties of the encoded protein or to provide codon optimization and thus increase the expression efficiency of the encoded protein.

[0051] immunostimulatory molecules The viruses of the present invention contain one or more immunostimulatory molecules and / or one or more genes encoding immunostimulatory molecules. Immunostimulatory molecules include proteins that can support the induction of an immune response, proteins that can mitigate inhibitory signals on the induction or effectiveness of an immune response, and RNA molecules (e.g., shRNA, antisense RNA, RNAi, or microRNA) that inhibit the expression of immune inhibitory molecules. Examples of immunostimulatory molecules include IL-2, IL-12, IL-15, IL-18, IL-21, IL-24, CD40 ligand, GITR ligand, 4-1-BB ligand, OX40 ligand, ICOS ligand, flt3 ligand, type I interferons including interferon alpha and interferon beta, interferon gamma, type III interferons (IL-28, IL-29), other cytokines such as TNF alpha or GM-CSF, TGF beta, or immune checkpoint antagonists. Immune checkpoint antagonists include antibodies, single-chain antibodies, and RNA1 / siRNA / microRNA / antisense RNA knockdown approaches. Agonists of immune enhancement / costimulatory pathways include mutated or wild-type, soluble, secreted and / or membrane-bound ligands, as well as agonistic antibodies, including single chain antibodies. With regard to targeting immune co-inhibitory or immune co-stimulatory pathways, proteins or other molecules (agonists or antagonists, as the case may be) that target CTLA-4 (antagonist), PD-1 (antagonist), PD-L1 (antagonist), LAG-3 (antagonist), TIM-3 (antagonist), VISTA (antagonist), CSF1R (antagonist), IDO (antagonist), CEACAM1 (antagonist), GITR (agonist), 4-1-BB (agonist), KIR (antagonist), SLAMF7 (antagonist), OX40 (agonist), CD40 (agonist), ICOS (agonist), or CD47 (antagonist) are particularly preferred. Thus, the viruses of the invention preferably encode one or more of these molecules.More preferably, the viruses of the invention encode GM-CSF and / or wild-type or modified versions of CD40L, ICOSL, 4-1-BBL, GITRL or OX40L, most preferably GM-CSF.

[0052] The inhibitor of the co-inhibitory pathway may be a CTLA-4 inhibitor. CTLA-4 inhibitors are typically molecules, such as peptides or proteins, that bind to CTLA-4 and reduce or block signaling by CTLA-4, such as reducing activation by B7. By reducing CTLA-4 signaling, the inhibitor reduces or eliminates blockage of the immune stimulatory pathway by CTLA-4.

[0053] The CTLA-4 inhibitor is preferably an antibody or an antigen-binding fragment thereof. The term "antibody" referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. An antibody refers to a glycoprotein or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (kappa) (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0054] The antibody is typically a monoclonal antibody. The antibody may be a chimeric antibody. The antibody is preferably a humanized antibody, more preferably a human antibody.

[0055] The term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to CTLA-4. Antigen-binding fragments also retain the ability to inhibit CTLA-4, thus reducing or eliminating CTLA-4 blockade of a stimulatory immune response. Examples of suitable fragments include Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, and isolated complementarity-determining regions (CDRs). Single-chain antibodies such as scFvs and heavy-chain antibodies such as VHHs and camelid antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. In a preferred embodiment, the antibody is an scFv. Examples of suitable scFv molecules are disclosed, for example, in WO2007 / 123737 and WO2014 / 066532, which are incorporated herein by reference. An scFv can be encoded by the nucleotide sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 35.

[0056] The viruses of the invention may encode one or more immunostimulatory molecules, preferably 1, 2, 3 or 4 immunostimulatory molecules, more preferably 1 or 2 immunostimulatory molecules.

[0057] The sequence of the gene encoding the immunostimulatory molecule can be codon-optimized to increase the expression level of the respective protein in the target cell compared to when the unmodified sequence is used.

[0058] Virus production Viruses of the invention are constructed using methods well known in the art. For example, a plasmid (for smaller viruses and single- and multi-genome component RNA viruses) or BAC (for larger DNA viruses, including herpesviruses) encoding the viral genome to be packaged, containing genes encoding fusogenic and immunostimulatory molecules under appropriate regulatory control, can be constructed by standard molecular biology techniques and transfected into permissive cells from which recombinant virus can be recovered.

[0059] Alternatively, in a preferred embodiment, a plasmid containing DNA regions flanking the intended insertion site can be constructed and then cotransfected with viral genomic DNA into permissive cells, resulting in homologous recombination between the flanking regions of the target insertion site in the plasmid and the same regions in the parent virus. Recombinant viruses can then be selected and purified through the loss or addition of functions inserted or deleted by the plasmid used for modification, such as by inserting or deleting a marker gene, such as GFP or lacZ, from the parent virus at the intended insertion site. In a most preferred embodiment, the insertion site is the HSV ICP34.5 locus, and thus the plasmid used for engineering contains HSV sequences flanking this insertion site, with expression cassettes encoding fusogenic proteins and immunostimulatory molecules between them. In this case, the parent virus can contain a cassette encoding GFP instead of ICP34.5, and recombinant virus plaques are selected by the loss of GFP expression. In a most preferred embodiment, the HSV US11 gene is also expressed as an IE gene. This can be achieved through deletion of the ICP47 coding region or by other means.

[0060] The sequences encoding the fusogenic protein and the immunostimulatory molecule are inserted into the viral genome under appropriate regulatory control. Depending on the species and insertion site, this may be under the regulatory control of the native promoter of the virus species used in the present invention, or preferably under the regulation of a heterologous promoter. Suitable heterologous promoters include mammalian promoters such as the IEF2a promoter or the actin promoter. Strong viral promoters such as the CMV IE promoter, RSV LTR, MMLV LTR, other retroviral LTR promoters, or promoters derived from SV40 are more preferred. Preferably, each foreign gene (i.e., encoding the fusogenic protein and the immunomodulatory molecule) is under the regulation of a separate promoter, but they can also be expressed from a single RNA transcript, for example, by inserting an internal ribosome entry site (IRES) between the protein-coding sequences. The RNA derived from each promoter is typically terminated using a polyadenylation sequence (e.g., a mammalian sequence such as the bovine growth hormone (BGH) polyA sequence, a synthetic polyadenylation sequence, a rabbit beta globin polyadenylation sequence, or a viral sequence such as the SV40 early or late polyadenylation sequence).

[0061] The present invention also provides a virus, such as a poxvirus or HSV, preferably HSV1, expressing at least three heterologous genes, each driven by a different promoter selected from a CMV promoter, a RSV promoter, an EF1a promoter, an SV40 promoter, and a retroviral LTR promoter. The virus can express, for example, four heterologous genes, each driven by a different promoter selected from a CMV promoter, a RSV promoter, an EF1a promoter, an SV40 promoter, and a retroviral LTR promoter. The retroviral LTR is preferably derived from MMLV (SEQ ID NO: 43), also known as MoMuLV. The heterologous genes can be terminated by a polyadenylation sequence, which may be the same or different. Preferably, each heterologous gene is terminated by a different polyadenylation sequence, preferably selected from BGH, SV40, HGH, and RBG polyadenylation sequences.

[0062] The present invention also provides a virus, such as a poxvirus or HSV, preferably HSV1, that expresses at least three heterologous genes, each terminated by a different polyadenylation sequence selected from BGH, SV40, HGH, and RBG polyadenylation sequences. The virus can, for example, express four heterologous genes, each terminated by a BGH, SV40, HGH, and RBG polyadenylation sequence, respectively.

[0063] Pharmaceutical Composition The present invention provides a pharmaceutical composition comprising a virus and a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents include isotonic saline, such as phosphate-buffered saline. The composition may further comprise other ingredients, such as sugars or proteins, to improve properties such as product stability. Alternatively, a lyophilized formulation may be used that is reconstituted in a pharmaceutically acceptable carrier or diluent before use.

[0064] If necessary, the choice of carrier is often a function of the delivery route of the composition. Within the scope of the present invention, the composition can be formulated for any suitable route and means of administration. Pharmaceutically acceptable carriers or diluents are those used in compositions suitable for intratumoral administration, intravenous / intraarterial administration, administration to the brain, or administration to a body cavity (e.g., bladder, pleural cavity, or intraperitoneal administration). The composition can be administered in any suitable form, preferably as a liquid.

[0065] The present invention also provides an article of manufacture comprising a virus of the invention in a sterile vial, ampoule, or syringe.

[0066] Medical Use / Method of Treatment The present invention provides a virus of the present invention for use in therapeutic treatment of the human or animal body, particularly for use in a method for treating cancer. Cancer typically occurs in mammals, preferably humans. The virus kills infected tumor cells by lysis and by fusing infected tumor cells with each other. The virus of the present invention also induces an enhanced systemic anti-tumor immune response due to the expression of immunostimulatory molecules that also kill cancer cells.

[0067] The present invention also provides a method of treating cancer, comprising administering a therapeutically effective amount of a virus of the present invention to an individual in need thereof.

[0068] The invention further provides the use of a virus of the invention in the manufacture of a medicament for treating cancer.

[0069] The viruses of the invention are particularly useful for treating any solid tumor, including any adenocarcinoma, carcinoma, melanoma, or sarcoma. For example, the viruses of the invention are useful for treating cancer of the head and neck, prostate, breast, ovary, lung, liver, endometrium, bladder, gallbladder, pancreas, colon, kidney, stomach / gastric, esophagus, or cervix, mesothelioma, melanoma, or other skin cancers, lymphoma, glioma, or other cancers of the nervous system, or sarcomas, such as soft tissue sarcomas.

[0070] The viruses of the invention can be used to treat malignant tumors, including tumors that have metastasized from the site of the original tumor. In this embodiment, the virus can be administered to the primary tumor or to one or more secondary tumors.

[0071] The viruses of the present invention can be administered in combination with other therapeutic agents, including chemotherapy, targeted therapy, immunotherapy (including one or more antagonists of immune co-inhibitory pathways and / or one or more agonists of immune co-stimulatory pathways), and / or in combination with radiation therapy, and / or in combination with any combination thereof. The therapeutic agent is preferably an anti-cancer agent.

[0072] The viruses of the invention can be administered in combination with a second virus, such as a second oncolytic virus.

[0073] For example, therapeutic agents may include immunogens (including recombinant or naturally occurring antigens, including combinations of such antigens or antigens delivered as DNA or RNA encoding them) to further stimulate an immune response, e.g., a cellular or humoral immune response, against tumor cells, particularly neoantigens. Therapeutic agents may be agents intended to increase or enhance the immune response, such as cytokines, agents intended to inhibit immune checkpoint pathways or stimulate immune enhancement pathways, or agents that inhibit the activity of regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs).

[0074] The therapeutic agent may be a drug known for use in existing cancer treatments. The therapeutic agent may be a radiotherapeutic agent or a chemotherapeutic agent. The therapeutic agent may be selected from cyclophosphamide, alkylating agents such as cisplatin or melphalan, plant alkaloids and terpenoids such as vincristine or paclitaxel (taxol), antimetabolites such as 5-fluorouracil, topoisomerase inhibitors type I or II such as camptothecin or doxorubicin, cytotoxic antibiotics such as actinomycin, anthracyclines such as epirubicin, glucocorticoids such as triamcinolone, inhibitors of protein, DNA, and / or RNA synthesis such as methotrexate and dacarbaxin, histone deacetylase (HDAC) inhibitors, or any other chemotherapeutic agent.

[0075] The therapeutic agent may be one or a combination of the following: an immunotherapeutic or immunomodulatory agent, such as a TLR agonist; an agent that downregulates T regulatory cells, such as cyclophosphamide; or an agent designed to block immune checkpoints or stimulate immune enhancing pathways, including, but not limited to, a monoclonal antibody, such as a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a VISTA inhibitor, a CSF1R inhibitor, an IDO inhibitor, a CEACAM1 inhibitor, a GITR agonist, a 4-1-BB agonist, a KIR inhibitor, an SLAMF7 inhibitor, an OX40 agonist, a CD40 agonist, an ICOS agonist, or a CD47 inhibitor. In a preferred embodiment, the therapeutic agent is a CTLA-4 inhibitor, such as an anti-CTLA-4 antibody, a PD1 inhibitor, such as an anti-PD-1 antibody, or a PD-L1 inhibitor, such as an anti-PD-L1 antibody. Such inhibitors, agonists and antibodies can be generated and tested by standard methods known in the art.

[0076] Immunotherapeutic agents may also include bispecific antibodies, dendritic cell-based cell-based therapies, NK cells, or engineered T cells, e.g., CAR-T cells or T cells expressing engineered T cell receptors. Immunotherapeutic agents also include agents that target specific genetic mutations occurring in tumors, agents intended to elicit an immune response against specific tumor antigens or combinations of tumor antigens, e.g., neoantigens, and / or agents intended to activate the STING / cGAS pathway, TLRs, or other innate immune responses and / or inflammatory pathways, e.g., intratumoral agents.

[0077] For example, the viruses of the invention may be used in combination with dacarbazine, a BRAF inhibitor and / or a CTLA-4, PD1 or PD-L1 blocker to treat melanoma; in combination with taxol, doxorubicin, vinorelbine, cyclophosphamide and / or gemcitabine to treat breast cancer; in combination with 5-fluorouracil, and optionally in combination with leucovorin, irinotecan and / or oxaliplatin to treat colorectal cancer; in combination with taxol, carboplatin, vinorelbine and / or gemcitabine, a PD-1 or PD-L1 blocker to treat lung cancer; and in combination with cisplatin and / or radiation therapy to treat head and neck cancer.

[0078] The therapeutic agent can be an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway. Examples of IDO inhibitors include epcadostat (INCB024360), 1-methyl-tryptophan, indoximod (1-methyl-D-tryptophan), GDC-0919, or F001287.

[0079] The mechanism of action of IDO in suppressing anti-tumor immune responses may also suppress immune responses that occur after oncolytic virotherapy. IDO expression is induced by Toll-like receptor (TLR) activation and interferon-γ, both of which may result from oncolytic virus infection. One embodiment of the use of oncolytic virotherapy for cancer treatment involves the combination of an oncolytic virus, including a virus expressing an immunostimulatory protein and / or a fusogenic protein, with an inhibitor of the IDO pathway, and optionally with one or more additional antagonists of immune co-inhibitory pathways, including those targeting CTLA-4, PD-1, and / or PD-L1, and / or one or more agonists of immune co-stimulatory pathways.

[0080] The present invention also provides a method for treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of an oncolytic virus, an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway and an additional antagonist of an immune co-inhibitory pathway and / or an agonist of an immune co-stimulatory pathway.

[0081] The oncolytic virus is preferably a modified clinical isolate. The oncolytic virus is preferably a poxvirus, more preferably HSV, such as HSV1 and / or HSV functionally inactivated against ICP34.5 and / or ICP47. The oncolytic virus may express an immunostimulatory molecule, such as GM-CSF, and / or a molecule encoding a costimulatory pathway, such as CD4OL, GITRL, OX4OL, 4-I-BBL, or ICOSL, and / or a CTLA-4 inhibitor, and / or a fusogenic protein, such as a GALV fusogenic glycoprotein with a mutated or deleted R sequence. The additional antagonist of the immune costimulatory pathway is preferably a CTLA-4 antagonist, a PD1 antagonist, or a PD-L1 antagonist. For example, the additional antagonist of the immune co-inhibitory pathway may be an inhibitor of the interaction between PD1 and PD-L1.

[0082] When a therapeutic agent and / or radiation therapy is used together with the virus of the present invention, the administration of the virus and the therapeutic agent and / or radiation therapy may be simultaneous or separated by time. The composition of the present invention may be administered before, simultaneously with, or after the therapeutic agent or radiation therapy. The method of treating cancer may include multiple administrations of the virus of the present invention and / or the therapeutic agent and / or radiation therapy. In a preferred embodiment, in combination with an immune checkpoint blocker or other immune enhancing agent, the virus of the present invention is administered once or multiple times before the subsequent simultaneous administration of the immune checkpoint blocker or other immune enhancing agent, or is administered simultaneously with the administration of the immune checkpoint blocker or other immune enhancing agent without prior administration of the virus of the present invention.

[0083] The virus of the present invention can be administered to a subject by any suitable route. Typically, the virus of the present invention is administered by direct intratumoral injection. Intratumoral injection includes direct injection into epidermal, subcutaneous, or nodular tumors, as well as image-guided (such as CT, MRI, or ultrasound) injection to localize deeper or more firmly deposits, for example, in visceral organs and other locations. The virus can be administered into a body cavity, for example, into the pleural cavity, bladder, or by intraperitoneal administration. The virus can be injected into a blood vessel, preferably into a blood vessel supplying a tumor.

[0084] Therapeutic agents that can be combined with the viruses of the present invention can be administered to human or animal subjects in vivo using a variety of known routes and techniques. For example, the compositions can be provided as injectable solutions, suspensions, or emulsions and administered via parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, or intravenous injection using a conventional needle and syringe or a liquid jet injection system. The compositions can be administered topically to the skin or mucosal tissue, such as nasal, intratracheal, intestinal, sublingual, rectal, or vaginal, or can be provided as a finely divided spray suitable for respiratory or pulmonary administration. In a preferred embodiment, the compositions are administered by intravenous infusion, orally, or directly to a tumor.

[0085] The virus and / or therapeutic agent can be administered to a subject in an amount compatible with a therapeutically effective dosage composition. The administration of the virus of the present invention is for "therapeutic" purposes. As used herein, the term "treatment" or "treatment" includes any one or more of the following purposes: preventing any metastasis or further metastasis that occurs, reducing or eliminating symptoms, reducing or completely eliminating tumors or cancer, increasing the time to cancer progression in a patient, increasing the time to recurrence after treatment, or increasing survival time.

[0086] Therapeutic treatment is given for stage I, II, III or IV cancer, preferably stage II, III or IV, more preferably stage III or IV, before or after surgical intervention (i.e., after recurrence or incomplete removal of tumor after surgery), preferably before any surgical intervention (either for resection of primary or recurrent / metastatic disease) or after recurrence after surgery or incomplete surgical removal of disease, i.e., while residual tumor remains.

[0087] Therapeutic treatment can be carried out after injecting the viral composition into the target tissue, which may be a tumor, into a body cavity, or directly into a blood vessel. As a guideline, the amount of virus administered can range from 10 to 100 mg / kg for HSV. 4 ~10 10 pfu, preferably 10 5 ~10 9 In the case of HSV, the initial low dose (e.g., 10 4 ~10 7 pfu) to seroconvert patients who are seronegative for HSV and to boost immunity in seropositive patients, followed by higher doses (e.g., 10 6 ~10 9pfu) can then be given. Typically, up to 20 ml of a pharmaceutical composition consisting essentially of virus and a suitable pharmaceutically acceptable carrier or diluent can be used for direct injection into a tumor, or up to 50 ml (which may be subjected to further dilution in a suitable diluent before administration) can be used for administration into a body cavity or into the bloodstream. However, for some oncolytic therapy applications, larger or smaller volumes can also be used, depending on the tumor and the route and site of administration.

[0088] The described routes of administration and dosages are intended as guidelines only, as a skilled physician can easily determine the optimal route of administration and dosage. The dosage can be determined according to various parameters, particularly the location of the tumor, tumor size, the age, weight, and condition of the patient being treated, and the route of administration. Preferably, the virus is administered by direct injection into the tumor. The virus can also be administered by injection into a blood vessel or a body cavity. The optimal route of administration depends on the location and size of the tumor. Multiple doses may be required to achieve an immunological or clinical effect, typically administered at intervals of 2 days to 12 weeks, preferably 3 days to 3 weeks, as needed. Repeated administrations can be given for up to 5 years or more, preferably 1 month to 2 years, depending on the response rate of the type of tumor being treated and the response of the particular patient, as well as any concomitant therapy that may be given. Various embodiments of the present invention are described below. 1. An oncolytic virus comprising (i) a gene encoding a fusogenic protein and (ii) an immunostimulatory molecule or a gene encoding an immunostimulatory molecule. 2. The virus described in 1 above, wherein the fusogenic protein is selected from the group consisting of vesicular stomatitis virus (VSV) G protein, syncytin-1, syncytin-2, simian virus 5 (SV5) F protein, measles virus (MV) H protein, MV F protein, respiratory syncytial virus (RSV) F protein, and glycoproteins derived from R peptide-deleted gibbon ape leukemia virus (GALV), murine leukemia virus (MLV), Mason-Pfizer simian virus (MPMV), or equine infectious anemia virus (EIAV). 3. The virus according to 1 or 2 above, wherein the immunostimulatory molecule is an antagonist of GM-CSF, IL-2, IL-12, IL-15, IL-18, IL-21, IL-24, type I interferon, interferon gamma, type III interferon, TNF alpha, TGF beta, an immune checkpoint antagonist, or an agonist of an immune enhancing pathway, such as CD40 ligand (CD40L), ICOS ligand, GITR ligand, 4-1-BB ligand, OX40 ligand, or flt3 ligand, or a modified version of any of them. 4. (a) the fusogenic protein is a glycoprotein derived from the gibbon ape leukemia virus (GALV) in which the R transmembrane peptide has been mutated or deleted (GALV-R-); and / or (b) The virus according to any one of 1 to 3 above, wherein the immunostimulatory molecule is (i) GM-CSF or CD40L, GITR ligand, 4-1-BB ligand, OX40 ligand, ICOS ligand or a modified version of any of them, or (ii) a CTLA-4 inhibitor. 5. The virus according to any one of 1 to 4 above, encoding more than one fusogenic protein and / or more than one immunostimulatory molecule. 6. The virus according to 5 above, wherein the immunostimulatory molecule is GM-CSF and one or more of (i) CD40L, GITR ligand, 4-1-BB ligand, OX40 ligand and ICOS ligand or modified versions of any of them, and / or (ii) a CTLA-4 inhibitor. 7. The virus according to any one of 4 to 6 above, wherein the CTLA-4 inhibitor is a CTLA-4 antibody or a fragment thereof. 8. A virus according to any one of 1 to 7 above, which is derived from a clinical isolate of the virus. 9. A virus according to any one of 1 to 8 above, which is a modified clinical isolate of the virus that kills two or more tumor cell lines in vitro more quickly and / or at lower doses than one or more reference clinical isolates of the same virus. 10. Clinical isolates strain RH018A, which has provisional accession number ECCAC16121904; strain RH004A, which has provisional accession number ECCAC16121902; strain RH031A, which has provisional accession number ECCAC16121907; strain RH040B, which has provisional accession number ECCAC16121908; strain RH015A, which has provisional accession number ECCAC16121903; strain RH021A, which has provisional accession number ECCAC16121905; strain RH023A, which has provisional accession number ECCAC16121906, or strain RH047A, which has provisional accession number ECCAC16121909 10. The virus according to claim 8 or 9, 11. The virus according to any one of 1 to 9 above, which is selected from the group consisting of herpesviruses, poxviruses, adenoviruses, retroviruses, rhabdoviruses, paramyxoviruses and reoviruses. 12. The virus according to any one of 1 to 11 above, which is a herpes simplex virus (HSV). 13. The virus according to claim 12, which is HSV1. 14.HSV (a) do not express functional ICP34.5; (b) does not express functional ICP47, and / or (c) expressing the US11 gene as an immediate early gene; 14. The virus according to any one of 10 to 13 above. 15. A virus according to any one of 12 to 14 above, in which a gene encoding a fusogenic protein and a gene encoding an immunostimulatory molecule have been inserted into the ICP34.5-encoding locus by either insertion or partial or complete deletion, each under separate regulatory control, optionally in a back-to-back orientation relative to each other. 16. A virus described in any one of 1 to 15 above, wherein the sequence of the gene encoding the fusogenic protein and / or the sequence of the gene encoding the immunostimulatory molecule have been codon-optimized to increase the expression level in target cells. 17. A virus that expresses three heterologous genes, each of which is driven by a different promoter selected from a CMV promoter, an RSV promoter, an SV40 promoter, and a retroviral LTR promoter. 18. A virus according to any one of 1 to 17 above, which expresses three heterologous genes, each of which is driven by a different promoter selected from a CMV promoter, an RSV promoter, an SV40 promoter, and a retroviral LTR promoter. 19. The virus according to 17 or 18 above, which expresses four heterologous genes driven by a CMV promoter, an RSV promoter, an SV40 promoter and a retroviral LTR promoter, respectively. 20. The virus according to any one of 17 to 19 above, wherein the retroviral LTR is derived from MMLV. 21. A virus expressing three heterologous genes, each of which is terminated by a different polyadenylation sequence selected from the group consisting of BGH, SV40, HGH, and RBG polyadenylation sequences. 22. A virus according to any one of 1 to 21 above, which expresses three heterologous genes, each of which is terminated by a different polyadenylation sequence selected from BGH, SV40, HGH and RBG polyadenylation sequences. 23. A virus according to claim 21 or 22, which expresses four heterologous genes terminated by BGH, SV40, HGH and RBG polyadenylation sequences, respectively. 24.(a) HSV, (b) HSV1, or (c) poxvirus 24. The virus according to any one of 17 to 23 above, 25. A pharmaceutical composition comprising the virus according to any one of 1 to 24 above and a pharmaceutically acceptable carrier or diluent. 26. A virus according to any one of 1 to 24 above for use in a method for treating the human or animal body by therapy. 27. The virus according to any one of 1 to 24 above for use in a method for treating cancer. 28. The virus for use according to 27 above, wherein the method comprises administering a further anti-cancer agent. 29. A virus for use according to claim 28, wherein the further anti-cancer agent is selected from agents targeting immune co-inhibitory or immune co-stimulatory pathways, radiotherapy and / or chemotherapy, agents targeting specific genetic mutations occurring in the tumor, agents intended to induce an immune response against one or more tumor antigens or neo-antigens, cellular products derived from T cells or NK cells, agents intended to stimulate STING, cGAS, TLR or other innate immune responses and / or inflammatory pathways, a second virus, optionally an oncolytic virus, and combinations thereof. 30. The virus for use according to claim 28 or 29, wherein the agent targeting the immune co-inhibitory pathway is a CTLA-4 inhibitor, PD-1 inhibitor, PD-L1 inhibitor, LAG-3 inhibitor, TIM-3 inhibitor, VISTA inhibitor, CSF1R inhibitor, IDO inhibitor, CEACAM1 inhibitor, KIR inhibitor, SLAMF7 or CD47 inhibitor, and / or the agent targeting the immune co-stimulatory pathway is a GITR agonist, 4-1-BB agonist, OX40 agonist, CD40 agonist or ICOS agonist. 31. A virus for use according to any one of 28 to 30 above, wherein the further anticancer agent is an antibody. 32. A virus for use according to any one of claims 28 to 31, wherein the method comprises administering an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway and a further antagonist of an immune co-inhibitory pathway or an agonist of an immune co-stimulatory pathway. 33. A virus for use according to any one of 28 to 32 above, wherein the virus and the further anticancer drug are administered separately. 34. A virus for use according to any one of 28 to 32 above, wherein the virus and a further anticancer drug are administered simultaneously. 35. A virus for use according to any one of 28 to 34 above, wherein the cancer is a solid tumor. 36. A product containing the virus according to any one of 1 to 35 above in a sterile vial, ampoule, or syringe. 37. A method for treating cancer, comprising administering a therapeutically effective amount of the virus according to any one of 1 to 24 above or the pharmaceutical composition according to 25 above to a patient in need thereof. 38. The method according to claim 37, further comprising administering a therapeutically effective amount of an additional anti-cancer agent to a patient in need thereof. 39. The method of claim 38, wherein the further anti-cancer agent is selected from the group consisting of agents targeting immune co-inhibitory or immune co-stimulatory pathways, radiotherapy and / or chemotherapy, agents targeting specific genetic mutations occurring in the tumor, agents intended to induce an immune response against one or more tumor antigens or neo-antigens, cellular products derived from T cells or NK cells, agents intended to stimulate STING, cGAS, TLR or other innate immune response and / or inflammatory pathways, a second virus, optionally an oncolytic virus, and combinations thereof. 40. The method of claim 39, wherein the agent targeting the immune co-inhibitory pathway is a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a VISTA inhibitor, a CSF1R inhibitor, an IDO inhibitor, a KIR inhibitor, an SLAMF7 inhibitor, a CEACAM1 inhibitor or a CD47 inhibitor, and / or the agent targeting the immune co-stimulatory pathway is a GITR agonist, a 4-1-BB agonist, an OX40 agonist, a CD40 agonist or an ICOS agonist. 41. The method according to any one of claims 38 to 40, wherein the further anticancer agent comprises an antibody. 42. The method according to any one of claims 38 to 41, wherein the virus and the further anticancer agent are administered separately. 43. The method according to any one of claims 38 to 41, wherein the virus and the further anticancer agent are administered simultaneously. 44. The method according to any one of 38 to 43 above, wherein the cancer is a solid tumor. 45. Use of a virus according to any one of 1 to 24 above in the manufacture of a medicament for use in a method for treating cancer. 46. ​​The use according to claim 45, wherein the method comprises administering a further anti-cancer agent.

[0089] The following examples illustrate the present invention. [Example]

[0090] [Example 1] Construction of the viruses of the invention The viral species used to illustrate the present invention is HSV, specifically HSV1. The HSV1 strain used for illustration was identified through a comparison of over 20 primary clinical isolates of HSV1 for their ability to kill a panel of human tumor-derived cell lines, and the selection of the viral strain with the highest ability to kill a wide range of these cells in a short time and at a low dose. The tumor cell lines used in this comparison include U87MG (glioma), HT29 (colorectal), LNCaP (prostate), MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu (squamous cell carcinoma), MCF7 (breast), A549 (lung), MIAPACA-2 (pancreatic), CAPAN-1 (pancreatic), and HT1080 (fibrosarcoma). Specifically, each primary clinical isolate of HSV is titrated onto each of the cell lines used for screening at MOIs such as 1, 0.1, 0.01, and 0.001, and each dose is assessed for the extent of cell death at time points such as 24 and 48 hours. The extent of cell death can be assessed, for example, by microscopic assessment of the proportion of surviving cells at each time point, or by a metabolic assay such as the MTT assay.

[0091] Next, an exemplary virus of the invention is constructed by deleting ICP47 from the viral genome using homologous recombination with a plasmid containing regions flanking HSV1 nucleotides 145300-145582 (HSV1 nucleotides 145300-145582 are the sequence to be deleted; HSV1 strain 17 sequence Genbank file NC 001806.2), between which GFP is encoded. GFP-expressing viral plaques are selected, followed by homologous recombination with the empty flanking regions, and plaques that do not express GFP are selected. This results in an ICP47-deleted virus in which US11 is expressed as an IE protein, since it is now under the control of the ICP47 promoter. Next, ICP34.5 is deleted using homologous recombination with a plasmid containing the regions flanking HSV1 nucleotides 124953-125727 (HSV1 nucleotides 124953-125727 are the sequence to be deleted; HSV1 strain 17 sequence Genbank file NC 001806.2), between which GFP is encoded. Viral plaques expressing GFP are again selected, and then GFP is removed by homologous recombination with the same flanking regions (but now with expression cassettes containing, in back-to-back orientation, a codon-optimized version of the mouse GM-CSF sequence and a codon-optimized version of the GALV R-sequence driven by the CMV IE promoter and the RSV promoter, respectively), and again viral plaques not expressing GFP are selected. This viral construction is performed using standard methods in the art.

[0092] The structure of the obtained virus is shown in Figure 1. The mGM-CSF and GALV-R sequences are shown in SEQ ID NOs: 2 and 8, respectively. The structure of the obtained virus is confirmed by PCR, GM-CSF expression by ELISA, and GALV-R expression by infection of human HT1080 tumor cells and observation of syncytial plaques.

[0093] Viruses were also constructed using similar procedures without any insertion into ICP34.5, or with only the mouse GM-CSF or GALV-R genes inserted. The structures of these viruses are also shown in FIG. 1.

[0094] For human use, hGM-CSF is used, the sequence of a codon-optimized version of which is shown in SEQ ID NO:4.

[0095] [Example 2] Expression of two immunostimulatory molecules from viruses expressing fusogenic proteins A virus similar to the GALV-R- and mGM-CSF-expressing viruses described above, but expressing a version of CD40L, was constructed. Here, instead of using a plasmid containing the ICP34.5 flanking regions and an expression cassette containing GM-CSF and GALV-R- driven by the CMV and RSV promoters, a plasmid containing the ICP34.5 flanking regions and an expression cassette containing GM-CSF, GALV, and CD40L driven by the CMV, RSV, and SV40 promoters was used for recombination with a virus containing GFP inserted into ICP34.5, and plaques not expressing GFP were again selected.

[0096] [Example 3] Effect of combined expression of oncolytic virus-derived fusogenic proteins and immunostimulatory molecules in mouse tumor models GALV R-protein induces cell-cell fusion in human cells but not in mouse cells. This is because the PiT-1 receptor required for cell fusion contains a sequence that does not allow cell fusion in mice. Consequently, mouse tumor cells expressing human PiT-1 are first prepared using standard methods in the art. Human PiT-1 is cloned into a lentiviral vector that also contains a selectable marker gene. The vector is transfected into target CT26 mouse colorectal cancer tumor cells, and clones resistant to the selectable marker are selected to generate CT26 / PiT-1 cells. PiT-1 expression is confirmed by Western blotting in untransfected cells and cells transfected with a lentivirus expressing PiT-1, and by transfection of a GALV-R-expressing plasmid and confirmation of cell fusion.

[0097] The utility of the present invention is demonstrated by administering CT26 / PiT-1 cells to both flanks of Balb / c mice and allowing CT26 / PiT-1 tumors to grow to approximately 0.5 cm in diameter.

[0098] The following treatments are then administered to groups of mice (5 per group) 3 times per week for 2 weeks, on one side of each mouse: - 50 μl of saline (group 1), - 10 μl of 50 μl of no gene inserted 5 pfu / ml, 10 6 pfu, or 10 7 pfu / ml HSV (group 3); - Mouse GM-CSF only inserted, 50 μl 10 5 pfu / ml, 10 6 pfu / ml, or 10 7 pfu / ml HSV (group 3); - GALV-R-only insert, 50 μl 10 5 pfu / ml, 10 6 pfu / ml, or 10 7pfu / ml of virus (group 3), or - Both mouse GM-CSF and GALV-R- are inserted, 50 μl 10 5 pfu / ml, 10 6 pfu / ml, or 10 7 pfu / ml of virus (group 3).

[0099] The effect on tumor growth is then observed for up to one month. Superior tumor control and shrinkage are observed, including through improved dose-response curves, in both injected and uninjected tumors using GM-CSF and GALV-R-expressing viruses compared to other groups.

[0100] [Example 4] Effect of combined expression of oncolytic virus-derived fusogenic proteins and immunostimulatory molecules on the therapeutic efficacy of immune checkpoint blockade in mouse tumor models The experiment in Example 3 above was repeated, except that mice were boosted every two weeks by intraperitoneal administration with an antibody targeting mouse PD-1 (10 mg / kg; Bioxcell RMP-1-14 on the same day as virus administration) or an antibody targeting mouse CTLA-4 (10 mg / kg; Bioxcell 9H10 on the same day as virus administration). Additional groups of mice were added that did not receive antibody treatment. More specifically, the groups of mice received (1) saline, (2) HSV without a gene insert, (3) HSV with GM-CSF and GALV-R- inserts as in Example 3, (4) PD-1 antibody, (5) CTLA-4 antibody, (6) HSV without a gene insert and PD-1 antibody, (7) HSV without a gene insert and CTLA-4 antibody, (8) HSV and PD-1 antibody with GM-CSF and GALV-R-, or (9) HSV and CTLA-4 antibody with GM-CSF and GALV-R-. Superior tumor control and shrinkage is observed, including through improved dose-response curves, in both injected and uninjected tumors using GM-CSF and GALV-R-expressing virus with anti-PD-1 or anti-CTLA-4 antibodies compared to other groups.

[0101] [Example 5] Recovery of clinical isolates The virus species used to illustrate the present invention is HSV, specifically HSV1. To illustrate the present invention, 181 volunteers suffering from recurrent cold sores were recruited. These volunteers were given sample collection kits (including Sigma Virovult collection tubes) to swab cold sores as they appeared, and these samples were then shipped to Replimune (Oxford, UK). Swabs were received from 72 volunteers between June 2015 and February 2016. Each swab sample was used to infect BHK cells. Of these, 36 live virus samples were recovered after seeding and growth on BHK cells. These samples are detailed in Table 1.

[0102] [Table 1] JPEG2025163138000002.jpg23489JPEG2025163138000003.jpg23689JPEG2025163138000004.jpg18689Designations A, B, C, etc. refer to multiple swabs from the same volunteer.

[0103] [Example 6] Identification of clinical isolates with improved antitumor efficacy The ability of primary clinical isolates of HSV1 to kill a panel of human tumor-derived cell lines was tested. The tumor cell lines used for this comparison were HT29 (colorectal), MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu (squamous cell carcinoma), MCF7 (breast), A549 (lung), MIAPACA-2 (pancreatic), and HT1080 (fibrosarcoma). The cell lines were used to test the CPE levels achieved for each primary clinical isolate at a range of MOIs and times postinfection.

[0104] Experiments were conducted in parallel using five to eight new virus strains. Virus strains were inoculated in duplicate at a range of MOIs (0.001 to 1), and the degree of CPE after crystal violet staining was assessed 24 and 48 hours after infection. The virus strains most effective at killing tumor cell lines were scored, and the two or three most effective strains from each of the five to eight screens were identified and compared in parallel in additional experiments to identify the top strains for further development.

[0105] Initial screening demonstrated substantial variability in the ability of different strains to kill various tumor cell lines. Of the initial 29 strains tested, eight strains of interest were identified in the initial screening for further comparison. These were strains RH004A, RH015A, RH018A, RH021A, RH023A, RH31A, RH040A, and RH047A.

[0106] For further comparison, eight strains were tested in parallel on a panel of tumor cell lines, and their relative ability to kill these tumor cell lines was assessed after crystal violet staining and observation for CPE. Figure 3 shows representative time points and MOIs for each virus on each cell line, demonstrating the different abilities of the viruses to kill the target tumor cell lines observed.

[0107] It was found that there was substantial variation between strains, and that while a particular strain may be particularly effective at killing one cell line, it was not necessarily particularly effective at killing other cell lines, further demonstrating the degree of variability in the ability of clinical strains of HSV to kill different types of tumor cells.

[0108] Figure 3 also shows which virus strains were best and second best at killing each of the cell lines, allowing the virus strains to be ranked in terms of their overall relative ability to kill the panel of cell lines as a whole. This analysis demonstrated that the RH004A, RH015A, RH018A, RH031A, and RH040A strains were relatively more effective than the other strains, and these five strains were selected for potential further development as oncolytic agents. Of these top five strains, the relative ranking based on their ability to kill the entire panel of cell lines was RH018A > RH004A > RH031A > RH040A > RH015A.

[0109] More specifically, in these experiments, tumor cell lines were seeded into multiwell tissue culture plates to approximately 80% confluence on the day of infection. Representative wells from each tumor cell line were trypsinized, and the number of cells in each well was determined. These cell counts were used to determine the volume of each clinical isolate required to achieve MOIs of 1, 0.1, 0.01, and 0.001. Separate wells of tumor cell lines were infected with the clinical isolates at these MOIs. All infections were performed in quadruplicate. Duplicate wells were incubated for 24 hours, and duplicate wells were incubated for 48 hours, both at 37°C and 5% CO2. Afterwards, the cells were fixed with glutaraldehyde and stained with crystal violet. The level of cell lysis was then assessed by macroscopic observation, microscopy (cell counting), and photography.

[0110] The RH018A strain was the first ranked strain of all strains tested and was compared to the "average" strain from the screen (i.e., a strain that was not in the top 8, but was also not in the set of strains that were least effective and did not kill the panel of tumor cell lines). This comparison showed that the RH018A strain was approximately 10 times more effective than the average strain (RH065A) at killing tumor cell lines (i.e., approximately one-tenth of the RH018A strain was required to kill an equal proportion of cells than was required for the RH065A strain). This is shown in Figure 4.

[0111] [Example 7] Modification of clinical isolates In this example, the clinical isolate selected in Example 6 was modified by deleting ICP34.5 from the viral genome by homologous recombination with a plasmid containing regions flanking the gene encoding ICP34.5 (nucleotides 143680-145300 and 145,582-147,083; HSV1 strain 17 sequence Genbank file NC 001806.2), between which it encodes GFP and the GALV-R-fusogenic glycoprotein. The structure of this virus (virus 10) is shown in Figure 5.

[0112] Additional viruses based on the RH018A strain were also constructed, in which both ICP34.5 and ICP47 (using the flanking regions containing nucleotides 123464-124953 and 125727-126781; HSV1 strain 17 sequence Genbank file NC 001806.2) were deleted (putting US11 under the control of the ICP47 promoter). To construct these viruses, GFP-expressing viral plaques were first selected, in which GFP was expressed instead of ICP47. Next, GFP was removed by homologous recombination using the empty flanking regions, and plaques that did not express GFP were selected. This resulted in an ICP47-deleted virus, in which US11 is now expressed as an IE protein because it is under the control of the ICP47 promoter. Next, ICP34.5 was deleted by homologous recombination with a plasmid containing the flanking regions of HSV1 (nucleotides 143680-145300 and 145,582-147,083; HSV1 strain 17 sequence GenBank file NC 001806.2), between which GFP was encoded. Viral plaques expressing GFP were again selected, and then GFP was removed by homologous recombination with the same flanking regions (but now with an expression cassette containing the inserted gene between them). The constructed viruses are shown in Figures 1 and 5. They contain, in back-to-back orientation, a codon-optimized version of the mouse GM-CSF sequence and a codon-optimized version of the GALV R-sequence driven by the CMV IE promoter and RSV promoter, respectively. Again, viral plaques not expressing GFP were selected. This viral construction was performed using standard methods in the art.

[0113] The mGM-CSF and GALV-R sequences are shown in SEQ ID NOs: 2 and 8, respectively. The structure of the resulting virus was confirmed by PCR, GM-CSF expression was confirmed by ELISA, and GALV-R expression was confirmed by infection of human HT1080 tumor cells and observation of syncytial plaques.

[0114] For human use, hGM-CSF is used, the sequence of a codon-optimized version of which is shown in SEQ ID NO: 4. The structure of this virus is shown in Figure 5. Expression of mouse or human GM-CSF from viruses 16, 17, and 19 is shown in Figure 6.

[0115] [Example 8] Viruses of the invention modified for oncolytic use and expressing fusogenic glycoproteins exhibit increased tumor cell killing in vitro compared to viruses that do not express fusogenic glycoproteins Virus 10, based on the clinical strain RH018A, which is ICP34.5 deleted and expresses GALVR and GFP (see Figure 5), was compared in vitro with a virus expressing only GFP (virus 12). As shown in Figure 7, virus 10 demonstrated enhanced killing in a panel of human tumor cell lines compared to virus 12.

[0116] [Example 9] Viruses of the invention modified for oncolytic use exhibit increased tumor cell killing compared to similarly modified viruses not of the invention. Virus 17 (see Figure 5), based on the clinical strain RH018A, which is deleted for ICP34.5 and ICP47 and expresses GALVR and GM-CSF, was compared in vitro with a known virus that is also deleted for ICP34.5 and ICP47 but is not derived from the strain of the present invention and expresses only GM-CSF. As shown in Figure 8, virus 17 demonstrated enhanced killing in a panel of human tumor cell lines compared to the previous virus.

[0117] [Example 10] Viruses of the invention modified for oncolytic use effectively treat mouse tumors in vivo Virus 16 was tested in mice bearing A20 lymphoma tumors on the left and right flanks. One million tumor cells were first implanted into both flanks of Balb / c mice, and tumors were allowed to grow to a diameter of 0.5-0.7 cm. Tumors on the right flank were then implanted with either vehicle (10 mice) or 5 x 10 cells of Virus 16. 6pfu (10 mice) three times (every other day) and the effect on tumor size was observed for an additional 30 days, demonstrating that both injected and uninjected tumors were effectively treated with virus 16 (see Figure 9).

[0118] [Example 11] Effect of combined expression of fusogenic proteins from the oncolytic viruses of the present invention and immunostimulatory molecules in a rat tumor model The GALV R-protein induces cell-cell fusion in human cells but not in mouse cells, however, GALV R- induces fusion in rat cells.

[0119] The utility of the present invention was further demonstrated by administering 9L cells to the flanks of Fischer 344 rats and allowing 9L tumors to grow to approximately 0.5 cm in diameter.

[0120] The following treatments were then administered to groups of rats (10 per group), 3 times per week for 3 weeks, to only one side of each rat: - 50 μl vehicle, - 50 μl 10 7 pfu / ml of virus 19 (expressing mGM-CSF but not GALV R-); - 50 μl 10 7 pfu / ml of virus 16 (expressing both murine GM-CSF and GALV-R-).

[0121] The effect on tumor growth was then observed for an additional 30 days. This demonstrated superior tumor control and shrinkage with the GALV-R-expressing virus in both injected and non-injected tumors, demonstrating improved systemic efficacy. This is shown in Figure 15. Figure 10 shows that the GALV-expressing virus (virus 15) also exhibited enhanced killing of rat 91 cells in vitro compared to the virus that did not express GALV (virus 24).

[0122] [Example 12] Viruses of the invention modified for oncolytic use are synergistic with immune checkpoint blockade in mouse tumor models Virus 16 was tested in mice bearing CT26 tumors on both the left and right flanks. One million tumor cells were first implanted into the flanks of Balb / c mice, and tumors were allowed to grow to a diameter of 0.5–0.6 cm.

[0123] Groups of 10 mice were then treated with: - vehicle (three injections into the right flank tumor every other day); - 5 x 10 injected into the right flank tumor every other day 6 pfu virus 16, - anti-mouse PD1 alone (10 mg / kg ip, every 3 days, BioXCell clone RMP1-14), - anti-mouse CTLA-4 (3 mg / kg ip, every 3 days, BioXCell clone 9D9), - virus 16 with anti-mouse PD1, - virus 16 with anti-mouse CTLA4, 1-methyltrypophan (IDO inhibitor (5 mg / ml in drinking water)), - 1-methyltryptophan with anti-mouse PD1, - 1-methyltryptophan and virus 16 together with anti-mouse PD1.

[0124] The effect on tumor size was observed for an additional 30 days. Greater tumor reduction was demonstrated in animals treated with the combination of virus and checkpoint blockade than in animals treated with a single treatment group (see Figure 11). Increased tumor reduction was also demonstrated with both anti-PD1 and IDO inhibition with virus 16 compared to anti-PD1 alone with virus 16 (see Figure 11).

[0125] Also, increased activity of virus 16 in combination with immune checkpoint blockade was seen in A20 tumors (Figure 12).

[0126] [Example 13] Effect of expression of fusogenic proteins from the oncolytic viruses of the present invention in a human xenograft model in immunodeficient mice The GALV R-protein induces cell-cell fusion in human cells but not in mouse cells. However, the effect of GALV expression on antitumor efficacy can be assessed using human xenograft tumors grown in immunodeficient mice.

[0127] Thus, the utility of the present invention was further demonstrated by injecting A549 human lung cancer cells into the flanks of nude mice and allowing tumors to grow to approximately 0.5 cm in diameter.

[0128] The following treatments were then administered to groups of mice (10 per group) three times over the course of one week into the tumor-bearing flank of each mouse: - 50 μl vehicle, - 50 μl 10 7 pfu / ml of virus 16 (expressing both murine GM-CSF and GALV-R-); - 50 μl 10 6 pfu / ml of virus 16; - 50 μl 10 5 pfu / ml of virus 16; - 50 μl 10 7 pfu / ml of virus 19 (expressing only murine GM-CSF); - 50 μl 10 6 pfu / ml of virus 19; - 50 μl 10 5 pfu / ml of virus 19.

[0129] The effect on tumor growth was then observed for an additional 30 days. This experiment demonstrated superior tumor control and regression with GALV-R-expressing virus in both tumor models (see Figure 14).

[0130] [Example 14] Expression of two immunostimulatory molecules from viruses expressing fusogenic proteins Viruses similar to the GALV-R- and mGM-CSF-expressing virus (virus 16) described above were constructed, but additionally expressing murine versions of CD40L (virus 32), ICOSL (virus 36), OX40L (virus 35), 4-1BBL (virus 33), and GITRL (virus 34). Here, instead of using a plasmid containing the ICP34.5 flanking regions and expression cassettes containing GM-CSF and GALV-R driven by the CMV and RSV promoters, a plasmid containing the ICP34.5 flanking regions and expression cassettes containing GM-CSF, GALV, and additional proteins driven by the CMV, RSV, and MMLV promoters, respectively, was used for recombination with a virus containing GM-CSF, GALV, and GFP inserted into ICP34.5. Plaques that did not express GFP were again selected. Correct insertion was confirmed by PCR, and expression was confirmed for the additional inserted genes by Western blotting and / or ELISA. These viruses are shown in Figure 5. Similarly, viruses expressing anti-mouse and anti-human CTLA-4 in addition to GALV and mGM-CSF were also constructed (see viruses 27 and 31 in Figure 5 and Figure 13). The in vivo effects of viruses expressing anti-mouse CTLA-4 (virus 27), mCD40L (virus 32), m4-1BBL (virus 33), or mOX40L (virus 35) in addition to mGM-CSF and GALVR are shown in Figure 16. This showed enhanced activity in A20 tumors compared with virus 16 (expressing mGM-CSF and GALVR). In these experiments, tumors were induced on both flanks of mice, and virus or vehicle was injected only into the tumor on the right flank. The dose of virus used was 5 x 10 4 pfu (1 × 10 in each case in 50 μl 6 pfu / ml) and given three times over a one-week period. This dose level of virus was subtherapeutic for virus 16 relative to uninjected tumors, demonstrating the clear benefit of delivery of the additional molecules encoded by viruses 27, 32, 33, and 35.

[0131] Deposit information The following HSV1 strains were deposited by Replimune Limited at the ECACC, Culture Collections, Public Health England, Porton Down, Salisbury, SP4 0JG, UK on 19 December 2016 and assigned the indicated provisional accession numbers: RH004A-provisional accession number 16121902 RH015A-provisional accession number 16121903 RH018A-provisional accession number 16121904 RH021A-provisional accession number 16121905 RH023A-provisional accession number 16121906 RH031A-provisional accession number 16121907 RH040B-provisional accession number 16121908 RH047A-provisional accession number 16121909

[0132] SEQUENCE LISTING <110> Replimune Limited <120> Modified oncolytic virus <130> PA25-333 <150> GB1600380.8 <151> 2016-01-08 <150> GB1600381.6 <151> 2016-01-08 <150> GB1600382.4 <151> 2016-01-08 <160> 45 <170> PatentIn version 3.5 <210> 1 <211> 426 <212> DNA <213> Mus musculus <400> 1 atgtggctgc agaatttact tttcctgggc attgtggtct acagcctctc agcacccacc 60 cgctcaccca tcactgtcac ccggccttgg aagcatgtag aggccatcaa agaagccctg 120 aacctcctgg atgacatgcc tgtcacattg aatgaagagg tagaagtcgt ctctaacgag 180 ttctccttca agaagctaac atgtgtgcag acccgcctga agatattcga gcagggtcta 240 cggggcaatt tcaccaaact caagggcgcc ttgaacatga cagccagcta ctaccagaca 300 tactgcccc caactccgga aacggactgt gaaacacaag ttaccaccta tgcggattc 360 atagacagcc ttaaaacctt tctgactgat atcccctttg aatgcaaaaa accagtccaa 420 aatga 426 <210> 2 <211> 426 <212> DNA <213> Mus musculus <400> 2 atgtggctcc agaacctcct cttctcggt atcgtcgtgt attcactctc cgcacctact 60 cgctcaccta tcactgtcac cagaccctgg aagcacgtgg aggccatcaa ggaggctctg 120 aacctgctgg acgatatgcc agtgaccctg aacgaggagg tggaggtggt gagcaacgag 180 ttctccttta agaagctgac ctgcgtgcag acaaggctga agatcttcga gcagggcctg 240 agaggaaact ttaccaagct gaagggcgcc ctgaacatga ccgcttctta ctaccagaca 300 tactgccccc ctacccccga gacagactgt gagacacagg tgaccacata cgccgacttc 360 attgatagcc tgaaaacatt cctgaccgac attccatttg agtgtaagaa gcccgtccag 420 aagtaa 426 <210> 3 <211> 435 <212> DNA <213> Homo sapiens <400> 3 atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc 60 cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg 120 cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc 180 tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag 240 cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac 300 tacaagcagc actgccctcc aaccccggaa acttcctgtg caacccagat tatcacctttt 360 gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag 420 ccagtccagg agtga 435 <210> 4 <211> 435 <212> DNA <213> Homo sapiens <400> 4 atgtggctgc agtccctgct gctgctgggc accgtcgcct gttctatttc cgcacccgca 60 aggtcaccaa gtccatctac tcagccttgg gagcacgtga acgcaatcca ggaggcacgg 120 cggctgctga acctgagccg ggacaccgcc gccgagatga acgagacagt ggaagtgatc 180 agcgagatgt tcgatctgca ggagcccacc tgcctgcaga caaggctgga gctgtacaag 240 cagggcctgc gcggctctct gaccaagctg aagggcccac tgacaatgat ggccagccac 300 tataagcagc actgcccccc taccccccgag acaagctgtg ccacccagat catcacattc 360 gagtccttta aggagaacct gaaggatttt ctgctggtca ttccatttga ttgttgggag 420 cccgtccagg agtaa 435 <210> 5 <211> 141 <212> PRT <213> Mus musculus <400> 5 Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser Leu 1 5 10 15 Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His 20 25 30 Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val 35 40 45 Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys 50 55 60 Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu 65 70 75 80 Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser 85 90 95 Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr 100 105 110 Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu 115 120 125 Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Val Gln Lys 130 135 140 <210> 6 <211> 144 <212> PRT <213> Homo sapiens <400> 6 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115 120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135 140 <210> 7 <211> 2010 <212> DNA <213> Gibbon leukemia virus <400> 7 atggtattgc tgcctgggtc catgcttctc acctcaaacc tgcaccacct tcggcaccag 60 atgagtcctg ggagctggaa aagactgatc atcctcttaa gctgcgtatt cggcggcggc 120 gggacgagtc tgcaaaataa gaacccccac cagcccatga ccctcacttg gcaggtactg 180 tcccaaactg gagacgttgt ctgggataca aaggcagtcc agcccccttg gacttggtgg 240 cccacactta aacctgatgt atgtgccttg gcggctagtc ttgagtcctg ggatatcccg 300 ggaaccgatg tctcgtcctc taaacgagtc agacctccgg actcagacta tactgccgct 360 tataagcaaa tcacctgggg agccataggg tgcagctacc ctcgggctag gactagaatg 420 gcaagctcta ccttctacgt atgtccccgg gatggccgga ccctttcaga agctagaagg 480 tgcggggggc tagaatccct atactgtaaa gaatgggatt gtgagaccac ggggaccggt 540 tattggctat ctaaatcctc aaaagacctc ataactgtaa aatgggacca aaatagcgaa 600 tggactcaaa aatttcaaca gtgtcaccag accggctggt gtaaccccct taaaatagat 660 ttcacagaca aagaaaatt atccaaggac tggataacgg gaaaaacctg gggattaaga 720 ttctatgtgt ctggacatcc aggcgtacag ttcaccattc gcttaaaaaat caccaacatg 780 ccagctgtgg footggtcc tgacctcgtc cttgtggaac areacctcc tagaacgtcc 840 ctcgctctcc cacctctct tcccccaagg gaagcgccac cgccatctct ccccgactct 900 aactccacag ccctggcgac tagtgcacaa actcccacgg tgagaaaaac aattgttacc 960 ctaaacactc cgcctcccac cacaggcgac agactttttg atcttgtgca gggggccttc 1020 ctaaccttaa atgctaccaa cccaggggcc actgagtctt gctggctttg tttggccatg 1080 ggcccccctt attatgaagc aatagcctca tcaggagagg tcgcctactc caccgacctt 1140 gaccggtgcc gctgggggac ccaaggaaag ctcaccctca ctgaggtctc aggacacggg 1200 ttgtgcatag gaaaggtgcc ctttacccat cagcatctct gcaatcagac cctatccatc 1260 aattcctccg gagaccatca gtatctgctc ccctccaacc atagctggtg ggcttgcagc 1320 actggcctca ccccttgcct ctccacctca gtttttaatc agactagaga tttctgtatc 1380 caggtccagc tgattcctcg catctattac tatcctgaag aagttttgtt acaggcctat 1440 gacaattctc accccaggac taaaagagag gctgtctcac ttaccctagc tgttttactg 1500 gggttgggaa tcacggcggg aataggtact ggttcaactg ccttaattaa aggacctata 1560 gacctccagc aaggcctgac aagcctccag atcgccatag atgctgacct ccgggccctc 1620 caagactcag tcagcaagtt agaggactca ctgacttccc tgtccgaggt agtgctccaa 1680 aataggagag gccttgactt gctgtttcta aaagaaggtg gcctctgtgc ggccctaaag 1740 gaagagtgct gtttttacat agaccactca ggtgcagtac gggactccat gaaaaaactc 1800 aaagaaaaac tggataaaag acagttagag cgccagaaaa gccaaaactg gtatgaagga 1860 tggttcaata actccccttg gttcactacc ctgctatcaa ccatcgctgg gcccctatta 1920 ctcctccttc tgttgctcat cctcgggcca tgcatcatca ataagttagt tcaattcatc 1980 aatgatagga taagtgcagt taaaatttaa 2010 <210> 8 <211> 2013 <212> DNA <213> Gibbon leukemia virus <400> 8 accatggtcc tgctgcctgg gtctatgctg ctgacttcta acctgcacca cctgcgacac 60 cagatgtctc ccggctcatg gaaacggctg atcatcctgc tgagctgcgt gttcggagga 120 ggaggcacct ccctgcagaa caagaatcct caccagccaa tgaccctgac atggcaggtg 180 ctgtcccaga caggcgacgt ggtgtgggat accaaggcag tgcagccacc ttggacatgg 240 tggcccaccc tgaagcctga cgtgtgcgcc ctggccgcct ccctggagtc ttgggacatc 300 cccggcacag acgtgagcag cagcaagagg gtgagaccac ccgactctga ttatacagcc 360 gcctacaagc agatcacctg gggcgccatc gggcttagct atcctcgggc ccgcacaagg 420 atggccagct ccacctttta cgtgtgccca cgcgacggaa ggaccctgtc tgaggcaagg 480 agatgtggcg gcctggagag cctgtattgc aaggagtggg attgtgagac cacaggcaca 540 ggctactggc tgtctaagtc tagcaaggac ctgatcaccg tgaagtggga tcaagaacagc 600 gagtggacac agaagttcca gcagtgccac cagaccggct ggtgtaatcc cctgaagaatc 660 gactttacag ataagggcaa gctgtccaag gactggatca ccggcaagac atggggcctg 720 agattctacg tgtctggcca ccctggcgtg cagtttacaa tccggctgaa gatcaccaac 780 atgccagcag tggcagtggg accagacctg gtgctggtgg agcagggacc tccacgcacc 840 tccctggccc tgccccctcc actgccccct agggaggccc caccccctag cctgcccgat 900 tctaacagca cagccctggc cacctccgcc cagaccccta cagtgcgcaa gaccatcgtg 960 acactgaata ccccaccccc taccacaggc gacaggctgt tcgatctggt gcagggcgcc 1020 tttctgacac tgaacgccac caatcctggc gcaaccgaga gctgctggct gtgcctggct 1080 atgggcccac cctactatga ggcaatcgcc tcctctggag aggtggcata ttccacagac 1140 ctggatagat gcagatgggg cacccagggc aagctgaccc tgacagaggt gtctggccac 1200 ggcctgtgca tcggcaaggt gccattcaca caccagcacc tgtgcaacca gaccctgagc 1260 atcaatagct ccggcgacca ccagtacctg ctgccaagca accactcctg gtgggcatgc 1320 tccacaggac tgaccccatg tctgagcacc agcgtgttca accagaccag agacttttgt 1380 atccaggtgc agctgatccc tcggatctac tattacccag aggaggtgct gctgcaggcc 1440 tatgataatt cccacccaag aacaaagagg gaggccgtgt ctctgaccct ggccgtgctg 1500 ctgggactgg gaatcacagc aggaatcggc acaggcagca ccgccctgat caagggacca 1560 atcgacctgc agcagggact gacctccctg cagatcgcca tcgacgccga tctgagagcc 1620 ctgcaggaca gcgtgtccaa gctggaggat tctctgacct ctctgagcga ggtggtgctg 1680 cagaacagga ggggcctgga cctgctgttc ctgaaggagg gaggactgtg cgccgccctg 1740 aaggaggagt gctgttttta tatcgaccac tctggcgccg tgcgggatag catgaagaag 1800 ctgaaggaga agctggataa gcgccagctg gagaggcaga agagccagaa ttggtacgag 1860 ggctggttca acaattcccc ctggtttacc acactgctgt ctaccatcgc aggacctctg 1920 ttattactgc tgctgctgct gatcctgggc ccatgtatca tcaacaagct ggtgcagttt 1980 atcaacgacc gaatctccgc agtgaaaatc taa 2013 <210> 9 <211> 669 <212> PRT <213> Gibbon leukemia virus <400> 9 Met Val Leu Leu Pro Gly Ser Met Leu Leu Thr Ser Asn Leu His His 1 5 10 15 Leu Arg His Gln Met Ser Pro Gly Ser Trp Lys Arg Leu Ile Ile Leu 20 25 30 Leu Ser Cys Val Phe Gly Gly Gly Gly Thr Ser Leu Gln Asn Lys Asn 35 40 45 Pro His Gln Pro Met Thr Leu Thr Trp Gln Val Leu Ser Gln Thr Gly 50 55 60 Asp Val Val Trp Asp Thr Lys Ala Val Gln Pro Pro Trp Thr Trp Trp 65 70 75 80 Pro Thr Leu Lys Pro Asp Val Cys Ala Leu Ala Ala Ser Leu Glu Ser 85 90 95 Trp Asp Ile Pro Gly Thr Asp Val Ser Ser Ser Lys Arg Val Arg Pro 100 105 110 Pro Asp Ser Asp Tyr Thr Ala Ala Tyr Lys Gln Ile Thr Trp Gly Ala 115 120 125 Ile Gly Cys Ser Tyr Pro Arg Ala Arg Thr Arg Met Ala Ser Ser Thr 130 135 140 Phe Tyr Val Cys Pro Arg Asp Gly Arg Thr Leu Ser Glu Ala Arg Arg 145 150 155 160 Cys Gly Gly Leu Glu Ser Leu Tyr Cys Lys Glu Trp Asp Cys Glu Thr 165 170 175 Thr Gly Thr Gly Tyr Trp Leu Ser Lys Ser Ser Lys Asp Leu Ile Thr 180 185 190 Val Lys Trp Asp Gln Asn Ser Glu Trp Thr Gln Lys Phe Gln Gln Cys 195 200 205 His Gln Thr Gly Trp Cys Asn Pro Leu Lys Ile Asp Phe Thr Asp Lys 210 215 220 Gly Lys Leu Ser Lys Asp Trp Ile Thr Gly Lys Thr Trp Gly Leu Arg 225 230 235 240 Phe Tyr Val Ser Gly His Pro Gly Val Gln Phe Thr Ile Arg Leu Lys 245 250 255 Ile Thr Asn Met Pro Ala Val Ala Val Gly Pro Asp Leu Val Leu Val 260 265 270 Glu Gln Gly Pro Pro Arg Thr Ser Leu Ala Leu Pro Pro Pro Leu Pro 275 280 285 Pro Arg Glu Ala Pro Pro Pro Ser Leu Pro Asp Ser Asn Ser Thr Ala 290 295 300 Leu Ala Thr Ser Ala Gln Thr Pro Thr Val Arg Lys Thr Ile Val Thr 305 310 315 320 Leu Asn Thr Pro Pro Pro Thr Thr Gly Asp Arg Leu Phe Asp Leu Val 325 330 335 Gln Gly Ala Phe Leu Thr Leu Asn Ala Thr Asn Pro Gly Ala Thr Glu 340 345 350 Ser Cys Trp Leu Cys Leu Ala Met Gly Pro Pro Tyr Tyr Glu Ala Ile 355 360 365 Ala Ser Ser Gly Glu Val Ala Tyr Ser Thr Asp Leu Asp Arg Cys Arg 370 375 380 Trp Gly Thr Gln Gly Lys Leu Thr Leu Thr Glu Val Ser Gly His Gly 385 390 395 400 Leu Cys Ile Gly Lys Val Pro Phe Thr His Gln His Leu Cys Asn Gln 405 410 415 Thr Leu Ser Ile Asn Ser Ser Gly Asp His Gln Tyr Leu Leu Pro Ser 420 425 430 Asn His Ser Trp Trp Ala Cys Ser Thr Gly Leu Thr Pro Cys Leu Ser 435 440 445 Thr Ser Val Phe Asn Gln Thr Arg Asp Phe Cys Ile Gln Val Gln Leu 450 455 460 Ile Pro Arg Ile Tyr Tyr Tyr Pro Glu Glu Val Leu Leu Gln Ala Tyr 465 470 475 480 Asp Asn Ser His Pro Arg Thr Lys Arg Glu Ala Val Ser Leu Thr Leu 485 490 495 Ala Val Leu Leu Gly Leu Gly Ile Thr Ala Gly Ile Gly Thr Gly Ser 500 505 510 Thr Ala Leu Ile Lys Gly Pro Ile Asp Leu Gln Gln Gly Leu Thr Ser 515 520 525 Leu Gln Ile Ala Ile Asp Ala Asp Leu Arg Ala Leu Gln Asp Ser Val 530 535 540 Ser Lys Leu Glu Asp Ser Leu Thr Ser Leu Ser Glu Val Val Leu Gln 545 550 555 560 Asn Arg Arg Gly Leu Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys 565 570 575 Ala Ala Leu Lys Glu Glu Cys Cys Phe Tyr Ile Asp His Ser Gly Ala 580 585 590 Val Arg Asp Ser Met Lys Lys Leu Lys Glu Lys Leu Asp Lys Arg Gln 595 600 605 Leu Glu Arg Gln Lys Ser Gln Asn Trp Tyr Glu Gly Trp Phe Asn Asn 610 615 620 Ser Pro Trp Phe Thr Thr Leu Leu Ser Thr Ile Ala Gly Pro Leu Leu 625 630 635 640 Leu Leu Leu Leu Leu Leu Ile Leu Gly Pro Cys Ile Ile Asn Lys Leu 645 650 655 Val Gln Phe Ile Asn Asp Arg Ile Ser Ala Val Lys Ile 660 665 <210> 10 <211> 759 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens <400> 10 atgatcgaga cctacaatca gacaagccca cggtccgccg caaccggact gcctatcagc 60 atgaagatct tcatgtacct gctgaccgtg tttctgatca cacagatgat cggctccgcc 120 ctgttcgccg tgtatctgca caggagactg gacaagatcg aggatgagcg caatctgcac 180 gaggacttcg tgtttatgaa gaccatccag cggtgcaaca caggcgagag gagcctgtct 240 ctgctgaatt gtgaggagat caagtcccag ttcgagggct ttgtgaagga tatcatgctg 300 aacaaggagg agacaaagaa ggacgaggat ccacagatcg cagcacacgt ggtgtccgag 360 gcaaactcta atgccgccag cgtgctgcag tgggccaaga agggctacta taccatgaag 420 tctaacctgg tgacactgga gaatggcaag cagctgaccg tgaagaggca gggcctgtac 480 tatatctatg cccaggtgac attctgctct aacagagagg caagctccca ggcacccttc 540 atcgtgggac tgtggctgaa gccctctagc ggcagcgaga ggatcctgct gaaggccgcc 600 aatacccact cctctagcca gctgtgcgag cagcagtcca tccacctggg aggcgtgttc 660 gagctgcagc ctggagccag cgtgttcgtg aacgtgacag acccatctca ggtgagccac 720 ggcaccggct tcacaagctt tggcctgctg aagctgtga 759 <210> 11 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens <400> 11 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Asp Glu Asp Pro Gln 100 105 110 Ile Ala Ala His Val Val Ser Glu Ala Asn Ser Asn Ala Ala Ser Val 115 120 125 Leu Gln Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys Ser Asn Leu Val 130 135 140 Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr 145 150 155 160 Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser 165 170 175 Gln Ala Pro Phe Ile Val Gly Leu Trp Leu Lys Pro Ser Ser Gly Ser 180 185 190 Glu Arg Ile Leu Leu Lys Ala Ala Asn Thr His Ser Ser Ser Gln Leu 195 200 205 Cys Glu Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro 210 215 220 Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His 225 230 235 240 Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu 245 250 <210> 12 <211> 1416 <212> DNA <213> Homo sapiens <400> 12 atgctgccct ttctgagcat gctggtgctg ctggtgcagc ctctgggaaa cctgggagcc 60 gagatgaaga gcctgtccca gagatctgtg cctaacacct gcacactggt catgtgcagc 120 cccaccgaga atggactgcc tggaagggac ggaagggatg gaagggaggg ccctcggggc 180 gagaagggcg acccaggact gcctggacca atgggactga gcggactgca gggaccaaca 240 ggacctgtgg gaccaaaggg agagaacgga tccgccggag agccaggccc taagggcgag 300 aggggcctgt ctggcccccc tggcctgcca ggcatcccag gccccgccgg caaggagggc 360 ccatccggca agcagggcaa tatcggcccc cagggcaagc ctggcccaaa gggcgaggca 420 ggaccaaagg gagaagtggg agcacctggc atgcagggat ccaccggagc aaagggatct acaggaccaa agggcgagcg cggcgcccca ggcgtgcagg gcgcccccgg caatgcagga gcagcaggac cagcaggacc tgcaggccca cagggcgccc ctggctctag gggcccaccc 600 ggcctgaagg gcgacagggg agtgcctggc gataggggca tcaaggaga gagcggactg 660 ccagattccg ccgccctgag gcagcagatg gaggccctga agggcaagct gcagaggctg gaggtggcct tctcccacta ccagaaggcc gccctgtttc cagacggcca caggagactg 780 gacaagatcg aggatgagcg caacctgcac gaggatttcg tgtttatgaa gaccatccag agatgcaaca caggcgagcg gtctctgagc ctgctgaatt gtgaggagat caagtctcag ttcgagggct ttgtgaagga catcatgctg aacaaggagg aacaagga ggagaatagc ttcgatgc agaagggcga tcagaatccc cagatcgcag cacacgtgat cagcgaggca agctccaaga ccacatccgt gctgcagtgg gccgagaagg gctactatac catgtccaac 1080 aatctggtga cactggagaa cggcaagcag ctgaccgtga agagacaggg cctgtactat 1140 atctatgccc aggtgacatt ctgctctaat cgggaggcct ctagccaggc cccttttatc 1200 gcctctctgt gcctgaagag cccaggcaga ttcgagcgga tcctgctgag ggccgccaac 1260 acccactcct ctgccaagcc atgcggacag cagagcatcc acctgggagg cgtgttcgag 1320 ctgcagccag gagcctccgt gtttgtgaat gtgacagacc catcccaggt gtctcacgga 1380 accggcttca catcctttgg cctgctgaag ctgtga 1416 <210> 13 <211> 471 <212> PRT <213> Homo sapiens <400> 13 Met Leu Pro Phe Leu Ser Met Leu Val Leu Leu Val Gln Pro Leu Gly 1 5 10 15 Donkey Leu Gly Here Glu Met Lys Ser Leu Ser Gln Arg Ser Val Pro Donkey 20 25 30 Thr Cys Thr Leu Val Met Cys Ser Pro Thr Glu Asn Gly Leu Pro Gly 35 40 45 Arg Asp Gly Arg Asp Gly Arg Glu Gly Pro Arg Gly Glu Lys Gly Asp 50 55 60 Pro Gly Leu Pro Gly Pro Met Gly Leu Ser Gly Leu Gln Gly Pro Thr 65 70 75 80 Gly Pro Val Gly Pro Lys Gly Glu Asn Gly Ser Ala Gly Glu Pro Gly 85 90 95 Pro Lys Gly Glu Arg Gly Leu Ser Gly Pro Pro Gly Leu Pro Gly Ile 100 105 110 Pro Gly Pro Ala Gly Lys Glu Gly Pro Ser Gly Lys Gln Gly Asn Ile 115 120 125 Gly Pro Gln Gly Lys Pro Gly Pro Lys Gly Glu Ala Gly Pro Lys Gly 130 135 140 Glu Val Gly Ala Pro Gly Met Gln Gly Ser Thr Gly Ala Lys Gly Ser 145 150 155 160 Thr Gly Pro Lys Gly Glu Arg Gly Ala Pro Gly Val Gln Gly Ala Pro 165 170 175 Gly Asn Ala Gly Ala Ala Gly Pro Ala Gly Pro Ala Gly Pro Gln Gly 180 185 190 Ala Pro Gly Ser Arg Gly Pro Pro Gly Leu Lys Gly Asp Arg Gly Val 195 200 205 Pro Gly Asp Arg Gly Ile Lys Gly Glu Ser Gly Leu Pro Asp Ser Ala 210 215 220 Ala Leu Arg Gln Gln Met Glu Ala Leu Lys Gly Lys Leu Gln Arg Leu 225 230 235 240 Glu Val Ala Phe Ser His Tyr Gln Lys Ala Ala Leu Phe Pro Asp Gly 245 250 255 His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp 260 265 270 Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser 275 280 285 Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe 290 295 300 Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser 305 310 315 320 Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val 325 330 335 Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu 340 345 350 Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly 355 360 365 Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln 370 375 380 Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile 385 390 395 400 Only Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu 405 410 415 Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser 420 425 430 Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe 435 440 445 Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr 450 455 460 Ser Phe Gly Leu Leu Lys Leu 465,470 <210> 14 <211> 1412 <212> DNA <213> Muscles <400> 14 atgctgccct tcctgagcat gctggtgctg ctggtgcagc ctctgggcaa tctgggcgcc 60 gagatgaagt ccctgtctca gaggagcgtg ccaacacct gcacactggt catgtgctt 120 ccaaccgaga atggactgcc aggaagggac ggaagagatg gaaggggg accaagggg 180 gagaagggcg accctggact gcctggacca atgggactgt ccggactgca gggaccaaca 240 ggccctgtgg gaccaaaggg agagaatgga agcgccggag agccaggacc taagggagag 300 aggggcctgt ccggcccccc tggcctgcct ggcatcccag gccccgccgg caaggagggc 360 ccttctggca agcagggcaa catcggacca cagggcaagc ctggaccaaa gggagaggca 420 ggaccaaagg gagaagtggg agcaccccggc atgcagggca gcaccggagc aaagggatcc 480 accggcccta agggagagag aggagcacct ggagtgcagg gcgccccagg caatgcagga 540 gcagcaggac cagcaggacc tgcaggccca cagggcgccc caggcagccg gggcccacccc 600 ggcctgaagg gcgacagggg agtgccaggc gataggggca tcaagggaga gtccggactg 660 ccagactctg ccgccctgag gcagcagatg gaggccctga agggcaagct gcagaggctg 720 gaggtggcct tctccacta ccagaaggcc gccctgtttc cagaggaca caggagactg 780 gataaggtgg aggaggaggt gaacctgcac gaggatttcg tgttcatcaa gaagctgaag 840 aggtgcaaca agggcgaggg cagcctgtcc ctgctgaatt gtgaggagat gcggcgccag 900 ttcgaggacc tggtgaagga tatcaccctg aacaaggagg agaagaagga gaattcttttt 960 gagatgcaga ggggcgacga ggatcctcag atcgcagcac acgtggtgtc cgaggcaaac 1020 tctaatgccg ccagcgtgct gcagtgggcc aagaagggct actataccat gaagtctaac 1080 ctggtcatgc tggagaatgg caagcagctg acagtgaaga gagagggcct gtactacgtg 1140 tacacccagg tgacattctg cagcaacaga gagcccagct cccagcggcc tttatcgtg 1200 ggcctgtggc tgaagccctc tatcggaagc gagaggatcc tgctgaaggc agccaatacc 1260 cactctagct cccagctgtg cgagcagcag tccgtgcacc tgggaggcgt gttcgagctg 1320 caggcaggag caagcgtgtt cgtgaacgga cagaggccag ccaggtcatc cacagagtgg 1380 gcttctctag ctttggcctg ctgaagctgt ga 1412 <210> 15 <211> 470 <212> PRT <213> Mus musculus <400> 15 Met Leu Pro Phe Leu Ser Met Leu Val Leu Leu Val Gln Pro Leu Gly 1 5 10 15 Asn Leu Gly Ala Glu Met Lys Ser Leu Ser Gln Arg Ser Val Pro Asn 20 25 30 Thr Cys Thr Leu Val Met Cys Ser Pro Thr Glu Asn Gly Leu Pro Gly 35 40 45 Arg Asp Gly Arg Asp Gly Arg Glu Gly Pro Arg Gly Glu Lys Gly Asp 50 55 60 Pro Gly Leu Pro Gly Pro Met Gly Leu Ser Gly Leu Gln Gly Pro Thr 65 70 75 80 Gly Pro Val Gly Pro Lys Gly Glu Asn Gly Ser Ala Gly Glu Pro Gly 85 90 95 Pro Lys Gly Glu Arg Gly Leu Ser Gly Pro Pro Gly Leu Pro Gly Ile 100 105 110 Pro Gly Pro Ala Gly Lys Glu Gly Pro Ser Gly Lys Gln Gly Asn Ile 115 120 125 Gly Pro Gln Gly Lys Pro Gly Pro Lys Gly Glu Ala Gly Pro Lys Gly 130 135 140 Glu Val Gly Ala Pro Gly Met Gln Gly Ser Thr Gly Ala Lys Gly Ser 145 150 155 160 Thr Gly Pro Lys Gly Glu Arg Gly Ala Pro Gly Val Gln Gly Ala Pro 165 170 175 Gly Asn Ala Gly Ala Ala Gly Pro Ala Gly Pro Ala Gly Pro Gln Gly 180 185 190 Ala Pro Gly Ser Arg Gly Pro Pro Gly Leu Lys Gly Asp Arg Gly Val 195 200 205 Pro Gly Asp Arg Gly Ile Lys Gly Glu Ser Gly Leu Pro Asp Ser Ala 210 215 220 Ala Leu Arg Gln Gln Met Glu Ala Leu Lys Gly Lys Leu Gln Arg Leu 225 230 235 240 Glu Val Ala Phe Ser His Tyr Gln Lys Ala Ala Leu Phe Pro Asp Gly 245 250 255 His Arg Arg Leu Asp Lys Val Glu Glu Glu Val Asn Leu His Glu Asp 260 265 270 Phe Val Phe Ile Lys Lys Leu Lys Arg Cys Asn Lys Gly Glu Gly Ser 275 280 285 Leu Ser Leu Leu Asn Cys Glu Glu Met Arg Arg Gln Phe Glu Asp Leu 290 295 300 Val Lys Asp Ile Thr Leu Asn Lys Glu Glu Lys Lys Glu Asn Ser Phe 305 310 315 320 Glu Met Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val 325 330 335 Ser Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys 340 345 350 Gly Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly Lys 355 360 365 Gln Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr Gln Val 370 375 380 Thr Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg Pro Phe Ile Val 385 390 395 400 Gly Leu Trp Leu Lys Pro Ser Ile Gly Ser Glu Arg Ile Leu Leu Lys 405 410 415 Ala Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu Gln Gln Ser Val 420 425 430 His Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala Ser Val Phe Val 435 440 445 Asn Val Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser Ser 450 455 460 Phe Gly Leu Leu Lys Leu 465 470 <210> 16 <211> 786 <212> DNA <213> Homo sapiens <400> 16 atgatcgaaa catacaacca aacttctccc cgatctgcgg ccactggact gcccatcagc 60 atgaaaattt tttgtattt acttactgtt tttcttatca cccagatgat tgggtcagca 120 cttttgctg tgtatcttca tagaaggttg gacaagatag aagatgaaag gaatcttcat 180 gaagattttg tattcatgaa aacgatacag agatgcaaca caggagaaag atccttatcc 240 ttactgaact gtgaggagat taaaagccag tttgaaggct ttgtgaagga tataatgtta 300 aacaaagagg agacgaagaa agaaaacagc tttgaaatgc aaaaaggtga tcagaatcct 360 caaattgcgg cacatgtcat aagtgaggcc agcagtaaaa caacatctgt gttacagtgg 420 gctgaaaaag gatactacac catgagcaac aacttggtaa ccctggaaaa tgggaaacag 480 ctgaccgtta aaagacaagg actctattat atctatgccc aagtcacctt ctgttccaat 540 cgggaagctt cgagtcaagc tccatttata gccagcctct gcctaaagtc ccccggtaga 600 ttcgagagaa tcttactcag agctgcaaat acccacagtt ccgccaaacc ttgcgggcaa 660 caatccattc acttgggagg agtatttgaa ttgcaaccag gtgcttcggt gtttgtcaat 720 gtgactgatc caagccaagt gagccatggc actggcttca cgtcctttgg cttactcaaa 780 ctctga 786 <210> 17 <211> 261 <212> PRT <213> Homo sapiens <400> 17 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> 18 <211> 783 <212> DNA <213> Mus musculus <400> 18 atgatagaaa catacagcca accttccccc agatccgtgg caactggact tccagcgagc 60 atgaagattt ttatgtattt acttactgtt ttccttatca cccaatgat tggatctgtg 120 ctttttgctg tgtatcttca tagagattg gataagtcg agaggaagt aaaccttcat 180 gagattttg tattcataa aaagctaag agatgcaca aaggagaagg atctttatcc 240 ttgctgaact gtgaggagat gagaaggcaa tttgagacc ttgtcagga tataacgtta 300 aaaaagaag agaaaaaaaacagcttt gaatgcaa gaggtgatga ggatcctcaa 360 attgcagcac acgttgtaag cgaagccaac agtaatgcag catccgttct acagtgggcc 420 aagaaaggat attackaccat gaaaagcaac ttggtaatgc ttgaaaatgg gaacagctg 480 acggttaaaa gagaggact ctattgtc tacactcag tcaccttg ctctaatcgg 540 gagccttcga gtcaaccccc attcatcgtc ggcctctggc tgaagcccag cagtggatct 600 gagagaatct tactcaggc ggcaatacc cacagttcct cccagctttg cgagcagcag 660 tctgttcact tgggcggagt gtttgaatta caagctggtg cttctgtgtt tgtcaacgtg 720 actgaagcaa gccaagtgat ccacagtt ggctctcat cttttggctt actcaactc 780 made 783 <210> 19 <211> 260 <212> PRT <213> Muscles <400> 19 With Glu Thr Tyr Ser Gln Pro Ser Pro Arg Ser Val Ala Thr Gly 1 5 10 15 Leu Pro Ala Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Val Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Val Glu Glu Glu Val Asn Leu His Glu Asp Phe Val 50 55 60 Phy Ile Lys Leu Lys Arg Cys Asn Lys Gly Glu Gly Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Met Arg Arg Gln Phe Glu Asp Leu Val Lys 85 90 95 Asp Ile Thr Leu Asn Lys Glu Glu Lys Lys Glu Asn Ser Phe Glu Met 100 105 110 Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val Ser Glu 115 120 125 Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys Gly Tyr 130 135 140 Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly Lys Gln Leu 145 150 155 160 Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr Gln Val Thr Phe 165 170 175 Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg Pro Phe Ile Val Gly Leu 180 185 190 Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg Ile Leu Leu Lys Ala Ala 195 200 205 Asn Thr His Ser Ser Ser Gln Leu Cys Glu Gln Gln Ser Val His Leu 210 215 220 Gly Gly Val Phe Glu Leu Gln Ala Gly Ala Ser Val Phe Val Asn Val 225 230 235 240 Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser Ser Phe Gly 245 250 255 Leu Leu Lys Leu 260 <210> 20 <211> 930 <212> DNA <213> Mus musculus <400> 20 atggatcagc acacactgga cgtggaggat accgctgacg ctaggcaccc agctggcacc 60 tcctgccctt ctgatgccgc tctgctgcgc gacacaggac tgctggccga tgccgctctg 120 ctgtctgaca cagtgcggcc aaccaacgcc gctctgccaa ccgatgctgc ttaccctgct 180 gtgaacgtga gggacagaga ggctgcttgg ccacctgccc tgaacttctg cagccgccac 240 cctaagctgt acggcctggt ggccctggtg ctgctgctgc tgatcgctgc ttgcgtgcca 300 atctttaccc ggacagagcc acgccccgct ctgacaatca ccacatcccc caacctgggc 360 accagggaga acaacgccga tcaggtgaca ccagtgtctc acatcggctg ccccaacacc 420 acacagcagg gaagcccagt gttcgccaag ctgctggcta agaaccaggc cagcctgtgc 480 aacaccacac tgaactggca cagccaggac ggagctggaa gctcctacct gtcccagggc 540 ctgagatacg aggaggataa gaaggagctg gtggtggact cccctggact gtactacgtg 600 ttcctggagc tgaagctgtc tccaaccttt acaaacaccg gccacaaggt gcagggatgg 660 gtgtctctgg tgctgcaggc taagccccag gtggacgatt tcgataacct ggccctgacc 720 gtggagctgt ttccttgtag catggagaac aagctggtgg acaggtcttg gagccagctg 780 ctgctgctga aggctggcca caggctgtcc gtgggactga gagcctacct gcacggcgcc 840 caggatgctt acagagactg ggagctgagc taccctaaca ccacatcctt cggactgttt 900 ctggtgaagc ctgacaaccc atgggagtga 930 <210> 21 <211> 765 <212> DNA <213> Homo sapiens <400> 21 atggagtacg cctctgacgc cagcctggat ccagaggccc cttggccacc tgcaccaagg 60 gcccgcgcct gccgcgtgct gccctgggcc ctggtggccg gcctgttatt actgctgctg 120 ctggccgccg cctgcgccgt gttcctggca tgtccttggg ccgtgagcgg agccagagcc 180 tccccaggct ctgccgccag ccctcggctg agagagggac cagagctgtc cccagacgat 240 ccagcaggcc tgctggacct gaggcaggga atgtttgccc agctggtggc ccagaacgtg 300 ctgctgatcg acggccccct gtcctggtac tctgatcctg gcctggccgg cgtgtctctg 360 accggcggcc tgagctataa ggaggataca aaggagctgg tggtggccaa ggccggcgtg 420 tactacgtgt tcttccagct ggagctgagg agagtggtgg caggaggg ctctggaagc 480 gtgtccctgg ccctgcacct gcagcccctg cggagccgccg caggagccgc cgccctggcc 540 ctgaccgtgg acctgccacc agccagctcc attccgcctt cggctttcag 600 ggcagactgc tgcacctgtc tgccggacag aggctgggag tgcacctgca caccgaggcc 660 agggcccgcc acgcatggca gctgacccag ggagcaacag tgctgggcct gttccgcgtg 720 acacctgaga tcccagcagg cctgcctagc ccacggtccg to <210> 22 <211> 1389 <212> DNA <213> Mus musculus <400> 22 atgctgcctt tcctgtccat gctggtgctg ctggtgcagc cactgggcaa cctgggagcc 60 gagatgaagt ctctgagcca gcgcagcgtg cctaacacct gcacactggt catgtgctcc cctacagaga acggcctgcc aggaagggac ggaagagatg gaagggaggg accaagggga gagaagggcg accccggact gcctggacca atgggactga gcggcctgca gggaccaacc 240 ggccccgtgg gacctaaggg agagaacgga tccgctggag agccaggacc taagggagag 300 agaggactgt ctggaccacc tggactgcca ggaatcccag gaccagctgg caggaggga 360 ccatccggca agcagggaaa catcggacca cagggaaagc ctggaccaaa gggagaggct 420 ggacctaagg gagaagtggg cgccccagga atgcagggct ctacaggagc taagggcagc 480 accggaccaa agggagagag gggagccccc ggagtgcagg gagcccctgg caacgctgga 540 gccgctggcc cagccggacc cgctggccct cagggagccc ccggctctag gggaccacca 600 ggcctgaagg gagacagagg cgtgcccgga gatcggggca tcaagggga gagcggcctg 660 cctgactccg ccgctctgag acagcagatg gaggctctga agggcaagct gcagcggctg 720 gaggtggcct tctccacta ccagaaggcc gctctgtttc ctgacggaag gacagagccc 780 aggcctgctc tgaccatcac cacatctcca aacctgggca caagagagaa caacgccgat 840 caggtgaccc ccgtgtctca catcggatgc cctaacacca cacagcaggg cagccccgtg 900 tttgccaagc tgctggctaa gaaccaggcc agcctgtgca acaccacact gaactggcac 960 tcccaggatg gcgccggaag ctcctacctg tctcagggcc tgcggtacga ggaggacaag 1020 aaggagctgg tggtggatag cccaggcctg tactacgtgt tcctggagct gaagctgtcc 1080 cccaccttta caaacaccgg acacaaggtg cagggatggg tgagcctggt gctgcaggct 1140 aagccccagg tggacgattt cgacaacctg gccctgaccg tggagctgtt tccttgctct 1200 atggagaaca agctggtgga tagatcctgg agccagctgc tgctgctgaa ggctggacac 1260 cgcctgagcg tgggcctgag ggcttacctg cacggagctc aggacgctta cagggattgg 1320 gagctgtcct accctaacac cacatctttc ggcctgtttc tggtgaagcc agacaacccc 1380 tgggagtga 1389 <210> 23 <211> 1389 <212> DNA <213> Homo sapiens <400> 23 atgctgctgt tcctgctgtc cgccctggtg ctgctgaccc agcctctggg ctacctggag 60 gccgagatga agacctattc tcaccggaca atgccaagcg cctgcacact ggtcatgtgc 120 agcagcgtgg agtctggcct gccaggaagg gacggaaggg atggaaggga gggacctaga 180 ggcgagaagg gcgaccctgg cctgccagga gcagcaggac aggcaggaat gcccggccag 240 gccggccccg tgggacctaa gggcgacaac ggaagcgtgg gagagccagg accaaagggc 300 gataccggcc cttccggacc acctggacca ccaggcgtgc ctggcccagc cggcagggag 360 ggccctctgg gcaagcaggg caatatcggc ccacagggca agcccggccc taagggcgag 420 gccggcccca agggcgaagt gggcgcccct ggcatgcagg gaagcgccgg agcccgcggc 480 ctggccggac ctaagggcga gagaggcgtg cctggagaga ggggcgtgcc aggaaacaca 540 ggcgcagcag gatctgccgg agcaatggga ccccagggca gccctggcgc caggggccct 600 ccaggcctga agggcgacaa gggcatccca ggcgataagg gagcaaaggg agagagcggc 660 ctgccagatg tggcctccct gcgccagcag gtggaggccc tgcagggcca ggtgcagcac 720 ctgcaggccg ccttctctca gtacaagaag gtggagctgt ttccaaacgg cgcctgcccc 780 tgggccgtga gcggagcccg ggcctcccca ggctctgccg ccagccctag gctgcgcgag 840 ggaccagagc tgagcccaga cgatccagca ggcctgctgg acctgagaca gggaatgttc 900 gcccagctgg tggcccagaa tgtgctgctg atcgacggcc cactgtcctg gtactctgat 960 ccaggcctgg ccggcgtgtc cctgaccggc ggcctgtctt ataaggagga tacaaaggag 1020 ctggtggtgg ccaaggccgg cgtgtactac gtgttcttcc agctggagct gaggagagtg 1080 gtggcaggag agggatccgg atctgtgagc ctggccctgc acctgcagcc cctgcggtcc 1140 gccgcaggag ccgccgccct ggccctgacc gtggacctgc cacctgcctc tagcgaggca 1200 cgcaattccg ccttcggctt tcagggccgg ctgctgcacc tgtctgccgg acagagactg 1260 ggagtgcacc tgcacaccga ggcccgggcc agacacgcct ggcagctgac ccagggagca 1320 acagtgctgg gcctgtttag ggtgacacct gagatcccag ccggcctgcc aagcccccgc 1380 tccgagtga 1389 <210> 24 <211> 522 <212> DNA <213> Mus musculus <400> 24 atggaggaga tgcctctgag ggagagctcc ccacagaggg ccgagagatg caagaagagc 60 tggctgctgt gcatcgtggc tctgctgctg atgctgctgt gctctctggg caccctgatc 120 tacacaagcc tgaagccaac cgccatcgag tcctgtatgg tgaagttcga gctgtctagc 180 tccaagtggc acatgacatc ccccaagcct cactgcgtga acaccacatc tgacggaaag 240 ctgaagatcc tgcagagcgg cacctacctg atctacggac aggtcatccc cgtggacaag 300 aagtacatca aggataacgc ccctttcgtg gtgcagatct acaagaagaa cgacgtgctg 360 cagacactga tgaacgattt tcagatcctg cccatcggcg gagtgtacga gctgcacgct 420 ggcgacaaca tctacctgaa gttcaactcc aggatcaca tccagaagac caacacatac 480 tgggggaatca tcctgatgcc agatctgccc tttatctctt ga 522 <210> 25 <211> 600 <212> DNA <213> Homo sapiens <400> 25 atgaccctgc acccaagccc catcacatgc gagttcctgt tttctaccgc cctgatcagc 60 ccaaagatgt gcctgagcca cctggagaat atgcccctgt cccactctcg gacacaggga 120 gcccagagaa gctcctggaa gctctggctg ttctgctcta tcgtgatgct gctgttcctg 180 tgcagctttt cctggctgat cttcatcttt ctgcagctgg agacagccaa ggagccttgc 240 atggccaagt ttggccctct gccatccaag tggcagatgg cctctagcga gcccccttgc 300 gtgaacaagg tgagcgactg gaagctggag atcctgcaga acggcctgta cctgatctat 360 ggccaggtgg cccccaacgc caattacaac gacgtggccc cttcgaggt gcggctgtat 420 aagaacaagg atatgatcca gaccctgaca aataagtcta agatccagaa cgtgggcggc 480 acatacgagc tgcacgtggg cgacaccatc gacctgatct tcaacagcga gcaccaggtg 540 ctgaagaaca atacatattg gggcatcatc ctgctggcca acccccagtt tatctcctga 600 <210> 26 <211> 1164 <212> DNA <213> Mus musculus <400> 26 atgctgcctt tcctgtctat gctggtgctg ctggtgcagc cactgggcaa cctgggagcc 60 gagatgaaga gcctgtccca gagatccgtg cccaacacct gcacactggt catgtgctct 120 cctaccgaga acggcctgcc aggaagggac ggaagagatg gaagggaggg acctcgggga 180 gagaagggcg acccaggact gcctggacca atgggactga gcggcctgca gggaccaaca 240 ggccccgtgg gacctaaggg agagaacgga agcgccggag agccaggacc taagggagag 300 aggggactgt ccggaccacc tggactgcct ggaatcccag gaccagctgg caaggaggga 360 ccatccggca agcagggaaa catcggacca cagggaaagc ctggaccaaa gggagaggct 420 ggaccaaagg gagaagtggg cgctcctgga atgcagggct ccaccggagc caagggctct 480 acaggaccaa aaggagagag gggagctccc ggagtgcagg gagcccctgg caacgctgga 540 gccgctggcc cagccggacc cgctggccct cagggagccc caggcagcag gggaccaccc 600 ggcctgaagg gcgacagggg cgtgccagga gataggggca tcaagggaga gtctggcctg 660 ccagacagcg ccgctctgag acagcagatg gaggccctga agggcaagct gcagcggctg 720 gaggtggctt tctccacta ccagaaggcc gctctgtttc cagatggcag cctgaagccc 780 accgccatcg agtcctgcat ggtgaagttt gagctgagct cctctaagtg gcacatgaca 840 tctcccaagc ctcactgcgt gaacaccaca tctgacggca agctgaagat cctgcagagc 900 ggcacctacc tgatctacgg ccaggtcatc cccgtggaca agaagtacat caaggataac 960 gcccctttcg tggtgcagat ctacaagaag aacgacgtgc tgcagacact gatgaacgat 1020 tttcagatcc tgccaatcgg cggagtgtac gagctgcacg ctggcgacaa catctacctg 1080 aagttcaact ctaaggatca catccagaag accaacacat actggggcat catcctgatg 1140 ccagatctgc cctttatcag ctga 1164 <210> 27 <211> 1152 <212> DNA <213> Homo sapiens <400> 27 atgctgctgt tcctgctgtc tgccctggtg ctgctgaccc agccactggg ctacctggag 60 gccgagatga agacctattc ccaccgcaca atgccttctg cctgcacact ggtcatgtgc 120 agcagcgtgg agagcggcct gccaggaagg gacggaaag atggaaggga gggacccaga 180 ggcgagaagg gcgaccctgg cctgccagga gcagcagcac aggcaggaat gccaggccag 240 gccggccccg tgggccctaa gggcgacaat ggatccgtgg gagagccagg accaaagggc 300 gataccggcc cttctggacc acctggacca ccaggcgtgc ctggaccagc aggaagagag 360 ggacctctgg gcaagcaggg aaacatcgga ccacagggca agccaggccc taagggcgag 420 gccggcccca agggcgaagt gggcgcccct ggcatgcagg gatccgccgg agccaggggc 480 ctggccggac ctaagggcga gcgcggcgtg cctggagaga ggggcgtgcc aggaataca 540 ggcgcagcag gatctgccgg agcaatggga ccacagggca gccccggcgc cagaggccct 600 ccaggcctga agggcgacaa gggaatccct ggcgataagg gagcaaaggg agagagcggc 660 ctgccagacg tggcctccct gaggcagcag gtggaggccc tgcagggaca ggtgcagcac 720 ctgcaggccg ccttcagcca gtacaagaag gtggagctt ttccaatgg cgagacagcc 780 aaggagccct gcatggccaa gttcggccca ctgcccagca agtggcagat ggcctctagc 840 gagcccctt gcgtgaacaa ggtgagcgat tggaagctgg agatcctgca gaacggcctg 900 tacctgatct atggccaggt ggccccaac gccaattaca acgacgtggc cccttttgag 960 gtgcggctgt atagaacaa ggatatgatc cagaccctga caataagtc windowsccag 1020 aacgtgggag gcacctacga gctgcacgtg ggcgacacaa tcgacctgat cttcacagc 1080 gagcaccagg tgctgaagaa caatacatat tggggcatca tcctgctggc caacccccag 1140 tttatctcct ga 1152 <210> 28 <211> 597 <212> DNA <213> Muscles <400> 28 atggagggcg agggagtgca gccctggat gagaacctgg agaacggctc ccggcctcgc 60 ttcaagtgga agaagaccct gcggctggtg gtgtctgga tcaagggcgc cggaatgctg ctgtgcttta tctacgtgtg cctgcagctg agctcctctc ccgccaagga tccccctatc 180 cagaggctga gaggagctgt gaccaggtgc gaggacggac agctgttcat cagctcctac 300. aagaacgagt accagacaat ggaggtgcag aacaacagcg tggtcatcaa gtgtgatggc 360. ctgtacatca tctacctgaa gggatccttc tttcaggagg tgaagatcga cctgcacttt cgggaggatc acaacccaat ctctatcccc atgctgaacg acggcaggag aatcgtgttc 420 acagtggtgg ccagcctggc ttttaaggac aaggtgtacc tgaccgtgaa cgccccagat 480 acactgtgcg agcacctgca gatcaacgac ggagagctga tcgtggtgca gctgacccct ggctactgtg ctccagaggg atcttaccac agcacagtga accaggtgcc cctgtga 597 <210> 29 <211> 552 <212> DNA <213> Homo sapiens <400> 29 atggagagggg tgcagcccct ggaggagaac gtgggaatg ccgcccggcc tegttcgag 60 aggaacaagc tgctgctgt ggctctgtg atccaggcc tgggctgct gctgtgcttc 120 acctacatct gtctgcactt ttctcctg caggtgagcc acagataccc ccgcatccag 180 agcatcagg tgcagttcac cgagtataag aaggaagg gctttatcct gatacccag 240 aaggaggacg agatcatgaa gtgcagac aattctgtga tcatcactg cgatggcttc 300 tacctgatct ccctgaaggg ctattttct caggaagtga atatcagcct gcactatcag 360 aaggacgagg agccactgtt tcagctgaag aaggtgcgga gcgtgaatttc cctgatgtg 420 gccagcctga cctacaagga caagtgtat ctgaacgtga ccacagataa tacatccctg 480 gacgatttcc acgtgaacgg cggcgagctg atcctgatcc accagaatcc cggcgagttt 540 tgcgtgctgt ga 552 <210> 30 <211> 1215 <212> DNA <213> Muscles <400> 30 atgctgccct tcctgtccat gctggtgctg ctggtgcagc ctctgggcaa cctgggagcc 60 gagatgaagt ctctgagcca gagatccgtg ccaaacacct gcaacactggt catgtgctct 120 cccaccgaga acggcctgcc tggaagggac ggagagatg gaagggaggg accccggga 180 gagaagggcg atcctggact gccaggacct atgggactga gcggcctgca gggaccaaca 240 ggccccgtgg gacctaaggg agagaacgga agcgccggag agccaggacc aaagggagag 300 aggggactgt ccggccccacc tggactgcct ggaatccctg gaccagctgg caaggaggga 360 ccttccggca agcagggaaa catcggacca cagggaaagc caggacctaa gggagaggct 420 ggaccaaagg gagaagtggg cgctcccgga atgcagggct ctaccggagc caagggcagc 480 agggaccta agggagagag gggagctcca ggagtgcagg gagccccccgg caacgctgga 540 gctgctggac cagctggacc agctggccct cagggagccc caggctctag gggaccacca 600 ggcctgaagg gcgacagggg cgtgccagga gataggggca tcaagggaga gagcggcctg 660 ccagattccg ccgctctgag acagcagatg gaggccctga agggcaagct gcagcggctg 720 gaggtggctt tcagccacta ccagaaggcc gctctgtttc ctgacggcag ctcctctcca 780 gccaaggatc ctccaatcca gcggctgcgc ggagctgtga ccaggtgcga ggatggccag 840 ctgttcatca gctcctacaa gaacgagtac cagacaatgg aggtgcagaa caactctgtg 900 gtcatcaagt gtgacggcct gtacatcatc tacctgaagg gcagcttctt tcaggaggtg 960 aagatcgacc tgcactttag agaggatcac aacccaatct ccatccccat gctgaacgac 1020 ggcaggagaa tcgtgttcac cgtggtggcc tctctggctt ttaaggacaa ggtgtacctg 1080 accgtgaacg cccccgatac actgtgcgag cacctgcaga tcaacgacgg cgagctgatc 1140 gtggtgcagc tgacccctgg atactgtgct ccagagggct cctaccactc tacagtgaac 1200 caggtgcctc tgtga 1215 <210> 31 <211> 1170 <212> DNA <213> Homo sapiens <400> 31 atgctgctgt tcctgctgag cgccctggtg ctgctgaccc agccactggg ctacctggag 60 gccgagatga agacctattc ccacagaaca atgccttctg cctgcacact ggtcatgtgc 120 agcagcgtgg agtccggcct gccaggaagg gacggcagag atggcaggga gggccccagg 180 ggcgagaagg gcgaccccgg cctgcctgga gcagcaggcc aggccggcat gccaggccag 240 gccggcccag tgggccccaa gggcgacaac ggcagcgtgg gcgagcccgg ccctaagggc 300 gataccggcc cctccggccc ccctggccca cccggcgtgc caggaccagc aggaagggag 360 ggaccactgg gcaagcaggg caatatcgga cctcagggca agcctggacc aaagggagag 420 gcaggaccaa agggagaagt gggcgcccct ggcatgcagg gatctgccgg agcccggggc 480 ctggccggcc ccaagggcga gagaggcgtg cccggcgaga ggggcgtgcc tggcaacaca 540 ggcgccgccg gctccgccgg cgccatggga cctcagggct ctccaggagc cagaggccct 600 ccaggcctga agggcgacaa gggaatccct ggcgataagg gagcaaaggg agagagcggc 660 ctgccagacg tggcctccct gcggcagcag gtggaggccc tgcagggcca ggtgcagcac 720 ctgcaggccg ccttcagcca gtacaagaag gtggagctgt ttcctaatgg cgtgtctcac 780 cgctacccac ggatccagag catcaaggtg cagttcaccg agtataagaa ggagaagggc 840 tttatcctga catctcagaa ggaggacgag atcatgaagg tgcagaacaa tagcgtgatc 900 atcaactgcg atggcttcta cctgatcagc ctgaagggct attttccca ggaagtgaat 960 atctctctgc actatcagaa ggatgaggag cctctgtttc agctgaagaa ggtgagatct 1020 gtgaacagcc tgatggtggc ctccctgacc tacaaggaca aggtgtatct gaacgtgacc 1080 acagataata catctctgga cgatttccac gtgaacggcg gcgagctgat cctgatccac 1140 cagaatcccg gcgagttttg cgtgctgtga 1170 <210> 32 <211> 969 <212> DNA <213> Mus musculus <400> 32 atgcagctga agtgtccatg cttcgtgtcc ctgggaacaa gacagcccgt ctggaagaaa 60 ctgcacgtga gctccggctt ctttagcggc ctggggctgt ttctgctgct gctgtctagt 120 ctgtgcgccg cttccgcaga gactgaagtc ggagccatgg tgggcagtaa cgtggtcctg 180 tcatgcatcg acccacaccg acggcatttc aacctgtctg gcctgtacgt gtattggcag 240 attgagaatc ccgaagtgtc agtcacctac tatctgcctt acaagagccc agggatcaac 300 gtggactcaa gctataaaaa tagggggcac ctgtccctgg attctatgaa gcagggaaac 360 ttcagcctgt acctgaaaaa tgtgacccct caggacacac aggagttcac ttgtcgcgtc 420 tttatgaaca ctgcaaccga actggtgaag attctggagg aagtggtccg gctgagagtc 480 gcagccaact ttagcactcc tgtgatctct accagtgatt cctctaatcc aggccaggag 540 cggacatata cttgcatgtc tagcgga taccccgac ctaatctgta ttggatcaac accacagaca attack tgataccgct ctgcagaaca attack cctgaacaag ctggggctgt atgacgtgat ctctactctg cggctgccat ggaccagtag aggagatgtg 720 ctgtgctgcg tggagaacgt ggccctgcac cagaatatca cctcaattag ccaggctgag tcctttaccg gcaacaatac aaagaatcct caggagacac aatacaatga actgaaagtg ctggtgccag tgctggccgt cctggctgca gcagctttcg tgtcttttat catctacaga900 aggacccgcc ctcaccgctc atacactgga cctaagaccg tgcagctgga actgacagac 960 catgcttga 969 <210> 33 <211> 909 <212> DNA <213> Homo sapiens <400> 33 atgcgtctgg gttcacctgg tctgctgtttt ctgctgtttt caagtctgcg tgctgatact caggagaagg aagtccgggc tatggtcgga agtgacgtgg agctgtcatg cgcttgtccc 120 gaagggtccc ggttcgacct gaacgatgtc tacgtgtatt ggcagacctc tgagagtaag 180 accgtggtca cataccacat ccctcagaac tccagcctgg aaaatgtgga ttcaaggtat 240 cggaacagag ccctgatgtc ccctgctggc atgctgcggg gagacttctc tctgagactg 300 tttaatgtga caccacagga tgagcagaaa ttccattgcc tggtcctgtc acagtccctg 360 ggatttcagg aggtgctgag tgtcgaagtg actctgcacg tcgccgctaa tttctccgtg 420 cctgtggtca gcgcaccaca tagcccctct caggacgagc tgacctttac atgtacttcc 480 atcaacggct acccccgccc taacgtgtac tggattaaca agactgacaa tagcctgctg 540 gatcaggcac tgcagaacga caccgtgttt ctgaatatgc gaggactgta cgatgtggtc 600 agcgtcctgc gtattgccag gaccccatct gtgaacatcg ggtgctgtat tgagaacgtc 660 ctgctgcagc agaatctgac agtggggagc cagactggta atgacatcgg cgagagggat 720 aagattaccg aaaaccccgt gagtacaggc gagaagaacg cagccacatg gtcaatcctg 780 gctgtgctgt gcctgctggt ggtcgtggct gtcgcaattg gctgggtgtg ccgcgatcgg 840 tgtctgcagc actcttatgc cggtgcttgg gcagtgagtc cagagactga actgaccggc 900 catgtctaa 909 <210> 34 <211> 1574 <212> DNA <213> Mus musculus <400> 34 cttaagatgg aaactgatac tctgctgctc tgggtgctgc tcctctgggt gcctggttca 60 actggggaca ttcgacgggc tgacattgtg atgacccaga ccacactgag cctgcccgtg 120 tccctgggcg accaggccag catctcctgc cggagctccc agtctatcgt gcacagcaac 180 ggaaacacat acctggagtg gtatctgcag aagcctggcc agtccccaaa gctgctgatc 240 tacaaggtgt ccaacaggtt cagcggcgtg cctgaccgct tttctggaag cggctccgga 300 acagatttca ccctgaagat cagcagggtg gaggctgagg acctgggcgt gtactactgc 360 ttccagggat cccacgtgcc ttacaccttt ggcggaggca caaagctgga gatcaagaga 420 gccgatgctg ctccaaccgt gtctggaagc ggaggcgggg gttctggagg cggtgggagc 480 ggtggcggag ggtctgaggc taagctgcag gagagcggcc ccgtgctggt gaagcctgga 540 gccagcgtga agatgtcctg taaggcttct ggatacacct tcacagacta ctacatgaac 600 tgggtgaagc agagccacgg caagtccctg gagtggatcg gagtgatcaa cccttacaac 660 ggcgacacct cttacaacca gaagtttaag ggcaaggcca ccctgacagt ggataagtct 720 agctccaccg cttacatgga gctgaacagc ctgacatccg aggattctgc cgtgtactac 780 tgtgctaggt actacggaag ctggttcgcc tactggggcc agggaacact gatcaccgtg 840 tccacagcca agaccacacc ccctagcgtg taccccctgg ctcctaggtc tagcagaggc 900 tgcaagccat gcatctgtac cgtgcccgag gtgagcagcg tgttcatctt tccacccaag 960 cccaaggacg tgctgaccat cacactgacc cctaaggtga catgcgtggt ggtggatatc 1020 agcaaggacg atccagaggt gcagttctcc tggtttgtgg acgatgtgga ggtgcacacc 1080 gcccagacac agccaaggga ggagcagttc aactccacct ttagatccgt gtctgagctg 1140 cccatcatgc accaggactg gctgaacgga aaggagttca agtgccgggt gaactccgcc 1200 gcttttcctg ctccaatcga gaagaccatc tctaagacaa agggccgccc aaaggctcca 1260 caggtgtaca ccatccctcc acccaaggag cagatggcta aggataaggt gagcctgacc 1320 tgtatgatca cagacttctt tcccgaggat atcacagtgg agtggcagtg gaacggacag 1380 cctgccgaga actacaagaa cacccagcca atcatggaca cagatggctc ttacttcgtg 1440 tacagcaagc tgaacgtgca gaagtctaac tgggaggctg gcaacacctt cacctgcagc 1500 gtgctgcacg aaggtctcca taatcaccac accgaaaaga gcctcagtca cagccctggg 1560 aaatgaggcg cgcc 1574 <210> 35 <211> 1484 <212> DNA <213> Homo sapiens <400> 35 cttaagatgg aaactgacac cctgctgctg tgggtcctgc tgctgtgggt gcctggatcc 60 accggcgata tcgtgctgac ccagtctcct ggcacactga gtctgtcacc aggggagcga 120 gcaacactgt cttgtagagc cagccagtct gtgggaagct cctacctggc ttggtatcag 180 cagaagccag gccaggcacc caggctgctg atctacggag ccttcagccg ggccactggc 240 attccagaca ggttctctgg aagtggctca gggaccgact tcaccctgac catcagccga 300 ctggagcccg aagacttcgc cgtgtactat tgccagcagt acggctctag tccttggact 360 tttggacagg gcaccaaagt ggagatcaag cgcggcgggg gaggctctgg gggaggcggg 420 agtggaggcg ggggatcaca ggtccagctg gtggaaagcg gcgggggagt ggtccagcca 480 ggccggagcc tgcggctgag ctgcgccgct tcaggattca cattttcaag ctataccatg 540 cactgggtcc ggcaggcacc agggaaggga ctggagtggg tgaccttcat cagctatgac 600 ggcaacaaca agtattacgc tgattccgtg aaagggaggt ttaccattag ccgcgacaac 660 tccaaaaata cactgtacct gcagatgaac agcctgcggg ccgaggatac tgctatctac 720 tattgcgcaa gaaccgggtg gctgggaccc ttcgactatt ggggccaggg gactctggtc 780 accgtgtcct ctgataagac acacacatgc cctccctgtc ctgcaccaga gctgctgggc 840 gggccatccg tgttcctgtt tccacccaag cctaaagaca ccctgatgat cagccggaca 900 cctgaagtca cttgcgtggt cgtggacgtg agtcacgagg atccagaagt caagtttaac 960 tggtacgtgg atggcgtcga ggtgcataat gccaagacca aacctcgcga ggaacagtac 1020 aatagcacat atcgagtcgt gtccgtcctg actgtgctgc atcaggattg gctgaacggc 1080 aaagagtata agtgcaaagt gagcaataag gcactgcctg ccccaatcga gaaaacaatt 1140 tccaaggcta aaggccagcc cagggaacct caggtgtaca ctctgcctcc aagtcgcgag 1200 gaaatgacca agaaccaggt gagcctgacc tgtctggtga aagggttcta tccatcagac 1260 attgcagtgg agtgggaaag caatggacag cccgaaaaca attacaagac cacaccccct 1320 gtgctggaca gcgatggctc cttctttctg tattctaagc tgactgtgga taaaagtcgc 1380 tggcagcagg ggaacgtctt tagctgttcc gtgatgcatg aggctctgca caatcattac 1440 acacagaagt ctctgagtct gtcacccggc aaatgaggcg cgcc 1484 <210> 36 <211> 632 <212> DNA <213> Artificial Sequence <220> <223> CMV promoter <400> 36 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420 agcggttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480 tttggcacca aaatcaacgg gactttccaa aatgcgtaa caactccgcc ccattgacgc 540 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600 gagaacccac tgcttactgg cttatcgaaa tt 632 <210> 37 <211> 394 <212> DNA <213> Artificial Sequence <220> <223> RSV promoter <400> 37 tgtacgggcc agatatacgc gtatctgagg ggactagggt gtgtttaggc gaaaagcggg 60 gcttcggttg tacgcggtta ggagtcccct caggatatag tagttcgct tttgcatagg 120 gagggggaaa tgtagtctta tgcaatacac ttgtagtctt gcaacatggt aacgatgagt 180 tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg 240 tggtacgatc gtgccttatt aggaaggcaa cagacaggtc tgacatggat tggacgaacc 300 actgaattcc gcattgcaga gataattgta tttaagtgcc tagctcgata caataaacgc 360 catttgacca ttcaccacat tggtgtgcac ctcc 394 <210> 38 <211> 188 <212> DNA <213> Artificial Sequence <220> <223> BGH polyA <400> 38 ctgtgccttc tagttgccag ccatctgttg tttgccccctc cccgtgcct tccttgaccc 60 tggaaggtgc cactcccact gtccttttcct aataaaatga ggaaattgca tcgcattgtc 120 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180 gggaagac 188 <210> 39 <211> 249 <212> DNA <213> Artificial Sequence <220> <223> SV40 late polyA <400> 39 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 60 tgctttatt gtgaaatttg tgatgctatt gctttatttg tgaaatttgt gatgctattg 120 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt 180 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca 240 aatgtggta 249 <210> 40 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> SV40 enhancer promoter <400> 40 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag 60 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc 120 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct 180 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 240 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa 300 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagct 345 <210> 41 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Rabbit beta-globin polyA <400> 41 gacctctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg 60 tctctcactc ggaaggacat atgggagggc aaatcattt 99 <210> 42 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> GFP <400> 42 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 60 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 120 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 180 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 240 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 300 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 360 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 420 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag 480 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc 540 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 600 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg 660 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 720 city ​​723 <210> 43 <211> 454 <212> DNA <213> Artificial Sequence <220> <223> MoMuLV LTR <400> 43 ttaattaagt aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagagaagt 60 tcagatcaag gtcaggaaca gatggacag ctgaatatgg gccaaacagg atatctgtgg 120 taagcagttc ctgccccggc tcaggccaa gaacagatgg aacagctgaa tatgggccaa 180 acaggatatc tgtgtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc 240 agatgcggtc cagccctcag cagttcttag agaaccatca gatgttttcca gggtgcccca 300 aggacctgaa atgaccctgt gccttattg aactaaccaa tcagttcgct tctcgctct 360 gttcgcgcgc ttctgctccc cgagctcaat aaagagccc acaacccctc actcggggcg 420 ccagtcctcc gattgactga gtcgcccgct tag 454 <210> 44 <211> 1349 <212> DNA <213> Artificial Sequence <220> <223> The EF1alpha promoter <400> 44 tttaataga gtaattcata CAAAggact cgcccctgcc ttggggaatc ccagggaccg 60 tcgttaaact cccactaacg tagaacccag agatcgctgc gttcccgccc cctcacccgc 120 ccgctctcgt catcactgag gtggagaaga gcatgcgtga ggctccggtg cccgtcagtg 180 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac 240 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg 300 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct 360 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc 420 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacgc ccctggctgc 480 agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt 540 gcggttaagg agccccttcg cctcgtgctt gagttgaggc ctggcttggg cgctggggcc 600 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag 660 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa 720 atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg 780 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa 840 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt 900 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa 960 gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag 1020 agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt 1080 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt 1140 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg 1200 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg 1260 cccttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagtttttt 1320 ttcttccatt tcaggtgtcg tgacttaag 1349 <210> 45 <211> 481 <212> DNA <213> Artificial Sequence <220> <223> HGH polyA <400> 45 gacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact 60 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg 120 tccttctata atattatggg gtggagggg gtggtatgga gcaaggggca agttgggaag 180 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct 240 tggctcactg caatctccgc ctcctgggtt caagcgattc tctgcctca gcctcccgag 300 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga 360 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca 420 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt 480 t 481

Claims

[Claim 1] The invention as described herein and / or in the drawings.

Images