Viral vectors for treating cancers refractory to immune checkpoint inhibitors

Abstract

The present invention generally relates to the use of replication-deficient HSV-1 vectors and one or more genes encoding recombinant proteins that stimulate immune destruction of tumors, for treating cancer in immune checkpoint inhibitor (ICI) refractory subjects.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests and priority of U.S. Provisional Patent Application No. 63 / 544,734 filed on 18 October 2023, U.S. Provisional Patent Application No. 63 / 574,364 filed on 4 April 2024, and U.S. Provisional Patent Application No. 63 / 700,851 filed on 30 September 2024, the disclosures of each of these applications being incorporated herein by reference in their entirety for all purposes.

[0002] This disclosure relates to a method for treating cancer in immune checkpoint inhibitor-refractory subjects using a replication-deficient HSV-1 vector that delivers one or more genes encoding recombinant proteins that stimulate tumor immune destruction. [Background technology]

[0003] The advancement of immuno-oncology has provided new therapies for treating cancers that have previously evaded successful treatment due to the evolution of mechanisms by which cancer cells have evaded the target immune system. Despite significant progress in the treatment of various cancers with immunotherapy, such as immune checkpoint inhibitors (ICIs) and CAR T-cell therapy, a considerable number of patients remain unresponsive to immunotherapy. For example, 40–60% of cutaneous melanoma patients achieve no significant therapeutic effect with ICIs, and a significant proportion of those who respond experience tumor recurrence within two years of treatment. Furthermore, treating solid tumors with immuno-oncological therapies can be more challenging due to the complexity of the tumor microenvironment (TME) of solid tumors, which includes epithelial cells, endothelial cells, mesenchymal cells, stromal cells, cancer cells, and immune cells. In certain solid tumors, cancer cells influence the activity or inactivation of immune cells in the TME. Viral vectors have been developed to deliver therapeutic drugs to cells. However, despite the advances made to date, there remains a need for methods and compositions that can be used alone or in combination with other cancer drugs to treat immunotherapy-refractory patients. [Overview of the project]

[0004] This invention is partly based on the discovery that oncolytic HSV-1 vectors may be useful in the treatment of cancer in subjects refractory to immune checkpoint inhibitors (ICIs) that require them. In particular, such treatment may reduce tumor size. The HSV-1 vectors described herein are intended to be useful in the treatment of cancer in ICI-refractory patients, for example, based on their ability to induce tertiary lymphoid structures (TLS) in the tumor bed and activate T cell and B cell survival and proliferation, and alternatively or additionally, by activating pathways that support the ICI response. Furthermore, the vectors can induce delayed tumor breakdown, enable sustained expression of therapeutic payloads (e.g., therapeutic polypeptides), exhibit increased immunogenicity, and increase immune activity against tumor cells. The therapeutic payloads may include, for example, factors known to regulate TLS.

[0005] The present invention is also, in part, based on the discovery that specific combinations of therapeutic polypeptides can be used to disrupt one or more pathways (e.g., parallel pathways) that affect the ability of immune cells to kill cancer cells, thereby reducing tumor size. For example, therapeutic polypeptides that target the tumor stroma or support the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment, induce TLS in the tumor bed, promote phagocytic innate immune surveillance, disrupt the Sirpα / CD47 axis, promote the upmodulation or downmodulation of one or more proteins related to the response to ICI, and / or reduce the viability of cancer cells can be expressed from viral vectors, e.g., viral vectors lacking functional ICP4 and functional ICP47 proteins, thereby enhancing the ability of immune cells to kill tumor cells and, for example, treating ICI-refractory target cancers. A vector may be designed to contain a specific combination of therapeutic polypeptides effective in treating a given type of tumor, such as selecting one or more therapeutic polypeptides that reduce inhibitory features of certain tumor microenvironments that may prevent the immune system from effectively attacking the tumor.

[0006] As a result, in one embodiment, the present disclosure relates to a method for treating cancer in ICI-refractory subjects, the method comprising administering a vector such as a herpes simplex virus (HSV) genome, wherein the HSV genome includes one or more modifications that interfere with the expression of functional ICP4 protein and / or functional ICP47 protein.

[0007] In another embodiment, the Disclosure relates to a method for expressing a therapeutic polypeptide in an ICI-refractory subject, the method comprising administering a vector containing an HSV genome to an ICI-refractory subject, wherein the HSV genome includes one or more modifications that interfere with the expression of functional ICP4 protein and / or functional ICP47 protein.

[0008] In another embodiment, the disclosure relates to a method for reducing tumor size in an ICI-refractory subject, the method comprising administering a vector comprising an ICI-refractory subject, wherein the HSV genome comprises one or more modifications that interfere with the expression of functional ICP4 protein and / or functional ICP47 protein, and the vector comprises a nucleic acid encoding a therapeutic polypeptide that functions to reduce tumor size.

[0009] In certain embodiments, the HSV-1 genome includes one or more modifications that inhibit the expression of two ICP4 proteins. In certain embodiments, the HSV genome includes one or more modifications that inhibit the expression of two ICP4 proteins and one ICP47 protein. In certain embodiments, the two ICP4 proteins are characterized as having the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4 (ICP4). In certain embodiments, the ICP47 protein is characterized as having the amino acid sequence of SEQ ID NO: 5 (ICP47).

[0010] In certain embodiments, the HSV genome is the HSV-1 genome.

[0011] In certain embodiments, the HSV-1 genome is the McKrae genome.

[0012] In certain embodiments, when administered to a subject, the vector results in (a) delayed tumor breakdown; (b) increased immunogenicity; (c) increased immune activation; (d) formation of TLS in the tumor bed; (e) upmodulation or downmodulation of one or more proteins related to the response to ICI; (f) decreased cancer cell viability; and (g) one or more combinations of any one or more of (a) to (f).

[0013] In certain embodiments, the vector comprises nucleic acids encoding one or more therapeutic polypeptides, wherein the one or more therapeutic polypeptides: (a) target the tumor stroma; (b) support the survival of T cells in the tumor microenvironment; (c) induce TLS in the tumor bed; (d) promote phagocytic innate immune surveillance (e.g., disrupting the Sirpα / CD47 axis); (e) promote the upmodulation or downmodulation of one or more proteins related to the response to ICI; (f) reduce the viability of cancer cells; or (g) perform any one or more combination of (a) to (f).

[0014] In certain embodiments, one or more therapeutic polypeptides target the tumor stroma by degrading extracellular matrix proteins. Examples of such therapeutic polypeptides that target the tumor stroma by degrading extracellular matrix proteins include MMP-9 or lysyl oxidase inhibitors.

[0015] In certain embodiments, one or more of the therapeutic polypeptides target the tumor stroma by activating local endothelium to increase T cell infiltration. Examples of such therapeutic polypeptides that target the tumor stroma by activating local endothelium to increase T cell infiltration include TNF, IL-1β, IL-6, or IL-18.

[0016] In certain embodiments, one or more therapeutic polypeptides support T cell survival in the tumor microenvironment by enhancing T cell recruitment to the tumor site. Examples of such therapeutic polypeptides that support T cell survival in the tumor microenvironment by enhancing T cell recruitment to the tumor site include CCL19 or CCL21.

[0017] In certain embodiments, the one or more therapeutic polypeptides described above support the survival of T cells in the tumor microenvironment by supporting T cell function. Examples of such therapeutic polypeptides that support the survival of T cells in the tumor microenvironment by supporting T cell function may include IL-7, IL-12, IL-15, IL-18, or IFN-γ.

[0018] In certain embodiments, the T cells are CAR T cells. Examples of such therapeutic polypeptides that support the survival of CAR T cells in the tumor microenvironment by supporting CAR T cell function may include soluble TGFβRII. Examples of such therapeutic polypeptides that support the survival of CAR T cells in the tumor microenvironment by supporting CAR T cell function may also include antigens recognized by CAR T cells, such as mesothelin.

[0019] In certain embodiments, the one or more therapeutic polypeptides described above include co-stimulatory molecules such as CD40L and OX40L.

[0020] In certain embodiments, the one or more therapeutic polypeptides described above induce TLS in the tumor bed. Examples of such therapeutic polypeptides that induce TLS in the tumor bed may include CCL19, lymphotoxin β, CXCL13, or TNF.

[0021] In certain embodiments, the one or more therapeutic polypeptides described above promote phagocytic innate immune surveillance. In certain embodiments, such therapeutic polypeptides that promote phagocytic innate immune surveillance disrupt the Sirpα / CD47 axis, such as a Sirpα-IgG fusion transgene or an anti-CD47 antibody.

[0022] In certain embodiments, the one or more therapeutic polypeptides described above support the polarization of anti-tumor macrophages. Examples of such therapeutic polypeptides that support anti-tumor macrophage polarization may include TNF, IL-1, IL-12, IL-17, or IFN-γ.

[0023] In certain embodiments, the one or more therapeutic polypeptides described above include hyaluronidase, MMP-9, inhibitors of lysyl oxidase, TNF, IL-1β, IL-6, IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesothelin, CD40L, OX40L, lymphotoxin β, CXCL13, Sirpα-IgG fusion transgene, anti-CD47 antibody, IL-1, or IL-17.

[0024] In certain embodiments, the one or more therapeutic polypeptides described above include IL-21, LTA, LTB, CXCL12, or VEGF-C.

[0025] In certain embodiments, the one or more therapeutic polypeptides described above include hyaluronidase, MMP-9, inhibitors of lysyl oxidase, TNF, IL-1β, IL-6, IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesothelin, CD40L, OX40L, lymphotoxin β, CXCL13, Sirpα-IgG fusion transgene, anti-CD47 antibody, IL-1, IL-17, IL-21, LTA, LTB, CXCL12, or VEGF-C.

[0026] In certain embodiments, examples of the one or more therapeutic polypeptides include: a) LTA, CCL19, TNF, IL-17, and IL-7; b) LTB, CCL19, CCL21, CXCL13, and IL-7; c) IFN-γ and IL-12; d) IFN-γ and IL-15; e) IL-12 and IL-15; f) IL-15 and IL-21; g) IFN-γ, IL-12, and IL-15; h) IFN-γ, IL-12, IL-15, and IL-21; or i) CCL19, IL-7, TNF, and IL-17.

[0027] In certain embodiments, the method further includes administering an ICI.

[0028] In certain embodiments, the ICI is selected from PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, and LAG-3 inhibitors.

[0029] In certain embodiments, the ICI may include anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab, or semiprimab); PD-L1 inhibitors (e.g., atezolizumab, avelumab, or durvalumab); CTLA-4 inhibitors (e.g., ipilimumab or tremelimumab); or PD-1 inhibitors such as LAG-3 inhibitors (e.g., relatlimab).

[0030] In certain embodiments, the method includes administering a plurality of ICIs selected from the group consisting of PD-1 inhibitors such as anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab, or semipirimumab) and CTLA-4 inhibitors (e.g., ipilimumab or tremelimumab).

[0031] In certain embodiments, one or more proteins associated with the response to the above-mentioned ICI include IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21.

[0032] In certain embodiments, inducing TLS in the tumor bed includes promoting the number, size, and / or organization of TLS structures.

[0033] In another embodiment, the present disclosure relates to a method for treating cancer in a subject requiring it, the method comprising administering a vector comprising an HSV genome, wherein the HSV genome comprises one or more modifications that interfere with the expression of functional ICP4 protein and / or functional ICP47 protein, expressing effective amounts of IL-12 and IL-15, thereby treating cancer.

[0034] In certain embodiments, the method further includes administering an ICI. Examples of ICIs include PD-1 inhibitors such as anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab, or semipirimab); PD-L1 inhibitors (e.g., atezolizumab, avelumab, or durvalumab); CTLA-4 inhibitors (e.g., ipilimumab or tremelimumab); or LAG-3 inhibitors (e.g., relatlimab).

[0035] In certain embodiments, the method includes administering a plurality of ICIs selected from the group consisting of PD-1 inhibitors such as anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab, or semipirimumab) and CTLA-4 inhibitors (e.g., ipilimumab or tremelimumab).

[0036] In certain embodiments, administration of a vector containing a herpes simplex virus (HSV) genome expressing IL-12 and IL-15 results in synergistic tumor regression compared to administration of a vector containing a herpes simplex virus (HSV) genome expressing (a) IL-12 but not IL-15, or (b) IL-15 only and not IL-12 (e.g., an otherwise identical vector).

[0037] These and other aspects and features of the present invention are described in the following detailed description and claims.

[0038] The foregoing and other purposes, features, and advantages of this disclosure will become apparent from the following description of preferred embodiments illustrated in the accompanying drawings. [Brief explanation of the drawing]

[0039] [Figure 1] This bar graph shows the effect of infection with a variant of the herpes simplex virus (HSV) type 1 (HSV-1) strain vector ("HSV-1 mICP4 / mICP47IFNγ") expressing an interferon-γ (IFN-γ) payload and containing mutant ICP4 and mutant ICP47 proteins ("HSV-1 mICP4 / mICP47") in breast cancer-derived Hs578 cells on the doubling rate of expression levels of genes involved in IFN-γ response characteristics, such as ISG15, IRF9, CCL2, and CXCL10, compared to an HSV-1 mICP4 / mICP47 green fluorescent protein (GFP) vector or an uninfected control group. *p<0.05, ****p<0.0001. [Figure 2] This bar graph shows the luminescence of breast cancer-derived Hs578 cells infected with HSV-1 mICP4 / mICP47 IFN-γ vectors at 0, 3, and 10 plaque-forming units (PFUs) per cell. Lower luminescence indicates higher tumor-lysing activity. [Figure 3] This is a series of bar graphs showing the quantification of mouse (m)CXCL13, mCCL19, or mIL-7 payload production in a prepared medium of breast cancer-derived Hs578T cells infected with 3 PFU / cell or 10 PFU / cell HSV-1 strain vectors expressing each payload and containing mutant ICP4 and mutant ICP47 proteins in their HSV-1 genome. [Figure 4] Figure 3 shows a bar graph illustrating the normalized luminescence of breast cancer-derived Hs578 T cells 6 days after infection with the HSV-1 vector. Lower luminescence indicates higher tumor-lysing activity. [Figure 5]This schematic diagram illustrates the activation of innate immune surveillance of cancer cells by macrophages via interference with the SIRPα / CD47 pathway by the HSV-1 mICP4 / mICP47 vector, which encodes the extracellular domain of SIRPα and the Fc fragment of the human IgG1 ("macro1") payload. [Figure 6] These are a series of flow cytograms representing the amount of phagocytosis measured by CD14 and carboxyfluorothene succinimimidyl ester (CFSE)-positive cells in lymphoma-derived Raji cells cultured in a modified medium (CM) derived from breast cancer-derived Hs578 T cells. (A) When treated with isotype control (IgG). (B) When treated with anti-CD47 antibody. (C) When infected with 3 PFU / cell control HSV-1 mICP4 / mICP47 GFP vector before co-culturing with M1 macrophages for 3 hours. (D) When infected with 3 PFU / cell HSV-1 mICP4 / mICP47 macro1 vector (encoding the extracellular domain of SIRPα and the Fc fragment of human IgG1) before co-culturing with M1 macrophages for 3 hours. [Figure 7] These bar graphs, obtained from the experiments described in Figures 6A-6D, show the percentage of phagocytosis in lymphoma-derived Raji cells. N=3; p<0.05. [Figure 8] This is a schematic diagram of the experimental administration regimen. Here, Balb / c nude mice were subcutaneously inoculated with 5 × 10⁶ lung cancer-derived A549 cells in the posterior ventral region. After the A549-derived tumor reached approximately 100 mm³, the mice were treated intratumorally (it) on days 1, 4, 7, 10, and 13 with 3 × 10⁷ PFU of HSV-1 mICP4 / mICP47 macro1 vector, or intraperitoneally (ip) with 10 mg / kg of anti-CD47 control antibody. [Figure 9]Figure 8 shows graphs illustrating the effect of treatment on tumor volume in Balb / c nude mice as described in the experiment. (A) Graphs showing tumor volume in mice infected with the control vector, HSV-1 mICP4 / mICP47 GFP vector, or HSV-1 mICP4 / mICP47 macro1 vector. (B) Graphs showing tumor volume in mice treated with the control IgG isotype control antibody or anti-CD47 antibody. [Figure 10] This heatmap shows computer-predicted statistical associations between gene expression from RNA-seq in tumor microenvironment (TME) ICI-treated samples (rows) and TME viability in the target population. Scores were calculated by combining the p-values ​​of TME vs. viability and TME vs. payload as -log10 meta-p-values ​​to generate "scores" for identifying potential payloads (columns) of the HSV-1 mICP4 / mICP47 vector described herein. The exemplary payload IFN-γ is highlighted with a black vertical rectangle and was present across the entire patient population (rows) in the analysis. [Figure 11] These graphs show a comparison of the top pathways identified in Gene Set Enrichment Analysis (GSEA) of RNA-seq data. (A) This graph shows the top pathways identified from stable / progressive (SD / PD) or partially / completely resolving (PR / CR) subjects in the SU2C-MARK cohort treated with immune checkpoint inhibitors (ICIs). (B) This graph shows the top pathways identified from cells infected with HSV-1 mICP4 / mICP47 for 72 hours. [Figure 12] Figures 11A-11B show the correlations of all pathways identified in the GSEA experiment in cells infected with HSV-1mICP4 / mICP47, compared to those treated with ICI. Rank: NES*(-log10(p-value)). [Figure 13]This graph shows the tumor size (as volume) over time in Balb / c mice treated with a medium control, a control antibody (10 mg / kg), an HSV-1 mICP4 / mICP47 GFP vector (3 × 10⁷ PFU), and the anti-PD-1 antibody clone RMP1-14 (10 mg / kg), followed by injection of 5 × 10⁵ mammary gland cancer EMT6 cells into the posterior ventral region. N=5-8 mice / group. [Figure 14] Graphs A and B show the therapeutic effect on tumor volume in Balb / c nude mice that were subcutaneously inoculated with mammary gland cancer EMT6 cells in the posterior ventral region. When EMT6-derived tumors reached approximately 100 mm3, mice were infected on days 1, 4, 7, and 10 with either 3 × 10⁷ control HSV-1 mICP4 / mICP47 GFP vectors, five HSV-1 mICP4 / mICP47 vectors expressing mCCL19, mL-7, mTNF, mL-17a, and GFP respectively (6 × 10⁶ PFU per vector; total dose 3 × 10⁷ PFU; "Multiplex A"), or five HSV-1 mICP4 / mICP47 vectors expressing mCCL19, mL-7, mCCL21, mCXCL13, and mLTB respectively (6 × 10⁶ PFU per vector, total dose 3 × 10⁷ PFU; "Multiplex B") (A), and tumor volume was measured (B). N=8 / group, *p<0.05, **p<0.01. [Figure 15] This bar graph shows the percentage of CD45-positive cells in the submandibular gland of mice transduced with the control medium or 3 × 10⁷ PFU of HSV-1 mICP4 / mICP47 GFP. [Figure 16]control HSV-1 mICP4 / mICP47 GFP vector (“GFP”), HSV-1 mICP4 / mICP47, uninfected or in vitro at 1 PFU / cell each; IFN-γ vector, or mIL-12 (“A”), IL-15 (“B”), IL-21 (“C”), CD40L (“D”), IFN-γ+IL-12 (“E”), IFN-γ+IL-15 (“F”), IL- HSV-1 encoding 12+IL-15 (“G”), IL-15+IL-21 (“H”), IFN-γ+IL-12+IL-15 (“I”), or IFN-γ+IL-12+IL-15+IL-21 (“J”) This bar graph shows the effect of treatment on the number of live breast cancer-derived Hs578T tumor cells infected with the mICP4 / mICP47 vector and co-cultured with peripheral blood mononuclear cells (PBMCs) for 72 hours ("+T cell activator"; groups A-J were co-cultured). In the experimental groups where IL-15 or IL-21 was present in the payload, recombinant proteins were provided, as well as GFP of HSV-1 mICP4 / mICP47, rather than HSV-1 mICP4 / mICP47 encoding IL-15 and / or IL-21, respectively. [Figure 17] This graph shows the Lasso regression analysis of immune cell populations that show a positive correlation (to the right of 0.0 on the x-axis) or a negative correlation (to the left of 0.0 on the x-axis) with PBMC-mediated tumor cell death of breast cancer-derived Hs578T tumor cells, as described in the experiment in Figure 16. [Figure 18] This graph shows the change in tumor volume over time in mice treated with a medium control; five HSV-1 mICP4 / mICP47 vectors expressing mCCL19, mIL-7, mTNF, mIL-17a, and GFP respectively (6 × 10⁶ PFU each; total dose 3 × 10⁷ PFU; "Multiplex A"); or five HSV-1 mICP4 / mICP47 vectors expressing mCCL19, mIL-7, mCCL21, mCXCL13, and mLTB respectively (6 × 10⁶ PFU each; total dose 3 × 10⁷ PFU; "Multiplex B"); and the anti-PD-1 antibody clone RMP1-14 (10 mg / kg), after which 5 × 10⁵ mammary carcinoma EMT6 cells were injected into the postventral region. [Figure 19] This graph shows the number of MHC class I (MHC-I) positive breast cancer-derived Hs578 T cells, measured by flow cytometry 24 hours after infection, after infection with either a control vector or HSV-1 mICP4 / mICP47 GFP at a rate of 10 PFU / cell. [Figure 20] This graph shows gene set enrichment analysis performed on RNA-seq data, illustrating the higher-level pathways enriched in cells infected with HSV-1 mICP4 / mICP47. [Figure 21] This is a series of bar graphs showing the quantification of interferon-gamma (IFN-γ) and IL-12 payload production in a prepared medium of breast cancer-derived Hs578 T cells infected with HSV-1 mICP4 / mICP47 expressing 3 PFU / cell or 10 PFU / cell respectively. [Figure 22A] Uninfected or in vitro control HSV-1 mICP4 / mICP47 GFP vector ("GFP"), HSV-1 mICP4 / mICP47 IFN-γ vector ("IFN-γ"), HSV-1 mICP4 / mICP47 IL-12 vector ("IL-12"), HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-15 ("IL-15"), HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-21 ("IL-21"), or HSV-1 encoding IFN-γ or IL-12. This graph shows the cytotoxic effects of breast cancer-derived Hs578T tumor cells infected with a combination of mICP4 / mICP47 vectors ("D1"), GFP or IFN-γ and recombinant human IL-15 ("D2"), GFP or IL-12 and recombinant human IL-15 ("D3"), GFP, IFN-γ, or IL-12 and recombinant human IL-15 ("T"), or GFP, IFN-γ, or IL-12 and recombinant human IL-15 and IL-21 ("Q"), or co-cultured with PBMCs ("activators"). Analysis was performed by flow cytometry. [Figure 22B]Uninfected or in vitro control HSV-1 mICP4 / mICP47 GFP vector ("GFP"), HSV-1 mICP4 / mICP47 IFN-γ vector ("IFN-γ"), HSV-1 mICP4 / mICP47 IL-12 vector ("IL-12"), HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-15 ("IL-15"), HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-21 ("IL-21"), or HSV-1 encoding IFN-γ or IL-12. This heatmap shows the cytotoxic effects of breast cancer-derived Hs578T tumor cells infected with or co-cultured with PBMCs ("activators"), using combinations of mICP4 / mICP47 vectors ("D1"); GFP or IFN-γ and recombinant human IL-15 ("D2"); GFP or IL-12 and recombinant human IL-15 ("D3"); GFP, IFN-γ, or IL-12 and recombinant human IL-15 ("T"); or GFP, IFN-γ, or IL-12 and recombinant human IL-15 and IL-21 ("Q"). Analysis was performed using standard least squares (OLS) regression. [Figure 23]Control PBMCs, or uninfected ("Uninf."), or control HSV-1 mICP4 / mICP47 GFP vector ("GFP"); HSV-1 mICP4 / mICP47 IL-12 vector ("IL-12"); HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-21 ("IL-21"); HSV-1 mICP4 / mICP47 IFN-γ vector ("IFN-γ"); combination of HSV-1 mICP4 / mICP47 vector encoding IFN-γ or IL-12 ("D1"); HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-15 ("IL-15"); or HSV-1 encoding GFP or IFN-γ This heatmap shows the composition measured by flow cytometry of PBMCs (Planetary Biomedical Cells) co-cultured with Hs578 T cells, or infected with mICP4 / mICP47 vectors, and recombinant human IL-15 ("D2"), recombinant human IL-12 and recombinant human IL-15 ("T"), GFP or IL-12 and recombinant human IL-15 ("D3"), or GFP, IFN-γ, or IL-12 and recombinant human IL-15 and IL-21 ("Q"), or co-cultured with PBMCs ("Activ."). [Figure 24] This graph shows the Lasso regression analysis of immune cell populations, indicating a positive correlation (to the right of 0.0 on the x-axis) or a negative correlation (to the left of 0.0 on the x-axis) with PBMC-mediated tumor cell death of breast cancer-derived Hs578T tumor cells, as described in the experiment in Figure 23. [Figure 25]Control PBMCs, or uninfected PBMCs, or in vitro, with control HSV-1 mICP4 / mICP47 GFP vector ("GFP"); HSV-1 mICP4 / mICP47 IFN-γ vector ("IFN-γ"); HSV-1 mICP4 / mICP47 IL-12 vector ("IL-12"); HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-15 ("IL-15"); HSV-1 mICP4 / mICP47 GFP vector and recombinant human IL-21 ("IL-21"); or HSV-1 encoding IFN-γ or IL-12. This is a series of graphs showing the total number of dendritic cells (DCs) and Ki67-positive / Gzmb-positive CD8-positive T cells in tumor cells infected with or co-cultured with PBMCs ("activators"), either with a combination of mICP4 / mICP47 vectors ("D1"), GFP or IFN-γ and recombinant human IL-15 ("D2"), GFP or IL-12 and recombinant human IL-15 ("D3"), GFP, IFN-γ, or IL-12 and recombinant human IL-15 and IL-21 ("Q"). [Figure 26A] This graph shows the volume of EMT6 tumor growth in Balb / c mice that were injected with 5 × 10⁵ mammary carcinoma EMT6 cells in the posterior ventral region and infected with 3 × 10⁷ PFU of a control HSV-1 mICP4 / mICP47 GFP vector, an HSV-1 mICP4 / mICP47 IL-15 vector, an HSV-1 mICP4 / mICP47 IL-12 vector, or a combination of HSV-1 mICP4 / mICP47 vectors encoding IL-15 or IL-12. [Figure 26B] This graph shows the volume of EMT6 tumor growth in individual Balb / c mice that were injected with 5 × 10⁵ mammary carcinoma EMT6 cells into the posterior ventral region and infected with 3 × 10⁷ PFU of a combination of HSV-1 mICP4 / mICP47 vectors encoding either IL-15 or IL-12. [Figure 26C]This graph shows the weight of EMT6 tumors in Balb / c mice that were injected with 5 × 10⁵ mammary carcinoma EMT6 cells in the posterior ventral region and infected with 3 × 10⁷ PFU of a control HSV-1 mICP4 / mICP47 GFP vector, an HSV-1 mICP4 / mICP47 IL-15 vector, an HSV-1 mICP4 / mICP47 IL-12 vector, or a combination of HSV-1 mICP4 / mICP47 vectors encoding IL-15 or IL-12. [Figure 26D] This graph shows the weight of LLC1 tumors in Balb / c mice that were injected with 5 × 10⁵ tumor-genic LLC1 cells in the posterior ventral region and infected with 3 × 10⁷ PFU of a control HSV-1 mICP4 / mICP47 GFP vector, an HSV-1 mICP4 / mICP47 IL-15 vector, an HSV-1 mICP4 / mICP47 IL-12 vector, or a combination of HSV-1 mICP4 / mICP47 vectors encoding IL-15 or IL-12. [Figure 27A] This is a series of graphs showing the percentages of Ki67-positive CD8-positive T cells and PD1-positive CD8-positive T cells (right and left panels, respectively) observed in PBMCs of mice infected with a control HSV-1 mICP4 / mICP47 GFP vector, an HSV-1 mICP4 / mICP47 IL-15 vector, an HSV-1 mICP4 / mICP47 IL-12 vector, or a combination of HSV-1 mICP4 / mICP47 vectors encoding IL-15 or IL-12, as measured by flow cytometry, in PBMCs of mice injected with mammary carcinoma EMT6 cells in the posterior ventral region. [Figure 27B] This graph shows the percentage of Ki67-positive CD8-positive T cells, as measured by flow cytometry, observed in tumors of mice infected with either a control HSV-1 mICP4 / mICP47 GFP vector, an HSV-1 mICP4 / mICP47 IL-15 vector, an HSV-1 mICP4 / mICP47 IL-12 vector, or a combination of HSV-1 mICP4 / mICP47 vectors encoding IL-15 or IL-12, after injecting tumor-genic LLC1 cells into the posterior ventral region. [Modes for carrying out the invention]

[0040] This invention is partly based on the discovery that oncolytic HSV-1 vectors may be useful in the treatment of cancer in subjects refractory to immune checkpoint inhibitors (ICIs) that require them. Such treatment may, in particular, reduce tumor size. The HSV-1 vectors described herein are intended to be useful in the treatment of cancer in ICI-refractory patients, for example, by inducing tertiary lymphoid structures (TLSs) in the tumor bed and activating T cell and B cell survival and proliferation, and by substituted or additionally activating pathways that support the ICI response. Furthermore, the vectors may induce delayed tumor lysis, release tumor-associated or tumor-specific antigens, enable sustained expression of therapeutic payloads (e.g., therapeutic polypeptides), exhibit increased immunogenicity, and increase immune activity against tumor cells. Therapeutic payloads may include, for example, factors known to control TLS formation in the tumor microenvironment or increase lymphocyte migration in tumors.

[0041] The present invention is also, in part, based on the discovery that specific combinations of therapeutic polypeptides can be used to disrupt one or more pathways (e.g., parallel pathways) that affect the ability of immune cells to kill cancer cells, thereby increasing cytotoxic activity and reducing tumor size. For example, therapeutic polypeptides that target the tumor stroma or support the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment, induce TLS in the tumor bed, promote phagocytic innate immune surveillance, disrupt the Sirpα / CD47 axis, promote the upmodulation or downmodulation of one or more proteins related to the response to ICI, and / or reduce the viability of cancer cells can be expressed from viral vectors, e.g., viral vectors lacking functional ICP4 and functional ICP47 proteins, thereby enhancing the ability of immune cells to kill tumor cells and, for example, treating ICI-refractory target cancers. A vector may be designed to contain a specific combination of therapeutic polypeptides effective in treating a given type of tumor, such as selecting one or more therapeutic polypeptides that reduce inhibitory features of certain tumor microenvironments that may prevent the immune system from effectively attacking the tumor.

[0042] I. Definition To facilitate understanding of this disclosure, many terms and phrases are defined below. Unless otherwise specified, all technical and scientific terms have the same meaning as they are generally understood by those skilled in the art in which the disclosed embodiments belong.

[0043] As used herein, the terms "a" and "an" mean "one or more" and include the plural form unless the context is appropriate.

[0044] As used herein, the term “agent” refers to a compound or entity of any chemical classification, such as polypeptides, nucleic acids, sugars, lipids, small molecules, or combinations thereof. In some embodiments, an agent is a natural product or includes such a natural product, in that it is found in nature and / or obtained from nature. In some embodiments, an agent is an entity or includes one or more entities that are artifacts, in that they are designed, manipulated and / or produced by human action and / or are not found in nature. Some specific embodiments of agents that may be utilized according to the present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNA, shRNA, DNA / RNA hybrids, antisense oligonucleotides, and ribozymes), peptides, peptide mimes, and the like.

[0045] The terms “about” and “approximately” indicate that a value includes inherent variations in error that exist between the apparatus, the method employed to determine the value, or the test subjects. In one non-limiting embodiment, the term is defined as within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%. When a value is expressed as an approximation, the use of the antecedent “about” is understood to mean that a particular value forms another aspect. Furthermore, it is understood that there are numerous values ​​disclosed in this disclosure, and each value is disclosed as “about” not only the value itself but also its specific value.

[0046] As used herein, the term “combination therapy” refers to a situation in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, two or more drugs may be administered concurrently, sequentially, or in a duplicate-dosing regimen.

[0047] As used herein, “expression” of a nucleic acid sequence means one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end formation); (3) translation of RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0048] As used herein, the terms “immune checkpoint inhibitor refractory subjects” or “ICI refractory subjects” refer to subjects who have received one or more ICI treatments and have not responded to or were unable to continue responding to one or more ICI treatments. Subjects who are considered unable to continue responding to ICI treatments may initially respond to ICI treatments for a period of time and then become unresponsive to ICIs. The one or more ICI treatments may include any dose or administration regimen of ICIs. Exemplary ICIs include PD-1 inhibitors (e.g., anti-PD-1 antibodies, e.g., pembrolizumab, nivolumab, or semipirimab), PD-L1 inhibitors (e.g., atezolizumab, avelumab, or durvalumab), CTLA-4 inhibitors (e.g., ipilimumab or tremelimumab), and LAG-3 inhibitors (e.g., Relatlimab).

[0049] As used herein, the terms “marker” or “marker protein” refer to a detectable or selectable agent. In some embodiments, the “marker” or “marker protein” is a detectable or selectable nucleic acid sequence. In some embodiments, the “marker” or “marker protein” is an expression product (e.g., RNA or protein) whose presence or absence is detectable and / or selectable in a cell. In some embodiments, the expression product is or comprises an enzyme. In some embodiments, the expression product is a fluorescent substance.

[0050] As used herein, the terms “subject” and “patient” refer to an organism treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, apes, horses, cattle, pigs, dogs, cats, etc.), and more preferably include humans.

[0051] When used herein, the term “pharmaceutically acceptable” applied to carriers, diluents, or excipients used to formulate compositions such as those disclosed herein means that the carrier, diluent, or excipient must be miscible with other components of the composition and must not be highly toxic to its recipient.

[0052] In this specification, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent encapsulation material, that is involved in the transport or delivery of a compound of interest from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is miscible with other components of the formulation and does not cause harm to the patient. Examples of substances that can function as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffers; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0053] As used in this disclosure, the terms “treat,” “treating,” or “treatment,” and other grammatical equivalents, are intended to include, and may include, reducing, improving, or preventing a disease, condition, or symptom; preventing further symptoms; improving or preventing underlying metabolic causes of symptoms; inhibiting a disease or condition, for example, preventing the onset of a disease or condition; mitigating a disease or condition; causing regression of a disease or condition; mitigating a condition caused by a disease or condition; or stopping symptoms of a disease or condition. The terms further include achieving therapeutic benefits and / or preventive benefits. The term “therapeutic benefit” means eradicating or improving an underlying disease being treated. A therapeutic benefit is also achieved by eradicating or improving one or more physiological symptoms associated with an underlying disease, such that improvement is observed in the patient, even though the patient may still have the underlying disease.

[0054] As used in this disclosure, the term “vector” refers to a nucleic acid molecule capable of transporting another ligated nucleic acid. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA ring, to which further DNA portions may be ligated. Another type of vector is a viral vector, to which a DNA portion may be ligated to a viral genome or a portion thereof. Certain types of vectors have the ability to autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with origins of replication, episomal mammalian vectors, and herpes simplex virus (HSV) vectors). Other vectors (e.g., non-episomal mammalian vectors), once introduced into a host cell, may be integrated into the host cell’s genome and thereby replicate with the host genome. Furthermore, certain types of vectors have the ability to direct the expression of genes to which they are operatively ligated. Such vectors are referred to herein as “expression vectors.”

[0055] The use of these clauses is not intended to limit the invention. Each clause may be applied to any aspect of the invention. In this application, the use of "or" means "and / or" unless otherwise stated or clearly distinguishable from the context.

[0056] Wherever a composition is described as having, including, or comprising a particular component, or where a process and method is described as having, including, or comprising a particular step, it is further intended that there exist compositions of the present invention that are essentially composed of or comprise the cited components, and that there exist processes and methods of the present invention that are essentially composed of or comprise the cited processing steps.

[0057] In this application, when it is said that an element or component is included in the list of cited elements or components and / or selected from the list of cited elements or components, it should be understood that the element or component may be any one of the cited elements or components, or it may be selected from a group consisting of two or more of the cited elements or components.

[0058] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether expressly or implicitly, can be combined in various ways without departing from the spirit and scope of the invention. For example, where a particular compound is mentioned, that compound can be used in various embodiments of the compositions of the invention and / or in the methods of the invention, unless otherwise understood from the context. In other words, while embodiments within this application are described and depicted in a manner that allows for clear and concise application to be described and depicted, it is intended and understood that embodiments can be combined or separated in various ways without departing from the teachings and inventions. For example, it should be understood that all features described and depicted herein may be applicable to all embodiments of the inventions described and depicted herein.

[0059] The expression "at least one of ~" should be understood to include each of the objects mentioned following it, and any various combinations of two or more of those objects, unless otherwise understood from the context and usage. Expressions like "and / or" relating to three or more objects should be understood to have the same meaning unless otherwise understood from the context.

[0060] Throughout this specification, where a composition is described as having, including, containing, incorporating, or comprising a particular component, or where a process and method is described as having, including, or comprising a particular step, the composition and method are intended to be comprehensive or unrestricted and not to exclude additional, implicit components or steps. Furthermore, it is intended that some compositions of this disclosure essentially contain or include cited components, and some processes and methods of this disclosure essentially contain or include cited steps.

[0061] Any examples or illustrative expressions herein, such as “such as” or “including,” are intended solely to better illustrate the invention and, unless claimed, do not limit the scope of the invention. Nothing in this specification should be construed as indicating that any unclaimed element is essential for the practice of the invention.

[0062] II. Viral vectors and herpes simplex virus type 1 (McKrae) Viral vectors can be used to facilitate the transfer of nucleic acids into cells. Herpes simplex virus (HSV) type 1 (HSV-1) vectors can typically accommodate foreign DNA sequences up to 25 kb. HSV-1 has a double-stranded linear DNA genome of approximately 152 kb that can be maintained episomalally within the cell nucleus. HSV-1 virions are enveloped and approximately 110 nm in diameter.

[0063] At least 17 strains of HSV-1 have been isolated, including, but are not limited to, McKrae, strain 17, strain F, strain H129, strain HF10, MacIntyre, strain HF, ATCC2011, and strain KOS (see Watson et al., (2012) Virology 433(2):528-537 for a review).

[0064] A. McKrae The McKrae strain was isolated from a patient with herpes simplex keratitis and subsequently passaged in tissue culture. A partial genome sequence of McKrae is provided as Sequence ID No. 1 (GenBank acceptance number: JQ730035.1).

[0065] Inter-strain differences in peripheral HSV-1 replication and pathogenicity are observed after injection into animals. For example, McKrae is one of the most pathogenic HSV-1 strains, exhibiting spontaneous or induced reactivation more frequently than other known strains.

[0066] HSV genes influence the characteristics and phenotype of the virus. The McKrae strain has at least nine unique genes and several non-coding sequences. In addition to those associated with pathogenicity and latent reactivation, such as RL1, RSI, and RL2, three UL genes (UL36, UL49A, and UL56) and three US genes (US7, US10, and US11) are unique to the McKrae strain. In addition to gene mutations, there are also McKrae strain-specific mutations in non-coding sequences such as LAT, "a" sequences, and miRNAs.

[0067] One or more of the following genes and non-coding sequences are thought to be characteristic of the McKrae strain. In McKrae, RLl(ICP34.5) has eight PAT repeats between residues 159 and 160, whereas in other strains, it has only three to five repeats. The PAT repeat is thought to affect the intracellular localization of the ICP34.5 protein (Mao et al. (2012) J. Biol. Chem. 277(13):11423-31). ICP34.5 is thought to be a neuroviral factor involved in viral replication and anti-host response.

[0068] The McKrae strain also contains a six-repeat elongation repeat element of the internal tandem repeat STPSTTT (SEQ ID NO: 38) located within the coding sequence of US07(gl). Furthermore, in McKrae, UL36 contains an immature stop codon introduced by a G nucleotide deletion in a mononucleotide string (nucleotide 72,535) encoding amino acid residue 2,453, and UL56 (amino acid 180) contains a single base pair insertion at nucleotide 115,992 (amino acid 97). The McKrae strain also contains an elongation ORF in US10 resulting from a 1 bp insertion at nucleotide 143,416, and this frameshift causes the loss of a stop codon in McKrae, resulting in a unique C-terminal protein sequence. In McKrae, the amino acids of residues 28 and 51 of UL49A also differ from those of other strains. While strain 17 has arginine and threonine, and other strains (e.g., KOS) have histidine and alanine, strain McKrae has histidine and threonine at residues 28 and 51, respectively. Strain McKrae also has a reduced number of tangent repeats at the UL-RL junction (181 bp in strains 17 and KOS, compared to 49 bp in McKrae), with approximately 330 nucleotides deleted immediately after the UL-RL junction repeat. McKrae also contains a unique mutation in the serial repeat 2 (DR2) sequence of the "a" sequence. Instead of a series of unbroken tangent repeats, the McKrae DR2 repeat is interrupted twice by the same guanine-rich sequence.

[0069] The main inter-strain differences within the LAT intron are due to variations in repeat elements (GCACCCCCACTCCCAC) (SEQ ID NO: 39) starting at nucleotide 119,482 in the McKrae strain, with different numbers of repeats. The McKrae strain contains 13 repeats, while strains F, H129, and 17 contain 9 repeats, and the KOS strain contains 15 repeats. Additionally, in McKrae, differences in tandem repeats starting at nucleotide 125,520 are observed between strains. The McKrae repeat elements include 12 repeats of CCCCAGCCCTCCCCAG (SEQ ID NO: 40) and 8 repeats of CCCCTCGCCCCCTCCCG (SEQ ID NO: 41). The first repeat unit differs from other strains in that it contains a GA transition, with the McKrae strain containing 3 more repeats than any other strain. The second repeat element in the McKrae strain is folded, missing 188 nucleotides compared to all other strains, and is separated from the upstream repeat by a 105 bp 100% conserved sequence containing miR-H5.

[0070] McKrae further contains a unique coding sequence for ICP4 not found in other known strains (Watson et al., (2012), cited above). ICP4 is a pre-early transcriptional regulator involved in reactivation. While other strains have an alanine-rich region (AASAPDAADALAAA) (SEQ ID NO: 42) between residues 707 and 720, in McKrae, this alanine-rich region is replaced by a serine-rich sequence (GPRRSSSSSGVAA) (SEQ ID NO: 43). The serine-rich substitution block present in McKrae is adjacent to the nuclear localization signal (NLS) (amino acids 728-734). Changes in the conformation of this region can alter the NLS, and consequently affect not only ICP4 but also the localization of other viral proteins affected by ICP4 localization (e.g., ICP0 and ICP8) (Knipe and Smith (1986) Molecular and Cellular Biology 6(7), 2371-2381). Therefore, this region may partially influence the viral phenotype by altering the localization of proteins in the nucleus.

[0071] Modification of B.HSV-1 vector Vectors containing the HSV-1 genome (e.g., derived from the McKrae strain) may render one or more HSV genes necessary for replication non-functional. For example, vectors containing the HSV genome may contain modifications that interfere with the expression of one or more functional ICP0, ICP4, ICP22, ICP27, and ICP47 genes. Examples of HSV genes necessary for replication include pre-early genes such as ICP4 and ICP47. ICP4 is a viral transcription factor that is expressed immediately after infection and maintains the HSV-1 viral cycle. HSV-1's ICP47 is an 87-amino acid cytoplasmic polypeptide, or 88 residues including the initiating methionine. In human cells, it binds to the TAP1-TAP2 heterodimer, but not in mouse cells, instead inhibiting peptide transport by blocking the peptide binding site of TAP. As a result, MHC class I molecules cannot be loaded with peptides. The resulting empty class I molecules are retained in the endoplasmic reticulum, and in HSV-infected human cells, the presentation of epitopes to cytotoxic T lymphocytes is lost. Mutations that inhibit the expression of one or more functional HSV genes (e.g., ICP4 and ICP47) include mutations in the gene's coding sequence or regulatory sequences that affect gene expression (e.g., promoters) (e.g., missense mutations, nonsense mutations, insertions, deletions, etc.). ICP47 mutations include, but are not limited to, mutations in A4, D27, K31, R32, R34, or R41 relative to SEQ ID NO: 5, and combinations thereof (as described in Mozzie et al. (2022) Mol Biol Evol. 39(7):msac142). ICP4 mutants and knockout mutants that disrupt the transcriptional activation ability of ICP4 include, but are not limited to, n208, d8-10, nd8-10, ΔSER, d120, n12, ΔSERn7, d3-8, nd3-8, m20, m20n7, m90, m90n7, d143, d143n7, and nd3-10 (Wagner et al. (2012) J Virol. 86(12):6862-74).Deletion of the region between N-terminal amino acids 30 and 210 of ICP4 is sufficient to eliminate transcriptional activation (Wagner et al. (2013) J Virol. 87(2):1010-1018). In some embodiments, the present disclosure provides replication-deficient HSV vectors having modifications to one or more of ICP0, ICP4, ICP22, ICP27, and ICP47.

[0072] The HSV-1 IE promoter contains one or more copies of the IE-specific regulatory sequence of consensus TAATGARAT (SEQ ID NO: 44) (where R is purine). These motifs are typically located within several hundred nucleotides of the proximal IE promoter sequence, but together with their flanking sequences, they are distinct functional entities capable of providing IE-specific control to other proximal promoter elements of different temporal classes. In some embodiments, replication-deficient viruses are created by deleting nucleotides in the IE-specific regulatory sequence that affect the expression of one or more of the IE-specific regulatory sequences, e.g., ICP0, ICP4, ICP22, ICP27, and ICP47. In some embodiments, the IE-specific regulatory sequence includes internal deletions. In some embodiments, the IE-specific regulatory sequence includes terminal deletions. In some embodiments, the IE-specific regulatory sequence is completely deleted.

[0073] In some embodiments, the Disclosure provides an HSV vector having non-functional ICP4 and ICP47 genes. In certain embodiments, the ICP4 gene or the regulatory sequence affecting ICP4 expression may include a mutant, and the ICP47 gene or the regulatory sequence affecting ICP47 expression may include a mutant. For example, the ICP4 gene may include a mutant, and the ICP47 gene may include a mutant. In another example, the regulatory sequence affecting ICP4 expression may include a mutant, and the regulatory sequence affecting ICP47 expression may include a mutant. In one specific example, the ICP4 gene or the regulatory sequence affecting ICP4 expression may include a deletion (e.g., a complete or partial deletion sufficient to disable activity), and the ICP47 gene or the regulatory sequence affecting ICP47 expression may include a deletion (e.g., a complete or partial deletion sufficient to disable activity). The ICP4 gene may include a deletion, and the ICP47 gene may include a deletion. Furthermore, the aforementioned combinations, for example, a deletion of ICP4 and a mutant of the ICP47 regulatory sequence, are intended.

[0074] In some embodiments, the disclosure provides an HSV vector or an HSV strain having a non-functional ICP47 gene. In some embodiments, the disclosure provides an HSV vector or an HSV strain having a non-functional ICP4 gene and an ICP47 gene. In some embodiments, the disclosure provides an HSV vector or HSV strain lacking ICP4 and ICP47. In some embodiments, the genes encoding ICP4 and ICP47 are completely or partially deleted without disrupting the expression of additional pre-initial genes.

[0075] HSV-1 vectors containing altered (e.g., mutated) HSV genes can be generated in cell lines expressing the deficiency protein in trans. In some embodiments, the HSV-1 vector is generated in mammalian cell lines, e.g., Vero line mammalian cell lines. In some embodiments, the cell line expresses ICP4. In some embodiments, the cell line expresses ICP47. In some embodiments, the cell line expresses ICP4 and ICP47. In some embodiments, the cell line expresses one or more of ICP0, ICP4, ICP22, ICP27, and ICP47. In some embodiments, the cell line expresses ICP4, ICP22, and ICP47. In some embodiments, the cell line expresses ICP4, ICP22, and UL55. In some embodiments, the cell line expresses ICP4, ICP27, and UL55. In some embodiments, the cell line contains a nucleic acid molecule having a Simian virus 40 polyadenylation signal (SV40 pA). Depending on the circumstances, viral vectors can be generated in Vero6-5C cells or Vero D cells.

[0076] In certain embodiments, the viral vector of this disclosure is a McKrae HSV-1 viral vector. Wild-type McKrae HSV-1 contains two copies of the gene encoding ICP4 and one copy of the gene encoding ICP47. In some embodiments, this disclosure provides an HSV vector having non-functional ICP4 and ICP47 genes. In certain embodiments, at least one ICP4 gene (i.e., one or both copies of the gene), or a regulatory sequence affecting the expression of ICP4, includes a mutation, and the ICP47 gene or a regulatory sequence affecting the expression of ICP47 includes a mutation. For example, at least one copy of the ICP4 gene (i.e., one or both copies of the ICP4 gene) may include a mutation, and the ICP47 gene may include a mutation. In another example, a regulatory sequence affecting the expression of at least one ICP4 gene (i.e., one or both copies of the ICP4 gene) includes a mutation, and a regulatory sequence affecting the expression of ICP47 includes a mutation. In one specific example, a regulatory sequence affecting the expression of at least one ICP4 gene (i.e., one or both copies of the ICP4 gene), or at least one ICP4 gene (i.e., one or both copies of the ICP4 gene), may contain a deletion, and a regulatory sequence affecting the expression of the ICP47 gene or ICP47 may contain a deletion.

[0077] In some embodiments, the Disclosure provides an HSV vector or an HSV strain having a non-functional ICP47 gene. In some embodiments, the Disclosure provides an HSV vector or an HSV strain having a non-functional ICP4 gene and an ICP47 gene. In some embodiments, the Disclosure provides an HSV vector or HSV strain having one or both ICP4-deficient and ICP47-deficient copies. In some embodiments, one or both copies of the ICP4-encoding gene are completely or partially deficient, and the ICP47-encoding gene is completely or partially deficient without disrupting the expression of additional pre-early genes.

[0078] Applications of C.HSV-1 vectors The viral vectors described herein (e.g., HSV-1 vectors) may exhibit characteristics that make them advantageous for use in the treatment of diseases in subjects, such as cancer in subjects refractory to ICI.

[0079] In certain embodiments, the viral vectors described herein exhibit delayed tumor lysis (for example, when administered to a subject). Delayed tumor lysis enables viral persistence in target (e.g., cancer) cells, sustained expression of the payload, exposure of tumor-associated antigens within the tumor microenvironment where the payload is primed, and limited nonspecific inflammation and tissue damage.

[0080] In certain embodiments, the viral vector exhibits increased immunogenicity (for example, when administered to a subject). Immunogenicity can be measured, for example, by detecting increased Tap1 gene expression and / or increased MHC class I expression (HLA). Methods for measuring Tap1 gene expression, as described in the examples herein, are known in the art and include, for example, RNA-seq analysis. Methods for measuring HLA expression are known in the art and include, for example, flow cytometry.

[0081] In certain embodiments, viral vectors exhibit increased immune activation (e.g., when administered to a subject). Immune activation can be measured by detecting increased or decreased RNA expression in pathways associated with immune cell activation, including, but not limited to, the IFNα response pathway, the inflammatory response pathway, the myc signaling pathway, and / or the MtorC1 signaling pathway. Genes involved in such pathways are listed on the Gene Set Enrichment Analysis (GSEA) website. See, for example, gsea-msigdb.org / gsea / msigdb / collections.jsp. Immune activation can also be detected by detecting increased expression of TNF superfamily members, including tumor necrosis factor α (TNF), lymphotoxin β (LTB), APRIL (TNFSF13), and LIGHT (TNFSF14), and / or by detecting changes in the expression of immunogenic cell death regulators that imply increased pyroptosis and / or apoptosis, such as detecting increased expression of PYCARD and caspase 10 (CASP10).

[0082] In certain embodiments, administration of a viral vector induces the formation of tertiary lymphoid structures (TLSs) in the tumor bed. TLSs can be measured, for example, by detecting localized increases in the expression of CD11c-positive dendritic cells, CD4-positive T cells, and B220-positive B cells.

[0083] In certain embodiments, administration of a viral vector causes a decrease in the viability of cancer cells. Cancer cell viability can be determined, for example, by measuring tumor volume or by counting the number of living cancer cells.

[0084] III. Payload A viral vector according to this disclosure comprises a nucleic acid molecule containing a vector payload. The payload contains a nucleic acid molecule encoding one or more polypeptides. The payload is intended to consist of nucleic acid molecules containing a sequence complementary to the nucleic acid sequence encoding the polypeptide. The payload may encode a regulatory nucleic acid molecule, such as a small interfering RNA (siRNA) polynucleotide or a microRNA (miRNA) polynucleotide.

[0085] The payload may be a target tissue to which the vector is administered, or a nucleic acid molecule encoding an exogenous protein in the target. For example, the payload may be a target tissue to which the vector is administered, or a nucleic acid molecule encoding an endogenous protein in the target. In some embodiments, the nucleic acid molecule is codon-optimized.

[0086] The nucleic acid containing the vector payload can, for example, encode one, two, three, four, five, six, seven, eight, nine, or ten therapeutic polypeptides. In a particular embodiment, the vector payload can encode 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, or 5-10 sets of therapeutic polypeptides.

[0087] A therapeutic polypeptide encoded on a vector may have one or more of the following functional attributes: (1) Target the tumor stroma; (2) Support the survival of T cells (e.g., CAR T cells) in the tumor microenvironment; (3) Induce TLS in the tumor bed; (4) Promote phagocytic innate immune surveillance (e.g., by disrupting the Sirpα / CD47 axis); (5) Promote the upregulation or downregulation of one or more proteins associated with the response to ICI (e.g., IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21); and / or (6) Reduces the viability of cancer cells.

[0088] One or more therapeutic polypeptides from one or more categories (1-6 above) may be encoded on the same vector to target multiple pathways to enhance immune activity against tumors. For example, a vector may encode one, two, or three therapeutic polypeptides from category 1 and one, two, or three therapeutic polypeptides from categories 2, 3, 4, 5, or 6. Alternatively, a vector may encode one, two, or three therapeutic polypeptides from category 2 and one, two, or three therapeutic polypeptides from categories 3, 4, 5, or 6. Alternatively, a vector may encode one, two, or three therapeutic polypeptides from category 3 and one, two, or three therapeutic polypeptides from categories 4, 5, or 6. Alternatively, a vector may encode one or two therapeutic polypeptides from category 1 and one or two therapeutic polypeptides from categories 2, 3, 4, 5, and / or 6. Alternatively, the vector may encode one or two therapeutic polypeptides from category 2 and one or two therapeutic polypeptides from categories 3, 4, 5, and / or 6. Similarly, the vector may encode one or two therapeutic polypeptides from category 3 and one or two therapeutic polypeptides from categories 4, 5, and / or 6.

[0089] In certain embodiments, the therapeutic polypeptide comprises TNF, IL-1β, IL-6, IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesoserine, CD40L, OX40L, lymphotoxin β, CXCL13, Sirpα-IgG fusion transgene (e.g., the extracellular domain of SIRPα and the Fc fragment of human IgG1), anti-CD47 antibody, IL-1, and / or IL-17.

[0090] In certain embodiments, the therapeutic polypeptide comprises IL-21, LTA, LTB, CXCL12, and / or VEGF-C.

[0091] In certain embodiments, the therapeutic polypeptide comprises hyaluronidase, MMP-9, lysyl oxidase inhibitors, TNF, IL-1β, IL-6, IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesoserine, CD40L, OX40L, lymphotoxin β, CXCL13, Sirpα-IgG fusion transgene (e.g., the extracellular domain of SIRPα and the Fc fragment of human IgG1), anti-CD47 antibody, IL-1, IL-17, IL-21, LTA, LTB, CXCL12, and / or VEGF-C.

[0092] In certain embodiments, the therapeutic polypeptide comprises LTA, CCL19, TNF, IL-17, and / or IL-7.

[0093] In certain embodiments, the therapeutic polypeptide comprises LTB, CCL19, CCL21, CXCL13, and / or IL-7.

[0094] In certain embodiments, the therapeutic polypeptide comprises IL-12 and IL-15.

[0095] Therapeutic polypeptides may be encoded on the same vector or on different vectors.

[0096] Therapeutic polypeptides in a payload may be cloned in tandem by linker sequences that separate individual polypeptides and driven from the same promoter. In certain embodiments, the linker sequence includes a ribosome skipping site (sequence), such as a 2A peptide. In an expression construct, if such a ribosome skipping site is contained in a linker located between two polypeptide sequences, translation of the transcribed mRNA produces the two polypeptides separately, rather than as a fusion protein. The 2A peptide sequence shares a core sequence motif of DXEXNPGP (SEQ ID NO: 45), where X is any amino acid. Non-limiting examples of suitable 2A peptide sequences include T2A (EGRGSLLTCGDVEENPGP; SEQ ID NO: 46), P2A (ATNFSLLKQAGDVEENPGP; SEQ ID NO: 49), E2A (QCTNYALLKLAGDVESNPGP; SEQ ID NO: 47), and F2A (VKQTLNFDLLKLAGDVESNPGP; SEQ ID NO: 48). Furthermore, efficiency can be increased by adding any Gly-Ser-Gly (GSG) tripeptide to the N-terminus of the 2A peptide.

[0097] A. Targeting the tumor stroma Viral vectors can encode one or more therapeutic polypeptides that target tumor stroma and / or activate local endothelium to increase T cell infiltration into the tumor, thereby reducing the viability of cancer cells within the tumor and decreasing tumor size.

[0098] Tumor stroma can be targeted by various mechanisms. For example, the extracellular matrix (ECM) can be lysed by proteins involved in ECM degradation, including but not limited to inhibitors of hyaluronidase, MMP-9, and lysyl oxidase. Consequently, in certain embodiments, the viral vector encodes one or more therapeutic polypeptides involved in ECM degradation, including but not limited to inhibitors of hyaluronidase, MMP-9, and lysyl oxidase.

[0099] Furthermore, cancer-associated fibroblasts can be targeted by proteins that interfere with the TGFβ pathway. As a result, in certain embodiments, the viral vector encodes one or more therapeutic polypeptides that interfere with the TGFβ pathway, for example, a polypeptide comprising the external domain of TGFβ (e.g., a non-transmembrane domain). In certain embodiments, the one or more therapeutic polypeptides comprise a fusion protein comprising the external domain of TGFβ, an IgG domain (e.g., an Fc domain), and the external domain of TGFβRII.

[0100] Local endothelium can be activated by expressing inflammatory cytokines, including but not limited to TNF, IL-1β, IL-6, and IL-18, which can promote T cell infiltration. The expression of enzymes that degrade the tumor ECM simultaneously breaks down the physical barrier within the tumor, allowing for the infiltration of immune cells. Consequently, in certain embodiments, the viral vector encodes one or more inflammatory cytokines, including, for example, TNF, IL-1β, IL-6, and IL-18.

[0101] Tumor stroma targeting and local endothelial activation can be regulated together or separately by the local expression of these therapeutic polypeptides.

[0102] In certain embodiments, hyaluronidase, MMP-9, a lysyl oxidase inhibitor, one or more therapeutic polypeptides that inhibit the TGFβ pathway, one or more genes encoding TNF, IL-1β, IL-6, or IL-18 are cloned into a non-replicating McKrae HSV-1 vector. The McKrae HSV-1 vector can be implemented into the McKrae HSV-1 virus, for example, using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting McKrae HSV-1 virus.

[0103] In certain embodiments, the effects of cancer cells that lyse the tumor stroma or secrete factors that activate local endothelium for T cell infiltration on the tumor stroma or local endothelium can be evaluated in vitro or in vivo using methods known in the art.

[0104] B. Supporting the survival of T cells (e.g., CAR T cells) in the tumor microenvironment. In certain embodiments, the viral vector encodes one or more therapeutic polypeptides that support the survival of T cells in the tumor microenvironment (TME). In the embodiments described herein, the T cells may be native T cells (e.g., T cells present in the subject to which the viral vector is administered) or CAR T cells (e.g., CAR T cells administered to the subject). In certain embodiments, the viral vector is administered together with T cells (e.g., CAR T cells) to treat a tumor, for example.

[0105] T cells in TMEs can be supported by various mechanisms. For example, the recruitment of T cells, such as native T cells or CAR T cells, to tumor sites can be improved by the presence of T cell recruitment factors in TMEs, such as the chemokine ligands CCL19 or CCL21.

[0106] Local T cells can be supported to elicit a potent and persistent antitumor response. For example, local expression of T cell trophic factors such as IL-7, IL-12, IL-15, IL-18, and IFN-γ can elicit a potent and durable antitumor response from local T cells or recruited T cells. As a result, in certain embodiments, the viral vector encodes one or more T cell trophic factors, including, for example, IL-7, IL-12, IL-15, IL-18, and IFN-γ. In certain embodiments, the viral vector encodes IL-12 and IL-15.

[0107] Furthermore, TGFβ signaling in tumor cells can be suppressed by the expression of soluble TGFβRII. Consequently, in certain embodiments, one or more therapeutic polypeptides comprise soluble TGFβRII.

[0108] Local expression of genes encoding costimulatory molecules such as CD40L or OX40L can stimulate local or recruited T cells depending on the target TME. As a result, in certain embodiments, one or more therapeutic polypeptides include CD40L or OX40L.

[0109] T cell support or T cell recruitment can be regulated together or separately by these therapeutic polypeptides.

[0110] In certain embodiments, one or more genes encoding CCL19, CCL21, IL-7, IL-12, IL-15, IL-18, IFNγ, soluble TGFβRII, CD40L, or OX40L are cloned into a non-replicating McKrae HSV-1 vector. The McKrae HSV-1 vector can be implemented into the McKrae HSV-1 virus, for example, using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting McKrae HSV-1 virus.

[0111] The effects of cancer cells secreting these factors on T cell recruitment or T cell function can be evaluated in vitro or in vivo using methods known in the art.

[0112] C. To support the survival of NK cells (e.g., CAR NK cells) in the tumor microenvironment. In certain embodiments, the viral vector encodes one or more therapeutic polypeptides that support the survival of NK cells in the tumor microenvironment (TME). In the embodiments described herein, the NK cells may be native NK cells (e.g., NK cells present in the subject to which the viral vector is administered) or CAR NK cells (e.g., CAR NK cells administered to the subject). In certain embodiments, the viral vector is administered together with NK cells (e.g., CAR NK cells) to treat a tumor, for example.

[0113] NK cells in TME can be supported by various mechanisms. For example, the recruitment of NK cells, such as native NK cells or CAR NK cells, to tumor sites can be enhanced by the presence of NK cell recruiting factors in TME, such as the chemokine ligands CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, and CXCL11.

[0114] Localized NK cells can induce a supported, potent, and sustained antitumor response. For example, localized expression of NK cell trophic factors such as IL-2, IL-15, IL-18, and IFN-α can elicit a potent and durable antitumor response from localized or recruited NK cells. Consequently, in certain embodiments, the viral vector encodes one or more NK cell trophic factors, including, for example, IL-2, IL-15, IL-18, and IFN-α.

[0115] Furthermore, TGFβ signaling in tumor cells can be suppressed by the expression of soluble TGFβRII. Consequently, in certain embodiments, one or more therapeutic polypeptides comprise soluble TGFβRII.

[0116] NK cell support or NK cell recruitment can be regulated together or separately by these therapeutic polypeptides.

[0117] In certain embodiments, one or more genes encoding CCL2, CX3CL1, CXCL16, CCL5, CXCL9, CXCL10, CXCL11, IL-2, IL-15, IL-18, and IFN-α, or soluble TGFβRII, are cloned into a non-replicating McKrae HSV-1 vector. The McKrae HSV-1 vector can be implemented into the McKrae HSV-1 virus, for example, using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting McKrae HSV-1 virus.

[0118] The effects of cancer cells secreting these factors on NK cell recruitment or function can be evaluated in vitro or in vivo using methods known in the art.

[0119] D. Induction of tertiary lymphocyte structures (TLS) in the tumor bed. The tumor microenvironment (TME) supports tumor growth and treatment resistance by providing physical and functional barriers to tumor-infiltrating lymphocytes (TILs), and includes heterogeneous cell aggregates, signaling molecules, blood vessels, and the extracellular matrix (ECM). Components of the TME often do not respond to classical immunotherapy because TILs are not effectively recruited and remain activated within the immunosuppressive TME. An increase in TILs within or near an immunoinfiltrating tumor is called an immunologically "active" tumor and is associated with the formation of TLSs. TLSs are ectopic lymphoid tissues that arise in non-lymphoid tissues and are structurally and functionally similar to secondary lymphoid organs (such as lymph nodes). TLSs include spatially defined B-cell and T-cell regions, high endothelial venules for naive T-cell migration, and, in some cases, fully formed germinal centers, hubs for B-cell affinity maturation, and clonal development. Functionally, TLS acts as a functional niche for the maturation of T-cell and B-cell responses to locally expressed tumor antigens, and TLS supports the organization of a robust immune response against solid tumors (Johansson-Percival et al. (2017) Nat Immunol., 18(11):1207-1217; and Tang et al. (2016) Cancer Cell, 30(3):500).

[0120] TLS formation has been associated with improved efficacy of immunotherapy in mice and humans, for example, by providing mobilization and survival signals to ICI-activated TILs within the tumor itself (Sautes-Fridman et al. (2019) Nat Rev Cancer, 19(6):307-325). Three independent research groups concluded that the presence of TLS in subjects treated with ICI therapy was associated with a strong B-cell response and extended patient survival in both melanoma and colorectal cancer, supporting the relevance of TLS to cancer progression and response to treatment (Cabrita et al:561-565; and Helmink et al. (2020) Nature, 577(7791):549-555). However, therapies that induce TLS in ICI-refractory "insensitive" tumors remain an unmet need.

[0121] The formation and function of TLSs in disease onset and / or progression have been shown to be dependent on antigen exposure in the context of generating a specific response to an antigen or autoantigen (Barone et al. (2016) Front Immunol., 7:477). Factors regulating TLSs include secreted and cell surface-expressed proteins in the cytokine-rich inflammatory microenvironment, such as TNF, IL-6, IL-17, and IL-22. Furthermore, it has been demonstrated that administration of adenovirus alone can induce transient TLS formation (Bombardieri et al. (2012) J Immunol. 189(7):3767-76), highlighting the spontaneous response to antigens released in the early stages of viral infection as a priming element for TLS formation. However, it is not well understood what combination of factors is sufficient to initiate TLS formation.

[0122] Provided herein are viral vectors encoding one or more therapeutic polypeptides that, in certain embodiments, induce TLS within a tumor bed. In certain embodiments, inducing TLS within a tumor bed includes promoting the number, size, and / or organization of TLS structures.

[0123] Thus, viral vectors encoding therapeutic polypeptides that induce TLS formation can be used according to the methods described herein. In certain embodiments, the viral vectors are used in combination with additional solid tumor therapeutics, including but not limited to CAR T cell therapy and ICIs.

[0124] In certain embodiments, one or more genes encoding one or more therapeutic polypeptides involved in the establishment and maintenance of TLS, including but not limited to CCL19, lymphotoxin β, CXCL13, and TNF, are cloned into an HSV-1 vector (e.g., including modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins). The HSV-1 vector can be implemented into the HSV-1 virus, for example, using a packaging strain. In certain embodiments, cancer cells are transduced with the resulting HSV-1 virus.

[0125] The impact of cancer cells secreting these factors on TLS within the tumor bed can be evaluated in vitro or in vivo using methods known in the art.

[0126] E. To promote phagocytic autoimmune surveillance (e.g., by disrupting the Sirpα / CD47 axis) In certain embodiments, the viral vector encodes one or more therapeutic polypeptides that promote phagocytic autoimmune surveillance and elimination of tumor cells, for example, by disrupting the signal regulatory protein α (SIRPα) / CD47 axis.

[0127] CD47 is an immunoglobulin that is overexpressed on the surface of many types of cancer cells. CD47 forms a signaling complex with SIRPα on macrophages, allowing cancer cells to evade macrophage-mediated phagocytosis. Phagocytic autoimmune surveillance and elimination of tumor cells can be induced by disruption of the Sirpα / CD47 axis, for example, by the presence of a Sirpα-IgG fusion transgene. Disruption of the Sirpα / CD47 axis can promote phagocytic autoimmune surveillance and elimination of tumor cells by exposing them to the tumor's "eat" signal. Consequently, in certain embodiments, a viral vector encodes one or more therapeutic polypeptides that disrupt the Sirpα / CD47 axis, thereby promoting phagocytic autoimmune surveillance and elimination of tumor cells. In certain embodiments, the one or more therapeutic polypeptides are anti-CD47 antibodies. In certain embodiments, the viral vector encodes a Sirpα-IgG fusion polypeptide (e.g., the extracellular domain of SIRPα and the Fc fragment of human IgG1).

[0128] Furthermore, the vectors can encode therapeutic polypeptides that support antitumor macrophage polarization, including but not limited to TNF, IL-1, IL-12, IL-17, and IFNγ.

[0129] In certain embodiments, one or more therapeutic polypeptides involved in the establishment and maintenance of TLS in tissues, including but not limited to Sirpα-IgG fusion transgenes, TNF, IL-1, IL-12, IL-17, and IFNγ, may be cloned into a non-replicating McKrae strain HSV-1 vector.

[0130] The impact of cancer cells secreting these factors on phagocytic autoimmune surveillance can be evaluated in vitro or in vivo using methods known in the art.

[0131] F. To promote the regulation of proteins related to the response to immune checkpoint inhibitors. The data provided herein (see, for example, Example 3 and Figure 12) demonstrate that, even without a functional payload, administration of an HSV-1 vector containing modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins promotes the upregulation or downregulation of one or more proteins related to the response to ICIs. As a result, it is intended that an HSV-1 vector containing modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins may be useful in treating ICI-refractory subjects. Furthermore, it is intended that an HSV-1 vector containing modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins may be used in combination with an ICI to treat cancer in subjects.

[0132] The viral vectors described herein may contain one or more therapeutic polypeptides that are predicted to promote a therapeutic response to ICI, including factors that have been reported to play a role in TLS formation, such as IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21.

[0133] The effects on protein expression related to responses to ICI can be evaluated using methods known in the art, such as the use of Gene Set Enrichment Analysis (GSEA) of RNA-seq data.

[0134] IV. Regulatory elements The nucleic acids of this disclosure are intended to include, as herein, non-natural regulatory sequences, gene regulatory sequences, promoters, non-coding sequences, introns, or coding sequences. They are further intended to include nucleic acids encoding nucleic acid tags or signaling sequences, or protein tags or protein signaling sequences. Typically, coding regions are operably linked to one or more regulatory nucleic acid components.

[0135] The promoters included in the nucleic acids of this disclosure may be tissue- or cell-type specific promoters, promoters specific to multiple tissues or cell types, organ-specific promoters, promoters specific to multiple organs, systemic or ubiquitous promoters, or nearly systemic or nearly ubiquitous promoters. Promoters having stochastic expression, inducible expression, conditional expression, or otherwise discontinuous, indeterminate, or unpredictable expression are also included in the scope of this disclosure. The promoters of this disclosure may include any of the above characteristics or other promoter characteristics known in the art.

[0136] Known examples of promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the CMV / human β3-globin promoter, the GFAP promoter, the chicken β-actin (CBA) promoter, the β-glucuronidase (GUSB) promoter, and ubiquitin promoters isolated from human ubiquitin A, human ubiquitin B, and human ubiquitin C.

[0137] In some embodiments, the promoter is a neuron-specific promoter in that it is a promoter that has specific expression in neurons, or in that it typically drives the expression of a relevant coding sequence in neurons or subpopulations of neurons, but not in one or more other tissues or cell types. Examples of such promoters include calcitonin gene-related peptide (CGRP), synapsin I (SYN), calcium / calmodulin-dependent protein kinase II, tubulin αI, neuron-specific enolase, microtubule-related protein IB (MAP1B), and platelet-derived growth factor β-chain promoters, as well as their derivatives. In some embodiments, the promoter is the calcitonin gene-related peptide (CGRP) promoter or a derivative thereof.

[0138] Other regulatory elements, such as enhancers and polyadenylation sites, may be operably linked to the payload. In some embodiments, the enhancer includes a human cytomegalovirus (HCMV) sequence. In some embodiments, the polyadenylation site includes a bovine growth hormone (BGH) polyadenylation signal.

[0139] In some embodiments, the promoter is a chimera of one or more promoters or regulators found in nature. In some embodiments, the viral vector contains a payload whose expression is driven by a CGRP promoter having an HCMV enhancer sequence.

[0140] V. Vector Preparation This disclosure relates in particular to replication-deficient McKrae virus vectors. Viral vectors can be produced by mutations (e.g., deletions) of one or more pre-initial genes or regulatory sequences that affect their expression. Viral genes can be mutated using methods of recombination techniques known in the art. Viral vectors of this disclosure can be replication-deficient as a result of homologous recombination events. Replication-deficient viral vectors can be produced by mutations in the ICP4 gene and the ICP47 gene. For example, replication-deficient viral vectors are produced by deletions in the ICP4 gene and the ICP47 gene.

[0141] The viral vectors of this disclosure can be constructed by deletion of a locus encoding one or more ICPs (e.g., ICP4 and ICP47) by homologous recombination. In some embodiments, the construction of the viral vectors of this disclosure includes a step of homologous recombination between a first plasmid and a second plasmid. In some embodiments, the first plasmid contains nucleic acid sequences homologous to a region of the HSV genome adjacent to the nucleic acid region of the HSV genome intended to be replaced. In some embodiments, the second plasmid contains the HSV genome or a fragment thereof. In some embodiments, the first plasmid contains nucleic acid sequences encoding a gene of interest between homologous nucleic acid sequences. In some embodiments, the gene of interest may be or contain a marker protein detectable by fluorescence, chemiluminescence, or other properties, which may be used to select a vector resulting from successful homologous recombination.

[0142] In some embodiments, the viral vector of this disclosure may be prepared by homologous recombination of a first plasmid containing a nucleic acid sequence homologous to the upstream region of the ICP4 promoter, which includes a viral origin in the short-chain reverse conversion circuit region of HSV, and a second plasmid containing the HSV McKrae strain genome.

[0143] The vector can be constructed by first replacing both copies of the ICP4 locus by homologous recombination using plasmid SASB3, and then screening for green fluorescent protein (GFP) expression plaques. For example, the plasmid can be constructed by cloning the SphI-AflIII (linker-added SalI) fragment (1928 bp) of the HSV-1 KOS strain genome (nucleotides 124,485-126,413, KT899744, KOS strain) into SphI / SalI digested pSP72, and then inserting a 695 bp BglII-BamHI fragment (GenBank acceptance number: KT899744.1 (SEQ ID NO: 2), KOS strain, nucleotides 131,931-132,626), which includes the upstream region of the ICP4 promoter containing the viral origin within a short reverse conversion circuit region, into the BglII-BamHI region of the vector plasmid. In some embodiments, the plasmid is constructed by cloning an HCMV-eGFP fragment into the BamHI region of the plasmid as described above. Such plasmids are then recombined into a specific locus of wild-type McKrae virus. The resulting viral vector is isolated using a stable cell line that expresses one or more genes that are missing or disrupted in the HSV genome necessary for replication.

[0144] Alternatively, the vector can be constructed by first replacing both copies of the ICP4 locus by homologous recombination using plasmid SDAXB and then screening for green fluorescent protein (GFP) expression plaques. For example, the plasmid can be constructed by cloning the SphI-AflIII fragment (1928 bp) of the HSV-1 KOS strain genome (GenBank acceptance number: KT899744.1 (SEQ ID NO: 2), nucleotides 124, 346-126, 273, KOS strain) into SphI / AflIII digested pSP72 to produce plasmid SDA, and then changing the AflIII site to the BamHI site (plasmid SDAB). A DNA PCR fragment from BamHI to BglII containing the upstream region of the ICP4 promoter, including the viral origin (nucleotides 144933-145534 of GenBank acceptance number: JQ730035.1 (SEQ ID NO: 1), McKrae strain) within a short inverse conversion circuit region, can be cloned into the BamHI region of plasmid SDAB to construct plasmid SDAXB. Next, the HCMV-eGFP fragment can be cloned into the BamHI region of the plasmid as described above. The plasmid may then be recombined into a specific locus of wild-type McKrae virus. The resulting vector can be isolated using a stable cell line expressing one or more genes that are deleted or disrupted in the HSV genome necessary for replication.

[0145] Alternatively, a vector containing a combination of ICP4 and ICP47 deletions can be constructed. For example, McKrae sequence nucleotides 143,521-144,562 and 144,640-145,534 (GenBank acceptance number: JQ730035.1 (SEQ ID NO: 1), McKrae strain) may be synthesized, and the SCMV promoter flanked at the Pme I site can be cloned between these flanking sequences. Next, a marker, such as a red fluorescent marker, can be cloned into a plasmid so that it is operablely linked to the SCMV promoter. Such a plasmid can be recombined into a specific locus of the McKrae virus having a deletion at the ICP4 locus. The fluorescent clone can be isolated, and insertion of the construct into the ICP47 locus can be confirmed by PCR. The resulting recombinant McKrae virus has a deletion of base pairs 144,562 to 144,640 according to the reference sequence (GenBank acceptance number: JQ730035.1 (SEQ ID NO: 1), McKrae strain). In some embodiments, the resulting viral vector is isolated using a stable cell line expressing one or more genes that are deleted or disrupted in the HSV genome necessary for replication.

[0146] VI. Vector Characterization Viral vectors conforming to this disclosure may be characterized by genome sequencing to determine whether the expected vector has been successfully constructed. Any sequencing method known in the art is acceptable for this purpose. Sequencing methods include, for example, nanopore sequencing, single-molecule real-time sequencing (SMRT), DNA nanoball (DNB) sequencing, pyrosequencing, and the use of DNA arrays.

[0147] The expression of a payload from a viral vector can be detected by any method known in the art for detecting proteins or nucleic acids. Methods for detecting protein expression include immunohistochemistry, flow cytometry, Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoelectron microscopy, immunoprecipitation (IP) of individual proteins, immunoprecipitation of protein complexes (Co-IP), chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), immunoelectrophoresis, spectrophotometric analysis, and bicinticoninate assay (BCA). Methods for detecting nucleic acid expression include Southern blotting, Northern blotting, polymerase chain reaction (PCR), quantitative PCR, and RT-PCR.

[0148] VII. Formulations and Administration Modes Compositions comprising viral vectors as described herein may be formulated for administration in solid or liquid forms, such as those applicable to: oral administration, e.g., liquids (aqueous or nonaqueous solutions or suspensions); tablets, e.g., buccal, sublingual, and systemic absorption; pills; powders; granules; pastes for application to the tongue; parenteral administration, e.g., as sterile solutions or suspensions, or as sustained-release formulations, by subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, e.g., as creams, ointments, or sustained-release patches or sprays applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., as pessaries, creams, or foams; sublingual administration; intraocular administration; transdermal administration; or administration to the nasal cavity, lungs, and other mucous membrane surfaces.

[0149] Administration may be intratumor, parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal, and vaginal. Preferred routes of delivery include intratumor administration. In some embodiments, the pharmaceutical composition comprises a viral vector combined with a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are suitable for pharmaceutically effective administration. Supplementary active compounds may also be incorporated into the composition. In some embodiments, the viral vector is formulated in glycerol. In some embodiments, the viral vector is formulated in about 10% glycerol in phosphate-buffered saline.

[0150] For ease of administration and dose uniformity, it is advantageous to formulate the composition in dose-unit form. As used herein, dose-unit form means a physically discontinuous unit suitable as a unit dose for the target to be treated, each unit containing a predetermined amount of viral vector calculated to produce the desired therapeutic effect in relation to a pharmaceutical carrier.

[0151] As used herein, the term “administration” refers to the administration of a composition to a subject. Administration to an animal subject (e.g., human) may be by any suitable route. For example, in some embodiments, administration may be bronchial (e.g., by bronchial infusion), buccal, enteral, intercutaneous, intra-arterial, intracutaneous, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intramedullary, intratumoral, intravenous, intra-organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal infusion), percutaneous, vaginal, and intravitreous. Depending on the circumstances, administration may involve intermittent administration and / or continuous administration (e.g., perfusion) for at least a selected period.

[0152] The pharmaceutical composition may be administered as needed at various intervals and over various periods, for example, once a week for about 1 to 10 weeks, about 2 to 8 weeks, about 3 to 7 weeks, or about 4, 5, or 6 weeks. Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other pre-existing conditions, may influence the dosage and timing adjustments required to effectively treat the subject. Treatment of a subject with a viral vector may consist of a single treatment or, more often, a series of treatments.

[0153] In certain embodiments, the pharmaceutical composition may be administered to a patient one or more times (e.g., two, three, four, five, six, seven, eight, nine, ten, or more times) as a result of the improved safety profile demonstrated by the vector described herein.

[0154] In some embodiments, the active agent, i.e., the viral vector of the Disclosure, and / or other agents administered together with the viral vector of the Disclosure, is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation including implants and microencapsulation delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid, can be used. Methods for preparing such compositions will be apparent to those skilled in the art. In some embodiments, the composition is targeted to cells of a specific cell type or cells infected with a virus.

[0155] VIII. Treatment method Viral vectors conforming to this disclosure are useful for a wide variety of therapeutic applications. In one embodiment, this disclosure relates to a method for treating cancer in subjects refractory to immune checkpoint inhibitors (ICIs). The method comprises administering a vector comprising a herpes simplex virus (HSV) genome, wherein the HSV genome includes modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins. The vector may comprise nucleic acids encoding one or more therapeutic polypeptides that function to reduce tumor size. For example, the therapeutic polypeptides may comprise one or more therapeutic polypeptides predicted to drive a therapeutic response to ICIs, including IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21.

[0156] In addition, the applicant has surprisingly discovered that administering a vector containing a herpes simplex virus (HSV) genome to a subject in need (wherein the HSV genome includes modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins, and expresses IL-12 and IL-15) results in a synergistic reduction in tumor size, thereby treating cancer. Accordingly, in certain embodiments, the present disclosure relates to a method for treating cancer, comprising administering a vector containing a herpes simplex virus (HSV) genome, wherein the HSV genome includes modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins, and expresses IL-12 and IL-15, resulting in a synergistic effect in reducing tumor size, thereby treating cancer. The tumor size reduction described above is synergistic compared to tumor size reduction induced by a vector containing a herpes simplex virus (HSV) genome (e.g., an otherwise identical vector), where the HSV genome includes modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins, and express IL-12 or IL-15, but not both IL-12 and IL-15. In certain embodiments, administration of a vector containing a herpes simplex virus (HSV) genome expressing IL-12 and IL-15 results in synergistic tumor regression compared to administration of herpes simplex virus alone. The tumor regression is synergistic compared to tumor regression induced by a vector containing a herpes simplex virus (HSV) genome (e.g., an otherwise identical vector), wherein the HSV genome includes modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins, and express IL-12 or IL-15, but not both IL-12 and IL-15.

[0157] In certain embodiments, the method further includes administering one or more ICIs.

[0158] In certain embodiments, the cancer described above is an epithelial carcinoma. In certain embodiments, the cancer is a solid tumor, a soft tissue tumor, a hematopoietic malignancy, or a metastatic lesion. In certain embodiments, a solid tumor is a sarcoma, adenocarcinoma, or carcinoma. In certain embodiments, a solid tumor is a tumor of the head and neck (e.g., pharynx), thyroid, lung (e.g., small cell lung cancer or non-small cell lung cancer (NSCLC)), breast, lymphatic system, gastrointestinal tract (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive tract or urogenital tract (e.g., kidney, urothelium, bladder, ovary, uterus, cervix, endometrium, prostate, testis), CNS (e.g., nerve cell or glial cell, e.g., neuroblastoma or glioma), or skin (e.g., melanoma). In certain embodiments, hematopoietic malignancies are leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell, or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) (e.g., transformed CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, pilocytic cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, multiple myeloma, or Richter syndrome (Richter transformation). In certain embodiments, cancers are selected from endometrial cancer, ovarian cancer, cervical cancer, vulvar cancer, uterine cancer, fallopian tube cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, urinary tract cancer, bladder cancer, head and neck cancer, oral cancer, and liver cancer. In certain embodiments, the cancer is breast cancer. Methods for expressing therapeutic polypeptides in subjects, for example, ICI-refractory subjects, are also provided. The method comprises administering a vector comprising an HSV genome, wherein the HSV genome includes modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins. The vector may comprise nucleic acids encoding one or more therapeutic polypeptides that function to reduce tumor size.For example, the therapeutic polypeptide may include one or more therapeutic polypeptides that are expected to drive a therapeutic response to an ICI, including IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21. In certain embodiments, the method further includes administering one or more ICIs.

[0159] A method for reducing tumor size in subjects, such as those refractory to ICI, is also provided. The method comprises administering a vector containing an HSV genome, wherein the HSV genome includes modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins. The vector may contain nucleic acids encoding therapeutic polypeptides that function to reduce tumor size. For example, the therapeutic polypeptides may include one or more therapeutic polypeptides predicted to drive a therapeutic response to ICI, including IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21.

[0160] In addition, the applicant has surprisingly discovered that administering a vector containing a herpes simplex virus (HSV) genome to a subject in need (wherein the HSV genome includes modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins, and expresses IL-12 and IL-15) results in a synergistic reduction in tumor size. Therefore, in certain embodiments, the disclosure relates to a method for treating cancer comprising administering a vector containing a herpes simplex virus (HSV) genome, wherein the HSV genome includes modifications that inhibit the expression of one or more functional ICP4 proteins and functional ICP47 proteins, expresses IL-12 and IL-15, and results in a synergistic effect in reducing tumor size.

[0161] In certain embodiments, the method further includes administering one or more ICIs.

[0162] The vectors described herein are useful for delivering one or more payloads to one or more target cells. In certain embodiments, the payloads persist in the target cells for up to 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, or more than 1 year.

[0163] In some embodiments, target cells may be located in tissues with poor blood vessels and limited access to systemic circulation. In some embodiments, target cells may be susceptible to infection by HSV. In some embodiments, target cells are particularly susceptible to infection by the McKrae strain of HSV.

[0164] Viral vectors according to this disclosure are useful for delivering one or more therapeutic polypeptides to cells, e.g., cells in a subject. For example, a viral vector comprising a heterologous nucleic acid segment operably linked to a promoter is useful for any disease or clinical condition associated with a decrease or absence of the protein encoded by the heterologous nucleic acid portion, or any disease or clinical condition that can be effectively treated by expressing the encoded protein in the subject. A viral vector comprising an expression cassette for synthesizing RNAi agents (e.g., one or more siRNAs or shRNAs) is useful for treating any disease or clinical condition associated with the overexpression of a transcript or the protein encoded therein in a subject, or any disease or clinical condition that can be treated by causing a decrease in a transcript or the protein encoded therein in a subject. A viral vector comprising an expression cassette for synthesizing one or more RNAs that self-mating or mating with each other to form RNAi agents targeting cytokine-encoding transcripts can be used to modulate immune system responses (e.g., responses that cause organ transplant rejection, allergies, autoimmune diseases, inflammation, etc.). Viral vectors that provide templates for the synthesis of one or more RNAs that self-mating or mate with each other to form RNAi agents that target the transcript of an infectious agent, or the cellular transcript whose encoded product is required for or contributes to any aspect of the infection process, may be used to treat infectious diseases.

[0165] The therapeutic polypeptides disclosed herein may include one or more of the following functional attributes: (a) Target the tumor stroma; (b) Support the survival of T cells (e.g., CAR T cells) and / or NK cells in the tumor microenvironment; (c) Induce tertiary lymphatic system structures (TLS) within the tumor bed; (d) Promote phagocytic innate immune surveillance; (e) Destroy the Sirpα / CD47 axis; (f) Promote the upregulation or downregulation of one or more proteins associated with the response to ICI (e.g., IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21); and / or (g) Reduces the viability of cancer cells.

[0166] Exemplary therapeutic polypeptides are described above in Section III and incorporated herein.

[0167] IX. Combination Therapy The compositions described herein may be administered in combination with one or more additional therapies, such as surgery, radiotherapy, or the administration of another therapeutic formulation. In certain embodiments, the additional therapy may include chemotherapy, such as cytotoxic agents. In certain embodiments, the additional therapy may include targeted therapies, such as tyrosine kinase inhibitors, proteasome inhibitors, or protease inhibitors. In certain embodiments, the additional therapy may include anti-inflammatory, anti-angiogenic, anti-fibrotic, or antiproliferative compounds, such as steroids, biological immunomodulators, monoclonal antibodies, antibody fragments, aptamers, siRNAs, antisense molecules, fusion proteins, cytokines, cytokine receptors, bronchodilators, statins, anti-inflammatory agents (such as methotrexate), or NSAIDs. In certain embodiments, the additional therapy may include combinations of different classes of therapeutic agents.

[0168] The viral vectors of this disclosure can be administered in combination with an ICI. The ICI may be selected from, for example, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, adenosine A2A receptor inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, BTLA inhibitors, KIR inhibitors, LAG3 inhibitors, TIM-3 inhibitors, VISTA inhibitors, and TIGIT inhibitors.

[0169] Exemplary ICIs include PD-1 inhibitors (e.g., anti-PD-1 antibodies, e.g., pembrolizumab, nivolumab, or semipirimab), PD-L1 inhibitors (e.g., atezolizumab, avelumab, or durvalumab), CTLA-4 inhibitors (e.g., ipilimumab or tremelimumab), and LAG-3 inhibitors (e.g., relatlimab).

[0170] ICIs can be PD-1 or PD-L1 inhibitors. PD-1 is a receptor present on the surface of T cells that functions as a checkpoint in the immune system, inhibiting or regulating T cell activity at the appropriate time to prevent excessive immune responses. However, cancer cells can exploit this checkpoint by expressing ligands that interact with PD-1 on the surface of T cells, such as PD-L1, to halt or regulate T cell activity. Exemplary PD-1 / PD-L1-based ICIs include antibody-based therapies. Exemplary therapies using PD-1 / PD-L1-based ICIs are described in U.S. Patents 8,728,474 and 9,073,994, and EP Patent 1537878B1, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, for example, in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patents 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149. Examples of anti-PD-L1 antibodies include, for example, atezolizumab (Tecentriq®, Genentech), durvalumab (AstraZeneca), MEDI4736, avelumab, and BMS936559 (Bristol Myers Squibb Co.).

[0171] The viral vectors of this disclosure can be administered in combination with CTLA-4 inhibitors. In the CTLA-4 pathway, the interaction between CTLA-4 on T cells and its ligands (e.g., CD80, also known as B7-1, and CD86) on the surface of antigen-presenting cells (not cancer cells) results in T cell inhibition. Exemplary CTLA-4-based ICI methods are described in U.S. Patents 5,811,097, 5,855,887, and 6,051,227. Exemplary anti-CTLA-4 antibodies are U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910, 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, and 8,143,379. This is described in Patent Nos. 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984, International Publication (PCT) Nos. WO98 / 42752, WO00 / 37504, and WO01 / 14424, and European Patent No. EP1212422B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0172] The viral vectors of this disclosure can be administered in combination with (i) a PD-1 or PD-L1 inhibitor, for example, a PD-1 or PD-L1 inhibitor disclosed herein, and (ii) a CTLA-4 inhibitor, for example, a CTLA-4 inhibitor disclosed herein.

[0173] The viral vectors of this disclosure can be administered in combination with an IDO inhibitor. Examples of IDO inhibitors include 1-methyl-D-tryptophan (known as Indoximod), epacadostat (INCB24360), Navoximod (GDC-0919), and BMS-986205.

[0174] Exemplary cytotoxic agents that can be administered in combination with the methods or compositions described herein include, for example, antimicrotubule agents, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors, alkylating agents, platinumating agents, nucleic acid synthesis inhibitors, histone deacetylase inhibitors (HDAC inhibitors), such as vorinostat (SAHA, MK0683), entinostat (MS-275), panobinostat (LBH589), and trichostatin. Examples of cytotoxic agents that can be administered by the methods or compositions described herein include A(TSA), Mocetinostat (MGCD0103), Belinostat (PXD101), Romidepsin (FK228, depsipeptide), DNA methyltransferase inhibitors, nitrogen mustard, nitrosourea, ethyleneimine, alkyl sulfonates, triazenes, folic acid analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epotilone, intercalating agents, agents that can interfere with signaling pathways, agents that promote apoptosis and radiation, or antibody molecular conjugates that bind to surface proteins to deliver toxic agents. In one embodiment, cytotoxic agents that can be administered by the methods or compositions described herein include platinum-based agents (such as cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluoro These include uracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacytidine, vorinostat, ixabepyrone, bortezomib, taxanes (e.g., paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vinorelbine, cortisine, anthracyclines (e.g., doxorubicin or epirubicin), daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, adriamycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, lysine, or maytansinoids.

[0175] In some embodiments, the provided composition comprises one or more such other activators. In some embodiments, such other activators are provided as part of a separate composition. In some embodiments, the combination therapy comprises the simultaneous administration of one or more doses or units of two or more different activators and / or modes of treatment. In some embodiments, the combination therapy comprises simultaneous exposure to two or more different activators and / or modes of treatment, for example, by a duplicate dosing regimen.

[0176] In some embodiments, the provided composition includes, or is administered in combination with, one or more other active agents, such as ICIs, that are useful for treating the associated disease, disorder, and / or condition.

[0177] While the implementation of the present invention will be better understood from the examples described herein, these examples are presented herein for illustrative purposes only and should not be construed as limiting the invention in any way. [Examples]

[0178] The following examples are given for illustrative purposes to illustrate various embodiments of the present invention and do not limit the invention in any way. These examples, together with the methods described herein, are representative and illustrative of currently preferred embodiments and do not limit the scope of the invention. Modifications and other uses therein that fall within the scope of the spirit of the invention as defined by the claims will be made by those skilled in the art.

[0179] Standard techniques can be used for recombinant DNA and oligonucleotide synthesis, as well as tissue culture and transformation (e.g., electroporation and lipofection). Enzymatic reactions and purification techniques may be carried out according to the manufacturer's specifications, as commonly achieved in the art, or as described herein. The aforementioned techniques and procedures may generally be carried out according to conventional methods well known in the art, as described in various general and more specific literature cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)).

[0180] Example 1: IFN-γ payload in HSV-1 mICP4 / mICP47 vector for tumor lysis This example describes the induction of IFN-γ response gene characteristics and the evaluation of tumor lysis by the HSV-1 mICP4 / mICP47 vector encoding the IFN-γ payload (HSV-1 mICP4 / mICP47 IFN-γ).

[0181] Hs578T human breast cancer cells were either uninfected or infected with HSV-1 mICP4 / mICP47 IFN-γ (3-10 plaque-forming units (PFUs) / cell), and the gene expression profile related to IFN-γ response characteristics was evaluated (Figure 1). Cell viability was assessed using the RealTime-Glo® assay (RealTime-Glo® MT Cell Viability Assay (Promega Corp., Madison, WI)) (Figure 2).

[0182] Hs578T cells were infected with 3 PFU / cell of HSV-1 mICP4 / mICP47 GFP or HSV-1 mICP4 / mICP47 IFN-γ and co-cultured with peripheral blood mononuclear cells (PBMCs) for 24 hours. Changes in the PBMC population were visualized using t-sne (t-distribution type stochastic neighborhood embedding) analysis and evaluated by flow cytometry. Dimensionality reduction (t-sne analysis) revealed robust phenotypic changes in several immune cell populations associated with HSV-1 mICP4 / mICP47 IFN-γ infection (data not shown).

[0183] In summary, these results indicate that the HSV-1 mICP4 / mICP47 IFN-γ vector mediates potent IFN-γ response gene properties, and that HSV-1 mICP4 / mICP47 IFN-γ infection mediates tumor lysis in a dose-dependent manner.

[0184] Example 2: mCXCL13, mCCL19, and mIL-7 payload in HSV-1 mICP4 / mICP47 vector for tumor lysis. This example describes the vector properties and evaluation of tumor lysis of three HSV-1 mICP4 / mICP47 vectors encoding mouse (m)CXCL13, mCCL19, and mIL-7, respectively (HSV-1 mICP4 / mICP47 mCXCL13, HSV-1 mICP4 / mICP47 mCCL19, and HSV-1 mICP4 / mICP47 mIL-7).

[0185] Hs578T human breast cancer cells were either uninfected or infected with HSV-1 mICP4 / mICP47 mCXCL13, HSV-1 mICP4 / mICP47 mCCL19, or HSV-1 mICP4 / mICP47 mIL-7 (0 PFU / cell, 3 PFU / cell, or 10 PFU / cell), and payload production rates were evaluated. As shown in Figure 3, payload production of mCXCL13, mCCL19, and mIL-7 increased in a dose-dependent manner, with both the 3 PFU / cell and 10 PFU / cell groups showing maximum production within 2 days after infection. Cell viability was evaluated using the RealTime Glo® MT cell viability assay, in which the luciferase reaction occurs in the culture medium. Luciferase and a proprietary viable substrate were added to the cell culture medium, and the viable substrate diffused into the cells, where it was reduced to a luciferase substrate, which then exited the cells and was rapidly utilized by luciferase in the culture medium. Only metabolically active cells reduced the substrate, and photoproduction was proportional to the number of viable cells in culture. Dead cells did not reduce the substrate. As shown in Figure 4, cell viability decreased with increasing vector dose, and HSV-1 mICP4 / mICP47 mCXCL13, HSV-1 mICP4 / mICP47 mCCL19, and HSV-1 mICP4 / mICP47 mIL-7 were shown to mediate tumor lysis in a dose-dependent manner.

[0186] Example 3: Anti-CD47 antibody payload in HSV-1 mICP4 / mICP47 vector for phagocytosis and tumor lysis The CD47 / SIRPα axis mediates a "don't ingest" signal that tumor cells use to evade macrophage-mediated immune surveillance (Figure 5). Anti-CD47 therapy has shown promising clinical outcomes in solid and hematological malignancies, but its efficacy is hindered by systemic toxicity. This example describes the evaluation of enhanced phagocytic and tumor-dissolving effects of an HSV-1 mICP4 / mICP47 vector (HSV-1 mICP4 / mICP47 macro1) with a Sirpα-IgG fusion polypeptide ("macro1") as its payload.

[0187] M1 macrophages were matured by culturing PBMCs containing 50 ng / mL macrophage colony-stimulating factor (m-CSF) with IFN-γ stimulation (50 ng / mL, 48 hours) for 8 days. Carboxyfluorescein succinimidyl (CFSE)-labeled Raji cells were cultured in Hs578T human breast cancer cell-derived modified medium (CM) infected with HSV-1 mICP4 / mICP47 macro1, or a control payload (GFP and unrelated antibodies; 3 PFU / cell and 10 PFU / cell, 48 hours), or 40 μg / mL isotype control (IgG), or anti-CD47 antibody, before co-culturing with M1 macrophages for 3 hours. Phagocytosis was quantified using flow cytometry analysis of cells positive for both CD14 and CSFE (Figure 6A-6D) (Figure 7). As shown in Figure 7, CM derived from HSV-1 mICP4 / mICP47 macro1-infected cells resulted in a dose-dependent increase in macrophage-mediated target cell phagocytosis, which was greater than the effect of anti-CD47 antibody. Furthermore, pretreatment with CM of an HSV-1 mICP4 / mICP47 vector encoding an unrelated payload did not significantly affect macrophage phagocytosis, demonstrating the specificity of the therapeutic payload response.

[0188] To test the antitumor effect of HSV-1 mICP4 / mICP47 macro1 in vivo, a mouse lung cancer model was used and HSV-1 treatment was administered as shown in the schematic diagram provided in Figure 8. Briefly, 5 × 10 6 A549 lung cancer cells were injected into the posterior ventral region of Balb / c nude mice. The tumor was approximately 100 mm in size. 3 Upon reaching a certain weight, intratumoral (it) administration of HSV-1 mICP4 / mICP47 macro1 (3 × 10⁷ PFU) or intraperitoneal (ip) administration of anti-CD47 antibody or isotype control antibody (10 mg / kg) was performed on days 1, 4, 7, 10, 13, and 14 (Figure 8), and body weight was assessed at the endpoint (Table 1). The results showed that HSV-1 mICP4 / mICP47 macro1 was well-tolerated. [Table 1]

[0189] The volume of A549 tumors was measured in response to various treatments (Figures 9A and 9B). Treatment with HSV-1 mICP4 / mICP47 macro1 resulted in suppression of tumor growth compared to the control group. This effect was dependent on the expression of the payload, and HSV-1 mICP4 / mICP47 GFP did not show statistically significant antitumor activity. Topical administration of HSV-1 mICP4 / mICP47 macro1 tended to show a higher efficacy compared to systemic administration of anti-CD47 antibody.

[0190] Tumor sections of A549 were collected on day 12 and stained with F4 / 80 to visualize intratumoral macrophages, but no change was observed in intratumoral macrophage infiltration (data not shown).

[0191] Example 4: Evaluation of the activation pathway after immune checkpoint inhibitor treatment or HSV-1 mICP4 / mICP47 vector infection. This example describes the evaluation of the pathways activated by HSV-1 mICP4 / mICP47 infection.

[0192] Computational analysis using RNA-seq datasets from all patients (Gide et al. (2019) Cancer Cell, 35(2):238-255.e6; Hugo et al. (2016) Cell, 165(1):35-44; Liu et al. (2019) Nat Med., 25(12):1916-1927; Riaz et al. (2017) Cell, 171(4):934-949.e16; and Van Allen et al. (2015) Science, 350(6257):207-211) defined statistical associations between tumor microenvironment (TME) characteristics / viability and TME characteristics / protein expression, which were identified as potential payloads for the HSV-1 mICP4 / mICP47 vector described herein. Next, the p-values ​​for TME vs. viability and TME vs. payload were combined to calculate the score as a -log10 meta-p-value (Figure 10). These analyses revealed that approximately half of the top 25 genes predicted to be driving forces of the treatment response to immune checkpoint inhibitor (ICI) therapy were factors reported to play a role in TLS formation, including CXCL13, IL-17a, LTA, LTB, CCL19, CCL21, and several TNF superfamily members.

[0193] In another study, using patient datasets from the SU2C-MARK cohort (Stand Up To Cancer-Mark Foundation non-small cell lung cancer (NSCLC) cohort) (Ravi et al. (2023) Nature Genetics 55.5:807-819), the top pathways showing decreased or increased expression in response to ICI (Figure 11A) or HSV-1 mICP4 / mICP47 GFP infection (Figure 11B) were identified by computer analysis. Similarities were observed in the induction of MYC and E2F targets, as well as the G2M cell cycle checkpoint response (normalized enrichment scores of 2.4, 3.0, and 3.2, respectively). These are pathways known to be associated with antitumor immune responses to ICI. Correlation analysis based on the identified pathways revealed a positive correlation between a characteristic Gene Set Enrichment Analysis (GSEA) pathway associated with the ICI response and a pathway controlled by HSV-1 mICP4 / mICP47 GFP infection (Figure 12), suggesting that the HSV-1 mICP4 / mICP47 vector organized changes in the tumor microenvironment that supported the ICI response.

[0194] In another similar study, computer analysis identified the top GSEA pathways upregulated in response to HSV-1 mICP4 / mICP47, including the IFN-γ response, IL-6 / JAK / STAT3 signaling, NF-κB-mediated TNF-α signaling, inflammatory response, and IFN-α response (Figure 20).

[0195] In another study, Hs578T cells were infected with either 10 PFU / cell of HSV-1 mICP4 / mICP47 or a control vector (HSV-1 mICP27). Flow cytometry was used to measure cell surface expression of MHC class I as an indicator of antigen presentation 24 hours post-infection (Figure 19). Hs578T cells infected with HSV-1 mICP4 / mICP47 showed a higher concentration of MHC class I-positive cells.

[0196] Example 5: PD-1 payload in HSV-1 mICP4 / mICP47 vector combined with an immune checkpoint inhibitor for tumor lysis This example describes the evaluation of HSV-1 mICP4 / mICP47 vector-mediated infection in combination with immune checkpoint inhibitor (ICI) antibody administration to reduce tumor size in vivo.

[0197] First, 5 x 10 5 Individual mammary gland cancer EMT6 cells were injected into the posterior ventral region of Balb / c mice. The tumor was approximately 100 mm. 3 After reaching this stage, the HSV-1 mICP4 / mICP47 GFP vector (3 × 10 7 PFU was administered intratumorally in combination with intraperitoneal administration of an anti-PD-1 antibody or an isotype control antibody RMP1-14 (10 mg / kg each). HSV-1 mICP4 / mICP47 GFP showed a slight antitumor effect as measured by tumor volume, but the effect of combination therapy (HSV-1 mICP4 / mICP47 GFP and anti-PD-1 antibody) was still slightly greater (Figure 13).

[0198] Example 6: Evaluation of single or multi-gene payloads This example describes the design and evaluation of a viral vector having a single gene payload, or multiple viral vectors, each encoding a single payload, thereby evaluating multi-gene payloads for reducing tumor size.

[0199] For example, based on the payload identified by the method described in Example 4, the payload was tested in vitro using a multiplex assay. Briefly, EMT6 mammary tumor cells were subcutaneously transplanted into the posterior ventral region of Balb / c mice. The tumor was approximately 100 mm. 3 Once it reaches this point, the mouse is placed in a medium (control) or 3x10 7PFU of HSV-1 mICP4 / mICP47 GFP; or a combination of HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 mCCL19, HSV-1 mICP4 / mICP47 mIL-7, HSV-1 mICP4 / mICP47 mTNF, and HSV-1 mICP4 / mICP47 mIL-17a ("Multiplex A"; 6×10 for each vector 6 PFU, total dose 3×10 7 PFU); or a combination of HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 mCCL19, HSV-1 mICP4 / mICP47 mIL-7, HSV-1 mICP4 / mICP47 mCCL21, HSV-1 mICP4 / mICP47 mCXCL13, and HSV-1 mICP4 / mICP47 mLTB ("Multiplex B"; 6×10 for each vector 6 PFU, total dose 3×10 7 PFU) were randomly assigned to the intratumoral injection group. Treatments were performed on day 1, day 4, day 7, and day 10 after randomization, and tumor growth was monitored over time (Figures 14A-14B). The HSV-1 mICP4 / mICP47 GFP virus backbone alone slightly reduced tumor growth, while administration of Multiplex A and Multiplex B, which express multiple factors involved in TLS formation, showed significant antitumor effects. Both Multiplex A and Multiplex B exhibited tumor growth inhibition compared to vehicle control mice (Multiplex A: 39.8±10.5%, p=0.004, Multiplex B: 38.6%±12.1, p=0.011, n=8 per group).

[0200] In another experiment, mice were treated with vehicle (control), 3×10 7PFU's HSV-1 mICP4 / mICP47 GFP, or combinations of HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 mCCL19, HSV-1 mICP4 / mICP47 mIL-7, HSV-1 mICP4 / mICP47 mTNF, and HSV-1 mICP4 / mICP47 mIL-17a ("Multiplex A"; 6 × 10¹⁶ vectors each) 6 PFU, total dose 3 × 10 7 Participants were randomly assigned to receive a combination of PFUs (Plant-Focused Uninfected Fuels) via intubation into the submandibular gland. Gland tissue was collected and analyzed by flow cytometry 15 days later. The HSV-1 mICP4 / mICP47 GFP viral backbone alone enhanced immune infiltration into infected glands, as identified by a significant increase in the proportion of CD45-positive cells compared to the excipient control (Figure 15). Histological collection of glands revealed the presence of clusters of CD11c-positive dendritic cells and CD4-positive T cells, demonstrating early TLS induction (data not shown). In the Multiplex A group, compared to the control HSV-1 mICP4 / mICP47 GFP vector, the number, size, and organization of TLS were increased, as indicated by staining with CD11c for dendritic cells, CD4 for T cells, and B220 for B cells (data not shown). Furthermore, in the Multiplex A group, TLS induction was accompanied by the novel expression of mature fibroblast reticular cells and CCL21 (as shown by staining fibroblast reticular cells co-localized with CD4 on T cells with GP38), as well as the novel expression of CCL21 co-localized with GP38 in fibroblast-like reticular cells and with B220 in B cells (data not shown).

[0201] In summary, these data demonstrate that the expression of a complex factor mediated by the virus induces TLS formation and antitumor immunity. This antitumor immunity is thought to have arisen through a combination of viral vector-dependent and payload-dependent effects.

[0202] In another series of experiments, Hs578T breast cancer cells were single- or multiple-infected (total 1 PFU / cell) with HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 IFN-γ, or other vectors encoding the payload, and then cultured for 72 hours with or without PBMCs ("T cell activators"). The viability of the tumor cells was evaluated by flow cytometry (Figure 16). Specific payload combinations, such as IL-15 ("B"), IFN-γ+IL-12 ("E"), IFN-γ+IL-15 ("F"), IL-12+IL-15 ("G"), IL-15+IL-21 ("H"), IFN-γ+IL-12+IL-15 ("I"), and IFN-γ+IL-12+IL-15+IL-21 ("J"), were shown to enhance PBMC-mediated cancer cell killing. Lasso regression analysis was used to identify immune cell populations that showed a positive correlation (right x-axis) or negative correlation (left x-axis) with PBMC-mediated tumor cell death (Figure 17).

[0203] Example 7: Evaluation of infection using an HSV-1 mICP4 / mICP47 vector expressing a tertiary lymphoid structure-related payload. Promoting TLS formation in inhibitory TMEs is expected to improve the response to ICIs and ultimately impact patient survival. However, there are limited therapeutic approaches developed and tested to successfully induce TLS formation in tumors. This example describes a novel HSV-1 mICP4 / mICP47 vector expressing a data-selected combination of factors that control TLS formation.

[0204] For example, a gene selection payload is selected and tested based on the payload identified by the method described in Example 4. For example, to induce tumor control via TLS, two combinations of HSV-1 mICP4 / mICP47 vectors can be used: Multiplex A (HSV-1 mICP4 / mICP47 LTA, HSV-1 mICP4 / mICP47 CCL19, HSV-1 mICP4 / mICP47 TNF, HSV-1 mICP4 / mICP47 IL-17, and HSV-1 mICP4 / mICP47 IL-7; or an HSV-1 mICP4 / mICP47 vector expressing LTA, CCL19, TNF, IL-17, and IL-7), and Multiplex B (HSV-1 mICP4 / mICP47 LTB, HSV-1 mICP4 / mICP47 CCL19, HSV-1 mICP4 / mICP47 CCL21, HSV-1 mICP4 / mICP47 CXCL13 and HSV-1 mICP4 / mICP47 IL-7 (or an HSV-1 mICP4 / mICP47 vector expressing LTB, CCL19, CCL21, CXCL13, IL-7) were selected and tested subcutaneously in 80 Balb / c mice (6-9 weeks old) with EMT6 tumors to define their ability to induce the formation of phenotypic mature TLS. Both the standard medium and GFP-expressing vectors were used as controls. One or more combinations of therapeutic polypeptides may be tested, including hyaluronidase, MMP-9, lysyl oxidase inhibitors, TNF, IL-1β, IL-6, IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesoserine, CD40L, OX40L, lymphotoxin β, CXCL13, Sirpα-IgG fusion transgene, anti-CD47 antibody, IL-1, IL-17, IL-21, LTA, LTB, CXCL12, or VEGF-C, or combinations thereof. Each experimental group will contain 20 tumor-bearing mice (10 females and 10 males). After randomization, the test substance (1 × 10⁶) will be administered on days 1, 4, and 7. 7 PFU (20 μL DPBS, 10% glycerol) was injected into the tumor. Tumor growth was monitored twice a week with calipers, body weight was measured, and clinical findings were recorded.

[0205] In another series of experiments, an intraperitoneal tumor development model (Rodriguez et al. (2021) Cell Rep, 36(3):109422) was used to model tumor lesions tolerant to TLS induction (e.g., lymph node metastasis). Additionally, yet another model has been tested: a genetically modified mouse model with slow tumor progression (Joshi et al. (2015) Immunity, 43(3):579-90).

[0206] Half of the animals in each group were euthanized on day 15 (time point 1), and the remaining animals were euthanized when their tumors reached 2,000 mm. 3Euthanasia was performed at the point in time (time point 2) to assess the time-dependent effects on TLS maturation and stability. At the end of the study, tumors, discharging lymph nodes, and blood (for plasma and blood cell isolation) were collected and analyzed. Histological and morphological characterization of TLS was completed using immunofluorescence staining for common lymphocyte markers of TLS maturation (T / B cells), follicular dendritic cell formation, secretion of survival factors, and expression of lymphoid chemokines, as well as the formation of high endothelial veins using fluorescently labeled antibodies such as CD3, CD4, CD19, B220, CD11c, CD31, podoplanin, CCL21, and CXCL13. Evaluation of tumors and tumor discharging lymph nodes based on flow cytometry was performed to measure viral infection, tumor composition, and payload expression. A set of genes associated with TLS characteristics were quantified in available samples by qPCR analysis. Immunophenotypic analysis based on flow cytometry of hematopoietic cells, including CD4-positive and CD8-positive T cells, T regulatory cells, B cells, NK cells and NKT cells, monocytes, and dendritic cells, was used to define changes in the peripheral immune response. TLS formation was detected within the tumor mesenteric cell (TME). Histological and morphological analyses revealed evidence of T / B cell region segregation and germinal center formation, confirming the formation of mature TLSs. Flow cytometry analysis demonstrated maturation of T and B cell responses, and transcriptome analysis confirmed genetic characteristics consistent with TLS formation. Phenotypic changes within the tumor were consistent with the progression of tumor lysis, manifested by a reduction in the area of ​​tumor-positive cells (histological) and a decrease in the number of tumor cells (flow cytometry). These changes were associated with an increase in the density of tumor-infiltrating lymphocytes (TILs) in the TME. In summary, these data characterize TLSs, their temporal evolution in tumors, and evaluate their relationship to phenotypic and transcriptional changes within tumors, peripheral immune responses, and their tumor burden.

[0207] Combination therapy with ICI will also be tested (see Example 8).

[0208] Example 8: Evaluation of tumor regression using HSV-1 mICP4 / mICP47 vectors expressing various payloads. This example describes the in vivo efficacy of a novel HSV-1 mICP4 / mICP47 vector expressing a data-selected payload.

[0209] A library of HSV-1 mICP4 / mICP47 vectors was constructed, each encoding one or more genes, each expressing one of the computer-predicted payload components (see, for example, Example 4 and Figure 10; e.g., IL-12, IL-15, IL-21, and IFN-γ).

[0210] As shown in Figure 21, IFN-γ and IL-12 payload production increased in a dose-dependent manner, with both the 3 PFU / cell and 10 PFU / cell groups showing maximum production within 2 days post-infection. Cell viability was assessed using the RealTime Glo® MT cell viability assay, in which the luciferase reaction occurs in the culture medium. Luciferase and a proprietary viable substrate were added to the cell culture medium, and the viable substrate diffused into the cells, where it was reduced to a luciferase substrate, which was then rapidly utilized by luciferase in the culture medium after exiting the cells. Only metabolically active cells reduced the substrate, and photoproduction was proportional to the number of viable cells in culture. Dead cells did not reduce the substrate. As shown in Figure 21 (right panel), cell viability decreased with increasing vector dose, indicating that HSV-1 mICP4 / mICP47 IFN-γ and HSV-1 mICP4 / mICP47 IL-12 mediate tumor lysis in a dose-dependent manner.

[0211] The ability of top-scoring viral gene constructs to induce immune activation and cancer cell death activity was tested using, for example, the method described in Example 6, with measurements based on an exovivo cancer / peripheral blood mononuclear cell (PBMC) co-culture system and flow cytometry. In vivo antitumor activity was evaluated using Hs578T, EMT6, and LLC1 tumor-bearing mice, using the methods described throughout the examples.

[0212] Infection of cancer cells with the HSV-1 mICP4 / mICP47 vector encoding a single gene demonstrated infection and payload-dependent induction of cancer cell killing via PBMCs (Figures 22A-22B). Cell death was associated with significant changes in the composition and function of immune cells in co-cultures (Figure 23). Correlation analysis identified payload-mediated regulation of multiple immune cell populations, accompanied by expansion and activation of NK cell-positive and CD8-positive T cell populations, which correlated with effective PBMC-mediated cancer cell killing (Lasso regression, α=0.1, R2=0.79) (Figure 24). Measurements of dendritic cells and Ki67-positive / Gzmb-positive CD8-positive T cells (Figure 25), as well as Ki67-positive and PD1-positive CD8-positive T cells (Figures 27A-27B), also revealed payload-mediated regulation of multiple immune cell populations. Subsequently, the combination of IL-12 and IL-15 was preferred for in vivo testing, and intratumor therapy with HSV-1 mICP4 / mICP47 vectors encoding IL-12 and IL-15 was shown to result in significant suppression of tumor growth in both tumor models tested (EMT6: 60.0% ± 12.6, p=0.002; LLC1: 37.2% ± 9.4, p=0.011) (Figures 26A-26D). Tumor regression was observed in 25% of mice with EMT6 tumors. Immune activation, both intratumor and peripheral, was associated with antitumor activity. In summary, these results demonstrate that a multi-gene payload, specifically IL-12 and IL-15, is identified through a multi-parameter determination matrix and possesses a synergistic antitumor effect.

[0213] Example 9: Evaluation of infection with HSV-1 mICP4 / mICP47 vector alone or in combination with anti-PD-1 therapy. The top-performing vectors from Example 7 will be tested alone and in combination with anti-PD-1 antibody therapy (clones RMP1-14 supplied from BioXCell). The experimental groups will include: 1. Medium + Isotype control 2. HSV-1 mICP4 / mICP47 GFP+ isotype control 3. Multiplex A from Example 6 or Example 7, or vector + isotype control from Example 7. 4. Multiplex B from Example 6 or Example 7, or vector + isotype control from Example 7. 5. Vehicle + anti-PD1 6.HSV-1 mICP4 / mICP47 GFP+anti-PD1 7. Multiplex A from Example 6 or Example 7, or vector + anti-PD1 from Example 7 8. Multiplex B from Example 6 or Example 7, or vector + anti-PD1 from Example 7.

[0214] Each experimental group contains eight tumor-bearing mice. Viral vector therapy was indicated by tumor caliper measurements three times a week when the tumor size was approximately 100 mm. 3 From the point at which this was reached, on days 1, 4, 7, and 10, 10% glycerol-containing DPBS was used as a medium control, with 1 × 10 in 20 μL of 10% glycerol-containing DPBS. 7 PFU was injected into the tumor. Anti-PD-1 antibody therapy was administered as 200 μg in PBS twice weekly intraperitoneally for two weeks, with rat IgG2a in PBS used as the isotype control. The endpoint of the study was when the tumor reached 1000 mm. 3 The endpoint is reached either (to allow time for the immunological response to progress) or three weeks, whichever comes first. Endpoint analysis includes histological analysis, morphological analysis, flow cytometry, and qPCR / RNASeq analysis, as is common in the art.

[0215] Using a similar experimental design, additional studies will be conducted on anti-CTLA-4 antibody therapy and anti-PD-1 + anti-CTLA-4 antibody therapy in combination with the vector(s) of Example 7.

[0216] Furthermore, experiments will be conducted to evaluate the timing of vector-mediated TLS induction by sequential treatment with the vector(s) from Example 7 and anti-PD-1 antibody therapy, anti-CTLA-4 antibody therapy, or anti-PD-1 + anti-CTLA-4 antibody therapy (for example, vector followed by antibody versus antibody followed by vector).

[0217] The addition of ICI therapy enhances the antitumor effects of multiple or multiple vectors in Examples 6 and 7, which is expected to be associated with immune-mediated tumor control and changes in TME indicating peripheral immune activation.

[0218] Example 10: Evaluation of survival after HSV-1 mICP4 / mICP47 vector infection in combination with anti-PD-1 therapy. This embodiment describes the evaluation of combinations of viral vectors, each encoding a payload, thereby evaluating multi-gene payloads for reducing tumor size.

[0219] EMT6 mammary gland tumor cells were subcutaneously transplanted into the posterior ventral region of Balb / c mice. The tumor was approximately 100 mm in size. 3 Once the mice reach a certain stage, they are randomly grouped into two groups and given either a medium or a combination of individual vectors: HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 mCCL19, HSV-1 mICP4 / mICP47 mIL-7, HSV-1 mICP4 / mICP47 mTNF, and HSV-1 mICP4 / mICP47 mIL-17a ("Multiplex A"; "6 × 10" for each vector). 6 PFU, total dose 3 × 10 7 PFU); or combinations of individual vectors HSV-1 mICP4 / mICP47 GFP, HSV-1 mICP4 / mICP47 mCCL19, HSV-1 mICP4 / mICP47 mIL-7, HSV-1 mICP4 / mICP47 mCCL21, HSV-1 mICP4 / mICP47 mCXCL13, and HSV-1 mICP4 / mICP47 mLTB ("Multiplex B"; "Each vector 6 × 10 6 PFU, total dose 3 × 107 Intratumoral injection of PFU was administered. Treatment was performed on days 1, 4, 7, and 10 after randomization. In combination with vector administration, mice were treated with anti-PD-1 antibody (clone: ​​RMP1-14) or isotype control antibody (IgG, 10 mg / kg), and tumor growth (Figure 18) and survival (Table 2) were monitored over time. Combination therapy of Multiplex A and Multiplex B with anti-PD-1 antibody increased the number of long-term survivors (over 40 days) compared to the control group. [Table 2]

[0220] In summary, these experiments demonstrated the in vivo antitumor effect of the HSV-1 mICP4 / mICP47 vector and its multiplexed payload, which control TLS induction, through improved long-term survival.

[0221] Embedding by reference The entirety of the disclosures in each patent document and scientific paper referenced herein is incorporated for reference purposes only.

[0222] Equal parts The present invention can be embodied in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described herein are considered illustrative in all respects and not limiting the invention as described herein. Accordingly, the scope of the invention is indicated not by the foregoing description but by the appended claims, and all modifications that fall within the meaning and equivalence of the claims are intended to be encompassed therewith. Sequence List Sequence ID 1 - GenBank Acceptance Number: JQ730035.1 starting point 1 gcagcccggg ccccccgcgc gcggggcggc gcgcaaaaaa ggcgggcggc ggtccgggcg 61 gcgtgcgcgc gcgcggcggg cgtggggggc ggggccgcgg gagcggggga ggagccccac 121 ccacagacgg ggaggagcgg gggaggagcg ggggaggagc gggggaggag ccccacccac 181 agacggggag gagcggggga ggagcggcca gaccccaaaa acgggccccc ccgaaacaca 241 ccccccgggg gtcgcgcgcg gccctttaaa gcgcggcggc gggcagcccg ggccccccgc 301 ggccgagact agcgagttag acaggcaagc actactcgcc tctgcacgca catgcttgcc 361 tgtcaaactc taccaccccg gcacgctctc tgtctccatg gcccgccgcc gccgccatcg 421 cggcccccgc cgcccccggc cgcccgggcc cacgggcgcc gtcccaaccg cacagtccca 481 ggtaacctcc acgcccaact cggaacccgc ggtcaggagc gcgcccgcgg ccgccccgcc 541 gccgcccccc gccggtgggc ccccgccttc ttgttcgctg ctgctgcgcc agtggctcca 601 cgttcccgag tccgcgtccg acgacgacga tgacgacgac tggccggaca gccccccgcc 661 cgagccggcg ccagaggccc ggcccaccgc cgccgccccc cggccccggt ccccaccgcc 721 cggcgtgggc ccggggggcg gggctgaccc ctcccacccc ccctcgcgcc ccttccgcct 781 tccgccgcgc ctcgccctcc gcctgcgcgt caccgcggag cacctggcgc gcctgcgcct 841 gcgacgcgcg ggcggggagg gggcgccgga gccccccgcg accccgcga cccccgcgac 901 ccccgcgacc cccgcgaccc ccgcgacccc cgcgacccccc gcgaccccg cgcgggtgcg 961 cttctcgccc cacgtccggg tgcgccacct ggtggtctgg gcctcggccg cccgcctggc 1021 gcgccgcggc tcgtgggccc gcgagcgggc cgaccgggct cggttccggc gccgggtggc 1081 ggaggccgag gcggtcatcg ggccgtgcct ggggcccgag gcccgtgccc gggccctggc 1141 ccgcggagcc ggcccggcga actcggtcta acgttacacc cgaggcgcct gggtcttccg 1201 cggagctccc gggagctccg caccaagccg ctctccggag agacgatggc aggagccgcg 1261 catatatacg ctgggagccg gtccgccccc aaggcgggcc cgcctcgggg gcgggactgg 1321 ccaatcggcg gccgccagcg cggcggggcc cggccaacca gcgtccgccg agtcttcggg 1381 gcccggccca ttgggcgga gttaccgccc aatgggccgg gccgcccact tcccggtatg 1441 gtaattaaaa acttgcaaga ggccttgttc cgcttcccgg tatggtaatt agaaactcat 1501 taatgggcgg ccccggccgc ccttcccgct tccggcaatt cccgcggccc ttaatgggca 1561 accccggtat tccccgcctc ccgcgccgcg cgtaaccact cccctggggt tccgggttat 1621 gctaattgct tttttggcgg aacacacggc ccctcgcgca ttggcccgcg ggtcgctcaa 1681 tgaacccgca ttggtcccct ggggttccgg gtatggtaat gagtttcttc gggaaggcgg 1741 gaagccccgg ggcaccgacg caggccaagc ccctgttgcg tcggcgggag gggcatgcta 1801 atggggttct ttgggggaca ccgggttggt cccccaaatc gggggccggg ccgtgcatgc 1861 taatgatatt ctttgggggc gccgggttgg tccccgggga cggggccgcc ccgcggtggg 1921 cctgcctccc ctgggacgcg cggccattgg gggaatcgtc actgccgccc ctttggggag 1981 gggaaaggcg tggggtataa gttagccctg gcccgacggt ctggtcgcat ttgcacctcg 2041 gcactcggag cgagacgcag cagccaggca gactcgggcc gccccctctc cgcatcacca 2101 cagaagcccc gcctacgttg cgacccccag ggaccctccg tccgcgaccc tccagccgca 2161 tacgaccccc atggagcccc gccccggagc gagtacccgc cggcctgagg gccgccccca 2221 gcgcgaggtg aggggccggg cgccatgtct ggggcgccat attgggggc gccatgttgg 2281 gggacccccg acccttaccc tggaaccggc ccccatgttg ggggaccccc actcatacac 2341 gggagccggg cgccatgttg gggcgccatg ttaggggcg tggaaccccg tgacactata 2401 tatacaggga ccgggggcgc catgttaggg ggcgcggaac cccctgaccc tatatataca 2461 gggaccgggg tcgccctgtt gggggtcgcc atgtgacccc ctgactttat atatacagac 2521 ccccaacaca tacacatggc ccctttgact cagacgcagg gcccggggtc gccgtgggac 2581 cccctgactc atacacagag acacgcccccc acaacaaaca cacagggacc ggggtcgccg 2641 tgttggggc gtggtcccca ctgactcata cgcaggcccc ccttactcac acgcatctag 2701 gggggtgggg aggagccgcc cgccatattt gggggacgcc gtgggacccc cgactccggt 2761 gcgtctggag ggcgggagaa gaggaagaa gaggggtcgg gatccaaagg acggacccag 2821 accacctttg gttgcagacc cctttctccc ccctcttccg aggccagcag gggggcagga 2881 ctttgtgagg cggggggga gaggggaac tcgtgggcgc tgattgacgc gggaaatccc 2941 cccccattct tacccgcccc ccttttttcc ccttagcccg ccccggatgt ctgggtgttt 3001 ccctgcgacc gagacctgcc ggacagcagc gactctgagg cggagaccga agtggggggg 3061 cggggggacg ccgaccacca tgacgacgac tccgcctccg aggcggacag cacggacacg 3121 gaactgttcg agacggggct gctggggccg cagggcgtgg atgggggggc ggtctcgggg 3181 gggagccccc cccgcgagga agaccccggc agttgcgggg gcgccccccc tcgagaggac 3241 ggggggagcg acgagggcga cgtgtgcgcc gtgtgcacgg atgagatcgc gccccacctg 3301 cgctgcgaca ccttcccgtg catgcaccgc ttctgcatcc cgtgcatgaa aacctggatg 3361 caattgcgca acacctgccc gctgtgcaac gccaagctgg tgtacctgat agtgggcgtg 3421 acgcccagcg ggtcgttcag caccatcccg atcgtgaacg acccccagac ccgcatggag 3481 gccgaggagg ccgtcagggc gggcacggcc gtggacttta tctggacggg caatcagcgg 3541 ttcgccccgc ggtacctgac cctggggggg cacacggtga gggccctgtc gcccacccac 3601 ccggagccca ccacggacga ggatgacgac gacctggacg acggtgaggc ggggggcggc 3661 aaggaccctg ggggagggg agggggg aatggggcgggg cggggcggg aggggcggggc 3721 cggggagggg gcgtaacctg atcgcgcccc ccgttgtctc ttgcagcaga ctacgtcccg 3781 cccgcccccc gccggacgcc ccgcgccccc cccgcagag gcaccgccgc gccccccgtg 3841 acggggcgggg cgtctaacgc agccccccag ccggccgcgg ctcggacagc gcccccctcg 3901 gcgcccatcg ggccacacgg cagcagtac accaacacca ccaccaacg cagcggcggc 3961 ggcggctccc gccagtcggcg agccggcggcg ccgcgggggg cgtctggccc ctccgggggg 4021 gttggggttg gggttggggt tgttgaagcg gaggcggggc ggccgagggg ccggacggggc 4081 ccccttgtca acagacccgc cccccttgca aacaacagag accccatagt gatcagcgac 4141 tcccccccgg cctctcccca caggcccccc gcggcgccca tgccaggctc cgccccccc 4201 cccggggcccc ccgcgtcctc ggccgcgtcg ggacccgcgc gccccgcc ggccgtggcc 4261 ccgtgcgtgc gagcgccgcc tccggggccc ggccccgccg ccccggcccc cggggcggag 4321 ccggccgccc gccccgcgga cggcgccgt gtgccccagt cgcactcgtc cctggctcag 4381 gccgcgaacc aagaacagag tctgtgccgg gcgcgtgcga cggtggcgcg cggctcgggg 4441 gggccgggcg tggagggtgg gcacgggccc tcccgcggcc gcaccccctc cggcgccgcc 4501 ccgctcccct ccgccgtctc tgtcgagcag gaggcggcgg tgcgtccgag gaagaggcgc 4561 gggtcgggcc aggaaaaccc ctccccccag tccacgcgtc cccccctcgc gccggcaggg 4621 gccaagaggg cggcgacgca ccccccctcc gactcagggc cgggggggcg cggccagggt 4681 gggcccggga cccccctgac gtcctcggcg gcctccgcct cttcctcctc tgcctcttcc 4741 tcctcggccc cgacccccgc gggggccgcc tcttccgccg ccggggccgc gtcctcctcc 4801 gcttccgcct cctcgggcgg ggccgtcggt gccctgggag ggagacaaga ggaaacctcc 4861 ctcggccccc gcgctgcttc tgggccgcgg gggccgagga agtgtgcccg gaagacgcgc 4921 cacgcggaga cttccggggc cgtccccgcg ggcggcctca cgcgctacct gcccatctcg 4981 ggggtctcta gcgtggtcgc cctgtcgcct tacgtgaaca agactatcac gggggactgc 5041 ctgcccatcc tggacatgga gacggggaac atcggggcgt acgtggtcct ggtggaccag 5101 acgggaaaca tggcgacccg gctgcgggcc gcggtccccg gctggagccg ccgcaccctg 5161 ctccccgaga ccgcgggtaa ccacgtgatg ccccccgagt acccgacggc ccccgcgtcg 5221 gagtggaaca gcctctggat gacccccgtg gggaacatgc tgttcgacca gggcacccta 5281 gtgggcgccc tggacttccg cagcctgcgg tctcggcacc cgtggtccgg ggagcagggg 5341 gcgtcgaccc gggacgaggg aaaacaataa gggacgcccc ccgtgtttgt ggggaggggg 5401 gtcgggtgct gggtggtctc tggccgcgcc cactacacca gccaatccgt gtcggggagg 5461 ggaaagtgaa agacacgggc accacacacc agcgggtctt tagtgttggc cctaataaaa 5521 aactcagggg atttttgctg tctattggga aataaaggtt tacttttgta tcttttccct 5581 gtctgtgttg gatggatctt gggggtgcgt gggagtgggg gtgcgtggga gtgggggtgc 5641 gtgggagtgg gggtgcgtgg gagtgggggt gcgtgggagt gggggtgcgt gggagtgggg 5701 gtgcgtggga gtgggggtgc gtgggagtgg gggtgcgtgg gagtgggggt gcgtgggagt 5761 gggggtgcgt gggagtgggg gtgcgtggga gtgggggtgc gtgggagtgg gggtgccatg 5821 ttgggcaggc tctggtgtta accacagagc cgcggcccgg gctgcctgac caccgatccc 5881 cgaaagcatc ctgccactgg catggagcca gaaccagat gggctgggtg tgggtgttaa 5941 gtttccgcga gcgcctgccc gcccggactg acctggcctc tggccgccac aaagggcggg 6001 gggggggtta actacactat agggcaacaa aggacgggag gggtggcggg acggggcgcc 6061 caaaaggggg tcggccacac cacagacgtg ggtgttgggg ggtggggcgg aggggtgggg 6121 gggagacaga aacaggaaca tagttagaaa acaagaatgc ggtgcagcca gagaatcaca 6181 ggagacgagg ggatgggcgt gttggttacc aacccacacc caggcatgct cggtggtatg 6241 aaggagggg ggcggtgctt cttagagacc gccgggggac gtggggttgg tgtgcaaagg 6301 cacgcgcacc cgcgtcggcc aggtgggccg gtaccccatc cccccctccc ccgacccttc 6361 cccccccgcg tgccagagat cacccccgtc ccccggcacc cgccactcct ccatatcctc 6421 gctttaggaa caactttggg gggggggtac acacgcgccg tgcatttcct tccacacccc 6481 ccctcccccg catcccccccc cccaggcagt aagacccaag catatagagc caggcacaaa 6541 aacacaggcg gggtgggaca catgccttct tggagtacgt gggtcattgg cgtggggggt 6601 tacagcgaca ccggccgacc ccctggcggt cttccagccg gcccttagat aagggggcag 6661 ttggtggtcg gacgggtaag taacagagtc tgactaaggg tgggaggggg ggaaaagaac 6721 gggctggtgt gctgtaacac gagcccaccc gcgagtggcg tggccgacct tagcctctgg 6781 ggcgccccct gtcgtttggg tccccccccc tctattgggg agaagcaggt gtctaaccta 6841 cctggaaacg cggcgtcttt gttgaacgac accggggcgc cctcgacgag tgggataacg 6901 ggggaggaag ggagggagga gggtactggg ggtgaagaag ggggggggga agaagcgaga 6961 acaggaaagg cgacggagcc cggcagaaca ccgaggaaaa aaaaaacaca gcgcatgcgc 7021 cgggccgttg tggggccccg ggccggggcc ccttgggtcc gccggggccc cgggccgggc 7081 cgccacgggg gccggccgtt ggcggtaacc ccgattgttt atctcaggcc ccgggccggg 7141 aacccggaaa agcctccggg gggccttttt cgcgtcgcgt gccggcgagc gggcccggac 7201 ggggcccgga ccgccgcggt cgggggcccc tcgtcccggg ccgtacgcgg ccttcgcccc 7261 gtgaggggcc gaacgaacga aacatcccgg cgacggaacg aaaaacaccc cagacgggtt 7321 taaaaaacag aaaccgtaac cccccccacc cccgaaacgg ggaaaacaaa aaacagacca 7381 gcggccggcc ggcgcttagg gggaggatgt cgccgacgcc ccttggccgc cccggctgca 7441 ggggggcccg gagagccgcg gcacccggac gcgcccggaa agtctttcgc accacccgcg 7501 atcggcacgg ccgcgccccc gcttttataa aggctcagat gacgcagcaa aaacaggcca 7561 cagcaccacg tgggtaggtg atgtaatttt attttcctcg tctgcggcct aatggatttc 7621 cgggcgcggt gcccctgtct gcagagcact taacggattg atatctcgcg ggcacgcgcg 7681 cccttaatgg accggcgcgg ggcggggggc cggataccca cacgggcggg ggggtgtcgc 7741 gggccgtctg ctggcccgcg gccacataaa caatgactcg gggcctttct gcctctgccg 7801 cttgtgtgtg cgcgcgccgg ctctgcggtg tcggcggcgg ctgcggcggc tgcggcggcc 7861 gccgtgttcg gtctcggtag ccggccggcg ggtggactcg cggggggccg gagggtggaa 7921 ggcagggggg tgtaggatgg gtatcaggac ttccacttcc cgtccttcca tccccccgttc 7981 ccctcggttg ttcctcgcct cccccaacac cccgccgctt tccgttgggg ttgttattgt 8041 tgtcgggatc gtgcgggccg ggggtcgccg gggcaggggc gggggcgggg gtgctcgtcg 8101 atcgaccggg ctcagtgggg gcgtggggtg ggtgggaaaa ggcgaggaga ctggggtggg 8161 gggtgtcggg ggtggctgtt tttttgtggt tgttttttgt gtctgttccc gtcccccgtc 8221 acccccctcc ctccgtcccc ccgtcgcggg tgtttgtgtt tgtttattcc gacatcggtt 8281 tatttaaata aacacagccg ttctgcgtgt ctgttcttgc gtgtggctgg gggcttatat 8341 gtggggtccc gggggcggga tggggtttag cggcgggggg cggcgcgccg gacggggcgc 8401 tggagataac ggcccccggg gaacggggga ccggggctgg gtctcccgcg gtgggtgggt 8461 gggcggcggt ggccgggccg ggccgggccg ggtgggcggg gtttggaaaa acgaggagga 8521 ggagaaggag gaggaggggg ggggagacgg ggggaaagca aggacacggc cccggggggg 8581 ggggagcgcg ggccgggccg cttggcaacc cccctgtttc ttccggaaac caggcttgtg 8641 gccccacccg acatcacaag ggacctcttg tcgggcctcc cgacgtacgc cgaggctatg 8701 tcggaccacc ccccaaccta agaggggaga ggggagagg gagaggggg aggggagagg 8761 ggagaggga ggagaggggg tatataaacc aacgaaaagc gcgggaacgg ggatacgggg 8821 cttgtgtggc acgacgtcgt ggttgtgtta ctgggcaaac acttggggac ttaggttttc 8881 tgtggtgccg accctaggcg ctatggggat tttgggttgg gttgggctta ttgccgttgg 8941 ggttttgtgt gtgcgggggg gcttgccttc aaccgaatat gttattcgga gtcgggtggc 9001 tcgagaggtg gggatatat taaaggtgcc ttgtgtgccg ctcccgtctg acgatcttga 9061 ttggcgctac gagaccccct cggctataaa ctatgctttg atagacggta tatttttgcg 9121 ttatcactgt cccggattgg acacggtctt gtgggatagg cacgcccaga gggcgtattg 9181 ggttaacccc tttttgtttg gggcgggttt tttggaggac ttgagtcatc ccgcgtttcc 9241 tgccgacacc caggaaacag aaacgcgctt ggccctttat aaaagagatac gccaggcgct 9301 ggacagtcgc aagcaggccg ccagccacac acctgtgaag gctgggtgtg tgaactttga 9361 ctattcgcgc acccgccgct gtgtagggcg ccaggatttg ggacttacca acagaacgtc 9421 tggacggacc ccggttctgc cgtcggacga tgaagcgggc ctgcagccga agcccctcac 9481 cacgccgtcg cccatcatcg ccacgtcgga ccccaccccg cgacgggacg ccgccacaaa 9541 aagcagacgc cgacgacccc attcccggcg catctaatga tgcctcgacg gaaaaccgtc 9601 cgggtttggg gggcgaaccg gccgcctgtc gctcgtcagg gccggcgggc gctcctcgcc 9661 gccctagagg ctgtcccgct ggtgtgacgt tttcctcgtc cgcgcccccc gaccctccca 9721 tggatttaac aaacgggggg gtgtcgcctg cggcgacctc ggcgcctctg gactggacca 9781 cgtttcggcg tgtgtttctg atcgacgacg cgtggcggcc cctgttggag cctgagctgg 9841 cgaacccctt aaccgcccac ctcctggccg aatataatcg tcggtgccag accgaagagg 9901 tgctgccgcc gcgggaggat gtgttttcgt ggactcgtta ttgcaccccc gacgaggtgc 9961 gcgtggttat catcggccag gacccatatc accaccccgg ccaggcgcac ggacttgcgt 10021 ttagcgtgcg cgcgaacgtg ccgcctcccc cgagtcttcg gaatgtcttg gcggccgtca 10081 agaactgtta tcccgaggca cggatgagcg gccacggttg cctggaaaag tgggcgcggg 10141 acggcgtcct gttactaaac acgaccctga ccgtcaagcg cggggcggcg gcgtcccact 10201 ctagaatcgg ttgggaccgc ttcgtgggcg gagttatccg ccggttggct gcgcgccgcc 10261 ccggcctggt gtttatgctc tggggcgcac atgcccagaa tgccatcagg ccggaccctc 10321 gggtccattg cgtcctcaag ttttcgcacc cgtcgcccct ctccaaggtt ccgttcggaa 10381 catgccagca tttcctcgtg gcgaatcgat atctcgagac ccggtcgatt tcacccatcg 10441 actggtcggt ttgaaaggca tcgacgtccg gggttttcgt ctgtgggggc ttttgggtat 10501 ttccgatgaa taaagacggt taatggttaa acctctggtc tcatacgggt cggtgatgtc 10561 gggcgtcggg ggagagggag ttccctctgc gcttgcgatt ctagcctcgt ggggctggac 10621 gttcgacacg ccaaaccacg agtcagggat atcgccagat acgactcccg cagattccat 10681 tcggggggcc gctgtggcct cacctgacca acctttacac gggggcccgg aacgggaggc 10741 cacagcgccg tctttctccc caacgcgcgc ggatgacggc ccgccctgta ccgacgggcc 10801 ctacgtgacg tttgataccc tgtttatggt gtcgtcgatc gacgaattag ggcgtcgcca 10861 gctcacggac accatccgca aggacctgcg gttgtcgctg gccaagttta gcattgcgtg 10921 caccaagacc tcctcgtttt cgggaaacgc cccgcgccac cacagacgcg gggcgttcca 10981 gcgcggcacg cgggcgccgc gcagcaacaa aagccttcag atgtttgtgt tgtgcaaacg 11041 cgcccacgcc gctcgagtgc gagagcagct tcgggtcgtt attcagtccc gcaagccgcg 11101 caagtattac acgcgatctt cggacgggcg gctctgcccc gccgtccccg tgttcgtcca 11161 cgagttcgtc tcgtccgagc caatgcgcct ccaccgagat aacgtcatgc tggcctcggg 11221 ggccgagtaa ccgccccccc cccgcgccac cctcactgcc cgtcgcgcgt gtttgatgtt 11281 aataaataac acataaattt ggctggttgt ttgttgtctt taatggaccg cccgcagggg 11341 gggtggcatt tcagtgtcgg gtgacgagcg cgatccggcc gggatcctag gaccccaaaa 11401 gtttgtctgc gtattccagg gcggggctca gttgaatctc ccgcagcacc tctaccagca 11461 ggtccgcggt gggctggaga aactcggccg tcccggggca ggcggtcgtc gggagtggag 11521 gcgcggcgcc caccccgtgt gccgcgcctg gcgtctcctc tgggggcgac ccgtaaatgg 11581 ttgcagtgat gtaaatggtg tccgcggtcc agaccacggt caaaatgccg gccgtggtgc 11641 tccgggcgct ttcgccgcgc gaggagctga cccaggagtc gaacggatac gcgtacatat 11701 gggcgtccca cccgcgttcg agcttctggt cgctgtcccg gcctataaag cggtaggcac 11761 aaaattcggc gcgacagtcg ataatcacca acagcccaat gggggtgtgc tggataacaa 11821 cgcctccgcg cggcaggcgg tcctggcgct cccggccccg taccataatc gcgcgggtgc 11881 cgtactcaaa aacatgcacc acctgcgcgg cgtcgggcag tgcgctggtc agcgaggccc 11941 tggcgtggca taggctatac gcgatggtcg tctgtggatt ggacatctcg cggtgggtag 12001 tgagtccccc gggccgggtt cggtagaact gtaaggggac ggcgggttaa tagacaatga 12061 ccacgttcgg atcgcgcaga gccgatagta tgtgctcact aatgacgtca tcgcgctcgt 12121 ggcgctcccg gagcggattt aagttcatgc gaaggaattc ggaggaggtg gtgcgggaca 12181 tggccacgta cgcgctgttg aggcgcaggt tgccgggcgt aaagcagatg gcgaccttgt 12241 ccaggctaag gccctgggag cgcgtgatgg tcatggcaag cttggagctg atgccgtagt 12301 cggcgtttat ggccatggcc agctccgtag agtcaatgga ctcgacaaac tcgctgatgt 12361 tggtgttgac gacggacatg aagccgtgtt ggtcccgcaa gaccacgtaa ggcagggggg 12421 cctcttccag taactcggcc acgttggccg tcgcgtgccg cctccgcagc tcgtccgcaa 12481 aggcaaacac ccgtgcgtac gtgtatccca tgagcgtata attgtccgtc tgcagggcga 12541 cggacatcag ccccccgcgc ggcgagccgg tcagcatctc gcagccccgg aagataacgt 12601 tgtccacgta cgtgctaaag ggggcgcctt caaatgcctc cccgaagagc tcttggagga 12661 ttcggaatct cccgaggaag gcccgcttca gcagcgcaaa ctgggtgtga acggcggcgg 12721 tggtctccgg ttccccgggg gtgtagtggc agtaaaacac gtcgagctgt tgttcgtcca 12781 gccccgcgaa aataacgtcg aggtcgtcgt cgggaaaatc gtccgggccc ccgtcccgcg 12841 gccccagttg cttaaaatca aacgcacgct cgccgggggc gcctgcgtcg gctattaccg 12901 acgcctgcgt cggcgccccc gaagatttgg ggcgcagaga cagaatctcc gccgttagtt 12961 ctcccatgcg ggcgtaggcg agggtcctct gggtcgcatc caggcccggg cgctgcagaa 13021 agttgtaaaa ggagataagc ccgctaaata tgagccgcga caggaacctg taggcaaact 13081 ccaccgaagt ctccccctga gtctttacaa agctgtcgtc acgcaacact gcctcgaagg 13141 cccggaacgt cccactaaac ccaaaaacca gttttcgcag gcgcgcggtc accgcgatct 13201 ggctgttgag gacgtaagtg acgtcgttgc gggccacgac cagctgctgt ttgctgtgca 13261 cctcgcagcg catgtgcccc gcgtcctggt cctggctctg cgagtagttg gtgatgcggc 13321 tggcgttggc cgtgagccac ttttcaatag tcaggccggg ctggtgtgtc agccgtcggt 13381 attcgtcaaa ctccttgacc gacacgaacg taagcacggg gagggtgaac acgacgaact 13441 ccccctcacg ggtcaccttc aggtaggcgt ggagcttggc catgtacgcg ctcacctctt 13501 tgtgggagga gaacagccgc gtccagccgg ggaggttggc ggggttggtg atgtagcttt 13561 ccgggacgac gaagcgatcc acgaactgca tgtgctcctc ggtgatgggc aggccgtact 13621 ccagcacctt catgaggtta ccgaactcgt gctcgacgca ccgtttgttg ttaataaaaa 13681 tggcccagct atacgagagg cgggcgtact cgcgcagcgt gcggttgcag atgaggtacg 13741 tgagcacgtt ctcgctctgg cggacggaac accgcagttt ctggtgctcg aaggtcgact 13801 ccagggacgc cgtctgcgtc ggcgagccca cacacaccaa cacgggccgc aggcgggccg 13861 catactgggg ggtgtggtac agggcgttaa tcatccacca gcaatacacc acggccgtga 13921 ggaggtgacg cccaaggagc ccggcctcgt ctatgacgat cacgttgctg cgggtaaagg 13981 ccggcagcgc cccgtgggtg gccggggcca accgcgtcag ggcgccctcg gccaacccca 14041 gggtccgttc cagggcggcc agggcgcgaa actcgttccg cgactcctcg cccccggagg 14101 cggccagggc gcgcttcgtg aggtccaaaa tcacctccca gtagtacgtc agatctcgtc 14161 gctgcaggtc ctccagcgag gcggggttgc tggtcagggt gtacgggtac tgtcccagtt 14221 gggcctggac gtgattcccg cgaaacccaa attcatgaaa gatggtgttg atgggtcggc 14281 tgagaaaggc gcccgagagt ttggcgtaca tgttttgggc cgcaatgcgc gtggcgcccg 14341 tcaccacaca gtccaagacc tcgttgattg tctgcacgca cgtgctcttt ccggagccag 14401 cgttgccggt gataagatac accgcgaacg gaaactccct gaggggcagg cctgcggggg 14461 actctaaggc cgccacgtcc cggaaccact gcagacgggg cacttgcgct ccgtcgagct 14521 gttgttgcga gagctctcgg atgcgcttaa ggattggctg caccccgtgc atagacgtaa 14581 aatttaaaaa ggcctcggcc ctccctggaa cggctggtcg gtccccgggt tgctgaaggt 14641 gcggcgggcc gggtctctgt ccgtctagct ggcgctcccc gccggccgcc gccatgaccg 14701 caccacgctc gcgggccccc actacgcgtg cgcgggggga cacggaagcg ctgtgctccc 14761 ccgaggacgg ctgggtaaag gttcacccca cccccggtac gatgctgttc cgtgagattc 14821 tccacgggca gctggggtat accgagggcc agggggtgta caacgtcgtc cggtccagcg 14881 aggcgaccac ccggcagctg caggcggcga tctttcacgc gctcctcaac gccaccactt 14941 accgggacct cgaggcggac tggctcggcc acgtggcggc ccgcggtctg cagccccaac 15001 ggctggttcg ccggtacagg aacgcccggg aggcggatat cgccggggtg gccgagcggg 15061 tgttcgacac gtggcggaac acgcttagga cgacgctgct ggactttgcc cacgggttgg 15121 tcgcctgctt tgcgccgggc ggcccgagcg gcccgtcaag cttccccaaa tatatcgact 15181 ggctgacgtg cctggggctg gtccccatat tacgcaagcg acaagaaggg ggtgtgacgc 15241 agggtctgag ggcgtttctc aagcagcacc cgctgacccg ccagctggcc acggtcgcgg 15301 aggccgcgga gcgcgccggc cccgggtttt ttgagctggc gctggccttc gactccacgc 15361 gcgtggcgga ctacgaccgc gtgtatatct actacaacca ccgccggggc gactggctcg 15421 tgcgagaccc catcagcggg cagcgcggag aatgtctggt gctgtggcct cccttgtgga 15481 ccggggaccg tctggtcttc gattcgcccg tccagcggct gtttcccgag atcgtcgcgt 15541 gtcactccct ccgggaacac gcgcacgtct gccggctgcg caataccgcg tccgtcaagg 15601 tgctgctggg gcgcaagagc gacagcgagc gcggggtggc cggcgccgcg cgggtcgtta 15661 acaaggtgtt gggggaggac gacgagacca aggccgggtc ggccgcctca cgcctcgtgc 15721 ggcttatcat caacatgaag ggcatgcgcc acgtaggcga cattaacgac accgtgcgtg 15781 cctacctcga cgaggccggg gggcacctga tagacgcccc ggccgtcgac ggtaccctcc 15841 ctggattcgg caagggcgga aacagccgcg ggtctgcggg ccaggaccag ggggggcggg 15901 cgccgcagct tcgccaggcc ttccgcacgg ccgtggttaa caacatcaac ggcgtgttgg 15961 agggctatat aaataacctg tttggaacca tcgagcgcct gcgcgagacc aacgcgggcc 16021 tggcgaccca attgcaggag cgcgaccgcg agctccggcg cgcaacagcg ggggccctgg 16081 agcgccagca gcgcgcggcc gacctggcgg ccgagtccgt gaccggtgga tgcggcagcc 16141 gccctgcggg ggcggacctg ctccgggccg actatgacat tatcgacgtc agcaagtcca 16201 tggacgacga catgtacgtc gccaacagct ttcagcaccc gtacatccct tcgtacgccc 16261 aggacctgga gcgcctgtcg cgcctctggg agcacgagct ggtgcgctgt tttaaaattc 16321 tgtgtcaccg caacaaccag ggccaagaga cgtcgatctc gtactccagc ggggcgatcg 16381 ccgcattcgt cgccccctac tttgaggcag tgcttcgggc cccccgggta ggcgcgccca 16441 tcacgggctc cgatgtcatc ctgggggagg aggagttatg ggatgcggtg tttaagaaaa 16501 cctgcctgca aacgtacctg acagacatcg cggccctgtt cgtcgcggac gtccagcacg 16561 cagcgctgcc cccgcccccc tccccggtcg gcgccgattt ccggcccggc gcgtccccgc 16621 ggggccggtc cagatcgcgg tcgcccggaa gaactgcgcg aggcgcaccg gaccagggcg 16681 ggggcatcgg gcaccgggat ggccgccgcg acggccgacg atgaggggtc ggccgtcacc 16741 atcctcaagc aggccatcgc cggggaccgc agcctggtcg aggcggccga ggcgattagc 16801 cagcagacgc tgctccgcct ggcctgcgag gtgcgccagg tcggcgaccg ccagccgcgg 16861 tttaccgcca ccagcatcgc gcgcgtcgac gtcgcgcctg ggtgccggtt gcggttcgtt 16921 ctggacggga gtcccgagga cgcctatgtg acgtcggagg attactttaa gcgctgctgc 16981 ggccagtcca gttatcgcgg cttcgcggtg gcggtcctga cggccaacga ggaccacgtg 17041 cacagcctgg ccgtgccccc cctcgttctg ctgcaccggt tctccctgtt caaccccagg 17101 gacctcctgg actttgagct tgcctgtctg ctgatgtacc tggagaactg cccccgaagc 17161 cacgccaccc cgtcgacctt tgccaaggtt ctggcgtggc tcggggtcgc gggtcgccgc 17221 acgtccccat tcgaacgcgt tcgctgcctt ttcatccgca gttgccactg ggtcctaaac 17281 acactcatgt tcatggtgca cgtaaaaccg ttcgacgacg agttcgtcct gccccactgg 17341 tacatggccc ggtacctgct ggccaacaac ccgccccccg ttctctcggc cctgttctgt 17401 gccaccccga cgagctcctc attccggctg ccggggccgc ccccccgctc cgactgcgtg 17461 gcctataacc ccgccgggat catggggagc tgctgggcgt cggaggaggt gcgcgcgcct 17521 ctggtctatt ggtggctttc ggagacccca aaacgacaga cgtcgtcgct gttttatcag 17581 ttttgttgaa ttttagtaaa taaacccggt tttgtttcta tggcctcctg acggatgcgc 17641 gtgtccttac tccgttttgg tgggtgggtg gctgtgtatg gcgtcccatc tgtgcgggga 17701 gggggcaagt cggcacgtat tcggacagac tcaagcacac acgggggagc gctcttggct 17761 cagggcaatg tttttattgg tcaaactcag gcaaacagaa acaacatctt gtcgtcaaag 17821 ggatacacaa acttcccccc ctcgccccat actcccgcca gcaccccggt aaacaccaac 17881 tcaatctcgc gcaggatttc gcgcaggtga tgagcgcagt ccacgggggg gagcacaagg 17941 ggccgcgggt atagatcgac ggggacgccg accgactccc cgcctccggg acagacacgc 18001 acgacgcgcc gccagtagtg ctctgcgtcc aacaaggcgc cgccgcggaa ggcagtgggg 18061 ggcaaggggt cgctggcctc aaagggggac acccgaacgc tccagtactc cgcgtccaac 18121 cgtttattaa acgcgtccac gataaggcgg tcgcaggcgt cctccataag gccccgggcc 18181 gtgagcgcgt cctcctccgg cacgcctgcc gttgtcaggc ccaggacccg tcgcagcgtg 18241 tcgcgtacga ccccggccgc cgtggtgtac gcgggcccgc ggagaggaaa tcccccaaga 18301 tggtcagtgt tgtcgcggga gttccagaac cacactcccg cttggctcca ggcgacggcg 18361 tgggtgtaga cgccctcgag cgccaggcac agtgggtgcc gcagccggag gccgttggcc 18421 ataagcacgg ctcccacggc cgtctcgatg gcccgccggg cgtcctcgat caccccggaa 18481 gccgcatccg cgtcttgggg gtccacgtta aagacacccc agaacgcacc cccatcgccc 18541 ccgcagaccg cgaacttaac cgagctggcc gtctcctcaa tctgcaggca gacggcggcc 18601 atcaccccgc ccaggagctg ccgcagcgca gggcaggcgt cgcacgtgtc cgggaccagg 18661 cgctccaaga cggccccggc ccagggctct gagggagcgg ccaccaccag cgcgtccagt 18721 cttgctaggc ccgtccggcc gtggggttcc gccagcccgc tccccccgag gtcggccagg 18781 gccgccagga gctgggcgcg aagtccgggg aagcaaaacc gcgccgtcca gacgggcccg 18841 acggccgcgg gcgggtctaa cagttggatg attttagtgg cgggatgcca ccgcgccacc 18901 gcctcccgca ccgcgggcag gaggcatccg gctgccgccg aggccacgcc gggccaggct 18961 cgcgggggga ggacgaccct ggcccccacc gcgggccagg cccccaggag cgcggcgtaa 19021 gcggccgcgg ccccgcgcac caggtcccgt gccgactcgg ccgtggccgg cacggtgaac 19081 gtgggccaac ccggaaaccc caggacggca aagtacggga cgggtccccc ccggacctca 19141 aactcgggcc ccagaaaggc aaagacgggg gccagggccc cgggggcggc gtggaccgtg 19201 gtatgccact gccggaaaag ggcgacgagc gccggcgcgg agaacttctc gccggcgctt 19261 acaaagtagt cgtaatcgcg gggcagcagc acccgtgccg tgactcgttg cgggtgcccg 19321 cgtggccgca ggcccacctc gcacacctcg accaggtccc cgaacgctcc ctccttcttg 19381 atcggcggaa acgcaagagt ctggtattcg cgcgcaaata gcgcggttcc ggtggtgatg 19441 ttaacggtca gcgaagcggc ggacgcgcac tggggggtgt cgcgaatggc cgccaggcgc 19501 gcccacgcca gccgcgcgtc gggatgctcg gcaacgcgcg ccgccagggc catagggtcg 19561 atgtcaatgt tggcctccgc gaccaggaga gcggcgcgag gggcggcggg cgggccccac 19621 gacgctctct caactttcac caccagtccc gtgcgtgggt ccgagccgat acgcagcggg 19681 gcgaacaggg ccaccggccc ggtctggcgc tccagggccg ccaggacgca cgcgtacagc 19741 gcccgccaca gagtcgggtt ctccaggggc tccagcgggg aggcggccgg cgtcgtcgcg 19801 gcgcgggcgg ccgccacgac ggcctggacg gagacgtccg cggagccgta gaaatcccgc 19861 agctccgtcg cggtgacgga gacctccgca aagcgcgcgc gaccctcccc tgcggcgttg 19921 19981 gcgtaaaggg taatggcgct gacgctctcc tccacccaca cgatatctgc ggtgtccatc 20041 gcacggcccc taggatcac gggcggtctg tgggtcccat gctgccgtgc ctggccgggc 20101 ccggtgggtc gcggaaaccg gtgacgggg ggggcggttt ttggggttgg ggtggggggtg 20161 ggaaacggcc cgggtccggg ggccaacttg gcccctcggt gcgttccggc aacagcgccg 20221 ccggtccgcg gacgaccacg taccgaacga gtgcggtccc gagacttata gggtgctaaa 20281 gttcaccgcc ccctgcatca tgggccaggc ctcggtgggg agctccgaca gcgccgcctc 20341 caggatgatg tcagcgttgg ggttggcgct ggatgagtgc gtgcgcaaac agcgccccca 20401 cgcgggcacg cgtagcttga agcgcgcgcc cgcaaactcc cgcttgtggg ccataagcag 20461 ggcgtacagc tgcctgtggg tccggcaggc gctgtggtcg atgtggtggg cgtccaacaa 20521 ccccacgatt gtctgtttgg tgaggttttt aacgcgcccc gccccgggaa acgtctgcgt 20581 gcttttggcc atctgcacgc caaacagttc gccccagatt atcttgaaca gcgccaccgc 20641 gtggtccgtc tcgctaacgg acccgcgcgg gggacagccg cttagggcgt cggcgacgcg 20701 cttgacggct tcctccgaga gcagaagtcc gtcggttacg ttacagtggc ccagttcgaa 20761 caccagctgc atgtagcggt cgtagtgggg ggtcagtagg tccagcacgt catcggggcc 20821 gaaggtcctc ccagatcccc cggccgccga gtcccaatgc aggcgcgcgg ccatggtgct 20881 gcacaggcac aacagctccc agacgggggt tacgttcagg gtggggggca gggccacgag 20941 ctccagctct ccggtgacgt tgatcgtggg gatgacgccc gtggcgtagt ggtcatagat 21001 ccgccgaaat atggcgctgc tgcgggtggc catgggaacg cggagacagg cctccagcaa 21061 cgccaggtaa ataaaccgcg tgcgtcccat caggctgttg aggttgcgca tgagcgcgac 21121 aatttccgcc ggcgcgacat cggaccggag gtatttttcg acgaaaagac ccacctcctc 21181 cgtctcggcg gcctgggccg gcagcgacgc ctcgggatcc cggcaccgca gctcccgtag 21241 atcgcgctgg gccctgaggg cgtcgaaatg tacgccccgc aaaaacagac agaagtcctt 21301 tggggtcagg gtatcgtcgt gtccccagaa gcgcacgcgt atgcagttta gggtcagcag 21361 catgtgaagg atgttaaggc tgtccgagag acacgccagc gtgcatctct caaagtagtg 21421 tttgtaacgg aatttgttgt agatgcgcga cccccgcccc agcgacgtgt cgcatgccga 21481 cgcgtcacag cgccccttga accggcgaca cagcaggttt gtgacctggg agaactgcgc 21541 gggccactgg ccgcaggaac tgaccacgtg gttcaggagc atgggcgtaa agacgggctc 21601 cgagcgcgcc ccggagccgt ccatgtaaat cagtagctcc cccttgcgga gggtgcgcac 21661 ccgtcccagg gactggtaca cggacaccat gtccggtccg tagttcatgg gtttcacgta 21721 ggcgaacatg ccatcaaagt gcaggggatc gaagctgagg cccacggtta cgaccgtcgt 21781 gtatataacc acgcggtatt ggccccacgt ggtcacgtcc ccgagggggg tgagcgagtg 21841 aagcaacagc acgcggtccg taaactgacg gcagaaccgg gccacgatct ccgcgaagga 21901 gaccgtcgat gaaaaaatgc agatgttatc gcccccgcca aggcgcgctt ccagctcccc 21961 aaagaacgtg gccccccggg cgtccggaga ggcgtccgga gacgggccgc ttggcggccc 22021 gggcggggcgc agggcagcct gcaggagctc ggtccccaga cgcgggagaa acaggcaccg 22081 gcgcgccgaa aacccgggca tggcgtactc gccgaccacc acatgcacgt ttttttcgcc 22141 ccggagaccg cacaggaagt ccaccaactg cgcgttggcg gttgcgtcca tggcgatgat 22201 ccgaggacag gtgcgcagca ggcgtagcat taacgcatcc acgcggccca gttgctgcat 22261 cgttggcga tagagctggc ccagcgtcga cataacctcg tccagacga ggacgtcgta 22321 gttgttcaga aggttggggc ccacgcgatg aaggctttcc acctggacga tagtcggtg 22381 gaggggcgg tcgttcataa tgtaattggt ggatgagaag taggtgacaa agtcgaccag 22441 gcctgactca gcgaaccgcg tcgccagggt ctgggtaaaa ctccgacgac aggagacgac 22501 gagcacactc gtgtccggag agtggatcgc ttcccgcagc cagcggatca gcgcggtagt 22561 ttttcccgac cccattggcg cgcggaccac agtcacgcac ctggccgtcg gggcgctcgc 22621 gttggggaag gtgacgggtc cgtgctgctg ccgctcgatc gttgttttcg ggtgaacccg 22681 gggcacccat tcggccaaat cccccccgta caacatccgc gctagcgata cgctcgacgt 22741 gtactgttcg cactcgtcgt ccccaatggg acgcccggcc cccagaggat cccccgactc 22801 cgcgcccccc acgaaaggca tgaccggggc gcggacggcg tggtgggtct ggtgtgtgca 22861 ggtggcgacg tttgtggtct ctgcggtctg cgtcacgggg cttctcgtcc tggcctctgt 22921 gttccgggca cggtttccct gcttttatgc cacggcgagc tcttatgccg gggtgaactc 22981 cacggccgag gtgcgcgggg gtgtagccgt gcccctcagg ttggacacgc agagccttgt 23041 gggcacttat gtaatcacgg ccgtgttgtt gttggccgcg gccgtgtatg ccgtggtcgg 23101 cgccgtgacc tcccgctacg accgcgccct ggacgcgggc cgccgtctgg ctgcggcccg 23161 catggccatg ccgcacgcca cgctgatcgc cggaaacgtc tgctcttggt tgctgcagat 23221 caccgtcctg ttgctggccc atcgcatcag ccagctggcc cacctggttt acgtcctgca 23281 ctttgcgtgt ctggtgtatt ttgcggccca tttttgcacc aggggggtcc tgagcgggac 23341 gtatctgcgt caggtgcacg gcctgatgga gccggccccg actcatcatc gcgtcgtcgg 23401 cccggctcga gccgtgctga caaacgcctt gctgttgggc gtcttcctgt gcacggccga 23461 cgccgcggta tccctgaata ccatcgccgc gttcaacttt aatttttcgg ccccgggcat 23521 gctcatatgc ctgaccgtgc tgttcgccct tctcgtcgta tcgctgttgt tggtggtcga 23581 gggggtgttg tgtcactacg tgcgcgtgtt ggtgggcccc cacctggggg ccgtggccgc 23641 cacgggcatc gtcggcctgg cctgcgagca ctattacacc aacggctact acgttgtgga 23701 gacgcagtgg ccgggggccc agacgggagt ccgcgtcgcc ctcgccctgg tcgccgcctt 23761 tgccctcggc atggccgtgc tccgctgcac ccgcgcctat ctgtatcaca ggcggcacca 23821 caccaaattt tttatgcgca tgcgcgacac gcgacaccgc gcacattccg ccctcaggcg 23881 cgtacgcagt tccatgcgcg gatcgcgaga cggccgccac aggcccgcac ccggcagccc 23941 gcccgggatt cccgaatatg cggaagaccc ctacgcgatc tcatacggcg gccagctcga 24001 ccggtacgga gattccgacg gggagccgat ttacgacgag gtggctgacg accaaaccga 24061 cgtattgtac gccaagatac aacacccgcg gcacctgccc gacgacgagc ccatctatga 24121 caccgttggg gggtacgacc ccgagcccgc cgaggacccc gtgtacagca ccgtccgccg 24181 ttggtagctg tttggttccg ttttaataaa ccgtttgtgt ttaacccgac cgtggtgtat 24241 gtctggtgtg tggcgtccga tcccgttact atcaccgttt cccccccccc cccctcaacc 24301 ccggcgattg tgggtttttt aaaaacgaca cgcgtgcgac cgtatacaga acattgtttt 24361 ggtttttatt cgctatcgga catggggggt ggaaactggg tggcggggca ggcgcctccg 24421 ggggtccgcc ggtgagtgtg gcgcgagggg gggtccgacg aacgcaggcg ctgtctcccc 24481 ggggcccgcg taaccacgcg catatccggg ggcacgtaga aattaccttc ctcttcggac 24541 tcgatatcca cgacatcaaa gtcgtgggcg gtcagcgaga cgacctcccc gtcgtcggtg 24601 atgaggacgt tgtttcggca gcagcagggc cgggccccgg agaacgagag gcccatagct 24661 cggcgagcgt gtcgtcgaac gccaggcggc tgcttcgctg gatggcctta tagatctccg 24721 gatcgatgcg gacgggggta atgatcaggg cgatcggaac ggcctggttc gggagaatgg 24781 acgccttgct gggtcctgcg gccccgagag ccccggcgcc gtcctccagg cggaacgtta 24841 cgccctcctc cgcgctggtg cggtgcctgc cgataaacgt caccagatgc gggtgggggg 24901 ggcagtcggg gaagtggctg tcgagcacgt agccctgcac caagatctgc ttaaagttcg 24961 ggtgacgggg gttcgcgaag acgggctcgc ggcggaccag atccccggag ctccaggaca 25021 cgggggagat ggtgtggcgt ccgaggtcgg gggcgccaaa cagaagcacc tccgagacaa 25081 cgccgctatt taactccacc aaggcccgat ccgcggcgga gcaccgcctt ttttcgcccg 25141 aggcgtgggc ctctgaccag gcctggtctt gcgtgacgag agcctcctcc gggccgggga 25201 cgcgcccggg cgcgaagtat cgcacgctgg gcttcgggat cgaccggata aatgcccgga 25261 acgcctccgg ggaccggtgt gtcatcaagt cctcgtacgc ggaggccgtg gggtcgctgg 25321 ggtccatggg gtcgaaagcg tacttggccc ggcatttgac ctcgtaaaag gccagggggg 25381 tcttggggac tggggccagg tagccgtgaa tgtcccgagg acagacgaga atatccaggg 25441 acgccccgac catccccgtg tgaccgtcca tgaggacccc acacgtatgc acgttctctt 25501 cggtgaggtc gctgggttcg tggaagataa agcgccgcgt gtcggcgccg gcctcgccgc 25561 cgtcgtccgc gcggcccacg cagtagcgaa acagcaggct tcgggccgtc ggctcgttca 25621 cccgcccgaa catcaccgcc gaagactgta catccggccg caggctggcg ttgtgcttca 25681 gccactgggg cgagaaacac ggaccctggg ggccccagcg gagggtggat gcggtcgtga 25741 ggccccgccg gagcagggcc catagctggc agtcggcctg gttttgcgtg gccgcctcgt 25801 aaaaccccat gaggggccgg ggcgccacgg cgtccgcggc ggccgggggc ccgcggcgcg 25861 tcaggcgcca taggtgccgg ccgagtccgc ggtccaccat acccgcctcc tcgaggacca 25921 cggccaggga acacagataa tccaggcggg cccagagggg accgatggcc agaggggcgc 25981 ggacgccgcg cagcaacccg cgcaggtggc gctcgaacgt ctcggctagt atatgggagg 26041 gcagcgcgtt ggggatcacc gacgccgacc acatagagtc aaggtccggg gagtcgggat 26101 cggcgtccgg gtcgcgggcg tgggtgcccc caggagatag cggaatgtct ggggtcggag 26161 gccctgaggc gtcagaaagt gccggcgacg cggcccgggg cttttcgtct gcggtgtcgg 26221 tggcgtgctg atcacgtggg gggttaacgg gcgaatggga gctcgggtcc acagctgacg 26281 tcgtctgggg tggggggggc aggggacgga aggtggttgt cagcggaaga ctgttagggc 26341 gggggcgctt gggggggctg tcggggccac gaggggtgtc ctcggccagg gcccagggac 26401 gcttagtcac ggtgcgtccc ggcggacatg ctgggcctac cgtggactcc atttccgaga 26461 cgacgtgggg gagcggtggt tgagcgcgcc gccgggtgaa cgctgattct cacgacagcg 26521 cgtgccgcgc gcacgggttg gtgtgacaca ggcgggacac cagcaccagg agaggcttaa 26581 gctcgggagg cagcgccacc gacgacagta tcgccttgtg tgtgtgctgg taatttatac 26641 accgatccgt aaacgcgcgc cgaatcttgg gattgcggag gtggcgccgg atgccctctg 26701 gtacgtcata cgccaggccg tgggtgttgg tctcggccga gttgacaaac agggctgggt 26761 gcagcacgca gcgataggcg agcagggcca gggcgaagtc cggcgacagc tggttgttga 26821 aatactggta accgggaaac cgggtcacgg gtacgcccag gctcggggcg acgtacacgc 26881 taaccaccaa ctccagcagc gtctggccaa gggcgtacag gtcaaccgct aacccgacgt 26941 cgtgcttcag gcggtggttg gtaaattcgg cccgttcgtt gttaaggtat ttcaccaaca 27001 gctccgggg ctggttatac ccgtgaccca ccagggtgtg aaagttggct gtggttaggg 27061 cggtgggcat gccaaacatc cggggggact tgaggtccgg ctcctggagg caaaactgcc 27121 cccgggcgat cgtggagttg gagttgaggg tgacgaggct aaagtcggcg aggacggccc 27181 gccggagcga gacggcgtcc gaccgcagca tgacgaggat gttggcgcac ttgatatcca 27241 ggtggctgat cccgcaggtg gtgtttaaaa acacaacggc gcgggccagc tccgtgaagc 27301 actggtggag ggccgtcgag accgaggggt ttgttgtgcg cagggacgcc agttggccga 27361 tatacttacc gaggtccatg tcgtacgcgg ggaacactat ctgtcgttgt tgcagcgaga 27421 acccgagggg cgcgatgaag ccgcggatgt tgtgggtgcg gccggcgcgt agagcgcact 27481 ccccgaccaa cagggtcgcg atgagctcaa cggcaaacca ctccttttcc tttatggtct 27541 taacggcaag cttatgttcg cgaatcagtt ggacgtcgcc gtatccccca gaccccccga 27601 agcttcgggc cccggggatc tcgagggtcg tgtagtgtag ggcggggttg atggcgaaca 27661 cggggctgca tagcttgcgg atgcgcgtga gggtaaggat gtgcgagggg gacgaggggg 27721 gtgcggttaa cgccgcctgg gatctgcgca ggggcgggcg gttcagtttg gccgccgtac 27781 cgggcgtctc gggggacgcg cggcgatgag acgagcggct cattcgccat cgggatagtc 27841 ccgcgcgaag ccgctcgcgg aggccggatc ggtggcggga cccgtgggag gagcgggagc 27901 cggcggcgtc ctggagagag gggccgctgg ggcgcccgga ggccccgtgt gggttggagt 27961 gtatgtagga tgcgagccaa tccttgaagg accgttggcg tgcaccttgg gggctgaggt 28021 tagctgccac atgaccagca ggtcgctgtc tgcgggactc atccatcctt cggccaggtc 28081 gccgtctccc cacagagaag cgttggtcgc tgcttcctcg agttgctcct cctggtccgc 28141 aagacgatcg tccacggcgt ccaggcgctc accaagcgcc ggatcgaggt accgtcggtg 28201 tgcggttaga aagtcacgac gcgccgcttg ctcctccacg cgaattttaa cacaggtcgc 28261 gcgctgtcgc atcatctcta agcgcgcgcg ggactttagc cgcgcctcca attccaagtg 28321 ggccgccttt gcagccataa aggcgccaac aaaccgagga tcttgggtgc tgacgccctc 28381 ccggtgcagc tgcagggtct ggtccttgta aatctcggct cggaggtgcg tctcggccag 28441 gcgtcggcgc agggccgcgt gggcggcatc tcggtccatt ccgccaccct gcgggcgacc 28501 cggggggtgc tctgatagtc tcgcgtgccc aaggcccgtg atcggggtac ttcgccgccg 28561 cgacccgcca cccggtgtgc gcgatgtttg gtcagcagct ggcgtccgac gtccagcagt 28621 acctggagcg cctcgagaaa cagaggcaac ttaaggtggg cgcggacgag gcgtcggcgg 28681 gcctcacaat gggcggcgat gccctacgag tgcccttttt agatttcgcg accgcgaccc 28741 ccaagcgcca ccagaccgtg gtcccgggcg tcgggacgct ccacgactgc tgcgagcact 28801 cgccgctctt ctcggccgtg gcgcggcggc tgctgtttaa tagcctggtg ccggcgcaac 28861 taaaggggcg tgatttcggg ggcgaccaca cggccaagct ggaattcctg gcccccgagt 28921 tggtacgggc ggtggcgcga ctgcggttta aggagtgcgc gccggcggac gtggtgcctc 28981 agcgtaacgc ctactatagc gttctgaaca cgtttcaggc cctccaccgc tccgaagcct 29041 ttcgccagct ggtgcacttt gtgcgggact ttgcccagct gcttaaaacc tccttccggg 29101 cctccagcct cacggagacc acgggccccc ccaaaaaacg ggccaaggtg gacgtggcca 29161 cccacggccg gacgtacggc acgctggagc tgttccaaaa aatgatcctt atgcacgcca 29221 cctactttct ggccgccgtg ctcctcgggg accacgcgga gcaggtcaac acgttcctgc 29281 gtctcgtgtt tgagatcccc ctgtttagcg acgcggccgt gcgccacttc cgccagcgcg 29341 ccaccgtgtt tctcgtcccc cggcgccacg gcaagacctg gtttctagtg cccctcatcg 29401 cgctgtcact ggcctccttt cgggggatca agatcggcta cacggcgcac atccgcaagg 29461 cgaccgagcc ggtgtttgag gagatcgacg cctgcctgcg gggctggttc ggttcggccc 29521 gagtggacca cgttaaaggg gaaaccatct ccttctcgtt tccggacggg tcgcgcagta 29581 ccatcgtgtt tgcctccagc cacaacacaa acgtaagtcc tcttttcttt cgcatggctc 29641 tcccaagggg ccccgggtcg acccgaccca cacccaccca cccacccaca tacacacaca 29701 accagacgcg ggaggaaagt ctgccccgtg ggcactgatt tttattcggg atcgcttgag 29761 gaggcccggg caacggcccg ggcaacggtg gggcaactcg tagcaaatag gcgactgatg 29821 tacgaagaga agacacacag gcgccacccg gcgctggtcg gggggatgtt gtccgcgccg 29881 caccgtcccc cgacgacctc ttgcagacgg tccgtgatgc aaggacggcg gggggcctgc 29941 agcagggtga ccgtatccac gggatggcca aagagaagcg gacacaggct agcatccccc 30001 tggaccgcca gggtacactg ggccatcttg gcccacagac acggggcgac gcagggacag 30061 gactccgtta cgacggagga gagccacagt gcgttggcgg aatcgatgtg gggcggcggg 30121 gcgcaggact cgcagccccc cgggtggttg gtgatcctgg ccaggagcca tcccagatgg 30181 cgggccctgc ttcccggtgg acagagcgac cccaggtcgc tgtccatggc ccagcagtag 30241 atctggccgc tggggaggtg ccaccaggcc cccgggccca aggcgcagca cgcgcccggc 30301 tccggggggg tcttcgcggg gaccagatac gcgccatcca gctcgccgac cactggctcc 30361 tccgcgagct gttcggtggt tgggtcgggg gtttcctccg ggggggtggc cgcccgtatg 30421 cgggcgaacg tgagggtgca caggagcggg gtcagggggt gcgtcacgct ccggaggtgg 30481 acgatcgcgc agtagcggcg ctcgcggtta aagaaaaaga gggcaaagaa ggtgttcggg 30541 ggcaaccgca gcgccttggg gcgcgtcaga tacagaaaaa tctcgcagaa gagggcgcgc 30601 ccggggtctg ggttaggaag ggccacctga cacagaggct cggtgaggac cgttagacac 30661 cgaaagatct tgagccgctc gtccgcccga acgacgcgcc acacaaagac ggagttgaca 30721 atgcgcgcga tagagtcgac gtccgtcccc aggtcgtcga ctctgtcgcg cgtgccgcga 30781 gctccggccc gggaatccgg ccggggcaag gtccccgggg gaccaggcgg cgccaggggc 30841 cgccggggtc ccagctgcgc catgccgggg gcggggggag ggcaaacccc agaggcgggg 30901 gccaacggcg cggggaggag tgggtggggcg aggtggccgg gggaaggcgc ccgctagcga 30961 gaacggccgt tcccggacga caccttgcga caaaacctaa ggacagcggc ccgcgcgacg 31021 gggtccgaga ggctaaggta ggccgcgatg ttaatggtga acgcaaagcc gccgggaaag 31081 acaactatgc cacagaggcg gcgataac cccaggcaga ggtaggcgta gctttccccg 31141 ggcaggtatt gctcgcagac cctgcgtggg gctgtggagg ggacggcctc catgaagcga 31201 catttactct gctcgcgttt actgacgtca ccatccatcg ccacggcgat tggacgattg 31261 ttaagccgca gcgtgtctcc gcttgtgctg tagtagtcaa aaacgtaatg gccgtcggag 31321 tcggcaaagc gggccggggag gtcgtcgccg agcgggacga cccgccgccc ccgaccgccc 31381 cgtcccccca ggtgtgccag gacggccagg gcatacgcgg tgtgaaaaaa ggagtcgggg 31441 gcggtcccct cgacggcgca catcaggttc tcgaggaga tggggagcg cctggtcacc 31501 tcccccaacc acgcgcgttg gtcggggcca aagtcatagc gcaggcgctg tgagattcgc 31561 gggccgccct gaagcgcggc ccggatggcc tggcccaggg cccggaggca cgccagatgt 31621 atgcgcgcgg taaaggcgac ctcggcggcg atgtcaaagg gcggcaggac ggggcgcggg 31681 tggcgcaggg gcacctcgag cgcgggaaag cgtagcagca gctccgcctg cccagcggga 31741 gacagctggt gggggcgcac gacgcgttct gcggcgcagg cctcggtcag ggccgtggcc 31801 agcgccgagg acagcagcgg agggcgggcg cgtcgcccgc cccacgccac ggagttctcg 31861 taggagacga cgacgaagcg ctgcttggtt ccgtagtggt ggcgcaggac cacggagata 31921 gaacgacggc tccacagcca gtccggccgg tcgccgccgg ccagggcttc ccatccgcga 31981 tccaaccact cgaccagcga ccgcggcttt gcggtaccag gggtcagggt tagaacgtcg 32041 ttcaggatgt cctcgccccc gggcccgtgg ggcacggggg ccacaaagcg gcccccgcct 32101 gggggctcca gacccgccaa caccgcatct gcgtcagccg cccccatggc gcccccgctg 32161 acggcctggt gaaccagggc gccctggcgg agccccgatg caacgccaca ggccgcacgc 32221 ccggtccgag cgcggaccgg gtggcggcgg gtgacgtcct gcactgcccg ctgaaccaac 32281 gcgaggatct cctcgttctc ctgcgcgatg gacacgtcct gggccgcggt cgtgtcgccg 32341 ccgggggccg tcagctgctc ctccggggag atgggggggt cggacgcccc gacgatgggc 32401 gggtctgcgg gcgcccccgc gtggggccgg gccaagggct gcggacgcgg ggacgcgctt 32461 tcccccagac ccatggacag gtgggccgca gcctccttcg cggccggcgg ggcggcggcg 32521 ccaagcagag cgacgtagcg gcacaaatgc cgacagacgc gcatgatgcg cgtgctgtcg 32581 gccgcgtagc gcgtgttggg ggggacgagc tcgtcgtaac taaacagaat cacgcgggca 32641 cagctcgccc ccgagcccca cgcaaggcgc agcgccgcca cggcgtacgg gtcatagacg 32701 ccctgcgcgt tacacaccac gggcagggag acgaacaacc ccccggcgct ggacgcacgc 32761 ggaaggaggc cagggtgtgc cggcacgacg ggggccagaa gctcccccac cgcatccgcg 32821 ggcacgtagg cggcaaacgc cgtgcaccac ggggtacagt cgccggtggc atgagcccga 32881 gtctggattt cgacctggaa gtttgcggcc gtcccgagtc cggggtggcc gcgcatcagg 32941 gcggccagag ggattcccgc ggccgccagg cactcgctgg atatgatgac gtgaaccaaa 33001 gacgagggcc gacccgggcc gtggccgaga tcgtactgga cctcgttggc caagtgcgcg 33061 ttcatggttc gggggtgggt gtgggtgtgt aggcgatgcg ggtcccccga gtccgcggga 33121 agggcgtggg tttggcgcgc gtatgcgtat tcgccaacgg aggcgtgcgt gcttatgcgc 33181 ggcgcgtttc ttctgtctct agggaatccg aggccaggac tttaacctgc tctttgtcga 33241 cgaggccaac tttattcgcc cggatgcggt ccagacgatt atgggctttc tcaaccaggc 33301 caactgcaag attatcttcg tgtcgtccac caacaccggg aaggccagta cgagcttttt 33361 gtacaacctc cgcggggccg cagacgagct tctcaacgtg gtgacctata tatgcgatga 33421 tcacatgccg agggtggtga cgcacacaaa cgccacggcc tgttcttgtt atatcctcaa 33481 caagcccgtt ttcatcacga tggacggggc ggttcgccgg accgccgatt tgtttctggc 33541 cgattccttc atgcaggaga tcatcggggg ccaggccagg gagaccggcg acgaccggcc 33601 cgttctgacc aagtctgcgg gggagcggtt tctgttgtac cgcccctcga ccaccaccaa 33661 cagcggcctc atggcccccg atttgtacgt gtacgtggat cccgcgttca cggccaacac 33721 ccgagcctcc gggaccggcg tcgctgtcgt cgggcggtac cgcgacgatt atatcatctt 33781 cgccctggag cacttttttc tccgcgcgct cacgggctcg gcccccgccg acatcgcccg 33841 ctgcgtcgtc cacagtctga cgcaggtcct ggccctgcat cccggggcgt ttcgcggcgt 33901 ccgggtggcg gtcgagggaa atagcagcca ggactcggcc gtcgccatcg ccacgcacgt 33961 gcacacagag atgcaccgcc tactggcctc ggagggggcc gacgcgggct cgggccccga 34021 gcttctcttc taccactgcg agcctcccgg gagcgcggtg ctgtacccct ttttcctgct 34081 caacaaacag aagacgcccg cctttgaaca ctttattaaa aagtttaact ccgggggcgt 34141 catggcctcc caggagatcg tttccgcgac ggtgcgcctg cagaccgacc cggtcgagta 34201 tctgctcgag cagctaaata acctcaccga aaccgtctcc cccaacactg acgtccgtac 34261 gtattccgga aaacggaacg gcgcctcgga tgaccttatg gtcgccgtca ttatggccat 34321 ctacctcgcg gcccaggccg gacctccgca cacattcgct cctatcacac gcgtcttgtg 34381 agcgcccaat aaacacaccc aggtatgcta cgcacgacca cggtgtcgtc tgttaagggg 34441 ggggggaagg gggtgttggc gggaagcgtg ggaacacggg ggattctctc acgaccggca 34501 ccagtaccac ccccctgtga acacagaaac cccaacccaa atcccataaa catacgacac 34561 acaggcatat tttggaattt cttaggtttt tatttattta ggtatgctgg ggtttctccc 34621 tggatgccca cccccacccc cccgtgggtc tagccgggcc ttagggatag cgtataacgg 34681 gggccatgtc tccggaccgc acaacggccg cgccgtcaaa ggtgcacacc cgaaccacgg 34741 gagccagggc caaggtgtct cctagttggc ccgcgtgggt cagccaggcg acgagcgcct 34801 cgtaaagcgg cagccttcgc tctccatcct gcatcagggc cggggcttcg gggtgaatga 34861 gctgggcggc ctcccgcgtg acactctgca tctgcaggag agcgttcacg tacccgtcct 34921 gggcacttag cgcaaagagc cgggggatta gcgtaaggat gatggtggtt ccctccgtga 34981 tcgagtaaac catgttaagg accagcgatc gcagctcggc gtttacggga ccgagttgtt 35041 ggacgtccgc cagcagcgag aggcgactcc cgttgtagta cagcacgttg aggtctggca 35101 gccctccggg gtttctgggg ctggggttca ggtcccggat gcccctggcc acgagccgcg 35161 ccacgatttc gcgcgccagg ggcgatggaa gcggaacggg aaaccgcaac gtgaggtcca 35221 gcgaatccag gcgcacgtcc gtcgcttggc cctcgaacac gggcgggacg aggctgatgg 35281 ggtccccgtt acagagatct acgggggagg tgttgcgaag gttaacggtg ccggcgtggg 35341 tgaggcccac gtccaggggg caggcgacga ttcgcgtggg aagcacccgg gtgatgaccg 35401 cggggaagcg ccttcggtac gccagcaaca accccaacgt gtcgggactg acgcctccgg 35461 agacgaagga ttcgtgcgcc acgtcggcca gcgtcagttg ccggcggatg gtcggcagga 35521 ataccacccg cccttcgcag cgctgcagcg ccgccgcatc ggggcgcgag atgcccgagg 35581 gtatcgcgat gtcagtttca aagccgtccg ccagcatggc gccgatccac gcggcaggga 35641 gtgcagtggt gggtcgggtg gcgggaggag cgcggtgggg gtcagcggcg tagcagagac 35701 gggcgaccaa cctcgcatag gacggggggt gggtcttagg gggttgggag gcgacaggga 35761 ccccagagca tgcgcgggga ggtctgtcgg gcccagacgc accgagagcg aatccgtccg 35821 cggagtcccg gcttgggttt tatggggccc ggccctcgga atcgcggctt gtcggcgggg 35881 acaaaggggg cggggctagg ggcttgcgga aacagaagac gcgtgggata aaagaatcgc 35941 actaccccaa ggaagggcgg ggcggtttat tacagagcca gtcccttgag cggggatgcg 36001 tcatagacga gatactgcgc gaagtgggtc tcccgcgcgt gggcttcccc gttgcgggcg 36061 ctgcggagga gggcggggtc gctggcgcag gtgagcgggt aggcctcctg aaacaggcca 36121 cacgggtcct ccacgagttc gcggcacccc ggggggcgct taaactgtac gtcgctggcg 36181 gcggtggccg tggacaccgc cgaacccgtc tccacgatca ggcgctccag gcagcgatgt 36241 ttggcggcga tgtcggccga cgtaaagaac ttaaagcagg ggctgagcac cggcgaggcc 36301 ccgttgaggt ggtaggcccc gttatagagc aggtccccgt acgaaaatcg ctgcgacgcc 36361 cacgggttgg ccgtggccgc gaaggcccgg gacgggtcgc tctggccgtg gtcgtacatg 36421 agggcggtga catccccctc cttgtccccc gcgtaaacgc ccccggcggc gcgtccccgg 36481 gggttgcagg gccggcggaa gtagttgacg tcggtcgaca cgggggtggc gataaactca 36541 cacacggcgt cctggccgtg gtccatccct gcgcgccgcg gcacctgggc gcacccgaac 36601 acggggacgg gctgggccgg ccccaggcgg tttcccgcca cgaccgcgtt ccgcaggtac 36661 acggctgccg cgttgtccag gagaggggga gccccgcggc ccaggtaaaa gttttgggga 36721 aggttgccca tgtcggtgac ggggttgcgg acggttgccg tggccacgac ggcggtgtag 36781 cccacgccca ggtccacgtt cccgcgcggc tgggtgagcg tgaagtttac ccccccgcca 36841 gtttcgtgcc gggccacctg gagctggccc aggaagtacg cctccgacgc gcgctccgag 36901 aacagcacgt tctcagtcac aaagcggtcc tgtcggacga cggtgaaccc aaacccggga 36961 tggaggcccg tcttgagctg atgatgcaag gccacgggac tgatcttgaa gtaccccgcc 37021 atgagcgcgt aggtcagcgc gttctccccg gccgcgctct cgcggacgtg ctgcacgacg 37081 ggctgtcgga tcgacgaaaa gtagttggcc cccagagccg gggggaccag ggggacctgc 37141 cgcgacaggt cgcgcagggc cggggggaaa ttgggcgcgt tcgccacgtg gtcggccccg 37201 gcgaacagcg cgtggacggg gagggggtaa aaatagtcgc cattttggat ggtatggtcc 37261 agatgctggg gggccatcag caggattccg gcgtgcaacg ccccgtcgaa tatgcgcatg 37321 ttggtggtgg acgcggtgtt ggcgcccgcg tcgggcgccg ccgagcagag cagcgccgtt 37381 gtgcgttcgg ccatgttgtg ggccagcacc tgcagcgtga gcatggcggg cccgtccact 37441 accacgcgcc cgttgtgaaa catggcgttg accgtgttgg ccaccagatt ggccgggtgc 37501 agggggtgcg cggggtccgt cacggggtcg ctggggcact cctcgccggg ggcgatctcc 37561 gggaccacca tgttctgcag ggtggcgtat acgcggtcga agcgaacccc cgcggtgcag 37621 cagcggcccc gcgagaaggc gggcaccatc acgtagtagt aaatcttgtg gtgcacggtc 37681 cagtccgccc cccggtgcgg ccggtcatcc gcggcgtccg cggctcgggc ctgggtgttg 37741 tgcagcagct ggccgtcgtt gcggttgaag tccgcggtcg ccacgttaca tgccgccgcg 37801 tacacggggt cgtggccccc cgcgctaacc cggcagtcgc gatggcggtc cagggccgcg 37861 cgccgcatca gggcgtcaca gtcccacacg aggggtggca gcagcgccgg gtctcgcatt 37921 aggtgattca gctcggcttg cgcctgcccg cccagctccg ggccggtcag ggtaaagtca 37981 tcaaccagct gggccagggc ctcgacgtgc gccaccaggt cccggtacac ggccatgcac 38041 tcctcgggaa ggtctccccc gaggtaggtc acgacgtacg agaccagcga gtagtcgttc 38101 acgaacgccg cgcaccgcgt gttgttccag tagctggtga tgcactggac cacgagccgg 38161 gccagggcgc agaagacgtg ctcgctgccg tgtatggcgg cctgcagcag gtaaaacacc 38221 gccgggtagt tgcggtcgtc gaacgccccg cgaacggcgg cgatggtggc gggggccatg 38281 gcgtggcgtc ccacccccag ctccaggccc cgggcgtccc ggaacgccgc cggacatagc 38341 gccaggggca agttgccgtt caccacgcgc caggtggcct ggatctcccc cgggccggcc 38401 gggggaacgt ccccccccgg cagctccacg tcggccaccc ccacaaagaa gtcgaacgcg 38461 gggtgcagct caagagccag gttggcgttg tcgggctgca taaactgctc cggggtcatc 38521 tggccttccg cgacccatcg gacccgcccg tgggccaggc gctgccccca ggcgttcaaa 38581 aacagctgct gcatgtctgc ggcggggccg gccggggccg ccacgtacgc cccgtacgga 38641 ttggcggctt cgacggggtc gcggttaagg cccccgaccg ccgcgtcaac gttcatcagc 38701 gaagggtggc acacggtccc gatcgcgtgt tccagagaca ggcgcagcac ctggcggtcc 38761 ttcccccaaa aaaacagctg gcggggcggg aaggcgcggg gatccgggtg gccgggggcg 38821 gggactaggt ccccggcgtg cgcggcaaac cgttccatga ccggattgaa caggcccagg 38881 ggcaggacga acgtcaggtc catggcgccc accaggggt agggaacgtt ggtggcggcg 38941 tagatgcgct tctccagggc ctccagaaag accagcttct cgccgatgga caccagatcc 39001 gcgcgcacgc gcgtcgtctg ggggggcgctc tcgagctcgt ccagcgtctg ccggttcagg 39061 tcgagctgct cctcctgcat ctccagcagg tggcggccca cgtcgtccag acttcgcacg 39121 gccttgccca tcacgagcgc cgtgaccagg ttggccccgt tcaggaccat ctcgccgtac 39181 gtcaccggca cgtcggcttc ggtgtcctcc gctttcagga aggactgcag gaggcgctgt 39241 ttgatcgggg cggtggtgac gagcaccccg tcgaccggcc gcccgcgcgt gtcggcatgc 39301 gtcagacggg gcacggccac ggagggctgc gtggccgtgg tgaggtccac gagccaggcc 39361 tcgacggcct cccggcggtg gcccgccttg cccaggaaaa agctcgtctc gcagaagctt 39421 cgctttagct cggcgaccag ggtcgcccgg gccaccctgg tggccaggcg gccgttgtcc 39481 aggtatcgtt gcatcggcaa caacaaagcc aggggcggcg ccttttccag cagcacgtgc 39541 agcatctggt cggccgtgcc gcgctcaaac gccccgagga cggcctggac gttgcgagcg 39601 agctgttgga tggcgcgcaa ctggcgatgc gcgccgatac ccgtcccgtc cagggcctcc 39661 cccgtgagca gggcgatggc ctcggtggcc aggctgaagg cggcgttcag ggcccggcgg 39721 tcgataatct tggtcatgta attgtgtgtg ggttgctcga tggggtgcgg gccgtcgcgg 39781 gcaatcagcg gctggtggac ctcgaactgt acgcgcccct cgttcatgta ggccagctcc 39841 ggaaacttgg tacacacgca cgccaccgac aacccgagct ccagaaagcg cacgagcgac 39901 agggtgttgc aatacgaccc cagcagggcg tcgaactcga cgtcgtacag gctgtttgca 39961 tcggagcgca cgcgggaaaa aaaatcgaac aggcgtcgat gcgacgccac ctcgatcgtg 40021 ctaaggaggg acccggtcgg caccatggcc gcggcatacc ggtatcccgg agggtcgcgg 40081 ttgggagctg ccatggggtc gcgtggagat cggctggatc tagcgatatt tgcccgggga 40141 ggctaagatc caccccaacg cccggccacc cgtgtacgtg cccgacggcc caaggtccac 40201 cgaaagacac gacgggcccg gacccaaaaa ggcgggggat gctgtgtgag gggccgggtg 40261 tcggtcgggg gggaaaggca ccgggagaag gctgcggcct cgttccagga gaacccagtg 40321 tccccaacag acccggggac gtgggatccc aggccttata tacccccccc gccccacccc 40381 cgttagaacg cgacgggtgc attcaagatg gccctggtcc aaaagcgtgc caggaagaaa 40441 ttggcagagg cggcaaagct gtccgccgcc gccacccaca tcgaggcccc ggccgcgcag 40501 gctatcccca gggcccgtgt gcgcagggga tcggtgggcg gcagcatttg gttggtggcg 40561 ataaagtgga aaagcccgtc cggactgaag gtctcgtggg cggcggcgaa caaggcacac 40621 agggccgtgc ctcccaaaaa cacggacatc ccccaaaaca ctggcgccga caacggcaga 40681 cgatccctct tgatgttaac gtacaggagg agcgcccgca ccgcccacgt aacgtagtag 40741 ccgacgatgg cggccaggat acaggccggc gccaccaccc ttccggtcag cccgtaatac 40801 atgcccgctg ccaccatctc caacggcttc aggaccaaaa acgaccaaag gaacagaatc 40861 acgcgctttg aaaagaccgg ctgggtatgg ggcggaagac gcgagtatgc cgaactgaca 40921 aaaaaatcag aggtgccgta cgaggacaat gaaaactgtt cctccagcgg cagttctccc 40981 tcctcccccc cgaaggcggc ctcgtcgacc agatctcgat ccaccagagg aaggtcatcc 41041 cgcatggtca tggggtgtgc ggtggaggtg gggagaccga aaccgcaaag ggtcgcttac 41101 gtcagcagga tcccgagatc aaagacaccc gggttcttgc acaaacacca cccgggttgc 41161 atccgcggag gcgagtgttt tgataaggcc gttccgcgcc ttgatataac ctttgatgtt 41221 gaccacaaaa cccggaattt acgcctacgc cccaatgccc acgcaagatg aggtaggtaa 41281 cccccccgtg ggtgtgacgt tgcgtttagt tcattggagg ccaaggggaa aaatggggtg 41341 gggaggaaac ggaaaaccca gtaggccgtg tcgggaacac gcccggggtt gtcctcaaaa 41401 ggcagggtcc atactacgga agccgtcgtt gtattcgaga cctgcctgtg cgacgcacgt 41461 cggggttgcc tgtgtccggt tcggccccca ccgcgtgcgg cacgcacgag gacgagtccg 41521 cgtgctttat tggcgttcca agcgttgccc tccagtttct gttgtcggtg ttcccccata 41581 cccacgccca catccaccgt agggggcctc tgggccgtgt tacgtcgccg cccgcgatgg 41641 agcttagcta cgccaccacc atgcactacc gggacgttgt gttttacgtc acaacggacc 41701 gaaaccgggc ctactttgtg tgcggggggt gtgtttattc cgtggggcgg ccgtgtgcct 41761 cgcagcccgg ggagattgcc aagtttggtc tggtcgttcg agggacaggc ccagacgacc 41821 gcgtggtcgc caactatgta cgaagcgagc tccgacaacg cggcctgcag gacgtgcgtc 41881 ccattgggga ggacgaggtg tttctggaca gcgtgtgtct tctaaacccg aacgtgagct 41941 ccgagctgga tgtgattaac acgaacgacg tggaagtgct ggacgaatgt ctggccgagt 42001 actgcacctc gctgcgaacc agcccgggtg tgctaatatc cgggctgcgc gtgcgggcgc 42061 aggacagaat catcgagttg tttgaacacc caacgatagt caacgtttcc tcgcactttg 42121 tgtatacccc gtccccatac gtgttcgccc tggcccaggc gcacctcccc cggctcccga 42181 gctcgctgga ggccctggtg agcggcctgt ttgacggcat ccccgcccca cgccagccac 42241 ttgacgccca caacccgcgc acggatgtgg ttatcacggg ccgccgcgcc ccacgaccca 42301 tcgccgggtc gggggcgggg tcggggggcg cgggcgccaa gcgggccacc gtcagcgagt 42361 tcgtgcaagt caaacacatt gaccgcgtgg gccccgctgg cgtttcgccg gcgcctccgc 42421 caaacaacac cgactcgagt tccctggtgc ccggggccca ggattccgcc ccgcccggcc 42481 ccacgctaag ggagctgtgg tgggtgtttt atgccgcaga ccgggcgctg gaggagcccc 42541 gcgccgactc tggcctcacc cgcgaggagg tacgtgccgt acgtgggttc cgggagcagg 42601 cgtggaaact gtttggctcc gcgggggccc cgcgggcgtt tatcggggcc gcgttgggcc 42661 tgagccccct ccaaaagctg gccgtttact actatatcat ccaccgagag aggcgcctgt 42721 cccccttccc cgcgctagtc cggctcgtag gccggtacac acagcgccac ggcctgtacg 42781 tccctcggcc cgacgaccca gtcttggccg atgccatcaa cgggctggtt cgcgacgcgc 42841 tggcggccgg aaccacagcc gagcagctcc tcatgttcga ccttctcccc ccaaaggacg 42901 tgccggtggg aagcgacgtg caggccgaca gcaccgctct gctgcgcttt atagaatcgc 42961 aacgtctcgc cgtccccggg ggggtgatct cccccgagca cgtcgcgtac cttggtgcgt 43021 tcctgagcgt gctgtacgct ggccgcgggc gcatgtccgc agccacgcac accgcgcggc 43081 tgacaggggt gacctccctg gtgctagcgg tgggtgacgt ggaccgtctt tccgcgtttg 43141 accgcggagc ggcgggcgcg gccagccgca cgcgggccgc cgggtacctg gatgtgcttc 43201 ttaccgttcg tctcgctcgc tcccaacacg gacagtctgt gtaaaagacc ccaataaacg 43261 tatgtcgcta ctacaccctt gtgtgtcaat ggacgcctct ccgggggggg gaagggaaag 43321 caaagagggg ctgggggagc ggcaccaccg gggcctgaac aaacaaacca cagacacggt 43381 tacagtttat tcggtcgggc ggagaaacgg ccgaagccac gcccacttta ttcgcgtctc 43441 caaaaaaacg ggacacttgt ccggagaacc tttaggatgc cagccagggc ggcggtaatc 43501 ataaccacgc ccagcgcaga ggcggccaga aacccgggcg caattgcggc cacgggctgc 43561 gtgtcaaagg ctagcaaatg aatgacggtt ccgtttggaa atagcaacaa ggccgtggac 43621 ggcacgtcgc tcgaaaacac gcttggggcg ccctccgtcg gcccggcggc gatttgctgc 43681 tgtgtgttgt ccgtatccac cagcaacaca gacatgacct ccccggccgg ggtgtagcgc 43741 ataaacacgg cccccacgag ccccaggtcg cgctggtttt gggtgcgcac cagccgcttg 43801 gactcgatat cccgggtgga gccttcgcat gtcgcggtga ggtaggttag gaacagtggg 43861 cgtcggacgt cgacgccggt gagcttgtag ccgatccccc ggggcagagg ggagtgggtg 43921 acgacgtagc tggcgctgtg ggtgatgggt accaggatcc gtggctcgac gttggcagac 43981 tgccccccgc accgatgtga ggcctcaggg acgaaggcgc ggatcagggc gttgtagtgt 44041 gcccaacgcg tcagggtcga ggcgaggccg tgggtctgct gggccaggac ttcgaccggg 44101 gtctcggatc gggtggcttg agccagcgcg tccaggataa acacgctctc gtctagatca 44161 aagcgcaggg aggccgcgca tggcgaaaag tggtccggaa gccaaaagag ggttttctgg 44221 tggtcggccc gggccagcgc ggtccggagg tcggcgttgg tcgctgcggc gacgtcggac 44281 gtacacaggg ccgaggctat cagaaggctc cggcgggcgc gttcccgctg caccgccgag 44341 gggacgccag ccaagaacgg ctgccggagg acagccgagg cgtaaaaatag cgcccggtgg 44401 acgaccgggg tggtcagcac gcggccccct agaaactcgg catacagggc gtcgatgaga 44461 tgggctgcgc tgggcgccac tgcgtcgtac gccgaggggc tatccagcac gaaggccagc 44521 tgatagccca gcgcgtgtaa tgccaagctc tgttcgcgct ccagaatctc ggccaccagg 44581 tgctggagcc gagcctctag ctgcaggcgg gccgtgggat ccaagactga cacattaaaa 44641 aacacagaat ccgcggcaca gcccgcggcc ccgcgggcgg ccaacccggc aagcgcgcgc 44701 gagtgggcca aaaagcctag caggtcggag aggcagaccg cgccgtttgc gtgggcggcg 44761 ttcacgaaag caaaacccga cgtcgcgagc agccccgtta ggcgccagaa gagagggggg 44821 cgcgggccct gctcggcgcc cgcgtccccc gagaaaaact ccgcgtatgc ccgcgacagg 44881 aactgggcgt agttcgtgcc ctcctccggg tagccgccca cgcggcggag ggcgtccagc 44941 gcggagccgt tgtcggcccg cgtcagggac cctaggacaa agacccgata ccgggggccg 45001 cccgggggcc cgggaagagc ccccgggggg ttttcgtccg cggggtcccc gacccgatct 45061 agcgtctggc ccgcggggac caccatcact tccaccggag ggctgtcgtg catggatatc 45121 acgagcccca tgaattcccg cccgtagcgc gcgcgcacca gcgcggcatc gcacccgagc 45181 accagctccc ccgtcgtcca gatgcccacg ggccacgtcg aggccgacgg ggagaaatac 45241 acgtacctac ctggggatct caacaggccc cgggtggcca accaggtcgt ggacgcgttg 45301 tgcaggtgcg tgatgtccag ctccgtcgtc gggtgccgcc gggccccaac cggcggtcgg 45361 gggggcggtg tatcacgcgg cccgctcggg tggctcgccg tcgccacgtt gtctccccgc 45421 gggaacgtca gggcctcggg gtcagggacg gccgaaaacg ttacccaggc ccgggaacgc 45481 agcaacacgg aggcggctgg attgtgcaag agacccttaa ggggggcgac cgagggggga 45541 ggctgggcgg tcggctcgac cgtggtgggg gcgggcaggc tcgcgttcgg gggccggccg 45601 agcaggtagg tcttcgggat gtaaagcagc tggccggggt cccgcggaaa ctcggccgtg 45661 gtgaccaata caaaacaaaa gcgctcctcg taccagcgaa gaaggggcag agatgccgta 45721 gtcaggttta gttcgtccgg cggcgccaga aatccgcgcg gtggtttttg ggggtcgggg 45781 gtgtttggca gccacagacg cccggtgttc gtgtcgcgcc agtacatgcg gtccatgccc 45841 aggccatcca aaaaccatgg gtctgtctgc tcagtccagt cgtggacctg accccacgca 45901 acgcccaaaa taataacccc cacgaaccat aaaccattcc ccatggggga ccccgtccct 45961 aacccacggg gcccgtggct atggcagggc ttgccgcccc gacgttggct gcgagccctg 46021 ggccttcacc cgaacttggg gggtggggtg gggaaaagga agaaacgcgg gcgtattggc 46081 cccaatgggg tctcggtggg gtatcgacag agtgccagcc ctgggaccga accccgcgtt 46141 tatgaacaaa cgacccaaca cccgtgcgtt ttattctgtc tttttattgc cgtcatagcg 46201 cgggttactt ccggtattgt ctccttccgt gtttcagtta gcctccccca tctcccgggc 46261 aaacgtgcgc gccaggtcgc agatcgtcgg tatggagccg ggggtggtga cgtgggtctg 46321 gaccatcccg gaggtaagtt gcagcagggc gtcccggcag ccggcgggcg attggtcgta 46381 atccaggata aagacgtgca tgggacggag gcgtttggcc aagacgtcca aggcccaggc 46441 aaacacgtta tacaggtcgc cgttgggggc cagcaactcg ggggcccgaa acagggtaaa 46501 taacgtgtcc ccgatatggg gttgtgggcc cgcgttgctc tggggctcgg caccctgggg 46561 cggcacggcc gtccccgaaa gctgtcccca atcctcccgc cacgacccgc cgccctgcag 46621 ataccgcacc gtattggcaa gcagctcgta aacgcggcga atcgcggcca acatagccag 46681 gtcaagccgc tcgccggggc gctggcgttt ggccaggcgg tcgatgtgtc tgtcctccgg 46741 aagggccccc aacacgatgt ttgtgccggg caaggtcggc gggatgaggg ccacgaacgc 46801 cagcacggcc tggggggtca tgctgcccat aaggtatcgc gcggccgggt aacacaggag 46861 ggcggcgatg ggatggcggt cgaagatgag ggtgagggcc gggggcgggg catgtgagct 46921 cccagcctcc cccccgatat gaggagccag aacggcgtcg gtcacggcat aaggcatgcc 46981 cattgttatc tgggcgcttg tcattaccac cgccgcgtcc ccggccgata tctcaccctg 47041 gtcgaggcgg tgttgtgtgg tgtagatgtt cgcgattgtc tcggaagccc ccaacacccg 47101 ccagtaagtc atcggctcgg gtacgtagac gatatcgtcg cgcgaaccca gggccaccag 47161 cagttgcgtg gtggtggttt tccccatccc gtggggaccg tctatataaa cccgcagtag 47221 cgtgggcatt ttctgctcca ggcggacttc cgtggctttt tgctgccggc gagggcgcaa 47281 cgccgtacgt cggttgttat ggccgcgaga acgcgcagcc tggtcgaacg cagacgcgtg 47341 ttgatggcag gggtacgaag ccatacgcgc ttctacaagg cgctggccga agaggtgcgg 47401 gagtttcacg ccaccaagat ctgcggcacg ctgttgacgc tgttaagcgg gtcgctgcag 47461 ggtcgctcgg tattcgaggc cacacgcgtc accttaatat gcgaagtgga cctgggaccg 47521 cgccgccccg actgcatctg cgtgttcgaa ttcgccaatg acaagacgct gggcggggtt 47581 tgtgtcatca tagaactaaa gacatgcaaa tatatttctt ccggggacac cgccagcaaa 47641 cgcgagcaac gggccacggg gatgaagcag ctgcgccact ccctgaagct cctgcagtcc 47701 ctcgcgcctc cgggtgacaa gatagtgtac ctgtgccccg tcctggtgtt tgtcgcccaa 47761 cggacgctcc gcgtcagccg cgtgacccgg ctcgtcccgc agaaggtctc cggtaatatc 47821 accgcagtcg tgcggatgct ccagagcctg tccacgtata cggtccccat tgagcctagg 47881 acccagcgag cccgtcgccg ccgcggcggc gccgcccggg ggtctgcgag cagaccgaaa 47941 aggtcacact ctggggcgcg cgacccgccc gagtcagcgg cccgccagtt accacccgcc 48001 gaccaaaccc ccgcctccac ggagggcggg ggggtgctta agaggatcgc ggcgctcttc 48061 tgcgtgcccg tggccaccaa gaccaaaccc cgagccgcct ccgaatgaga gtgtttcgtt 48121 ccttccccct ccccccgcgt cagacaaacc ctaaccaccg cttaagcggc ccccgcgagg 48181 tccgaagact catttggatc cggcgggagc cacccgacaa cagcccccgg gttttcccac 48241 gccagacgcc ggtccgctgt gccatcgcgc cccctcatcc caccccccat cttgtcccca 48301 aataaaacaa ggtctggtag ttaggacaac gaccgcagtt ctcgtgtgtt attttcgctc 48361 tccgcctctc gcagatggac ccgtactgcc catttgacgc tctggacgtc tgggaacaca 48421 ggcgcttcat agtcgccgat tcccgaaact tcatcacccc cgagttcccc cgggactttt 48481 ggatgtcgcc cgtctttaac ctcccccggg agacggcggc ggagcaggtg gtcgtcctac 48541 aggcccagcg cacagcggct gccgctgccc tggagaacgc cgccatgcag gcggccgagc 48601 tccccgtcga tatcgagcgc cggttacgcc cgatcgaacg gaacgtgcac gagatcgcag 48661 gcgccctgga ggcgctggag acggcggcgg ccgccgccga agaggcggat gccgcgcgcg 48721 gggatgagcc ggcgggtggg ggcgacgggg gggcgccccc gggtctggcc gtcgcggaga 48781 tggaggtcca gatcgtgcgc aacgacccgc cgctacgata cgacaccaac ctccccgtgg 48841 atctgctaca catggtgtac gcgggccgcg gggcgaccgg ctcgtcgggg gtggtgttcg 48901 ggacctggta ccgcactatc caggaccgca ccatcacgga ctttcccctg accacccgca 48961 gtgccgactt tcgggacggc cggatgtcca agaccttcat gacggcgctg gtcctgtccc 49021 tgcagtcgtg cggccggctg tatgtgggcc agcgccacta ttccgccttc gagtgcgccg 49081 tgttgtgtct ctacctgctg taccgaaaca cgcacggggc cgccgacgat agcgaccgcg 49141 ctccggtcac gttcggggat ctgctgggcc ggctgccccg ctacctggcg tgcctggccg 49201 cggtgatcgg gaccgagggc ggccggccac agtaccgcta ccgcgacgac aagctcccca 49261 agacgcagtt cgcggccggc gggggccgct acgaacacgg agcgctggcg tcgcacatcg 49321 tgatcgccac gctgatgcac cacggggtgc tcccggcggc cccgggggac gtcccccggg 49381 acgcgagtac ccacgttaac cccgacggcg tggcgcacca cgacgacata aaccgcgccg 49441 ccgccgcgtt cctcagccgg ggccacaacc tattcctgtg ggaggaccag actctgctgc 49501 gggcaaccgc gaacaccata acggccctgg gcgttatcca gcggctcctc gcgaacggca 49561 acgtgtacgc ggaccgcctc aacaaccgcc tgcagctggg catgctgatc cccggagccg 49621 tcccttcgga ggccatcgcc cgtggggcct ccgggtccga ctcgggggcc atcaagagcg 49681 gagacaacaa tctggaggcg ctatgtgcca attacgtgct tccgctgtac cgggccgacc 49741 cggcggtcga gctgacccag ctgtttcccg gcctggccgc cctgtgtctt gacgcccagg 49801 cggggcggcc ggtcgggtcg acgcggcggg tggtggatat gtcatcgggg gcccgccagg 49861 cggcgctggt gcgcctcacc gccctggaac tcatcaaccg cacccgcaca aaccccaccc 49921 ccgtggggga ggttatccac gcccacgacg ccctggcgat ccaatacgaa caggggcttg 49981 gcctgctggc gcagcaggca cgcattggct tgggctccaa caccaagcgt ttctccgcgt 50041 tcaacgttag cagcgactac gacatgttgt actttttatg tctggggttc attccacagt 50101 acctgtcggc ggtttagtgg gtggtgggcg aggggggagg gggcattagg gagaaagaac 50161 aagagcctcc gttgggtttt ctttgtgcct gtactcaaaa ggtcataccc cgtaaacggc 50221 gggctccagt cccggcccgg tggttggcgt gaacgcaacg gcgggagctg ggttagcgtt 50281 tagtttagca ttcgctctcg cctttccgcc cgccccccga ccgttgcgcc ttttttttcg 50341 tccaccaaag tctctgtggg tgcgcgcatg gcagccgatg ccccgggaga ccggatggag 50401 gagcccctgc ccgacagggc cgtgcccatt tacgtggctg ggtttttggc cctgtatgac 50461 agcggggact cgggcgagtt ggcattggat ccggatacgg tgcgggcggc cctgcctccg 50521 gataacccac tcccgattaa cgtggaccac cgcgctggct gcgaggtggg gcgggtgctg 50581 gccgtggtcg acgacccccg cgggccgttt tttgtggggc tgatcgcctg cgtgcagctg 50641 gagcgcgtcc tcgagacggc cgccagcgct gcgattttcg agcgccgcgg gccgccgctc 50701 tcccgggagg agcgcctgtt gtacctgatc accaactacc tgccctcggt ctccctggcc 50761 acaaaacgcc tggggggcga ggcgcacccc gatcgcacgc tgttcgcgca cgtcgcgctg 50821 tgcgcgatcg ggcggcgcct cggcactatc gtcacctacg acaccggtct cgacgccgcc 50881 atcgcgccct ttcgccacct gtcgccggcg tctcgcgagg gggcgcggcg actggccgcc 50941 gaggccgaga tcgcgctgtc cgggcgcacc tgggcgcccg gcgtggaggc gctgacccac 51001 acgctgcttt ccaccgccgt taacaacatg atgctgcggg accgctggag cctggtggcc 51061 gagcggcggc ggcaggccgg gatcgccgga cacacctacc tccaggcgag cgaaaaattc 51121 aaaatgtggg gggcggagcc tgtttccgcg ccggcgcgcg ggtataagaa cggggccccg 51181 gagtccacgg acataccgcc cggctcgatc gctgccgcgc cgcagggtga ccggtgccca 51241 atcgtccgtc agcgcggggt cgccttgtcc ccggtactgc cccccatgaa ccccgttccg 51301 acatcgggca ccccggcccc cgcgccgccc ggcgacggga gctacctgtg gatcccggcc 51361 tcccattaca accagctcgt cgccggccat gccgcgcccc aaccccagcc gcattccgcg 51421 tttggtttcc cggctgcggc gggggccgtg gcctatgggc ctcacggcgc gggtctttcc 51481 cagcattacc ctccccacgt cgcccatcag tatcccgggg tgctgttctc gggacccagc 51541 ccactcgagg cgcagatagc cgcgttggtg ggggccatag ccgcggaccg ccaggcgggc 51601 ggtcagccgg ccgcgggaga ccctggggtc cgggggtcgg gaaagcgtcg ccggtacgag 51661 gcggggccgt cggagtccta ctgcgaccag gacgaaccgg acgcggacta cccgtactac 51721 cccggggagg ctcgaggcgg gccgcgcggg gtcgactctc ggcgcgcggc ccgccagtct 51781 cccgggacca acgagaccat cacggcgctg atgggggcgg tgacgtcttt gcagcaggaa 51841 ctggcgcaca tgcgggctag gaccagcgcc ccctatggga tgtacacgcc ggtggcgcac 51901 tatcgccctc aggtggggga gccggaacca acaacgaccc acccggccct ttgtcccccg 51961 gaggccgtgt atcgcccccc accacacagc gccccctacg gtcctcccca gggtccggcg 52021 tcccatgccc ccactccccc gtatgcccca gctgcctgcc cgccaggccc gccaccgccc 52081 ccatgtcctt ccacccagac gcgcgcccct ctaccgacgg agcccgcgtt cccccccgcc 52141 gccaccggat cccaaccgga ggcatccaac gcggaggccg gggcccttgt caacgccagc 52201 agcgcagcac acgtggacgt tgacacggcc cgcgccgccg atttgttcgt ctctcagatg 52261 atgggggccc gctgattcgc cccggtcttt ggtaccatgg gatgtcttac tgtatatctt 52321 tttaaataaa ccaggtaata ccaaagaaga cccattggtg tatgttcttt ttttattggg 52381 aggcgcgggt aggcgggtag ctttacaatg caaaagcctt cgacgtggag gaaggcgtgg 52441 gggggaatcg gcactgacca agggggtccg ttttgtcacg ggaaaggaaa gaggaaacag 52501 gccgcggaca cccgggggag tttatgtgtt cccttttctt tcttcccaca cacacaaaag 52561 gcgtaccaaa caaacaaacc aaaagatgca catgcggttt aacacccgtg gtttttattt 52621 acaacaaacc ccccgtcaca ggtcgtcctc gtcggcgtca ccgtctttgt tgggaacttg 52681 ggtgtagttg gtgttgcggc gcttgcgcat gaccatgtcg gtgaccttgg cgctgagcag 52741 cgcgctcgtg cccttcttct tggccttgtg ttccgtgcgc tccatggcag acaccagggc 52801 catgtaccgt atcatctccc gggcctcggc tagcttggcc tcgtcaaagt cgccgccctc 52861 ctcgccctcc ccggacgcgt ccgggttggt ggggttcttg agctccttgg tggttagcgg 52921 gtacagggcc ttcatggggt tgctctgcag ccgcatgacg tagcgaaagg cgaagaaggc 52981 cgccgccagg ccggccagga ccaacagacc cacggccagc gccccaaagg ggttggacat 53041 gaaggaggac acgcccgaca cggccgatac cacgccgccc acgatgccca tcaccacctt 53101 gccgaccgcg cgccccaggt cgcccatccc ctcgaagaac gcgcccaggc ccgcaaacat 53161 ggcggcgttg gcgtcggcgt ggatgaccgt gtcgatgtcg gcgaagcgca ggtcgtgcag 53221 ctggttgcgg cgctggacct ccgtgtagtc cagcaggccg ctgtccttga tctcgtggcg 53281 ggtgtacacc tccaggggga caaactcgtg atcctccagc atggtgatgt tgaggtcgat 53341 gaaggtgctg acggtggtga tgtcggcgcg gctcagctgg tgggagtacg cgtactcctc 53401 gaagtacacg tagcccccac cgaaggtgaa gtagcgccgg tgtcccacgg tacacggctc 53461 gatcgcatcg cgcgtcagcc gcagctcgtt gttctccccc agctgcccct cgaccaacgg 53521 gccctggtct tcgtaccgaa agctgaccag ggggcggctg tagcaggccc cgggccgcga 53581 gctgatgcgc atcgagtttt ggacgatcac gttgtccgcg gcgaccggca cgcacgtgga 53641 gacggccatc acgtcgccga gcatccgcgc gctcacccgc cggcccacgg tggccgaggc 53701 gatggcgttg gggttcagct tgcgggcctc gttccacagg gtcagctcgt gattctgcag 53761 ctcgcaccac gcgatggcaa cgcggcccaa catatcgttg acatggcgct gtatgtggtt 53821 gtacgtaaac tgcagcctgg cgaactcgat ggaggaggtg gtcttgatgc gctccacgga 53881 cgcgttggcg ctggccccgg gcggcggggg cgtggggttt gggggcttgc ggctctgctc 53941 tcggaggtgt tcccgcacgt acagctccgc gagcgtgttg ctgagaaggg gctggtacgc 54001 gatcagaaag cccccattgg ccaggtagta ctgcggctgg cccaccttga tgtgcgtcgc 54061 gttgtacctg cgggcgaaga tgcggtccat ggcgtcgcgg gcgtccttgc cgatgcagtc 54121 ccccaggtcc acgcgcgaga gcgggtactc ggtcaggttg gtggtgaagg tggtggatat 54181 ggcgtcggag gagaatcgga aggagccgcc gtactcggag cgcagcatct cgtccacctc 54241 ctgccacttg gtcatggtgc agaccgacgg gcgctttggc acccagtccc aggccacggt 54301 gaacttgggg gtcgtgagca ggttccgggt ggtcggcgcc gtggcccggg ccttggtggt 54361 gaggtcgcgc gcgtagaagc cgtcgacctg cttgaagcgg tcggcggcgt agctggtgtg 54421 ttcggtgtgc gacccctccc ggtagccgta aaacggggac atgtacacaa agtcgccagt 54481 cgccagcaca aactcgtcgt acgggtacac cgagcgcgcg tccacctcct cgacgatgca 54541 gtttaccgtc gtcccgtacc ggtggaacgc ctccacccgc gaggggttgt acttgaggtc 54601 ggtggtgtgc cagccccggc tcgtgcgggt cgcggcgttg gccggtttca gctccatgtc 54661 ggtctcgtgg tcgtcccggt gaaacgcggt ggtctccagg ttgttgcgca cgtacttggc 54721 cgtggaccga cagaccccct tggcgttgat cttgtcgatc acctcctcga aggggacggg 54781 ggcgcggtcc tcaaagatcc ccataaactg ggagtagcgg tggccgaacc acacctgcga 54841 aacggtgacg tctttgtagt acatggtggc cttgaacttg tacggggcga tgttctcctt 54901 gaagaccacc gcgatgccct ccgtgtagtt ctgaccctcg ggccgggtcg ggcagcggcg 54961 cggctgctcg aactgcacca ccgtggcgcc cgtggggggt gggcacacgt aaaagtttgc 55021 atcggtgttc tccgccttga tgtcccgcag gtgctcgcgc agggtggcgt ggcccgcggc 55081 gacggtcgcg ttgtcgccgg cggggcgcgg cggctttggg ggtttcggtt ttttgttctt 55141 cttcggtttc gggtcccccg ttgggggggc gccaggggcg ggcggcgccg gagtggcagg 55201 gcccccgttc gccgcctggg tcgcggccgc gaccccaggc gtgccggggg aactcggagc 55261 cgccgacacc accaggaccc ccagcgtcaa ccccaagagc gcccatacga cgaaccaccg 55321 gcgcccccgc gcgggggcgc cctggcgcat ggcgggacta cgggggcccg tcgtgccccc 55381 cgtcaggtag cctgggggcg aggtgctgga ggaccgagta gaggatcgag aaaacgtctc 55441 ggtcgtagac cacgaccgac cgggggccga tacagccgtc gggggcgctc tcgacgatgg 55501 ccaccagcgg acagtcggag tcgtacgtga gatatacgcc gggcgggtaa cggtaacgac 55561 cttcggaggt cgggcggctg cagtccgggc ggcgcaactc gagctccccg caccggtaga 55621 ccgaggcaaa gagtgtggtg gcgataatca gctcgcgaat atatcgccag gcggcgcgct 55681 gagtgggcgt tattccggaa atgccgtcaa aacagtaaaa cctctgaaat tcgctgacgg 55741 cccaatcagc acccgagccc cccgccccca tgatgaaccg ggcgagctcc tccttcaggt 55801 gcggcaggag ccccacgttc tcgacgctgt aatacagcgc ggtgttgggg ggctgggcga 55861 agctgtgggt ggagtgatca aagaggggcc cgttgacgag ctcgaagaag cgatgggtga 55921 tgctgggggag cagggccggg tccacctggt gtcgcaggag agacgctcgc atgaaccggt 55981 gcgcgtcgaa cacgccccggc gccgagcggt tgtcgatgac cgtgcccgcg cccgccgtca 56041 gggcgcagaa gcgcgcgcgc gccgcaaagc cgttggcgac cgcggcgaac gtcgcgggca 56101 gcacctcgcc gtggacgctg acccgcagca tcttctcgag ctccccgcgc tgctcgcgga 56161 cgcagcgccc caggctggcc aacgaccgct tcgtcaggcg gtccgcgtac agccgccgtc 56221 gctcccgcac gtccgcggcc gcttgcgtgg cgatgtcccc ccacgtctcg ggcccctgcc 56281 ccccgggccc gcggcgacgg tcttcgtcct cgccccccgcc cccgggagct cccaaccccc 56341 gtgccccttc ctctacggcg acacggtccc cgtcgtcgtc ggggcccgcg ccgcccttgg 56401 gcgcgtccgc cgcgcccccc gcccccatgc gcgccagcac gcgacgcagc gcctcctcgt 56461 cgcactgttc ggggctgacg aggcgccgca agagcggcgt cgtcaggtgg tggtcgtagc 56521 acgcgcggat gagcgcctcg atctgatcgt cgggtgacgt ggcctgaccg ccgattatta 56581 gggcgtccac catatccagc gccgccaggt ggctcccgaa cgcgcgatcg aaatgctccg 56641 cccgccgccc gaacagcgcc agttccacgg ccaccgcggc ggtctcctgc tgcaactcgc 56701 gccgcgccag cgcggtcagg ttgctggcaa acgcgtccat ggtggtctgg ccggcgcggt 56761 cgccggacgc gagccagaat cgcaattcgc tgatggcgta caggccgggc gtggtggcct 56821 gaaacacgtc gtgcgcctcc agcagggcgt cggcctcctt gcggaccgag tcattctcgg 56881 gcgacgggtg gggctgcccg tcgccccccg cggtccgggc cagcgcatgg tccaacacgg 56941 agagcgcccg cgcgcggtcg gcgtccgaca gcccggcggc gtggggcagg taccgccgca 57001 gctcgttggc gtccagccgc acctgcgcct gctgggtgac gtggttacag atacggtccg 57061 ccaggcggcg ggcgatcgtc gccccctggt tcgccgtcac acacagttcc tcgaaacaga 57121 ccgcgcaggg gtgggacggg tcgctaagct ccggggggac gataaggccc gaccccaccg 57181 cccccaccat aaactcccga acgcgctcca gcgcggcggt ggcgccgcgc gagggggtga 57241 tgaggtggca gtagtttagc tgctttagaa agttctcgac gtcgtgcagg aaacacagct 57301 ccatatggac ggtcccgcca tacgtatcca gcctgacccg ttggtgatac ggacagggtc 57361 gggccaggcc catggtctcg gtgaaaaacg ccgcgacgtc tcccgcggtc gcgaacgtct 57421 ccaggctgcc caggagccgc tcgccctcgc gccacgcgta ctctagcagc aactccaggg 57481 tgaccgacag cggggtgaga aaggccccgg cctgggcctc caggcccggc ctcagacgac 57541 gccgcagcgc ccgcacctga agcgcgttca gcttcagttg ggggagcttc ccccgtccga 57601 tgtgggggtc gcaccgccgg agcagctcta tctgaaacac ataggtctgc acctgcccga 57661 gcagggctaa caacttttga cgggccacgg tgggctcgga caccggggcg gccatctcgc 57721 ggcgccgatc tgtaccgcgg ccggagtatg cggtggaccg aggcggtccg tacgctaccc 57781 ggcgtctggc tgagccccgg ggtccccctc ttcggggcgg cctcccgcgg gcccgccgac 57841 cggcaagccg ggagtcggcg gcgcgtgcgt ttctgctcta ttcccagaca ccgcggagag 57901 gaatcacggc ccgcccagag atatagacac ggaacacaaa caagcacgga tgtcgtagca 57961 ataatttatt ttacacacat tccccgcccc gccctaggtt cccccacccc caacccctca 58021 cagcatatcc aacgtcaggt ctcccttttt gtcggggggc ccctccccaa acgggtcatc 58081 cccgtggaac gcccgtttgc ggccggcaaa tgccggtccc ggggcccccg ggccgccgaa 58141 cggcgtcgcg ttgtcgtcct cgcagccaaa atccccaaag ttaaacacct ccccggcgtt 58201 gccgagttgg ctgactaggg cctcggcctc gtgcgccacc tccagggccg cgtccgtcga 58261 ccactcgccg ttgccgcgct ccagggcacg cgcggtcagc tccatcatct cctcgcttag 58321 gtactcgtcc tccaggagcg ccagccagtc ctcgatctgc agctgctggg tgcggggccc 58381 caggcttttc acggtcacca cgaacacgct actggcgacg gccgccccgc cctcggagat 58441 aatgccccgg agctgctcgc acagcgagct ttcgtgcgct ccgccgccga ggctcgaggc 58501 cgcgcacaca aacccggccc ggggacaggc caggacgaac ttgcgggtgc ggtcaaaaaat 58561 aaggagcggg cacgcgtttt tgccgcccat caggctggcc cagttcccgg cctgaaacac 58621 acggtcgttg ccggccatgc cgtagtactt gctgatgctc aaccccaaca cgaccatggg 58681 gcgcgccgcc atgacgggcc gcagcaggtt gcagctggcg aacatggacg tccacgcgcc 58741 cggatgcgcg tccacggcgt ccatcagcgc gcgggccccg gcctccaggc ccgccccgcc 58801 ctgcgcggac cacgcggccg cagcctgcac gctggggga cggcgggacc ccgcgatgat 58861 ggccgtaagg gtgttgatga agtacgtcga gtgatcgcag taccgcagaa tctggtttgc 58921 catgtagtac atcgccagct cgctcacgtt gttggggggcc aggttaataa agtttatcgc 58981 gccgtagtcc agggaaaact tttaatgaa cgcgatggtc tcgatgtcct cgcgcgacag 59041 gagccgggcg ggaagctggt tgcgttggag ggccgtccag aaccactgcg ggttcggctg 59101 gttggacccc gggggcttgc cgttgggggaa gatggccgcg tggaactgct tcagcagaaa 59161 gcccagcggt ccgaggagga tgtccacgcg cttgtcgggc ttctggtagg cgctctggag 59221 gctggcgacc cgcgccttgg cggcctcgga cgcgttggcg ctcgcgcccg cgaacaacac 59281 gcggctcttg acgcgcagct ccttgggaaa ccccagggtc acgcgggcaa cgtcgccctc 59341 gaagctgctc tcggcggggg ccgtctggcc ggccgttagg ctgggggcgc agatagccgc 59401 cccctccgag agcgcgaccg tcagcgtttt ggccgacaga aacccgttgt taaacatgtc 59461 catcacgcgc cgccgcagca ccggttggaa ttgattgcga aagttgcgcc cctcgaccga 59521 ctgcccggcg aacaccccgt ggcactggct cagggccagg tcctgataca cggcgaggtt 59581 ggatcgccgc ccgagaagct gaagcagggg gcatggcccg cacgcgtacg ggtccagcgt 59641 cagggacatg gcgtggttgg cctcgcccag accgtcgcga aacttgaagt tcctcccctc 59701 caccaggttg cgcatcagct gctccacctc gcggtccacg acctgcctga cgttgttcac 59761 caccgtatgc agggcctcgc ggttggtgat gatggtctcc agccgcccca tggccgtggg 59821 gaccgcctgg tccacgtact gcagggtctc gagttcggcc atgacgcgct cggtcgccgc 59881 gcggtacgtc tcctgcatga tggtccgggc ggtctcggat ccgtccgcgc gcttcagggc 59941 cgagaaggcg gcgtagtttc ccagcacgtc gcagtcgctg tacatgctgt tcatggtccc 60001 gaagacgccg atggctccgc gggcggcgct ggcgaacttg ggatggcgcg cccggaggcg 60061 catgagcgtc gtgtgtacgc aggcgtggcg cgtgtcgaag gtgcacaggt tgcagggcac 60121 gtcggtctgg ttggagtccg cgacgtatcg aaacacgtcc atctcctggc gcccgacgat 60181 cacgccgccg tcgcagcgct ccaggtaaaa cagcatcttg gccagcagcg ccggggaaaa 60241 cccacacagc atggccaggt gctcgccggc aaattcctgg gttccgccga cgaggggcgc 60301 ggtgggccga ccctcgaacc cgggcaccac gtgtccctcg cggtccacct gtgggttggc 60361 cgccacgtgg gtcccgggca cgaggaagaa gcggtaaaag gagggtttgc tgtggtcctt 60421 tgggtccgcc ggaccggcgt cgtccacctc ggtgagatgg agggccgagt tggtgctaaa 60481 taccatggcc cccacgagtc ccgcggcgcg cgccaggtac gccccgacgg cgttggcgcg 60541 ggccgcggcc gtgtcctggc cctcgcacag cggccatgcg gagatgtcgg tgggcggctc 60601 gtcgaagacg gccatcgaca cgatagactc gagggccagg gcggcgtctc cggccatgac 60661 ggaggccagg cgctgttcga acccgcccgc cgggcccttg ccgccgccgt cgcgcccacc 60721 ccgcggggtc ttaccctggc tggcttcgaa ggccgtgaac gtaatgtcgg cggggagggc 60781 ggcgccctcg tggttttcgt caaacgccag gtgggcggcc gcgcgggcca cggcgtccac 60841 gtttcggcat cgcagtgcca cggcggcggg tcccacgacc gcctcgaaca ggaggcggtt 60901 gagggggcgg ttaaaaaacg gaagcgggta ggtaaaattc tccccgatcg atcggtggtt 60961 ggcgttgaac ggctcggcga tgacccggct aaaatccggc atgaacagct gcaacggata 61021 cacgggtatg cggtgcacct ccgccccgcc tatggttacc ttgtccgagc ctcccaggtg 61081 cagaaaggtg ttgttgatgc acacggcctc cttgaagccc tcggtaacga ccagatacag 61141 gagggcgcgg tccgggtcca ggccgaggcg ctcacacagc gcctcccccg tcgtctcgtg 61201 tttgaggtcg ccgggccggg gggtgtagtc cgaaaagcca aaatggcggc gtgcccgctc 61261 gcagagtcgc gtcaggtttg gggcctgggt gttggggtcc aggtgccggc cgccgtgaaa 61321 gacgtacacg gacgagctgt agtgcgatgg cgtcagtttc agggacaccg cggtaccccc 61381 gagccccgtc gtgcgagaac ccacgaccac ggctacgttg gcctcaaagc cgctctccac 61441 ggtcaggccc acgaccaggg gcgccacggc gacgtcggca tcgccgctgc gcgccgacag 61501 taacgccaga agctcgatgc cttcggatgg acacgcgcga gcgtacacgt atcccagggg 61561 cccggggggg accttgatgg tggttgccgt cttgggcttt gtctccatgt cctcctggca 61621 atcggtccgc aaacggaggt aatcccggca cgacgacgga cgcccgacga ggtatgtctc 61681 ccgagcgtca aaatccgggg ggggcggcga cggtcaaggg gagggtggga gaccggggtt 61741 ggggaatgaa tccctaccct tcaccgacaa cccccgggta accacggggt gccgatgaac 61801 cccggcggct ggcaacgcgg ggtccctgcg agaggcacag atgcttacgg tcaggtgctc 61861 cgggccgggt gcgtctgata tgcggttggt atatgtacac tttacctggg ggcgtgccgg 61921 accgcaccag cccctcccac accccgcgcg tcatcagccg gtgggcg [Water102 bp] Expand Ns 62070 t ttttatata gcggccacgc ccaccggcta 62101 cgtcacgctc ctgtcggccg ccggcggtcc onecccgg ccggccgggc cgacgcgat 62161 aaaccgggcc gccggccggg gcgccgcgca gcagctcgcc gcccggatcc gccagacaaa 62221 caaggccctt gcacatgccg gcccggggcga gcctgggggt ccggtaattt tgccatccca 62281 cccaagcggc ttttggggtt tttcctcttc ccccctcccc acctcccccc tctttagggg 62341 ttcgggtggg grandmotherccgcg atgttttccg gtggcggcgg cccgctgtcc cccggagga 62401 agtcggcggc cagggcggcg tccggggtttt ttgcgcccgc cggccctcgc ggagccggcc 62461 ggggaccccc gccttgtttg aggcaaaact tttacaaccc ctacctcgcc ccagtcgggga 62521 cgcaacaga gccgaccggg ccaacccagc gccatacgta ctatagcga tgcgatgaat 62581 ttcgattcat cgccccgcgg gtgctggacg aggatgcccc cccggagaag cgcgccgggg 62641 tgcacgacgg tcacctcaag cgcgccccca aggtgtactg cggggggac gagcgcgacg 62701 tcctccgcgt cgggtcgggc ggcttctggc cgcggcgctc gcgcctgtgg ggcggcgtgg 62761 accacgcccc ggcggggttc gaccccaccg tcaccgtctt tcacgtgtat gacatcctgg 62821 agaacgtgga gcacgcgtac ggcatgcgcg cggcccagtt ccacgcgcgg tttatggacg 62881 ccatcacacc gacggggacc gtcatcacgc tcctgggcct gactccggaa ggccaccggg 62941 tggccgttca cgtttacggc acgcggcagt acttttacat gaacaaggag gaggttgaca 63001 ggcacctaca atgccgcgcc ccacgagatc tctgcgagcg catggccgcg gccctgcgcg 63061 agtccccggg cgcgtcgttc cgcggcatct ccgcggacca cttcgaggcg gaggtggtgg 63121 agcgcaccga cgtgtactac tacgagacgc gccccgctct gttttaccgc gtctacgtcc 63181 gaagcgggcg cgtgctgtcg tacctgtgcg acaacttctg cccggccatc aagaagtacg 63241 agggtggggt cgacgccacc acccggttca tcctggacaa ccccgggttc gtcaccttcg 63301 gctggtaccg tctcaaaccg ggccggaaca acacgctagc ccagccgcgg gccccgatgg 63361 ccttcgggac atccagcgat gtcgagttta actgtacggc ggacaacctg gccatcgagg 63421 ggggcatgag cgacctaccg gcatacaagc tcatgtgctt cgatatcgaa tgcaaggcgg 63481 ggggggagga cgagctggcc tttccggtgg ccgggcaccc ggaggacctg gtcatccaga 63541 tatcctgtct gctctacgac ctgtccacca ccgccctgga gcacgtcctc ctgttttcgc 63601 tcggttcctg cgacctcccc gaatcccacc tgaacgagct ggcggccagg ggcctgccca 63661 cgcccgtggt tctggaattc gacagcgaat tcgagatgct gttggccttc atgacccttg 63721 tgaaacagta cggccccgag ttcgtgaccg ggtacaacat catcaacttc gactggccct 63781 tcttgctggc caagctgacg gacatttaca aggtccccct ggacgggtac ggccgcatga 63841 acggccgggg cgtgtttcgc gtgtgggaca taggccagag ccacttccag aagcgcagca 63901 agataaaggt gaacggcatg gtgaacatcg acatgtacgg gattataacc gacaagatca 63961 agctctcgag ctacaagctc aacgccgtgg ccgaagccgt cctgaaggac aagaagaagg 64021 acctgagcta tcgcgacatc cccgcctact acgccgccgg gccgcgcaa cgcggggtga 64081 tcggcgagta ctgcatacag gattccctgc tggtgggcca gctgtttttt aagtttttgc 64141 cccatctgga gctctcggcc gtcgcgcgct tggcgggtat taacatcacc cgcaccatct 64201 acgacggcca gcagatccgc gtctttacgt gcctgctgcg cctggccgac cagaagggct 64261 ttattctgcc ggacacccag gggcgattta ggggcgccgg gggggaggcg cccaagcgtc 64321 cggccgcagc ccgggaggac gaggagcggc cagaggagga gggggaggac gagaacgaac 64381 gcgaggaggg cgggggcgag cgggagccgg agggcgcgcg ggagaccgcc ggccggcacg 64441 tggggtacca gggggccagg gtccttgacc ccacttccgg gtttcacgtg aaccccgtgg 64501 tggtgttcga ctttgccagc ctgtacccca gcatcatcca ggcccacaac ctgtgcttca 64561 gcacgctctc cctgagggcc gacgcagtgg cgcacctgga ggcgggcaag gactacctgg 64621 agatcgagat gggggggcga cggctgttct tcgtcaaggc tcacgtgcga gagagcctcc 64681 tcagcatcct cctgcgggac tggctcgcca tgcgaaagca gatccgctcg cggattcccc 64741 agagcagccc cgaggaggcc gtgctcctgg acaagcagca ggccgccatc aaggtcgtgt 64801 gtaactcggt gtacgggttc acgggagtgc agcacggact cctgccgtgc ctgcatgttg 64861 ccgcgacggt gacgaccatc ggccgcgaga tgctgctcgc gacccgcgag tacgtccacg 64921 cgcgctgggc ggccttcgaa cagctcctgg ccgatttccc ggaggcggcc gacatgcgcg 64981 cccccgggcc ctattccatg cgcatcatct acggggacac ggactccata tttgtgctgt 65041 gccgcggcct cacggccgcc gggctgacgg ccatgggcga caagatggcg agccacatct 65101 cgcgcgcgct gtttctgccc cccatcaaac tcgagtgcga aaagacgttc accaagctgc 65161 tgctgatcgc caagaaaaag tacatcggcg tcatctacgg gggtaagatg ctcatcaagg 65221 gcgtggatct ggtgcgcaaa aacaactgcg cgtttatcaa ccgcacctcc agggccctgg 65281 tcgacctgct gttttacgac gataccgtat ccggagcggc cgccgcgtta gccgagcgcc 65341 ccgcagagga gtggctggcg cgacccctgc ccgagggact gcaggcgttc ggggccgtcc 65401 tcgtagacgc ccatcggcgc atcaccgacc cggagaggga catccaggac tttgtcctca 65461 ccgccgaact gagcagacac ccgcgcgcgt acaccaacaa gcgcctggcc cacctgacgg 65521 tgtattacaa gctcatggcc cgccgcgcgc aggtcccgtc catcaaggac cggatcccgt 65581 acgtgatcgt ggcccagacc cgcgaggtag aggagacggt cgcgcggctg gccgccctcc 65641 gcgagctaga cgccgccgcc ccaggggacg agcccgcccc ccccgcggcc ctgccctccc 65701 cggccaagcg cccccgggag acgccgtcgc atgccgaccc cccgggaggc gcgtccaagc 65761 cccgcaagct gctggtgtcc gagctggccg aggatcccgc atacgccatt gcccacggcg 65821 tcgccctgaa cacggactat tacttctccc acctgttggg ggcggcgtgc gtgacattca 65881 aggccctgtt tgggaataac gccaagatca ccgagagtct gttaaaaagg tttattcccg 65941 aagtgtggca ccccccggac gacgtggccg cgcggctccg ggccgcaggg ttcggggcgg 66001 tgggtgccgg cgctacggcg gaggaaactc gtcgaatgtt gcatagagcc tttgatactc 66061 tagcatgagc cccccgtcga agctgatgtc cctcatttta caataaatgt ctgcggccga 66121 cacggtcgga atctccgcgt ccgtgggttt ctctgcgttg cgccggacca cgagcacaaa 66181 cgtgctctgc cacacgtggg cgacgaacct gtaccccggg cacgcggtga gcatccggtc 66241 tatgagccgg tagtgcaggt gggcggacgt gccgggaaag atgacgtaca gcatgtggcc 66301 cccgtaagtg gggtccgggt aaaaaacag ccgcgggtcg cacgccccgc ctccgcgcag 66361 gatcgtgtgg acgaaaaaaa gctcgggttg gccaagaatc ccggccaaga ggtcctggag 66421 gggggcgttg tggcggtcgg ccaacacgac caaggaggcc aggaagcgc gatgctcgaa 66481 tatcgtgttg atctgctgca cgaaggccag gattagggcc tcgcggctgg tggcggcgaa 66541 ccgccccgtct cccgcgttgc acgcgggaca gcaacccccg atgcctaggt agtagcccat 66601 cccggagagg gtcaggcagt tgtcggccac ggtctggtcc agagaagg gcagcgacac 66661 gggagtggtc ttcaccaggg gcaccgagag cgagcgcacg atggcgatct cctcggaggg 66721 cgtctgggcg agggcggcga aaaggccccg atagcgctgg cgctcgtgta aacacagctc 66781 ctgtttgcgg gcgtgaggcg gcaggctctt ccgggaggcc cgacgcacca cgcccagagt 66841 cccgccggcc gcagaggagc gcgaccgccg gcgctccttg ccgtgatagg gcccgggccg 66901 ggagccgcgg cgatgggggt cggtatcata cataggtaca cagggtgtgc tccagggaca 66961 ggagcgagat cgagtggcgt ctaagcagcg cgcccgcctc acggacaaat gtggcgagcg 67021 cggtgggctt tggtacaaat acctgatacg tcttgaaggt gtagatgagg gcacgcaacg 67081 ctatgcagac acgcccctcg aactcgttcc cgcaggccag cttggccttg tggagcagca 67141 gctcgtcggg atgggtggcg gggggatggc cgaacagaac ccaggggtca acctccatct 67201 ccgtgatggc gcacatgggg tcacagaaca tgtgcttaaa gatggcctcg ggccccgcgg 67261 cccgcagcag gctcacaaac cggcccccgt ccccgggctg cgtctcgggg tccgcctcga 67321 gctggtcgac gacgggtacg atacagtcga agaggctcgt gttgttttcc gagtagcgga 67381 ccacggaggc ccggagtctg cgcagggcca gccagtaagc ccgcaccagt aacaggttac 67441 acagcaggca ttctccgccg gtgcgcccgc gcccccggcc gtgtttcagc acggtggcca 67501 tcagagggcc caggtcgagg tcgggctggg catcgggttc ggtaaactgc gcaaagcgcg 67561 gagccacgtc gcgcgtgcgt gccccgcgat gcgcttccca ggactggcgg accgtggcgc 67621 gacgggcctc cgcggcagcg cgcagctggg gccccgactc ccagacggcg ggggtgccgg 67681 cgaggagcag caggaccaga tccgcgtacg cccacgtatc cggcgactcc tccggctcgc 67741 ggtccccggc gaccgtctcg aattccccgt tgcgagcggc ggcgcgcgta cagcagctgt 67801 ccccgccccc gcgccgaccc tccgtgcagt ccaggagacg ggcgcaatcc ttccagttca 67861 tcagtgcggt ggtaagcgac ggctgcgtgc cggataccgc cgccgacccc gccccctcct 67921 cgcccccgga ggccaaggtt ccgatgaggg cccgggtggc agactgcgcc aggaacgagt 67981 agttggagta ctgcaccttg gcggctcccg gggagggcga gggcttgggt tgcttctggg 68041 catgccgccc gggcaccccg ccgtcggtac ggaagcagca gtggagaaaa aagtgccggt 68101 ggatgtcgtt tatggtgagg gcaaagcgtg cgaaggagcc gaccagggtc gccttcttgg 68161 tgcgcagaaa gtggcggtcc atgacgtaca caaactcgaa cgcggccacg aagatgctag 68221 cggcgcagtg gggcgccccc aggcatttgg cacagagaaa cgcgtaatcg gccacccact 68281 gaggcgagag gcggtaggtt tgcttgtaca gctcgatggt gcggcagacc agacagggcc 68341 ggtccagcgc gaaggtgtcg atggccgccg cggaaaaggg cccggtgtcc aaaagcccct 68401 ccccacaggg atccgggggc gggttgcggg gtcctccgcg cccgcccgaa ccccctccgt 68461 cgcccgcccc cccgcgggcc cttgaggggg cggtgaccac gtcggcggcg acgtcctcgt 68521 cgagcgtacc gacgggcggc acacctatca cgtgactggc cgtcaggagc tcggcgcaga 68581 gagcctcgtt aagagccagg aggctgggat cgaaggccac atacgcgcgc tcgaacgccc 68641 ccgccttcca gctgctgccg ggggactctt cgcacaccgc gacgctcgcc aggaccccgg 68701 ggggcgaagt tgccatggct gggcgggagg ggcgcacgcg ccagcgaact ttacgggaca 68761 caatccccga ctgcgcgctg cggtcccaga ccctggagag tctagacgcg cgctacgtct 68821 cgcgagacgg cgcgcatgac gcggccgtct ggttcgagga tatgaccccc gccgagctgg 68881 aggttgtctt cccgactacg gacgccaagc taaactacct gtcgcggacg cagcggctgg 68941 cctccctcct gacgtacgcc gggcctataa aagcgcccga cgacgccgcc gccccgcaga 69001 ccccggacac cgcgtgtgtg cacggcgagc tgctcgcccg caagcgggaa agattcgcgg 69061 cggtcattaa ccggttcctg gacctgcacc agattctgcg gggctgacgc gcgcgctgtt 69121 gggcgggacg gttcgcgaac cctttggtgg gtttacgcgg gcacgcacgc tcccatcgcg 69181 ggcgccatgg cgggactggg caagccctac accggccacc caggtgacgc cttcgagggt 69241 ctcgttcagc gaattcggct tatcgtccca tctacgttgc ggggcgggga cggggaggcg 69301 ggcccctact ctccctccag cctcccctcc aggtgcgcct ttcagtttca tggccatgac 69361 gggtccgacg agtcgtttcc catcgagtat gtactgcggc ttatgaacga ctgggccgag 69421 gtcccgtgca acccttacct gcgcatacag aacaccggcg tgtcggtgct gtttcagggg 69481 ttttttcatc gcccacacaa cgcccccggg ggcgcgatta cgccagagcg gaccaatgtg 69541 atcctggggt ccaccgagac gacggggttg tccctcggcg acctggacac catcaagggg 69601 cggctcggcc tggatgcccg gccgatgatg gccagcatgt ggatcagctg ctttgtgcgc 69661 atgccccgcg tgcagctcgc gtttcggttc atgggccccg aagatgccgg acggacgaga 69721 cggatcctgt gccgcgccgc cgagcaggct attacccgtc gccgccgaac ccggcggtcc 69781 cgggaggcgt acggggccga ggccgggctg ggggtggccg gaacgggttt ccgggccagg 69841 ggggacggtt ttggcccgct ccccttgtta acccaagggc cctcccgccc gtggcaccag 69901 gccctgcggg gtcttaagca cctacggatt ggcccccccg cgctcgtttt ggcggcggga 69961 ctcgtcctgg gggccgctat ttggtgggtg gttggtgctg gcgcgcgcct ataaaaaagg 70021 acgcaccgcc gccctaatcg ccagtgcgtt ccggacgcct tcgccccaca cagccctccc 70081 gaccgacacc cccatatcgc tccccgacct ccggtcccga tggccgtccc gcaatttcac 70141 cgccccagca ccgttaccac cgatagcgtc cgggcgcttg gcatgcgcgg gctcgtcttg 70201 gccaccaata actctcagtt tatcatggat aacaaccacc cacaccccca gggcacccaa 70261 ggggccgtgc gggagtttct ccgcggtcag gcggcggcac tgacggacct tggtctggcc 70321 cacgcaaaca acacgtttac cccgcagcct atgttcgcgg gcgacgcacc ggccgcctgg 70381 ttgcggcccg cgtttggcct gcggcgcacc tattcacctt ttgtcgttcg agaaccttcg 70441 acgcccggga ccccgtgagg cccagggagt tccttctggg gtgttttaat caataaaaga 70501 ccacaccaac gcacgagcct tgcgtttaat gtcgtgttta ttcaagggag tgggataggg 70561 ttcgacggtt cgaaacttaa cacaccaaat aatcgagcgc gtctagccca gtaacatgcg 70621 cacgtgatgt aggctggtca gcacggcgtc gctgtgatga agcagcgccc ggcgggtccg 70681 ctgtaactgc tgttgtaggc ggtaacaggc gcggatcagc accgccaggg cgctacgacc 70741 ggtgcgttgc acgtagcgtc gcgacagaac tgcgtttgcc gatacgggcg gggggccgaa 70801 ttgtaagcgc gtcacctctt gggagtcatc ggcgtataac gcactgaatg gttcgttggt 70861 tatgggggag tgtggttccc cagggagtgg gtcgagcgcc tcggcctcgg aatccgagag 70921 gaacaacgag gtggcgtcgg agtcttcgtc gtcagagaca tacagggtct gaagcagcga 70981 cacgggcgtg ggggtagcgt cgatgtgtag cgcgagggag gatgcccacg aagacacccc 71041 agacaaggag ctgcccgtgc gtggatttgt ggaagacgcg gaagccggga cggatggggcg 71101 gttttgcggt gcccggacc gaaccgccgg atactccccg ggtgctacat gccccgttttg 71161 gggctggggt tggggctggg gcgcggacag gcggctgacg gtcaaatgcc cccggggggcg 71221 cgcagatgtg gcgggcgtgg ccaccggctg ccgtgtagtg gggcggcggg aaaccggggcc 71281 tccggggcgta acaccgccct ccagcgtcaa gtatgtgggg ggcggggcctg acgtcgggggg 71341 cggggtgacg ggttggaccg cgggaggcgg gggagagg cctgcgggag aggatgaggt 71401 cggctcggcc gggttgcggc ctaaaacagg ggccgtgggg tcggcggggt cccagggtga 71461 aggagggat tcccgcgatt cggacagcga cgcgacagcg gggcgcgtaa ggcgccgctg 71521 cggcccgcct acggggaccc tggggggggt tggcggcggga cccgaggtta gcggggggcg 71581 gcggttttcg cccccgggca aaaccgtgcc ggttgcgacc gggggcgga cgggatcgat 71641 agggagagcg ggagaagcct ggccggcgga ctggggaccg agcgggaggg gcacaccaga 71701 caccaaagcg tggggcgctg gctctggggg tttggggaggg gccggggggc gcgcgaaatc 71761 ggtaaccggg gcgaccgtgt cggggagggc aggcggccgc caaccctggg tggtcgcgga 71821 agcctgggtg gcgcgcgcca gggagcgtgc ccggcggtgt cggcgcgcgc gcgacccgga 71881 cgaagaagcg gcagaagcgc gggaggaggc gggggggcgg ggggcggtgg catcgggggg 71941 cgccgggga ctttgggggg acggcaagcg ccggacgtcg tcgcggggc ccacggggcgc 72001 cggccgcgtg ctttcggccg ggacgcccgg tcgtgcttcg cgagccgtga ctgccggccc 72061 agggggccgc ggtgcacact gggatgtggg gacggactga tcggcggtgg gcgaaagggg 72121 gtccggggca aggaggggcg cggggccgcc ggagtcgtca gacgcgagct cctccaggcc 72181 gtgaatccat gcccacatgc gaggggggac gggctcgccg ggggtggcgt cggtgaatag 72241 cgtgggggcc aggcttccgg gccccaacga gccctccgtc ccaacaaggt ccgccgggcc 72301 gggggtcggg ttcgggaccg aggggctctg gtcgtcgggg gcggcgctggt acaccggatg 72361 ccccgggaat agctcccccg acaggaggga ggcgtcgaac ggccgcccga ggatagctcg 72421 tgcgaggaag gggtcctcgt cggtggcgct ggcggcgagg acgtcctcgc cgcccgccac 72481 aaacgggagc tcctcggtgg cctcgctgcc aacaaaccgc acgtcggggg ggccgggggg 72541 tccgggtttt cccacaacac cgcgaccggg gtcatggaga tgtccacgag caccagacac 72601 ggcgggcccc gggcgagggg ccgctcggcg atgagcgcgg acaggcgcgg gagctgtgcc 72661 gccagacacg cgttttcgat cgggttcagg tcggcgtgca ggaggcggac ggcccacgtc 72721 tcgatgtcgg acgacacggc atcgcgcaag gcggcgtccg gcccgcgagc gcgtgagtca 72781 aacagcgtga ggcacagctc cagctccgac tcgcgggaaa aggccgtggt gttgcggagc 72841 gccacgacga cgggcgcgcc caggagcact gccgccagca ccaggtccat ggccgtaacg 72901 cgcgccgcgg gggtgcggtg ggtggcggcg gccggcacgg cgacgtgctg gcccgtgggc 72961 cggtagaggg cgttgggggg agcggggggt gacgcctcgc gcccccccga ggggctcagc 73021 gtctgcccag attccagacg cgcggtcaga agggcgtcga aactgtcata ctctgtgtag 73081 tcgtccggaa acatgcaggt ccaaagagcg gccagcgcgg tgcttgggag acacatgcgc 73141 ccgaggacgc tcaccgccgc cagcgcctgg gcgggactca gctttcccag cgcggcgccg 73201 cgctcggttc ccagctcggg gaccgagcgc cagggcgcca gggggtcggt ttcggacaac 73261 ttgccgcggc gccagtctgc cagccgcgtg ccgaacatga ggccccgggt cggagggcct 73321 ccggccgaaa acgctggcag cacgcggatg cgggcgtctg gatgcggggt caggcgctgc 73381 agaatagca tggaatctgc tgcgttctga aacgcacggg ggagggtgag atgcatgtac 73441 tcgtgttggc ggaccagatc caggcgccaa aaggtgtaaa tgtgttccgg ggagctggcc 73501 accagcgcca ccagcacgtc gttctcgtta aaggaaacgc ggtgcctagt ggagctctgg 73561 ggtccgagcg gcggccccgg ggccgccgcg tcacccccc attccagctg ggcccagcga 73621 cacccaaact cgcgcgtgag agtggtcgcg acgagggcga cgtagagctc ggccgccgca 73681 tccatcgagg ccccccatct cgcctggcgg tggcgcacaa agcgtccgaa gagctgaaag 73741 ttggcggcct gggcgtcgct gagggccagc tgaagccggt tgatgacggt gaggacgtac 73801 atggccgtga cggtcgaggc cgactccagg gtgtccgtcg gaagcggggg gcgaatgcat 73861 gccgcctcgg gacacatcag cagcgcgccg agcttgtcgg tcacggccgg gaagcagagc 73921 gcgtactgca gtggcgttcc atccgggacc aaaaagctgg gggcgaacgg ccgatccagc 73981 gtactggtgg cctcgcgcag caccaggggc cccgggcctc cgctcactcg caggtacgcc 74041 tcgccccggc ggcgcagcat ctgcgggtcg gcctcttggc cgggtggggc ggacgcccgg 74101 gcgcgggcgt ctagggcgcg aagatccacg agcaggggcg cgggcgcggc ggccgcgccc 74161 gcgcccgtct ggcctgtggc cttggcgtac gcgctatata agcccatgcg gcgttggatg 74221 agctcccgcg cgccccggaa ctcctccatc gcccatgggg ccaggtcccc ggccaccgcg 74281 tcgaattccg ccaacaggcc ccccagggta tcaaagttca tctcccaggc cacccttggc 74341 accacctcgt cccgcagccg ggcgctcagg tcggcgtgtt gggccacgcg ccccccgagc 74401 tcctccacgg ccccggcccg ctcggcgctc ttggcgccca ggacgccctg gtacttggcg 74461 ggaaggcgct cgtagtcccg ctgggctcgc agccccgaca cagtgttggt ggtgtcctgc 74521 agggcgcgaa gctgctcgca tgccgcgcga aatccctcgg gcgatttcca ggcccccccg 74581 cgaacgcggc cgaagcgacc ccatacctcg tcccactccg cctcggcctc ctcgagagac 74641 ctccgcaggg cctcgacgcg gcgacgggtg tcgaagagcg cctgcaggcg cgcgccctgt 74701 cgcgtcagga ggcccgggcc gtcgctgctg gccgcgctta gcgggtgcgt ctcaaaggta 74761 cgctgggcat gttccaacca ggcgaccgcc tgcacgtcga gctcgcgcgc cttctccgtc 74821 tggtccaaca gaatttcgac ctgatccgcg atctcctccg ccgagcgcgc ctggtccagc 74881 gtcttggcca cggtcgccgg gacggcgacc accttcagca gggtcttcag attggccaga 74941 ccctcggcct cgagctgggc ccggcgctcg cgcgcggcca gcacatcccg cagccccgcc 75001 gtgacccgct cggtggcttc ggcgcgctgc tgtttggcgc gcaccacggc gtccttggta 75061 tcggccaggt cctgtcgggt cacgaatgcg acgtagtcgg agtacgccgt gtccttcacg 75121 gggctctggt ccacgcgctc cagcgccgcc acgcacgcca ccagcgcgtc ctcgctcggg 75181 cagggcaggg tgacccctgc ccggacaagc tcggcggccg ccgccgggtc gttgcgcacc 75241 gcggatatct cctccgcggc ggcggccagg tccagcgcca cgcttccgat cgcgcgccgc 75301 gcgtcggccc ggagggcgtc caggcgatcg cggatatcca cgtactcggc gtagcccttt 75361 tgaaaaaacg gcacgtactg gcgcagggcc ggcacgcccc ccaagtcttc cgacaggtgt 75421 agtacggcct cgtggtagtc gataaacccg tcgttcgcct gggcccgctc cagcagcccc 75481 cccgcgagcc gcagaagccg cgccaggggc tcggtgtcca cccgaaacat gtcggcgtac 75541 gtgtcggccg cggccccaaa ggccgcgctc cagtcgatgc ggtgaatggc tgcgagcggg 75601 gggagcatgg ggtggcgctg gttctcgggg gtgtatgggt taaacgcaag ggccgtctcc 75661 agggcaaggg tcaccgcctt ggcgttggtt cccagcgcct gctcggcccg ctttcggaag 75721 tcccgggggt tgtagccgtg cgtgcccgcc agcgcctgca ggcgacggag ctcgaccacg 75781 tcaaactcgg cactgctttc cacgcggtcc agcacggcct ccacgtcggc ggcccagcgc 75841 tcgtggctac tgcgggcgcg ctgggccgcc atcttctctc tgaggtcggc ggtggcggcc 75901 tcaagttcgt cggcgcggcg tcgcgtggcg ccgatgacct ttcccagctc ctgcagggcg 75961 cgcccgctgg gggagtggtc cccggccgtc ccttcggcgt gcaacaggcc cccgaacctg 76021 ccctcgtggc ccgcgaggct ttcccgcgcg ccggtggtcg cgcgcgtcgc ggcttggatc 76081 agggaggcat gctctccctt cggttggttg gcggcccggc gcacctggac gacaaggtcg 76141 gcggcagccg accctaaggt cgtgagctgg gcgatggccc cccgcgcgtc cagggccaac 76201 cgagtcgcct tgacgtatcc cgcggcgctg tcggccatgg ccgctaggaa ggccagggg 76261 gaggccgggt cgctggcggc cgcgcccagg gccgtcaccg cgtcgaccag gacgcggtgc 76321 gcccgcacgg ccgcatccac cgtcgacgcg gggtctgccg tcgcgacggc ggcgctgccg 76381 gcgttgatgg cgttcgagac ggcgtgggct atgatcgggg cgtgatcggc gaagaactgc 76441 aagagaaacg gagtctcggg ggcgtcggcg aacaggttct tcagcaccac cacgaagctg 76501 ggatgcaagc cggacagagc cgtcgccgtg tccggagtcg ggtgctccag ggcatctcgg 76561 tactgcccca gcagccccca catgtccgcc cgcagcgccg ccgtaacctc agggggcgcc 76621 ccccgaacgg cctcggggag gtccgaccag cccgccggca gggaggcccg cagggtcgcc 76681 aggacggccg gacaggcctt tagccccaca aagtcaggga gggggcgcag gaccccctgg 76741 agtttgtgca agaacttctc ccgggcgtcg cgggccacct tcgcccgctc ccgcgctccc 76801 tcgagcattg cctccaggga gcgcgcgcgc tcccgcaaac ggacacgcgc atcgggggcg 76861 agctctgccg tcagcttggc ggcatccatg gcccgcgcct gccgcagcgc ttcctcggcc 76921 atgcgcgtgg cctctggcga cagcccgccg tcgtcggggt agggcgacgc gccgggcgca 76981 ggaacaaagg ccgcgtcgct gtccagctgc tggcccaggg ccgcatctag ggcgtcgaag 77041 cgccgcagct cggccagacc cgagctgcgg cgcgcctgct ggtcgttaat gtcgcggatg 77101 ctgcgcgcca gctcgtccag cggcttgcgt tctatcagcc cttggttggc ggcgtccgtc 77161 aggacggaga gccaggccgc caggtcctcg ggggcgtcca gcgtctggcc ccgctgtatc 77221 agatcccgca acaggatggc cgtggggctg gtcgcgatcg ggggcggggc gggaatggcg 77281 gcgcgctgcg cgatgtcccg cgtgtgctgg tcgaagacag gcagggactc gagcagctgg 77341 accacgggca cgacggcggc cgaagccacg tgaaaccggc ggtcgttgtt gtcgctggcc 77401 tgtagagcct tggcgctgta tacggcccccc cggtaaaagt actccttaac cgccccctcg 77461 atcgcccgac gggcctgggt ccgcacctcc tccagccgaa cctgaacggc ctcggggccc 77521 agggggggtg ggcgcggagc cccctgcggg gccgccccgg ccggggcggg cattacgccg 77581 aggggcccgg cgtgctgtga gaccgcgtcg accccgcgag cgagggcgtc gagggcctcg 77641 cgcatctggc gatcctccgc ctccacccta atctcttcgc cacgggcaaa tttggccaga 77701 gcctggactc tatacagaag cggttctggg tgcgtcgggg tggcgggggc aaaaagggtg 77761 tccgggtggg cctgcgagcg ctccagaagc cactcgccga ggcgtgtata cagattggcc 77821 ggcggggccg cgcgaagctg cagctccagg tccgcgagtt ccccgtaaaa ggcgtccgtc 77881 tcccgaatga catccctagc cacaaggatc agcttcgcca gcgccaggcg accgatcaga 77941 gagttttcgt ccagcacgtg ctggacgagg ggcagatggg cggccacgtc ggccaggctc 78001 aggcgcgtgg aggccagaaa gtcccccacg gccgttttcc ggggcagcat gctcagggta 78061 aactccaaca gggcggcggc cgggccggcc accccggcct gggtgtgcgt ccgggccccg 78121 ttctcgatga gaaaggcgag gacgcgttca aagaaaaaaa taacacagag ctccagcagc 78181 cccggagaag ccggatacgg cgaccgtaag gcgctgatgg tgagccgcga acacgcggcg 78241 acctcgcggg ccagggcggc ggagcacgcg gtgaacttaa ccgccgtggc ggccacgttt 78301 gggtgggcct cgaacagctg ggcaaggtct gcgcccgggg gctcgggtga gcggcgagtc 78361 ttcagcgcct cgagggcctg cgaggacgcc ggaaccgtgg gcccgtcgtc ctcgcccgcc 78421 tcggcgaccg gcggcccggc cgggtcgggg ggtgccgagg cgaggacagg ctccggaacg 78481 gaggcgggga ccgcggcccc gacgggggtt ttgcctttgg gggtggattt cttcttggtt 78541 ttggcagggg gggccgagcg tttcgttttc tcccccgaag tcaggtcttc gacgctggaa 78601 ggcggagtcc aggtgggtcg gcggcgcttg ggatggccgg ccgagtagcg tgcccggtgc 78661 cgaccaaccg ggacgacgcc catctccagg acccgcatgt cgtcgtcatc ttcttcggcc 78721 gcctctgcgg cggggggctt gggggcggag ggaggcggtg gtgggatcgc ggagggtggg 78781 tcggcggagg ggggatccgt gggtggggta cccttcaggg ccaccgccca tacatcgtcg 78841 ggcgcccgat tcgggcgctt ggcctctggt tttgccgacg gaccggccgt cccccgggat 78901 gtctcggagg ccctgtcgtc gcgacgggcc cgggtcggtg gcggcgactg ggcggctgtg 78961 ggcgggtgtg gccccgtgcc ccctaccccc tcccgggggc ccacgccgac gcagggctcc 79021 cccaggcccg cgatctcgcc ccgcaggggg tgcgtgatgg ccacgcgccg ttcgctgaac 79081 gcttcgtcct gcaggtaagt ctcgctggcc ccgtaaagat gcagagccgc ggccgtcaag 79141 tccgcaggag ccgcgggttc cgggcccgac ggcacgaaaa acaccatggc tcccgcccac 79201 cgtacgtccg ggcgatcgcg ggtgtaatac gtcaggtatg gatacatgtc ccccgcccgc 79261 actttggcga tgaacgcggg ggtgccctcc ggaaggccgt gcgggtcaaa aaggtatgcg 79321 gtgtcgccgt ccctgaacag ccccatccct aggggccaa tggttaggag cgtgtacgac 79381 aggggcgca gggcccacgg gccggcgaag aacgtgtgtg cggggcattg tgtctccagc 79441 aggcccgccg cgggctcccc gaaagccc acctcgccgt atacgcgcga gaagacacag 79501 ​​cgcagtccgc cgcgcgcccc tgggtactcg aggaagttgg ggagctcgac gatcgaacac 79561 atgcgcggcg gcccagggcc cgcggtcgcg cgcgtccact cgcccccctc gaccaaaaa 79621 ccctcgatgg cctccgcgga cagaacgtcg cgagggccca catcaaatat gaggctgaga 79681 areacagcg acgagcgcat gcacgatacc gacccccccg gctccaggtc gggcgcgaac 79741 tggttccgag caccggtgac cacgatgtcg cgatcccccc cgcgttccat cgtggagtgc 79801 ggtggggtgc ccgcgatcat atgtgcccta ctggccagag acccggcctg tttatggacc 79861 ggacccccgg ggttagtgtt gtttccgcca cccatgcccc cgtaccatgg ccccggttcc 79921 cctgattagg ctacgagtcg cggtgatcgc ttcccaaaaa ccgagctgcg tttgtctgtc 79981 ttgatcttcc cccccccccc cgcccgcccg cccgcccgcc cgcccgcaca ccataacacc 80041 gagaacaaca cacgggggtg ggcgtaacat aataaagctt tattggtaac tagttaacgg 80101 caagtccgtg ggtggcgcga cggtgtcctc cgggctcatc tcgtcgtcct cgacgggggt 80161 gttggaatga ggcgccccct cgcggtccgc ctggcgtggg ccgtgcccat aggcctccgg 80221 cttctgtgcg tccatgggca taggcgcggg gagactgttt ccggcgtcgc ggacctccag 80281 gtccctggga gactccggtc cggctaacgg acgaaacgcg gaagcgcgaa acacgccgtc 80341 ggtgacccgc aggagctcgt tcatcagtaa ccaatccata ctcagcgtaa cggccagccc 80401 ctggcgagac agatccacgg agtccggaac cgcggtcgtc tggcccaggg ggccgaggct 80461 gtagtccccc caggccccta ggtcgcgacg gctcgtaagc acgacgcggt cggccgcggg 80521 gctttgcggg ggggcgtcct cgggcgcatg cgccattacc tctcggatgg ccgcggcgcg 80581 ctggtcggcc gagctgacca agggcgccac gaccacggcg cgctccgtct gcaggccctt 80641 ccacgtgtcg tggagttcct ggacaaactc ggccacgggc tcgggtcccg cggccgcgcg 80701 cgcggcttga tagcaggccg agagacgccg ccagcgcgct agaaactgac ccatgaagca 80761 aaacccgggg acctggtctc ccgacagcag cttcgacgcc cgggtgtgaa tgccggacac 80821 aacggacaga aacccgtgaa tttcgcgccg gaccacggcc agcacgttgt cctcgtgcga 80881 cacctgggcc gccagctcgt cgcacacctc caggtgcgcc gtggtttcgg tgatgacgga 80941 acgcaggctc gcgagggacg cgaccagcgc gcgcttggcg tcgtgataca tgctgcagta 81001 ctgactcacc gcgtccccca tggcctcggg gggccagggc cccaggcggt cgggagtgtc 81061 cccgaccacc gcatacaggc ggcgcccgtc gctctcgaac cgacactcga aaaaggcgga 81121 gagcgtgcgc atgtgcagcc gcagcagcac gatggcgtcc tccagttggc gaatcagggg 81181 gtctgcgcgc tcggcgaggt cctgcagcac cccccgggcg gccagggcgt acatgctaat 81241 caacaggagg ctggtgccca cctcgggggg cggggggggc tgcagctgga ccaggggccg 81301 cagctgctcg acggcacccc tggagatcac gtacagctcc cggagcagct gctctatgtt 81361 gtcggccatc tgcatagtgg ggccgaggcc gccccgggcg gccggttcga ggagggtaat 81421 cagcgcgccc agtttggtgc gatggccctc gaccgtgggg agatagccca gcccaaagtc 81481 ccgggcccag gccaacacac gcagggcgaa ctcgaccggg cgtggaaggt aggccgcgct 81541 acacgtggcc cccaacgcgt ccccgaccac cagggccaga acgtagggga cgaagcccgg 81601 gtcggcgagg acgttggggt gaatgccctc gagggcgggg aagcggatct gggtcgccgc 81661 ggccaggtgg acagaggggg cgtggctggg ctgcccgacg gggagaagcg cggacagcgg 81721 cgtggccggg gtggtggggg tgatgtccca gtgggtctga ccatacacgt cgatccagat 81781 gagcgccgtc tcgcggagaa ggctgggttg accggaacta aagcggcgct cggccgtctc 81841 aaactccccc acgagcgccc gccgcaggct cgccagatgt tccgtcggca cggccggacc 81901 catgatacgc gccagtgtct ggctcagaac gccccccgac aggccgaccg cctcgcagag 81961 ccgcccgtgc gtgtgctcgc tggcgccctg gacccgcctg aaagttttta cgtagttggc 82021 atagtacccg tattcccgcg ccagaccaaa cacgttcgac cccgcgaggg caatgcaccc 82081 aaagagctgc tggacttcgc cgagtccgtg gccggcgggc gtccgcgcgg ggacgcccgc 82141 cgccagaaac ccctccaggg ccgaaaggta gtgcgtgcag tgcgagggcg tgaacccagc 82201 gtcgatcagg gtgttgatca ccacggaggg cgaattggta ttctggatca acgtccacgt 82261 ctgctgcagc agagccagca gccgctgctg ggcgccggcg gagggctgct ccccgagctg 82321 cagcaggctg gagacggcag gctggaagac tgccagtgcc gacgaactca ggaacggcac 82381 gtcgggatca aacacggcca cgtccgtccg cacgcgcgcc attagcgtcc ccgggggcgc 82441 acaggccgag cgcgggctga cgcggctgag ggccgtcgac acgcgcacct cctcgcggct 82501 gcgaaccatc ttgttggcct ccagtggcgg aatcattatg gccgggtcga tctcccgcac 82561 ggtgtgctga aactgcgcca acaggggcgg cgggaccaca gccccccgct cgggggtcgt 82621 caggtactcg tccaccaggg ccaacgtaaa gagggcccgt gtgaggggag tgagggtcgc 82681 gtcgtctatg cgctggaggt gcgccgagaa cagcgtcacc cgattactca ccagggccaa 82741 gaaccggagg ccctcttgca cgaacggggc ggggaagagc aggctgtacg ccggggtggt 82801 aaggttcgcg ctgggctgcc ccaacgggac cggcgccatc ttgagtgacg tctccccaag 82861 ggcctcgatg gaggtccgcg ggctcatggc caagcagctc ttggtgacgg tttgccagcg 82921 gtctatccac tccacggcgc actggcggac gcggaccggc cccagggccg ccgcggtgcg 82981 caggccggcg gaatccagcg catgggacgt gtcggagccg gtgaccgcga ggatggtgtc 83041 cttgatgacc tccatctccc ggaaggcctg gtcgggggcc tcggggagag ccaccaccaa 83101 gcggtgtacg agcaacccgg ggaggttctc ggccaagagc gccgtctccg gaagcccgtg 83161 ggcccggtgg agcgcgcaca ggtgttccag cagcggccgc cagcatgccc gcgcgtctac 83221 cggggcaatg gccgttcccg acaacagaaa cgccgccatg gcggcgcgca gcttggccgt 83281 ggccagaaac gccgggtcgt ccgccccgtt tgccgtctcg gccgtggggg ttggcggttg 83341 gcgaaggccg gctaggctcg ccaataggcg ctgcataggt ccgtccgagg gcggaccggc 83401 gggtgaggtc gtgacgacgg gggcctcgga cgggagaccg cggtctgcca tgacgcccgg 83461 ctcgcgtggg tgggggacag cgtagaccaa cgacgagatc gggcgggaat gactgtcgtg 83521 cgctgtaggg agcggcgaat tatcgatccc ccgcggccct ccaggaaccc cgcaggcgtt 83581 gcgagtaccc cgcgtcttcg cggggtgtta tacggccact taagtcccgg catcccgttc 83641 gcggacccag gcccggggga ttgtccggat gtgcgggcag cccggacggc gtgggttgcg 83701 gactttctgc ggggcggccc aaatggccct ttaaacgtgt gtatacggac gcgccgggcc 83761 agtcggccaa cacaacccac cggaggcggt agccgcgttt ggctgtgggg tgggtggttc 83821 cgccttgcgt gagtgtcctt tcgacccccc tcccccgggt tttgttaggt cgcgatctgc 83881 agtcgcaatg aagaccaatc cgctacccgc aaccccttcc gtgtggggcg ggagtaccgt 83941 ggaactcccc cccaccacac gcgataccgc gggacagggc ctgcttcggc gcgtcctgcg 84001 ccccccgatc tctcgccgcg acggcccagt gctccccagg gggtcgggac cccggagggc 84061 ggccagcacg ctgtggttgc ttggcctgga cggcacagac gcgccccctg gggcgctgac 84121 ccccaacgac gataccgaac aggccctgga caagatcctg cggggcacca tgcgcggggg 84181 ggcggccctg atctgctccc cgcgccatca tctaacccgc caagtgatcc tgacggatct 84241 gtgccaaccc aacgcggatc gtgccgggac gctgcttctg gcgctgcggc accccgccga 84301 cctgcctcac ctggcccacc agcgcgcccc gccaggccgg cagaccgagc ggctgggcga 84361 ggcctggggc cagctgatgg aggcgaccgc cctggggtcg gggcgagccg agagcgggtg 84421 cacgcgcgcg ggcctcgtgt cgtttaactt cctggtggcg gcgtgtgccg cctcgtacga 84481 cgcgcgcgac gccgccgatg cggtacgggc ccacgtcacg gccaactacc gcgggacgcg 84541 ggtgggggcg cgcctggatc gtttttccga gtgtctgcgc gccatggttc acacgcacgt 84601 cttcccccac gaggtcatgc ggtttttcgg ggggctggtg tcgtgggtca cccaggacga 84661 gctagcgagc gtcaccgccg tgtgcgccgg gccacaggag gcggcgcaca ccggccaccc 84721 gggccggccc cgctcggccg tgatcctccc ggcgtgtgcg ttcgtggacc tggacgccga 84781 gctggggctg gggggcccgg gtgcggcgtt tctgtacctg gtattcactt accgccagcg 84841 ccgggaccag gagctgtgtt gtgtgtacgt gatcaagagc cagctccccc cgcgcgggtt 84901 ggagccggcc ctggagcggc tgtttgggcg cctccggatc accaacacga ttcacggcac 84961 cgaggacatg acgcccccgg ccccaaaccg aaaccccgac ttccccctcg cgggcctggc 85021 cgccaatccc caaaccccgc gttgctcggc tggccaggtc acgaaccccc agttcgccga 85081 caggctgtac cgctggcagc cggacctgcg ggggcgcccc accgcacgca cctgtacgta 85141 cgccgccttt gcagagctcg gcatgatgcc cgaggatagt ccccgctgcc tgcaccgcac 85201 cgagcgcttt ggggcggtca gcgtccccgt tgtcatcctg gaaggcgtgg tgtggcgccc 85261 cggcgagtgg cgggcatgcg cgtgagcgta gcaaacgccc cgcccacaca acgctccgcc 85321 cccaacccct tccccgctgt cactcgtggt tcgttgaccc ggacgtccgc caaataaagc 85381 cactgaaacc cgaaacgcga gtgttgtaac gtcctttggg cgggaggaag ccacaaaatg 85441 caaatgggat acatggaagg aacacacccc cgtgactcag gacatcggcg tgtccttttg 85501 ggtttcactg aaactggccc gcgccccacc cctgcgcgat gtggataaaa agccagcgcg 85561 ggtggtttag ggtaccacag gtgggtgctt tggaaacttg tcggtcgccg tgctcctgtg 85621 agcttgcgtc cctccccggt ttcctttgcg ctcccgcctt ccggacctgc tcttgcctat 85681 cttctttggc tctcggtgcg attcgtcagg cagcggcctt gtcgaatctc gaccccacca 85741 ctcgccggac ccgccgacgt cccctctcga gcccaccgaa acccgccgcg tctgttgaaa 85801 tggccagccg cccagccgca tcctctcccg tcgaagcgcg ggccccggtt gggggacagg 85861 aggccggcgg ccccagcgca gccacccagg gggaggccgc cggggcccct ctcgcccacg 85921 gccaccacgt gtactgccag cgagtcaatg gcgtgatggt gctttccgac aagacgcccg 85981 ggtccgcgtc ctaccgcatc agcgatagca actttgtcca atgtggttcc aactgcacca 86041 tgatcatcga cggagacgtg gtgcgcgggc gcccccagga cccgggggcc gcggcatccc 86101 ccgctccctt cgttgcggtg acaaacatcg gagccggcag cgacggcggg accgccgtcg 86161 tggcattcgg gggaacccca cgtcgctcgg cggggacgtc taccggtacc cagacggccg 86221 acgtccccac cgaggccctt gggggccccc ctcctcctcc ccgcttcacc ctgggtggcg 86281 gctgttgttc ctgtcgcgac acacggcgcc gctctgcggt attcgggggg gagggggatc 86341 cagtcggccc cgcggagttc gtctcggacg accggtcgtc cgattccgac tcggatgact 86401 cggaggacac ggactcggag acgctgtcac acgcctcctc ggacgtgtcc ggcggggcca 86461 cgtacgacga cgcccttgac tccgattcgt catcggatga ctccctgcag atagatggcc 86521 ccgtgtgtcg cccgtggagc aatgacaccg cgcccctgga tgtttgcccc gggacccccg 86581 gcccgggcgc cgacgccggt ggtccctcag cggtagaccc acacgcgccg acgccagagg 86641 ccggcgctgg tcttgcggcc gatcccgccg tggcccggga cgacgcggag gggctttcgg 86701 acccccggcc acgtctggga acgggcacgg cctaccccgt ccccctggaa ctcacgcccg 86761 agaacgcgga ggccgtggcg cgctttctgg gagatgccgt gaaccgcgaa cccgcgctca 86821 tgctggagta cttttgccgg tgcgcccgcg aggaaaccaa gcgtgtcccc cccaggacat 86881 tcggcagccc ccctcgcctc acggaggacg actttgggct tctcaactac gcgctcgtgg 86941 agatgcagcg cctgtgtctg gacgttcctc cggtcccgcc gaacgcatac atgccctatt 87001 atctcaggga gtatgtgacg cggctggtca acgggttcaa gccgctggtg agccggtccg 87061 ctcgccttta ccgcatcctg ggggttctgg tgcacctgcg gatccggacc cgggaggcct 87121 cctttgagga gtggctgcga tccaaggaag tggccctgga ttttggcctg acggaaaggc 87181 ttcgcgagca cgaagcccag ctggtgatcc tggcccaggc tctggaccat tacgactgtc 87241 tgatccacag cacaccacac acgctggtcg agcggggct gcaatcggcc ctgaagtatg 87301 aggagtttta cctaaagcgt tttggcgggc actacatgga gtccgtcttc cagatgtaca 87361 cccgcatcgc cggctttttg gcctgccggg ccacgcgcgg catgcgccac atcgccctgg 87421 ggcgagagg gtcgtggtgg gaaatgttca agttcttttt ccaccgcctc tacgaccacc 87481 agatcgtacc gtcgaccccc gccatgctga acctggggac ccgcaactac tacacctcca 87541 gctgctacct ggtaaacccc caggccacca caaacaaggc gaccctgcgg gccatcacca 87601 gcaacgtcag tgccatcctc gcccgcaacg ggggcatcgg gctatgcgtg caggcgttta 87661 acgactccgg ccccgggacc gccagcgtca tgcccgccct caaggtcctt gactcgctgg 87721 tggcggcgca caacaaagag agcgcgcgtc cgaccggcgc gtgcgtgtac ctggagccgt 87781 ggcacaccga cgtgcgggcc gtgctccgga tgaagggggt cctcgccggc gaagaggccc 87841 agcgctgcga caatatcttc agcgccctct ggatgccaga cctgtttttc aagcgcctga 87901 ttcgccacct ggacggcgag aagaacgtca catggaccct gttcgaccgg gacaccagca 87961 tgtcgctcgc cgactttcac ggggaggagt tcgagaagct ctaccagcac ctcgaggtca 88021 tggggttcgg cgagcagata cccatccagg agctggccta tggcattgtg cgcagcgcgg 88081 ccacgaccgg gagccccttc gtcatgttca aagacgcggt gaaccgccac tacatctacg 88141 acacccaggg ggcggccatc gccggctcca acctctgcac cgagatcgtc catccggcct 88201 ccaagcgatc cagtggggtc tgcaacctgg gaagcgtgaa tctggcccga tgcgtctcca 88261 ggcagacgtt tgactttggg cggctccgcg acgccgtgca ggcgtgcgtg ctgatggtga 88321 acatcatgat cgacagcacg ctacaaccca cgccccagtg cacccgcggc aacgacaacc 88381 tgcggtccat gggaatcggc atgcagggcc tgcacacggc ctgcctgaag ctggggctgg 88441 atctggagtc tgccgaattt caggacctga aaacacat cgccgaggtg atgctgctgt 88501 cggcgatgaa gaccagcaac gcgctgtgcg ttcgcggggc ccgtcccttc aaccacttta 88561 agcgcagcat gtatcgcgcc ggccgctttc actgggagcg ctttccggac gcccggccgc 88621 ggtacgagg cgagtgggag atgctacgcc agagcatgat gaaacggc ctgcgcaaca 88681 gccagtttgt cgcgctgatg cccaccgccg cctcggcgca gatctcggac gtcagcgagg 88741 gctttgcccc cctgttcacc aacctgttca gcaaggtgac ccgggacggc gagacgctgc 88801 gccccaacac gctcctgcta aaagactgg aacgcacgtt tagcgggaag cgcctcctgg 88861 aggtgatgga cagtctcgac gccaagcagt ggtccgtggc gcaggcgctc ccgtgcctgg 88921 agcccaccca ccccctccgg cgattcaaga ccgcgtttga ctacgaccag aagttgttga 88981 tcgacctgtg tgcggaccgc gccccctacg tcgaccatag ccaatccatg accctgtatg 89041 tcacggagaa ggcggacggg accctcccag cctccaccct ggtccgcctt ctggtccacg 89101 catataagcg cggactaaaa acagggatgt actactgcaa ggttcgcaag gcgaccaaca 89161 gcggggtctt tggcggcgac gacaacattg tctgcacgag ctgcgcgctg tgaccgacaa 89221 accccctccg cgccaggccc gccgccactg tcgtcgccgt cccacgctct cccctgctgc 89281 catggattcc gcggccccag ccctctcccc cgctctgacg gcccttacgg gccagagcgc 89341 gacggcggac ctggcgatcc agattccaaa gtgccccgac cccgagaggt acttctacac 89401 ctcccagtgt cccgacatta accacctgcg ctccctcagc atccttaacc gctggctgga 89461 aaccgagctt gttttcgtgg gggacgagga ggacgtctcc aagctttccg agggcgagct 89521 cagcttttac cgcttcctct tcgctttcct gtcggccgcc gacgacctgg ttacggaaaa 89581 cctgggcggc ctctccggcc tgtttgagca gaaggacatt ctccactact acgtggagca 89641 ggaatgcatc gaagtcgtac actcgcgcgt gtacaacatc atccagctgg tgcttttcca 89701 caacaacgac caggcgcgcc gcgagtacgt ggccggcacc atcaaccacc cggccatccg 89761 cgccaaggtg gactggttgg aagcgcgggt gcgggaatgc gcctccgttc cggaaaagtt 89821 cattctcatg atcctcatcg agggcatctt ttttgccgcc tcgtttgccg ccatcgccta 89881 ccttcgcacc aacaaccttc tgcgggtcac ctgccagtca aacgacctca tcagccggga 89941 cgaggccgtg cacacgacgg cctcgtgtta catctacaac aactacctcg gcgggcacgc 90001 caagcccccg cccgaccgcg tgtacgggct gttccgccag gcggtcgaga tcgagatcgg 90061 atttatccga tcccaggcgc cgacggacag ccatatcctg agcccggcgg cgctggcggc 90121 catcgaaaac tacgtgcgat tcagcgcgga tcgcctgttg ggccttatcc acatgaagcc 90181 actgttttcc gccccacccc ccgacgccag ctttccgctg agcctcatgt ccaccgacaa 90241 acacaccaat tttttcgagt gtcgcagcac ctcctacgcc ggggcggtcg tcaacgatct 90301 gtgagtgtcg cggcgcgctt ctaccccgtgt ttgcccataa taaacctctg aaccaaactt 90361 tgggtctcat tgtgatctt gtcagggacg cggggtggg agaggataaa agggcggcca 90421 aaagcagta accaggtccg tccagattct gcggcatag ataccata ttttattggt 90481 gggtcgttg ttcggggaca agcgcgctcg tctgacgttt gggctactcg tcccagaatt 90541 tggccaggac gtccttgtag aacgcgggtg gggggcctg gttccgcac tgctccagaa 90601 acctgtcggc gatatcaggg gccgtgat gccggggtcac gatatcgc gccaggtttt 90661 cgtcgcggat gtcctggtag ataggcaggc gttcagaag agtccacggc ccccgctcct 90721 tggggct aagcgatatg acgtacttaa tgtagcggtg ttccaccagc tcggtgatgg 90781 tcatgggatc ggggagccag tccaggact ctggggcgtc gtggatgacg tggcgtcgcc 90841 gttggccac atactgcgg tgcttcca gcagctgcgc gttcgggacc tggacgacc 90901 cgggcggggt gagtatctcc gaggaggacg acctggggcc ggggtggccc ccggtaacgt 90961 cccggggatc cagggggagg tcctcgtcgt cttcgtatcc gccggcgatc tgttgggtta 91021 gaatttcggt ccacgagacg cgcgtctcgg tgccgccggc ggccggcggc agagggggcc 91081 tggtttccgt ggagcgcgag ctggtgtgtt cccggcggat ggcccgccgg gtctgagagc 91141 gactcggggg ggtccagtga cattcgcgca gcacatcctc cacggaggcg taggtgttat 91201 tgggatggag gtcggtgtgg cagcggacaa agagggccag gaactggggg tagctcatct 91261 taaagtactt cagtatatcg cgacagttga tcgtgggaat gtagcaggcg ctaatatcca 91321 acacaatatc acagcccatc aacaggaggt cagtgtccgt ggtgtacacg tacgcgaccg 91381 tgttggtgtg atagaggttg gcgcaggcat cgtccgcctc caactgaccc gagttaatgt 91441 aggcgtaccc cagggcccgg agaacgcgaa tacagaacag atgcgccaga cgcagggccg 91501 gcttcgaggg cgcggcggac ggcagcgcgg ctccggaccc ggccgtcccc cgggtccccg 91561 aggccagaga ggtgccgcgt cggcgcatgt tggaaaaggc agagctgggt ctggagtcgg 91621 tgatggggga aggcggtgga gaggcgtcca cgtcactggc ctcctcgtcc gtccggcact 91681 gggccgtcgt gcgggccagg atggccttgg ctccaaacac aaccggctcc atacaattga 91741 ccccgcgatc ggtaacgaag atggggaaaa gggacttttg ggtaaacacc tttaataagc 91801 gacagaggca gtgtagcgta atggcctcgc ggtcgtaact ggggtatcgg cgctgatatt 91861 tgaccaccaa cgtgtacatg acgttccaca ggtccacggc aatgggggtg aagtacccgg 91921 ccggggcccc aaggccccgg cgcttgacca gatggtgtgt gtgggcaaac ttcatcatcc 91981 cgaacaaacc catgtcaggt cgattgtaac tgcggatcgg cctaactaag gcgtggttgg 92041 tgcgacggtc cgggacaccc gagcctgtct ctctgtgtat ggtgacccag acaacaacac 92101 cgacacaaga ggacaataat ccgttagggg acgctcttta taatttcgat ggcccaactc 92161 cacgcggatt ggtgcagcac cctgcatgcg ccggtgcggg ccaaccttcc ccccgctcat 92221 tgcctcttcc aaaagggtgt ggcctaacga gctgggggcg tatttaatca ggctagcgcg 92281 gcgggcctgc cgtagtttct ggctcggtga gcgacggtcc ggttgcttgg gtcccctggc 92341 tgccatcaaa accccaccct cgcagcggca tacgccccct ccgcgtcccg cacccgagac 92401 cccggcccgg ctgccctcac caccgaagcc cacctcgtca ctgtggggtg ttcccagccc 92461 gcgttgggat gacggattcc cctggcggtg tggcccccgc ctcccacgtg gaggacgcgt 92521 cggacgcgtc cctcgggcag ccggaggagg gggcgccctg ccaggtggtc ctgcagggcg 92581 ccgaacttaa tggaatccta caggcgtttg ccccgctgcg cacgagcctt ctggactcgc 92641 ttctggttat gggcgaccgg ggcatcctta tccataacac gatctttggg gagcaggtgt 92701 tcctgcccct ggaacactcg caattcagtc ggtatcgctg gcgcggaccc acggcggcgt 92761 tcctgtctct cgtggaccag aagcgctccc tcctgagcgt gtttcgcgcc aaccagtacc 92821 cggacctacg tcgggtggag ttggcgatca cgggccaggc cccgtttcgc acgctggttc 92881 agcgcatatg gacgacgacg tccgacggcg aggccgttga gctagccagc gagacgctga 92941 tgaagcgcga actgacgagc tttgtggtgc tggttcccca gggaaccccc gacgttcagt 93001 tgcgcctgac gaggccgcag ctcaccaagg tccttaacgc gaccggggcc gatagtgcca 93061 cgcccaccac gttcgagctc ggggttaacg gcaaaatttc cgtgttcacc acgagtacct 93121 gcgtcacatt tgctgcccgc gaggagggcg tgtcgtccag caccagcacc caggtccaga 93181 tcctgtccaa cgcgctcacc aaggcgggcc aggcggccgc caacgccaag acggtgtacg 93241 gggaaaatac ccatcgcacc ttctctgtgg tcgtcgacga ttgcagcatg cgggcggtgc 93301 tccggcgact gcaggtcggc gggggcaccc tcaagttctt cctcacgacc cccgtcccca 93361 gtctgtgcgt caccgccacc ggtcccaacg ctgtatcggc ggtatttctc ctgaaacccc 93421 agaagatttg cctggactgg ctgggtcata gccaggggtc tccttcagcc gggagctcgg 93481 cctcccgggc ctctgggagc gagccaacag acagcaagga ctccgcgtcg gacgcggtca 93541 gccacggcga tccggaagac ctcgatggcg ctgcccgggc gggagaggcg ggggcctcgc 93601 acgcctgtcc gatgccgtcg tcgaccacgc gggtcactcc cacgaccaag cgggggcgct 93661 cggggggcga ggatgcgcgc gcggacacgg ccctaaagaa acctaagacg gggtcgccca 93721 ccgcaccccc gcccgcagat ccagtccccc tggacacgga ggacgactcc gatgcggcgg 93781 acgggacggc ggcccgtccc gccgctccag acgcccggag cggaagccgt tacgcgtgtt 93841 actttcgcga cctcccgacc ggagaagcaa gccccggcgc cttctccgcc ttccgggggg 93901 ggccccaaac cccgtatggt tttggattcc cctgacgggg cggggccttg gcggccgccc 93961 aactctcgca ccatcccggg ttaatgtaaa taaacttggt attgcccaac actctcccgc 94021 gtgtcgcgtg tggttcatgt gtgtgcctgg cgtcccccac cctcgggttc gtgtatttcc 94081 tttccctgtc cttataaaag ccgtatgtgg ggcgctgacg gaaccacccc gcgtgccatc 94141 acggccaagg cgcgggatgc tccgcaacga cagccaccgg gccgcgtccc cggaggacgg 94201 ccagggacgg gtcgacgacg gacggccaca cctcgcgtgc gtgggggccc tggcgcgggg 94261 gttcatgcat atctggcttc aggccgccac gctgggtttt gcgggatcgg tcgttatgtc 94321 gcgcgggccg tacgcgaatg ccgcgtctgg ggcgttcgcc gtcgggtgcg ccgtgctggg 94381 ctttatgcgc gcaccccctc ccctcgcgcg gcccaccgcg cggatatacg cctggctcaa 94441 actggcggcc ggtggagcgg cccttgttct gtggagtctc ggggagcccg gcacgcagcc 94501 gggggccccg gccccgggcc cggccaccca gtgcctggcg ctgggcgccg cctatgcggc 94561 gctcctggtg ctcgccgatg acgtctatcc gctctttctc ctcgccccgg ggcccctgtt 94621 cgtcggcacc ctggggatgg tcgtcggcgg gctgacgatc ggaggcagcg cgcgctactg 94681 gtggatcggt gggcccgccg cggccgcctt ggccgcggcg gtgttggcgg gcccgggggc 94741 gaccaccgcc agggactgct tctccagggc gtgccccgac caccgccgcg tctgcgtcat 94801 cgtcgcaggc gagtctgttt cccgccgccc cccggaggac ccagagcgac ccggggaccc 94861 cgggccaccg tcccccccga caccccaacg atcccagggg ccgccggccg atgaggtcgc 94921 accggccggg gtagcgcggc ccgaaaacgt ctgggtgccc gtggtcacct ttctgggggc 94981 gggcgcgctc gccgtcaaga cggtgcgaga acatgcccgg ggaacgccgg gcccgggcct 95041 gccgctgtgg ccccaggtgt ttctcggagg ccatgtggcg gtggccctga cggagctgtg 95101 tcaggcgctt gcgccctggg accttacgga cccgctgctg tttgttcacg ccggactgca 95161 ggtcatcaac ctcgggttgg tgtttcggtt ttccgaggtt gtcgtgtatg cggcgctagg 95221 gggtgccgtg tggatttcgt tggcgcaggt gctggggctc cggcgtcgcc tgcgcaggaa 95281 ggaccccggg gacggggccc ggttggcggc gacgcttcgg ggcctcttct tctccgtgta 95341 cgcgctgggg tttggggtgg gggcgctgct gtgccctccg gggtcaacgg gcgggcggtc 95401 gggcgattga tatatttttc aataaaaggc attagtcccg aagaccgccg gtgtgtgatg 95461 atttcgccat aacacccaaa ccccggatgg ggcccgggta taaattccgg aaggggacac 95521 gggctaccct cactaccgag ggcgcttggt cgggaggccg catcgaacgc acacccccat 95581 ccggtggtcc gtgtggaggt cgttttcagt gcccggtctc gctttgccgg gaacgctagc 95641 cgatccctcg cgagggggag gcgtagggca tggccccggg gcgggtgggc cttgccgtgg 95701 tcctgtggag cctgttgtgg ctcggggcgg gggtgtccgg gggctcggaa actgcctcca 95761 ccgggcccac gatcaccgcg ggagcggtga cgaacgcgag cgaggccccc acatcggggt 95821 cccccgggtc agccgccagc ccggaagtca cccccacatc gaccccaaac cccaacaatg 95881 tcacacaaaa caaaaccacc cccaccgagc cggccagccc cccaacaacc cccaagccca 95941 cctccacgcc caaaagcccc cccacgtcca cccccgaccc caaacccaag aacaacacca 96001 cccccgccaa gtcgggccgc cccactaaac cccccgggcc cgtgtggtgc gaccgccgcg 96061 acccattggc ccggtacggc tcgcgggtgc agatccgatg ccggtttcgg aattccaccc 96121 gcatggagtt ccgcctccag atatggcgtt actccatggg tccgtccccc ccaatcgctc 96181 cggctcccga cctagaggag gtcctgacga acatcaccgc cccacccggg ggactcctgg 96241 tgtacgacag cgcccccaac ctgacggacc cccacgtgct ctgggcggag ggggccggcc 96301 cgggtgccga ccctccgttg tattctgtca ccgggccgct gccgacccag cggctgatta 96361 tcggcgaggt gacgcccgcg acccagggaa tgtattactt ggcctggggc cggatggaca 96421 gcccgcacga gtacgggacg tgggtgcgcg tccgcatgtt ccgccccccg tctctgaccc 96481 tccagcccca cgcggtgatg gagggtcagc cgttcaaggc gacgtgcacg gccgacgcct 96541 actacccgcg taaccccgtg gagttggtct ggttcgagga cgaccgccag gtgtttaacc 96601 cgggccagat cgacacgcag acgcacgagc accccgacgg gttcaccacc gtctctaccg 96661 tgacctccga ggctgtcggc ggccaggtcc ccccgcggac cttcacctgc cagatgacgt 96721 ggcaccgcga ctccgtgaca ttctcgcgac gcaatgccac cgggctggcc ctggtgctgc 96781 cgcggccaac catcaccatg gaatttgggg tccggcatgt ggtctgcacg gccggctgcg 96841 tccccgaggg cgtgacgttt gcctggttcc tgggggacga cccctcaccg gcggctaagt 96901 cggccgttac ggcccaggag tcatgcgacc accccgggct ggctacggtc cggtccaccc 96961 tgcccatttc gtacgactac agcgagtaca tctgtcggtt gaccggatat ccggccggga 97021 ttcccgttct agaacaccac ggcagtcacc agcccccacc cagggacccc accgagcggc 97081 aggtgatcga ggcgatcgag tgggtgggga ttggaatcgg ggttctcgcg gcgggggtcc 97141 tggtcgtaac ggcgatcgtg tacgtcgtcc gcacatcaca gtcgcggcag cgtcatcggc 97201 ggtaacgcga gacccccccg ttaccttttt aatatctata tagtttggtc ccccctctat 97261 cccgcccacc gctgggcgct ataaagccgc caccctctct tccctcaggt catccttggt 97321 cgatcccgaa cgacacacgg cgtggagcaa aacgcctccc cctgagccgc tttcctacca 97381 acacaccggc atgcctctgc gggcatcgga acacgcctac cggcccctgg gccccgggac 97441 accccccatg cgggctcggc tccccgccgc ggcctgggtt ggcgtcggga ccatcatcgg 97501 gggagttgtg atcattgccg cgttggtcct cgtgccctcg cgggcctcgt gggcactttc 97561 cccatgcgac agcggatggc acgagttcaa cctcgggtgc atatcctggg atccgacccc 97621 catggagcac gagcaggcgg tcggcggctg tagcgccccg gcgaccctga tcccccgcgc 97681 ggctgccaaa cagctggccg ccgtcgcacg cgtccagtcg gcaagatcct cgggctactg 97741 gtgggtgagc ggagacggca ttcgggcctg cctgcggctc gtcgacggcg tcggcggtat 97801 tgaccagttt tgcgaggagc ccgcccttcg catatgctac tatccccgca gtcccggggg 97861 ctttgttcag tttgtaactt cgacccgcaa cgcgctgggg ctgccgtgag gcgcgtgtac 97921 tgcggtctgt ctcgtctcct cttctcccct tccctccccc tccgcatccc aggatcacac 97981 cggccaacga gggttggggg gtccggcacg gacccaaaat aataaacaca caatcacgtg 98041 cgataaaaag aacacgcggt cccctgtggt gtttttggtt atttttatta aatctcgtcg 98101 acaaacaggg ggaaaggggc gtggtctagc gacggcagca cgggcggagg cgttcaccgg 98161 ctccggcgtc cttcgcgttt aagcttggtc aggagggcgc tcagggcggc gacgttggtc 98221 gggccgtcgt tggtcagggc gttggctcga tggcgggcga ggacgggcga ggggctcaac 98281 ggcgggggcg ggggcccggt gcggcccggg ggggaaaata gggcggatcc cccccagtcg 98341 tacaggggat tttccgcctc aatgtacggg gaggccggcg ctgcattcgc cgtgttcacg 98401 cagacgtttt cgtagacccg catccatggt atttcctcgt agacacgccc cccgtcctcg 98461 cgcaccgtct cgtatattga ctcgtcgtcc tcgtaggggg cgtgccgttc gcgggccgag 98521 gcggcgtggg tggctttgcg gcgggcgtcg tcgtcgtcgt cgtcggccgt cagatacgtg 98581 gcttccatct ggtcgggttc tccctccggg gcgggtcccc acacccgtgg ccgatcgagg 98641 ctccccagag acgcgcgccg gacgaggagg gggcacgtcg ccgccggcgg tcgcctgtcg 98701 ggtcccgcga cgttacgggc cgggaggcgc gggggcacct cccccatgtg cgtgtaatac 98761 gtggccggct gtgcggccgc agcggggggc tcggcgaccg ggtcgttcgc atccggaagc 98821 gggggccccg cgccgtccgc gcggcgcctc cggaacctcc gggtggacgc gggggtcgag 98881 tgtaggcgag gtcgggggag gggcgggggc tcgttgtcgc gccgcgcccg ctgaatcttt 98941 tcccgacagg tcccaccccc cgcgcgatgc ccccccgggc cgctggccat gtcgtccggg 99001 ggaggccccg cggaccacgt cgtccggcga gacgccacga gccgcaggat ggactcgtag 99061 tggagcgacg gcgccccgct gcggagcaga tccgcggcca gggcggcccc gaaccaagcc 99121 ttgatgctca actccatccg ggcccagctg ggggcggtca tcgtggggaa caggggggcg 99181 gtggtccgac agaaacgctc ctggctgtcc accgcggccc gcagatactc gttgttcagg 99241 ctgtcggtgg cccagacgcc gtacccggtg agggtcgcgt tgatgatata ctgggcgtgg 99301 tgatggacga tcgacagaac ctccaccgtg gatacgacgg tatccacggt cccgtacgta 99361 ccgccgctcc gcttgccggt ctgccacagg ttggctaggc gcgtcaggtg gcccaggacg 99421 tcgctgaccg ccgccctgag cgccatgcac tgcatggagc cggttgtgcc gctgggaccc 99481 cggtccagat ggcgcgcgaa cgtttccgcg ggcgcctccg ggctgccgcc gagcgggagg 99541 aaccggcgat tggagggact cagccggtga catacgtgct tgtctgtcgt ccacagcatc 99601 caggacgccc accggtacag cacggagacg taggccagga gctcgttgag ccgcagtgcg 99661 gtgtcggtgc tggggcggct tgggtccgcc gggcgcataa agaacatgta ctgctgaatc 99721 cgatggaggg cgtcgcgcag gccggccacg gtggcggcgt acttggccgc cacggccccg 99781 ctcttgaacg gggtgcgcgc cagcagcttt ggcgccaggg tgggccgcag cagcacgtga 99841 aggctggggt cgcagtcgcc cacggggtcc tcggggacgt ccaggccgct gggcaccacc 99901 gtctgcaggt acttccagta ctgcgtgagg atggcgcggc tcaactggcc gccgggcagc 99961 tccacctcgc ccagcgcctg ggtggcggcc gaagcgtagt gccggatgta ctcgtagtgc 100021 gggtcgctgg cgagcccgtc cacgatcaaa ctctcgggaa ccgtgttgtg ttgccgcgcg 100081 gccaaccgga cgctgcgatc ggtgcaggtc agaaacgccg gctcgcggtc gtcggagcgc 100141 tgccgcaagg cgcccacggc cgcgctaagg agcccctccg gggtggggag cagacacccg 100201 ccgaagatgc gccgctcggg aacgcccgcg ttgtcgccgc ggatcaggtt ggcaggcgtc 100261 aggcaccgcg ccagccgcag ggagctcgcg ccgcgcgtcc ggcgctgcat ggtgacgccc 100321 gttcggtcgg gacccgccgg tcggagttat gccgcgtcca gggccatcgg ggcgcttttt 100381 atcgggagga gcttatgggc gtggcgggcc tcccagcccg gtcgcgcgcc tccccgacac 100441 gtgcgcccgc agggcggcgg ccccctcgtc tcccatcagc agtttcctaa actgggacat 100501 gatgtccacc acgcggaccc gcgggccccaa cacggacccg ccgcttacgg ggggcgggggg 100561 gaagggctcc aggtccttga gaaaggc ggggtctgcc gtcccggaca cggggcccg 100621 gggcgctgag gaggcggggc gcagatccac gtgctccgcg gccgcgcgga cgtccgccca 100681 gaacttggcg ggggtggtgc gcgcgtacag gggctgggtc gctcggagga cgcacgcgta 100741 gcgcaggggg gtgtacgtgc ccacctcggg ggccgtgaat cccccgtcaa acgcggccag 100801 tgtcacgcac gccaccacgg tgtcggcaaa gcccagcagc cgctgcagga cgagcccggc 100861 ggccagaatg gcgcgcgtgg ccgccgcgtc gtcccggcgc cggtgcgcgt ccccgcacgc 100921 ccggggcgtac tttaaggtca cggtcgccag ggccgtgtgc agcgcgtaca ccgcagcgcc 100981 cagcacggcg ttgagcccgc tgttggcgag cagccggcgc gctgcggtgt cgcccagcgc 101041 ctcgtgctcg gcccccacga ccgcggggct tcccaggggc agggcgcga acagctcctc 101101 ccgcgccacg tccgcaaagg cggggtggtg cacgtgcggg tgcaggcgcg cccccacgac 101161 caccgagagc cactggaccg tctgctccgc catcaccgcc agcacatcca gcacgcgccc 101221 caggaaggcg gcctcccgcg tcaaaacgca ccggacggcg tcgggattga agcggcgag 101281 cagggccccg gtggccaggt acgtcatgcg gccggcatag cgggcggcca cgcgacagtc 101341 gcggtccagc agcgcgcgca ccccgggcca gtacagcagg gaccccagcg agctgcggaa 101401 caccgcggcg tcggggccgg attgggggga cactaacccc cccgcgctca gtaacggcac 101461 ggccgcggcc ccgacgggac gcaacgccgt gaggctcgcg aactgccgcc tcagctcggc 101521 cgccctgtcg tccaggtcag acccgcgcgc ctccgcgtga aggcgcgtcc cgcacaccca 101581 cccgttgatg gccagccgca cgacggcatc cgccaaaaag ctcatcgcct gggcggggct 101641 ggtttttgtt cgacgatccg tcaggtcaag aatcccatcg cccgtgatat accaggccaa 101701 cgcctcgccc tgctgcaggg tttggcggaa aaacaccgcg gggttgtcgg gggaggcgaa 101761 gtgcatgacc cccacgcgcg ataacccgaa cgcgctatcc ggacacgggt aaaacccggc 101821 cggatgcccc agggctaggg cggagcgcac ggactcgtcc cacacggcaa cctgaggggc 101881 cagtcgatcc aacgggaatg ccgcccggag ctccgggccc ggcacgcgtc cctccagaac 101941 ctccaccttg ggcggggaac gggccccgcc gccgtcctcc ggcccgacgg cttccgggta 102001 gtcgtcctcc tcgtactgca gctcctctag gaacagcggc gacggcgcca cccgcgaacc 102061 gccgacccgc cccaaaatag cccgcgcgtc gacgggaccc aggtatcccc cctgccgggc 102121 ctgcggagga ccgcggggaa cctcatcatc atcgtccagg cgaccgcgca ccgactggct 102181 acgggccgca tcgggcccgg ggcgctgccg ggacgctcgg cgatgggatg tgggcggggc 102241 ttccgacgcg cgccgtcgtc gggctcgcgg gccttcccgt cgacggcgca cgggcggctc 102301 gtcgcccgcc atctcctcca gagcctctag ctcgctgtcg tcatccccgc ggaacaccgc 102361 acgcaggtac cccatgaacc ccaccccatc gcccgctggc tcgtccgcca cgggcgaggc 102421 gcgggggcgg gtggatgcgc gcctcctgcg ccccgcgggt tcgcgagccg acatggtggc 102481 gatagacgcg ggttatcgga tgtccgctac cccccaaaaa agaaaaagac cccacagcgc 102541 ggatggaggc cggggtaggt gccgccggac cccctcgcga tgggaatgga cgggagcgac 102601 ggggccggcg caaaaaaacg cagtatctcc cgcgaaggct acccgccgcc ccagcccccg 102661 gccaaatgcg gaaacggtcc cgcgctctcg cctttatacg cgggccgccc tgcgacacaa 102721 tcacccgtcc gtggtttcga atctacacga caggcccgca gacgcggcta acacacacgc 102781 cggcaaccca gaccccagtg ggttggttgc gcggtcccgt ctcctggcta gttctttccc 102841 ccaccaccaa ataatcagac gacaaccgca ggttttgtaa tgtatgtgct cgtgtttatt 102901 gtggatacga accggtgacg ggaggggaaa acccagacgg gggatgcggg tccggtcgcg 102961 ccccctaccc accgtactcg tcaattccaa gggcatcggt aaacatctgc tcaaactcga 103021 agtcggccat atccagagcg ccgtaggggg cggagtcgtg gggggtaaat cccggccccg 103081 gggaatcccc gtcccccaac atgtccagat cgaaatcgtc tagcgcgtcg gcatgcgcca 103141 tcgccacgtc ctcgccgtct aagtggagct cgtcccccag gctgacatcg gtcggggggg 103201 ccgtcgacag tctgcgcgtg tgtcccgcgg ggagaaagga caggcgcgga gccgccagcc 103261 ccgcctctc gggggcgtcg tcgtccggga gatcgagcag gccctcgatg gtagacccgt 103321 aattgttttt cgtacgcgcg cggctgtacg cgtgttcccg catgaccgcc tcggagggcg 103381 aggtcgtgaa gctggaatac gagtccaact tcgcccgaat caacaccata aagtacccag 103441 aggcgcggc ctgggtgcca tgcagggtgg gaggggtcgt caacggcgcc cctggctcct 103501 ccgtagccgc gctgcgcacc agcgggaggt taaggtgctc gcgaatgtgg tttagctccc 103561 gcagccggcg ggcctcgatt ggcactcccc ggacggtgag cgctccgttg acgaacatga 103621 agggctggaa cagacccgcc aactgacgcc agctctccag gtcgcaacag aggcagtcaa 103681 acaggtcggg ccgcatcatc tgctcggcgt acgcggccca taggatctcg cgggtcaaaa 103741 atagatacaa atgcaaaaac agaacacgcg ccagacgagc ggtctctcgg tagtacctgt 103801 ccgcgatcgt ggcgcgcagc attctccca ggtcgcgatc gcgtcgcgc atgtgcgcct 103861 ggcggtgcag ctgccggacg ctggcgcgca ggtaccggta cagggccgag cagaagttgg 103921 ccaacacggt tcgatagctc tcctcccgcg cccgtagctc ggcgtggaag aaacgagaga 103981 gcgcttcgta gtagagcccg aggccgtcgc gggtggccgg aagcgtcggg aaggccacgt 104041 cgccgtgggc gcgaatgtcg atttgggcgc gttcggggac gtacgcgtcc ccccattcca 104101 ccacatcgct gggcagcgtt gataggaatt tacactcccg gtacaggtcg gcgttggtcg 104161 gtagcgccga aaacagatcc tcgttccagg tatcgagcat ggtacatagc gcggggcccg 104221 cgctaaagcc caagtcgtcg aggagacggt taaagagggc ggcggggggg acgggcatgg 104281 gtggggaggg catgagctgg gcctggctca ggcgccccgt tgcgtacagc gggggggccg 104341 ccggggtgtt tttgggaccc ccggctgggc gggggggcgg tggcgaagcg ccgtccgcgt 104401 tcatgtcggc aaacagctcg tcgaccaaga ggtccattgg gtggggttga tacgggaaag 104461 acgatatcgg gcttttgatg cgatcgtccc cgcccgccca gagagtgtgg gacgcccgac 104521 ggcgcgggaa gagaaaaacc cccaaacgcg ttagaggacc ggacggacct tatgggggga 104581 agtgggcagc gggaaccccg tccgttcccg aggaatgaca gcccgtggtc gccaccacgc 104641 atttaagcaa cccgcacggg ccgccccgta cctcgtgact tccccccaca ttggctcctg 104701 tcacgtgaag gcgaaccgag ggcggctgtc caacccaccc cccgccaccc agtcccggtc 104761 cccgtcggat tgggaaacaa aggcacgcaa cgccaacacc gaatgaaccc ctgttggtgc 104821 tttattgtct gggtacggaa gttttcactc gacgggccgt ctggggcgag aagcggagcg 104881 ggctggggct cgaggtcgct cggtggggcg cgacgccgca gaacgccctc gagtcgccgt 104941 ggccgcgtcg acgtcctgca ccacgtctgg attcaccaac tcgttggcgc gctgaagcag 105001 gtttttgccc tcgcagaccg tcacgcggat ggtggtgatg ccaaggagtt cgttgaggtc 105061 ttcgtctgtg cgcggacgcg acatgtccca gagctggacc gccgccatcc gggcatgcat 105121 ggccgccagg cgcccgaccg cggcgcagaa gacgcgcttg ttaaagccgg ccacccgggg 105181 ggtccatggc gcgtcggggt ttgggggggc ggtgctaaag tgcagctttc tggccagccc 105241 ctgcgcgggt gtcttggatc gggttggcgc cgtcgacgcg ggggcgtctg ggagtgcggc 105301 ggattctggc tgggccgatt tcctgccgcg ggtggtctcc gccgccgggg ccgcgggggc 105361 cttagtcgcc acccgctggg ttcggggggc ccggggggcg gtggtgggtg tgcgtccggc 105421 ccctccggac ccagcgggtg gcggaggtgc ccgcgcaggc cccgggccgg acaaaaccgc 105481 cccggaaacg ggacgccgcg tccgggggac ctccgggtgt tcgtcgtctt cggatgacga 105541 gcccccgtag agggcataat ccgactcgtc gtactggacg aaacggacct cgcccctctg 105601 gcgcgagcgt gtctgtaggg cgccacggcg ggaggtgtca ggcggactat cgggactcgc 105661 catacctgaa gacggggtgt agtacagatc ctcgtactca tcgcgcggaa cctcccgcgg 105721 acccgacttc acggagcggc gagaggtcat ggttccacga acacgctagg gtcggatgcg 105781 cggacaatta ggcctgggtt cggacggcgg gggtggtgca ggtgtggaga ggtcgagcga 105841 taggggcggc ccgggagaga agagagggtc cgcaaaaccc actggggatg cgtgagtggc 105901 cctctgtggg cggtggggga gagtcttata ggaagtgcat ataaccacaa cccatgggtc 105961 taaccaatcc ccaggggcca agaaacagac acgccccaaa cggtctcggt ttccgcgagg 106021 aaggggaagt cctgggacac cctccacccc cacccctcac cccacacagg gcgggttcag 106081 gcgtgcccgg cagccagtag cctctggcag atctgacaga cgtgtgcgat aatacacacg 106141 cccatcgagg ccatgcctac ataaaagggc accagggccc ccggggcaga catttggcca 106201 gtgttttggg tctcgcaccg cgcgcccccg atcccatcgc gcccgccctc ctcgccgggc 106261 ggctccccgt gcgggcccgc gtctcccgcc gctaaggcga cgagcaagac aaacaacagg 106321 cccgcccgac agacccttct gggggggccc atcgtcccta acaggaagat gagtcagtgg 106381 ggatccgggg cgatccttgt ccagccggac agcttgggtc gggggtacga tggcgactgg 106441 cacacggccg tcgctactcg cgggggcgga gtcgtgcaac tgaacctggt caacaggcgc 106501 gcggtggctt ttatgccgaa ggttagcggg gactccggat gggccgtcgg gcgcgtctct 106561 ctggacctgc gaatggctat gccggctgac ttttgcgcga ttattcacgc ccccgcgcta 106621 gccagccccg ggcaccacgt aatactgggt cttatcgact cggggtaccg cggaaccgtt 106681 atggccgtgg tcgtagcgcc taaaaggacg cgggaatttg cccccgggac cctgtgggtc 106741 gacgtgacgt tcctggacat cctggcgacc cccccggccc tcaccgagcc gatttccctg 106801 cggcagttcc cgcaactggc gcccccccct ccaaccgggg ccgggatacg cgaagatcct 106861 tggttggagg gggcgctcgg ggccccaagc gtgactacgg ccctaccggc gcgacgccga 106921 gggcggtccc tcgtctatgc cggcgagctg acgccggttc agacggaaca cggggacggc 106981 gtacgagaag ccatcgcctt ccttccaaaa cgcgaggagg atgccggttt cgacattgtc 107041 gtccgtcgcc cggtcaccgt cccggcaaac ggcaccacgg tcgtgcagcc atccctccgc 107101 atgctccacg cggacgccgg gcccgcggcc tgctatgtgt tggggcggtc gtcgctcaac 107161 gcccgcggcc tcctggtcgt tcctacgcgc tggctccccg ggcacgtatg tgcgtttgtt 107221 gtttacaacc ttacgggggt tcctgtgacc ctcgaggccg gcgccaaggt cgcccagctc 107281 ctggttgcgg gggcggacgc tcttccttgg atccccccgg acaactttca cgggaccaaa 107341 gcgcttcgaa actaccccag gggtgttccg gactcaaccg ccgaacccag gaacccgccg 107401 ctcctggtgt ttacgaacga gtttgacgcg gaggcccccc cgagcgagcg cgggaccggg 107461 ggttttggct ctaccggtat ttagcccaca gctttgggtt cgttccgggc aataaaaaac 107521 gtttgtatcg catctttcct gtgtgtagtt gtttatgttg gatgcctgtg ggtctatcac 107581 acccgcccct ccatcccaca aacacaaaac acacgggttg gatgaaaaca cgcatttatt 107641 gacccaaaac acacggagct gctcgagatg ggccagggcg aggtgcggtt ggggaggctg 107701 taggtctggg aacggacacg cggggacacg attccggttt ggggtccggg agggcgtcgc 107761 cgtttcgggc ggcaggcgcc agcgtaacct ccgggggcgg cgtgtggggg tgccccaagg 107821 agggcgcctc ggtcacccca atcccccccg accgggttcc cccggcaacc ccgaaggcgg 107881 agaggccaag ggcccgttcg gcgatggcca catcctccat gaccacgtca ctctcggcca 107941 tgctccgaat agcctgggag acgagcacat ccgcggactt gtcagccgcc cccacggaca 108001 tgtacatctg caggatggtg gccatacacg tgtccgccag gcgccgcatc ttgtcctgat 108061 gggccgccac ggccccgtcg atcgtggggg cctcgagccc ggggtggtgg cgcgccagtc 108121 gttctaggtt caccatgcag gcgtggtacg tgcgggccaa ggcgcgggcc ttcacgaggc 108181 gtcgggtgtc gtccagggac cccagggcgt catcgagcgt gatgggggcg ggaagtagcg 108241 cgttaacgac cgccagggcc tcctgcagcc gcggctccgc ctccgagggc ggaacggccg 108301 cgcggatcat ctcatattgt tcctcggggc gcgctcccca gccacatata gccccgagaa 108361 gagaagccat cgcgggcggg tactggccct tgggcgcgcg gacgcaatgg ggcaggaaga 108421 cgggaaccgc ggggagaggc gggcggccgg gactcccgtg gaggtgaccg cgctttatgc 108481 gaccgacggg tgcgttatta cctcttcgat cgccctcctc acaaactctc tactgggggc 108541 cgagccggtt tatatattca gctacgacgc atacaggcac gatggccgtg ctgacgggcc 108601 cacggagcaa gacaggttcg aagagagtcg ggcgctctac caagcgtcgg gcgggctaaa 108661 tggcgactcc ttccgagtaa ccttttgttt attggggacg gaagtgggtg ggacccacca 108721 ggcccgcggg cgaacccgac ccatgttcgt ctgtcgcttc gagcgagcgg acgacgtcgc 108781 cgcgctacag gacgccctgg cgcacgggac cccgctacaa ccggaccaca tcgccgccac 108841 cctggacgcg gaggccacgt tcgcgctgca tgcgaacatg atcctggctc tcaccgtggc 108901 catcaacaac gccagccccc gcaccggacg cgacgccgcc gcggcgcagt atgatcaggg 108961 cgcgtcccta cgctcgctcg tggggcgcac gtccctggga caacgcggcc ttaccacgct 109021 atacgtccac cacgaggcgc gcgtgcttgc cgcgtaccgc agggcgtatt atggaagcgc 109081 gcagagtccc ttctggtttc ttagcaaatt cgggccggac gaaaaaagcc tggtgctcac 109141 cactcggtac tacctgcttc aggcccagcg tctggggggc gcggggcca cgtacgacct 109201 gcaggccatc aggacatct gcgccaccta cgcgattccc cacgcccccc gccccgacac 109261 cgtcagcgct gcgtccctga cctcgtttgc cgccatcacg cggttctgtt gcakgagcca 109321 gtacgcccgc ggggccgcgg cggccgggtt tccgctttac gtggagcgcc gtattgcggc 109381 cgacgtccgc gagaccagtg cgctggagaa gttcataacc cacgatcgca gttgcctgcg 109441 cgtgtccgac cgtgaattca ttacgtacat ctacctggcc cattttgagt gtttcagccc 109501 cccgcgccta gccacgcatc ttcgggccgt gacgacccac gaccccaacc ccgcggccag 109561 cacggagcag ccctcgcccc tgggcaggga ggccgtggaa caatttttt gtcacgtgcg 109621 cgcccaactg aatatcgggg agtacgtcaa acacaacgtg accccccggg agaccgtcct 109681 ggatggcgat acggccaagg cctacctgcg cgctcgcacg tacgcgcccg gggccctgac 109741 gcccgcccc gcgtattgcg gggccgtgga ctccgccacc aaaatgatgg ggcgtttggc 109801 ggacgccgaa aagctcctgg tcccccgcgg gtggcccgcg tttgcgcccg ccagtcccgg 109861 ggaggacacg gcgggcggca cgccgccccc acagacctgc ggaattgtca agcgcctcct 109921 gagactggcc gccacggaac agcagggcac cacacccccg gcgatcgcgg cgcttatccg 109981 taatgcggcg gtgcagactc ccctgcccgt ctaccggata tccatggtcc ccacgggaca 110041 ggcatttgcc gcgctggcct gggacgactg ggcccgcata acgcgggacg ctcgcctggc 110101 cgaagcggtc gtgtccgccg aagcggcggc gcaccccgac cacggcgcgc tgggcaggcg 110161 gctcacggat cgcatccgcg cccagggccc cgtgatgccc cctggcggcc tggatgccgg 110221 ggggcagatg tacgtgaatc gcaacgagat attcaacggc gcgctggcaa tcacaaacat 110281 catcctggat ctcgacatcg ccctgaagga gcccgtcccc tttcgccggc tccacgaggc 110341 cctgggccac tttaggcgcg gggctctggc tgcggttcag ctcctgtttc ccgcggcccg 110401 cgtggacccc gacgcatatc cctgttattt tttcaaaagc gcatgtcggc ccggcccggc 110461 gtccgtgggt tccggcagcg gactcggcaa cgacgacgac ggggactggt ttccctgcta 110521 cgacgacgcc ggtgatgagg agtgggcgga ggacccgggc gccatggaca catcccacga 110581 tccccccggac gacgaggttg cctactttga cctgtgccac gaagtcggcc ccacggcgga 110641 acctcgcgaa acggattcgc ccgtgtgttc ctgcaccgac aagatcggac tgcgggtgtg 110701 catgcccgtc cccgccccgt acgtcgtcca cggttctcta acgatgcggg gggtggcacg 110761 ggtcatccag caggcggtgc tgttggaccg agattttgtg gaggccatcg ggagctacgt 110821 aaaaaacttc ctgttgatcg atacgggagt gtacgcccac ggccacagcc tgcgcttgcc 110881 gtattttgcc aaaatcgccc ccgacgggcc tgcgtgcgga aggctgctgc cagtgtttgt 110941 gatccccccc gcctgcaaag acgttccggc gtttgtcgcc gcgcacgccg acccgcggcg 111001 cttccatttt cacgccccgc ccacctatct cgcttccccc cgggagatcc gtgtcctgca 111061 cagcctgggt ggggactatg tgagcttctt tgaaaggaag gcgtcccgca acgcgctgga 111121 acactttggg cgacgcgaga ccctgacgga ggtcctgggt cggtacaacg tacagccgga 111181 tgcgggaggg accgtcgagg ggttcgcatc ggaactgctg gggcggatag tcgcgtgcat 111241 cgaaacccac tttcccgaac acgccggcga atatcaggcc gtatccgtcc ggcgggccgt 111301 fotaaggac gactgggtcc tcctacagct agtccccgtt cgcggtaccc tgcagcaaag 111361 cctgtcgtgt ctgcgcttta agcacggccg ggcgagtcgc gccacggcgc ggacattcgt 111421 cgcgctgagc gtcggggcca acaaccgcct gtgcgtgtcc ttgtgtcagc agtgctttgc 111481 cgccaaatgc gacagcaacc gcctgcacac gctgtttacc attgacgccg gtacgccatg 111541 ctcgccgtcc gttccctgca gcacctctca accgtcgtct tgataacggc gtacggcctc 111601 gtgctcgtgt ggtacaccgt cttcggtgcc agtccgctgc accgatgtat ttacgcggta 111661 cgcccccaccg gcaccaacaa cgacaccgcc ctcgtgtgga tgaaaatgaa ccagacccta 111721 ttgtttctgg gggccccgac gcccccccc aacggggct ggcgcaacca cgcccatatc 111781 tgctacgcca atcttatcgc gggtagggtc gtgcccttcc aggtcccacc cgacgccatg 111841 aatcgtcgga tcatgaacgt ccacgaggca gttaactgtc tggagaccct atggtacaca 111901 cgggtgcgtc tggtggtcgt agggtggttc ctgtatctgg cgttcgtcgc cctccaccaa 111961 cgccgatgta tgtttggtgt cgtgagtccc gcccacaaga tggtggcccc ggccacctac 112021 ctcttgaact acgcaggccg catcgtatcg agcgtgttcc tgcagtaccc ctacacgaaa 112081 attacccgcc tgctctgcga gctgtcggtc cagcggcaaa acctggttca gttgtttgag 112141 acggacccgg tcaccttctt gtaccaccgc cccgccatcg gggtcatcgt aggctgcgag 112201 ttgatgctac gctttgtggc cgtgggtctc atcgtcggca ccgctttcat atcccggggg 112261 gcatgtgcga tcacataccc cctgtttctg accatcacca cctggtgttt tgtctccacc 112321 atcggcctga cagagctgta ttgtattctg cggcggggcc cggcccccaa gaacgcagac 112381 aaggccgccg ccccggggcg atccaagggg ctgtctggcg tctgcgggcg ctgttgttcc 112441 atcatcctct cgggcatcgc agtgcgattg tgttatatcg ccgtggtggc cggggtggtg 112501 ctcgtggcgc ttcactacga gcaggagatc cagaggcgcc tgtttgatgt atgacgtcac 112561 atccaggccg gcggaaaccg gaacggcata tgcaaattg aaactgtcct gtcttggggc 112621 ccacccaccc gacgcgtcat atgcaaatga aaatcggtcc cccgaggcca cgtgtagcct 112681 ggatcccaac gaccccgccc atgggtccca attggccgtc ccgttaccaa gaccaaccca 112741 gccagcgtat ccaccccgc ccgggtcccc gcggaagcgg aacggtgtat gtgatatgct 112801 attaaatac atgccacgta cttatggtgt ctgattggtc cttgtctgtg ccggaggtgg 112861 ggcggggcc ccgcccgggg ggcggaacga ggaggggttt gggagagccg gccccggcac 112921 cacgggtata aggacatcca ccacccggcc ggtggtggtg tgcagccgtg ttccaaccac 112981 ggtcacgctt ctgtgcctct ccccgattcg ggcccggtcg ctcgctaccg gtgcaccacc 113041 accagaggcc atatccgaca ccccagcccc gacggcagcc gacagcccgg tcatggcgac 113101 tgacattgat atgctaattg acctcggcct ggacctctc gacagcgatc tggacgagga 113161 cccccccgag ccggcggaga gccgccgcga cgacctggca tcggacagca gcggggagtg 113221 ttcctcgtcg gacgaggaca tggaagaccc ccacggagag gacggaccgg agccgatact 113281 cgacgccgct cgcccggcgg tccgcccgtc tcgtccagaa gaccccggcg tacccagcac 113341 ccagacgcct cgtccgacgg agcggcaggg ccccaacgat cctcaaccag cgccccacag 113401 tgtgtggtcg cgcctcgggg cccggcgacc gtcttgctcc cccgagcagc acgggggcaa 113461 ggtggcccgc ctccaacccc caccgaccaa agcccagcct gcccgcggcg gacgccgtgg 113521 gcgtcgcagg ggtcggggtc gcggtggtcc cggggccgcc gatggtttgt cggacccccg 113581 ccggcgtgcc cccagaacca atcgcaaccc ggggggaccc cgccccgggg cggggtggac 113641 ggacggcccc ggcgcccccc atggcgaggc gtggcgcgga agtgagcagc ccgacccacc 113701 cggaggcccg cggacacggg gcgtgcgcca agcacccccc ccgctaatga cgctggcgat 113761 tgcccccccg cccgcggacc cccgcgcccc ggccccggag cgaaaggcgc ccgccgccga 113821 caccatcgac gccaccacgc ggttggtcct gcgctccatc tccgagcgcg cggcggtcga 113881 ccgcatcagc gagagctttg gccgcagcgc acaggtcatg cacgacccct ttggggggca 113941 gccgtttccc gccgcgaata gcccctgggc cccggtgttg gcgggccaag gagggccctt 114001 tgacgccgag accagacggg tctcctggga aaccttggtc gcccacggcc cgagcctcta 114061 tcgcactttt gccggcaatc ctcgggccgc atcgaccgcc aaggccatgc gcgactgcgt 114121 gctgcgccaa gaaaatttca tcgaggcgct ggcctccgcc gacgagacgc tggcgtggtg 114181 caagatgtgc atccaccaca acctgccgct gcgcccccag gaccccatta tcgggacggc 114241 cgcggctgtg ctggataacc tcgccacgcg cctgcggccc tttctccagt gctacctgaa 114301 ggcgcgaggc ctgtgcggcc tggacgaact gtgttcgcgg cggcgtctgg cggacattaa 114361 ggacattgca tccttcgtgt ttgtcattct ggccaggctc gccaaccgcg tcgagcgtgg 114421 cgtcgcggag atcgactacg cgacccttgg tgtcggggtc ggagagaaga tgcatttcta 114481 cctccccggg gcctgcatgg cgggcctgat cgaaatccta gacacgcacc gccaggagtg 114541 ttcgagtcgt gtctgcgagt tgacggccag tcacatcgtc gccccccccgt acgtgcacgg 114601 caaatatttt tattgcaact ccctgtttta ggtacaataa aaacaaaaca tttcaaacaa 114661 atcgccccac gtgttgtcct tctttgctca tggccggcgg ggcgtgggtc acggcagatg 114721 gcgggggtgg gcccggcgta cggcctgggt gggcggaggg aactaaccca acgtataaat 114781 ccgtccccgc tccaaggccg gtgtcatagt gcccttagga gcttcccgcc cgggcgcatc 114841 cccccttttg cactatgaca gcgacccccc tcaccaacct gttcttacgg gccccggaca 114901 taacccacgt tgccccccct tactgcctca acgccacctg gcaggccgaa acggccatgc 114961 acaccagcaa aactgactcc gcttgcgtgg ccgtgtggag ttacctggtc cgcgcctcct 115021 gtgagaccag cggcacaatc cactgctttt tctttgtggt atacaaggac accaccata 115081 cccctccgct gattaccgag ctccgcaact ttgcggacct ggttaaccac ccgccggtcc 115141 tacgcgact ggaggataag cgcggggtgc ggctgcggtg tgcgcggccg tttagcgtcg 115201 ggacgatta ggacgtct gggtccggcg cgtcctcggc gggagtac acgataacg 115261 ggatcgtgta ccactgccac tgtcggtatc cgttctcaa aacatgctgg atgggggcct 115321 cggcggccct acagcacctg cgctcatca gctccaggcgg catgccgcc cgcgcggcag 115381 agcatcgacg cgtcagatt aaattaagg cgtgatttcc aaccccat gatgtgtgt 115441 aacccccc aaaaaaata agagccgtaa cccaccaaa ccaggcgtgg tgtgagtttg 115501 tggacccaaa gcctcagag acacgcgac agccagtat ggaccgtgag acttttatt 115561 attackcac agggggctt accgccacag gataccaga ataatgacca ccacaatcgc 115621 gaccacccca atacagcat ggcgccccac cacgccaca cagccctgtc gccggtatgg 115681 ggcatgatca gacgagccgc gagccgcgcg ttggccctg tacagctcgc gcgaattgac 115741 cctaggaggc cgccacgcgc ccgagttg cgttcgtcgc tggtcgtcgg gcaccaagc 115801 cccggacgg tgttcggtcg aacgaacgg cacgacagtg ccataggtttg gggggtggtc 115861 cgacatagcc tcggcgtacg tcgggaggcc cgacaagagg tcccttgtga tgtcgggtgg 115921 ggccacaagc ctggtttccg gaagaaacag gggggttgcc aataacccgc cagggccaaa 115981 actccggccc tggcgcacgt cgttcggcgc ggcgccgggc gcgccgagcg gctcgctggg 116041 cggcttggcg tgagcggccc cgctccgacg cctcgccctc tccggaggag gttggtggaa 116101 ttggcacgga cgacaggggc ccagcagagt acggtggagg tgggtccgtg ggggtgtcca 116161 gatcaataac gacaaacggc ccctcgttcc taccagacaa gctatcgtag ggggggcgggg 116221 gatcaacaaa cgcgttcccc gcgctccata gacccgcgtc gggttgcgcc gcctccgaag 116281 ccatggatgc gccccaaagc cacgactccc gcgcgctagg tccttggggt aagggaaaag 116341 gccctactcc ccatccaagc cagccaagtt aacgggctac gccttgggg atgggactgg 116401 caccccggcg gattttgttg ggctggcatg cgtcgcccaa ccgagggccg cgtccacggg 116461 acgcgccttt tataaccccg ggggtcattc ccaacgatca catgcaatct aactggctcc 116521 cctctcctcc cctctcccct ctcccctctc ccctctcccc tctcccctct cccctcttag 116581 gttggggggt ggtccgacat agcctcggcg tacgtcggga ggcccgacaa gaggtccctt 116641 gtgatgtcgg gtggggccac aagcctggtt tccggaagaa acaggggggt tgccaagcgg 116701 cccggcccgc gctccccccc ccccggggcc gtgtccttgc tttccccccg tctccccccc 116761 cctcctcctc cttctcctcc tcctcgtttt tccaaacccc gcccacccgg cccggcccgg 116821 cccggccacc gccgcccacc cacccaccgc gggagaccca gccccggtcc cccgttcccc 116881 gggggccgtt atctccagcg ccccgtccgg cgcgccgccc cccgccgcta aaccccatcc 116941 cgcccccggg accccacata taagccccca gccacacgca agaacagaca cgcagaacgg 117001 ctgtgtttat ttaaataaac cgatgtcgga ataaacaaac acaaacaccc gcgacggggg 117061 gacggaggga ggggggtgac gggggacggg aacagacaca aaaaacaacc acaaaaaaac 117121 agccaccccc gacacccccc accccagtct cctcgccttt tcccacccac cccacgcccc 117181 cactgagccc ggtcgatcga cgagcacccc cgcccccgcc cctgccccgg cgacccccgg 117241 cccgcacgat cccgacaaca ataacaaccc caacggaaag cggcggggtg ttgggggagg 117301 cgaggaacaa ccgaggggaa cgggggatgg aaggacggga agtggaagtc ctgataccca 117361 tcctacaccc ccctgccttc caccctccgg ccccccgcga gtccacccgc cggccggcta 117421 ccgagaccga acacggcggc cgccgcagcc gccgcagccg ccgccgacac cgcagagccg 117481 gcgcgcgcac acacaagcgg cagaggcaga aaggccccga gtcattgttt atgtggccgc 117541 gggccagcag acggcccgcg acaccccccc gcccgtgtgg gtatccggcc ccccgccccg 117601 cgccggtcca ttaagggcgc gcgtgcccgc gagatatcaa tccgttaagt gctctgcaga 117661 caggggcacc gcgcccggaa atccattagg ccgcagacga ggaaaataaa attacatcac 117721 ctacccacgt ggtgctgtgg cctgtttttg ctgcgtcatc tgagccttta taaaagcggg 117781 ggcgcggccg tgccgatcgc gggtggtgcg aaagactttc cgggcgcgtc cgggtgccgc 117841 ggctctccgg gcccccctgc agccggggcg gccaaggggc gtcggcgaca tcctccccct 117901 aagcgccggc cggccgctgg tctgtttttt gttttccccg tttcgggggt ggggggggtt 117961 acggtttctg ttttttaaac ccgtctgggg tgtttttcgt tccgtcgccg ggatgtttcg 118021 ttcgttcggc ccctcacggg gcgaaggccg cgtacggccc gggacgaggg gcccccgacc 118081 gcggcggtcc gggccccgtc cgggcccgct cgccggcacg cgacgcgaaa aaggcccccc 118141 ggaggctttt ccgggttccc ggcccggggc ctgagataaa caatcggggt taccgccaac 118201 ggccggcccc cgtggcggcc cggcccgggg ccccggcgga cccaaggggc cccggcccgg 118261 ggccccacaa cggcccggcg catgcgctgt gttttttttt tcctcggtgt tctgccgggc 118321 tccgtcgcct ttcctgttct cgcttcttcc cccccccctt cttcaccccc agtaccctcc 118381 tccctccctt cctcccccgt tatcccactc gtcgagggcg ccccggtgtc gttcaacaaa 118441 gacgccgcgt ttccaggtag gttagacacc tgcttctccc caatagaggg ggggggaccc 118501 aaacgacagg gggcgcccca gaggctaagg tcggccacgc cactcgcggg tgggctcgtg 118561 ttacagcaca ccagcccgtt cttttccccc cctcccaccc ttagtcagac tctgttactt 118621 acccgtccga ccaccaactg cccccttatc taagggccgg ctggaagacc gccagggggt 118681 cggccggtgt cgctgtaacc ccccacgcca atgacccacg tactccaaga aggcatgtgt 118741 cccaccccgc ctgtgttttt gtgcctggct ctctatgctt gggtcttact gcctgggggg 118801 ggggatgcgg gggagggggg gtgtggaagg aaatgcacgg cgcgtgtgta cccccccccc 118861 aaagttgttc ctaaagcgag gatatggagg agtggcgggt gccgggggac gggggtgatc 118921 tctggcacgc ggggggggaa gggtcggggg agggggggat ggggtaccgg cccacctggc 118981 cgacgcgggt gcgcgtgcct ttgcacacca accccacgtc ccccggcggt ctctaagaag 119041 caccgccccc cctccttcat accaccgagc atgcctgggt gtgggttggt aaccaacacg 119101 cccatcccct cgtctcctgt gattctctgg ctgcaccgca ttcttgtttt ctaactatgt 119161 tcctgtttct gtctcccccc cacccctccg ccccaccccc caacacccac gtctgtggtg 119221 tggccgaccc ccttttgggc gccccgtccc gccacccctc ccgtcctttg ttgccctata 119281 gtgtagttaa cccccccccc gccctttgtg gcggccagag gccaggtcag tccgggcggg 119341 caggcgctcg cggaaactta acacccacac ccagcccact gtggttctgg ctccatgcca 119401 gtggcaggat gctttcgggg atcggtggtc aggcagcccg ggccgcggct ctgtggttaa 119461 caccagagcc tgcccaacat ggcaccccca ctcccacgca cccccactcc cacgcacccc 119521 cactcccacg cacccccact cccacgcacc cccactccca cgcaccccca ctcccacgca 119581 cccccactcc cacgcacccc cactcccacg cacccccact cccacgcacc cccactccca 119641 cgcaccccca ctcccacgca cccccactcc cacgcacccc cactcccacg cacccccaag 119701 atccatccaa cacagacagg gaaaagatac aaaagtaaac ctttatttcc caatagacag 119761 caaaaatccc ctgagttttt tattagggcc aacactaaag acccgctggt gtgtggtgcc 119821 cgtgtctttc actttcccct ccccgacacg gattggctgg tgtagtgggc gcggccagag 119881 accacccagc acccgacccc cctccccaca aacacgggg gcgtccctta ttgttttccc 119941 tcgtcccggg tcgacgcccc ctgctccccg gaccacgggt gccgagaccg caggctgcgg 120001 aagtccaggg cgcccactag ggtgccctgg tcgaacagca tgttccccac gggggtcatc 120061 cagaggctgt tccactccga cgcggggcc gtcgggtact cggggggcat cacgtggtta 120121 cccgcggtct cggggagcag ggtgcggcgg ctccagccgg ggaccgcggc ccgcagccgg 120181 gtcgccatgt ttcccgtctg gtccaccagg accacgtacg ccccgatgtt ccccgtctcc 120241 atgtccagga tgggcaggca gtccccgtg atagtcttgt tcacgtaagg cgacagggcg 120301 accacgctag agaccccga gatgggcagg tagcgcgtga ggccgcccgc ggggacggcc 120361 ccggaagtct ccgcgtggcg cgtcttccgg gcacattcc tcggcccccg cggcccagaa 120421 gcagcgcggg ggccgaggga ggtttcctct tgtctccctc ccagggcacc gacggccccg 120481 cccgaggagg cggaagcgga ggaggacgcg gccccggcgg cggaagaggc ggcccccgcg 120541 ggggtcgggg ccgaggagga agaggcagag gaggaagagg cggaggccgc cgaggacgtc 120601 aggggggtcc cgggcccacc ctggccgcgc ccccccggcc ctgagtcgga gggggggtgc 120661 gtcgccgccc tcttggcccc tgccggcgcg agggggggac gcgtggactg gggggagggg 120721 ttttcctggc ccgacccgcg cctcttcctc ggacgcaccg ccgcctcctg ctcgacagag 120781 acggcggagg ggagcggggc ggcgccggag ggggtgcggc cgcgggaggg cccgtgccca 120841 ccctccacgc ccggcccccc cgagccgcgc gccaccgtcg cacgcgcccg gcacagactc 120901 tgttcttggt tcgcggcctg agccagggac gagtgcgact ggggcacacg gcgcgcgtcc 120961 gcggggcggg cggccggctc cgccccgggg gccggggcgc gggggccggg ccccggaggc 121021 ggcgctcgca cgcacggggc cacggccgcg cgggggcgcg cgggtcccga cgcggccgag 121081 gacgcggggg gcccggggcg gggggcggag cctggcatgg gcgccgcggg gggcctgtgg 121141 ggagaggccg ggggggagtc gctgatcact atggggtctc tgttgtttgc aaggggggcg 121201 ggtctgttga caagggggcc cgtccggccc ctcggccgcc ccgcctccgc ttcaacaacc 121261 ccaaccccaa ccccaacccc cccggagggg ccagacgccc cccgcggcgc cgcggctcgc 121321 gactggcggg agccgccgcc gccgctgctg ttggtggtgg tgttggtgtt actgctgccg 121381 tgtggcccga tgggcgccga ggggggcgct gtccgagccg cggccggctg gggggctgcg 121441 ttagacgccc cgcccgtcac ggggggcgcg gcggtgcctc tgcgtggggg ggcgcggggc 121501 gtccggcggg gggcgggcgg gacgtagtct gctgcaagag acaacggggg gcgcgatcag 121561 gttacgcccc ctccccggcc cgccctttcc tcgcccgccc gcccattcct ccctcctcct 121621 cctcccccag ggtccttgcc gccccccgcc tcaccgtcgt ccaggtcgtc gtcatcctcg 121681 tccgtggtgg gctccgggtg ggtgggcgac agggccctca ccgtgtgccc ccccagggtc 121741 aggtaccgcg gggcgaaccg ctgattgccc gtccagataa agtccacggc cgtgcccgcc 121801 ctgacggcct cctcggcctc catgcgggtc tgggggtcgt tcacgatcgg gatggtgctg 121861 aacgacccgc tgggcgtcac gcccactatc aggtacacca gcttggcgtt gcacagcggg 121921 caggtgttgc gcaattgcat ccaggttttc atgcacggga tgcagaagcg gtgcatgcac 121981 gggaaggtgt cgcagcgcag gtggggcgcg atctcatccg tgcacacggc gcacacgtcg 122041 ccctcgtcgc tccccccgtc ctctcgaggg ggggcgcccc cgcaactgcc ggggtcttcc 122101 tcgcgggggg ggctcccccc cgagaccgcc cccccatcca cgccctgcgg ccccagcagc 122161 cccgtctcga acagttccgt gtccgtgctg tccgcctcgg aggcggagtc gtcgtcatgg 122221 tggtcggcgt ccccccgccc ccccacttcg gtctccgcct cagagtcgct gctgtccggc 122281 aggtctcggt cgcagggaaa cacccagaca tccggggcgg gctaagggga aaaaaggggg 122341 gcgggtaaga atgggggggg atttcccgcg tcaatcagcg cccacgagtt ccccctctcc 122401 ccccccgcct cacaaagtcc tgcccccctg ctggcctcgg aagagggggg agaagggggt 122461 ctgcaaccaa aggtggtctg ggtccgtcct ttggatcccg acccctcttc ttccctcttc 122521 tcccgccctc cagacgcacc ggagtcgggg gtcccacggc gtcccccaaa tatggcgggc 122581 ggctcctccc caccccccta gatgcgtgtg agtaaggggg gcctgcgtat gagtcagtgg 122641 ggaccacgcc cccaacacgg cgaccccggt ccctgtgtgt ttgttgtggg ggcgtgtctc 122701 tgtgtatgag tcaggggtc ccacggcgac cccgggccct gcgtctgagt caaaggggcc 122761 atgtgtatgt gttgggggtc tgtatatata aagtcagggg gtcacatggc gacccccaac 122821 agggcgaccc cggtccctgt atatataggg tcaggggggtt ccgcgccccc taacatggcg 122881 cccccggtcc ctgtatatat agtgtcacgg ggttccacgc cccctaacat ggcgccccaa 122941 catggcgcccc ggctcccgtg tatgagtggg ggtcccccaa catggggcc ggttccaggg 123001 taagggtcgg gggtccccca acatggcgcc ccccaatatg gcgccccaga catggcgccc 123061 ggcccctcac ctcgcgctgg gggcggccct caggccggcg ggtactcgct ccggggcggg 123121 gctccatggg ggtcgtatgc ggctggaggg tcgcggacgg agggtccctg ggggtcgcaa 123181 cgtaggcggg gcttctgtgg tgatgcggag agggggcggc ccgagtctgc ctggctgctg 123241 cgtctcgctc cgagtgccga ggtgcaaatg cgaccagacc gtcgggccag ggctaactta 123301 taccccacgc ctttcccctc cccaaagggg cggcagtgac gattccccca atggccgcgc 123361 gtcccagggg aggcaggccc accgcggggc ggccccgtcc ccggggacca acccggcgcc 123421 cccaaagaat atcattagca tgcacggccc ggcccccgat ttgggggacc aacccggtgt 123481 cccccaaaga accccattag catgcccctc ccgccgacgc aacaggggct tggcctgcgt 123541 cggtgccccg gggcttcccg ccttcccgaa gaaactcatt accatacccg gaaccccagg 123601 ggaccaatgc gggttcattg agcgacccgc gggccaatgc gcgaggggcc gtgtgttccg 123661 ccaaaaaagc aattagcata acccggaacc ccaggggagt ggttacgcgc ggcgcgggag 123721 gcggggaata ccggggttgc cattaaggg ccgcggggaat tgccggaagc gggaagggcg 123781 gccggggccg cccattaatg agtttctaat taccataccg ggaagcggaa caaggcctct 123841 tgcaagtttt taattaccat accgggaagt gggcggcccg gcccattggg cggtaactcc 123901 cgcccaatgg gccgggcccc gaagactcgg cggacgctgg ttggccgggc cccgccgc 123961 tggcggccgc cgattggcca gtcccgcccc cgaggcgggc ccgccttggg ggcggaccgg 124021 ctcccagcgt atatatgcgc ggctcctgcc atcgtctctc cggagagcgg cttggtgcgg 124081 agctcccggg agctccgcgg aagacccagg cgcctcgggt gtaacgttag accgagttcg 124141 ccgggccggc tccgcggcc agggcccggg cacgggcctc gggccccagg cacggccga 124201 tgaccgcctc ggcctccgcc acccggcgcc ggaaccgagc ccggtcggcc cgctcgcggg 124261 cccacgagcc gcggcgcgcc aggcgggcgg ccgaggccca gaccaccagg tggcgcaccc 124321 ggacgtgggg cgagaagcgc acccgcgcgg gggtcgcggg ggtcgcgggg gtcgcgggg 124381 tcgcgggggt cgcgggggtc gcgggggtcg cgggggtcgc ggggggctcc ggcgccccct 124441 ccccgcccgc gcgtcgcagg cgcaggcgcg ccaggtgctc cgcggtgacg cgcaggcgga 124501 gggcgaggcg cggcggaagg cggaaggggc gcgagggggg gtgggagggg tcagccccgc 124561 cccccgggcc cacgccgggc ggtggggacc ggggccgggg ggcggcggcg gtgggccggg 124621 cctctggcgc cggctcgggc ggggggctgt ccggccagtc gtcgtcatcg tcgtcgtcgg 124681 acgcggactc gggaacgtgg agccactggc gcagcagcag cgaacaagaa ggcgggggcc 124741 caccggcggg gggcggcggc ggggcggccg cgggcgcgct cctgaccgcg ggttccgagt 124801 tgggcgtgga ggttacctgg gactgtgcgg ttgggacggc gcccgtgggc ccgggcggcc 124861 gggggcggcg ggggccgcga tggcggcggc ggcgggccat ggagacagag agcgtgccgg 124921 ggtggtagag tttgacaggc aagcatgtgc gtgcagaggc gagtagtgct tgcctgtcta 124981 actcgctagt ctcggccgcg gggggcccgg gctgcccgcc gccgcgcttt aaagggccgc 125041 gcgcgacccc cggggggtgt gtttcggggg ggcccgtttt tggggtctgg ccgctcctcc 125101 cccgctcctc cccgtctgtg ggtggggctc ctcccccgct cctcccccgc tcctcccccg 125161 ctcctccccg tctgtgggtg gggctcctcc cccgctcccg cggccccgcc ccccacgccc 125221 gccgcgcgcg cgcacgccgc ccggaccgcc gcccgccttt tttgcgcgcc gccccgcgcg 125281 cggggggccc gggctgccac aggtgtaaca acaccaacag aacaccaaca gcacggcgca 125341 ccggcgactc cggttcctca tccacacgtc acgtcatcca acacacctgc ccaacaacac 125401 aactcacagc gacaactcac cgcgcaacaa ctcctgttcc tcatccacac gtcaccgcgc 125461 accccccgct cctccagacg tcccccagcg caacacgccg ctcctgtcac acaccacagc 125521 cccagccctc cccagcccca gccctcccca gccccagccc tccccagccc cagccctccc 125581 cagccccagc cctccccagc cccagccctc cccagcccca gccctcccca gccccagccc 125641 tccccagccc cagccctccc cagccccagc cctccccagc cccagccctc cccagcccca 125701 gccctcccca gccgcgtccc gcgctccctc gggggggttc gggcatctct acctcagtgc 125761 cgccaatctc aggtcagaga tccaaaccct ccgggggcgc ccgcgcacca ccaccgcccc 125821 tcgccccctc ccgcccctcg ccccctcccg cccctcgccc cctcccgccc ctcgccccct 125881 cccgcccctc gccccctccc gcccctcgcc ccctcccgcc cctcgccccc tcccgcccct ...

Claims

1. A method for treating cancer in a patient refractory to an immune checkpoint inhibitor (ICI), the method comprising administering a vector comprising a herpes simplex virus (HSV) genome, wherein the HSV genome comprises one or more modifications that inhibit the expression of a functional ICP4 protein and / or a functional ICP47 protein.

2. A method for expressing a therapeutic polypeptide in an ICI-refractory subject, the method comprising administering a vector containing an HSV genome to the ICI-refractory subject, wherein the HSV genome includes one or more modifications that inhibit the expression of a functional ICP4 protein and / or a functional ICP47 protein.

3. A method for reducing tumor size in an ICI-refractory subject, the method comprising administering a vector comprising an HSV genome to the ICI-refractory subject, wherein the HSV genome comprises one or more modifications that inhibit the expression of functional ICP4 protein and / or functional ICP47 protein, and the vector comprises a nucleic acid encoding a therapeutic polypeptide that functions to reduce tumor size.

4. The method according to any one of claims 1 to 3, wherein the HSV-1 genome comprises one or more modifications that inhibit the expression of two ICP4 proteins.

5. The method according to any one of claims 1 to 4, wherein the HSV genome comprises one or more modifications that inhibit the expression of two ICP4 proteins and one ICP47 protein.

6. The method according to claim 4 or 5, characterized in that the two ICP4 proteins have the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4 (ICP4).

7. The method according to any one of claims 1 to 6, characterized in that the ICP47 protein has the amino acid sequence of SEQ ID NO: 5 (ICP47).

8. The method according to any one of claims 1 to 7, characterized in that the HSV genome is an HSV-1 genome.

9. The method according to any one of claims 1 to 7, wherein the HSV-1 genome is the McKrae genome.

10. The method according to any one of claims 1 to 9, wherein when the vector is administered to the subject, it results in one or more of the following: (a) Delay in tumor lysis; (b) Increased immunogenicity; (c) Increased immune activation; (d) Formation of TLS in the tumor bed; (e) Upregulation or downregulation of one or more proteins related to the response to ICI; (f) Reduced viability of cancer cells; and (g) A combination of one or more of (a) to (f).

11. The method according to any one of claims 1 to 10, wherein the vector comprises nucleic acids encoding one or more therapeutic polypeptides, wherein the one or more therapeutic polypeptides are as follows: (a) Target the tumor stroma; (b) Support the survival of T cells in the tumor microenvironment; (c) Induce tertiary lymphoid structures (TLS) within the tumor bed; (d) Promote phagocytic innate immune surveillance (e.g., disrupt the Sirpα / CD47 axis); (e) Promote the upregulation or downregulation of one or more proteins related to the response to ICI; (f) to reduce the viability of cancer cells; or (g) A combination of one or more of (a) to (f).

12. The method according to claim 11, wherein the one or more therapeutic polypeptides target the tumor stroma by degrading extracellular matrix proteins.

13. The method according to claim 12, wherein the one or more therapeutic polypeptides include an inhibitor of hyaluronidase, MMP-9, or lysyl oxidase.

14. The method according to claim 11, wherein the one or more therapeutic polypeptides target the tumor stroma by activating local endothelium and increasing T cell infiltration.

15. The method according to claim 14, wherein the one or more therapeutic polypeptides include an inflammatory cytokine.

16. The method according to claim 15, wherein the inflammatory cytokine comprises TNF, IL-1β, IL-6, or IL-18.

17. The method according to claim 11, wherein the one or more therapeutic polypeptides support the survival of T cells in the tumor microenvironment by enhancing the recruitment of T cells to the tumor site.

18. The method according to claim 17, wherein the one or more therapeutic polypeptides include CCL19 or CCL21.

19. The method according to claim 11, wherein the one or more therapeutic polypeptides support the survival of T cells in the tumor microenvironment by supporting the function of T cells.

20. The method according to claim 19, wherein the one or more therapeutic polypeptides include a T cell trophic factor.

21. The method according to claim 20, wherein the T cell trophic factor is selected from IL-7, IL-12, IL-15, IL-18, and IFN-γ.

22. The method according to any one of claims 11 and 17 to 21, wherein the T cells are CAR T cells.

23. The method according to claim 22, wherein the one or more therapeutic polypeptides include soluble TGFβRII.

24. The method according to claim 22, wherein the one or more therapeutic polypeptides include an antigen recognized by CAR T cells.

25. The method according to claim 24, wherein the antigen comprises mesoserine.

26. The method according to claim 11, wherein the one or more therapeutic polypeptides include a co-stimulatory molecule.

27. The method according to claim 26, wherein the co-stimulatory molecule is selected from CD40L and OX40L.

28. The method according to claim 11, wherein the one or more therapeutic polypeptides induce tertiary lymphoid structures (TLS) in the tumor bed.

29. The method according to claim 28, wherein the one or more therapeutic polypeptides include CCL19, lymphotoxin β, CXCL13, or TNF.

30. The method according to claim 29, wherein the one or more therapeutic polypeptides promote phagocytic innate immune surveillance.

31. The method according to claim 30, wherein the one or more therapeutic polypeptides disrupt the Sirpα / CD47 axis.

32. The method according to claim 30, wherein the one or more therapeutic polypeptides comprise a Sirpα-IgG fusion gene.

33. The method according to claim 30, wherein the one or more therapeutic polypeptides include an anti-CD47 antibody.

34. The method according to claim 11, wherein one or more therapeutic polypeptides support antitumor macrophage polarization.

35. The method according to claim 34, wherein the one or more therapeutic polypeptides include TNF, IL-1, IL-12, IL-17, or IFN-γ.

36. The method according to claim 11, wherein the one or more therapeutic polypeptides include hyaluronidase, MMP-9, a lysyl oxidase inhibitor, TNF, IL-1β, IL-6, IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesoserine, CD40L, OX40L, lymphotoxin β, CXCL13, Sirpα-IgG fusion transgene, anti-CD47 antibody, IL-1, or IL-17.

37. The method according to claim 11, wherein the one or more therapeutic polypeptides include IL-21, LTA, LTB, CXCL12, or VEGF-C.

38. The method according to claim 11, wherein the one or more therapeutic polypeptides include hyaluronidase, MMP-9, a lysyl oxidase inhibitor, TNF, IL-1β, IL-6, IL-18, CCL19, CCL21, IL-7, IL-12, IL-15, IFN-γ, soluble TGFβRII, mesoserine, CD40L, OX40L, lymphotoxin β, CXCL13, Sirpα-IgG fusion transgene, anti-CD47 antibody, IL-1, IL-17, IL-21, LTA, LTB, CXCL12, or VEGF-C.

39. The method according to claim 38, wherein the one or more therapeutic polypeptides include the following: a) LTA, CCL19, TNF, IL-17, and IL-7; b) LTB, CCL19, CCL21, CXCL13, and IL-7; c) IFN-γ and IL-12; d) IFN-γ and IL-15; e) IL-12 and IL-15; f) IL-15 and IL-21; g) IFN-γ, IL-12, and IL-15; h) IFN-γ, IL-12, IL-15, and IL-21; or i) CCL19, IL-7, TNF, and IL-17.

40. The method according to any one of claims 1 to 39, further comprising administering an immune checkpoint inhibitor (ICI).

41. The method according to claim 40, wherein the ICI is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor.

42. The method according to claim 41, wherein the ICI comprises a PD-1 inhibitor.

43. The method according to claim 42, wherein the PD-1 inhibitor comprises an anti-PD-1 antibody.

44. The method according to claim 43, wherein the anti-PD-1 antibody comprises pembrolizumab, nivolumab, or semipirimab.

45. The method according to claim 41, wherein the ICI comprises a PD-L1 inhibitor.

46. The method according to claim 45, wherein the PD-L1 inhibitor comprises atezolizumab, avelumab, or durvalumab.

47. The method according to claim 41, wherein the ICI comprises a CTLA-4 inhibitor.

48. The method according to claim 47, wherein the CTLA-4 inhibitor comprises ipilimumab or tremelimumab.

49. The method according to claim 41, wherein the ICI comprises a LAG-3 inhibitor.

50. The method according to claim 49, wherein the LAG-3 inhibitor comprises Relatlimab.

51. The method according to claim 41, wherein the method comprises administering a plurality of ICIs selected from the group consisting of PD-1 inhibitors and CTLA-4 inhibitors.

52. The method according to claim 51, wherein the PD-1 inhibitor comprises an anti-PD-1 antibody.

53. The method according to claim 52, wherein the anti-PD-1 antibody comprises pembrolizumab, nivolumab, or semipirimab, and the CTLA-4 inhibitor comprises ipilimumab or tremelimumab.

54. The method according to claim 8, wherein one or more proteins related to the reaction with the ICI include IL-21, CD40L, IL-2, IL-16, CXCL9, CXCL10, CCL5, ICAM3, ICAM2, CCL22, CCL20, IL-10, CXCL13, IL-17a, LTA, LTB, CCL19, and CCL21.

55. The method according to claim 8, wherein inducing tertiary lymphoid structures (TLS) in the tumor bed promotes the number, size, and / or organization of TLS structures.

56. A method for treating cancer in a subject requiring such treatment, the method comprising administering a vector comprising a herpes simplex virus (HSV) genome, wherein the HSV genome comprises one or more modifications that inhibit the expression of functional ICP4 protein and / or functional ICP47 protein, expressing effective amounts of IL-12 and IL-15, thereby treating cancer.

57. The method according to claim 56, further comprising administering an immune checkpoint inhibitor (ICI).

58. The method according to claim 57, wherein the ICI is selected from a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor.

59. The method according to claim 57, wherein the ICI comprises a PD-1 inhibitor.

60. The method according to claim 58, wherein the PD-1 inhibitor comprises an anti-PD-1 antibody.

61. The method according to claim 60, wherein the anti-PD-1 antibody comprises pembrolizumab, nivolumab, or semipirimab.

62. The method according to claim 58, wherein the ICI comprises a PD-L1 inhibitor.

63. The method according to claim 62, wherein the PD-L1 inhibitor comprises atezolizumab, avelumab, or durvalumab.

64. The method according to claim 58, wherein the ICI comprises a CTLA-4 inhibitor.

65. The method according to claim 64, wherein the CTLA-4 inhibitor comprises ipilimumab or tremelimumab.

66. The method according to claim 58, wherein the ICI comprises a LAG-3 inhibitor.

67. The method according to claim 66, wherein the LAG-3 inhibitor comprises Relatlimab.

68. The method according to claim 58, wherein the method comprises administering a plurality of ICIs selected from the group consisting of PD-1 inhibitors and CTLA-4 inhibitors.

69. The method according to claim 68, wherein the PD-1 inhibitor comprises an anti-PD-1 antibody.

70. The method according to claim 69, wherein the anti-PD-1 antibody comprises pembrolizumab, nivolumab, or semipirimab, and the CTLA-4 inhibitor comprises ipilimumab or tremelimumab.

71. The method according to any one of claims 56 to 70, wherein administration of a vector containing a herpes simplex virus (HSV) genome expressing IL-12 and IL-15 results in synergistic tumor regression compared to administration of a vector containing a herpes simplex virus (HSV) genome expressing IL-12 but not IL-15, or (b) expressing only IL-15 but not IL-12.

72. The method according to any one of claims 56 to 71, wherein the subject is ICI-insensitive.

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