Compositions and methods to treat cancer

Abstract

The technology described herein is directed to a codon-optimized nucleic acid sequence encoding an adenosine degrading enzyme (ADE), e.g., an adenosine kinase (ADK), or an adenosine Deaminase (ADA), as well as secretable ADEs and vectors (e.g., oncolytic virus) expressing such a ADE One such oncolytic virus is a Herpes Simplex Virus (oHSV). Also disclosed are therapeutic formulations comprising the nucleic acid sequence or the vector. Also disclosed are methods for treating cancer in a subject by administering a therapeutic formulation comprising a vector described herein.

Description

Attorney Docket No: 030258-000107WOPTCOMPOSITIONS AND METHODS TO TREAT CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U. S. C. § 119 of U. S. Provisional Application No. 63 / 776,235 filed March 23 2025, the contents of which are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] Described herein are methods and compositions relating to the treatment of excess adenosine, e.g., to oncology and cancer therapy. In some embodiments of any of the aspects, the compositions relate the codon-optimized and / or secretable adenosine-degrading enzymes (ADE). Also provided are methods and uses of ADEs for treating diseases and conditions, such as tumors or cancer, and, in particular, any disease or condition associated with elevated adenosine or other associated markers.BACKGROUND

[0003] Glioblastoma (GBM) is a fatal cancer of the brain with only a 3-4% five-year survival showing no change in outcome over multiple decades of numerous clinical trials. One feature that has stymied progress is the presence of GBM stem-like cells (GSCs), which drive tumor progression and therapy evasion. While immune checkpoint inhibitor (ICI) clinical trials show efficacy in some peripheral tumors, the efficacy of ICI therapy has not been demonstrated, including in GBM patients in phase III clinical trials. Thus, there is an unmet need for effective cancer therapies that can target tumor cells associated with GBM while avoiding deleterious effects on normal tissues.SUMMARY

[0004] Described herein are engineered enzymes that reduce the level of adenosine in tumors (and other pathological tissues). This improves endogenous immune responses and permits more effective immunotherapy.

[0005] In one aspect of any of the embodiments, described herein is a composition comprising a nucleic acid encoding a fusion protein, the fusion protein comprising: a) a signal peptide (SP) polypeptide; and b) an Adenosine Degrading Enzyme (ADE) polypeptide.1Attorney Docket No: 030258-000107WOPT

[0006] In some embodiments of any of the aspects, the ADE polypeptide is mammalian ADE. In some embodiments of any of the aspects, the ADE polypeptide is human ADE.

[0007] In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL). In some embodiments of any of the aspects, the ADE polypeptide is ADKS. In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence comprises the sequence of SEQ ID NO: 6.

[0008] In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADKS, and the codon-optimized nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADKS, and the codon-optimized nucleic acid sequence comprises the sequence of SEQ ID NO: 6.

[0009] In some embodiments of any of the aspects, the ADE polypeptide is an adenosine deaminase 1 (AD Al) or an adenosine deaminase 2 (ADA2). In some embodiments of any of the aspects, the ADE polypeptide is ADA1. In some embodiments of any of the aspects, the nucleic acid encodes ADA1, and the nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes ADA1, and the nucleic acid sequence comprises the sequence of SEQ ID NO: 10.

[0010] In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADA1, and the codon-optimized nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADA1, and the codon-optimized nucleic acid sequence comprises the sequence of SEQ ID NO: 10.

[0011] In some embodiments of any of the aspects, the ADE has a Km for adenosine of 1 μM or less. In some embodiments of any of tire aspects, the ADE has a Km for adenosine within the range of 1 nM to 1 μM.

[0012] In one aspect of any of the embodiments, described herein is a composition comprising a nucleic acid encoding a fusion protein, the fusion protein comprising: a) a signal peptide (SP); and b) a polypeptide comprising a sequence having at least 80% sequence identity to the sequence of one of SEQ ID NOs: 1-4 and 100-106. In some embodiments of any of the aspects, the polypeptide comprises the sequence of one of SEQ ID NO: 1-4 and 100-106.

[0013] In some embodiments of any of the aspects, the SP is selected from the group of: Human interleukin-2 (IL-2), Human tissue plasminogen activator tPA (PLAT), Human Ig K-chain leader2Attorney Docket No: 030258-000107WOPT(IgK / IGKC), Mouse Ig K-chain leader (mlgK), Human albumin (ALB), HSV-1 glycoprotein D (gD / US6), HSV-1 glycoprotein B (gB / UL27), Human growth hormone (GH1), CD33, Human preprotrypsin trypsinogen signal peptide, BM40 / SPARC (osteonectin), Gaussia luciferase, and Human transferrin (TF). In some embodiments of any of the aspects, the SP is selected from the group consisting of: IgG K chain signal peptide; IgG heavy chain signal peptide; human IgG K chain signal peptide; human IgG heavy chain signal peptide; murine Ig K chain signal peptide; interleukin-2 signal peptide; interleukin-7 signal peptide; interleukin- 12 signal peptide; granulocyte colony-stimulating factor signal peptide; insulin signal peptide; growth hormone signal peptide; prolactin signal peptide; a secreted plasma protein signal peptide; albumin signal peptide; transferrin signal peptide; fibrinogen signal peptide; tissue plasminogen activator signal peptide; epidermal growth factor signal peptide; vascular endothelial growth factor signal peptide; fibroblast growth factor signal peptide; influenza hemagglutinin signal peptide; HIV gp120 signal peptide; VSV glycoprotein signal peptide; SARS-CoV-2 spike signal peptide; a yeast leader sequence; a-mating factor prepro leader; invertase signal peptide; acid phosphatase signal peptide; killer toxin signal peptide; GP67 signal peptide; honeybee melittin signal peptide; a synthetic Ig leader; an optimized tPA variant; a hybrid signal peptide; Interleukin- 1; fibroblast growth factor 2; and galectin-1. In some embodiments of any of the aspects, the SP is an Interleukin -2 (IL-2) SP.

[0014] In some embodiments of any of the aspects, the nucleic acid is operably linked to a promoter. In some embodiments of any of the aspects, the nucleic acid is operably linked to a viral promoter. In some embodiments of any of the aspects, the viral promoter is selected from the group consisting of: a Cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, a Herpes Simplex Virus (HSV) promoter (HSV-1 US12, IE, and IE 4 / 5). In some embodiments of any of the aspects, the viral promoter is CMVA or CMVB.

[0015] In some embodiments of any of the aspects, the nucleic acid is operably linked to a tissuespecific promoter. In some embodiments of any of the aspects, the tissue-specific promoter is selected from the group consisting of: stromelysin 3 promoter, the surfactant protein A promoter, the secretory leukoprotease inhibitor (SLPI) promoter, the tyrosinase promoter, which is specific for melanoma cells, the stress inducible grp78 / BiP promoter, the AP2 adipose enhancer, the a-1 antitrypsin transthyretin promoter, the interleukin- 10 promoter, which is specific for glioblastoma multiform cells, the c-erbB-2 promoter, the a-B-crystallin / heat shock protein promoter, the basic fibroblast growth factor promoter, the epidermal growth factor receptor promoter, the mucin-like glycoprotein (DF3. MUC1) promoter, the mtsl promoter, the nestin promoter-enhancer, the NSE promoter, the somatostatin receptor promoter, the c-erbB-3 and c-erbB-2 promoters, the c-erbB4 promoter, the thyroglobulin promoter, the a-fetoprotein promoter, and the villin promoter.

[0016] In some embodiments of any of the aspects, the nucleic acid is a DNA molecule, an expression cassette, a transgene, an RNA molecule, or an mRNA molecule.3Attorney Docket No: 030258-000107WOPT

[0017] In one aspect of any of the embodiments, described herein is a composition comprising a vector comprising the nucleic acid described herein.

[0018] In some embodiments of any of the aspects, the vector is a plasmid, cosmid, or viral vector. In some embodiments of any of the aspects, the viral vector is selected from the group consisting of: recombinant retrovirus, a recombinant herpes simplex virus, an adenovirus, an adeno-associated virus, an alphavirus, an oncolytic virus, and a lentivirus.

[0019] In one aspect of any of the embodiments, described herein a lipid nanoparticle (LNP) comprising the composition described herein.

[0020] In one aspect of any of the embodiments, described herein is a virus particle comprising the composition described herein.

[0021] In some embodiments of any of the aspects, the virus is a DNA virus or RNA virus. In some embodiments of any of the aspects, the virus is an oncolytic virus. In some embodiments of any of the aspects, the virus is an oncolytic DNA virus selected from the group consisting of: herpes simplex virus (HSV), adenovirus, and vaccinia virus. In some embodiments of any of the aspects, the virus is an oncolytic RNA virus selected from the group consisting of: reovirus, vesicular stomatitis virus (VSV), poliovirus, Seneca Valley Virus (SVV) or senecavirus A (SVA). measles virus (MV). flavivirus, and Semliki Forest virus. In some embodiments of any of the aspects, the oncolytic virus is herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). In some embodiments of any of the aspects, the HSV is a HSV variant selected from the group consisting of: G207, G47A, MG18L. A68H-6, R3616, and R47A. In some embodiments of any of the aspects, the HSV is a G47A variant. In some embodiments of any of the aspects, the viral vector or virus particle comprises an inactivating mutation of one, two. three, or four genes selected from the group of: Infected Cell Protein 34.5 gene (y34.5 / ICP34.5), Infected Cell Protein 47 gene (ICP47), Infected Cell Protein 6 gene (ICP6), and Unique Short 3 gene (US3). In some embodiments of any of the aspects, the viral vector or virus particle comprises an inactivating mutation of ICP47.

[0022] In some embodiments of any of the aspects, the viral vector or virus particle comprises an HSV genome and the nucleic acid is inserted into the HSV genome at the ICP6 locus.

[0023] In one aspect of any of the embodiments, described herein is a polypeptide encoded by the nucleic acid described herein.

[0024] In one aspect of any of the embodiments, described herein is a system comprising a mammalian cell infected with a viral particle described herein. In some embodiments of any of the aspects, the mammalian cell secretes the ADE. In some embodiments of any of the aspects, the mammalian cell is selected from the group consisting of: mesenchymal stem cells (MSCs), T cells, and neural stem cells.

[0025] In one aspect of any of the embodiments, described herein is a serum isolated from a mammalian cell infected with a viral particle described herein.4Attorney Docket No: 030258-000107WOPT

[0026] In one aspect of any of the embodiments, described herein is a method to reduce the level of extracellular adenosine, the method comprising contacting a cell with tire composition described herein.

[0027] In one aspect of any of the embodiments, described herein is a method for reducing extracellular adenosine in a subject in need thereof, the method comprising administering the composition described herein. In some embodiments of any of the aspects, the subject is a subject in need of treatment for atrial fibrillation, syncope, hypoxia, sleep apnea, altitude hypoxia, hypertension, and heart failure.

[0028] In one aspect of any of the embodiments, described herein is a method for treating cancer in a subject in need thereof, the method comprising administering the composition described herein. In some embodiments of any of the aspects, the cancer is a solid tumor.

[0029] In some embodiments of any of the aspects, the cancer is selected from the group consisting of: a pancreatic tumor, a primary tumor of the nervous system, a central nervous system (CNS) tumor, a peripheral nervous system (PNS) tumor, a primary central nervous system lymphoma (PCNSL), a brainstem glioma, and a combination thereof. In some embodiments of any of the aspects, the primary tumor of the nervous system is selected from the group consisting of: diffuse glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic mixed oligoastrocytoma, glioblastoma, ependymoma, and anaplastic ependymoma. In some embodiments of any of the aspects, the pancreatic tumor is selected from the group consisting of: pancreatic ductal adenocarcinoma (PDAC), pancreatic neuroendocrine tumor, solid pseudopapillary neoplasm, acinar cell carcinoma, pancreatoblastoma, pancreatic lymphoma.

[0030] In some embodiments of any of the aspects, the composition is administered by intratumoral, intraperitoneal, or intravenous administration. In some embodiments of any of the aspects, the composition is administered directly into the solid tumor. In some embodiments of any of the aspects, the composition is administered intracranially. In some embodiments of any of the aspects, the composition is administered by multiple inoculations. In some embodiments of any of the aspects, the composition is administered at a dose of 1×103pfu to 1×1010pfu. In some embodiments of any of the aspects, the composition is administered at a dose of 1×103pfu to 1×107pfu. In some embodiments of any of the aspects, the composition is administered at a dose of 1×103pfu to 1×106pfu. In some embodiments of any of the aspects, the composition is administered at a dose of at least 1×103pfu. In some embodiments of any of the aspects, the composition is administered at a dose of at least 1×105pfu. In some embodiments of any of the aspects, the composition is administered as mono- or combination therapy.5Attorney Docket No: 030258-000107WOPTBRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1: Graphical representation of the adenosine pathway. Cell membrane showing ADO canonical and non-canonical pathways. eADO interacts with the adenosine A2a receptor (A2a) and adenosine A2b receptor (A2b) expressed in different immune cells leading to multiple immunosuppressive effects.

[0032] FIG. 2: Schematic of G47AmADE (mADAl and mADKS) genomic structure. G47A with insertion of eGFP-mADE cassettes (lower) in ICP6 and lacZ, leading to loss of ICP6, LacZ, and addition ofeGFP.

[0033] FIGs. 3A-3B: Western blot of cellular and secreted mADAl or mADKS after shuttle plasmid transfection. Transfection of Vero cells with 2.5 ug of shuttle plasmids encoding the two different proteins ADA1 (FIG. 3A) and ADKS (FIG. 3B) under CMV-A or CMV-B promoters. Vinculin is a marker cytosolic protein. CTRL = non-transfected.

[0034] FIG. 4: Genomic validation of the G47AmADEs viruses. Validation of genomic structure by restriction enzyme digestion of purified viral DNA with Hindlll and AsiSI enzymes. The new G47A-CMV-BmADAl (B-mADAl) and G47A-CMV-BmADKS (B-mADKS) are compared with the G47A genome. Picture taken after 15h of electrophoresis. Arrows indicate the difference in the restriction pattern between ADE viruses and G47A. MW1= 1Kb Plus DNA ladder #10787018, Invitrogen. MW2= GeneRuler High Range DNA ladder # SM1351. Invitrogen.

[0035] FIGs. 5A-5B: Western Blot of cellular and secreted mADAl and mADKS (CMV-A or CMV-B virus). FIG. 5A. ADA1 is expressed from both the A and B promoters after infection with G47A-mADAl virus (MOI of 1.5), both intracellularly in cell lysates (lanes 1 and 2) and extracellularly in the supernatants (lanes 5 and 6) from Vero cells cultured for 16h post-infection (p.i). FIG. 5B. ADKS is expressed from both the A and B promoters after infection with G47A-mADKS virus (MOI of 1.5), both intracellularly (lanes 1 and 12) and in the supernatant (lanes 5 and 6) of Vero cells cultured for 16 h.

[0036] FIGs. 6A-6B: ADA activity assay, measured by inosine levels in the supernatants of infected Vero cells using an Incucyte™ instrument. FIG. 6A. Kinetic absorbance measurements of inosine production every 10 minutes. FIG. 6B. Calculation of nanomole production of inosine by ADA1 after Ih or 2h of adenosine addition (Substrate; Abeam ab211093). ADA1 is secreted by the two new ADE viruses, G47A-CMV-mADAl and G47A-CMV-BmADAl. Positive control: Recombinant ADA enzyme (Abeam ab211093). Negative controls: Infected cell supernatant with G47A not armed, noninfected cell media, and media alone. Note that inosine levels doubled w ith the doubling in time.

[0037] FIG. 7: Cytotoxicity of the G47AmADA and G47AmADK compared to G47A in two different KPC pancreatic cancer cell models. The MTS assay was used to determine the viability of cells 4 days after infection with different MOIs of G47AmADA, G47AmADK, and G47A. Dose response curves were generated with Prism™ and the IC50 values determined by nonlinear fit of log(inhibitor) vs.6Attorney Docket No: 030258-000107WOPTresponse variable slope (four parameters). 6419c5 KPC cells were not permissive to HSV infection, likely due to the lack of receptor expression.

[0038] FIGs. 8A-8C: Survival of mice bearing orthotopic 005 mGSC-derived tumors after treatment with ADA and ADKS viruses. FIG. 8A. Schematic representation of the experimental timeline. 005 mGSCs were implanted intracerebrally in C57BL / 6 mice, and the mice treated intratum orally with G47A-BmADAl, G47A-BmADKS, G47A, or PBS at a dose of 1.6×106pfu / mouse on the days indicated. FIG. 8B-8C. Kaplan-Meier survival plots of G47AmADK compared to G47A or PBS treatment, and G47AmADA compared to G47A or PBS treatment. (ns> 0.5, *p <0.05, ** p<0.002, ***p<0.0002, **** pO.0001).

[0039] FIGs. 9A-9D: Plot of subcutaneous KPC tumor growth after G47A-ADA1 and G47A-ADKS. FIG. 9A. Schematic representation of the experiment timeline. C57BL / 6 mice after implantation of 200,000 KPC (2838c3 model) cells were treated with intratumoral G47A-BmADAl. G47A-BmADKS, G47A, or PBS at a dose of 107pfu / mouse. FIG. 9B-9C. Follow-up of tumor growth is represented as the mean of tumor volume ± SEM (n = 8) for each individual group. FIG. 9D. Follow-up of tumor growth is represented as the mean of tumor volume ± SEM (n = 8) for all groups together. Statistical analysis is based on a Two-way ANOVA Turkey’s Multiple comparison test. (ns> 0.5. *p <0.05, ** p<0.002. ***p<0.0002. **** p<0.0001).

[0040] FIGs. 10A-10B: Genetic maps for mouse ADA1 plasmids. FIG. 10A. pIS-ICP6-GFP-CMVA-mADAl construct. FIG. 10B. pJS-ICP6-GFP-CMVB-mADAl construct.

[0041] FIGs. 11A-11B: Genetic maps formouse ADKS plasmids. FIG. HA. pIS-ICP6-GFP-CMVA-mADKS construct. FIG. 1 IB. pJS-ICP6-GFP-CMVB-mADKS construct.

[0042] FIGs. 12A-12B: Genetic maps for theoretical human ADA1 plasmids. FIG. 12A. Genetic map of pJS-ICP6-GFP-CMVA-hADAl plasmid. FIG. 12B. Genetic map of pJS-ICP6-GFP-CMVB-hADAl plasmid. IL-2 signal peptide is the human signal peptide. ADA1 sequence is human ADA isoform 1 and is encoded by transcript variant 1.

[0043] FIGs. 13A-13B: Genetic maps for theoretical human ADKS plasmids. FIG. 13A. pJS-ICP6-GFP-CMVA-hADKS plasmid. FIG. 13B. pJS-ICP6-GFP-CMVB-hADKS plasmid. IL-2 signal peptide is the human signal peptide. ADKS sequence is human ADK isofonn short or also referred as isoform a. ADKS is encoded by transcript variant 1.

[0044] Figs. 14A-14B depict Western Blot of cellular and secreted mADAl and mADKS (CMV-A or CMV-B virus). Fig. 14A. MADAl is expressed from both the A and B promoters 16h p.i. of G47A-mADAl (MOI of 1.5) in Vero cells, both intracellularly in cell lysates (lanes 1 and 2) and extracellularly in the supernatant (lanes 5 and 6). Fig. 14B. ADKS is expressed by both the A and B promoters by infection with G47D-mADKS (MOI of 1.5) virus both intracellularly (lanes 1 and 2) and in the supernatant (lanes 5 and 6) of Vero cells in culture at 16h p.i..7Attorney Docket No: 030258-000107WOPT

[0045] Figs. 15A-15E depict growth plots of subcutaneous KPC tumors after ADA and ADK treatment. Fig 15A. Schematic representation of the experiment timeline. C57BL / 6 mice bearing 200,000 KPC (2838c3 model) cells were treated with intratumoral G47AmADA, G47AmADK, G47A, or PBS. Figs. 15B-15E depict plots of tumor volumes of individual mice per group. Volume is calculated using V=(length*width2) / 2 formula.

[0046] Figs. 16A-16B depict an assay of ADA activity, measurement of inosine in supernatants. Fig. 16A depicts kinetic absorbance measurements of inosine levels every 10 minutes in Vero cells. Fig. 16B. Production of inosine (nanomoles / min) by ADA1, Ih after adenosine addition (Substrate). ADA1 is secreted after G47AmADAl infection of Vero cells and 3 different mGSC cells. Positive control: Recombinant ADA enzyme. Negative controls: Infected cell supernatant with G47A not armed, noninfected cell media, and media alone.

[0047] Fig. 17 depicts Kaplan Meier survival curves of treated 005 mGSC-derived tumors. 2×104005 cells were intracranially implanted in C57BL / 6 mice. When tumors were established, 5×104pfu of G47 mADAl. G47A, or PBS were stereotactically injected at the same coordinates as tumor implantation (days 8 and 14 post-implantation). Animals were followed for neurologic deficits and survival (Kaplan-Meier analysis) plotted (*p-value < 0.05, **p-value < 0. 01).

[0048] Figs. 18A-18F depict an assay of ADA activity, measurement of inosine in supernatants. Kinetic absorbance measurement of inosine levels every 10 minutes in Vero cells and 5 different mGSC cell lines (C3, RIG-C1, 005, NF53, and C1). Production of inosine (nanomoles / min) by ADA1 Ih after adenosine addition (Substrate). ADA1 is secreted after G47AmADAl infection of Vero cells and all tested mGSCs. Positive control: Recombinant ADA enzyme. Negative controls: Infected cell supernatant with G47A not armed, non-infected cell media, and media alone.

[0049] Fig. 19 depicts ADA activity assay, inosine detection in supernatant. Kinetic absorbance measurement of inosine production every 10 minutes in 3 different mGSC cells, as in Fig. 18.

[0050] Fig. 20 depicts a homology comparison of active sites of ADA1 between species. 8 taxa were analyzed. Conserved: 583 aa / 1581aa.

[0051] Fig. 21 depicts a phylogenetic tree of ADA1 similarities between species. Evolutionary analysis by the Maximum Likelihood method. The phylogeny was inferred using the Maximum Likelihood method and Tamura-Nei (1993) model of nucleotide substitutions and the tree with the highest log likelihood (-5,837.08) is shown. The tree is drawn to scale with branch lengths (shown next to the branches) computed using the Maximum Likelihood method and measured in the number of substitutions per site. The initial tree for the heuristic search was selected by choosing the tree with the superior log-likelihood between a Neighbor-Joining (NJ) tree and a Maximum Parsimony (MP) tree. The NJ tree was generated using a matrix of pairwise distances computed using the Tamura-Nei (1993) model. The MP tree had the shortest length among 10 MP tree searches, each performed with a randomly generated starting tree. The analytical procedure encompassed 8 coding nucleotide8Attorney Docket No: 030258-000107WOPTsequences using 1st, 2nd, 3rd, and non-coding positions with 1,581 positions in the final dataset. Evolutionary analyses were conducted in MEGA12™ utilizing up to 3 parallel computing threads.

[0052] Fig. 22 depicts a homology comparison of active sites of ADKS between species. 8 taxa were analyzed. Conserved: 275aa / 391aa.

[0053] Fig. 23 depicts a phylogenetic tree of ADKS similarities between species. The phylogeny was inferred using the Maximum Likelihood method and Jones-Taylor-Thomton (1992) model of amino acid substitutions and the tree with the highest log likelihood (-1,850.49) is shown. The tree is drawn to scale with branch lengths (shown next to the branches) computed using the Maximum Likelihood method and measured in the number of substitutions per site. The initial tree for the heuristic search was selected by choosing the tree with the superior log-likelihood between a Neighbor-Joining (NJ) tree and a Maximum Parsimony (MP) tree. The NJ tree was generated using a matrix of pairwise distances computed using the p-distance. The MP tree had the shortest length among 10 MP tree searches, each performed with a randomly generated starting tree. The analytical procedure encompassed 8 amino acid sequences with 389 positions in the final dataset. Evolutionary analyses were conducted in MEGA12™ utilizing up to 3 parallel computing threads.DETAILED DESCRIPTION

[0054] In one aspect of any of the embodiments, described herein is a composition comprising a nucleic acid encoding a fusion protein, the fusion protein comprising: a signal peptide (SP) polypeptide; and an Adenosine Degrading Enzyme (ADE) polypeptide. In one aspect of any of the embodiments, described herein is a fusion protein comprising: a signal peptide (SP) polypeptide; and an Adenosine Degrading Enzyme (ADE) polypeptide. The inventors have demonstrated the compositions provided herein provide high levels of extracellular ADEs, which can be effective to reduce immunosuppressive ADO in the tumor microenvironment and other pathological environments.

[0055] As used herein, “adenosine degrading enzyme” or “ADE” refers to a protein that catalyze the conversion of adenosine to another molecule. ADEs include Adenosine Deaminase (ADA) and Adenosine Kinase (ADK). ADA irreversibly converts adenosine to inosine, while ADK phosphory lates adenosine to AMP.

[0056] In some embodiments of any of the aspects, the ADE polypeptide is a mammalian ADE. In some embodiments of any of the aspects, the ADE polypeptide is a human ADE. In some embodiments of any of the aspects, the ADE polypeptide is a canine ADE. In some embodiments of any of the aspects, the ADE polypeptide is a feline ADE. In some embodiments of any of the aspects, the ADE polypeptide is a bovine ADE. In some embodiments of any of the aspects, the ADE polypeptide is an9Attorney Docket No: 030258-000107WOPTovine ADE. In some embodiments of any of the aspects, the ADE polypeptide is an equine ADE. In some embodiments of any of the aspects, the ADE polypeptide is a porcine ADE.

[0057] As used herein, “adenosine deaminase’’ or “ADA” refers to a polypeptide whose monomer comprises eight central β-barrels and eight peripheral a-helices as well as five additional helices: three helices located between pi and al folds; and two antiparallel carboxy -terminal helices are located across the amino-terminal of the -barrel. In some embodiments of any of the aspects, ADA converts adenosine to inosine.

[0058] In some embodiments of any of any of the aspects, the ADA is ADA1. In some embodiments of any of any of the aspects, the ADA is ADA2. In some embodiments of any of any of the aspects, the ADA is ADA1 or ADA2.

[0059] ADA sequences, e.g., nucleic acid sequences and polypeptide sequences) are known for a number of species. For example, human ADA1 (UniProt accession No. P00813; NCBI Gene ID: 100), has the amino acid sequence set forth in SEQ ID NOs: 2. 105, and 106 and mouse ADA1, (UniProt accession No. P03958; NCBI Gene ID: 11486) has amino acid sequence set forth in SEQ ID NO: 4. Human ADA2 (NCBI Gene ID: 51816), has the amino acid sequence set forth in SEQ ID NOs: 101-104. The human and mouse AD Al orthologs share approximately 83.5% amino acid sequence identity, as determined by pairwise alignment using [alignment tool, e.g., BLAST or ClustalW] with default parameters. Accordingly, it will be understood by those skilled in the art that the technology encompasses not only the human and mouse forms of ADA1, but also orthologs and variants from other species that share at least 80% sequence identity with the human sequence set forth in SEQ ID NO: 2, 105. or 106. In some embodiments of any of the aspects, the technology encompasses not only the human and mouse forms of ADA1, but also orthologs and variants from other species that share at least 80% sequence identity with the human sequence set forth in SEQ ID NO: 2, 105, or 106 and retain adenosine deaminase activity. In some embodiments of any of the aspects, the technology encompasses not only the human and mouse forms of ADA1, but also orthologs and variants from other species that share at least 85% sequence identity with the human sequence set forth in SEQ ID NO: 2, 105, or 106. In some embodiments of any of the aspects, the technology encompasses not only the human and mouse forms of AD Al, but also orthologs and variants from other species that share at least 85% sequence identity with the human sequence set forth in SEQ ID NO: 2, 105, or 106 and retain adenosine deaminase activity.

[0060] In some embodiments of any of the aspects, the ADA1 polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 2, 105, or 106. In some embodiments of any of the aspects, the ADA1 polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 2, 105. or 106. In some embodiments of any of the aspects, the ADA1 polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2. 105, or 106. In some embodiments of any of the aspects, the ADA1 polypeptide comprises a sequence10Attorney Docket No: 030258-000107WOPTwith at least 95% sequence identity to the sequence of SEQ ID NO: 2, 105, or 106. In some embodiments of any of the aspects, the ADA1 polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 2, 105, or 106. In some embodiments of any of the aspects, the ADA1 polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 2. 105, or 106. In some embodiments of any of the aspects, the ADA1 polypeptide comprises a sequence which is identical to the sequence of SEQ ID NO: 2, 105, or 106.

[0061] In some embodiments of any of the aspects, the ADA1 polypeptide comprises the sequence of SEQ ID NO: 2, 105, or 106. In some embodiments of any of the aspects, the ADA1 polypeptide consists essentially of the sequence of SEQ ID NO: 2, 105. or 106. In some embodiments of any of the aspects, the ADA1 polypeptide consists of the sequence of SEQ ID NO: 2, 105. or 106.

[0062] In some embodiments of any of the aspects, the ADA1 polypeptide comprises the sequence of SEQ ID NO: 2, 105, or 106 and a SP. In some embodiments of any of the aspects, the ADA1 polypeptide consists essentially of the sequence of SEQ ID NO: 2. 105, or 106 and a SP. In some embodiments of any of the aspects, the ADA1 polypeptide consists of the sequence of SEQ ID NO: 2, 105, or 106 and a SP.

[0063] In some embodiments of any of the aspects, the ADA2 polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 101, 102, 103, or 104. In some embodiments of any of the aspects, the ADA2 polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 101. 102, 103, or 104. In some embodiments of any of the aspects, the ADA2 polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 101, 102, 103, or 104. In some embodiments of any of the aspects, the ADA2 polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 101, 102, 103, or 104. In some embodiments of any of the aspects, the ADA2 polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 101, 102, 103, or 104. In some embodiments of any of the aspects, the ADA2 poly peptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 101, 102, 103, or 104. In some embodiments of any of the aspects, the ADA2 polypeptide comprises a sequence which is identical to the sequence of SEQ ID NO: 101, 102, 103, or 104.

[0064] In some embodiments of any of the aspects, the ADA2 polypeptide comprises the sequence of SEQ ID NO: 101, 102, 103, or 104. In some embodiments of any of the aspects, the ADA2 polypeptide consists essentially of the sequence of SEQ ID NO: 101, 102, 103, or 104. In some embodiments of any of the aspects, the ADA2 polypeptide consists of the sequence of SEQ ID NO: 101, 102. 103, or 104.

[0065] In some embodiments of any of the aspects, the ADA2 polypeptide comprises the sequence of SEQ ID NO: 101, 102, 103, or 104 and a SP. In some embodiments of any of the aspects, the ADA2 polypeptide consists essentially of the sequence of SEQ ID NO: 101, 102, 103, or 104 and a SP. In11Attorney Docket No: 030258-000107WOPTsome embodiments of any of the aspects, the ADA2 polypeptide consists of the sequence of SEQ ID NO: 101, 102, 103, or 104 and a SP.

[0066] In some embodiments of any of the aspects, the nucleic acid encodes ADA1, and the nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes ADA1, and the nucleic acid sequence comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes ADA1, and the nucleic acid sequence comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes ADA1, and the nucleic acid sequence comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes ADA1, and the nucleic acid sequence comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes ADA1, and the nucleic acid sequence comprises a sequence identical to the sequence of SEQ ID NO: 10.

[0067] In some embodiments of any of the aspects, the nucleic acid encodes ADA1, and the nucleic acid sequence comprises the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes ADA1, and the nucleic acid sequence consists essentially of the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes ADA1, and the nucleic acid sequence consists of the sequence of SEQ ID NO: 10.

[0068] In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADA1, and the codon -optimized nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADA1, and the codon-optimized nucleic acid sequence comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADA1, and the codon-optimized nucleic acid sequence comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADA1, and the codon-optimized nucleic acid sequence comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized AD Al, and the codon-optimized nucleic acid sequence comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADA1, and the codon-optimized nucleic acid sequence comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADA1, and the codon-optimized nucleic acid sequence is identical to the sequence of SEQ ID NO: 10.12Attorney Docket No: 030258-000107WOPT

[0069] In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADA1, and the codon-optimized nucleic acid sequence comprises the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADA1, and the codon-optimized nucleic acid sequence consists essentially of the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADA1, and the codon-optimized nucleic acid sequence consists of the sequence of SEQ ID NO: 10.

[0070] As used herein, “Adenosine Kinase" or “ ADK" refers to a polypeptide comprising one large a / p domain with nine -strands, eight a-helices, and one small a / p-domain with five P-strands and two a-helices. The active site is formed along the edge of the p-sheet in the large domain while the small domain acts as a lid to cover the upper face of the active site. ADK exists in two isoforms, ADK-long (ADKL) and ADK-short (ADKS), which differ in the length of the N-terminus. In some embodiments of any of the aspects, ADK converts adenosine to 5 '-adenosine monophosphate (AMP) via phosphorylation,

[0071] ADK sequences, e.g., nucleic acid sequences and polypeptide sequences) are known for a number of species. For example, human (hADKS), (UniProt accession No. P55263; NCBI Gene ID: 132), has the amino acid sequence set forth in SEQ ID NO: 1 and the mouse ADKS, (UniProt accession No. P55264; NCBI Gene ID: 11534) has the amino acid sequence set forth in SEQ ID NO: 3. The human and mouse ADKS orthologs share approximately 78% amino acid sequence identity, as determined by pairwise alignment using [alignment tool, e.g., BLAST or ClustalW] with default parameters. Accordingly, it will be understood by those skilled in the art that the technology encompasses not only the human and mouse forms of ADKS, but also orthologs and variants from other species that share at least 80% sequence identity with the human sequence set forth in SEQ ID NO: 1. In some embodiments of any of the aspects, the technology encompasses not only the human and mouse forms of ADKS. but also orthologs and variants from other species that share at least 80% sequence identity with the human sequence set forth in SEQ ID NO: 1 and retain adenosine kinase activity.

[0072] For example, human (hADKL) has the amino acid sequence set forth in SEQ ID NO: 100. It will be understood by those skilled in the art that the technology described herein encompasses not only the human and mouse forms of ADKL, but also orthologs and variants from other species that share at least 80% sequence identity with the human sequence set forth in SEQ ID NO: 100 and retain adenosine kinase activity, In some embodiments of any of the aspects, the technology described herein encompasses not only the human and mouse forms of ADKL, but also orthologs and variants from other species that share at least 80% sequence identity with the human sequence set forth in SEQ ID NO: 100 and retain adenosine kinase activity.

[0073] In some embodiments of any of the aspects, the ADE polypeptide comprises Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL). In some embodiments of any of the aspects, the ADE polypeptide consists of Adenosine Kinase Short isoform (ADKS) or Adenosine13Attorney Docket No: 030258-000107WOPTKinase Long isofonn (ADKL). In some embodiments of any of the aspects, the ADE polypeptide consists essentially of Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL). In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL).

[0074] In some embodiments of any of the aspects, the ADE polypeptide comprises Adenosine Kinase Short isoform (ADKS). In some embodiments of any of the aspects, the ADE polypeptide consists of Adenosine Kinase Short isoform (ADKS). In some embodiments of any of the aspects, the ADE polypeptide consists essentially of Adenosine Kinase Short isoform (ADKS). In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS).

[0075] In some embodiments of any of the aspects, the ADE polypeptide comprises Adenosine Kinase Long isoform (ADKL). In some embodiments of any of the aspects, the ADE polypeptide consists of Adenosine Kinase Long isoform (ADKL). In some embodiments of any of the aspects, the ADE polypeptide consists essentially of Adenosine Kinase Long isoform (ADKL). In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Long isoform (ADKL).

[0076] In some embodiments of any of the aspects, the ADKS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the ADKS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the ADKS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the ADKS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the ADKS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the ADKS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the ADKS polypeptide comprises a sequence which is identical to the sequence of SEQ ID NO: 1.

[0077] In some embodiments of any of the aspects, the ADKS polypeptide comprises the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the ADKS polypeptide consists essentially of the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the ADKS polypeptide consists of the sequence of SEQ ID NO: 1.

[0078] In some embodiments of any of the aspects, the ADKS polypeptide comprises the sequence of SEQ ID NO: 1 and a SP. In some embodiments of any of the aspects, the ADKS polypeptide consists essentially of the sequence of SEQ ID NO: 1 and a SP. In some embodiments of any of the aspects, the ADKS polypeptide consists of the sequence of SEQ ID NO: 1 and a SP.

[0079] In some embodiments of any of the aspects, the ADKL polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 100. In some embodiments of any of the14Attorney Docket No: 030258-000107WOPTaspects, the ADKL polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 100. In some embodiments of any of the aspects, the ADKL polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 100. In some embodiments of any of the aspects, the ADKL polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 100. In some embodiments of any of the aspects, the ADKL polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 100. In some embodiments of any of the aspects, the ADKL polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 100. In some embodiments of any of the aspects, the ADKL polypeptide comprises a sequence which is identical to the sequence of SEQ ID NO: 100.

[0080] In some embodiments of any of the aspects, the ADKL polypeptide comprises the sequence of SEQ ID NO: 100. In some embodiments of any of the aspects, the ADKL polypeptide consists essentially of the sequence of SEQ ID NO: 100. In some embodiments of any of the aspects, the ADKL polypeptide consists of the sequence of SEQ ID NO: 100.

[0081] In some embodiments of any of the aspects, the ADKL polypeptide comprises the sequence of SEQ ID NO: 100 and a SP. In some embodiments of any of the aspects, the ADKL polypeptide consists essentially of the sequence of SEQ ID NO: 100 and a SP. In some embodiments of any of the aspects, the ADKL polypeptide consists of the sequence of SEQ ID NO: 100 and a SP.

[0082] In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isofonn (ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isofonn (ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform15Attorney Docket No: 030258-000107WOPT(ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence comprises a sequence identical to the sequence of SEQ ID NO: 6.

[0083] In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence comprises the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence consists essentially of the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL), and the nucleic acid sequence consists of the sequence of SEQ ID NO: 6.

[0084] In some embodiments of any of the aspects, the nucleic acid encodes a codon-optimized ADKS, and the codon-optimized nucleic acid sequence comprises the sequence of SEQ ID NO: 6.

[0085] In one aspect of any of the embodiments, described herein is a composition comprising a nucleic acid encoding a fusion protein, the fusion protein comprising: a) a signal peptide (SP); and b) a polypeptide comprising a sequence having at least 80% sequence identity to the sequence of one of SEQ ID NOs: 1-4 and 100-106. In some embodiments of any of the aspects, the polypeptide comprises a sequence having at least 85% sequence identity to the sequence of one of SEQ ID NOs: 1-4 and 100-106. In some embodiments of any of the aspects, the polypeptide comprises a sequence having at least 90% sequence identity to the sequence of one of SEQ ID NOs: 1-4 and 100-106. In some embodiments of any of the aspects, the polypeptide comprises a sequence having at least 95% sequence identity to the sequence of one of SEQ ID NOs: 1-4 and 100-106. In some embodiments of any of the aspects, the polypeptide comprises a sequence having at least 98% sequence identity to the sequence of one of SEQ ID NOs: 1-4 and 100-106. In some embodiments of any of the aspects, the polypeptide comprises a sequence having at least 99% sequence identity to the sequence of one of SEQ ID NOs: 1-4 and 100-106. In some embodiments of any of the aspects, the polypeptide comprises a sequence identical to the sequence of one of SEQ ID NOs: 1-4 and 100-106. In some embodiments of any of the aspects, the polypeptide comprises the sequence of one of SEQ ID NOs: 1-4 and 100-106. In some embodiments of any of the aspects, the polypeptide consists essentially of the sequence of one of SEQ ID NOs: 1-4 and 100-106. In some embodiments of any of the aspects, the polypeptide consists of the sequence of one of SEQ ID NOs: 1-4 and 100-106. In some embodiments of any of the aspects, the sequence of one of SEQ ID NOs: 1-4 and 100-106 is the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the sequence of one of SEQ ID NOs: 1-4 and 100-106 is the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the sequence of one of SEQ ID NOs: 1-4 and 100-106 is the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the sequence of one of SEQ ID NOs: 1-4 and 100-106 is the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the sequence of one of SEQ ID NOs: 1-4 and 100-16Attorney Docket No: 030258-000107WOPT106 is the sequence of SEQ ID NO: 100. In some embodiments of any of the aspects, the sequence of one of SEQ ID NOs: 1-4 and 100-106 is the sequence of SEQ ID NO: 101. In some embodiments of any of the aspects, the sequence of one of SEQ ID NOs: 1-4 and 100-106 is the sequence of SEQ ID NO: 102. In some embodiments of any of the aspects, the sequence of one of SEQ ID NOs: 1-4 and 100-106 is the sequence of SEQ ID NO: 103. In some embodiments of any of the aspects, the sequence of one of SEQ ID NOs: 1-4 and 100-106 is the sequence of SEQ ID NO: 104. In some embodiments of any of the aspects, the sequence of one of SEQ ID NOs: 1-4 and 100-106 is the sequence of SEQ ID NO: 105. In some embodiments of any of the aspects, the sequence of one of SEQ ID NOs: 1-4 and 100-106 is the sequence of SEQ ID NO: 106.

[0086] In some embodiments of any of the aspects, the nucleic acid sequences encoding ADE further comprise a heterologous secretion signal sequence operably linked at the 5' end of the coding sequence, such that the expressed fusion product is directed for secretion out of the cell via the endoplasmic reticulum-Golgi secretory pathway. In some embodiments of any of the aspects, the encoded protein is Interleukin-2 (IL-2), and the secretion signal is positioned N-terminally and is cleaved upon translocation, yielding a mature secreted IL-2 polypeptide. The human IL-2 sequence is exemplified by SEQ ID NO: 5. Suitable secretion signals include, but are not limited to. the native IL-2 signal peptide (UniProt accession No. P60568), the IgG kappa chain leader sequence, the CD33 signal peptide, and the murine Ig kappa-chain V-J2-C signal peptide, among others known in the art. For example, see the Signal Peptide Database on the world wide web at signalpeptide.de. In some embodiments of any of the aspects, the secretion signal sequence is heterologous to IL-2 and is selected to optimize expression levels and secretion efficiency in a given host cell system, including but not limited to mammalian, insect, or yeast expression systems. The skilled artisan will appreciate that any functional secretion signal that directs translocation of the IL-2 polypeptide across the endoplasmic reticulum membrane and results in extracellular secretion of the mature protein falls within the scope of the compositions and methods described herein.

[0087] Table. 1 Amino acid sequences.SEQ ID Description Amino acid sequenceNO1 hADKS mtsvrenilf gmgnplldis awdkdfldk yslkpndqil aedkhkelfd elvkkfkvey haggstqnsi kvaqwmiqqp hkaatffgci gidkfgeilk rkaaeahvda hyyeqneqpt gtcaacitgd nrslianlaa ancykkekhl dleknwmlve karveyiagf lltvspesvl kvahhasenn riftlnlsap fisqfykesl mkvmpyvdil fgneteaatf areqgfetkd17Attorney Docket No: 030258-000107WOPTikeiakktqa Ipkmnskrqr iviftqgrdd timatesevt afavldqdqk eiidtngagd afvggflsql vsdkplteci raghyaasii irrtgctfpe kpdflihADAl isoform MAQTPAFDKPKVELHVHLDGSIKPETILYYGRRRGIALPANTAEG 1 LLNVIGMDKPLTLPDFLAKFDYYMPAIAGCREAIKRIAYEFVEM KAKEGVVYVEVRYSPHLLANSKVEPIPWNQAEGDLTPDEVVAL VGQGLQEGERDFGVKARSILCCMRHQPNWSPKWELCKKYQQQ TVVAIDLAGDETIPGSSLLPGHVQAYQEAVKSGIHRTVHAGEVG SAEVVKEAVDILKTERLGHGYHTLEDQALYNRLRQENMHFEICP WSSYLTGAWKPDTEHAVIRLKNDQANYSLNTDDPLIFKSTLDTD YQMTKRDMGFTEEEFKRLNINAAKSSFLPEDEKRELLDLLYKAY GMPPSASAGQNLHuman ADA1 mwrcgtvrtc wptpkwsqsp gtrlnwspkv velckkyqqq tvvaidlagd etipgssllp isofonn 2 ghvqayqeav ksgihrtvha gevgsaevvk eavdilkter Ighgyhtled qalynrlrqe nmhfeicpws syltgawkpd tehavirlkn dqanyslntd dplifkstld tdyqmtkrdm gfteeefkrl ninaakssfl pedekrelld llykaygmpp sasagqnlHuman ADA1 maqtpafdkp kvelhvhldg sikpetilyy grrrgialpa ntaegllnvi gmdkpltlpd isoform 3 flakfdyymp aiagcreaik riayefvemk akegwyvev rysphllans kvepipwnqa egdltpdew alvgqglqeg erdfgvkars ilccmrhqpn wspkvvelck kyqqqtwai dlagdetipg ssllpghvqa yqavdilkte rlghgyhtle dqalynrlrq emnhfeicpw ssyltgawkp dtehavirlk ndqanyslnt ddplifkstl dtdyqmtkrd mgfteeefkr Ininaakssf Ipedekrell dllykaygmp psasagqnlmADKS (e.g.. MTSTSENVLFGMGNPLLDISAVVDKDFLDKYSLKPNDQILAEDK NM 001243041 HKELFDELVKKFKVEYHAGGSTQNSMKVAQWLIQEPHKAATFF •1) GCIGIDKFGEILKRKAADAHVDAHYYEQNEQPTGTCAACITGGN RSLVANLAAANCYKKEKHLDLERNWVLVEKARVYYIAGFFLTV SPESVLKVARYAAENNRVFTLNLSAPFISQFFKEALMDVMPYVD1 LFGNETEAATFAREQGFETKDIKEIAKKAQALPKVNSKRQRTVIF TQGRDDTIVAAENDVTAFPVLDQNQEEIIDTNGAGDAFVGGFLS QLVSDKPLTECIRAGHYAASVIIRRTGCTFPEKPDFHmADAl MAQTPAFNKPKVELHVHLDGAIKPETILYFGKKRGIALPADTVEE LRNIIGMDKPLSLPGFLAKFDYYMPVIAGCREAIKRIAYEFVEMK AKEGWYVEVRYSPHLLANSKVDPMPWNQTEGDVTPDDVVDL18Attorney Docket No: 030258-000107WOPTVNQGLQEGEQAFGIKVRSILCCMRHQPSWSLEVLELCKKYNQKT VVAMDLAGDETIEGSSLFPGHVEAYEGAVKNGIHRTVHAGEVG SPEVVREAVDILKTERVGHGYHTIEDEALYNRLLKENMHFEVCP WSSYLTGAWDPKTTHAVVRFKNDKANYSLNTDDPLIFKSTLDT DYQMTKKDMGFTEEEFKRLNINAAKSSFLPEEEKKELLERLYRE YQ IL-2 SP MYRMQLLSCIALSLALVTNSHuman ADKL maaaeeepkp kklkveapqa Irenilfgmg nplldisaw dkdfldkysl kpndqilaed khkelfdelv kkfkveyliag gstqnsikva qwmiqqphka atffgcigid kfgeilkrka aeahvdahyy eqneqptgtc aacitgdnrs lianlaaanc ykkekhldle knwmlvekar vcyiagfflt vspesvlkva hhasemirif tlnlsapfis qfykeslmkv mpyvdilfgn eteaatfare qgfetkdike iakktqalpk mnskrqrivi ftqgrddtim atesevtafa vldqdqkeii dtngagdafv ggflsqlvsd kpltecirag hyaasiiirr tgctfpekpd fli Human ADA2 mlvdgpserp alcflllava msffgsalsi detrahlllk ekmmrlggrl vlntkeelan isoform a erlmtlkiae mkeamrtlif ppsmhffqak hliersqvfn ilrmmpkgaa Ihlhdigivt mdwlvmvty rphchicftp rgimqfrfah ptprpsekcs kwilledyrk rvqnvtefdd sllrnftlvt qhpeviytnq nwwskfeti fftisglihy apvfrdyvfr smqefyednv lymeirarll pvvclsgelih deewsvktyq evaqkfveth pefigikiiy sdhrskdvav iaesirmamg Irikfptwa gfdlvghedt ghslhdykea Imipakdgvk Ipyffhaget dwqgtsidrn ildalmlntt righgfalsk hpavrtyswk kdipievcpi snqvlklvsd Imhpvatlm atghpmviss ddpamfgakg Isydfyevfm giggmkadlr tlkqlamnsi kystllesek ntfmeiwkkr wdkfiadvat kHuman ADA2 mdslewnwal vyelsgehhd eewsvktyqe vaqkfvethp efigikiiys dhrskdvavi isofonn b aesimiamgl rikfptwag fdlvgliedtg hslhdykeal mipakdgvkl pyffhagetd wqgtsidrni Idalmlnttr ighgfalskh pavrtyswkk dipievcpis nqvlklvsdl mhpvatlma tghpmvissd dpamfgakgl sydfyevfmg iggmkadlrt Ikqlamnsik ystllesekn tfmeiwkkrw dkfiadvatkHuman ADA2 mmrlggrlvl ntkeelaner Imtlkiaemk eamrtlifpp smhffqakhl iersqvfnil isoform c nnmpkgaalhlhdigivtmdwlvmvtyrp hchicftprg imqfrfahpt prpsekcskw illedyrkrv qnvtefddsl Irnftlvtqh peviytnqnv vwskfetiff tisglihyap vfrdyvfrsm qefyednvly meirarllpv yelsgehhde ewsvktyqev aqkfvethpe figikiiy sd hrskdvavia esimiamglr ikfptwagf dlvghedtgh slhdykealm19Attorney Docket No: 030258-000107WOPTipakdgvklp yffhagetdw qgtsidmil dalmlnttri ghgfalskhp avrtyswkkd ipievcpisn qvlklvsdlr nhpvatlmat ghpmvissdd pamfgakgls ydfyevfmgi ggmkadlrtl kqlamnsiky stlleseknt fmeiwkkrwd kfiadvatk104 Human ADA2 mdwlvrnvty rphchicftp rgimqfrfah ptprpsekcs kwilledyrk rvqnvtefdd isoform d sllrnftlvt qhpeviytnq nwwskfeti fftisglihy apvfrdyvfr smqefyednv lymeirarll pvyclsgchh deewsvktyq evaqkfveth pefigikiiy sdhrskdvav iaesirmamg Irikfptwa gfdlvghedt ghslhdykea Imipakdgvk Ipyffhaget dwqgtsidrn ildalmlntt righgfalsk hpavrtyswk kdipievcpi snqvlklvsd Imhpvatlm atghpmviss ddpamfgakg Isydfyevfm giggmkadlr tlkqlamnsi kystllesek ntfmeiwkkr wdkfiadvat k

[0088] Table 2. DNA nucleic acid sequences encoding amino acid sequences.SEQ ID Description SequenceNO6 hADKS GCCCAGACGCCCGCCTTCGACAAGCCCAAAGTGGAACTGCAT (NM_001123.4) GTCCACCTAGACGGATCCATCAAGCCTGAAACCATCTTATACT ATGGCAGGAGGAGAGGGATCGCCCTCCCAGCTAACACAGCAG AGGGGCTGCTGAACGTCATTGGCATGGACAAGCCGCTCACCC TTCCAGACTTCCTGGCCAAGTTTGACTACTACATGCCTGCTAT CGCGGGCTGCCGGGAGGCTATCAAAAGGATCGCCTATGAGTT TGTAGAGATGAAGGCCAAAGAGGGCGTGGTGTATGTGGAGGT GCGGTACAGTCCGCACCTGCTGGCCAACTCCAAAGTGGAGCC AATCCCCTGGAACCAGGCTGAAGGGGACCTCACCCCAGACGA GGTGGTGGCCCTAGTGGGCCAGGGCCTGCAGGAGGGGGAGC GAGACTTCGGGGTCAAGGCCCGGTCCATCCTGTGCTGCATGC GCCACCAGCCCAACTGGTCCCCCAAGGTGGTGGAGCTGTGTA AGAAGTACCAGCAGCAGACCGTGGTAGCCATTGACCTGGCTG GAGATGAGACCATCCCAGGAAGCAGCCTCTTGCCTGGACATG TCCAGGCCTACCAGGAGGCTGTGAAGAGCGGCATTCACCGTA CTGTCCACGCCGGGGAGGTGGGCTCGGCCGAAGTAGTAAAAG AGGCTGTGGACATACTCAAGACAGAGCGGCTGGGACACGGCT ACCACACCCTGGAAGACCAGGCCCTTTATAACAGGCTGCGGC20Attorney Docket No: 030258-000107WOPTAGGAAAACATGCACTTCGAGATCTGCCCCTGGTCCAGCTACC TCACTGGTGCCTGGAAGCCGGACACGGAGCATGCAGTCATTC GGCTCAAAAATGACCAGGCTAACTACTCGCTCAACACAGATG ACCCGCTCATCTTCAAGTCCACCCTGGACACTGATTACCAGAT GACCAAACGGGACATGGGCTTTACTGAAGAGGAGTTTAAAAG GCTGAACATCAATGCGGCCAAATCTAGTTTCCTCCCAGAAGA TGAAAAGAGGGAGCTTCTCGACCTGCTCTATAAAGCCTATGG GATGCCACCTTCAGCCTCTGCAGGGCAGAACCTCTGA SP+hADKS ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTG CACTTGTCACAAACAGTACGTCAGTCAGAGAAAATATTCTCTT TGGAATGGGAAATCCTCTGCTTGACATCTCTGCTGTAGTGGAC AAAGATTTCCTTGATAAGTATTCTCTGAAACCAAATGACCAA ATCTTGGCTGAAGACAAACACAAGGAACTGTTTGATGAACTT GTGAAAAAATTCAAAGTCGAATATCATGCTGGTGGCTCTACC CAGAATTCAATTAAAGTGGCTCAGTGGATGATTCAACAGCCA CACAAAGCAGCAACATTTTTTGGATGCATTGGGATAGATAAA TTTGGGGAGATCCTGAAGAGAAAAGCTGCTGAAGCCCATGTG GATGCTCATTACTACGAGCAGAATGAGCAGCCAACAGGAACT TGTGCTGCATGCATCACTGGTGACAACAGGTCCCTCATAGCTA ATCTTGCTGCTGCCAATTGTTATAAAAAGGAAAAACATCTTGA TCTGGAGAAAAACTGGATGTTGGTAGAAAAAGCAAGAGTTTG TTATATAGCAGGCTTTTTTCTTACAGTTTCCCCAGAGTCAGTA TTAAAGGTGGCTCACCATGCTTCTGAAAACAACAGGATTTTCA CTTTGAATCTATCTGCACCGTTTATTAGCCAGTTCTACAAGGA ATCATTGATGAAAGTTATGCCTTATGTTGATATACTTTTTGGA AATGAGACAGAAGCTGCCACTTTTGCTAGAGAGCAAGGCTTT GAGACTAAAGACATTAAAGAGATAGCCAAAAAGACACAAGC CCTGCCAAAGATGAACTCAAAGAGGCAGCGAATCGTGATCTT CACCCAAGGGAGAGATGACACTATAATGGCTACAGAAAGTGA AGTCACTGCTTTTGCTGTCTTGGATCAAGACCAGAAAGAAATT ATTGATACCAATGGAGCTGGAGATGCATTTGTTGGAGGTTTTC TGTCTCAACTGGTCTCTGACAAGCCTCTGACTGAATGTATCCG TGCTGGCCACTATGCAGCAAGCATCATAATTAGACGGACTGG CTGCACCTTTCCTGAGAAGCCAGACTTCCACTG21Attorney Docket No: 030258-000107WOPTFragment from CGAAACCCGCCGCGTCTGTTGAAATGGCCAGCCGCCCCGCC Plasmid pJS- GCATCCTCTCCCGTCGAAGCGCGGGCCCCGGTTGGGGGA ICP6-GFP- CAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAG CMVA-hADAl GCCGCCGGGGCCCCTCTCGCCCGCGGCCACCACGTGTAC TGCCAGCGAGTCAATGGCGTGATGGTGCTTTCCGACAAGAPositions 24- CGCCCGGGTCCGCGTCCTACCGCATCAGCGATAGCAACTT1181: ICP65’TGTCCAATGTGGTTCCAACTGCACCATGATCATAGACGGAPositions 1203- GACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCG 1922: GFP GCATCCCCCGCTCCCTTCGTTGCGGTGACAAACATCGGAG CCGGCAGCGACGGCGGGACCGCCGTCGTGGCATTCGGGGPositions 2074- GAACCCCACGTCGCTCGGCGGGGACGTCTACCGGTACCC 2129: betaAGACGACCGACGTCCCCACCGAGGCCCTTGGGGGCCCCC globin poly (A) CTCCTCCTCCCCGCTTCACCCTGGGTGGCGGCTGTTGTTC signal CTGTCGCGACACACGGCGCCGCTCTGCGGTATTCGGGGG GGAGGGGGATCCCGTCGGCCCCGCGGAGTTCGTCTCGGAPositions 2500- CGACCGGTCGTCCGATTCCGACTCGGATGACTCGGAGGAC3382: CMVACCGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTenhancer ACCGGCGGGGCCACGTACTCAGCGGTAGACCCACACGCACPositions 3401- CGACGCCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCCG 3460: Human CCGTGGCCCGGGACGACGCGGAGGGGCTTTCGGACCCCC IL-2 signal GGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCC peptide TGGAACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCT TTCTGGGAGATGCCGTGAACCGCGAACCCGCGCTCATGCTPositions 3461- GGAGTACTTTTGCCGGTGCGCCCGCGAGGAAACCAAGCG 4494: Human TGTCCCCCCCAGGACATTCTGCAGCCCCCCTCGCCTCACG ADA isoform 1 GAGGACGACTTTGGGCTTCTCAACTACGCGCTCGTGGAGA TGCAGCGCCTGTGTCTGGACGTTCCTCCGGTCCCGCCGAAPositions 4677- CGCATACATGCCCTATTATCTCAGGGAGTATGTGACGCGG4676: SV40CTGGTCAACGGGTTCAAGCCGCTGGTGAGCCGGTCCGCTCpoly (A) signal GCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGCGGATPositions 4780- CCTATCTGCGATCGCAATGGTGAGCAAGGGCGAGGAGCTG 5695: ICP63’ TTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGC GACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCA TCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT CGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGC22Attorney Docket No: 030258-000107WOPTTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCG CCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAA GTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCG CCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCC CGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG GCATGGACGAGCTGTACAAGTAAAGCGGCCGCACTCCTCAG GTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAAT GCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCA AAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTT CTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTG GAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCA AATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGG CAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGC TATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCC ATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTT TTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAA ATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCT GACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC CCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGAAACGCGT GGATCCACATCATGAAGCCCCTTGAGCATCTGACTTCTGG CTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGA ATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGC AAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTT TGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGG TTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCT GCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAG GTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTT TAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGA TTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCAttorney Docket No: 030258-000107WOPTTCTTCTCTTATGGAGCTCCCCCCGCTCCTCCCCCCGCTCCT CCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCC CCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGC TCCTCCCCCCCCCGCTCCCGCGGCCCCGCCCCCAACGCCC GCTCCTCCCCCCGCTCCCGCGGCCCCGCCCCCAACGCCCG CCGCGCGCGCGCACGCCGCCCTTGACTCACGGGGATTTCC AAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGG CACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACT CCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGT GGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGA ACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACT ATAGGGAGACCCAAGCTTACCGGCGAAGGAGGGCCACCG GATCCCTCGAGGCCACCATGTACAGGATGCAACTCCTGTCTTG CATTGCACTAAGTCTTGCACTTGTCACAAACAGTACGTCAGT CAGAGAAAATATTCTCTTTGGAATGGGAAATCCTCTGCTT GACATCTCTGCTGTAGTGGACAAAGATTTCCTTGATAAGT ATTCTCTGAAACCAAATGACCAAATCTTGGCTGAAGACAA ACACAAGGAACTGTTTGATGAACTTGTGAAAAAATTCAAA GTCGAATATCATGCTGGTGGCTCTACCCAGAATTCAATTA AAGTGGCTCAGTGGATGATTCAACAGCCACACAAAGCAGC AACATTTTTTGGATGCATTGGGATAGATAAATTTGGGGAG ATCCTGAAGAGAAAAGCTGCTGAAGCCCATGTGGATGCTC ATTACTACGAGCAGAATGAGCAGCCAACAGGAACTTGTGC TGCATGCATCACTGGTGACAACAGGTCCCTCATAGCTAAT CTTGCTGCTGCCAATTGTTATAAAAAGGAAAAACATCTTG ATCTGGAGAAAAACTGGATGTTGGTAGAAAAAGCAAGAGT TTGTTATATAGCAGGCTTTTTTCTTACAGTTTCCCCAGAGT CAGTATTAAAGGTGGCTCACCATGCTTCTGAAAACAACAG GATTTTCACTTTGAATCTATCTGCACCGTTTATTAGCCAGT TCTACAAGGAATCATTGATGAAAGTTATGCCTTATGTTGAT ATACTTTTTGGAAATGAGACAGAAGCTGCCACTTTTGCTA GAGAGCAAGGCTTTGAGACTAAAGACATTAAAGAGATAGC CAAAAAGACACAAGCCCTGCCAAAGATGAACTCAAAGAGG CAGCGAATCGTGATCTTCACCCAAGGGAGAGATGACACTA TAATGGCTACAGAAAGTGAAGTCACTGCTTTTGCTGTCTT GGATCAAGACCAGAAAGAAATTATTGATACCAATGGAGCTAttorney Docket No: 030258-000107WOPTGGAGATGCATTTGTTGGAGGTTTTCTGTCTCAACTGGTCT CTGACAAGCCTCTGACTGAATGTATCCGTGCTGGCCACTA TGCAGCAAGCATCATAATTAGACGGACTGGCTGCACCTTT CCTGAGAAGCCAGACTTCCACTGAGGTACCACCCAGCTTTC TTGTACAAAGTGGTGATGGCCGGCCGCTTCGAGCAGACATGA TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGC AGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGC TTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAAC AACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGT GGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA TTAATTAAGAACGGATCCGGACCCGGGAGGCCTCCTTTGA GGAGTGGCTGCGATCCAAGGAAGTGGCCCTGGACTTTGG CCTGACGGAAAGGCTTCGCGAGCACGAAGCCCAGCTGGT GATCCTGGCCCAGGCTCTGGACCATTACGACTGTCTGATC CACAGCACACCGCACACGCTGGTCGAGCGGGGGCTGCAA TCGGCCCTGAAGTATGAGGAGTTTTACCTAAAGCGCTTTG GCGGGCACTACATGGAGTCCGTCTTCCAGATGTACACCCG CATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGCGGCATG CGCCACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGAA ATGTTCAAGTTCTTTTTCCACCGCCTCTACGACCACCAGAT CGTACCGTCGACCCCCGCCATGCTGAACCTGGGGACCCGC AACTACTACACCTCCAGCTGCTACCTGGTAAACCCCCAGG CCACCACAAACAAGGCGACCCTGCGGGCCATCACCAGCAA CGTCAGCGCCATCCTCGCCCGCAACGGGGGCATCGGGCT ATGCGTGCAGGCGTTTAACGACTCCGGCCCCGGGACCGCT AGCGTCATACCCGCCCTCAAGGTCCTCGACTCGCTGGTGG CGGCGCACAACAAAGAGAGCGCGCGTCCAACCGGCGCGT GCGTGTACCTGGAGCCGTGGCACACCGACGTGCGGGCCG TGCTCCGGATGAAGGGGGTCCTCGCCGGCGAAGAGGCCC AGCGCTGCGACAATATCTTCAGCGCCCTCTGGATGCCAGA CCTGTTTTTCAAGCGCCTGATTCGCCACCTGGACGGCGAG AAGAACGTCACATGGACCCTGTTCGACCGGGACACCAGCA TGTCGCTCGCCGACTTTCACGGGGAGGAGTTCGAGAAGCT CTACCAGCACCTCGACTCTAGAGGGAttorney Docket No: 030258-000107WOPTFragment from CGAAACCCGCCGCGTCTGTTGAAATGGCCAGCCGCCCCGCC Plasmid pJS- GCATCCTCTCCCGTCGAAGCGCGGGCCCCGGTTGGGGGA ICP6-GFP- CAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAG CMVB-liADKS GCCGCCGGGGCCCCTCTCGCCCGCGGCCACCACGTGTAC TGCCAGCGAGTCAATGGCGTGATGGTGCTTTCCGACAAGAPositions 24- CGCCCGGGTCCGCGTCCTACCGCATCAGCGATAGCAACTT1181: ICP65’TGTCCAATGTGGTTCCAACTGCACCATGATCATAGACGGAPositions 1203- GACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCG 1922: GFP GCATCCCCCGCTCCCTTCGTTGCGGTGACAAACATCGGAG CCGGCAGCGACGGCGGGACCGCCGTCGTGGCATTCGGGGPositions 2074- GAACCCCACGTCGCTCGGCGGGGACGTCTACCGGTACCC 2129: betaAGACGACCGACGTCCCCACCGAGGCCCTTGGGGGCCCCC globin poly (A) CTCCTCCTCCCCGCTTCACCCTGGGTGGCGGCTGTTGTTC signal CTGTCGCGACACACGGCGCCGCTCTGCGGTATTCGGGGG GGAGGGGGATCCCGTCGGCCCCGCGGAGTTCGTCTCGGAPositions 2499- CGACCGGTCGTCCGATTCCGACTCGGATGACTCGGAGGAC3184: CMVACCGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTenhancer BCCGGCGGGGCCACGTACTCAGCGGTAGACCCACACGCACPositions 3197- CGACGCCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCCG 3256: Human CCGTGGCCCGGGACGACGCGGAGGGGCTTTCGGACCCCC IL-2 signal GGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCC peptide TGGAACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCT TTCTGGGAGATGCCGTGAACCGCGAACCCGCGCTCATGCTPositions 3257- GGAGTACTTTTGCCGGTGCGCCCGCGAGGAAACCAAGCG 4290: Human TGTCCCCCCCAGGACATTCTGCAGCCCCCCTCGCCTCACG ADKS GAGGACGACTTTGGGCTTCTCAACTACGCGCTCGTGGAGA TGCAGCGCCTGTGTCTGGACGTTCCTCCGGTCCCGCCGAAPositions 4351- CGCATACATGCCCTATTATCTCAGGGAGTATGTGACGCGG4472: SV40CTGGTCAACGGGTTCAAGCCGCTGGTGAGCCGGTCCGCTCpoly (A) signal GCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGCGGATPositions 4576- CCTATCTGCGATCGCAATGGTGAGCAAGGGCGAGGAGCTG 5491: ICP63’ TTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGC GACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCA TCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT CGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGC26Attorney Docket No: 030258-000107WOPTTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCG CCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAA GTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCG CCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCC CGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG GCATGGACGAGCTGTACAAGTAAAGCGGCCGCACTCCTCAG GTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAAT GCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCA AAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTT CTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTG GAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCA AATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGG CAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGC TATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCC ATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTT TTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAA ATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCT GACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC CCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGAAACGCGT TGACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATT AGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACAttorney Docket No: 030258-000107WOPTATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTG GCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAAC TCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGT GGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGA ACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACT ATAGGGAGACCCAAGCTTACCGGCGAAGGAGGGCCACCG GATCCCTCGAGGCCACCATGTACAGGATGCAACTCCTGTCTT GCATTGCACTAAGTCTTGCACTTGTCACAAACAGTACGTCAG TCAGAGAAAATATTCTCTTTGGAATGGGAAATCCTCTGCT TGACATCTCTGCTGTAGTGGACAAAGATTTCCTTGATAAG TATTCTCTGAAACCAAATGACCAAATCTTGGCTGAAGACA AACACAAGGAACTGTTTGATGAACTTGTGAAAAAATTCAA AGTCGAATATCATGCTGGTGGCTCTACCCAGAATTCAATT AAAGTGGCTCAGTGGATGATTCAACAGCCACACAAAGCAG CAACATTTTTTGGATGCATTGGGATAGATAAATTTGGGGA GATCCTGAAGAGAAAAGCTGCTGAAGCCCATGTGGATGCT CATTACTACGAGCAGAATGAGCAGCCAACAGGAACTTGTG CTGCATGCATCACTGGTGACAACAGGTCCCTCATAGCTAA TCTTGCTGCTGCCAATTGTTATAAAAAGGAAAAACATCTT GATCTGGAGAAAAACTGGATGTTGGTAGAAAAAGCAAGAG TTTGTTATATAGCAGGCTTTTTTCTTACAGTTTCCCCAGAG TCAGTATTAAAGGTGGCTCACCATGCTTCTGAAAACAACA GGATTTTCACTTTGAATCTATCTGCACCGTTTATTAGCCAG TTCTACAAGGAATCATTGATGAAAGTTATGCCTTATGTTGA TATACTTTTTGGAAATGAGACAGAAGCTGCCACTTTTGCT AGAGAGCAAGGCTTTGAGACTAAAGACATTAAAGAGATAG CCAAAAAGACACAAGCCCTGCCAAAGATGAACTCAAAGAG GCAGCGAATCGTGATCTTCACCCAAGGGAGAGATGACACT ATAATGGCTACAGAAAGTGAAGTCACTGCTTTTGCTGTCT TGGATCAAGACCAGAAAGAAATTATTGATACCAATGGAGC TGGAGATGCATTTGTTGGAGGTTTTCTGTCTCAACTGGTC TCTGACAAGCCTCTGACTGAATGTATCCGTGCTGGCCACT ATGCAGCAAGCATCATAATTAGACGGACTGGCTGCACCTT TCCTGAGAAGCCAGACTTCCACTGAGGTACCACCCAGCTTT CTTGTACAAAGTGGTGATGGCCGGCCGCTTCGAGCAGACATGAttorney Docket No: 030258-000107WOPTATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATG CAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTG CTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAA CAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTG TGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGT ATTAATTAAGAACGGATCCGGACCCGGGAGGCCTCCTTTG AGGAGTGGCTGCGATCCAAGGAAGTGGCCCTGGACTTTG GCCTGACGGAAAGGCTTCGCGAGCACGAAGCCCAGCTGG TGATCCTGGCCCAGGCTCTGGACCATTACGACTGTCTGAT CCACAGCACACCGCACACGCTGGTCGAGCGGGGGCTGCA ATCGGCCCTGAAGTATGAGGAGTTTTACCTAAAGCGCTTT GGCGGGCACTACATGGAGTCCGTCTTCCAGATGTACACCC GCATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGCGGCAT GCGCCACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGA AATGTTCAAGTTCTTTTTCCACCGCCTCTACGACCACCAGA TCGTACCGTCGACCCCCGCCATGCTGAACCTGGGGACCCG CAACTACTACACCTCCAGCTGCTACCTGGTAAACCCCCAG GCCACCACAAACAAGGCGACCCTGCGGGCCATCACCAGC AACGTCAGCGCCATCCTCGCCCGCAACGGGGGCATCGGG CTATGCGTGCAGGCGTTTAACGACTCCGGCCCCGGGACCG CTAGCGTCATACCCGCCCTCAAGGTCCTCGACTCGCTGGT GGCGGCGCACAACAAAGAGAGCGCGCGTCCAACCGGCGC GTGCGTGTACCTGGAGCCGTGGCACACCGACGTGCGGGC CGTGCTCCGGATGAAGGGGGTCCTCGCCGGCGAAGAGGC CCAGCGCTGCGACAATATCTTCAGCGCCCTCTGGATGCCA GACCTGTTTTTCAAGCGCCTGATTCGCCACCTGGACGGCG AGAAGAACGTCACATGGACCCTGTTCGACCGGGACACCAG CATGTCGCTCGCCGACTTTCACGGGGAGGAGTTCGAGAAG CTCTACCAGCACCTCGACTCTAGAGGGhADAl GCCCAGACGCCCGCCTTCGACAAGCCCAAAGTGGAACTGCAT (NM 000022.4) GTCCACCTAGACGGATCCATCAAGCCTGAAACCATCTTATACT ATGGCAGGAGGAGAGGGATCGCCCTCCCAGCTAACACAGCAG AGGGGCTGCTGAACGTCATTGGCATGGACAAGCCGCTCACCC TTCCAGACTTCCTGGCCAAGTTTGACTACTACATGCCTGCTAT CGCGGGCTGCCGGGAGGCTATCAAAAGGATCGCCTATGAGTT29Attorney Docket No: 030258-000107WOPTTGTAGAGATGAAGGCCAAAGAGGGCGTGGTGTATGTGGAGGT GCGGTACAGTCCGCACCTGCTGGCCAACTCCAAAGTGGAGCC AATCCCCTGGAACCAGGCTGAAGGGGACCTCACCCCAGACGA GGTGGTGGCCCTAGTGGGCCAGGGCCTGCAGGAGGGGGAGC GAGACTTCGGGGTCAAGGCCCGGTCCATCCTGTGCTGCATGC GCCACCAGCCCAACTGGTCCCCCAAGGTGGTGGAGCTGTGTA AGAAGTACCAGCAGCAGACCGTGGTAGCCATTGACCTGGCTG GAGATGAGACCATCCCAGGAAGCAGCCTCTTGCCTGGACATG TCCAGGCCTACCAGGAGGCTGTGAAGAGCGGCATTCACCGTA CTGTCCACGCCGGGGAGGTGGGCTCGGCCGAAGTAGTAAAAG AGGCTGTGGACATACTCAAGACAGAGCGGCTGGGACACGGCT ACCACACCCTGGAAGACCAGGCCCTTTATAACAGGCTGCGGC AGGAAAACATGCACTTCGAGATCTGCCCCTGGTCCAGCTACC TCACTGGTGCCTGGAAGCCGGACACGGAGCATGCAGTCATTC GGCTCAAAAATGACCAGGCTAACTACTCGCTCAACACAGATG ACCCGCTCATCTTCAAGTCCACCCTGGACACTGATTACCAGAT GACCAAACGGGACATGGGCTTTACTGAAGAGGAGTTTAAAAG GCTGAACATCAATGCGGCCAAATCTAGTTTCCTCCCAGAAGA TGAAAAGAGGGAGCTTCTCGACCTGCTCTATAAAGCCTATGG GATGCCACCTTCAGCCTCTGCAGGGCAGAACCTCTGA SP+hADAl ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTG CACTTGTCACAAACAGTGCCCAGACGCCCGCCTTCGACAAGC CCAAAGTGGAACTGCATGTCCACCTAGACGGATCCATCAAGC CTGAAACCATCTTATACTATGGCAGGAGGAGAGGGATCGCCC TCCCAGCTAACACAGCAGAGGGGCTGCTGAACGTCATTGGCA TGGACAAGCCGCTCACCCTTCCAGACTTCCTGGCCAAGTTTGA CTACTACATGCCTGCTATCGCGGGCTGCCGGGAGGCTATCAA AAGGATCGCCTATGAGTTTGTAGAGATGAAGGCCAAAGAGGG CGTGGTGTATGTGGAGGTGCGGTACAGTCCGCACCTGCTGGC CAACTCCAAAGTGGAGCCAATCCCCTGGAACCAGGCTGAAGG GGACCTCACCCCAGACGAGGTGGTGGCCCTAGTGGGCCAGGG CCTGCAGGAGGGGGAGCGAGACTTCGGGGTCAAGGCCCGGTC CATCCTGTGCTGCATGCGCCACCAGCCCAACTGGTCCCCCAAG GTGGTGGAGCTGTGTAAGAAGTACCAGCAGCAGACCGTGGTA GCCATTGACCTGGCTGGAGATGAGACCATCCCAGGAAGCAGC30Attorney Docket No: 030258-000107WOPTCTCTTGCCTGGACATGTCCAGGCCTACCAGGAGGCTGTGAAG AGCGGCATTCACCGTACTGTCCACGCCGGGGAGGTGGGCTCG GCCGAAGTAGTAAAAGAGGCTGTGGACATACTCAAGACAGA GCGGCTGGGACACGGCTACCACACCCTGGAAGACCAGGCCCT TTATAACAGGCTGCGGCAGGAAAACATGCACTTCGAGATCTG CCCCTGGTCCAGCTACCTCACTGGTGCCTGGAAGCCGGACAC GGAGCATGCAGTCATTCGGCTCAAAAATGACCAGGCTAACTA CTCGCTCAACACAGATGACCCGCTCATCTTCAAGTCCACCCTG GAC ACT GATT ACC AGATGACCAAACGGGACATGGGCTTT ACT GAAGAGGAGTTTAAAAGGCTGAACATCAATGCGGCCAAATCT AGTTTCCTCCCAGAAGATGAAAAGAGGGAGCTTCTCGACCTG CTCTATAAAGCCTATGGGATGCCACCTTCAGCCTCTGCAGGGC AGAACCTCTGAFragment from CGAAACCCGCCGCGTCTGTTGAAATGGCCAGCCGCCCCGCC Plasmid pJS- GCATCCTCTCCCGTCGAAGCGCGGGCCCCGGTTGGGGGACAG ICP6-GFP- GAGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAGGCCGC CMVA-hADAl CGGGGCCCCTCTCGCCCGCGGCCACCACGTGTACTGCCAGCG AGTCAATGGCGTGATGGTGCTTTCCGACAAGACGCCCGGGTCPositions 24- CGCGTCCTACCGCATCAGCGATAGCAACTTTGTCCAATGTGGT1181: ICP65’TCCAACTGCACCATGATCATAGACGGAGACGTGGTGCGCGGGPositions 1203- CGCCCCCAGGACCCGGGGGCCGCGGCATCCCCCGCTCCCTTC 1922: GFP GTTGCGGTGACAAACATCGGAGCCGGCAGCGACGGCGGGACC GCCGTCGTGGCATTCGGGGGAACCCCACGTCGCTCGGCGGGGPositions 2074- ACGTCTACCGGTACCCAGACGACCGACGTCCCCACCGAGGCC 2129: betaCTTGGGGGCCCCCCTCCTCCTCCCCGCTTCACCCTGGGTGGCG globin poly (A) GCTGTTGTTCCTGTCGCGACACACGGCGCCGCTCTGCGGTATT signal CGGGGGGGAGGGGGATCCCGTCGGCCCCGCGGAGTTCGTCTC GGACGACCGGTCGTCCGATTCCGACTCGGATGACTCGGAGGAPositions 2450- CACCGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTC3382: CMVCGGCGGGGCCACGTACTCAGCGGTAGACCCACACGCACCGACenhancer AGCCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCCGCCGTGGCPositions 3401- CCGGGACGACGCGGAGGGGCTTTCGGACCCCCGGCCACGTCT 3459: Human GGGAACGGGCACGGCCTACCCCGTCCCCCTGGAACTCACGCC CGAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGAGATGCCGT GAACCGCGAACCCGCGCTCATGCTGGAGTACTTTTGCCGGTG31Attorney Docket No: 030258-000107WOPTIL-2 signal CGCCCGCGAGGAAACCAAGCGTGTCCCCCCCAGGACATTCTG peptide CAGCCCCCCTCGCCTCACGGAGGACGACTTTGGGCTTCTCAAC TACGCGCTCGTGGAGATGCAGCGCCTGTGTCTGGACGTTCCTCPositions 3460- CGGTCCCGCCGAACGCATACATGCCCTATTATCTCAGGGAGT4549: Human ATGTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGGTGAGCC ADA isoform 1GGTCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTPositions 4609- GCGGATCCTATCTGCGATCGCAATGGTGAGCAAGGGCGAG 4730: SV40 GAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC poly(A) signal GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCPositions 4834- TGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTG 5749: ICP63’ ACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCG ACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCG AAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACG GCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACA CCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGG AGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACA ACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACG GCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACG GCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCA TCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGA GCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCG ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCA CTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCACT CCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGT GTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTT TTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCT TGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCAT TGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGG ACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGT ATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGC TGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTAT ATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAA AAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTT GTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACAT GTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCC32Attorney Docket No: 030258-000107WOPTAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGAC CTGCAGCCCAAGCTTGGCGTAATCATGAAACGCGTGGATC CACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAA GGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGT CTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAA CATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCC ATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTC ATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCA TAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTT TTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACAT GTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGT CATAGCTGTCCCTCTTCTCTTATGGAGCTCCCCCCGCTCCTCCC CCCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGCTCC TCCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCCCCG CTCCTCCCCCCCCCGCTCCCGCGGCCCCGCCCCCAACGCCCGC TCCTCCCCCCGCTCCCGCGGCCCCGCCCCCAACGCCCGCCGCG CGCGCGCACGCCGCCCTTGACTCACGGGGATTTCCAAGTCTCC ACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCA ACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGAC GCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAG CAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTT ATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTTAC CGGCGAAGGAGGGCCACCGGATCCCTCGAGGCCACCATGTAC AGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTG TCACAAACAGTGCCCAGACGCCCGCCTTCGACAAGCCCAAAG TGGAACTGCATGTCCACCTAGACGGATCCATCAAGCCTGAAA CCATCTTATACTATGGCAGGAGGAGAGGGATCGCCCTCCCAG CTAACACAGCAGAGGGGCTGCTGAACGTCATTGGCATGGACA AGCCGCTCACCCTTCCAGACTTCCTGGCCAAGTTTGACTACTA CATGCCTGCTATCGCGGGCTGCCGGGAGGCTATCAAAAGGAT CGCCTATGAGTTTGTAGAGATGAAGGCCAAAGAGGGCGTGGT GTATGTGGAGGTGCGGTACAGTCCGCACCTGCTGGCCAACTC CAAAGTGGAGCCAATCCCCTGGAACCAGGCTGAAGGGGACCT CACCCCAGACGAGGTGGTGGCCCTAGTGGGCCAGGGCCTGCA GGAGGGGGAGCGAGACTTCGGGGTCAAGGCCCGGTCCATCCT GTGCTGCATGCGCCACCAGCCCAACTGGTCCCCCAAGGTGGTAttorney Docket No: 030258-000107WOPTGGAGCTGTGTAAGAAGTACCAGCAGCAGACCGTGGTAGCCAT TGACCTGGCTGGAGATGAGACCATCCCAGGAAGCAGCCTCTT GCCTGGACATGTCCAGGCCTACCAGGAGGCTGTGAAGAGCGG CATTCACCGTACTGTCCACGCCGGGGAGGTGGGCTCGGCCGA AGTAGTAAAAGAGGCTGTGGACATACTCAAGACAGAGCGGCT GGGACACGGCTACCACACCCTGGAAGACCAGGCCCTTTATAA CAGGCTGCGGCAGGAAAACATGCACTTCGAGATCTGCCCCTG GTCCAGCTACCTCACTGGTGCCTGGAAGCCGGACACGGAGCA TGCAGTCATTCGGCTCAAAAATGACCAGGCTAACTACTCGCTC AACACAGATGACCCGCTCATCTTCAAGTCCACCCTGGACACT GATTACCAGATGACCAAACGGGACATGGGCTTTACTGAAGAG GAGTTTAAAAGGCTGAACATCAATGCGGCCAAATCTAGTTTC CTCCCAGAAGATGAAAAGAGGGAGCTTCTCGACCTGCTCTAT AAAGCCTATGGGATGCCACCTTCAGCCTCTGCAGGGCAGAAC CTCTGAGGTACCACCCAGCTTTCTTGTACAAAGTGGTGATGGC CGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTT GGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATT TGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAA GCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTAT GTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAA GTAAAACCTCTACAAATGTGGTATTAATTAAGAACGGATCCG GACCCGGGAGGCCTCCTTTGAGGAGTGGCTGCGATCCAAGGA AGTGGCCCTGGACTTTGGCCTGACGGAAAGGCTTCGCGAGCA CGAAGCCCAGCTGGTGATCCTGGCCCAGGCTCTGGACCATTA CGACTGTCTGATCCACAGCACACCGCACACGCTGGTCGAGCG GGGGCTGCAATCGGCCCTGAAGTATGAGGAGTTTTACCTAAA GCGCTTTGGCGGGCACTACATGGAGTCCGTCTTCCAGATGTAC ACCCGCATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGCGGC ATGCGCCACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGAA ATGTTCAAGTTCTTTTTCCACCGCCTCTACGACCACCAGATCG TACCGTCGACCCCCGCCATGCTGAACCTGGGGACCCGCAACT ACTACACCTCCAGCTGCTACCTGGTAAACCCCCAGGCCACCA CAAACAAGGCGACCCTGCGGGCCATCACCAGCAACGTCAGCG CCATCCTCGCCCGCAACGGGGGCATCGGGCTATGCGTGCAGG CGTTTAACGACTCCGGCCCCGGGACCGCTAGCGTCATACCCG CCCTCAAGGTCCTCGACTCGCTGGTGGCGGCGCACAACAAAGAttorney Docket No: 030258-000107WOPTAGAGCGCGCGTCCAACCGGCGCGTGCGTGTACCTGGAGCCGT GGCACACCGACGTGCGGGCCGTGCTCCGGATGAAGGGGGTCC TCGCCGGCGAAGAGGCCCAGCGCTGCGACAATATCTTCAGCG CCCTCTGGATGCCAGACCTGTTTTTCAAGCGCCTGATTCGCCA CCTGGACGGCGAGAAGAACGTCACATGGACCCTGTTCGACCG GGACACCAGCATGTCGCTCGCCGACTTTCACGGGGAGGAGTT CGAGAAGCTCTACCAGCACCTCGACTCTAGAGGGFragment from CGAAACCCGCCGCGTCTGTTGAAATGGCCAGCCGCCCCGCC Plasmid pJS- GCATCCTCTCCCGTCGAAGCGCGGGCCCCGGTTGGGGGA ICP6-GFP- CAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAG CMVA-mADAl GCCGCCGGGGCCCCTCTCGCCCGCGGCCACCACGTGTAC TGCCAGCGAGTCAATGGCGTGATGGTGCTTTCCGACAAGAPositions 24- CGCCCGGGTCCGCGTCCTACCGCATCAGCGATAGCAACTT1181: ICP65’TGTCCAATGTGGTTCCAACTGCACCATGATCATAGACGGAPositions 1203- GACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCG 1922: GFP GCATCCCCCGCTCCCTTCGTTGCGGTGACAAACATCGGAG CCGGCAGCGACGGCGGGACCGCCGTCGTGGCATTCGGGGPositions 2074- GAACCCCACGTCGCTCGGCGGGGACGTCTACCGGTACCC 2129: betaAGACGACCGACGTCCCCACCGAGGCCCTTGGGGGCCCCC globin poly (A) CTCCTCCTCCCCGCTTCACCCTGGGTGGCGGCTGTTGTTC signal CTGTCGCGACACACGGCGCCGCTCTGCGGTATTCGGGGG GGAGGGGGATCCCGTCGGCCCCGCGGAGTTCGTCTCGGAPositions 2450- CGACCGGTCGTCCGATTCCGACTCGGATGACTCGGAGGAC3382: CMVACCGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTenhancer ACCGGCGGGGCCACGTACTCAGCGGTAGACCCACACGCACPositions 3401- CGACGCCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCCG 3460: Human CCGTGGCCCGGGACGACGCGGAGGGGCTTTCGGACCCCC IL-2 signal GGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCC peptide, codon TGGAACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCT optimized for TTCTGGGAGATGCCGTGAACCGCGAACCCGCGCTCATGCT mus musculus GGAGTACTTTTGCCGGTGCGCCCGCGAGGAAACCAAGCG TGTCCCCCCCAGGACATTCTGCAGCCCCCCTCGCCTCACGPositions 3464- GAGGACGACTTTGGGCTTCTCAACTACGCGCTCGTGGAGA 4516: Murine TGCAGCGCCTGTGTCTGGACGTTCCTCCGGTCCCGCCGAACGCATACATGCCCTATTATCTCAGGGAGTATGTGACGCGG35Attorney Docket No: 030258-000107WOPTcodon optimized CTGGTCAACGGGTTCAAGCCGCTGGTGAGCCGGTCCGCTC ADA isoform 1 GCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGCGGAT CCTATCTGCGATCGCAATGGTGAGCAAGGGCGAGGAGCTGPositions 4576- TTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGC4697: SV40GACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCpoly(A) signal GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCAPositions 4801- TCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT 5716: ICP63’ CGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGC TACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCG CCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAA GTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCG CCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCC CGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG GCATGGACGAGCTGTACAAGTAAAGCGGCCGCACTCCTCAG GTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAAT GCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCA AAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTT CTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGT TGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAG GGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAG TTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGG TTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGC TGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAG ATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCC CTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCT CTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGA GATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGAAAC GCGTGGATCCACATCATGAAGCCCCTTGAGCATCTGACTTC TGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTT36Attorney Docket No: 030258-000107WOPTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAG GGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAG AGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACA AAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCC CCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTT GAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTT CTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCC AGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGT CCCTCTTCTCTTATGGAGCTCCCCCCGCTCCTCCCCCCGC TCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCT CCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCC CCGCTCCTCCCCCCCCCGCTCCCGCGGCCCCGCCCCCAAC GCCCGCTCCTCCCCCCGCTCCCGCGGCCCCGCCCCCAACG CCCGCCGCGCGCGCGCACGCCGCCCTTGACTCACGGGGA TTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGT TTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAA CAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTA CGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTA GAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGAC TCACTATAGGGAGACCCAAGCTTACCGGCGAAGGAGGGC CACCGGATCCCTCGAGGCCACCATGTACAGGATGCAACTCC TGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG GCTCAGACACCAGCCTTCAACAAGCCCAAGGTGGAACTGC ATGTGCACCTGGACGGCGCCATCAAGCCTGAGACAATCCT GTACTTCGGCAAGAAGAGAGGAATCGCCCTGCCTGCCGAC ACCGTGGAAGAACTGAGAAACATCATCGGCATGGACAAGC CCCTGAGCCTGCCTGGCTTCCTGGCCAAGTTCGACTACTA CATGCCTGTGATCGCCGGCTGCAGAGAGGCCATCAAGAG AATCGCCTACGAGTTCGTGGAAATGAAGGCCAAAGAAGGC GTGGTCTACGTCGAAGTGCGGTACAGCCCTCATCTGCTGG CCAACTCTAAGGTGGACCCCATGCCTTGGAACCAGACAGA GGGCGACGTGACACCTGACGACGTGGTGGATCTGGTCAA CCAGGGACTGCAAGAGGGCGAGCAGGCCTTCGGAATCAA AGTGCGGAGCATCCTGTGCTGCATGAGGCACCAACCATCT TGGAGCCTGGAAGTGCTGGAACTGTGCAAGAAGTACAACC AGAAAACCGTGGTGGCCATGGACCTGGCTGGCGACGAAAAttorney Docket No: 030258-000107WOPTCAATCGAGGGCAGCTCTCTGTTCCCCGGCCACGTGGAAGC TTATGAGGGCGCTGTGAAGAACGGCATCCACAGAACAGTG CACGCTGGCGAAGTGGGCTCTCCTGAGGTTGTCAGAGAA GCCGTGGACATCCTGAAAACCGAGAGAGTCGGCCACGGC TACCACACCATCGAGGATGAGGCCCTGTACAACAGACTGC TGAAAGAAAACATGCACTTTGAAGTGTGCCCCTGGTCCAG CTACCTGACAGGCGCTTGGGACCCTAAGACAACACACGCT GTCGTGCGGTTCAAGAACGACAAGGCCAACTACAGCCTGA ACACAGACGACCCTCTGATCTTCAAGAGCACCCTGGACAC CGACTACCAGATGACCAAGAAAGACATGGGCTTCACCGAG GAAGAGTTCAAGAGGCTGAACATCAACGCCGCCAAGAGC AGCTTCCTGCCTGAGGAAGAGAAGAAAGAGCTGCTGGAA AGGCTGTACAGAGAGTACCAGTAAGGTACCACCCAGCTTTC TTGTACAAAGTGGTGATGGCCGGCCGCTTCGAGCAGACATGA TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAAT GCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCT ATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGT TAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGG GAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAA TGTGGTATTAATTAAGAACGGATCCGGACCCGGGAGGCCTC CTTTGAGGAGTGGCTGCGATCCAAGGAAGTGGCCCTGGA CTTTGGCCTGACGGAAAGGCTTCGCGAGCACGAAGCCCA GCTGGTGATCCTGGCCCAGGCTCTGGACCATTACGACTGT CTGATCCACAGCACACCGCACACGCTGGTCGAGCGGGGG CTGCAATCGGCCCTGAAGTATGAGGAGTTTTACCTAAAGC GCTTTGGCGGGCACTACATGGAGTCCGTCTTCCAGATGTA CACCCGCATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGC GGCATGCGCCACATCGCCCTGGGGCGAGAGGGGTCGTGG TGGGAAATGTTCAAGTTCTTTTTCCACCGCCTCTACGACC ACCAGATCGTACCGTCGACCCCCGCCATGCTGAACCTGGG GACCCGCAACTACTACACCTCCAGCTGCTACCTGGTAAAC CCCCAGGCCACCACAAACAAGGCGACCCTGCGGGCCATC ACCAGCAACGTCAGCGCCATCCTCGCCCGCAACGGGGGC ATCGGGCTATGCGTGCAGGCGTTTAACGACTCCGGCCCCG GGACCGCTAGCGTCATACCCGCCCTCAAGGTCCTCGACTC GCTGGTGGCGGCGCACAACAAAGAGAGCGCGCGTCCAACAttorney Docket No: 030258-000107WOPTCGGCGCGTGCGTGTACCTGGAGCCGTGGCACACCGACGT GCGGGCCGTGCTCCGGATGAAGGGGGTCCTCGCCGGCGA AGAGGCCCAGCGCTGCGACAATATCTTCAGCGCCCTCTGG ATGCCAGACCTGTTTTTCAAGCGCCTGATTCGCCACCTGG ACGGCGAGAAGAACGTCACATGGACCCTGTTCGACCGGG ACACCAGCATGTCGCTCGCCGACTTTCACGGGGAGGAGTT CGAGAAGCTCTACCAGCACCTCGACTCTAGAGGGFragment from CGAAACCCGCCGCGTCTGTTGAAATGGCCAGCCGCCCCGCC Plasmid pJS- GCATCCTCTCCCGTCGAAGCGCGGGCCCCGGTTGGGGGA ICP6-GFP- CAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAG CMVB-mADAl GCCGCCGGGGCCCCTCTCGCCCGCGGCCACCACGTGTAC TGCCAGCGAGTCAATGGCGTGATGGTGCTTTCCGACAAGAPositions 24- CGCCCGGGTCCGCGTCCTACCGCATCAGCGATAGCAACTT1181: ICP65’TGTCCAATGTGGTTCCAACTGCACCATGATCATAGACGGAPositions 1203- GACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCG 1922: GFP GCATCCCCCGCTCCCTTCGTTGCGGTGACAAACATCGGAG CCGGCAGCGACGGCGGGACCGCCGTCGTGGCATTCGGGGPositions 2074- GAACCCCACGTCGCTCGGCGGGGACGTCTACCGGTACCC 2128: betaAGACGACCGACGTCCCCACCGAGGCCCTTGGGGGCCCCC globin poly (A) CTCCTCCTCCCCGCTTCACCCTGGGTGGCGGCTGTTGTTC signal CTGTCGCGACACACGGCGCCGCTCTGCGGTATTCGGGGG GGAGGGGGATCCCGTCGGCCCCGCGGAGTTCGTCTCGGAPositions 2499- CGACCGGTCGTCCGATTCCGACTCGGATGACTCGGAGGAC3184: CMVACCGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTenhancer BCCGGCGGGGCCACGTACTCAGCGGTAGACCCACACGCACPositions 3198- CGACGCCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCCG 3256: Human CCGTGGCCCGGGACGACGCGGAGGGGCTTTCGGACCCCC IL-2 signal GGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCC peptide, codon TGGAACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCT optimized for TTCTGGGAGATGCCGTGAACCGCGAACCCGCGCTCATGCT mus musculus GGAGTACTTTTGCCGGTGCGCCCGCGAGGAAACCAAGCG TGTCCCCCCCAGGACATTCTGCAGCCCCCCTCGCCTCACGPositions 3260- GAGGACGACTTTGGGCTTCTCAACTACGCGCTCGTGGAGA 4312: Murine TGCAGCGCCTGTGTCTGGACGTTCCTCCGGTCCCGCCGAACGCATACATGCCCTATTATCTCAGGGAGTATGTGACGCGG39Attorney Docket No: 030258-000107WOPTcodon optimized CTGGTCAACGGGTTCAAGCCGCTGGTGAGCCGGTCCGCTC ADA isoform 1 GCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGCGGAT CCTATCTGCGATCGCAATGGTGAGCAAGGGCGAGGAGCTGPositions 4372- TTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGC4493: SV40GACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCpoly(A) signal GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCAPositions 4597- TCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT 5512: ICP63’ CGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGC TACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCG CCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAA GTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCG CCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCC CGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG GCATGGACGAGCTGTACAAGTAAAGCGGCCGCACTCCTCAG GTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAAT GCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCA AAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTT CTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGT TGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAG GGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAG TTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGG TTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGC TGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAG ATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCC CTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCT CTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGA GATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGAAAC GCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAA TTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCG40Attorney Docket No: 030258-000107WOPTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATG TTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCA ATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTA CATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACG TCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTAC GTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGG ATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTG TTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA ACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGT ACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACT AGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGA CTCACTATAGGGAGACCCAAGCTTACCGGCGAAGGAGGG CCACCGGATCCCTCGAGGCCACCATGTACAGGATGCAACTCC TGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGGC TCAGACACCAGCCTTCAACAAGCCCAAGGTGGAACTGCAT GTGCACCTGGACGGCGCCATCAAGCCTGAGACAATCCTGT ACTTCGGCAAGAAGAGAGGAATCGCCCTGCCTGCCGACAC CGTGGAAGAACTGAGAAACATCATCGGCATGGACAAGCCC CTGAGCCTGCCTGGCTTCCTGGCCAAGTTCGACTACTACA TGCCTGTGATCGCCGGCTGCAGAGAGGCCATCAAGAGAAT CGCCTACGAGTTCGTGGAAATGAAGGCCAAAGAAGGCGT GGTCTACGTCGAAGTGCGGTACAGCCCTCATCTGCTGGCC AACTCTAAGGTGGACCCCATGCCTTGGAACCAGACAGAGG GCGACGTGACACCTGACGACGTGGTGGATCTGGTCAACCA GGGACTGCAAGAGGGCGAGCAGGCCTTCGGAATCAAAGT GCGGAGCATCCTGTGCTGCATGAGGCACCAACCATCTTGG AGCCTGGAAGTGCTGGAACTGTGCAAGAAGTACAACCAGA AAACCGTGGTGGCCATGGACCTGGCTGGCGACGAAACAA TCGAGGGCAGCTCTCTGTTCCCCGGCCACGTGGAAGCTTA TGAGGGCGCTGTGAAGAACGGCATCCACAGAACAGTGCA CGCTGGCGAAGTGGGCTCTCCTGAGGTTGTCAGAGAAGC CGTGGACATCCTGAAAACCGAGAGAGTCGGCCACGGCTA CCACACCATCGAGGATGAGGCCCTGTACAACAGACTGCTGAttorney Docket No: 030258-000107WOPTAAAGAAAACATGCACTTTGAAGTGTGCCCCTGGTCCAGCT ACCTGACAGGCGCTTGGGACCCTAAGACAACACACGCTGT CGTGCGGTTCAAGAACGACAAGGCCAACTACAGCCTGAAC ACAGACGACCCTCTGATCTTCAAGAGCACCCTGGACACCG ACTACCAGATGACCAAGAAAGACATGGGCTTCACCGAGGA AGAGTTCAAGAGGCTGAACATCAACGCCGCCAAGAGCAG CTTCCTGCCTGAGGAAGAGAAGAAAGAGCTGCTGGAAAG GCTGTACAGAGAGTACCAGTAAGGTACCACCCAGCTTTCTT GTACAAAGTGGTGATGGCCGGCCGCTTCGAGCAGACATGATA AGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAG TGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTT TATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACA ACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTG GGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATT AATTAAGAACGGATCCGGACCCGGGAGGCCTCCTTTGAGG AGTGGCTGCGATCCAAGGAAGTGGCCCTGGACTTTGGCCT GACGGAAAGGCTTCGCGAGCACGAAGCCCAGCTGGTGAT CCTGGCCCAGGCTCTGGACCATTACGACTGTCTGATCCAC AGCACACCGCACACGCTGGTCGAGCGGGGGCTGCAATCG GCCCTGAAGTATGAGGAGTTTTACCTAAAGCGCTTTGGCG GGCACTACATGGAGTCCGTCTTCCAGATGTACACCCGCAT CGCCGGCTTTTTGGCCTGCCGGGCCACGCGCGGCATGCG CCACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGAAAT GTTCAAGTTCTTTTTCCACCGCCTCTACGACCACCAGATC GTACCGTCGACCCCCGCCATGCTGAACCTGGGGACCCGCA ACTACTACACCTCCAGCTGCTACCTGGTAAACCCCCAGGC CACCACAAACAAGGCGACCCTGCGGGCCATCACCAGCAAC GTCAGCGCCATCCTCGCCCGCAACGGGGGCATCGGGCTA TGCGTGCAGGCGTTTAACGACTCCGGCCCCGGGACCGCTA GCGTCATACCCGCCCTCAAGGTCCTCGACTCGCTGGTGGC GGCGCACAACAAAGAGAGCGCGCGTCCAACCGGCGCGTG CGTGTACCTGGAGCCGTGGCACACCGACGTGCGGGCCGT GCTCCGGATGAAGGGGGTCCTCGCCGGCGAAGAGGCCCA GCGCTGCGACAATATCTTCAGCGCCCTCTGGATGCCAGAC CTGTTTTTCAAGCGCCTGATTCGCCACCTGGACGGCGAGA AGAACGTCACATGGACCCTGTTCGACCGGGACACCAGCATAttorney Docket No: 030258-000107WOPTGTCGCTCGCCGACTTTCACGGGGAGGAGTTCGAGAAGCTC TACCAGCACCTCGACTCTAGAGGGFragment from CGAAACCCGCCGCGTCTGTTGAAATGGCCAGCCGCCCCGCCG Plasmid pJS- CATCCTCTCCCGTCGAAGCGCGGGCCCCGGTTGGGGGACAGG ICP6-GFP- AGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAGGCCGCC CMVA- GGGGCCCCTCTCGCCCGCGGCCACCACGTGTACTGCCAGCGA mADKS GTCAATGGCGTGATGGTGCTTTCCGACAAGACGCCCGGGTCC GCGTCCTACCGCATCAGCGATAGCAACTTTGTCCAATGTGGTTPositions 24- CCAACTGCACCATGATCATAGACGGAGACGTGGTGCGCGGGC1181: ICP65’GCCCCCAGGACCCGGGGGCCGCGGCATCCCCCGCTCCCTTCGPositions 1203- TTGCGGTGACAAACATCGGAGCCGGCAGCGACGGCGGGACCG 1922: GFP CCGTCGTGGCATTCGGGGGAACCCCACGTCGCTCGGCGGGGA CGTCTACCGGTACCCAGACGACCGACGTCCCCACCGAGGCCCPositions 2074- TTGGGGGCCCCCCTCCTCCTCCCCGCTTCACCCTGGGTGGCGG 2129: betaCTGTTGTTCCTGTCGCGACACACGGCGCCGCTCTGCGGTATTC globin poly (A) GGGGGGGAGGGGGATCCCGTCGGCCCCGCGGAGTTCGTCTCG signal GACGACCGGTCGTCCGATTCCGACTCGGATGACTCGGAGGAC ACCGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTCCPositions 2450- GGCGGGGCCACGTACTCAGCGGTAGACCCACACGCACCGACG3382: CMVCCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCCGCCGTGGCCenhancer ACGGGACGACGCGGAGGGGCTTTCGGACCCCCGGCCACGTCTGPositions 3401- GGAACGGGCACGGCCTACCCCGTCCCCCTGGAACTCACGCCC 3460: Human GAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGAGATGCCGTG IL-2 signal AACCGCGAACCCGCGCTCATGCTGGAGTACTTTTGCCGGTGC peptide, codon GCCCGCGAGGAAACCAAGCGTGTCCCCCCCAGGACATTCTGC optimized for AGCCCCCCTCGCCTCACGGAGGACGACTTTGGGCTTCTCAACT mus musculus ACGCGCTCGTGGAGATGCAGCGCCTGTGTCTGGACGTTCCTCC GGTCCCGCCGAACGCATACATGCCCTATTATCTCAGGGAGTATPositions 3461- GTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGGTGAGCCGG 4495: Murine TCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGC codon optimized GGATCCTATCTGCGATCGCAATGGTGAGCAAGGGCGAGGAGC ADK isofomi S TGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCG (short) ACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGG GCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCA CCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCA43Attorney Docket No: 030258-000107WOPTPositions 4555- CCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCA 4676: SV40 CATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG poly (A) signal CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAA CTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTPositions 4780- GGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGA5695: ICP63’CGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAG CCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCAT CAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAG CGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGG CGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCAC CCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTC GGCATGGACGAGCTGTACAAGTAAAGCGGCCGCACTCCTCAG GTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAAT GCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCA AAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTT CTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTG GAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCA AATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGG CAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGC TATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCC ATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTT TTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAA ATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCT GACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC CCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGAAACGCGT GGATCCACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTA ATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTT TTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCAT TTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACAT ATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAA GAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTT ATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATA TTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCC TTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACT CCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGCTCCCCCCGC44Attorney Docket No: 030258-000107WOPTTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCC CCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCT CCCCCCGCTCCTCCCCCCCCCGCTCCCGCGGCCCCGCCCCCAA CGCCCGCTCCTCCCCCCGCTCCCGCGGCCCCGCCCCCAACGCC CGCCGCGCGCGCGCACGCCGCCCTTGACTCACGGGGATTTCC AAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCAC CAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCC CCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC TATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTT ACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC AAGCTTACCGGCGAAGGAGGGCCACCGGATCCCTCGAGGCCA CCATGTACAGAATGCAGCTGCTGAGCTGTATCGCCCTGTCTCT GGCCCTGGTCACCAACAGCACAAGCACCTCTGAGAACGTGCT GTTCGGCATGGGCAACCCTCTGCTGGATATCAGCGCCGTGGT GGACAAGGACTTCCTGGACAAGTACAGCCTGAAGCCTAACGA CCAGATCCTGGCCGAGGACAAGCACAAAGAGCTGTTCGACGA GCTGGTCAAGAAGTTCAAGGTCGAGTACCACGCTGGCGGCAG CACCCAGAACTCTATGAAGGTTGCCCAGTGGCTGATCCAAGA GCCTCACAAGGCCGCCACATTCTTCGGCTGTATCGGCATCGAC AAGTTCGGCGAGATCCTGAAGAGAAAGGCCGCCGATGCTCAC GTGGACGCCCACTACTATGAGCAGAACGAGCAGCCTACAGGC ACCTGTGCCGCTTGTATCACCGGCGGCAATAGAAGCCTGGTG GCCAATCTGGCCGCTGCCAACTGCTACAAGAAAGAGAAGCAC CTGGACCTGGAACGGAACTGGGTGCTCGTGGAAAAGGCCAGA GTGTACTATATCGCCGGATTCTTCCTGACCGTGTCTCCCGAGT CTGTGCTGAAGGTGGCCAGATACGCCGCCGAGAACAACAGAG TGTTCACCCTGAACCTGAGCGCCCCTTTCATCAGCCAGTTCTT CAAAGAGGCCCTGATGGACGTGATGCCCTACGTGGACATCCT GTTTGGCAACGAGACAGAGGCCGCTACCTTCGCTAGAGAGCA GGGCTTCGAGACAAAGGACATCAAAGAGATCGCCAAGAAGG CCCAGGCTCTGCCCAAAGTGAACAGCAAGAGGCAGAGAACC GTGATCTTCACCCAGGGCAGAGATGACACAATCGTGGCTGCC GAGAACGACGTGACCGCCTTTCCTGTGCTGGACCAGAATCAA GAGGAAATCATCGACACAAACGGCGCTGGCGACGCTTTCGTC GGCGGATTTCTGTCTCAGCTGGTGTCCGACAAGCCCCTGACCG AGTGTATCAGAGCTGGCCACTATGCCGCCTCCGTGATCATCAGAttorney Docket No: 030258-000107WOPTAAGAACCGGCTGCACATTCCCCGAGAAGCCCGACTTTCACTG AGGTACCACCCAGCTTTCTTGTACAAAGTGGTGATGGCCGGC CGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGAC AAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTG AAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTG CAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTT CAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAA AACCTCTACAAATGTGGTATTAATTAAGAACGGATCCGGACC CGGGAGGCCTCCTTTGAGGAGTGGCTGCGATCCAAGGAAGTG GCCCTGGACTTTGGCCTGACGGAAAGGCTTCGCGAGCACGAA GCCCAGCTGGTGATCCTGGCCCAGGCTCTGGACCATTACGACT GTCTGATCCACAGCACACCGCACACGCTGGTCGAGCGGGGGC TGCAATCGGCCCTGAAGTATGAGGAGTTTTACCTAAAGCGCTT TGGCGGGCACTACATGGAGTCCGTCTTCCAGATGTACACCCG CATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGCGGCATGCGC CACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGAAATGTTC AAGTTCTTTTTCCACCGCCTCTACGACCACCAGATCGTACCGT CGACCCCCGCCATGCTGAACCTGGGGACCCGCAACTACTACA CCTCCAGCTGCTACCTGGTAAACCCCCAGGCCACCACAAACA AGGCGACCCTGCGGGCCATCACCAGCAACGTCAGCGCCATCC TCGCCCGCAACGGGGGCATCGGGCTATGCGTGCAGGCGTTTA ACGACTCCGGCCCCGGGACCGCTAGCGTCATACCCGCCCTCA AGGTCCTCGACTCGCTGGTGGCGGCGCACAACAAAGAGAGCG CGCGTCCAACCGGCGCGTGCGTGTACCTGGAGCCGTGGCACA CCGACGTGCGGGCCGTGCTCCGGATGAAGGGGGTCCTCGCCG GCGAAGAGGCCCAGCGCTGCGACAATATCTTCAGCGCCCTCT GGATGCCAGACCTGTTTTTCAAGCGCCTGATTCGCCACCTGGA CGGCGAGAAGAACGTCACATGGACCCTGTTCGACCGGGACAC CAGCATGTCGCTCGCCGACTTTCACGGGGAGGAGTTCGAGAA GCTCTACCAGCACCTCGACTCTAGAGGGFragment from CGAAACCCGCCGCGTCTGTTGAAATGGCCAGCCGCCCCGCCG Plasmid pJS- CATCCTCTCCCGTCGAAGCGCGGGCCCCGGTTGGGGGACAGG ICP6-GFP- AGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAGGCCGCC CMVB-mADKS GGGGCCCCTCTCGCCCGCGGCCACCACGTGTACTGCCAGCGAGTCAATGGCGTGATGGTGCTTTCCGACAAGACGCCCGGGTCC46Attorney Docket No: 030258-000107WOPTPositions 24- GCGTCCTACCGCATCAGCGATAGCAACTTTGTCCAATGTGGTT 1181: ICP65’ CCAACTGCACCATGATCATAGACGGAGACGTGGTGCGCGGGC GCCCCCAGGACCCGGGGGCCGCGGCATCCCCCGCTCCCTTCGPositions 1203- TTGCGGTGACAAACATCGGAGCCGGCAGCGACGGCGGGACCG1922: GFPCCGTCGTGGCATTCGGGGGAACCCCACGTCGCTCGGCGGGGAPositions 2074- CGTCTACCGGTACCCAGACGACCGACGTCCCCACCGAGGCCC 2128: betaTTGGGGGCCCCCCTCCTCCTCCCCGCTTCACCCTGGGTGGCGG globin poly (A) CTGTTGTTCCTGTCGCGACACACGGCGCCGCTCTGCGGTATTC signal GGGGGGGAGGGGGATCCCGTCGGCCCCGCGGAGTTCGTCTCG GACGACCGGTCGTCCGATTCCGACTCGGATGACTCGGAGGACPositions 2499- ACCGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTCC 3184: CMV GGCGGGGCCACGTACTCAGCGGTAGACCCACACGCACCGACG enhancer B CCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCCGCCGTGGCC CGGGACGACGCGGAGGGGCTTTCGGACCCCCGGCCACGTCTGPositions 3198- GGAACGGGCACGGCCTACCCCGTCCCCCTGGAACTCACGCCC3256: Human GAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGAGATGCCGTG IL-2 signalAACCGCGAACCCGCGCTCATGCTGGAGTACTTTTGCCGGTGCpeptide, codon GCCCGCGAGGAAACCAAGCGTGTCCCCCCCAGGACATTCTGCoptimized for AGCCCCCCTCGCCTCACGGAGGACGACTTTGGGCTTCTCAACTmus musculus ACGCGCTCGTGGAGATGCAGCGCCTGTGTCTGGACGTTCCTCCPositions 3257- GGTCCCGCCGAACGCATACATGCCCTATTATCTCAGGGAGTAT 4291: Murine GTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGGTGAGCCGG codon optimized TCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGC ADK isoform S GGATCCTATCTGCGATCGCAATGGTGAGCAAGGGCGAGGAGC (short) TGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCG ACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGPositions 4351- GCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCA 4472: SV40 CCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCA poly(A) signal CCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCA CATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGPositions 4576- CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAA5492: ICP63’CTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCT GGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGA CGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAG CCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCAT CAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAG47Attorney Docket No: 030258-000107WOPTCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGG CGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCAC CCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTC GGCATGGACGAGCTGTACAAGTAAAGCGGCCGCACTCCTCAG GTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAAT GCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCA AAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTT CTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTG GAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCA AATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGG CAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGC TATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCC ATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTT TTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAA ATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCT GACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC CCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGAAACGCGT TGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGG GGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATA ACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCC CCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACG CCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTAC GGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC CTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACT TGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGA TGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTG ACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGG GAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGT CGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGT GTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACT AGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTC ACTATAGGGAGACCCAAGCTTACCGGCGAAGGAGGGCCACCG GATCCCTCGAGGCCACCATGTACAGAATGCAGCTGCTGAGCT GTATCGCCCTGTCTCTGGCCCTGGTCACCAACAGCACAAGCACAttorney Docket No: 030258-000107WOPTCTCTGAGAACGTGCTGTTCGGCATGGGCAACCCTCTGCTGGAT ATCAGCGCCGTGGTGGACAAGGACTTCCTGGACAAGTACAGC CTGAAGCCTAACGACCAGATCCTGGCCGAGGACAAGCACAAA GAGCTGTTCGACGAGCTGGTCAAGAAGTTCAAGGTCGAGTAC CACGCTGGCGGCAGCACCCAGAACTCTATGAAGGTTGCCCAG TGGCTGATCCAAGAGCCTCACAAGGCCGCCACATTCTTCGGCT GTATCGGCATCGACAAGTTCGGCGAGATCCTGAAGAGAAAGG CCGCCGATGCTCACGTGGACGCCCACTACTATGAGCAGAACG AGCAGCCTACAGGCACCTGTGCCGCTTGTATCACCGGCGGCA ATAGAAGCCTGGTGGCCAATCTGGCCGCTGCCAACTGCTACA AGAAAGAGAAGCACCTGGACCTGGAACGGAACTGGGTGCTC GTGGAAAAGGCCAGAGTGTACTATATCGCCGGATTCTTCCTG ACCGTGTCTCCCGAGTCTGTGCTGAAGGTGGCCAGATACGCC GCCGAGAACAACAGAGTGTTCACCCTGAACCTGAGCGCCCCT TTCATCAGCCAGTTCTTCAAAGAGGCCCTGATGGACGTGATGC CCTACGTGGACATCCTGTTTGGCAACGAGACAGAGGCCGCTA CCTTCGCTAGAGAGCAGGGCTTCGAGACAAAGGACATCAAAG AGATCGCCAAGAAGGCCCAGGCTCTGCCCAAAGTGAACAGCA AGAGGCAGAGAACCGTGATCTTCACCCAGGGCAGAGATGACA CAATCGTGGCTGCCGAGAACGACGTGACCGCCTTTCCTGTGCT GGACCAGAATCAAGAGGAAATCATCGACACAAACGGCGCTG GCGACGCTTTCGTCGGCGGATTTCTGTCTCAGCTGGTGTCCGA CAAGCCCCTGACCGAGTGTATCAGAGCTGGCCACTATGCCGC CTCCGTGATCATCAGAAGAACCGGCTGCACATTCCCCGAGAA GCCCGACTTTCACTGAGGTACCACCCAGCTTTCTTGTACAAAG TGGTGATGGCCGGCCGCTTCGAGCAGACATGATAAGATACAT TGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAA ATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAA CCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCA TTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTT TTAAAGCAAGTAAAACCTCTACAAATGTGGTATTAATTAAGA ACGGATCCGGACCCGGGAGGCCTCCTTTGAGGAGTGGCTGCG ATCCAAGGAAGTGGCCCTGGACTTTGGCCTGACGGAAAGGCT TCGCGAGCACGAAGCCCAGCTGGTGATCCTGGCCCAGGCTCT GGACCATTACGACTGTCTGATCCACAGCACACCGCACACGCT GGTCGAGCGGGGGCTGCAATCGGCCCTGAAGTATGAGGAGTTAttorney Docket No: 030258-000107WOPTTTACCTAAAGCGCTTTGGCGGGCACTACATGGAGTCCGTCTTC CAGATGTACACCCGCATCGCCGGCTTTTTGGCCTGCCGGGCCA CGCGCGGCATGCGCCACATCGCCCTGGGGCGAGAGGGGTCGT GGTGGGAAATGTTCAAGTTCTTTTTCCACCGCCTCTACGACCA CCAGATCGTACCGTCGACCCCCGCCATGCTGAACCTGGGGAC CCGCAACTACTACACCTCCAGCTGCTACCTGGTAAACCCCCAG GCCACCACAAACAAGGCGACCCTGCGGGCCATCACCAGCAAC GTCAGCGCCATCCTCGCCCGCAACGGGGGCATCGGGCTATGC GTGCAGGCGTTTAACGACTCCGGCCCCGGGACCGCTAGCGTC ATACCCGCCCTCAAGGTCCTCGACTCGCTGGTGGCGGCGCAC AACAAAGAGAGCGCGCGTCCAACCGGCGCGTGCGTGTACCTG GAGCCGTGGCACACCGACGTGCGGGCCGTGCTCCGGATGAAG GGGGTCCTCGCCGGCGAAGAGGCCCAGCGCTGCGACAATATC TTCAGCGCCCTCTGGATGCCAGACCTGTTTTTCAAGCGCCTGA TTCGCCACCTGGACGGCGAGAAGAACGTCACATGGACCCTGT TCGACCGGGACACCAGCATGTCGCTCGCCGACTTTCACGGGG AGGAGTTCGAGAAGCTCTACCAGCACCTCGACTCTAGAGGGFragment from GAATCTCGACCCCACCACTCGCCGGACCCGCCGACGTCCCCTC G47A-CMVB- TGGAGCCCGCCGAAACCCGCCGCGTCTGTTGAAATGGCCAGC mADAl CGCCCAGCCGCATCCTCTCCCGTCGAAGCGCGGGCCCCG GTTGGGGGACAGGAGGCCGGCGGCCCCAGCGCAGCCACCPositions 77- CAGGGGGAGGCCGCCGGGGCCCCTCTCGCCCACGGCCAC1234: ICP65’CACGTGTACTGCCAGCGAGTCAATGGCGTGATGGTGCTTTPositions 1256- CCAACAAGACGCCCGGGTCCGCGTCCTACCGCATCAGCGA 1975: GFP TAGCAACTTTGTCCAATGTGGTTCCAACTGCACCATGATC ATAGACGGAGACGTGGTGCGCGGGCGCCCCCAGGACCCGPositions 2127- GGGGCCGCGGCATCCCCCGCTCCCTTCGTTGCGGTGACAA 2181: betaACATCGGAGCCGGCAGCGACGGCGGGACCGCCGTCGTGG globin poly (A) CATTCGGGGGAACCCCACGTCGCTCGGCGGGGACGTCTA signal CCGGTACCCAGACGACCGACGTCCCCACCGAGGCCCTTG GGGGCCCCCCTCCTCCTCCCCGCTTCACCCTGGGTGGCGGPositions 2552- CTGTTGTTCCTGTCGCGACACACGGCGCCGCTCTGCGGTA3135: CMVTTCGGGGGGGAGGGGGATCCCGTCGGCCCCGCGGAGTTCenhancer BGTCTCGGACGACCGGTCGTCCGATTCCGACTCGGATGACT CGGAGGACACCGACTCGGAGACGCTGTCACACGCCTCCTC50Attorney Docket No: 030258-000107WOPTPositions 3250- GGACGTGTCCGGCGGGGCCACGTACTCAGCGGTAGACCC 3312: Human ACACGCACCGACGCCAGGGGCCGGCGCTGGTCTTGCGGC IL-2 signal CGATCCCGCCGTGGCCCGGGACGACGCGGAGGGGCTTTC peptide, codon GGACCCCCGGCCACGTCTGGGAACGGGCACGGCCTACCC optimized for CGTCCCCCTGGAACTCACGCCCGAGAACGCGGAGGCCGT mus musculus GGCGCGCTTTCTGGGAGATGCCGTGAACCGCGAACCCGC GCTCATGCTGGAGTACTTTTGCCGGTGCGCCCGCGAGGAAPositions 3313- ACCAAGCGTGTCCCCCCCAGGACATTCTGCAGCCCCCCTC4365: Murine GCCTCACGGAGGACGACTTTGGGCTTCTCAACTACGCGCTcodon optimized CGTGGAGATGCAGCGCCTGTGTCTGGACGTTCCTCCGGTC ADA isoform 1CCGCCGAACGCATACATGCCCTATTATCTCAGGGAGTATGPositions 4425- TGACGCGGCTGGTCAACGGGTTCAAGCCGCTGGTGAGCC 4546: SV40 GGTCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGTGCA poly (A) signal CCTGCGGATCCTATCTGCGATCGCAATGGTGAGCAAGGGCG AGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTPositions 4650- GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGG 4691: ICP63’ CGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCT GAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGG CCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCT TCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTT CAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCAC CATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCC GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATC GAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATC CTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACG TCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGT GAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTG CAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCAC CCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATC ACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCA CTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTG TGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCC CTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCA TCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATA51Attorney Docket No: 030258-000107WOPTGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATG GGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTT AGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACA AAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCC CTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGG TTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAAC ATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCC TCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTA TGGAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATG AAACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATC AATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGC GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCA TAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG AGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA TCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA CCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGG ATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCC ATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAAC GGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGAC GCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATAT AAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTG GCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGC TTACCGGCGAAGGAGGGCCACCGGATCCCTCGAGGCCACCAT GTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCA CTTGTCACGAATTCGGCTCAGACACCAGCCTTCAACAAGCC CAAGGTGGAACTGCATGTGCACCTGGACGGCGCCATCAA GCCTGAGACAATCCTGTACTTCGGCAAGAAGAGAGGAATC GCCCTGCCTGCCGACACCGTGGAAGAACTGAGAAACATCA TCGGCATGGACAAGCCCCTGAGCCTGCCTGGCTTCCTGGC CAAGTTCGACTACTACATGCCTGTGATCGCCGGCTGCAGA GAGGCCATCAAGAGAATCGCCTACGAGTTCGTGGAAATGA AGGCCAAAGAAGGCGTGGTCTACGTCGAAGTGCGGTACA GCCCTCATCTGCTGGCCAACTCTAAGGTGGACCCCATGCCAttorney Docket No: 030258-000107WOPTTTGGAACCAGACAGAGGGCGACGTGACACCTGACGACGT GGTGGATCTGGTCAACCAGGGACTGCAAGAGGGCGAGCA GGCCTTCGGAATCAAAGTGCGGAGCATCCTGTGCTGCATG AGGCACCAACCATCTTGGAGCCTGGAAGTGCTGGAACTGT GCAAGAAGTACAACCAGAAAACCGTGGTGGCCATGGACCT GGCTGGCGACGAAACAATCGAGGGCAGCTCTCTGTTCCCC GGCCACGTGGAAGCTTATGAGGGCGCTGTGAAGAACGGC ATCCACAGAACAGTGCACGCTGGCGAAGTGGGCTCTCCTG AGGTTGTCAGAGAAGCCGTGGACATCCTGAAAACCGAGA GAGTCGGCCACGGCTACCACACCATCGAGGATGAGGCCC TGTACAACAGACTGCTGAAAGAAAACATGCACTTTGAAGT GTGCCCCTGGTCCAGCTACCTGACAGGCGCTTGGGACCCT AAGACAACACACGCTGTCGTGCGGTTCAAGAACGACAAGG CCAACTACAGCCTGAACACAGACGACCCTCTGATCTTCAA GAGCACCCTGGACACCGACTACCAGATGACCAAGAAAGAC ATGGGCTTCACCGAGGAAGAGTTCAAGAGGCTGAACATCA ACGCCGCCAAGAGCAGCTTCCTGCCTGAGGAAGAGAAGA AAGAGCTGCTGGAAAGGCTGTACAGAGAGTACCAGTAAGG TACCACCCAGCTTTCTTGTACAAAGTGGTGATGGCCGGCCGCT TCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAAC CACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAAT TTGTGATGCTATTGCTTTATTTGTAACCATTATAGCTGCAATA AACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGT TCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCT CTACAAATGTGGTATTAATTAAGAACGGATCCGGACCCGGG AGGCCTCCTTTGAGGAGTGGCTGCGATFragment from GAATCTCGACCCCACCACTCGCCGGACCCGCCGACGTCCCCTC G47A-CMVB- TGGAGCCCGCCGAAACCCGCCGCGTCTGTTGAAATGGCCAGC mADKS CGCCCAGCCGCATCCTCTCCCGTCGAAGCGCGGGCCCCGGTT GGGGGACAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGGPositions 77- GGAGGCCGCCGGGGCCCCTCTCGCCCACGGCCACCACGTGTA1234: ICP65’CTGCCAGCGAGTCAATGGCGTGATGGTGCTTTCCAACAAGACPositions 1256- GCCCGGGTCCGCGTCCTACCGCATCAGCGATAGCAACTTTGTC 1975: GFP CAATGTGGTTCCAACTGCACCATGATCATAGACGGAGACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCGGCATCCCCC53Attorney Docket No: 030258-000107WOPTPositions 2127- GCTCCCTTCGTTGCGGTGACAAACATCGGAGCCGGCAGCGAC 2181: betaGGCGGGACCGCCGTCGTGGCATTCGGGGGAACCCCACGTCGC globin poly (A) TCGGCGGGGACGTCTACCGGTACCCAGACGACCGACGTCCCC signal ACCGAGGCCCTTGGGGGCCCCCCTCCTCCTCCCCGCTTCACCC TGGGTGGCGGCTGTTGTTCCTGTCGCGACACACGGCGCCGCTCPositions 2552- TGCGGTATTCGGGGGGGAGGGGGATCCCGTCGGCCCCGCGGA3135: CMVGTTCGTCTCGGACGACCGGTCGTCCGATTCCGACTCGGATGACenhancer BTCGGAGGACACCGACTCGGAGACGCTGTCACACGCCTCCTCGPositions 3250- GACGTGTCCGGCGGGGCCACGTACTCAGCGGTAGACCCACAC 3309: Human GCACCGACGCCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCC IL-2 signal GCCGTGGCCCGGGACGACGCGGAGGGGCTTTCGGACCCCCGG peptide, codon CCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCCTGGAA optimized for CTCACACCCGAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGA mus musculus GATGCCGTGAACCGCGAACCCGCGCTCATGCTGGAGTACTTTT GCCGGTGCGCCCGCGAGGAAACCAAGCGTGTCCCCCCCAGGAPositions 3310- CATTCTGCAGCCCCCCTCGCCTCACGGAGGACGACTTTGGGCT 4344: Murine TCTCAACTACGCGCTCGTGGAGATGCAGCGCCTGTGTCTGGAC codon optimized GTTCCTCCGGTCCCGCCGAACGCATACATGCCCTATTATCTCA ADK isoform S GGGAGTATGTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGG (short) TGAGCCGGTCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGT GCACCTGCGGATCCTATCTGCGATCGCAATGGTGAGCAAGGGPositions 4404- CGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT4525: SV40GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGApoly (A) signal GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTPositions 4629- CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTC 4670: ICP63’ GTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGC CCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACG ACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCG ACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACT ACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGA ACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACC TGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGC54Attorney Docket No: 030258-000107WOPTGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGA TCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCA CTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTG TGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCC CTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCA TCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATA GTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATG GGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTT AGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACA AAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCC CTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGG TTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAAC ATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCC TCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTA TGGAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATG AAACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATC AATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGC GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCA TAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG AGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA TCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA CCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGAT AGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGC GGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTC TGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTA ATACGACTCACTATAGGGAGACCCAAGCTTACCGGCGAAGGA GGGCCACCGGATCCCTCGAGGCCACCATGTACAGAATGCAGC TGCTGAGCTGTATCGCCCTGTCTCTGGCCCTGGTCACCAACAG CACAAGCACCTCTGAGAACGTGCTGTTCGGCATGGGCAACCC TCTGCTGGATATCAGCGCCGTGGTGGACAAGGACTTCCTGGA CAAGTACAGCCTGAAGCCTAACGACCAGATCCTGGCCGAGGAAttorney Docket No: 030258-000107WOPTCAAGCACAAAGAGCTGTTCGACGAGCTGGTCAAGAAGTTCAA GGTCGAGTACCACGCTGGCGGCAGCACCCAGAACTCTATGAA GGTTGCCCAGTGGCTGATCCAAGAGCCTCACAAGGCCGCCAC ATTCTTCGGCTGTATCGGCATCGACAAGTTCGGCGAGATCCTG AAGAGAAAGGCCGCCGATGCTCACGTGGACGCCCACTACTAT GAGCAGAACGAGCAGCCTACAGGCACCTGTGCCGCTTGTATC ACCGGCGGCAATAGAAGCCTGGTGGCCAATCTGGCCGCTGCC AACTGCTACAAGAAAGAGAAGCACCTGGACCTGGAACGGAA CTGGGTGCTCGTGGAAAAGGCCAGAGTGTACTATATCGCCGG ATTCTTCCTGACCGTGTCTCCCGAGTCTGTGCTGAAGGTGGCC AGATACGCCGCCGAGAACAACAGAGTGTTCACCCTGAACCTG AGCGCCCCTTTCATCAGCCAGTTCTTCAAAGAGGCCCTGATGG ACGTGATGCCCTACGTGGACATCCTGTTTGGCAACGAGACAG AGGCCGCTACCTTCGCTAGAGAGCAGGGCTTCGAGACAAAGG ACATCAAAGAGATCGCCAAGAAGGCCCAGGCTCTGCCCAAAG TGAACAGCAAGAGGCAGAGAACCGTGATCTTCACCCAGGGCA GAGATGACACAATCGTGGCTGCCGAGAACGACGTGACCGCCT TTCCTGTGCTGGACCAGAATCAAGAGGAAATCATCGACACAA ACGGCGCTGGCGACGCTTTCGTCGGCGGATTTCTGTCTCAGCT GGTGTCCGACAAGCCCCTGACCGAGTGTATCAGAGCTGGCCA CTATGCCGCCTCCGTGATCATCAGAAGAACCGGCTGCACATTC CCCGAGAAGCCCGACTTTCACTGAGGTACCACCCAGCTTTCTT GTACAAAGTGGTGATGGCCGGCCGCTTCGAGCAGACATGATA AGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAG TGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTT TATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACA ACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTG GGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATT AATTAAGAACGGATCCGGACCCGGGAGGCCTCCTTTGAGGAG TGGCTGCGATFragment from CGAAACCCGCCGCGTCTGTTGAAATGGCCAGCCGCCCCGCC Plasmid pJS- GCATCCTCTCCCGTCGAAGCGCGGGCCCCGGTTGGGGGA ICP6-GFP- CAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAG CMVA-hADKS GCCGCCGGGGCCCCTCTCGCCCGCGGCCACCACGTGTACTGCCAGCGAGTCAATGGCGTGATGGTGCTTTCCGACAAGA56Attorney Docket No: 030258-000107WOPTPositions 24- CGCCCGGGTCCGCGTCCTACCGCATCAGCGATAGCAACTT 1181: ICP65’ TGTCCAATGTGGTTCCAACTGCACCATGATCATAGACGGA GACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCGPositions 1203- GCATCCCCCGCTCCCTTCGTTGCGGTGACAAACATCGGAG1922: GFPCCGGCAGCGACGGCGGGACCGCCGTCGTGGCATTCGGGGPositions 2074- GAACCCCACGTCGCTCGGCGGGGACGTCTACCGGTACCC 2129: betaAGACGACCGACGTCCCCACCGAGGCCCTTGGGGGCCCCC globin poly (A) CTCCTCCTCCCCGCTTCACCCTGGGTGGCGGCTGTTGTTC signal CTGTCGCGACACACGGCGCCGCTCTGCGGTATTCGGGGG GGAGGGGGATCCCGTCGGCCCCGCGGAGTTCGTCTCGGAPositions 2500- CGACCGGTCGTCCGATTCCGACTCGGATGACTCGGAGGAC 3382: CMV ACCGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGT enhancer A CCGGCGGGGCCACGTACTCAGCGGTAGACCCACACGCAC CGACGCCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCCGPositions 3401- CCGTGGCCCGGGACGACGCGGAGGGGCTTTCGGACCCCC3460: Human GGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCC IL-2 signalTGGAACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCTpeptideTTCTGGGAGATGCCGTGAACCGCGAACCCGCGCTCATGCTPositions 3461- GGAGTACTTTTGCCGGTGCGCCCGCGAGGAAACCAAGCG 4494: Human TGTCCCCCCCAGGACATTCTGCAGCCCCCCTCGCCTCACG ADK isofomi GAGGACGACTTTGGGCTTCTCAACTACGCGCTCGTGGAGA short TGCAGCGCCTGTGTCTGGACGTTCCTCCGGTCCCGCCGAA CGCATACATGCCCTATTATCTCAGGGAGTATGTGACGCGGPositions 4555- CTGGTCAACGGGTTCAAGCCGCTGGTGAGCCGGTCCGCTC 4676: SV40 GCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGCGGAT poly (A) signal CCTATCTGCGATCGCAATGGTGAGCAAGGGCGAGGAGCTG TTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCPositions 4780- GACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC5695: ICP63’GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCA TCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT CGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGC TACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCG CCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAA GTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA57Attorney Docket No: 030258-000107WOPTCAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCG CCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCC CGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG GCATGGACGAGCTGTACAAGTAAAGCGGCCGCACTCCTCAG GTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAAT GCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCA AAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTT CTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTG GAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCA AATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGG CAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGC TATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCC ATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTT TTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAA ATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCT GACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC CCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGAAACGCGT GGATCCACATCATGAAGCCCCTTGAGCATCTGACTTCTGG CTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGA ATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGC AAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTT TGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGG TTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCT GCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAG GTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTT TAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGA TTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCC TCTTCTCTTATGGAGCTCCCCCCGCTCCTCCCCCCGCTCCT CCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCC CCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGC TCCTCCCCCCCCCGCTCCCGCGGCCCCGCCCCCAACGCCC GCTCCTCCCCCCGCTCCCGCGGCCCCGCCCCCAACGCCCGAttorney Docket No: 030258-000107WOPTCCGCGCGCGCGCACGCCGCCCTTGACTCACGGGGATTTCC AAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGG CACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACT CCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGT GGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGA ACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACT ATAGGGAGACCCAAGCTTACCGGCGAAGGAGGGCCACCG GATCCCTCGAGGCCACCATGTACAGGATGCAACTCCTGTCTTG CATTGCACTAAGTCTTGCACTTGTCACAAACAGTACGTCAGT CAGAGAAAATATTCTCTTTGGAATGGGAAATCCTCTGCTT GACATCTCTGCTGTAGTGGACAAAGATTTCCTTGATAAGT ATTCTCTGAAACCAAATGACCAAATCTTGGCTGAAGACAA ACACAAGGAACTGTTTGATGAACTTGTGAAAAAATTCAAA GTCGAATATCATGCTGGTGGCTCTACCCAGAATTCAATTA AAGTGGCTCAGTGGATGATTCAACAGCCACACAAAGCAGC AACATTTTTTGGATGCATTGGGATAGATAAATTTGGGGAG ATCCTGAAGAGAAAAGCTGCTGAAGCCCATGTGGATGCTC ATTACTACGAGCAGAATGAGCAGCCAACAGGAACTTGTGC TGCATGCATCACTGGTGACAACAGGTCCCTCATAGCTAAT CTTGCTGCTGCCAATTGTTATAAAAAGGAAAAACATCTTG ATCTGGAGAAAAACTGGATGTTGGTAGAAAAAGCAAGAGT TTGTTATATAGCAGGCTTTTTTCTTACAGTTTCCCCAGAGT CAGTATTAAAGGTGGCTCACCATGCTTCTGAAAACAACAG GATTTTCACTTTGAATCTATCTGCACCGTTTATTAGCCAGT TCTACAAGGAATCATTGATGAAAGTTATGCCTTATGTTGAT ATACTTTTTGGAAATGAGACAGAAGCTGCCACTTTTGCTA GAGAGCAAGGCTTTGAGACTAAAGACATTAAAGAGATAGC CAAAAAGACACAAGCCCTGCCAAAGATGAACTCAAAGAGG CAGCGAATCGTGATCTTCACCCAAGGGAGAGATGACACTA TAATGGCTACAGAAAGTGAAGTCACTGCTTTTGCTGTCTT GGATCAAGACCAGAAAGAAATTATTGATACCAATGGAGCT GGAGATGCATTTGTTGGAGGTTTTCTGTCTCAACTGGTCT CTGACAAGCCTCTGACTGAATGTATCCGTGCTGGCCACTA TGCAGCAAGCATCATAATTAGACGGACTGGCTGCACCTTT CCTGAGAAGCCAGACTTCCACTGAGGTACCACCCAGCTTTC TTGTACAAAGTGGTGATGGCCGGCCGCTTCGAGCAGACATGAAttorney Docket No: 030258-000107WOPTTAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGC AGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGC TTT ATTTGTAACC ATTAT AAGCTGC AATAAACAAGTTA AC A AC AACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGT GGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA TTAATTAAGAACGGATCCGGACCCGGGAGGCCTCCTTTGA GGAGTGGCTGCGATCCAAGGAAGTGGCCCTGGACTTTGG CCTGACGGAAAGGCTTCGCGAGCACGAAGCCCAGCTGGT GATCCTGGCCCAGGCTCTGGACCATTACGACTGTCTGATC CACAGCACACCGCACACGCTGGTCGAGCGGGGGCTGCAA TCGGCCCTGAAGTATGAGGAGTTTTACCTAAAGCGCTTTG GCGGGCACTACATGGAGTCCGTCTTCCAGATGTACACCCG CATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGCGGCATG CGCCACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGAA ATGTTCAAGTTCTTTTTCCACCGCCTCTACGACCACCAGAT CGTACCGTCGACCCCCGCCATGCTGAACCTGGGGACCCGC AACTACTACACCTCCAGCTGCTACCTGGTAAACCCCCAGG CCACCACAAACAAGGCGACCCTGCGGGCCATCACCAGCAA CGTCAGCGCCATCCTCGCCCGCAACGGGGGCATCGGGCT ATGCGTGCAGGCGTTTAACGACTCCGGCCCCGGGACCGCT AGCGTCATACCCGCCCTCAAGGTCCTCGACTCGCTGGTGG CGGCGCACAACAAAGAGAGCGCGCGTCCAACCGGCGCGT GCGTGTACCTGGAGCCGTGGCACACCGACGTGCGGGCCG TGCTCCGGATGAAGGGGGTCCTCGCCGGCGAAGAGGCCC AGCGCTGCGACAATATCTTCAGCGCCCTCTGGATGCCAGA CCTGTTTTTCAAGCGCCTGATTCGCCACCTGGACGGCGAG AAGAACGTCACATGGACCCTGTTCGACCGGGACACCAGCA TGTCGCTCGCCGACTTTCACGGGGAGGAGTTCGAGAAGCT CTACCAGCACCTCGACTCTAGAGGGFragment from CGAAACCCGCCGCGTCTGTTGAAATGGCCAGCCGCCCCGCCG Plasmid pJS- CATCCTCTCCCGTCGAAGCGCGGGCCCCGGTTGGGGGACAGG ICP6-GFP- AGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAGGCCGCC CMVB-hADAl GGGGCCCCTCTCGCCCGCGGCCACCACGTGTACTGCCAGCGA GTCAATGGCGTGATGGTGCTTTCCGACAAGACGCCCGGGTCC GCGTCCTACCGCATCAGCGATAGCAACTTTGTCCAATGTGGTT60Attorney Docket No: 030258-000107WOPTPositions 24- CCAACTGCACCATGATCATAGACGGAGACGTGGTGCGCGGGC 1181: ICP65’ GCCCCCAGGACCCGGGGGCCGCGGCATCCCCCGCTCCCTTCG TTGCGGTGACAAACATCGGAGCCGGCAGCGACGGCGGGACCGPositions 1203- CCGTCGTGGCATTCGGGGGAACCCCACGTCGCTCGGCGGGGA1922: GFPCGTCTACCGGTACCCAGACGACCGACGTCCCCACCGAGGCCCPositions 2074- TTGGGGGCCCCCCTCCTCCTCCCCGCTTCACCCTGGGTGGCGG 2129: betaCTGTTGTTCCTGTCGCGACACACGGCGCCGCTCTGCGGTATTC globin poly (A) GGGGGGGAGGGGGATCCCGTCGGCCCCGCGGAGTTCGTCTCG signal GACGACCGGTCGTCCGATTCCGACTCGGATGACTCGGAGGAC ACCGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTCCPositions 2499- GGCGGGGCCACGTACTCAGCGGTAGACCCACACGCACCGACG 3184: CMV CCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCCGCCGTGGCC enhancer B CGGGACGACGCGGAGGGGCTTTCGGACCCCCGGCCACGTCTG GGAACGGGCACGGCCTACCCCGTCCCCCTGGAACTCACGCCCPositions 3197- GAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGAGATGCCGTG3256: HumanAACCGCGAACCCGCGCTCATGCTGGAGTACTTTTGCCGGTGC IL-2 signalGCCCGCGAGGAAACCAAGCGTGTCCCCCCCAGGACATTCTGCpeptideAGCCCCCCTCGCCTCACGGAGGACGACTTTGGGCTTCTCAACTPositions 3257- ACGCGCTCGTGGAGATGCAGCGCCTGTGTCTGGACGTTCCTCC 4345: human GGTCCCGCCGAACGCATACATGCCCTATTATCTCAGGGAGTAT ADA1 isoform 1 GTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGGTGAGCCGG TCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGCPositions 4405- GGATCCTATCTGCGATCGCAATGGTGAGCAAGGGCGAGGAGC 4526: SV40 TGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCG poly(A) signal ACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGG GCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAPositions 4630- CCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCA5545: ICP63’CCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCA CATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAA CTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCT GGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGA CGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAG CCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCAT CAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAG CGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGG61Attorney Docket No: 030258-000107WOPTCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCAC CCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTC GGCATGGACGAGCTGTACAAGTAAAGCGGCCGCACTCCTCAG GTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAAT GCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCA AAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTT CTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTG GAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCA AATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGG CAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGC TATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCC ATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTT TTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAA ATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCT GACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC CCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGAAACGCGT TGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGG GGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATA ACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCC CCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACG CCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTAC GGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC CTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACT TGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGA TGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTG ACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGG GAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGT CGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGT GTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACT AGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTC ACTATAGGGAGACCCAAGCTTACCGGCGAAGGAGGGCCACCG GATCCCTCGAGGCCACCATGTACAGGATGCAACTCCTGTCTTG CATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCCCAGACG CCCGCCTTCGACAAGCCCAAAGTGGAACTGCATGTCCACCTAAttorney Docket No: 030258-000107WOPTGACGGATCCATCAAGCCTGAAACCATCTTATACTATGGCAGG AGGAGAGGGATCGCCCTCCCAGCTAACACAGCAGAGGGGCTG CTGAACGTCATTGGCATGGACAAGCCGCTCACCCTTCCAGACT TCCTGGCCAAGTTTGACTACTACATGCCTGCTATCGCGGGCTG CCGGGAGGCTATCAAAAGGATCGCCTATGAGTTTGTAGAGAT GAAGGCCAAAGAGGGCGTGGTGTATGTGGAGGTGCGGTACA GTCCGCACCTGCTGGCCAACTCCAAAGTGGAGCCAATCCCCT GGAACCAGGCTGAAGGGGACCTCACCCCAGACGAGGTGGTG GCCCTAGTGGGCCAGGGCCTGCAGGAGGGGGAGCGAGACTTC GGGGTCAAGGCCCGGTCCATCCTGTGCTGCATGCGCCACCAG CCCAACTGGTCCCCCAAGGTGGTGGAGCTGTGTAAGAAGTAC CAGCAGCAGACCGTGGTAGCCATTGACCTGGCTGGAGATGAG ACCATCCCAGGAAGCAGCCTCTTGCCTGGACATGTCCAGGCC TACCAGGAGGCTGTGAAGAGCGGCATTCACCGTACTGTCCAC GCCGGGGAGGTGGGCTCGGCCGAAGTAGTAAAAGAGGCTGT GGACATACTCAAGACAGAGCGGCTGGGACACGGCTACCACAC CCTGGAAGACCAGGCCCTTTATAACAGGCTGCGGCAGGAAAA CATGCACTTCGAGATCTGCCCCTGGTCCAGCTACCTCACTGGT GCCTGGAAGCCGGACACGGAGCATGCAGTCATTCGGCTCAAA AATGACCAGGCTAACTACTCGCTCAACACAGATGACCCGCTC ATCTTCAAGTCCACCCTGGACACTGATTACCAGATGACCAAA CGGGACATGGGCTTTACTGAAGAGGAGTTTAAAAGGCTGAAC ATCAATGCGGCCAAATCTAGTTTCCTCCCAGAAGATGAAAAG AGGGAGCTTCTCGACCTGCTCTATAAAGCCTATGGGATGCCA CCTTCAGCCTCTGCAGGGCAGAACCTCTGAGGTACCACCCAG CTTTCTTGTACAAAGTGGTGATGGCCGGCCGCTTCGAGCAGAC ATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGA ATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTA TTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAA CAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAG GTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGT GGTATTAATTAAGAACGGATCCGGACCCGGGAGGCCTCCTTT GAGGAGTGGCTGCGATCCAAGGAAGTGGCCCTGGACTTTGGC CTGACGGAAAGGCTTCGCGAGCACGAAGCCCAGCTGGTGATC CTGGCCCAGGCTCTGGACCATTACGACTGTCTGATCCACAGCA CACCGCACACGCTGGTCGAGCGGGGGCTGCAATCGGCCCTGAAttorney Docket No: 030258-000107WOPTAGTATGAGGAGTTTTACCTAAAGCGCTTTGGCGGGCACTACA TGGAGTCCGTCTTCCAGATGTACACCCGCATCGCCGGCTTTTT GGCCTGCCGGGCCACGCGCGGCATGCGCCACATCGCCCTGGG GCGAGAGGGGTCGTGGTGGGAAATGTTCAAGTTCTTTTTCCAC CGCCTCTACGACCACCAGATCGTACCGTCGACCCCCGCCATGC TGAACCTGGGGACCCGCAACTACTACACCTCCAGCTGCTACCT GGTAAACCCCCAGGCCACCACAAACAAGGCGACCCTGCGGGC CATCACCAGCAACGTCAGCGCCATCCTCGCCCGCAACGGGGG CATCGGGCTATGCGTGCAGGCGTTTAACGACTCCGGCCCCGG GACCGCTAGCGTCATACCCGCCCTCAAGGTCCTCGACTCGCTG GTGGCGGCGCACAACAAAGAGAGCGCGCGTCCAACCGGCGC GTGCGTGTACCTGGAGCCGTGGCACACCGACGTGCGGGCCGT GCTCCGGATGAAGGGGGTCCTCGCCGGCGAAGAGGCCCAGCG CTGCGACAATATCTTCAGCGCCCTCTGGATGCCAGACCTGTTT TTCAAGCGCCTGATTCGCCACCTGGACGGCGAGAAGAACGTC ACATGGACCCTGTTCGACCGGGACACCAGCATGTCGCTCGCC GACTTTCACGGGGAGGAGTTCGAGAAGCTCTACCAGCACCTC GACTCTAGAGGGHuman IL-2 ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTG signal peptide CACTTGTCACAAACAGTmADKS ACAAGCACCTCTGAGAACGTGCTGTTCGGCATGGGCAACCCT CTGCTGGATATCAGCGCCGTGGTGGACAAGGACTTCCTGGAC AAGTACAGCCTGAAGCCTAACGACCAGATCCTGGCCGAGGAC AAGCACAAAGAGCTGTTCGACGAGCTGGTCAAGAAGTTCAAG GTCGAGTACCACGCTGGCGGCAGCACCCAGAACTCTATGAAG GTTGCCCAGTGGCTGATCCAAGAGCCTCACAAGGCCGCCACA TTCTTCGGCTGTATCGGCATCGACAAGTTCGGCGAGATCCTGA AGAGAAAGGCCGCCGATGCTCACGTGGACGCCCACTACTATG AGCAGAACGAGCAGCCTACAGGCACCTGTGCCGCTTGTATCA CCGGCGGCAATAGAAGCCTGGTGGCCAATCTGGCCGCTGCCA ACTGCTACAAGAAAGAGAAGCACCTGGACCTGGAACGGAACT GGGTGCTCGTGGAAAAGGCCAGAGTGTACTATATCGCCGGAT TCTTCCTGACCGTGTCTCCCGAGTCTGTGCTGAAGGTGGCCAG ATACGCCGCCGAGAACAACAGAGTGTTCACCCTGAACCTGAG64Attorney Docket No: 030258-000107WOPTCGCCCCTTTCATCAGCCAGTTCTTCAAAGAGGCCCTGATGGAC GTGATGCCCTACGTGGACATCCTGTTTGGCAACGAGACAGAG GCCGCTACCTTCGCTAGAGAGCAGGGCTTCGAGACAAAGGAC ATCAAAGAGATCGCCAAGAAGGCCCAGGCTCTGCCCAAAGTG AACAGCAAGAGGCAGAGAACCGTGATCTTCACCCAGGGCAG AGATGACACAATCGTGGCTGCCGAGAACGACGTGACCGCCTT TCCTGTGCTGGACCAGAATCAAGAGGAAATCATCGACACAAA CGGCGCTGGCGACGCTTTCGTCGGCGGATTTCTGTCTCAGCTG GTGTCCGACAAGCCCCTGACCGAGTGTATCAGAGCTGGCCAC TATGCCGCCTCCGTGATCATCAGAAGAACCGGCTGCACATTCC CCGAGAAGCCCGACTTTCACTGA SP+mADKS ATGTACAGAATGCAGCTGCTGAGCTGTATCGCCCTGTCTCTGG CCCTGGTCACCAACAGCACAAGCACCTCTGAGAACGTGCTGT TCGGCATGGGCAACCCTCTGCTGGATATCAGCGCCGTGGTGG ACAAGGACTTCCTGGACAAGTACAGCCTGAAGCCTAACGACC AGATCCTGGCCGAGGACAAGCACAAAGAGCTGTTCGACGAGC TGGTCAAGAAGTTCAAGGTCGAGTACCACGCTGGCGGCAGCA CCCAGAACTCTATGAAGGTTGCCCAGTGGCTGATCCAAGAGC CTCACAAGGCCGCCACATTCTTCGGCTGTATCGGCATCGACAA GTTCGGCGAGATCCTGAAGAGAAAGGCCGCCGATGCTCACGT GGACGCCCACTACTATGAGCAGAACGAGCAGCCTACAGGCAC CTGTGCCGCTTGTATCACCGGCGGCAATAGAAGCCTGGTGGC CAATCTGGCCGCTGCCAACTGCTACAAGAAAGAGAAGCACCT GGACCTGGAACGGAACTGGGTGCTCGTGGAAAAGGCCAGAGT GTACTATATCGCCGGATTCTTCCTGACCGTGTCTCCCGAGTCT GTGCTGAAGGTGGCCAGATACGCCGCCGAGAACAACAGAGTG TTCACCCTGAACCTGAGCGCCCCTTTCATCAGCCAGTTCTTCA AAGAGGCCCTGATGGACGTGATGCCCTACGTGGACATCCTGT TTGGCAACGAGACAGAGGCCGCTACCTTCGCTAGAGAGCAGG GCTTCGAGACAAAGGACATCAAAGAGATCGCCAAGAAGGCC CAGGCTCTGCCCAAAGTGAACAGCAAGAGGCAGAGAACCGT GATCTTCACCCAGGGCAGAGATGACACAATCGTGGCTGCCGA GAACGACGTGACCGCCTTTCCTGTGCTGGACCAGAATCAAGA GGAAATCATCGACACAAACGGCGCTGGCGACGCTTTCGTCGG CGGATTTCTGTCTCAGCTGGTGTCCGACAAGCCCCTGACCGAG65Attorney Docket No: 030258-000107WOPTTGTATCAGAGCTGGCCACTATGCCGCCTCCGTGATCATCAGAA GAACCGGCTGCACATTCCCCGAGAAGCCCGACTTTCACTGAmADAl (e.g.. CAGACACCAGCCTTCAACAAGCCCAAGGTGGAACTGCATGTG NM_001272052 CACCTGGACGGCGCCATCAAGCCTGAGACAATCCTGTACTTC ■2) GGCAAGAAGAGAGGAATCGCCCTGCCTGCCGACACCGTGGAA GAACTGAGAAACATCATCGGCATGGACAAGCCCCTGAGCCTG CCTGGCTTCCTGGCCAAGTTCGACTACTACATGCCTGTGATCG CCGGCTGCAGAGAGGCCATCAAGAGAATCGCCTACGAGTTCG TGGAAATGAAGGCCAAAGAAGGCGTGGTCTACGTCGAAGTGC GGTACAGCCCTCATCTGCTGGCCAACTCTAAGGTGGACCCCAT GCCTTGGAACCAGACAGAGGGCGACGTGACACCTGACGACGT GGTGGATCTGGTCAACCAGGGACTGCAAGAGGGCGAGCAGG CCTTCGGAATCAAAGTGCGGAGCATCCTGTGCTGCATGAGGC ACCAACCATCTTGGAGCCTGGAAGTGCTGGAACTGTGCAAGA AGTACAACCAGAAAACCGTGGTGGCCATGGACCTGGCTGGCG ACGAAACAATCGAGGGCAGCTCTCTGTTCCCCGGCCACGTGG AAGCTTATGAGGGCGCTGTGAAGAACGGCATCCACAGAACAG TGCACGCTGGCGAAGTGGGCTCTCCTGAGGTTGTCAGAGAAG CCGTGGACATCCTGAAAACCGAGAGAGTCGGCCACGGCTACC ACACCATCGAGGATGAGGCCCTGTACAACAGACTGCTGAAAG AAAACATGCACTTTGAAGTGTGCCCCTGGTCCAGCTACCTGAC AGGCGCTTGGGACCCTAAGACAACACACGCTGTCGTGCGGTT CAAGAACGACAAGGCCAACTACAGCCTGAACACAGACGACC CTCTGATCTTCAAGAGCACCCTGGACACCGACTACCAGATGA CCAAGAAAGACATGGGCTTCACCGAGGAAGAGTTCAAGAGG CTGAACATCAACGCCGCCAAGAGCAGCTTCCTGCCTGAGGAA GAGAAGAAAGAGCTGCTGGAAAGGCTGTACAGAGAGTACCA GTAA SP+mADAl ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTG CACTTGTCACGAATTCGGCTCAGACACCAGCCTTCAACAAGC CCAAGGTGGAACTGCATGTGCACCTGGACGGCGCCATCAAGC CTGAGACAATCCTGTACTTCGGCAAGAAGAGAGGAATCGCCC TGCCTGCCGACACCGTGGAAGAACTGAGAAACATCATCGGCA TGGACAAGCCCCTGAGCCTGCCTGGCTTCCTGGCCAAGTTCGA66Attorney Docket No: 030258-000107WOPTCTACTACATGCCTGTGATCGCCGGCTGCAGAGAGGCCATCAA GAGAATCGCCTACGAGTTCGTGGAAATGAAGGCCAAAGAAG GCGTGGTCTACGTCGAAGTGCGGTACAGCCCTCATCTGCTGGC CAACTCTAAGGTGGACCCCATGCCTTGGAACCAGACAGAGGG CGACGTGACACCTGACGACGTGGTGGATCTGGTCAACCAGGG ACTGCAAGAGGGCGAGCAGGCCTTCGGAATCAAAGTGCGGA GCATCCTGTGCTGCATGAGGCACCAACCATCTTGGAGCCTGG AAGTGCTGGAACTGTGCAAGAAGTACAACCAGAAAACCGTGG TGGCCATGGACCTGGCTGGCGACGAAACAATCGAGGGCAGCT CTCTGTTCCCCGGCCACGTGGAAGCTTATGAGGGCGCTGTGA AGAACGGCATCCACAGAACAGTGCACGCTGGCGAAGTGGGCT CTCCTGAGGTTGTCAGAGAAGCCGTGGACATCCTGAAAACCG AGAGAGTCGGCCACGGCTACCACACCATCGAGGATGAGGCCC TGTACAACAGACTGCTGAAAGAAAACATGCACTTTGAAGTGT GCCCCTGGTCCAGCTACCTGACAGGCGCTTGGGACCCTAAGA CAACACACGCTGTCGTGCGGTTCAAGAACGACAAGGCCAACT ACAGCCTGAACACAGACGACCCTCTGATCTTCAAGAGCACCC TGGACACCGACTACCAGATGACCAAGAAAGACATGGGCTTCA CCGAGGAAGAGTTCAAGAGGCTGAACATCAACGCCGCCAAG AGCAGCTTCCTGCCTGAGGAAGAGAAGAAAGAGCTGCTGGAA AGGCTGTACAGAGAGTACCAGTAAAmplicon GAATCTCGACCCCACCACTCGCCGGACCCGCCGACGTCCCCTC sequencing TGGAGCCCGCCGAAACCCGCCGCGTCTGTTGAAATGGCCAGC G47A-CMV-B CGCCCAGCCGCATCCTCTCCCGTCGAAGCGCGGGCCCCGGTT mADAl clone GGGGGACAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGG 2-A4-A4-F3 GGAGGCCGCCGGGGCCCCTCTCGCCCACGGCCACCACGTGTA CTGCCAGCGAGTCAATGGCGTGATGGTGCTTTCCAACAAGAC GCCCGGGTCCGCGTCCTACCGCATCAGCGATAGCAACTTTGTC CAATGTGGTTCCAACTGCACCATGATCATAGACGGAGACGTG GTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCGGCATCCCCC GCTCCCTTCGTTGCGGTGACAAACATCGGAGCCGGCAGCGAC GGCGGGACCGCCGTCGTGGCATTCGGGGGAACCCCACGTCGC TCGGCGGGGACGTCTACCGGTACCCAGACGACCGACGTCCCC ACCGAGGCCCTTGGGGGCCCCCCTCCTCCTCCCCGCTTCACCC TGGGTGGCGGCTGTTGTTCCTGTCGCGACACACGGCGCCGCTC67Attorney Docket No: 030258-000107WOPTTGCGGTATTCGGGGGGGAGGGGGATCCCGTCGGCCCCGCGGA GTTCGTCTCGGACGACCGGTCGTCCGATTCCGACTCGGATGAC TCGGAGGACACCGACTCGGAGACGCTGTCACACGCCTCCTCG GACGTGTCCGGCGGGGCCACGTACTCAGCGGTAGACCCACAC GCACCGACGCCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCC GCCGTGGCCCGGGACGACGCGGAGGGGCTTTCGGACCCCCGG CCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCCTGGAA CTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGA GATGCCGTGAACCGCGAACCCGCGCTCATGCTGGAGTACTTTT GCCGGTGCGCCCGCGAGGAAACCAAGCGTGTCCCCCCCAGGA CATTCTGCAGCCCCCCTCGCCTCACGGAGGACGACTTTGGGCT TCTCAACTACGCGCTCGTGGAGATGCAGCGCCTGTGTCTGGAC GTTCCTCCGGTCCCGCCGAACGCATACATGCCCTATTATCTCA GGGAGTATGTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGG TGAGCCGGTCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGT GCACCTGCGGATCCTATCTGCGATCGCAATGGTGAGCAAGGG CGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGA GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTC GTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGC CCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACG ACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCG ACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACT ACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGA ACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACC TGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGC GCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGA TCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCA CTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTG TGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCC CTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCAAttorney Docket No: 030258-000107WOPTTCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATA GTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATG GGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTT AGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACA AAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCC CTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGG TTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAAC ATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCC TCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTA TGGAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATG AAACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATC AATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGC GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCA TAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG AGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA TCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA CCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGAT AGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGC GGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTC TGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTA ATACGACTCACTATAGGGAGACCCAAGCTTACCGGCGAAGGA GGGCCACCGGATCCCTCGAGGCCACCATGTACAGGATGCAAC TCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTC GGCTCAGACACCAGCCTTCAACAAGCCCAAGGTGGAACTGCA TGTGCACCTGGACGGCGCCATCAAGCCTGAGACAATCCTGTA CTTCGGCAAGAAGAGAGGAATCGCCCTGCCTGCCGACACCGT GGAAGAACTGAGAAACATCATCGGCATGGACAAGCCCCTGAG CCTGCCTGGCTTCCTGGCCAAGTTCGACTACTACATGCCTGTG ATCGCCGGCTGCAGAGAGGCCATCAAGAGAATCGCCTACGAG TTCGTGGAAATGAAGGCCAAAGAAGGCGTGGTCTACGTCGAA GTGCGGTACAGCCCTCATCTGCTGGCCAACTCTAAGGTGGACAttorney Docket No: 030258-000107WOPTCCCATGCCTTGGAACCAGACAGAGGGCGACGTGACACCTGAC GACGTGGTGGATCTGGTCAACCAGGGACTGCAAGAGGGCGAG CAGGCCTTCGGAATCAAAGTGCGGAGCATCCTGTGCTGCATG AGGCACCAACCATCTTGGAGCCTGGAAGTGCTGGAACTGTGC AAGAAGTACAACCAGAAAACCGTGGTGGCCATGGACCTGGCT GGCGACGAAACAATCGAGGGCAGCTCTCTGTTCCCCGGCCAC GTGGAAGCTTATGAGGGCGCTGTGAAGAACGGCATCCACAGA ACAGTGCACGCTGGCGAAGTGGGCTCTCCTGAGGTTGTCAGA GAAGCCGTGGACATCCTGAAAACCGAGAGAGTCGGCCACGGC TACCACACCATCGAGGATGAGGCCCTGTACAACAGACTGCTG AAAGAAAACATGCACTTTGAAGTGTGCCCCTGGTCCAGCTAC CTGACAGGCGCTTGGGACCCTAAGACAACACACGCTGTCGTG CGGTTCAAGAACGACAAGGCCAACTACAGCCTGAACACAGAC GACCCTCTGATCTTCAAGAGCACCCTGGACACCGACTACCAG ATGACCAAGAAAGACATGGGCTTCACCGAGGAAGAGTTCAAG AGGCTGAACATCAACGCCGCCAAGAGCAGCTTCCTGCCTGAG GAAGAGAAGAAAGAGCTGCTGGAAAGGCTGTACAGAGAGTA CCAGTAAGGTACCACCCAGCTTTCTTGTACAAAGTGGTGATG GCCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGT TTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTA TTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATA AGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTT ATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGC AAGTAAAACCTCTACAAATGTGGTATTAATTAAGAACGGATC CGGACCCGGGAGGCCTCCTTTGAGGAGTGGCTGCGATAmplicon GAATCTCGACCCCACCACTCGCCGGACCCGCCGACGTCCCCTC sequencing TGGAGCCCGCCGAAACCCGCCGCGTCTGTTGAAATGGCCAGC G47A-CMV-B CGCCCAGCCGCATCCTCTCCCGTCGAAGCGCGGGCCCCGGTT mADKS clone GGGGGACAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGG 4-E1-H3-B1 GGAGGCCGCCGGGGCCCCTCTCGCCCACGGCCACCACGTGTA CTGCCAGCGAGTCAATGGCGTGATGGTGCTTTCCAACAAGAC GCCCGGGTCCGCGTCCTACCGCATCAGCGATAGCAACTTTGTC CAATGTGGTTCCAACTGCACCATGATCATAGACGGAGACGTG GTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCGGCATCCCCC GCTCCCTTCGTTGCGGTGACAAACATCGGAGCCGGCAGCGAC70Attorney Docket No: 030258-000107WOPTGGCGGGACCGCCGTCGTGGCATTCGGGGGAACCCCACGTCGC TCGGCGGGGACGTCTACCGGTACCCAGACGACCGACGTCCCC ACCGAGGCCCTTGGGGGCCCCCCTCCTCCTCCCCGCTTCACCC TGGGTGGCGGCTGTTGTTCCTGTCGCGACACACGGCGCCGCTC TGCGGTATTCGGGGGGGAGGGGGATCCCGTCGGCCCCGCGGA GTTCGTCTCGGACGACCGGTCGTCCGATTCCGACTCGGATGAC TCGGAGGACACCGACTCGGAGACGCTGTCACACGCCTCCTCG GACGTGTCCGGCGGGGCCACGTACTCAGCGGTAGACCCACAC GCACCGACGCCAGGGGCCGGCGCTGGTCTTGCGGCCGATCCC GCCGTGGCCCGGGACGACGCGGAGGGGCTTTCGGACCCCCGG CCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCCTGGAA CTCACACCCGAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGA GATGCCGTGAACCGCGAACCCGCGCTCATGCTGGAGTACTTTT GCCGGTGCGCCCGCGAGGAAACCAAGCGTGTCCCCCCCAGGA CATTCTGCAGCCCCCCTCGCCTCACGGAGGACGACTTTGGGCT TCTCAACTACGCGCTCGTGGAGATGCAGCGCCTGTGTCTGGAC GTTCCTCCGGTCCCGCCGAACGCATACATGCCCTATTATCTCA GGGAGTATGTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGG TGAGCCGGTCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGT GCACCTGCGGATCCTATCTGCGATCGCAATGGTGAGCAAGGG CGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGA GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTC GTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGC CCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACG ACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCG ACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACT ACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGA ACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACC TGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGC GCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGAAttorney Docket No: 030258-000107WOPTTCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCA CTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTG TGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCC CTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCA TCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATA GTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATG GGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTT AGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACA AAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCC CTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGG TTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAAC ATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCC TCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTA TGGAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATG AAACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATC AATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGC GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCA TAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG AGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA TCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA CCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGAT AGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGC GGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTC TGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTA ATACGACTCACTATAGGGAGACCCAAGCTTACCGGCGAAGGA GGGCCACCGGATCCCTCGAGGCCACCATGTACAGAATGCAGC TGCTGAGCTGTATCGCCCTGTCTCTGGCCCTGGTCACCAACAG CACAAGCACCTCTGAGAACGTGCTGTTCGGCATGGGCAACCC TCTGCTGGATATCAGCGCCGTGGTGGACAAGGACTTCCTGGA CAAGTACAGCCTGAAGCCTAACGACCAGATCCTGGCCGAGGA CAAGCACAAAGAGCTGTTCGACGAGCTGGTCAAGAAGTTCAAAttorney Docket No: 030258-000107WOPTGGTCGAGTACCACGCTGGCGGCAGCACCCAGAACTCTATGAA GGTTGCCCAGTGGCTGATCCAAGAGCCTCACAAGGCCGCCAC ATTCTTCGGCTGTATCGGCATCGACAAGTTCGGCGAGATCCTG AAGAGAAAGGCCGCCGATGCTCACGTGGACGCCCACTACTAT GAGCAGAACGAGCAGCCTACAGGCACCTGTGCCGCTTGTATC ACCGGCGGCAATAGAAGCCTGGTGGCCAATCTGGCCGCTGCC AACTGCTACAAGAAAGAGAAGCACCTGGACCTGGAACGGAA CTGGGTGCTCGTGGAAAAGGCCAGAGTGTACTATATCGCCGG ATTCTTCCTGACCGTGTCTCCCGAGTCTGTGCTGAAGGTGGCC AGATACGCCGCCGAGAACAACAGAGTGTTCACCCTGAACCTG AGCGCCCCTTTCATCAGCCAGTTCTTCAAAGAGGCCCTGATGG ACGTGATGCCCTACGTGGACATCCTGTTTGGCAACGAGACAG AGGCCGCTACCTTCGCTAGAGAGCAGGGCTTCGAGACAAAGG ACATCAAAGAGATCGCCAAGAAGGCCCAGGCTCTGCCCAAAG TGAACAGCAAGAGGCAGAGAACCGTGATCTTCACCCAGGGCA GAGATGACACAATCGTGGCTGCCGAGAACGACGTGACCGCCT TTCCTGTGCTGGACCAGAATCAAGAGGAAATCATCGACACAA ACGGCGCTGGCGACGCTTTCGTCGGCGGATTTCTGTCTCAGCT GGTGTCCGACAAGCCCCTGACCGAGTGTATCAGAGCTGGCCA CTATGCCGCCTCCGTGATCATCAGAAGAACCGGCTGCACATTC CCCGAGAAGCCCGACTTTCACTGAGGTACCACCCAGCTTTCTT GTACAAAGTGGTGATGGCCGGCCGCTTCGAGCAGACATGATA AGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAG TGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTT TATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACA ACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTG GGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATT AATTAAGAACGGATCCGGACCCGGGAGGCCTCCTTTGAGGAG TGGCTGCGATHuman IL-2 ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTG signal peptide CACTTGTCACGAATTCGGCTICP65’ ATGGCCAGCCGCCCAGCCGCATCCTCTCCCGTCGAAGCGCGG GCCCCGGTTGGGGGACAGGAGGCCGGCGGCCCCAGCGCAGCC ACCCAGGGGGAGGCCGCCGGGGCCCCTCTCGCCCACGGCCAC73Attorney Docket No: 030258-000107WOPTCACGTGTACTGCCAGCGAGTCAATGGCGTGATGGTGCTTTCCA ACAAGACGCCCGGGTCCGCGTCCTACCGCATCAGCGATAGCA ACTTTGTCCAATGTGGTTCCAACTGCACCATGATCATAGACGG AGACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCGGC ATCCCCCGCTCCCTTCGTTGCGGTGACAAACATCGGAGCCGGC AGCGACGGCGGGACCGCCGTCGTGGCATTCGGGGGAACCCCA CGTCGCTCGGCGGGGACGTCTACCGGTACCCAGACGACCGAC GTCCCCACCGAGGCCCTTGGGGGCCCCCCTCCTCCTCCCCGCT TCACCCTGGGTGGCGGCTGTTGTTCCTGTCGCGACACACGGCG CCGCTCTGCGGTATTCGGGGGGGAGGGGGATCCCGTCGGCCC CGCGGAGTTCGTCTCGGACGACCGGTCGTCCGATTCCGACTCG GATGACTCGGAGGACACCGACTCGGAGACGCTGTCACACGCC TCCTCGGACGTGTCCGGCGGGGCCACGTACTCAGCGGTAGAC CCACACGCACCGACGCCAGGGGCCGGCGCTGGTCTTGCGGCC GATCCCGCCGTGGCCCGGGACGACGCGGAGGGGCTTTCGGAC CCCCGGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCC CTGGAACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCTTT CTGGGAGATGCCGTGAACCGCGAACCCGCGCTCATGCTGGAG TACTTTTGCCGGTGCGCCCGCGAGGAAACCAAGCGTGTCCCC CCCAGGACATTCTGCAGCCCCCCTCGCCTCACGGAGGACGAC TTTGGGCTTCTCAACTACGCGCTCGTGGAGATGCAGCGCCTGT GTCTGGACGTTCCTCCGGTCCCGCCGAACGCATACATGCCCTA TTATCTCAGGGAGTATGTGACGCGGCTGGTCAACGGGTTCAA GCCGCTGGTGAGCCGGTCCGCTCGCCTTTACCGCATCCTGGGG GTTCTGGTGCACCTG GGATCCGGACCCGGGAGGCCTCCTTTGAGGAGTGGCTGCGAT CCAAGGAAGTGGCCCTGGACTTTGGCCTGACGGAAAGGCTTC GCGAGCACGAAGCCCAGCTGGTGATCCTGGCCCAGGCTCTGG ACCATTACGACTGTCTGATCCACAGCACACCGCACACGCTGG TCGAGCGGGGGCTGCAATCGGCCCTGAAGTATGAGGAGTTTT ACCTAAAGCGCTTTGGCGGGCACTACATGGAGTCCGTCTTCCA GATGTACACCCGCATCGCCGGCTTTTTGGCCTGCCGGGCCACG CGCGGCATGCGCCACATCGCCCTGGGGCGAGAGGGGTCGTGG TGGGAAATGTTCAAGTTCTTTTTCCACCGCCTCTACGACCACC AGATCGTACCGTCGACCCCCGCCATGCTGAACCTGGGGACCCAttorney Docket No: 030258-000107WOPTGCAACTACTACACCTCCAGCTGCTACCTGGTAAACCCCCAGG CCACCACAAACAAGGCGACCCTGCGGGCCATCACCAGCAACG TCAGCGCCATCCTCGCCCGCAACGGGGGCATCGGGCTATGCG TGCAGGCGTTTAACGACTCCGGCCCCGGGACCGCTAGCGTCA TACCCGCCCTCAAGGTCCTCGACTCGCTGGTGGCGGCGCACA ACAAAGAGAGCGCGCGTCCAACCGGCGCGTGCGTGTACCTGG AGCCGTGGCACACCGACGTGCGGGCCGTGCTCCGGATGAAGG GGGTCCTCGCCGGCGAAGAGGCCCAGCGCTGCGACAATATCT TCAGCGCCCTCTGGATGCCAGACCTGTTTTTCAAGCGCCTGAT TCGCCACCTGGACGGCGAGAAGAACGTCACATGGACCCTGTT CGACCGGGACACCAGCATGTCGCTCGCCGACTTTCACGGGGA GGAGTTCGAGAAGCTCTACCAGCACCTCGA GFP ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCC ATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTC AGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAG CTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGC CCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTG CTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTC AAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATC TTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTG AAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG GGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAG CTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCC GACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGC CACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTAC CAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCC GACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGAC CCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTG ACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG TAAb-globin polyA AATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTT signal TTTGTGTCTCTCA75Attorney Docket No: 030258-000107WOPTCMVA ATCCACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATpromoter / enhanc AAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTT er GTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTT AAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATA TGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAG AGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTA TTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATAT TTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCT TACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTC CCAGTCATAGCTGTCCCTCTTCTCTTATGGAGCTCCCCCCGCT CCTCCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCCC CGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTCCCCCCGCTCCTC CCCCCGCTCCTCCCCCCCCCGCTCCCGCGGCCCCGCCCCCAAC GCCCGCTCCTCCCCCCGCTCCCGCGGCCCCGCCCCCAACGCCC GCCGCGCGCGCGCACGCCGCCCTTGACTCACGGGGATTTCCA AGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACC AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCC CATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCT ATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTT ACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC AAGCTTACCGGCGAAGGAGGGCCACC CMV B GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGpromoter / enhanc GTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA er CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCC CGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGC CAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACG GTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCA AGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCC TGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTT GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGAT GCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGA CTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGG AGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTC GTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTG TACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTA76Attorney Docket No: 030258-000107WOPTGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCA CTATAGGGAGACCCAAGCTTACCGGCGAAGGAGGGCCACCGG ATCC32 CMV B GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGG enhancer GTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCC CGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGC CAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACG GTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCA AGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCC TGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTT GGCAGTACATCTACGTATTAGTCATCGCTATTACCATG33 CMV B GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG promoter TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCA ATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAA AATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT34 SV40 TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGC poly(A)signal AGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTT

[0089] In some embodiments of any of the aspects, the ADE polypeptide comprises a conserved ADK catalytic active site. In some embodiments of any of the aspects, the ADK comprises conserved residues corresponding to Asnl4 (N14), Aspl8 (D18), Gly65 (G65), and Asn68 (N68) of SEQ ID NO: 1. In some embodiments of any of the aspects, the ADK comprises conserved residues corresponding to Asnl4 (N14), Aspl8 (D18), Ser32 (S32), Gly65 (G65), Asn68 (N68), Aspl30 (D130), Asnl31 (N131), Asn223-Glu224-Thr225-Glu226 (NETE), and Asp300 (D300) of SEQ ID NO: 1. In some embodiments of any of the aspects, the ADK comprises conserved residues corresponding to Asnl4 (N14). Aspl8 (D18). Ser32 (S32), Gly65 (G65), Asn68 (N68). Asnl31 (N131), Asn223-Glu224-Thr225-Glu226 (NETE). and Asp300 (D300) of SEQ ID NO: 1.Attorney Docket No: 030258-000107WOPT

[0090] In some embodiments of any of the aspects, the ADE polypeptide comprises a conserved ADA catalytic active site. In some embodiments of any of the aspects, the ADA comprises conserved residues corresponding to Hisl5 (H15), Hisl7 (H17), Aspl9 (D19), Leu58 (L58), Leu62 (L62), Glyl84 (G184), His214 (H214), Glu217 (E217), His238 (H238), Asp295 (D295), and Asp296 (D296) of SEQ ID NO: 2. In some embodiments of any of the aspects, the ADE polypeptide comprises a conserved ADA catalytic active site. In some embodiments of any of the aspects, the ADA comprises conserved residues corresponding to Hisl5 (H15), Hisl7 (H17), His214 (H214), and Asp295 (D295) of SEQ ID NO: 2. In some embodiments of any of the aspects, the ADE polypeptide comprises a conserved ADA catalytic active site. In some embodiments of any of the aspects, the ADA comprises conserved residues corresponding to Hisl7 (H17). Aspl9 (D19), Glyl84 (G184), and Asp296 (D296) of SEQ ID NO: 2. In some embodiments of any of the aspects, the ADE polypeptide comprises a conserved ADA catalytic active site. In some embodiments of any of the aspects, the ADA comprises conserved residues corresponding to His238 (H238) of SEQ ID NO: 2. In some embodiments of any of the aspects, the ADE polypeptide comprises a conserved ADA catalytic active site. In some embodiments of any of the aspects, the ADA comprises conserved residues corresponding to Glu217 (E217) of SEQ ID NO: 2.

[0091] As used herein “conserved” refers to a residue, amino acid, or sequence which is identical to a corresponding residue, amino acid, or sequence in a reference sequence.

[0092] In some embodiments of any of the aspects, the ADE has a Km for adenosine of 10 pM or less. In some embodiments of any of the aspects, the ADE has a Km for adenosine of 1 pM or less. In some embodiments of any of the aspects, the ADE has a Km for adenosine of 0.5 pM or less. In some embodiments of any of the aspects, the ADE has a Km for adenosine of 0.1 pM or less. In some embodiments of any of the aspects, the ADE has a Km for adenosine of 0.01 pM or less.

[0093] In some embodiments of any of the aspects, the ADE has a Km for adenosine within the range of 0.1 nM to 10 pM. In some embodiments of any of the aspects, the ADE has a Km for adenosine within the range of 1 nM to 1 μM. In some embodiments of any of the aspects, the ADE has a Km for adenosine within the range of 10 nM to 100 nM. In some embodiments of any of the aspects, the ADE has a Km for adenosine within the range of 10 nM to 1 pM. In some embodiments of any of the aspects, the ADE has a Km for adenosine within the range of 1 nM to 100 nM.

[0094] In one aspect of any of the embodiments, described herein is a composition comprising: a codon-optimized nucleic acid encoding an Adenosine-Degrading Enzyme (ADE) polypeptide. In some embodiments of any of the aspects, the codon-optimization is optimization for viral expression. In some embodiments of any of the aspects, the codon-optimization is optimization for human expression. In some embodiments of any of the aspects, the codon-optimization is optimization for expression in the species of a subject. In some embodiments of any of the aspects, the ADE polypeptide is a fusion polypeptide further comprising a signal peptide (SP).78Attorney Docket No: 030258-000107WOPT

[0095] As used herein, “codon-optimization” refers to a sequence in which, e.g., tire native or wild-ty pe sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that the altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native / wild-type sequence (or a cell obtained from such organism).

[0096] Secreted ADEs can be generated by fusion of a secretion signal sequence to the ADE, e.g., ADA1 or ADKS. As used herein, the terms “signal peptide”, “SP”, "secretion signal sequence," "secretion sequence." "secretion signal peptide," “leader sequence”, or "signal sequence," refer to a sequence that directs the transport of a propeptide to the endoplasmic reticulum and through the secretory pathway during protein translation. A signal peptide is usually 3-60 amino acids long. As used herein a signal peptide does not refer to a sequence that targets a protein to the nucleus or other organelles, such as mitochondria. In some embodiments of any of the aspects, the SP comprises 5 to 15 amino acids with hydrophobic side chains that are recognized by a cytosolic protein, SRP (Signal Recognition Particle), which stops translation and aids in the transport of an mRNA-ribosome complex to a translocon in the membrane of the endoplasmic reticulum. In some embodiments of any of the aspects, the SP comprises at least three regions: an amino- terminal polar region (N region), where frequently positive charged amino acid residues are observed, a central hydrophobic region (H region) of 7-8 amino acid residues and a carboxy- terminal region (C region) that includes the cleavage site. Commonly, the signal peptide is cleaved from the mature protein with cleavage occurring at this cleavage site.

[0097] Exemplary’ SPs include, but are not limited to. the human interleukin-2 signal peptide (IL-2), die human tissue plasminogen activator signal peptide (tPA; encoded by PLAT), the human immunoglobulin kappa chain leader sequence (IgK / IGKC), the mouse immunoglobulin kappa chain leader sequence (mlgK), the human serum albumin signal peptide (ALB), the herpes simplex virus ty pe 1 (HSV-1) glycoprotein D signal peptide (gD / US6), the herpes simplex virus type 1 (HSV-1) glycoprotein B signal peptide (gB / UL27), the human growth hormone signal peptide (GH1), the CD33 signal peptide, the human preprotrypsin trypsinogen signal peptide, the BM40 / SPARC signal peptide (osteonectin), the Gaussia luciferase signal peptide, and the human transferrin signal peptide (TF). In some embodiments of any of the aspects, the SP is selected from the group consisting of: IgG K chain signal peptide; IgG heavy chain signal peptide; human IgG K chain signal peptide; human IgG heavy chain signal peptide: murine Ig K chain signal peptide; cytokine SPs (e.g.. interleukin-2 signal peptide; interleukin-7 signal peptide; interleukin- 12 signal peptide; granulocyte colony-stimulating factor signal peptide); hormone SPs (e.g. insulin signal peptide: growth hormone signal peptide; prolactin signal peptide: a secreted plasma protein signal peptide; albumin signal peptide; transferrin signal peptide; fibrinogen signal peptide); growth factor SPs (e.g.. tissue plasminogen activator signal peptide;79Attorney Docket No: 030258-000107WOPTepidermal growth factor signal peptide; vascular endothelial grow th factor signal peptide; fibroblast growth factor signal peptide); viral SPs (e.g., influenza hemagglutinin signal peptide; HIV gpl20 signal peptide; VSV glycoprotein signal peptide; SARS-CoV-2 spike signal peptide); a yeast secretion SP (e.g., a yeast leader sequence; a-mating factor prepro leader; invertase signal peptide; acid phosphatase signal peptide; killer toxin signal peptide); an insect SP (e.g., GP67 signal peptide; honeybee melittin signal peptide); an engineered or synthetic SP (e.g., a synthetic Ig leader; an optimized tPA variant; a hybrid signal peptide); and / or a leaderless or non-classical secretion protein (e.g.. Interleukin- 1|3; fibroblast growth factor 2; and galectin-1). The sequences of these peptides are known in the art.

[0098] In additional embodiments, the signal peptide can be a synthetic or optimized signal peptide designed to enhance secretion efficiency in a particular host cell system, including but not limited to mammalian, insect, yeast, or bacterial expression systems. One of ordinary skill in the art will appreciate that the selection of an appropriate signal peptide can vary depending on the host cell used for expression and the desired localization or secretion of the encoded protein.

[0099] The SP polypeptide is operably linked to the ADE such that the tw o sequences are joined in the correct reading frame and positioned to direct the newly synthesized fusion protein into the secretory pathway of the host cell. SP polypeptides are commonly positioned 5' to the nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences can be positioned elsewhere in the nucleotide sequence of interest. In some embodiments of any of the aspects, the SP polypeptide is located N-terminus of the ADE polypeptide.

[0100] In some embodiments of any of the aspects, the SP polypeptide is derived from human interleukin-2 (IL-2). In some embodiments of any of the aspects, the IL-2 SP polypeptide comprises the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the IL-2 SP polypeptide comprises a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the IL-2 SP polypeptide comprises a sequence with at least 98% sequence identity7to SEQ ID NO: 5. In some embodiments of any of the aspects, the IL-2 SP polypeptide comprises a sequence with at least 99% sequence identity to SEQ ID NO: 5. In some embodiments of any of die aspects, the IL-2 SP polypeptide consists of the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, die IL -2 SP polypeptide consists essentially of the sequence of SEQ ID NO: 5.

[0101] In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADKS, die nucleic acid encoding the fusion protein a sequence w ith at least 80% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADKS, the nucleic acid encoding the fusion protein a sequence with at least 85% sequence identity7to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADKS, the nucleic acid encoding the fusion protein a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of80Attorney Docket No: 030258-000107WOPTthe aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADKS, the nucleic acid encoding the fusion protein a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADKS, the nucleic acid encoding the fusion protein a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADKS, the nucleic acid encoding the fusion protein a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADKS, the nucleic acid encoding the fusion protein a sequence identical to the sequence of SEQ ID NO: 7.

[0102] In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADKS, the nucleic acid encoding the fusion protein a sequence comprising the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADKS, the nucleic acid encoding the fusion protein a sequence consisting essentially of the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADKS. the nucleic acid encoding the fusion protein a sequence consisting of the sequence of SEQ ID NO: 7.

[0103] In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADA1. the nucleic acid encoding the fusion protein a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADA1, the nucleic acid encoding the fusion protein a sequence w ith at least 85% sequence identity' to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADA1, the nucleic acid encoding the fusion protein a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADA1, the nucleic acid encoding the fusion protein a sequence with at least 95% sequence identity' to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADA1, the nucleic acid encoding the fusion protein a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and AD Al, the nucleic acid encoding the fusion protein a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and AD Al, the nucleic acid encoding the fusion protein a sequence identical to the sequence of SEQ ID NO: 11.81Attorney Docket No: 030258-000107WOPT

[0104] In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADA1, the nucleic acid encoding the fusion protein a sequence comprising the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADA1, the nucleic acid encoding the fusion protein a sequence consisting essentially of the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the nucleic acid described herein encodes a fusion protein comprising an SP and ADA1, the nucleic acid encoding the fusion protein a sequence consisting of the sequence of SEQ ID NO: 11.

[0105] In some embodiments of any of the aspects, a polypeptide, e.g., a fusion protein with a SP polypeptide and adenosine degrading enzyme polypeptide (e.g., SP+ADE), as described herein can comprise at least one peptide bond replacement. An adenosine degrading enzyme polypeptide (e.g., SP+ADE) as described herein can comprise one type of peptide bond replacement or multiple types of peptide bond replacements, e.g. 2 types. 3 types, 4 types. 5 types, or more types of peptide bond replacements. Non-limiting examples of peptide bond replacements include urea, thiourea, carbamate, sulfonyl urea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid, para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronic ester, olefinic group, and derivatives thereof.

[0106] In some embodiments of any of the aspects, a polypeptide, e.g., an adenosine degrading enzyme polypeptide (e.g., SP+ADE), as described herein can comprise naturally occurring amino acids commonly found in polypeptides and / or proteins produced by living organisms, e.g. Ala (A), Vai (V), Leu (L). lie (I), Pro (P), Phe (F), Trp (W), Met (M). Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q), Asp (D). Glu (E), Lys (K), Arg (R), and His (H). In some embodiments of any of the aspects, an adenosine degrading enzy me polypeptide (e.g., SP+ADE) as described herein can comprise alternative amino acids. Non-limiting examples of alternative amino acids include, D-amino acids; beta-amino acids; homocysteine, phosphoserine, phospho threonine, phosphotyrosine, hydroxy proline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1, 2,3,4, -tetrahydroisoquinoline-3 -carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citruline, alpha-methyl-alanine, para-benzoylphenylalanine. para-amino phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine), diaminobutyric acid, 7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine, biphenylalanine, cyclohexylalanine, aminoisobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylgtycine, 1-amino-l-cyclopentanecarboxylic acid, 1-amino-l-cyclohexanecarboxylic acid, amino-benzoic acid, amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine. nipecotic acid, alpha-amino butyric acid, thienyl-alanine, t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs;82Attorney Docket No: 030258-000107WOPTazidc-modificd amino acids; alkvnc-modificd amino acids; cyano-modificd amino acids; and derivatives thereof.

[0107] In some embodiments of any of the aspects, a polypeptide, e.g. an adenosine degrading enzyme polypeptide (e.g., SP+ADE), can be modified, e g. by addition of a moiety' to one or more of the amino acids that together comprise the peptide. In some embodiments of any of the aspects, a polypeptide as described herein can comprise one or more moiety molecules, e.g. 1 or more moiety molecules per polypeptide. 2 or more moiety molecules per polypeptide, 5 or more moiety molecules per polypeptide, 10 or more moiety molecules per polypeptide or more moiety molecules per polypeptide. In some embodiments of any of the aspects, a polypeptide as described herein can comprise one more types of modifications and / or moieties, e.g. 1 type of modification, 2 types of modifications. 3 types of modifications or more types of modifications. Non-limiting examples of modifications and / or moieties include PEGylation; glycosylation; HESylation; ELPylation; lipidation; acetylation; amidation; endcapping modifications; cyano groups; phosphorylation; albumin, and cyclization. In some embodiments of any of the aspects, an end-capping modification can comprise acetylation at the N-terminus, N-terminal acylation, and N-terminal formylation. In some embodiments of any of the aspects, an endcapping modification can comprise amidation at the C-terminus. introduction of C-terminal alcohol, aldehyde, ester, and thioester moieties. The half-life of a polypeptide can be increased by the addition of moieties, e.g. PEG, albumin, or other fusion partners (e.g. Fc fragment of an immunoglobulin).

[0108] A polypeptide or protein described herein (e.g., SP+ADE) can be encoded and / or expressed by nucleic acids and / or vectors. Accordingly, in one aspect of any of the embodiments described herein is a nucleic acid encoding an ADE polypeptide (e.g., SP+ADE) as described herein. In one aspect of any of the embodiments, described herein is a vector encoding or comprising an ADE polypeptide (e.g., SP+ADE) as described herein.

[0109] In some embodiments of any of the aspects, the nucleic acid encoding an ADE polypeptide (e.g., SP+ADE) as described herein comprises DNA. In some embodiments of any of the aspects, die nucleic acid encoding an ADE polypeptide (e.g., SP+ADE) as described herein consists essentially of DNA. In some embodiments of any of the aspects, the nucleic acid encoding an ADE polypeptide (e.g., SP+ADE) as described herein consists of DNA.

[0110] In some embodiments of any of the aspects, a DNA molecule encoding an ADE polypeptide (e.g., SP+ADE)as described herein comprises one of SEQ ID NOs: 1-4 or a nucleic acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%. at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least at least about 98%, at least about 99%, or 100%, identical to one of SEQ ID NOs: 6-25. In some embodiments of any of the aspects, a DNA molecule encoding an ADE polypeptide (e.g., SP+ADE)as described herein comprises one of SEQ ID NOs: 1-4 or a nucleic acid sequence that is at least about 75%. at least about 80%, at least about 85%, at least about 90%. at least about 91%, at least83Attorney Docket No: 030258-000107WOPTabout 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least at least about 98%, at least about 99%, or 100%, identical to one of SEQ ID NOs: 6-25 that maintains the same function (e.g., adenosine degradation), or a codon-optimized version thereof.

[0111] In some embodiments of any of the aspects, a DNA molecule encoding an ADE and / or fusion polypeptide (e.g., SP+ADE) as described herein comprises at least one regulatory sequence upstream of the encoded ADE and / or fusion polypeptide (e.g., SP+ADE). In some embodiments of any of the aspects, a DNA molecule encoding an ADE polypeptide (e g., SP+ADE) as described herein comprises a promoter for transcription of the ADE and / or fusion polypeptide (e.g., SP+ADE) using an RNA polymerase. In some embodiments of any of the aspects, a DNA molecule encoding an ADE polypeptide (e.g., SP+ADE) as described herein comprises a T7 promoter.

[0112] In some embodiments of any of the aspects, the sequence encoding an ADE polypeptide (e.g., SP+ADE) is operably linked to a viral promoter. In some embodiments of any of the aspects, the viral promoter can be a Cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, a Herpes Simplex Virus (HSV) promoter (HSV-1 IE and IE 4 / 5). In some embodiments of any of the aspects, the viral promoter is CMVA or CMVB.

[0113] In some embodiments of any of the aspects, the nucleic acid encoding an ADE polypeptide (e.g., SP+ADE) is operably linked to a tissue-specific promoter. In some embodiments of any of the aspects, the tissue-specific promoter is be stromelysin 3 promoter, the surfactant protein A promoter, the secretory leukoprotease inhibitor (SLPI) promoter, the tyrosinase promoter, which is specific for melanoma cells, the stress inducible grp78 / BiP promoter, the AP2 adipose enhancer, the a-1 antitrypsin transthyretin promoter, the interleukin- 10 promoter, which is specific for glioblastoma multiform cells, the c-erbB-2 promoter, the a-B-crystallin / heat shock protein promoter, the basic fibroblast growth factor promoter, the epidermal growth factor receptor promoter, the mucin-like glycoprotein (DF3, MUC1) promoter, die mtsl promoter, the nestin promoter-enhancer, the NSE promoter, the somatostatin receptor promoter, the c-erbB-3 and c-erbB-2 promoters, the c-erbB4 promoter, the thyroglobulin promoter, the a-fetoprotein promoter, or the villin promoter.

[0114] When the nucleic acid molecule that encodes an ADE polypeptide (e.g., SP+ADE) described herein is expressed in a cell, a variety of transcription control sequences (e g., promotcr / cnhanccr sequences) can be used to direct its expression. The promoter can be a native promoter, e.g., the promoter of the at least one adenosine degrading enzyme in its endogenous context, which provides normal regulation of expression of the adenosine degrading enzyme. In some embodiments the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of the ADE and / or fusion polypeptide (e.g., SP+ADE). A variety of conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule.

[0115] The precise nature of the regulatory sequences needed for expression can vary between species or cell types, but in general can include, as necessary, 5'non-transcribed and 5' non-translated sequences84Attorney Docket No: 030258-000107WOPTinvolved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. In particular, such 5' non-transcribed regulatory sequences can include a promoter region which includes a promoter sequence for transcriptional control of the ADE and / or fusion polypeptide (e.g., SP+ADE). Regulatory sequences can also include enhancer sequences or upstream activator sequences as desired.

[0116] A nucleic acid molecule that encodes an ADE polypeptide (e.g., SP+ADE) as described herein can be introduced into a cell or cells using methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc. Expressing the nucleic acid molecule encoding an ADE polypeptide (e.g., SP+ADE) as described herein can also be accomplished by integrating the nucleic acid molecule into the genome.

[0117] In some embodiments of any of tire aspects, one or more of the nucleic acids encoding an ADE polypeptide (e.g., SP+ADE) described herein is expressed in a recombinant expression vector or plasmid. FIGs. 10-13 contain schematics of exemplary vectors used. In some embodiments of any of the aspects, the vector is plasmid, cosmid, or viral vector. In one aspect of any of the embodiments, described herein is a vector comprising the nucleic acid described herein, e.g., a nucleic acid encoding an ADE polypeptide (e.g., SP+ADE).

[0118] In some embodiments of any of the aspects, the vector is a viral vector derived from recombinant retrovirus, an adenovirus, an adeno-associated virus, an alphavirus, an oncolytic virus, or a lentivirus. In some embodiments of any of the aspects, the vector is a viral vector derived from herpes simplex virus. In some embodiments of any of the aspects, the vector is a viral vector derived from replication-deficient herpes simplex virus vectors, e.g., NP2, EG110A, B-Vec, and JANI.

[0119] As used herein, the tenn "vector" refers to a polynucleotide sequence suitable for transferring nucleic acids (e.g., DNA encoding an ADE polypeptide (e.g., SP+ADE) as described herein) into a host cell. The vector can encompass any genetic element that is capable of replication when associated with the proper control elements and that can transfer nucleic acid sequences to cells. The term "vector” includes a plasmid, a cloning vector, an expression vector, naked DNA, a mini-chromosome, a chromosome, a transposon, a cosmid, a virus, virion, phage, and the like. See, for example. U. S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670, and, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments are ligated. Another type of vector is a viral vector, wherein additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g.. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In85Attorney Docket No: 030258-000107WOPTgeneral, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the technology described herein is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0120] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least tw o different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g.. a promoter, suppressor, activator, enhancer, response element, or the like).

[0121] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized. A cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector can be cut in a determinable fashion and into which a desired DNA sequence can be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence can occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication can occur actively during a lytic phase or passively during a lysogenic phase.

[0122] An expression vector is one into which a desired DNA sequence can be inserted by restriction and ligation such that it is operably joined to regulatory sequences and can be expressed as a transcript. Vectors can further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds (e.g., ampicillin resistance), genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., [5-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein), hi some embodiments of any of the aspects, the vectors used herein are capable of autonomous replication and expression of the ADE and / or fusion polypeptide (e.g., SP+ADE) present in the DNA segments to which they are operably joined. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector can comprise additional elements, for example, the expression vector can have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.86Attorney Docket No: 030258-000107WOPT

[0123] Expression vectors containing all the necessary elements for expression arc commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (or RNA). That heterologous DNA (or RNA) is placed under operable control of transcriptional elements to pennit the expression of heterologous DNA in the host cell.

[0124] As used herein, the tenn “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and / or particle can be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Non-limiting examples of a viral vector include an AAV vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpesvirus vector, an alphavirus vector, a poxvirus vector, a baculovirus vector, and a chimeric virus vector.

[0125] It should be understood that the vectors described herein can, in some embodiments of any of the aspects, be combined with other suitable compositions and therapies. In some embodiments of any of the aspects, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleic acid of interest (e.g., encoding an ADE polypeptide (e g., SP+ADE) as described herein) in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0126] Provided herein are engineered viruses and compositions thereof, e.g., for treating cancer.

[0127] In one aspect of any of the embodiments, described herein is a viral vector comprising a nucleic acid encoding an ADE polypeptide (e.g., a SP+ADE). In one aspect of any of the embodiments, described herein is a virus particle comprising a nucleic acid encoding an ADE polypeptide (e.g., a SP+ADE). In one aspect of any of the embodiments, described herein is a virus particle comprising a viral vector comprising a nucleic acid encoding an ADE polypeptide (e.g., a SP+ADE).

[0128] As used herein, “viral particle’’ refers to a particle comprising at least one viral capsid polypeptide and a nucleic acid molecule, e.g., a viral genome and / or viral vector. Viral vectors are discussed elsewhere herein.

[0129] In some embodiments of any of the aspects, the virus is a DNA virus. In some embodiments of any of the aspects, the virus is an RNA virus.

[0130] In some embodiments of any of the aspects, the virus is an oncolytic virus. Oncolytic viruses that can be administered according to the methods described herein include, without limitation, large DNA viruses such as herpes simplex virus (HSV).

[0131] In some embodiments of any of the aspects, the virus is an oncolytic DNA virus selected from the group consisting of: herpes simplex virus (HSV), adenovirus, and vaccinia virus. In some87Attorney Docket No: 030258-000107WOPTembodiments of any of the aspects, the virus is an oncolytic RNA virus selected from the group consisting of: reovirus, vesicular stomatitis virus (VSV), poliovirus, Seneca Valley Virus (SVV) or senecavirus A (SVA), measles virus (MV), flavivirus, and Semliki Forest virus. In some embodiments of any of the aspects, the oncolytic virus is herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). In some embodiments of any of the aspects, the oncolytic virus is herpes simplex virus type 1 (HSV-1). In some embodiments of any of the aspects, the oncolytic virus is herpes simplex virus ty pe 2 (HSV-2).

[0132] In some embodiments of any of the aspects, the oncolytic herpes simplex virus is G47A, a safe and efficacious agent for direct intracranial inoculation in humans. In some embodiments of any of the aspects, the viruses that are described herein are engineered to express an adenosine-degrading enzyme (ADE).

[0133] In some embodiments of any of the aspects, a replication-competent HSV-1 vector can be used for tumor therapy because it can replicate and spread within tumors and directly lyse infected cancer cells. The oncolytic HSV-1 described herein has been developed with mutations that attenuate neurovirulence and / or impair viral DNA replication in normal cells, thereby enhancing tumor-selective replication and improving safety.

[0134] Engineered viruses can also harbor one or more introduced genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the viral genome or on an endogenous plasmid, wherein the genetic change can result in the alteration, disruption, removal, or addition of one or more protein coding genes, non-protein-coding genes, gene regulatory regions, or any combination thereof, and wherein such change can be a fusion of two or more separate genomic regions or can be synthetically derived.

[0135] In some embodiments of any of the aspects, the oncolytic HSV-1 vector is derived from die wild-type HSV-1 strain F. It has deletions in both copies of the major determinant of HSV neurovirulence, the y34.5 gene, and an inactivating insertion of the E. coli lacZ gene in UL39, which encodes the infected-cell protein 6 (ICP6). ICP6 is the large subunit of ribonucleotide reductase, a key enzy me for nucleotide metabolism and viral DNA synthesis in non-dividing cells, but not in dividing cells. In addition to being the major determinant of HSV neurovirulence, ICP34.5 also functions by blocking host cell-induced shutoff of protein synthesis in response to viral infection. This is likely responsible for the less efficient growth of y34.5 mutants compared to wild-type HSV. This double mutation confers important advantages: a minimal chance of reverting to wild ty pe, preferential replication in tumor cells, attenuated neurovirulence, and hypersensitivity to ganciclovir and acyclovir. In some embodiments of any of the aspects, the ICP47 gene is mutated since HSV-1 infection down-regulates major histocompatibility complex (MHC) class I expression on the surface of infected host cells. The binding of 1CP47 to the transporter associated with antigen presentation (TAP) blocks antigenic peptide transport in the endoplasmic reticulum and loading of MHC class I molecules. The88Attorney Docket No: 030258-000107WOPTbinding of ICP47 is species-specific for TAPs from large mammals, with the affinity for murine TAP about 100-fold less than for human.

[0136] In one aspect of any of the embodiments, described herein is an adeno-associated virus (AAV) viral vector comprising a nucleic acid encoding an ADE polypeptide (e.g., a SP+ADE). In one aspect of any of the embodiments, described herein is a virus particle comprising a nucleic acid encoding an ADE polypeptide (e.g., a SP+ADE). In one aspect of any of the embodiments, described herein is an adeno-associated virus (AAV) virus particle comprising an AAV viral vector comprising a nucleic acid encoding an ADE polypeptide (e.g., a SP+ADE).

[0137] In one aspect of any of the embodiments, described herein is an adeno-associated virus (AAV) viral vector comprising a nucleic acid encoding an ADA polypeptide (e.g., a SP+ADA). In one aspect of any of the embodiments, described herein is a virus particle comprising a nucleic acid encoding an ADA polypeptide (e.g., a SP+ADA). In one aspect of any of the embodiments, described herein is an adeno-associated virus (AAV) virus particle comprising an AAV viral vector comprising a nucleic acid encoding an ADA polypeptide (e.g., a SP+ADA).

[0138] Engineered viruses can be produced using techniques including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins. polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), CRISPR, or any combination thereof.

[0139] In some embodiments of any of the aspects, the oncolytic HSV is engineered to preferentially replicate in tumor cells while being attenuated in normal tissue. Following rescue or infection, viral plaques are isolated from cells under a semisolid overlay, and plaque -purified virus is expanded to generate working stocks for further analysis.

[0140] Alternative technologies for generating herpes simplex virus (HSV) vectors include cloning the HSV genome as a bacterial artificial chromosome (BAC) for bacterial recombineering and reconstitution in mammalian cells, and yeast-based genome assembly / maintenance approaches such as yeast artificial chromosome (YAC) systems that leverage yeast homologous recombination to engineer large HSV DNA constructs.

[0141] In some embodiments of any of tire aspects, the HSV is a HSV variant selected from the group consisting of: G207, G47A, MG18L, A68H-6, R3616, and R47A. These variants and their sequences are known in the art and described in more detail, e.g., in Nguyen et al. Oncolytic Virother 10:1-27 (2012); Bemstock et al. Viruses 13:1158 (2021); Dambach et al. Mol Ther 13:891-8 (2006); Ning et al. Front Micro 5:303 (2014); and Fukuhara et al. Cancer sci 112:3293-3301 (2021); each of which is incorporated by reference herein in its entirety. In some embodiments of any of the aspects, the HSV is G47A.

[0142] In some embodiments of any of the aspects, the viral vector and / or viral particle comprises an inactivating mutation of one. two, three, or four genes selected from the group of: Infected Cell89Attorney Docket No: 030258-000107WOPTProtein 34.5 gene (y34.5 / ICP34.5), Infected Cell Protein 47 gene (ICP47), Infected Cell Protein 6 gene (ICP6), and Unique Short 3 gene (US3). In some embodiments of any of the aspects, the viral vector and / or viral particle comprises an inactivating mutation of each of: Infected Cell Protein 34.5 gene (y34.5 / ICP34.5), Infected Cell Protein 47 gene (ICP47), Infected Cell Protein 6 gene (ICP6), and Unique Short 3 gene (US3). In some embodiments of any of the aspects, the viral vector and / or viral particle comprises an inactivating mutation of ICP47.

[0143] As used herein “inactivating mutation” refers to a modification of a wild-type sequence which results in partial or complete reduction of the expression or activity of a protein encoded by an endogenous DNA sequence such that the protein can no longer accomplish its function. In some embodiments of any of the aspects, an inactivating mutation comprises an engineered modification. A “modification” in a nucleic acid sequence refers to any detectable change in the genetic material, e.g., a change or alteration relative to a reference sequence, e.g., the wild-type sequence.Modifications can be insertions, deletions, replacements, indels, SNPs, mutations, substitutions, or the like. A modification is usually a change of one or more deoxyribonucleotides, the modification being obtained by, for example, adding, deleting, inverting, or substituting nucleotides.

[0144] In some embodiments of any of the aspects, an inactivating mutation comprises, consists of, or consists essentially of an engineered excision of at least part of a coding or regulatory sequence. In some embodiments of any of the aspects, an inactivating mutation comprises, consists of, or consists essentially of an engineered excision of a gene’s promoter. In some embodiments of any of the aspects, an inactivating mutation comprises, consists of, or consists essentially of an engineered excision of at least 5%, at least 10%, at least 20%, at least 30% or more of a gene's coding sequence. In some embodiments of any of the aspects, an inactivating mutation comprises, consists of, or consists essentially of an engineered excision of at least 90%, at least 95%, or 100% of a gene’s coding sequence. In some embodiments of any of the aspects, a loss-of-function allele comprises, consists of, or consists essentially of an engineered missense or nonsense mutation within the first 10% of the coding sequence of a gene.

[0145] In some embodiments of any of the aspects, an inactivating mutation comprises, consists of, or consists essentially of an engineered knock-out modification. As used herein, “knock-out” refers to partial or complete reduction of the expression of a protein encoded by an endogenous DNA sequence such that the protein can no longer accomplish its function. In some embodiments of any of the aspects, the “knock-out” can be produced by targeted deletion of the whole or part of a gene encoding a protein. In some embodiments of any of the aspects, the deletion can prevent or reduce the expression of the functional protein.

[0146] In some embodiments of any of the aspects, a knock-out comprises a deletion of the whole or part of a gene encoding a protein. In some embodiments of any of the aspects, a knock-out modification comprises deletion of the entire coding sequence of the relevant gene. In some90Attorney Docket No: 030258-000107WOPTembodiments of any of the aspects, a knock-out does not comprise any of the coding sequence of the relevant gene. In some embodiments of any of the aspects, a knock-out modification comprises deletion of a part of the coding sequence of the relevant gene, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the coding sequence of the relevant gene. In some embodiments of any of the aspects, a knock-out modification comprises a non-sense mutation of the relevant gene, e.g., in the first 10%, first 20%, first 30%, first 40%, first 50%, first 60%, or first 70% of the coding sequence of the relevant gene. In some embodiments of any of the aspects, a knock-out modification comprises a missense mutation of the relevant gene, e.g., in the first 10%, first 20%. first 30%, first 40%. first 50%, first 60%, or first 70% of the coding sequence of the relevant gene. In some embodiments of any of the aspects, a knock-out modification comprises the introduction of a stop codon in the relevant gene, e.g., in the first 10%, first 20%. first 30%. first 40%, first 50%, first 60%, or first 70% of the coding sequence of the relevant gene. In some embodiments of any of the aspects, a knock-out modification comprises deletion of the promoter of the relevant gene, e.g., deletion of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of the promoter of the relevant gene.

[0147] In some embodiments of any of the aspects, the nucleic acid encoding an ADE polypeptide (e.g., SP+ADE) is inserted into a viral genome at the ICP6 locus. In some embodiments of any of the aspects, the nucleic acid encoding an ADE polypeptide (e.g., SP+ADE) is inserted into a HSV genome at the ICP6 locus. In some embodiments of any of the aspects the viral vector or viral particle comprises an HSV genome and the nucleic acid encoding an ADE polypeptide (e.g.. SP+ADE) is inserted into the HSV genome at the ICP6 locus.

[0148] The recombinant oncolytic virus (e.g., oHSV-SP+ADE) described herein can be comprised by compositions, such as pharmaceutical compositions, as described further herein, hi some embodiments of any of the aspects, the recombinant oncolytic virus (e.g., oHSV-SP+ADE) described herein can be comprised by cells, such as eukaryotic cells. In some embodiments the cells are VERO, GSC, KPC-Y, mesenchymal stem / stromal cells and neural stem cell–type carriers.

[0149] In one aspect of any of the embodiments, described herein is a viral vector or virus particle comprising a nucleic acid encoding an ADE polypeptide (e.g., SP+ADE) as described herein. In some embodiments of any of the aspects, die virus particle is a recombinant retrovirus, a herpes simplex virus, a recombinant herpes simplex virus, an adenovirus, an adeno-associated virus, an alphavirus, an oncolytic virus, or a lentivirus. In some embodiments of any of the aspects, the virus particle is an oncolytic virus (oV) such as a Herpes Simplex Virus (oHSV).

[0150] In one aspect of any one of the embodiments, described herein is a mammalian cell infected by virus particles comprising a nucleic acid encoding an ADE polypeptide (e.g.. SP+ADE) as described herein. In one aspect of any one of the embodiments, described herein is system comprising a mammalian cell infected by virus particles comprising a nucleic acid encoding an ADE polypeptide91Attorney Docket No: 030258-000107WOPT(e.g., SP+ADE) as described herein. In some embodiments of any of the aspects, the mammalian cell secretes the ADE and / or fusion polypeptide. In some embodiments of any of the aspects, the mammalian cell is selected from the group consisting of: mesenchymal stem cells (MSCs), T cells, and neural stem cells

[0151] In one aspect of any one of the embodiments, described herein is serum isolated from a mammalian cell or subject infected by virus particles comprising a nucleic acid encoding an ADE polypeptide (e.g., SP+ADE) as described herein.

[0152] In one aspect of any of the embodiments, described herein is a pharmaceutical composition comprising a nucleic acid encoding an ADE polypeptide (e.g., SP+ADE), vector, viral vector, viral particle, or cell as described herein.

[0153] In some embodiments of any of the aspects, a pharmaceutical composition described herein comprises a pharmaceutically acceptable carrier. In some embodiments of any of tire aspects, the active ingredients of the pharmaceutical composition comprise a nucleic acid encoding an ADE polypeptide (e.g., SP+ADE), vector, viral vector, viral particle, or cell as described herein. In some embodiments of any of the aspects, the active ingredients of the pharmaceutical composition consist essentially of a nucleic acid encoding an ADE polypeptide (e.g.. SP+ADE), vector, viral vector, viral particle, or cell as described herein. In some embodiments of any of the aspects, the active ingredients of the pharmaceutical composition consist of a nucleic acid encoding an ADE polypeptide (e.g.. SP+ADE), vector, viral vector, viral particle, or cell as described herein. Pharmaceutically acceptable carriers for include sterile aqueous solutions such as water for injection, isotonic saline, and buffered solutions (e.g., phosphate-buffered formulations), optionally with tonicity agents and stabilizers (e.g., sorbitol and protein stabilizers such as albumin), provided that the excipients maintain viral infectivity and are suitable for the intended route of administration.

[0154] In some embodiments of any of the aspects, the pharmaceutical composition as described herein can be a parenteral dose form (i.e., administered or occurring elsewhere in the body than die mouth and alimentary canal). Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or are typically manufactured aseptically prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, and suspensions ready for injection. In addition, controlled or sustained local delivery approaches can also be used, for example via repeated administration, catheter-based delivery, or biomaterial depots (e.g., hydrogels), provided viral infectivity is maintained.

[0155] Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Non-limiting examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride92Attorney Docket No: 030258-000107WOPTinjection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection.

[0156] Vectors and virus particles can be administered using cellular carriers, including mesenchymal stem / stromal cells and neural stem cell-type carriers, which have been reported to exhibit tumor-tropic migration in glioma models and have been explored as vehicles to deliver oHSV to tumors (including by intratumoral injection of virus-loaded carrier cells).

[0157] In one aspect of any of the embodiments, described herein is a Lipid nanoparticle (LNP) comprising a nucleic acid encoding an ADE polypeptide (e.g., SP+ADE) as described herein. The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle. Exemplary liposomes can comprise, e.g., DSPC. DPPC, DSPG. Cholesterol, hydrogenated soy phosphatidylcholine, soy phosphatidyl choline, methoxypolyethylene glycol (mPEG-DSPE) phosphatidyl choline (PC), phosphatidyl glycerol (PG), distearoylphosphatidylcholine, and combinations thereof. Lipid nanoparticles can comprise multiple components, including, e.g.. ionizable lipids, pegylated lipids, phospholipids, and cholesterol.

[0158] In some embodiments of any of the aspects, the methods described herein comprise administering an effective amount of a composition described herein to a subject in order to alleviate a symptom of. e.g., cancer. As used herein, "alleviating a symptom” of a disease or condition is ameliorating any condition or symptom associated with the disease or condition. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%. 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art.

[0159] In some embodiments of any of the aspects, a virus particle (e.g., oHSV-SP+ADE) is administered at a dose of lx 103pfu to IxlO10pfu. In some embodiments of any of the aspects, a virus particle is administered at a dose of 1 * 103pfu to 1 * 107pfu. In some embodiments of any of the aspects, a virus particle is administered at a dose of 1 * 103pfu to 1 * 106pfu. In some embodiments of any of the aspects, a virus particle is administered at a dose of at least 1* 103pfu. In some embodiments of any of the aspects, a virus particle is administered at a dose of at least 1 * 105pfu. In some embodiments of any of the aspects, a virus particle is formulated at a dose of about 3* 109pfu. In some embodiments of any of the aspects, a virus particle is formulated at a dose of about 108PFU / mL. In some embodiments of any of the aspects, a virus particle is formulated at a dose of about 108PFU / mL.

[0160] Virus particles as described herein, e.g., oncolytic viruses, can be administered in a single administration or multiple administrations. The virus can be administered at a dosage of lx 103pfu, at least 1 x 103pfu, 1 x 105PFU, at least 1 x 105PFU. 5 x 105PFU, at least 1 x 106PFU, 5 x 106or about 5 x 106PFU, 1 x 107. at least 1 x 107PFU, 1 x 108or about 1 x 108PFU, at least 1 x 108PFU, about or at least 5 x 108PFU, 1 x 109or at least 1 x 109PFU, 5xl09or at least 5xl09PFU, or 1 x 1010PFU. For example, the virus93Attorney Docket No: 030258-000107WOPTcan be administered at a dosage of between about 1×103and 1×1010pfu, between about 1×104and 1×107pfu, or between about 1×103and 106pfu.

[0161] The term “effective amount" as used herein refers to the amount of a composition needed to alleviate at least one or more symptoms of the disease or disorder and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of an active ingredient that is sufficient to provide a particular therapeutic effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0162] Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. extracellular adenosine levels or tumor volume (contrast-enhancing tumor volume on MRI) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.

[0163] The dosage ranges for the administration of compositions, according to the methods described herein depend upon, for example, the form of the active ingredient, its potency (e.g., PFU), and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for tumor volume or extracellular adenosine levels or the extent to which, for example, anti-tumor immune responses are desired to be induced. The dosage should not be so large as to cause adverse side effects, such as excessive local inflammation / edema, systemic flu-like symptoms, or unintended herpetic infection. Generally, the dosage will vary with the age, condition, and sex of the patient and can be detennined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0164] The efficacy of a composition, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. anti-cancer) can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. MRI-assessed tumor burden using RANO criteria, or extracellular adenosine levels. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill94Attorney Docket No: 030258-000107WOPTin the art and / or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e g. tumor volume on MRI); or (2) relieving die severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for diat disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. neurologic function / performance status (e.g.. KPS), seizure frequency, corticosteroid requirement, overall survival, progression-free survival). It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of orthotopic glioblastoma. When using an experimental animal model, efficacy of treatment is evidenced w hen a statistically significant change in a marker is observed, e.g. reduced tumor volume by imaging or histology; prolonged survival in the animal model; extracellular adenosine (eADO) degradation in the tumor microenvironment (TME).

[0165] In vitro and animal model assays are provided herein w hich allow the assessment of a given dose a composition described herein. By way of non-limiting example, the effects of a dose of a composition described herein can be assessed by can be assessed by measuring viral infectivity and replication (e.g., plaque assay / TCID50 and replication kinetics) and / or oncolytic activity in tumor cells in vitro, and by assessing tumor burden and / or survival in an appropriate animal tumor model. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen.

[0166] In some embodiments of any of the aspects, an effective dose of a composition as described herein can be administered to a patient once. In some embodiments of any of the aspects, an effective dose of a composition described herein can be administered to a patient repeatedly.

[0167] The dosing schedule can vary depending on clinical factors (including tolerability and route of administration). In some embodiments of any of the aspects, a composition described herein is administered as intermittent treatments (e.g., weekly to every few weeks) and can be repeated over multiple cycles. Where delivered by catheter-based intratumoral infusion, administration can occur over a defined period (e.g., minutes to hours) according to the delivery protocol. In some embodiments of any of the aspects, administration can be one or more doses and / or treatments daily over a period of weeks or months. Examples of dosing and / or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week. 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. In some embodiments of any95Attorney Docket No: 030258-000107WOPTof the aspects, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer.

[0168] A variety of means for administering the compositions described herein to subjects can be used depending on tumor type and clinical context and are known to those of skill in the art. Such methods can include, but are not limited to, parenteral, intravenous (IV), intramuscular (IM), subcutaneous (SC), injection, intraperitoneal (IP), or intratumoral administration. In some embodiments of any of the aspects, the composition is administered locally, for example by intratumoral or intralesional injection (including injection into or adjacent to a tumor or tumor resection cavity).

[0169] In some embodiments of any of the aspects, the composition is administered by intratumoral, intraperitoneal, or intravenous administration. In some embodiments of any of the aspects, the composition is administered directly into a solid tumor. In some embodiments of any of the aspects, the composition is administered intracranially.

[0170] In some embodiments of any of the aspects, the composition is administered by multiple inoculations or injections.

[0171] In some embodiments of any of the aspects, the composition described herein is administered as a monotherapy, e.g.. another treatment for the cancer is not administered to the subject. In some embodiments of any of the aspects, the recombinant oncolytic virus (e g.. oHSV-SP+ADE) described herein is administered as a monotherapy, e.g.. another treatment for the cancer is not administered to the subject.

[0172] In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and / or treatment to the subject, e.g. as part of a combinatorial therapy. Non-limiting examples of a second agent and / or treatment can include a cancer therapy selected from the group consisting of: radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide and trimethylol melamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide. estramustine, ifosfamide. mechlorethamine, mechlorethamine oxide hydrochloride, melphalan. novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as96Attorney Docket No: 030258-000107WOPTcarmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as die enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin. caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin. esorubicin, idarubicin. marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin. puromycin, quelamycin. rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetre xate; purine analogs such as fludarabine, 6-mercaptopurine. thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine. 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine. enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane. testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (" Ara-C"); cyclophosphamide; thiotepa; taxoids. e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N. J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC -alpha, Raf, H-Ras, EGFR (e.g.,97Attorney Docket No: 030258-000107WOPTerlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation (e.g., pazopanib, sunitinib, sorafenib, regorafenib, cabozantinib. lenvatinib, ponatinib, ziv-aflibercept, axitinib, tivozanib, vandetanib, ramucirumab); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0173] In some embodiments of any of the aspects, the cancer treatment method further comprises administering an immune checkpoint inhibitor. In some embodiments of any of the aspects, the immune checkpoint inhibitor comprises an immune checkpoint inhibitor antibody. In some embodiments of any of the aspects, the checkpoint inhibitor immunotherapy is an inhibitor of a checkpoint molecule selected from the group consisting of: programmed cell death 1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Adenosine A2A receptor (A2AR), CD276, CD39, CD73. B7 family immune checkpoint molecules. V-set domain-containing T-cell activation inhibitor 1 (B7H4), B and T Lymphocyte Attenuator (BTLA), Indoleamine 2,3-dioxygenase (IDO), Killer-cell Immunoglobulin-like Receptor (KIR), Lymphocyte Activation Gene-3 (LAG-3), nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain Ig suppressor of T cell activation (VISTA), and Sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7).

[0174] Non-limiting examples of immune checkpoint inhibitors (ICIs) include: pembrolizumab (Keytruda®), nivolumab (Opdivo®), cemiplimab (Libtayo®), spartalizumab, camrelizumab (AiRuiKa™), sintilimab (TYVYT®), tislelizumab, toripalimab (Tuoyi™), dostarlimab (JEMPERLI), INCMGA00012, AMP-224, AMP-514 (MEDI0608), atezolizumab (Tecentriq®), avelumab (Bavencio®), envafolimab (KN035), cosibelimab (CK-301), AUNP12, CA-170, BMS-986189, BMS-936559 (MDX-1105), durvalumab (IMFINZI®), tremelimumab, and ipilimumab (Yervoy®). See e.g., US Patents US5811097, US5855887, US6051227, US6682736, US6984720, US7595048, US7605238, US7943743, US8008449, US8217149, US8354509, US8383796, US8728474, US8735553, US8779105, US8779108, US8907053, US8900587, US8952136, US9067999, US9073994, US9683048, US9987500, US10160736, US10316089, US10441655. US10590199, US11225522, US Patent Publication US2014341917; Storz et al., MAbs. 2016 Jan; 8(1): 10-26; the contents of each of which are incorporated herein by reference in their entireties.

[0175] One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr.. Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff’s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

[0176] In addition, the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.98Attorney Docket No: 030258-000107WOPT

[0177] In some embodiments of any of the aspects, described herein is a method to reduce a level of extracellular adenosine, the method comprising contacting a cell with the composition described herein. In some embodiments of any of the aspects, the cell is a cancer cell. In some embodiments of any of the aspects, the cell is a neuron. In some embodiments of any of the aspects, the cell is a cardiac cell. In some embodiments of any of tire aspects, the cell is a vascular cell.

[0178] In some embodiments of any of the aspects, described herein is a method for reducing extracellular adenosine in a subject in need thereof, the method comprising administering the composition described herein to the subject. In some embodiments of any of the aspects, the subject is a subject in need of treatment for atrial fibrillation, syncope, hypoxia, sleep apnea, altitude hypoxia, hypertension, and heart failure. In some embodiments of any of the aspects, the subject is a subject in need of treatment for atrial fibrillation. In some embodiments of any of the aspects, the subject is a subject in need of treatment for syncope. In some embodiments of any of the aspects, the subject is a subject in need of treatment for hypoxia. In some embodiments of any of the aspects, the subject is a subject in need of treatment for sleep apnea. In some embodiments of any of the aspects, the subject is a subject in need of treatment for altitude hypoxia. In some embodiments of any of the aspects, the subject is a subject in need of treatment for hypertension. In some embodiments of any of the aspects, the subject is a subject in need of treatment for heart failure.

[0179] In some embodiments of any of the aspects, the methods described herein relate to treating a subject having or diagnosed as having cancer with the compositions described herein.

[0180] In some embodiments of any of the aspects, the cancer is a solid tumor.

[0181] In some embodiments of any of the aspects, the cancer is selected from the group consisting of: a pancreatic tumor, a primary tumor of the nervous system, a central nervous system (CNS) tumor, a peripheral nervous system (PNS) tumor, a primary central nervous system lymphoma (PCNSL), a brainstem glioma, and a combination thereof.

[0182] In some embodiments of any of the aspects, the cancer is a pancreatic cancer. In some embodiments of any of the aspects, the pancreatic tumor is selected from the group consisting of: pancreatic ductal adenocarcinoma (PDAC), pancreatic neuroendocrine tumor, solid pseudopapillary neoplasm, acinar cell carcinoma, pancreatoblastoma, pancreatic lymphoma. In some embodiments of any of the aspects, the cancer is pancreatic ductal adenocarcinoma (PDAC).

[0183] In some embodiments of any of the aspects, the cancer is a primary tumor of the nervous system. In some embodiments of any of the aspects, the primary tumor of the nervous system is selected from the group consisting of: diffuse glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic mixed oligoastrocytoma, glioblastoma, ependymoma, and anaplastic ependymoma. In some embodiments of any of the aspects, the cancer is glioblastoma.

[0184] Subjects having cancer can be identified by a physician using current methods of diagnosing cancer. Symptoms and / or complications of cancer which characterize these conditions and aid in99Attorney Docket No: 030258-000107WOPTdiagnosis arc well known in the art and include but arc not limited to, the presence of a tumor or neoplastic mass, unexplained weight loss, fatigue, persistent pain, abnormal bleeding or discharge, changes in bowel or bladder function, persistent cough or hoarseness, lymphadenopathy, and organ dysfunction resulting from tumor invasion or metastasis. Tests that can aid in a diagnosis of cancer include, but are not limited to. histopathological examination and biopsy of suspected tumor tissue, immunohistochemistry (IHC), flow cytometry, blood-based biomarker assays including measurement of circulating tumor DNA (ctDNA) and tumor-associated antigens, complete blood count (CBC), imaging studies such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasound, as well as molecular and genomic profiling of tumor tissue. A family history of cancer, or exposure to risk factors for cancer, including but not limited to tobacco use, chronic exposure to ionizing radiation or ultraviolet radiation, exposure to carcinogenic chemicals or environmental toxins, chronic viral infections such as human papillomavirus (HPV), hepatitis B virus (HBV). or hepatitis C virus (HCV), immunosuppression, obesity, and chronic inflammation, can also aid in determining if a subject is likely to have cancer or in making a diagnosis of cancer.

[0185] The compositions described herein can be administered to a subject having or diagnosed as having cancer. In some embodiments of any of the aspects, the methods described herein comprise administering an effective amount of a composition described herein to a subject in order to alleviate a symptom of cancer. As used herein, "alleviating a symptom of cancer" means ameliorating any condition or symptom associated with cancer, including but not limited to reduction in tumor size or volume, inhibition of tumor growth or metastasis, reduction in cancer cell proliferation, alleviation of cancer-associated pain, fatigue, or organ dysfunction, and improvement in overall survival or progression-free survival. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique, including but not limited to radiographic imaging, histopathological assessment, measurement of circulating tumor biomarkers, or clinically validated tumor response criteria such as the Response Evaluation Criteria in Solid Tumors (RECIST)

[0186] As used herein, the term “cancer’’ relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.100Attorney Docket No: 030258-000107WOPT

[0187] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term “malignant’' refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. As used herein, the tenn “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

[0188] A "cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0189] As used herein the term "neoplasm" refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues. Thus, a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.

[0190] A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micro metastases. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.

[0191] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and101Attorney Docket No: 030258-000107WOPTsarcomas; as well as B-cell lymphoma (including low gradc / follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phacomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome.

[0192] A “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation / cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.

[0193] The term “inhibiting tumor cell growth or proliferation” means decreasing A tumor cell's growth or proliferation by at least 10%, 20%, 30%, 40%. 50%, 60%, 70%, 80%, 90%. 95%, or 100%, and includes inducing cell death in a cell or cells within a cell mass.

[0194] The term “tumor progression” refers to all stages of a tumor, including tumorigenesis, tumor growth and proliferation, invasion, and metastasis.

[0195] The term “inhibiting tumor progression” means inhibiting the development, growth, proliferation, or spreading of a tumor, including without limitation the following effects: inhibition of growth of cells in a tumor, (2) inhibition, to some extent, of tumor growth, including slow ing down or complete growth arrest; (3) reduction in the number of tumor cells; (4) reduction in tumor size; (5) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into adjacent peripheral organs and / or tissues; (6) inhibition (i.e. reduction, slowing down or complete stopping) of metastasis; (7) increase in the length of survival of a patient or patient population following treatment for a tumor; and / or (8) decreased mortality of a patient or patient population at a given timepoint following treatment for a tumor.

[0196] A tumor “responds” to a particular agent if tumor progression is inhibited as defined above.

[0197] The term “respond” generally means that a patient exhibits a complete or partial response to the oncolytic therapy as defined in the Response Evaluation Criteria in Solid Tumors (RECIST) criteria (Eisenhauer et al., European Journal of Cancer, 45:228-247 (2009), incorporated herein by reference). A complete response means a disappearance of all target lesions. A partial response means at least a102Attorney Docket No: 030258-000107WOPT30% decrease in the sum of the longest diameter (LD) of target lesions, taking as a reference the baseline sum LD. In particular, a response generally refers to a complete or partial change in tumor size. Similarly, patients who fail to respond to oncolytic therapy are those that exhibit stable disease (neither sufficient shrinkage to qualify as a partial response nor sufficient increase to qualify as progressive disease) or progressive disease (at least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD since the treatment started). The skilled clinician / radiologist will understand the appropriate methods for determining tumor measurements using e.g. computed tomography (CT) or magnetic resonance imaging (MRI), in conjunction with clinical assessment.

[0198] In one respect, the present technology relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising). In some embodiments of any of the aspects, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments of any of the aspects, the compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.

[0199] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all teclmical and scientific terms used herein have die same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0200] The term "oncolytic virus" refers to any virus that is typically able to kill a tumor cell (non-resistant) by infecting the tumor cell. Preferably, the virus is engineered, e.g., to increase tumor cell selectivity. Representative examples of oncolytic virus include without limitation, adenovirus, reovirus, herpes simplex virus (HSV), Newcastle disease virus, poxvirus, myxoma virus, rhabdovirus, picomavirus, influenza virus, coxsackievirus and parvovirus.103Attorney Docket No: 030258-000107WOPT

[0201] An oncolytic virus is "rcplication-sclcctivc" if it is more capable of replicating or is capable of replicating to a greater extent (e.g., burst size) in a tumor cell of a subject than in a non-tumor cell of the subject.

[0202] The term “in expressible form’’ when used in the context of a DNA molecule means operably linked (e.g., located within functional distance) to sequences necessary for transcription of the DNA into RNA by the RNA polymerase transcription machinery found in eukaryotic cells (e.g., promoter sequences, and other 5' regulatory' sequences). One example is a DNA molecule in the context of an expression vector. Expression can refer to transcription of DNA into RNA, and when protein coding sequences are involved, expression can also encompass translation of the mRNA into protein. Viral expression vectors can comprise the viral genome in the context of a virion that is used to infect a cell.

[0203] The term “operably linked” is used herein to refer to a functional relationship of one nucleic acid sequence to another nucleic acid sequence. Nucleic acid sequences are “operably linked” when placed into a functional relationship with one another. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. The nucleic acid sequences being linked can be contiguous, or separated by intervening sequences, and when necessary in the same reading phase and / or appropriate orientation. Linking is accomplished, for example, by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0204] The term “heterologous” is used herein to describe the relationship of one nucleic acid or amino acid sequence to one or more different nucleic acid or amino acid sequences, respectively. The term heterologous, in reference to two or more such sequences, indicates that the different sequences are found in nature within separate, different and distinct larger nucleic acids or polypeptides. The joining of heterologous sequences creates a non-naturally occurring juxtaposition of sequences. Such joining is the product of engineering performed in the laboratory. When such amino acid sequences are joined, the resulting protein is referred to herein as a fusion protein. The products of such joining are referred to as “recombinant”.

[0205] The term “isolated” when used in reference to a substance refers to the fact diat the substance is removed from the context of other substances or entities in which it is admixed with prior to the isolation. The term “isolated” when used in reference to a nucleic acid sequence refers to the fact that the nucleic acid sequence is removed from the context of other nucleic acid sequences in which it is present in nature (e.g., in the context of a chromosome).

[0206] An “effective amount” as the term is used herein, is used to refer to an amount that is sufficient to produce at least a reproducibly detectable amount of the desired results. An effective amount will vary with the specific conditions and circumstances. Such an amount can be determined by the skilled practitioner for a given situation.104Attorney Docket No: 030258-000107WOPT

[0207] The term “therapeutically effective amount' ’ refers to an amount that is sufficient to produce a therapeutically significant reduction in one or more symptoms of the condition when administered to a typical subject who has the condition. A therapeutically significant reduction in a symptom is, e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more as compared to a control or non-treated subject.

[0208] The tenns “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%. at least about 25%. at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%. at least about 55%, at least about 60%. at least about 65%, at least about 70%. at least about 75%, at least about 80%, at least about 85%. at least about 90%, at least about 95%, at least about 98%, at least about 99%. or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal, e.g., for an individual without a given disorder.

[0209] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%. or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%. or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0210] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g.. dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g.. a primate, e.g., a human. The terms, “individual.” “patient” and “subject” are used interchangeably herein.

[0211] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans105Attorney Docket No: 030258-000107WOPTcan be advantageously used as subjects that represent animal models of, c.g., glioblastoma and pancreatic cancer. A subject can be male or female.

[0212] The term “glioma” refers to a tumor originating in the neuroglia of the brain or spinal cord. Gliomas are derived from the glial cell types such as astrocytes and oligodendrocytes, thus gliomas include astrocytomas and oligodendrogliomas, as well as anaplastic gliomas, glioblastomas, and ependymomas. Astrocytomas and ependymomas can occur in all areas of the brain and spinal cord in both children and adults. Oligodendrogliomas typically occur in the cerebral hemispheres of adults. Other brain tumors are meningiomas, ependymomas, pineal region tumors, choroid plexus tumors, neuroepithelial tumors, embryonal tumors, peripheral neuroblastic tumors, tumors of cranial nerves, tumors of the hemopoietic system, germ cell tumors, and tumors of the stellar region.

[0213] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.

[0214] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0215] As used herein, the terms “protein" and “polypeptide" are used interchangeably to designate a series of amino acid residues, coimected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphory lated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. " Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "poly peptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0216] The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, e.g. adenosine degradation, of the full length polypeptide, e.g., one of SEQ ID NO: 1-4 and 100-106, e.g. as measured using a colorimetric assay for adenosine degradation. Such assays are commercially available, e.g., the Adenosine Deaminase (ADA) Activity Assay Kit (Colorimetric) (ab204695). Conservative substitution variants that maintain the activity of a wildty pe ADE will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of106Attorney Docket No: 030258-000107WOPTthe wildtype is guided by, for example, sequence alignment with ADE homologs or paralogs from other species. Amino acids that are identical between ADE homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant. Variants, fragments, and / or fusion proteins can be tested for activity, for example, by contacting a variant with adenosine as described herein. Further discussion of the structure of ADEs can be found, e.g. in Long et al. Biochem Pharmacol. 2008 Feb 2;75(8):1588-600; Park et al. Cell Mol Life Sci. 2008 Sep:65(18):2875-96; Zaialov et al. J Biol Chem.2010 Feb 9;285(16): 12367-77; and Ma et al. Acta Crystallogr D Struct Biol. 2022 Jan L78(Pt 1): 91 -103, which are incorporated by reference herein in their entireties.

[0217] In some embodiments of any of the aspects, a nucleic acid or polypeptide has a stated sequence identity to the entire length of the reference sequence. In some embodiments of any of the aspects, a domain of a nucleic acid or polypeptide has a stated sequence identity to the entire length of the reference sequence.

[0218] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" w here the alteration results in the substitution of an ammo acid w ith a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0219] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity' characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confinn that a desired activity, e.g. adenosine degrading activity and specificity of a native or reference polypeptide is retained.

[0220] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger. in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W). Met (M); (2) uncharged polar: Gly (G), Ser (S). Thr (T), Cys (C), Tyr (Y). Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on107Attorney Docket No: 030258-000107WOPTcommon side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr. Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai. into lie or into Leu.

[0221] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide can be a functional fragment of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a polypeptide which retains at least 50% of the wild-type reference polypeptide's activity according to the following assay. For example, activity of the polypeptides described herein can be measured using the Adenosine Deaminase (ADA) Activity Assay Kit (Colorimetric) (ab204695), an assay in which inosine formed from the breakdown of adenosine is detected via a multi-step reaction, resulting in the formation of an intermediate that reacts with the ADA convertor and developer to generate uric acid that can be easily quantified at OD293 nm. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0222] In some embodiments, the polypeptide described herein can be a variant of a polypeptide sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant." as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide -encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a protein or fragment thereof that retains activity of the native or reference polypeptide. A wide variety of, for example, PCR-based, site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan to generate and test artificial variants.

[0223] A variant amino acid or DNA sequence can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).108Attorney Docket No: 030258-000107WOPT

[0224] A variant amino acid sequence can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to a native or reference sequence. As used herein, "similarity" refers to an identical amino acid or a conservatively substituted amino acid, as described herein. Accordingly, the percentage of “sequence similarity’’ is the percentage of amino acids which is either identical or conservatively changed: e.g., “sequence similarity” = (% sequence identity)+(% conservative changes). It should be understood that a sequence that has a specified percent similarity to a reference sequence necessarily encompasses a sequence with the same specified percent identity to that reference sequence. The skilled person will be aware of various computer programs, using different mathematical algorithms, that are available to determine the identity or similarity between two sequences. For instance, use can be made of a computer program employing the Needleman and Wunsch algorithm (Needleman et al. (1970)); the GAP program in the Accelrys GCG software package (Accelerys Inc.. San Diego U. S. A.); the algorithm of E. Meyers and W. Miller (Meyers et al. (1989)) which has been incorporated into the ALIGN program (version 2.0); or more preferably the BLAST (Basic Local Alignment Tool using default parameters); see e.g., US Patent 10,023,890, the content of which is incorporated by reference herein in its entirety.

[0225] Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally w ith serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0226] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established. Alterations of the original amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by109Attorney Docket No: 030258-000107WOPTKhudyakov ct al. “Artificial DNA: Methods and Applications” CRC Press, 2002; Braman “In Vitro Mutagenesis Protocols” Springer, 2004; and Rapley “The Nucleic Acid Protocols Handbook” Springer 2000; which are herein incorporated by reference in their entireties. In some embodiments, a polypeptide as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.

[0227] As used herein, the tenn “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[0228] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the molecules described herein and / or to the translation of mRNA into a polypeptide.

[0229] " Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g.5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0230] As used herein, “vector” means a construct, which is capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, bacterial artificial chromosomes (BAC), Pl-derived artificial chromosome (PAC), yeast artificial chromosome (YAC), human artificial chromosome (HAC), DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. Expression can be achieved in any appropriate host cell that has been transformed, transfected or infected with the expression vector. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably the host cells employed are E. coli, yeast or a mammalian cell, such as a primary cell or cell line such as COS or CHO.110Attorney Docket No: 030258-000107WOPT

[0231] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0232] In some embodiments of any of the aspects, a polypeptide described herein is a fusion protein or fusion polypeptide. As used herein, a “fusion protein’’ or “fusion polypeptide” refers to a non-naturally occurring protein or polypeptide comprising one or more domains from or derived from one source (e.g.. synthetic or a first naturally occurring protein) in combination with one or more domains from or derived from another source (e.g., synthetic or a second naturally occurring protein) to create a synthetic protein. Such domains include naturally occurring domains, as well as mutated or engineered domains derived from naturally occurring domains, or portions of a naturally occurring domain having a desired activity.

[0233] In some embodiments of any of the aspects, the nucleic acid or polypeptide described herein is exogenous. In some embodiments of any of the aspects, the nucleic acid or polypeptide described herein is ectopic. In some embodiments of any of the aspects, the nucleic acid or polypeptide described herein is not endogenous.

[0234] The term "exogenous" refers to a substance present in a cell or molecule other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell. As used herein, “ectopic” refers to a substance that is found in an unusual location and / or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and / or at a different time. Ectopic also includes a substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural enviromnent.

[0235] In some embodiments of any of the aspects, a vector, viral vector, or virus particle comprises at least one functional heterologous gene. As used herein, the term “heterologous” refers to that which is not endogenous to, or naturally occurring in, a referenced sequence, molecule (including e.g., a111Attorney Docket No: 030258-000107WOPTprotein), virus, cell, tissue, or organism. For example, a heterologous sequence of the present disclosure can be derived from a different species, or from the same species but substantially modified from an original form. Also for example, a nucleic acid sequence that is not normally expressed in a cell or a virus is a heterologous nucleic acid sequence with regard to that cell or virus. The term "heterologous" can refer to DNA, RNA, or protein that does not occur naturally as part of the organism in which it is present or which is found in a location or locations in the genome that differ from that in which it occurs in nature. It is DNA, RNA, or protein that is not endogenous to the virus or cell and has been artificially introduced into the virus or cell.

[0236] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. cancer. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a cancer. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is. “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to w hat would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (z.e., not w orsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0237] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry’. The phrase "pharmaceutically acceptable" is employ ed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate ith a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in or within nature.

[0238] As used herein, the term "administering." refers to the placement of a composition or compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the composition, compound, or metabolite thereof at a desired site. Pharmaceutical112Attorney Docket No: 030258-000107WOPTcompositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g.. by a medical professional and / or the subject being treated.

[0239] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, transfection, transduction, perfusion, injection, or other delivery method known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g.. an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine. A cell in a subject can be contacted with any of the compositions described herein described herein following administration of a composition as described herein to the subject. “Contacting” of a cell can be performed in vitro, ex vivo, or in vivo.

[0240] As the term is used herein, “transfection” refers to the introduction of nucleic acid into a cell (e.g., for the purpose of propagation and / or expression of the nucleic acid by the cell). Examples of methods of transfection are electroporation, calcium phosphate, lipofection, and viral infection utilizing a viral vector. Often nucleic acid is introduced into a cell in expressible form. That means that the nucleic acid is in the appropriate context of regulatory sequences such that the cellular machinery will recognize it and process it (e.g., transcribe RNA from DNA. translate protein from RNA). In one embodiment, a nucleic acid is in expressible form when it is inserted into an expression vector in the proper orientation to confer expression.

[0241] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference or a p-value of less than 0.05.

[0242] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0243] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0244] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0245] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.113Attorney Docket No: 030258-000107WOPT

[0246] As used herein, the term “corresponding to’’ refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.

[0247] All reference to NCBI and UNIPROT database information such as Ref Seq's and Gene ID’s herein, refer to the sequence information present in the databases as of March 20, 2026.

[0248] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in tire practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0249] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in. or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0250] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to tire particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common tenns in cell biology, immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc.. 1995 (ISBN 1-56081-569-8); Immunology' by Werner Luttmann, published by Elsevier. 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.). W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XL published by Jones & Bartlett Publishers. 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook. Molecular Cloning: A Laboratory Manual. 4th ed.. Cold Spring Harbor Laboratory' Press, Cold Spring Harbor, N. Y., USA (2012) (ISBN114Attorney Docket No: 030258-000107WOPT1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.). John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735. 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0251] In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identify of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0252] Other terms are defined herein within the description of the various aspects of the invention. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0253] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on die information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0254] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due115Attorney Docket No: 030258-000107WOPTto biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0255] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0256] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:1. A composition comprising a nucleic acid encoding a fusion protein, the fusion protein comprising:a) a signal peptide (SP) polypeptide; andb) an Adenosine Degrading Enzyme (ADE) polypeptide.2. The composition of any one of the preceding paragraphs, wherein the ADE polypeptide is mammalian ADE.3. The composition of any one of the preceding paragraphs, wherein the ADE polypeptide is human ADE.4. The composition of any one of the preceding paragraphs, wherein the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL). 5. The composition of any one of the preceding paragraphs, wherein the ADE polypeptide is ADKS.6. The composition of any one of the preceding paragraphs, wherein the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL), and wherein the nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6.7. The composition of any one of the preceding paragraphs, wherein the ADE polypeptide is Adenosine Kinase Short isofomi (ADKS) or Adenosine Kinase Long isoform (ADKL), and wherein the nucleic acid sequence comprises the sequence of SEQ ID NO: 6.8. The composition of any one of the preceding paragraphs, wherein the nucleic acid encodes a codon-optimized ADKS. andwherein the codon-optimized nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6.116Attorney Docket No: 030258-000107WOPTThe composition of any one of the preceding paragraphs, wherein the nucleic acid encodes a codon-optimized ADKS, andwherein the codon-optimized nucleic acid sequence comprises the sequence of SEQ ID NO: 6. The composition of any one of the preceding paragraphs, wherein the ADE polypeptide is an adenosine deaminase 1 (ADA1) or an adenosine deaminase 2 (ADA2).The composition of any one of the preceding paragraphs, wherein the ADE polypeptide is ADA1.The composition of any one of the preceding paragraphs, wherein the nucleic acid encodes AD Al, andwherein the nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10.The composition of any one of the preceding paragraphs, wherein the nucleic acid encodes AD Al, andwherein the nucleic acid sequence comprises the sequence of SEQ ID NO: 10.The composition of any one of the preceding paragraphs, wherein the nucleic acid encodes a codon-optimized AD Al, andwherein the codon-optimized nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10.The composition of any one of the preceding paragraphs, wherein the nucleic acid encodes a codon-optimized AD Al, andwherein the codon-optimized nucleic acid sequence comprises the sequence of SEQ ID NO:The composition of any one of the preceding paragraphs, wherein the ADE has a Km for adenosine of 1 pM or less.The composition of any one of the preceding paragraphs, wherein the ADE has a Km for adenosine witiiin the range of 1 nM to 1 pM.A composition comprising a nucleic acid encoding a fusion protein, the fusion protein comprising:a) a signal peptide (SP): andb) a polypeptide comprising a sequence having at least 80% sequence identity to the sequence of one of SEQ ID NOs: 1-4 and 100-106.The composition of any one of the preceding paragraphs, wherein the polypeptide comprises the sequence of one of SEQ ID NO: 1-4 and 100-106.The composition of any one of the preceding paragraphs, wherein the SP is selected from the group of:117Attorney Docket No: 030258-000107WOPTHuman intcrlcukin-2 (IL-2), Human tissue plasminogen activator tPA (PLAT), Human Ig K-chain leader (IgK / IGKC), Mouse Ig K-chain leader (mlgK), Human albumin (ALB), HSV-1 glycoprotein D (gD / US6), HSV-1 glycoprotein B (gB I UL27), Human growth hormone (GH1), CD33, Human preprotrypsin trypsinogen signal peptide, BM40 / SPARC (osteonectin), Gaussia luciferase, and Human transferrin (TF).The composition of any one of the preceding paragraphs, wherein the SP is selected from the group consisting of:IgG K chain signal peptide; IgG heavy chain signal peptide: human IgG K chain signal peptide; human IgG heavy chain signal peptide; murine Ig K chain signal peptide; interleukin-2 signal peptide; interleukin-7 signal peptide; interleukin- 12 signal peptide; granulocyte colony -stimulating factor signal peptide; insulin signal peptide; growth hormone signal peptide; prolactin signal peptide; a secreted plasma protein signal peptide; albumin signal peptide; transferrin signal peptide; fibrinogen signal peptide; tissue plasminogen activator signal peptide; epidermal growth factor signal peptide; vascular endothelial growth factor signal peptide; fibroblast growth factor signal peptide; influenza hemagglutinin signal peptide; HIV gp120 signal peptide; VSV glycoprotein signal peptide; SARS-CoV-2 spike signal peptide; a yeast leader sequence; a-mating factor prepro leader; invertase signal peptide; acid phosphatase signal peptide; killer toxin signal peptide; GP67 signal peptide; honeybee melittin signal peptide; a synthetic Ig leader; an optimized tPA variant; a hybrid signal peptide; Interleukin- 1; fibroblast growth factor 2; and galectin-1.The composition of any one of the preceding paragraphs, wherein the SP is an Interleukin -2 (IL-2) SP.The composition of any one of the preceding paragraphs, wherein the nucleic acid is operably linked to a promoter.The composition of any one of the preceding paragraphs, wherein the nucleic acid is operably linked to a viral promoter.The composition of any one of the preceding paragraphs, wherein the viral promoter is selected from the group consisting of:a Cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, a Herpes Simplex Virus (HSV) promoters (HSV-1 US 12, IE, and IE 4 / 5).The composition of any one of the preceding paragraphs, wherein the viral promoter is CMVA or CMVB.The composition of any one of the preceding paragraphs, wherein the nucleic acid is operably linked to a tissue-specific promoter.118Attorney Docket No: 030258-000107WOPTThe composition of any one of the preceding paragraphs, wherein the tissue-specific promoter is selected from the group consisting of:stromelysin 3 promoter, the surfactant protein A promoter, the secretory leukoprotease inhibitor (SLPI) promoter, the tyrosinase promoter, which is specific for melanoma cells, the stress inducible grp78 / BiP promoter, the AP2 adipose enhancer, the a-1 antitrypsin transthyretin promoter, the interleukin- 10 promoter, which is specific for glioblastoma multiform cells, the c-erbB-2 promoter, the a-B-crystallin / hcat shock protein promoter, the basic fibroblast growth factor promoter, the epidermal growth factor receptor promoter, the mucin-like glycoprotein (DF3. MUC1) promoter, the mtsl promoter, the nestin promoter-enhancer, the NSE promoter, the somatostatin receptor promoter, the c-erbB-3 and c-erbB-2 promoters, the c-erbB4 promoter, the thyroglobulin promoter, the a-fetoprotein promoter, and the villin promoter.The composition of any one of the preceding paragraphs, wherein the nucleic acid is a DNA molecule, an expression cassette, a transgene, an RNA molecule, or an mRNA molecule.A composition comprising a vector comprising the nucleic acid of any one of the preceding paragraphs.The composition of any one of the preceding paragraphs, wherein the vector is a plasmid, cosmid, or viral vector.The composition of any one of the preceding paragraphs, wherein the viral vector is selected from the group consisting of:recombinant retrovirus, a recombinant herpes simplex virus, an adenovirus, an adeno-associated virus, an alphavirus, an oncolytic virus, and a lentivirus.A lipid nanoparticle (LNP) comprising the composition of any one of the preceding paragraphs.A virus particle comprising the composition of any one of the preceding paragraphs.The viral vector or virus particle of any one of the preceding paragraphs, wherein the virus is a DNA virus or RNA virus.The viral vector or virus particle of any one of the preceding paragraphs, wherein the virus is an oncolytic virus.The viral vector or virus particle of any one of the preceding paragraphs, wherein the virus is an oncolytic DNA virus selected from the group consisting of:herpes simplex virus (HSV), adenovirus, and vaccinia virus.The viral vector or virus particle of any one of the preceding paragraphs, wherein the virus is an oncolytic RNA virus selected from the group consisting of:119Attorney Docket No: 030258-000107WOPTreovirus, vesicular stomatitis virus (VSV), poliovirus, Seneca Valley Virus (SVV) or senecavirus A (SVA), measles virus (MV), flavivirus, and Semliki Forest virus. The viral vector or virus particle of any one of the preceding paragraphs, wherein the oncolytic virus is herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2).The viral vector or virus particle of any one of the preceding paragraphs, wherein the HSV is a HSV variant selected from the group consisting of:G207, G47A, MG18L, A68H-6. R3616, and R47A.The viral vector or virus particle of any one of the preceding paragraphs, wherein the HSV is a G47A variant.The viral vector or virus particle of any one of the preceding paragraphs, comprising an inactivating mutation of one, tw o. three, or four genes selected from the group of:Infected Cell Protein 34.5 gene (y34.5 / ICP34.5). Infected Cell Protein 47 gene (ICP47), Infected Cell Protein 6 gene (ICP6), and Unique Short 3 gene (US3).The viral vector or virus particle of any one of the preceding paragraphs, comprising an inactivating mutation of ICP47.The viral vector or virus particle of any one of the preceding paragraphs, comprising an HSV genome and wherein the nucleic acid is inserted into the HSV genome at the ICP6 locus. A polypeptide encoded by the nucleic acid of any one of the preceding paragraphs.A system comprising a mammalian cell infected with a viral particle of any one of the preceding paragraphs.The system of paragraph 55, wherein the mammalian cell secretes the ADE.The system of paragraphs of any one of the preceding paragraphs, wherein the mammalian cell is selected from the group consisting of:mesenchymal stem cells (MSCs), T cells, and neural stem cells.A serum isolated from a mammalian cell infected with a viral particle of any one of the preceding paragraphs.A method to reduce the level of extracellular adenosine, the method comprising contacting a cell with die composition of any one of the preceding paragraphs.A method for reducing extracellular adenosine in a subject in need thereof, the method comprising administering the composition of any one of the preceding paragraphs to the subject.The method of paragraph 60, wherein the subject is a subject in need of treatment for atrial fibrillation, syncope, hypoxia, sleep apnea, altitude hypoxia, hypertension, and heart failure.120Attorney Docket No: 030258-000107WOPTA method for treating cancer in a subject in need thereof, the method comprising administering the composition of any one of the preceding paragraphs to the subject.The method of paragraph 62, wherein the cancer is a solid tumor.The method of any one of paragraphs 59-63, wherein the cancer is selected from the group consisting of:a pancreatic tumor, a primary tumor of the nervous system, a central nervous system (CNS) tumor, a peripheral nervous system (PNS) tumor, a primary central nervous system lymphoma (PCNSL). a brainstem glioma, and a combination thereof.The method of any one of paragraphs 59-63, wherein the primary tumor of the nervous system is selected from the group consisting of:diffuse glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic mixed oligoastrocytoma, glioblastoma, ependymoma, and anaplastic ependymoma.The method of any one of paragraphs 54-56, wherein the pancreatic tumor is selected from the group consisting of:pancreatic ductal adenocarcinoma (PDAC). pancreatic neuroendocrine tumor, solid pscudopapillary neoplasm, acinar cell carcinoma, pancreatoblastoma. pancreatic lymphoma.The method of any one of the preceding paragraphs, wherein the composition is administered by intratumoral, intraperitoneal, or intravenous administration.The method of any one of the preceding paragraphs, wherein the composition is administered directly into the solid tumor.The method of any one of the preceding paragraphs, wherein the composition is administered intracranially.The method of any one of the preceding paragraphs, wherein the composition is administered by multiple inoculations.The method of any one of the preceding paragraphs, wherein the composition is administered at a dose of 1 x 103pfu to 1*10’ " pfu.The method of any one of the preceding paragraphs, wherein the composition is administered at a dose of 1 * 103pfu to 1 * 107pfu.The method of any one of the preceding paragraphs, wherein the composition is administered at a dose of 1 * 103pfu to 1 * 106pfu.The method of any one of the preceding paragraphs, wherein the composition is administered at a dose of at least 1* 103pfu.121Attorney Docket No: 030258-000107WOPTThe method of any one of the preceding paragraphs, wherein the composition is administered at a dose of at least lx10’ pfu.The method of any one of the preceding paragraphs, wherein the composition is administered as mono- or combination therapy.The composition of any one of the preceding paragraphs, for use in treating cancer.The composition of paragraph 68, wherein the cancer is a solid tumor.The composition of any one of paragraphs 68-69, wherein the cancer is selected from the group consisting of:a pancreatic tumor, a primary tumor of the nervous system, a central nervous system (CNS) tumor, a peripheral nervous system (PNS) tumor, a primary central nervous system lymphoma (PCNSL). a brainstem glioma, and a combination thereof.The composition of any one of paragraphs 68-70, wherein the primary tumor of the nervous system is selected from the group consisting of:diffuse glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic mixed oligoastrocytoma, glioblastoma, ependymoma, and anaplastic ependymoma.The composition of any one of paragraphs 68-70, wherein the pancreatic tumor is selected from the group consisting of:pancreatic ductal adenocarcinoma (PDAC), pancreatic neuroendocrine tumor, solid pseudopapillarv neoplasm, acinar cell carcinoma, pancreatoblastoma. pancreatic lymphoma.The composition of any one of paragraphs 68-72, wherein the composition is administered by intratumoral, intraperitoneal, or intravenous administration.The composition of any one of paragraphs 68-72, wherein the composition is administered directly into the solid tumor.The composition of any one of paragraphs 68-72, wherein the composition is administered intracranially.The composition of any one of paragraphs 68-75, wherein the composition is administered by multiple inoculations.The composition of any one of paragraphs 68-76, wherein the composition is administered at a dose of 1×103pfu to 1×1010pfu.The composition of any one of paragraphs 68-76, wherein the composition is administered at a dose of R IO’’ pfu to R IO pfu.The composition of any one of paragraphs 68-76, wherein the composition is administered at a dose of 1×103pfu to 1×106pfu.122Attorney Docket No: 030258-000107WOPT80. The composition of any one of paragraphs 68-76, wherein the composition is administered at a dose of at least 1 * 103pfu.81. The composition of any one of paragraphs 68-76, wherein the composition is administered at a dose of at least 1 x 105pfu.82. The composition of any one of paragraphs 68-81, wherein the composition is administered as mono- or combination therapy.

[0257] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESExample 1

[0258] The development of oncolytic viruses (OV) is iterative and extends from our first description of a genetically -engineered OV (1) to our G-series of oncolytic herpes simplex viruses (oHSV) that we developed (2). The current clinically-relevant G-series iteration, G47A (3) has deletions in ICP34.5 and ICP6 as well as a deletion of ICP47 which overcomes inhibition of MHC class I presentation setting the stage for improved oncolytic immunovirotherapy against glioblastoma (GBM) and other cancers and it has been demonstrated that sequential inoculations of G47A prolonged the 1 year survival of patients with recurrent GBM from a historical 15% to 84% leading to conditional approval for clinical use by Japan Ministry of Health, Labour and Welfare (MHLW) (4, 5) (6). Further therapeutic improvements are clearly needed but because G47A has been shown to be very safe after high-dose inoculation in human brain (4. 5) we are using it as a base virus to develop the next and improved virus iteration.

[0259] GBM is a fatal cancer of the brain with only a 3-4% 5 year survival showing no change over multiple decades of numerous clinical trials (7). One feature that has stymied progress is the presence of glioblastoma stem-like cells (GSCs), which drive intratumoral heterogeneity and evolution, tumor progression, tumor microenvironment (TME) constituents and immunophenotypes, and therapy evasion (8). In addition, GSCs provide more representative GBM models for evaluation of experimental therapeutics (9, 10). While immune checkpoint inhibitor (ICI) clinical trials show efficacy in some peripheral tumors, several concerning factors for extending their use in GBM have emerged: 1, Not even all peripheral tumors respond. (11, 12); 2, Some tumors need immune stimulation in addition to ICI (12); 3, Most GBMs do not have the high mutational load of responsive melanomas and lung cancers (13); 4, GBM is very immunosuppressive (14); 5. Efficacy of ICI therapy has not been demonstrated in GBM patients123Attorney Docket No: 030258-000107WOPT(15)(16). Indeed, even double checkpoint blockade has not had a significant effect on longevity for GBM (17)(18, 19), with the exception of the rare GBMs with germline bMMRD mutations (20). Therefore, we are using this iterative process of an intracranially -safe oHSV to continually improve outcomes. We note that studies by Ott et al. (21) demonstrated that the most expressed immune inhibitor in human GBM is the adenosine (ADO) pathway, including the receptor A2aR. Coy et al. have also shown that GBMs strongly express ADO pathway ectonucleotidases CD39 and CD73 (22). At a single-cell level. CD39 is primarily expressed by microglia and CD73 by the GBM tumor cells and there is a strong spatial proximity between these two which is associated with a poor outcome (22). Of importance, this increased level of ADO expression also exists for multiple other cancers (23).

[0260] It would be beneficial to be able to reduce the levels of ADO in the tumor microenvironment (TME) in order to improve immunovirotherapy against GBM. ADO-receptor inhibitors and multiple inhibitors of ADO-pathway enzymes (Figure 1) such as CD39 and CD73 have been developed but thus far have had little clinical impact. An alternative strategy would be to degrade extracellular ADO (eADO) but no such clinically -effective strategy has been developed. Moreover, systemic reduction of ADO could have deleterious side-effects. In contrast, an intratumoral oncolytic virus could be use...

Claims

Attorney Docket No: 030258-000107WOPTWhat is claimed herein is:

1. A composition comprising a nucleic acid encoding a fusion protein, the fusion protein comprising:a) a signal peptide (SP) polypeptide; andb) an Adenosine Degrading Enzyme (ADE) polypeptide.

2. The composition of any one of the preceding claims, wherein die ADE poly peptide is mammalian ADE.

3. The composition of any one of the preceding claims, wherein die ADE polypeptide is human ADE.

4. The composition of any one of the preceding claims, wherein the ADE polypeptide is Adenosine Kinase Short isofomi (ADKS) or Adenosine Kinase Long isoform (ADKL).

5. The composition of any one of the preceding claims, wherein the ADE polypeptide is ADKS.

6. The composition of any one of the preceding claims, wherein the ADE polypeptide is Adenosine Kinase Short isofomi (ADKS) or Adenosine Kinase Long isoform (ADKL), and wherein the nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6.

7. The composition of any one of the preceding claims, wherein the ADE polypeptide is Adenosine Kinase Short isoform (ADKS) or Adenosine Kinase Long isoform (ADKL), and wherein the nucleic acid sequence comprises the sequence of SEQ ID NO: 6.

8. The composition of any one of the preceding claims, wherein the nucleic acid encodes a codon- optimized ADKS, andwherein the codon-optimized nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6.

9. The composition of any one of the preceding claims, wherein the nucleic acid encodes a codon- optimized ADKS, andwherein the codon-optimized nucleic acid sequence comprises the sequence of SEQ ID NO: 6.

10. The composition of any one of the preceding claims, wherein die ADE polypeptide is an adenosine deaminase 1 (ADA1) or an adenosine deaminase 2 (ADA2).

11. The composition of any one of the preceding claims, wherein die ADE polypeptide is ADA1.

12. The composition of any one of the preceding claims, wherein the nucleic acid encodes ADA1, andwherein the nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10.139Attorney Docket No: 030258-000107WOPT13. The composition of any one of the preceding claims, wherein the nucleic acid encodes ADA1, andwherein die nucleic acid sequence comprises die sequence of SEQ ID NO: 10.

14. The composition of any one of the preceding claims, wherein the nucleic acid encodes a codon- optimized ADA1, andwherein the codon-optimized nucleic acid sequence comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10.

15. The composition of any one of the preceding claims, wherein the nucleic acid encodes a codon- optimized ADA1, andwherein the codon-optimized nucleic acid sequence comprises the sequence of SEQ ID NO: 10.

16. The composition of any one of the preceding claims, wherein the ADE has a Km for adenosine of 1 pM or less.

17. The composition of any one of the preceding claims, wherein the ADE has a Km for adenosine within the range of 1 nM to 1 μM.

18. A composition comprising a nucleic acid encoding a fusion protein, the fusion protein comprising:a) a signal peptide (SP); andb) a polypeptide comprising a sequence having at least 80% sequence identity to the sequence of one of SEQ ID NOs: 1-4 and 100-106.

19. The composition of any one of the preceding claims, wherein die polypeptide comprises the sequence of one of SEQ ID NO: 1-4 and 100-106.

20. The composition of any one of the preceding claims, wherein the SP is selected from the group of:Human interleukin-2 (IL-2), Human tissue plasminogen activator tPA (PLAT), Human Ig K-chain leader (IgK / IGKC). Mouse Ig K-chain leader (mlgK), Human albumin (ALB), HSV-1 glycoprotein D (gD / US6), HSV-1 glycoprotein B (gB / UL27), Human growth hormone (GH1). CD33, Human preprotrypsin trypsinogen signal peptide, BM40 / SPARC (osteonectin), Gaussia luciferase, and Human transferrin (TF).

21. The composition of any one of the preceding claims, wherein the SP is selected from the group consisting of:IgG K chain signal peptide; IgG heavy chain signal peptide: human IgG K chain signal peptide; human IgG heavy chain signal peptide; murine Ig K chain signal peptide; interleukin-2 signal peptide; interleukin-7 signal peptide; interleukin- 12 signal140Attorney Docket No: 030258-000107WOPTpeptide; granulocyte colony -stimulating factor signal peptide; insulin signal peptide; growth hormone signal peptide; prolactin signal peptide; a secreted plasma protein signal peptide; albumin signal peptide; transferrin signal peptide; fibrinogen signal peptide; tissue plasminogen activator signal peptide; epidermal growth factor signal peptide; vascular endothelial growth factor signal peptide; fibroblast growth factor signal peptide; influenza hemagglutinin signal peptide; HIV gpl20 signal peptide; VSV glycoprotein signal peptide; SARS-CoV-2 spike signal peptide; a yeast leader sequence; a-mating factor prepro leader; invertase signal peptide; acid phosphatase signal peptide; killer toxin signal peptide; GP67 signal peptide; honeybee melittin signal peptide; a synthetic Ig leader; an optimized tPA variant; a hybrid signal peptide; Interleukin- 1|3; fibroblast growth factor 2; and galectin-1.

22. The composition of any one of the preceding claims, wherein the SP is an Interleukin -2 (IL- 2) SP.

23. The composition of any one of the preceding claims, wherein the nucleic acid is operably linked to a promoter.

24. The composition of any one of the preceding claims, wherein the nucleic acid is operably linked to a viral promoter.

25. The composition of any one of the preceding claims, wherein the viral promoter is selected from the group consisting of:a Cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, a Herpes Simplex Virus (HSV) promoters (HSV-1 US 12, IE, and IE 4 / 5).

26. The composition of any one of the preceding claims, wherein the viral promoter is CMVA or CMVB.

27. The composition of any one of the preceding claims, wherein the nucleic acid is operably linked to a tissue-specific promoter.

28. The composition of any one of the preceding claims, wherein the tissue-specific promoter is selected from the group consisting of:stromelysin 3 promoter, the surfactant protein A promoter, the secretory leukoprotease inhibitor (SLPI) promoter, the tyrosinase promoter, which is specific for melanoma cells, the stress inducible grp78 / BiP promoter, the AP2 adipose enhancer, the a-1 antitrypsin transthyretin promoter, the interleukin- 10 promoter, which is specific for glioblastoma multiform cells, the c-erbB-2 promoter, the a-B-crystallin / hcat shock protein promoter, the basic fibroblast growth factor promoter, the epidermal growth factor receptor promoter, the mucin-like glycoprotein (DF3. MUC1) promoter, the mtsl promoter, the nestin promoter-enhancer, the NSE promoter, the somatostatin receptor141Attorney Docket No: 030258-000107WOPTpromoter, the c-crbB-3 and c-crbB-2 promoters, the c-crbB4 promoter, the thyroglobulin promoter, the a-fetoprotein promoter, and the villin promoter.

29. The composition of any one of the preceding claims, wherein the nucleic acid is a DNA molecule, an expression cassette, a transgene, an RNA molecule, or an mRNA molecule.

30. A composition comprising a vector comprising the nucleic acid of any one of the preceding claims.

31. The composition of any one of the preceding claims, wherein the vector is a plasmid, cosmid, or viral vector.

32. The composition of any one of the preceding claims, wherein the viral vector is selected from the group consisting of:recombinant retrovirus, a recombinant herpes simplex virus, an adenovirus, an adeno-associated virus, an alphavirus, an oncolytic virus, and a lentivirus.

33. A lipid nanoparticle (LNP) comprising the composition of any one of the preceding claims.

34. A virus particle comprising the composition of any one of the preceding claims.

35. The viral vector or virus particle of any one of the preceding claims, wherein the virus is a DNA virus or RNA virus.

36. The viral vector or virus particle of any one of the preceding claims, wherein the virus is an oncolytic virus.

37. The viral vector or virus particle of any one of the preceding claims, wherein the virus is an oncolytic DNA virus selected from the group consisting of:herpes simplex virus (HSV), adenovirus, and vaccinia virus.

38. The viral vector or virus particle of any one of the preceding claims, wherein the virus is an oncolytic RNA virus selected from the group consisting of:reovirus. vesicular stomatitis virus (VSV), poliovirus, Seneca Valley Virus (SVV) or senecavirus A (SVA), measles virus (MV), flavivirus, and Semliki Forest virus.

39. The viral vector or virus particle of any one of the preceding claims, wherein the oncolytic virus is herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2).

40. The viral vector or virus particle of any one of the preceding claims, wherein the HSV is a HSV variant selected from the group consisting of:G207. G47A, MG18L, A68H-6. R3616, and R47A.

41. The viral vector or virus particle of any one of the preceding claims, wherein the HSV is a G47A variant.142Attorney Docket No: 030258-000107WOPT42. The viral vector or virus particle of any one of the preceding claims, comprising an inactivating mutation of one, two, three, or four genes selected from the group of:Infected Cell Protein 34.5 gene (y34.5 / ICP34.5), Infected Cell Protein 47 gene (ICP47), Infected Cell Protein 6 gene (ICP6), and Unique Short 3 gene (US3).

43. The viral vector or virus particle of any one of the preceding claims, comprising an inactivating mutation of ICP47.

44. The viral vector or virus particle of any one of the preceding claims, comprising an HSV genome and wherein the nucleic acid is inserted into the HSV genome at the ICP6 locus.

45. A polypeptide encoded by the nucleic acid of any one of the preceding claims.

46. A system comprising a mammalian cell infected with a viral particle of any one of the preceding claims.

47. The system of claim 46, wherein the mammalian cell secretes the ADE.

48. The system of claims of any one of the preceding claims, wherein the mammalian cell is selected from the group consisting of:mesenchymal stem cells (MSCs), T cells, and neural stem cells.

49. A serum isolated from a mammalian cell infected with a viral particle of any one of the preceding claims.

50. A method to reduce the level of extracellular adenosine, the method comprising contacting a cell with the composition of any one of the preceding claims.

51. A method for reducing extracellular adenosine in a subject in need thereof, the method comprising administering the composition of any one of the preceding claims to the subject.

52. The method of claim 51, wherein the subject is a subject in need of treatment for atrial fibrillation, syncope, hypoxia, sleep apnea, altitude hypoxia, hypertension, and heart failure.

53. A method for treating cancer in a subject in need thereof, the method comprising administering the composition of any one of the preceding claims to the subject.

54. The method of claim 53, wherein the cancer is a solid tumor.

55. The method of any one of claims 53-54, wherein the cancer is selected from the group consisting of:143Attorney Docket No: 030258-000107WOPTa pancreatic tumor, a primary tumor of the nervous system, a central nervous system (CNS) tumor, a peripheral nervous system (PNS) tumor, a primary central nervous system lymphoma (PCNSL), a brainstem glioma, and a combination thereof.

56. The method of any one of claims 53-55, wherein the primary tumor of the nervous system is selected from the group consisting of:diffuse glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic mixed oligoastrocytoma, glioblastoma, ependymoma, and anaplastic ependymoma.

57. The method of any one of claims 53-55. wherein the pancreatic tumor is selected from the group consisting of:pancreatic ductal adenocarcinoma (PDAC), pancreatic neuroendocrine tumor, solid pseudopapillary neoplasm, acinar cell carcinoma, pancreatoblastoma, pancreatic lymphoma.

58. The method of any one of the preceding claims, wherein the composition is administered by intratumoral. intraperitoneal, or intravenous administration.

59. The method of any one of the preceding claims, wherein the composition is administered directly into the solid tumor.

60. The method of any one of the preceding claims, wherein the composition is administered intracranially.

61. The method of any one of the preceding claims, wherein the composition is administered by multiple inoculations.

62. The method of any one of the preceding claims, w herein the composition is administered at a dose of 1x103pfu to 1x1010pfu.

63. The method of any one of the preceding claims, wherein the composition is administered at a dose of 1 * 103pfu to 1 x 107pfu.

64. The method of any one of the preceding claims, wherein the composition is administered at a dose of 1 x 103pfu to 1 x 106pfu.

65. The method of any one of the preceding claims, wherein the composition is administered at a dose of at least 1x 103pfu.

66. The method of any one of the preceding claims, wherein the composition is administered at a dose of at least 1x10’ pfu.

67. The method of any one of the preceding claims, wherein the composition is administered as mono- or combination therapy.

68. The composition of any one of the preceding claims, for use in treating cancer.144Attorney Docket No: 030258-000107WOPT69. The composition of claim 68, wherein the cancer is a solid tumor.

70. The composition of any one of claims 68-69, wherein the cancer is selected from the group consisting of:a pancreatic tumor, a primary tumor of the nervous system, a central nervous system (CNS) tumor, a peripheral nervous system (PNS) tumor, a primary central nervous system lymphoma (PCNSL). a brainstem glioma, and a combination thereof.

71. The composition of any one of claims 68-70, wherein the primary tumor of the nervous system is selected from the group consisting of:diffuse glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic mixed oligoastrocytoma, glioblastoma, ependymoma, and anaplastic ependymoma.

72. The composition of any one of claims 68-70, wherein the pancreatic tumor is selected from the group consisting of:pancreatic ductal adenocarcinoma (PDAC), pancreatic neuroendocrine tumor, solid pseudopapillary neoplasm, acinar cell carcinoma, pancreatoblastoma. pancreatic lymphoma.

73. The composition of any one of claims 68-72. wherein the composition is administered by intratumoral. intraperitoneal, or intravenous administration.

74. The composition of any one of claims 68-72, wherein the composition is administered directly into the solid tumor.

75. The composition of any one of claims 68-72, wherein the composition is administered intracranially.

76. The composition of any one of claims 68-75, wherein the composition is administered by multiple inoculations.

77. The composition of any one of claims 68-76, wherein the composition is administered at a dose of 1 x 103pfu to 1 * 1010pfu.

78. The composition of any one of claims 68-76, wherein the composition is administered at a dose of 1 x 103pfu to 1 x 107pfu.

79. The composition of any one of claims 68-76, wherein the composition is administered at a dose of 1 x 103pfu to 1 x 106pfu.

80. The composition of any one of claims 68-76, wherein the composition is administered at a dose of at least 1x 103pfu.

81. The composition of any one of claims 68-76, wherein the composition is administered at a dose of at least 1 x IO3pfu.145Attorney Docket No: 030258-000107WOPT82. The composition of any one of claims 68-81, wherein the composition is administered as mono- or combination therapy.146

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