Zika viruses and uses thereof

Abstract

Provided herein are methods of treating a cancer (e g., a glioblastoma, a breast cancer, or a melanoma) in a subject in need thereof. The methods comprise administering to the subject a zika virus, wherein the zika virus comprises a nucleotide sequence at least 99.6% identical with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2.

Description

ZIKA VIRUSES AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 607,147, filed December 7, 2023, the disclosure of which is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under project number 1ZIAAI000891- 23 by the National Institutes of Health, National Institute of Allergy and Infectious Diseases. The Government has certain rights in the invention.FIELD OF THE INVENTION

[0003] This invention relates to oncolytic virus therapy for treating cancer (e.g., a glioblastoma, a breast cancer, or a melanoma) in a subject in need thereof.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0004] This application contains a sequence listing, which is submitted electronically. The contents of the electronic sequence listing (070281.1 WO 1 Sequence Listing.xml; size: 27,676 bytes; and creation date of November 26, 2024) is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0005] Glioblastoma (GBM) is the most common and deadliest high-grade primary malignant brain tumor, with median survival averaging 14-16 months. Given the poor survival with currently approved treatments for GBM, new therapeutic strategies are urgently needed.BRIEF SUMMARY OF THE INVENTION

[0006] In one general aspect, the invention relates to methods of treating a cancer (e.g., glioblastoma, a breast cancer, or a melanoma) in a subject in need thereof. The methods comprise administering to the subject a zika virus, wherein the zika virus comprises a nucleotide sequence at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2. In certain embodiments, the zika virus comprises thenucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2. In certain embodiments, the zika virus comprises the nucleotide sequence of SEQ ID NO: 1.

[0007] Also provided are methods of treating a cancer (e.g., glioblastoma, a breast cancer, or a melanoma) in a subject in need thereof, the method comprising administering a Zika Virus Sao Jose do Rio Preto strain (ZIKVS) (SEQ ID NO: 1) or a progeny thereof or a Zika Virus Nicaragua strain (ZIKVN) (SEQ ID NO:2) or a progeny thereof.

[0008] In certain embodiments, the zika virus or progeny thereof is administered at a dose of about 102to about 107plaque forming units (PFU). In certain embodiments, the zika virus or progeny thereof is administered at a dose of about 104PFU, about 1CF PFU, about 1 C to about 106PFU, or about 105 7PFU.

[0009] In certain embodiments, the zika virus or progeny thereof is administered intratumorally, intravenously, subcutaneously, intranasally, or intracranially. The zika virus or progeny thereof can, for example, be administered intravenously.

[0010] In certain embodiments, the zika virus or progeny thereof is administered to the subject in a pharmaceutical composition comprising the zika virus or progeny thereof and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises a buffer, a salt, and an isotonic agent. The buffer can, for example, be a combination of potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4). In certain embodiments, the salt is mono-sodium glutamate. In certain embodiments, the isotonic agent is a sugar. The sugar can, for example, be sucrose.

[0011] In certain embodiments, the pharmaceutical composition comprises (a) the zika virus or progeny thereof; (b) the buffer, wherein the buffer is a combination of potassium dihydrogen phosphate (KH2PO4) at a concentration of about 0.001M to about 0.01M and a dipotassium hydrogen phosphate (K2HPO4) at a concentration of about 0.001M to about 0.01M; (c) the salt is mono-sodium glutamate at a concentration of about 0.001M to about 0.01M; and (d) the isotonic agent is a sugar, wherein the sugar is sucrose at a concentration of about 0.05M to about 0.25M. The pharmaceutical composition can, for example, comprise (a) the zika virus or progeny thereof; (b) KH2PO4 at a concentration of about 0.0038M and K2HPO4 at a concentration of about 0.0072M; (c) mono-sodium glutamate at a concentration of about 0.0054M; and (d) sucrose at a concentration of about 0.128M.

[0012] In certain embodiments, the cancer is a solid cancer, e.g., a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, a glioma, a glioblastoma, and other solid tumors. In certain embodiments, the cancer is a breast cancer, a metastatic melanoma, an ovarian cancer, or a glioblastoma. In certain embodiments, the cancer is a hematologic malignancy, e.g., a lymphoma, a myeloma, a leukemia, and other hematologic malignancies.

[0013] In certain embodiments, the zika virus or progeny thereof is administered with at least one other therapy. The at least one other therapy can, for example, be selected from a radiation therapy, an adjuvant temozolomide (TMZ) therapy, a carmustine (BCNU) therapy, a lomustine (CCNU) therapy, an immune checkpoint blockade therapy, a chimeric antigen receptor T cell (CAR-T) therapy, an oncolytic viral therapy, a vaccine therapy, a tumor-lysate loaded dendritic cell vaccine (DCVax-L) therapy, a focused ultrasound therapy, and / or combinations thereof. In certain embodiments, the immune checkpoint blockade therapy comprises at least one agent that targets an immune checkpoint peptide, wherein the immune checkpoint peptide is selected from cytotoxic-T- lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), PD-1 ligand 1 (PD-L1), indoleamine 2,3 dioxygenase 1 (IDO), cluster of differentiation 47 (CD47), or cluster of differentiation 73 (CD73). In certain embodiments, in the immune checkpoint blockade therapy comprises administration of atezolizumab, avelumab, cemiplimab, dostarlimab, durvalumab, nivolumab, ipilimumab, or pembrolizumab.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The foregoing summary, as well as the following detailed description of embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings.

[0015] FIGs.1 A-1D show Zika virus (ZIKV) MR766 infects patient derived glioblastoma cell lines. FIG. 1 A shows an image of a Western blot confirming AXL protein expression in several patient derived glioblastoma (GBM) cell lines. FIG. IB. shows a graph demonstrating that ZIKV variably replicates in different GBM cell lines. ZIKV incubated with cell lines for 6 hours at an MOI of 10. Using qRT-PCR, the relative expression of ZIKV RNA (NS3) was quantified. FIG. 1Cshows images and graphs demonstrating that consistent with FIG. IB, GBM cell lines are variably permissive to ZIKV infection. The percent of infected cells, as quantified by flow cytometry, increases over time. ZIKV MOI 1, evaluated at 24, 48, and 72 hours post infection. FIG. ID shows a graph demonstrating that consistent with FIG. IB and 1C, after infection, ZIKV replication in the GBM cell lines varies (plaque assay).

[0016] FIGs. 2A-2M show CRISPR knockout of AXL prevents ZIKV MR766 infection. FIG. 2A shows an image of aWestern blot confirming the absence of AXL protein expression following CRISPR knockout. 8034 and 8049 are patient derived GBM cell lines. FIGs. 2B and 2C show graphs demonstrating AXL knockout prevents ZIKV infection (flow cytometry). FIG. 2D shows images of immunofluorescence confirming that AXL CRISPR knockout prevents ZIKV entry into GBM cell lines. Compared to the parent cell lines, the knockout cell lines show few, if any, ZIKV staining. FIG. 2E shows a graph demonstrating the percent of infected cells as determined by immunofluorescence image counting. CRISPR knockout of AXL largely prevents ZIKV infection of patient derived GBM cell lines. FIGs. 2F and 2G show graphs demonstrating qRT-PCR results confirming reduced levels of ZIKV RNA in AXL knockout cell lines. FIG. 2H shows a plaque assay confirming that AXL knockout produces significantly less infectious ZIKV compared to the control parental patient derived GBM cell line 8049. Cells were infected with serial dilution (10‘2to 10'6) of virus. FIGs. 21 and 2J show graphs of PFU count from supernatant 24, 48, 72, and 96 hours post infection. Parental cell lines produce more infectious virus than their AXL knockout clones. FIG. 2K shows images of differentiated glioma cells (DGC) derived from parental patient derived GBM cell lines. Immunofluorescence imaging confirms that the DGCs express GFAP while the parental lines, principally stem cells, do not. FIG. 2L shows graphs demonstrating GFAP expression is significantly higher in DGCs as compared to parental cell lines as determined by immunofluorescence image counting. FIG. 2M shows graphs demonstrating ZIKV infection is significantly lower in DGCs than in parental cells as shown by flow cytometry.

[0017] FIGs. 3A-3C show CRISPR AXL knockout prevents ZIKV infection and cytotoxicity. FIGs. 3 A and 3B show parental and AXL CRISPR knockout cell lines are exposed to ZIKVN (A) and ZIKVS (B) for up to 7 days. FIGs. 3C shows results of annexin V flow cytometry assay for apoptosis. 8034 and 8049 parental and AXL knockout cell lines are exposed to ZIKVS (MOI of 1). Annexin V assay is performed 7 days post infection.

[0018] FIG. 4 show screening of candidate oncolytic ZIKVS and ZIKVN wild type strains using patient derived GBM cell lines to compare the infection efficacy.

[0019] FIGs. 5A-5G show intratumoral treatment of heterotopic patient derived xenografts. FIGs. 5 A and 5D show graphs of patient derived GBM cells (2 x 1068034 or 8049) used to grow subcutaneous tumors in the flanks of immunocompromised mice. Mice were then randomly assigned to receive mock treatment, ZIKVN, or MR766 (5 x IO3virus) directly into the tumor. Tumor volumes were measured before treatment and for 100 days after. Both MR766 and ZIKVN treatment led to rapid and persistent resolution of tumor. FIGs. 5B and 5E show Kaplan-Meier survival curves for the mice treated in FIG. 5A and 5D above. FIGs 5C and 5F show the average weight of mice in each treatment group over time. The average weight of MR766 treated mice was lower at each time point compared to mock and ZIKVN treated mice. FIG. 5G shows a graph of the biodistribution of ZIKV following intratumoral treatment of subcutaneous flank GBM. Compared to mock infection, very little ZIKV RNA was recovered from vital organs.

[0020] FIGs. 6A-6E show treatment of orthotopic patient-derived GBM xenograft tumors with ZIKVN and ZIKVS prolongs survival. FIG. 6A show images of luciferase tagged GBM cells (8034, 20,000 cells) stereotactically implanted in the right frontal lobe of immunocompromised mice. Mice were followed by live imaging. Once tumors were established, the mice were randomized to receive mock treatment, 5.6 x 104PFU of ZIKVN or 3.7 x 104PFU of ZIKVS delivered into the tumor stereotactically. FIG. 6B shows a Kaplan-Meier survival curve for the mice described in FIG. 6A. FIG. 6C shows a graph demonstrating the change in luminescence over time. This is a measure of viable tumor cells. FIG. 6D shows a graph demonstrating the change in tumor area over time. Tumor area continues to increase following mock treatment, while tumor area steadily declines following ZIKVN or ZIKVS treatment. FIG. 6E shows a graph demonstrating the change in mouse weight over time. ZIKVN treated mice consistently weighed less than ZIKVS treated mice. This difference increased over time.

[0021] FIGs. 7A-7D show the determination of the lowest effective dose of ZIKVS for intratumoral delivery. FIG. 7A shows a Kaplan-Meier survival curve. Intracranial patient derived GBM tumors were established in immunodeficient mice. The mice were randomized to receive mock treatment or intratumoral ZIKVS at 102, 103, or 104PFU. Ten mice were in each treatment group. Mice were followed with bioluminescence imaging. Within 21 days, there were 8 complete responses in the 104group, and 7 complete responses in the 102and 103groups. Survival was 80%at 87 days post treatment in the 104group, and 70% in the 102and 103groups. FIG. 7B shows a graph demonstrating tumor area over time. Tumors continued to grow for several days after treatment, then they steadily decreased in size. FIG. 7C shows a graph demonstrating the change in luminescence over time. Consistent with FIG. 7B, bioluminescence peaked, then steadily decreased for all active treatment groups. FIG. 7D shows a graph demonstrating the change in weight over time. All mice experienced a nadir around day 20. This was followed by a steady increase in weight in the active treatment groups.

[0022] FIGs. 8A-8D show intravenous delivery of ZIKVS prolongs the survival of mice bearing intracranial patient derived GBM xenografts. FIG. 8A shows a Kaplan-Meier survival curve. Within 11 days of ZIKVS treatment, there were six complete responses, one partial response, and one tumor related death. At 80 days post treatment, 75% of mice were alive. FIG. 8B shows a graph demonstrating the change in tumor area over time. Tumors continued to grow for several days after treatment, then steadily declined. FIG. 8C shows a graph demonstrating the change in bioluminescence over time is consistent with FIG. 8B. FIG. 8D is a graph demonstrating the change in weight over time. Following a nadir at day 23 (6 days post treatment), treated mice steadily gained weight.

[0023] FIGs. 9A-9E show the characterization of ZIKV MR766 infection of 8049 parental and 8049 CRISPR AXL knockout organoids. FIGs. 9A and 9B show bright field images of parental and AXL knockout organoids (scale bar, 2mm). FIG. 9C shows images of immunofluorescence staining for Sox2, Nestin, OLIG2, P-Tubulin III, and Ki67 in 8049 parental and 8049 AXL knockout whole organoid sections at 7-8 weeks old (scale bar, 200 pm). FIG. 9D show representative immunofluorescence images of mock and ZIKV infected 8-week old organoids of 8049 parental and 8049 AXL knockout at 7- and 14-days post-infection, staining for ZIKV MR766 and DAPI (scale bar, 200 mm). FIG. 9E shows a graph providing quantification of parental and AXL knockout organoid cells infected with ZIKV MR766 at 7- and 14-days post-infection (n = 3 measurements per cell line).

[0024] FIG. 10 shows a graph demonstrating that melanoma cell lines are susceptible to ZIKV infection.

[0025] FIGs. 11A-11B show that ZIKV infection of melanoma cell lines led to apoptosis.

[0026] FIG. 12 shows a graph demonstrating that ZIKV strains infected breast cancer cell lines.

[0027] FIG. 13 shows a graph demonstrating that ZIKV strains were cytotoxic to breast cancer cell lines.

[0028] FIGs 14A-14B show that ZIKV can treat breast cancer in mouse model. FIG. 14A shows a schematic for the ZIKV treatment of breast cancer and a distant metastasis. FIG. 14B shows a graph demonstrating tumor volume over time with tumors being measured daily.DETAILED DESCRIPTION OF THE INVENTION

[0029] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification.

[0031] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0032] Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of about 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0033] As used herein, the terms “comprises,” “comprising,” “includes,” “include,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list ofelements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0034] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0035] As used herein, the term “consists of,” or variations such as “consist of’ or “consisting of,” as used throughout the specification and claims excludes any element, step, or ingredient not specified in the listed claim elements, where such element, step or ingredient is related to the claimed invention.

[0036] As used herein, the term “consists essentially of,” or variations such as “consist essentially of’ or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition.

[0037] As used herein, “subject” means any animal, preferably a mammal, most preferably a human. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human.

[0038] The words “right,” “left,” “lower,” and “upper” designate directions in the drawings to which reference is made.

[0039] It should also be understood that the terms “approximately,” “generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component, indicatethat the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc ), would not vary the least significant digit.

[0040] The terms “identical” or percent “identity,” in the context of two or more nucleic acids (e g., zika virus nucleotide sequences) or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence. Percent identity or identical to a reference nucleic acid sequence can be determined, for example, by (1) aligning a test sequence to a reference providing for maximal alignment taking into account additions, deletions, or nucleotide differences; (2) determining the number of nucleotides that are the same in the maximal aligned sequence; (3) dividing by the number of bases in the reference sequence; and (4) multiplying by 100. Percent identity or identical to a reference amino acid sequence can be determined in an analogous fashion. Additional, percent identity or identical can be determined using an algorithm.

[0041] For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0042] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat’ 1. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0043] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0044] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0045] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0046] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0047] As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and doublestranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0048] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation. Also included within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0049] The peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of the amino acids are known, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.

[0050] The Zika virus is a single-stranded RNA virus of the Flaviviridae family, genus Flavivirus. The virus genome provides a 5’ UTR, a 3’ UTR and encodes a single polyprotein of about 3419 amino acids that is cleaved by the viral serine and cellular furin proteases into the functional domains: the Capsid (C), Precursor of membrane (prM), Envelope (E) and 7 non- structural proteins (NS).

[0051] Methods of use

[0052] Preferred Zika virus are those cytotoxic to cancer cells, while at the same time not unacceptably toxic to the host. Zika virus that are highly virulent may be effective against a cancer but be unacceptably toxic to the host. On the other hand, less virulent Zika virus may not be effective in treating cancer. Potential toxicity can be evaluated using different techniques, such as clinical observation and / or measuring weight loss or other toxic indicators in animal models.

[0053] Provided herein are methods of treating a cancer (e.g., a glioblastoma, a breast cancer, or a melanoma) in a subject in need thereof. The methods comprise administering to the subject a zika virus comprising a nucleotide sequence at least 99.6% identical with the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2. SEQ ID NO: 1 and SEQ ID NO: 2, have a sequence identity of about 99.6% (42 nucleotide differences). In certain embodiments, the zika virus is at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to SEQ ID NO: 1. In certain embodiments the zika virus differs from SEQ ID NO: 1 by up to 44 nucleotides, up to 42 nucleotides, up to 40 nucleotides, up to 35 nucleotides, up to 30 nucleotides, up to 25 nucleotides or up to 10 nucleotides. In certain embodiments, the zika virus is at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to SEQ ID NO: 2. In certain embodiments the zika virus differs from SEQ ID NO: 2 by up to 44 nucleotides, up to 42 nucleotides, up to 40 nucleotides, up to 35 nucleotides, up to 30 nucleotides, up to 25 nucleotides or up to 10 nucleotides. Each nucleotide difference can be a substitution, addition, and / or deletion. Preferably, any nucleotide change to a coding region is a substitution.

[0054] In certain embodiments, the zika virus strains comprise a nucleotide sequence comprising 100% identity to nucleotides 1-105, 1300-1400, 1400-1500, 1550-1650, 1900-2000, 2500-2600, 3000-3100, 3500-3600, 3900-4000, 6300-6400, 6590-6690, 8300-8400, 9200-9300, and / or 9800- 9900 of SEQ ID NO: 1 or SEQ ID NO:2. In certain embodiments, the zika virus strains comprise a nucleotide sequence comprising 100% identity to nucleotides 1300-1400, 1400-1500, 1550-1650, 1900-2000, 2500-2600, 3000-3100, 3500-3600, 3900-4000, 6300-6400, 6590-6690, 8300-8400, 9200-9300, and / or 9800-9900 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0055] In certain embodiments, the zika virus strains comprise a nucleotide sequence comprising 100% identity to nucleotides 400-500, 600-700, 1400-1500, 1500-1600, 1700-1800, 1900-2000, 2000-2100, 2500-2600, 2700-2800, 2900-3000, 3200-3300, 3300-3400, 3600-3700, 3800-3900, 3900-4000, 4600-4700, 4700-4800, 5100-5200, 5300-5400, 5400-5500, 5700-5800, 6200-6300, 6300-6400, 6600-6700, 6700-6800, 7500-7600, 7800-7900, 8100-8200, 8300-8400, 8400-8500, 8500-8600, 9200-9300, 9400-9500, and / or 10600-10700 of SEQ ID NO: 1. In certain embodiments, the zika virus strains comprise a nucleotide sequence comprising 100% identity to nucleotides 400- 500, 600-700, 1400-1500, 1500-1600, 1700-1800, 1900-2000, 2000-2100, 2500-2600, 2700-2800, 2900-3000, 3200-3300, 3300-3400, 3600-3700, 3800-3900, 3900-4000, 4600-4700, 4700-4800, 5100-5200, 5300-5400, 5400-5500, 5700-5800, 6200-6300, 6300-6400, 6600-6700, 6700-6800, 7500-7600, 7800-7900, 8100-8200, 8300-8400, 8400-8500, 8500-8600, 9200-9300, 9400-9500, and / or 10600-10700 of SEQ ID NO:2.

[0056] In certain embodiments, the methods comprise administering to the subject a zika virus comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2.

[0057] In certain embodiments, the methods comprise administering to the subject a Zika Virus Sao Jose do Rio Preto strain (ZIKVS) or a progeny thereof or a Zika Virus Nicaragua strain (ZIKVN) or a progeny thereof. In certain embodiments, the Sao Jose do Rio Preto Strain (ZIKVS) can comprise SEQ ID NO: 1. In certain embodiments, the Zika Virus Nicaragua strain (ZIKVN) can comprise SEQ ID NO:2.

[0058] By “progeny thereof’ it is meant a virus produced from replication of the original parent strains (e.g., SEQ ID NO: 1 or SEQ ID NO:2). During viral replication, mutations can spontaneously be produced and / or selected for. The progeny can, for example, have 100% identity to the parent strains, or, alternatively, the progeny can, for example, have less than 100% identity to the parentstrains. By way of an example, the progeny can have at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity to the parent strains.

[0059] The wildtype ZIKV Nicaragua / 2016-UCB 7420 (ZIKVN) strain was isolated from serum received from a patient in Nicaragua.

[0060] The wildtype ZIKV-SJRP / 2016-184 (ZIKVS) was isolated from serum obtained from a patient in Sao Jose de Rio Preto.

[0061] In certain embodiments, the zika virus strains disclosed herein possess one or more desirable properties, including, but not limited to, being capable of treating a cancer (e.g., a glioblastoma, a breast cancer, or a melanoma) in a subject in need thereof, wherein upon treatment, the zika virus is capable of targeting cancer cells (e.g., glioblastoma cancer cells, breast cancer cells, or melanoma cancer cells) or cancer stem cells (e.g., glioblastoma cancer stem cells, breast cancer stem cells, or melanoma cancer cells) at a higher rate than non-cancer neural cells. As illustrated in the examples below, ZIKVS or ZIKVN provide significant tumor toxicity while exhibiting minimal to no toxic effects on the non-tumor cells in the employed animal models.

[0062] As used herein a control zika virus strain can, for example, be the MR766 strain. The MR766 strain was isolated from an infected rhesus monkey and deposited in the American Type Culture Collection (ATCC) (Manassas, Virginia). Additional commercially available zika virus strains can also be used as a control.

[0063] According to embodiments of the invention, the pharmaceutical composition comprises a therapeutically effective amount of a zika virus or progeny thereof. As used herein, the term “therapeutically effective amount” refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject. A therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose.

[0064] As used herein with reference to the zika virus, a therapeutically effective amount means an amount of the zika virus or progeny thereof that reduces tumor size (e.g., glioblastoma, breast cancer, or melanoma), inhibits growth of a tumor (e.g., glioblastoma, breast cancer, or melanoma), and / or reduces one or more symptoms associated with a tumor (e.g., glioblastoma, breast cancer, or melanoma) in a subject in need thereof. Also as used herein with reference to the zika virus, a therapeutically effective amount means an amount of the zika virus or progeny thereof that results in treatment of a disease, disorder, or condition; prevents or slows the progression of the disease, disorder, or condition; or reduces or completely alleviates symptoms associated with the disease,disorder, or condition. Treatment of a glioblastoma and methods for monitoring treatment of a glioblastoma are described in, for example, Stupp et al., NEJM 352:987-96 (2005), which is incorporated by reference herein in its entirety.

[0065] The therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0066] In certain embodiments, a therapeutically effective amount of the zika virus can, for example, comprise about 102to about 107plaque forming units (PFU) as determined, for example, in examples provided below. For example, a therapeutically effective amount of the zika virus can comprise about 102, about 102 5, about 103, about IO3 3, about 104, about IO4 5, about 105, about 103 1, about IO52, about 105 3, about 105 4, about 105 5, about IO3 6, about 1057, about IO5 8, about 105 9, about 106, about 1064, about IO6 2, about 1063, about 106 4, about 106 5, about 1066, about IO67, about IO6 8, about IO6 9, or about 107PFU. By way of another example a therapeutically effective amount of the zika virus can comprise about 102to about 107PFU, about 102to about 106PFU, about 102to about 105PFU, about 102to about 104PFU, about 102to about 103PFU, about 103to about 107PFU, about 103to about 106PFU, about 103to about 105PFU, about 103to about 104PFU, about 104to about 107PFU, about 104to about 106PFU, about 104to about 105PFU, about 105to about 107PFU, about 103to about 106PFU, or about 106to about 107PFU.

[0067] According to particular embodiments, the disease, disorder or condition to be treated is cancer. The cancer can, for example, be a solid cancer, e.g., a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, a sarcoma, a glioma, a glioblastoma, and other solid tumors. In certain embodiments, the cancer is a breast cancer, a metastatic melanoma, an ovarian cancer, or a glioblastoma. The cancer can, for example, be a hematologic malignancy, e.g., a lymphoma, a myeloma, a leukemia, and other hematologic malignancies.

[0068] According to particular embodiments, a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or asymptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (ix) increase the survival of a subject with the disease, disorder or condition to be treated, or a symptom associated therewith; (xi) inhibit or reduce the disease, disorder or condition to be treated, or a symptom associated therewith in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0069] According to particular embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for intratumoral, intravenous, subcutaneous, intranasal, or intracranial administration. In certain embodiments, the zika virus is administered intravenously.

[0070] As used herein, the terms “treat,” “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to a cancer (e.g., a glioblastoma, a breast cancer, or a melanoma). The terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as a tumor or more preferably a cancer (e.g., a glioblastoma, a breast cancer, or a melanoma). In a particular embodiment, “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.

[0071] According to particular embodiments, provided are compositions used in the treatment of a cancer (e.g., a glioblastoma, a breast cancer, or a melanoma). For glioblastoma cancer therapy, in certain embodiments, the compositions can be used in combination with another treatment and / or therapy including, but not limited to, surgery, a radiation therapy, an adjuvant temozolomide (TMZ) therapy, a carmustine (BCNU) therapy, a lomustine (CCNU) therapy, an immune checkpoint blockade therapy, a chimeric antigen receptor T cell (CAR-T) therapy, an oncolytic viral therapy, a vaccine therapy, a tumor-lysate loaded dendritic cell vaccine (DCVax-L) therapy, a focused ultrasound therapy, and / or combinations thereof. Therapies for treating glioblastoma multiforme are known in the art, see, e.g., Rong et al., J. Exp. Clin. Can. Res. 41(1): 142 (2022), which is incorporated by reference herein in its entirety. In certain embodiments, the immune checkpoint blockade therapy comprises at least one agent that targets an immune checkpoint peptide (e.g., receptor or ligand). In certain embodiments the immune checkpoint peptide is selected from cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), PD-1 ligand 1 (PD-L1), indoleamine 2,3 dioxygenase 1 (IDO), cluster of differentiation 47 (CD47), or cluster of differentiation 73 (CD73). The immune checkpoint therapy can, for example comprise administration of atezolizumab, avelumab, cemiplimab, dostarlimab, durvalumab, nivolumab, ipilimumab, or pembrolizumab.

[0072] As used herein, the term “in combination,” in the context of the administration of two or more therapies to a subject, refers to the use of more than one therapy. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. For example, a first therapy (e.g., a zika virus composition described herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a radiation therapy, an adjuvant temozolomide (TMZ) therapy, a lomustine-TMZ chemotherapy, an immune checkpoint blockade therapy, a chimeric antigen receptor T cell (CAR-T) therapy, an oncolytic viral therapy, a vaccine therapy, and / or a focused ultrasound therapy) to a subject.

[0073] Pharmaceutical Compositions

[0074] In another general aspect, the invention relates to a pharmaceutical composition, comprising the zika virus or progeny thereof of the invention and a pharmaceutically acceptable carrier. The term “pharmaceutical composition” as used herein means a product comprising a zika virus or progeny thereof of the invention together with a pharmaceutically acceptable carrier. Zika viruses or progeny thereof of the invention and compositions comprising them are also useful in the manufacture of a medicament for therapeutic applications mentioned herein.

[0075] As used herein, the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term “pharmaceutically acceptable carrier” refers to a sterile nontoxic material suitable for administration to a subject. The carrier should not interfere with the effectiveness of a composition according to the invention or the biological activity of a composition according to the invention. According to particular embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in a zika virus pharmaceutical composition can be used in the invention.

[0076] The formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, e.g., Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005), and any later editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tonicity regulating agents, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used in formulating the pharmaceutical compositions of the invention.

[0077] In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation comprising water. The liquid formulation can comprise a solution, a suspension, an emulsion, a microemulsion, a gel, and the like. An aqueous formulation typically comprises at least 50% w / w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w / w of water.

[0078] In one embodiment, the pharmaceutical composition can be formulated as an injectable which can be injected, for example, via an injection device (e.g., a syringe or an infusion pump). The injection can be delivered intratum orally, intravenously, subcutaneously, intranasally, or intracranially, for example.

[0079] In another embodiment, the pharmaceutical composition is a solid formulation, e.g., a freeze-dried or spray-dried composition, which can be used as is, or whereto the physician or the patient adds solvents, and / or diluents prior to use. Solid dosage forms can include tablets, such as compressed tablets, and / or coated tablets, and capsules (e.g., hard or soft gelatin capsules). The pharmaceutical composition can also be in the form of sachets, dragees, powders, granules, lozenges, or powders for reconstitution, for example.

[0080] The dosage forms can be immediate release, in which case they can comprise a water- soluble or dispersible carrier, or they can be delayed release, sustained release, or modified release, in which case they can comprise water-insoluble polymers that regulate the rate of dissolution of the dosage form in the gastrointestinal tract or under the skin.

[0081] In other embodiments, the pharmaceutical composition can be delivered intratumorally, intravenously, subcutaneously, intranasally, or intracranially.

[0082] The pH in an aqueous formulation can be between pH 3 and pH 10. In one embodiment of the invention, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is from about 3.0 to about 7.0.

[0083] In another embodiment of the invention, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, dipostassium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, potassium dihyrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine, and tris(hydroxymethyl)-aminomethane, and mixtures thereof. The buffer can be present individually or in the aggregate, in a concentration from about 0.01 mg / ml to about 50 mg / ml, for example from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific buffers constitute alternative embodiments of the invention. In certain embodiments, the buffer is a combination of potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4). The combination of potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4) can comprise KH2PO4 at a concentration of about 0.001M to about 0.01M, preferably about 0.038M, and K2HPO4 at a concentration of about 0.001M to about 0.01M, preferably about 0.0072M.

[0084] In another embodiment of the invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include: benzethonium chloride, benzoic acid,benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorohexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidurea, methyl 4- hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof. The preservative can be present individually or in the aggregate, in a concentration from about 0.01 mg / ml to about 50 mg / ml, for example from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific preservatives constitute alternative embodiments of the invention.

[0085] In another embodiment of the invention, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of the isotonic agents include a salt (such as sodium chloride), an amino acid (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), an alditol (such as glycerol, 1,2-propanediol propylene glycol), 1,3 -propanediol, and 1,3- butanediol), polyethylene glycol (e.g., PEG400), and mixtures thereof. Another example of an isotonic agent includes a sugar. Non-limiting examples of sugars may be mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose. Another example of an isotonic agent is a sugar alcohol, wherein the term “sugar alcohol” is defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. The isotonic agent can be present individually or in the aggregate, in a concentration from about 0.01 mg / ml to about 50 mg / ml, for example from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific isotonic agents constitute alternative embodiments of the invention. In certain embodiments, the pharmaceutical composition comprises a salt. The salt can, for example, be mono-sodium glutamate. In certain embodiments, the mono-sodium glutamate is present in the pharmaceutical composition at a concentration of about 0.001M to about 0.01M, preferably about 0.0054M. In certain embodiments, the isotonic agent is a sugar. The sugar can, for example, be sucrose. In certain embodiments, the sucrose is present in the pharmaceutical composition at a concentration of about 0.05M to about 0.25M, preferably about 0.128M.

[0086] In another embodiment of the invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, salts of ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agent can be presentindividually or in the aggregate, in a concentration from about 0.01 mg / ml to about 50 mg / ml, for example from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific chelating agents constitute alternative embodiments of the invention.

[0087] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0088] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, wherein said stabilizer is carboxy- / hydroxycellulose and derivates thereof (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, 2-methylthioethanol, polyethylene glycol (e.g., PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone, salts (e.g., sodium chloride), sulphur- containing substances such as monothioglycerol), or thioglycolic acid. The stabilizer can be present individually or in the aggregate, in a concentration from about 0.01 mg / ml to about 50 mg / ml, for example from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific stabilizers constitute alternative embodiments of the invention.

[0089] In further embodiments of the invention, the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant, or two different surfactants. The term “surfactant” refers to any molecules or ions that are comprised of a water-soluble (hydrophilic) part, and a fat-soluble (lipophilic) part. The surfactant can, for example, be selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants. The surfactant can be present individually or in the aggregate, in a concentration from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific surfactants constitute alternative embodiments of the invention.

[0090] In a further embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as, e.g., EDTA, and / or benzamidine hydrochloric acid (HC1). The protease inhibitor can be present individually or in the aggregate, in a concentration from about 0. 1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific protease inhibitors constitute alternative embodiments of the invention.EMBODIMENTS

[0091] The invention provides also the following non-limiting embodiments.

[0092] Embodiment 1 is a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a zika virus comprising a nucleotide sequence at least 99.6% identical with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2. In further embodiments, the zika virus is at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to SEQ ID NO: 1. In certain embodiments the zika virus differs from SEQ ID NO: 1 by up to 44 nucleotides, up to 42 nucleotides, up to 40 nucleotides, up to 35 nucleotides, up to 25 nucleotides or up to 10 nucleotides. In certain embodiments, the zika virus is at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to SEQ ID NO: 2. In certain embodiments the zika virus differs from SEQ ID NO: 2 by up to 44 nucleotides, up to 42 nucleotides, up to 40 nucleotides, up to 35 nucleotides, up to 25 nucleotides or up to 10 nucleotides

[0093] Embodiment 2 is the method of embodiment 1, wherein the zika virus comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2.

[0094] Embodiment 3 is the method of embodiment 1 or 2, wherein the zika virus comprises the nucleotide sequence of SEQ ID NO: 1.

[0095] Embodiment 4 is a method of treating a cancer in a subject in need thereof, the method comprising administering a Zika Virus Sao Jose do Rio Preto strain (ZIKVS) or a progeny thereof or a Zika Virus Nicaragua strain (ZIKVN) or a progeny thereof.

[0096] Embodiment 5 is the method of any one of embodiments 1-4, wherein the zika virus or progeny thereof is administered at a dose of about 102to about 107plaque forming units (PFU).

[0097] Embodiment 6 is the method of embodiment 5, wherein the zika virus or progeny thereof is administered at a dose of about 104PFU, about 105PFU, about 105to about 106PFU, or about 105 7PFU.

[0098] Embodiment 7 is the method of any one of embodiments 1-6, wherein the zika virus or progeny thereof is administered intratumorally, intravenously, subcutaneously, intranasally, or intracranially.

[0099] Embodiment 8 is the method of embodiment 7, wherein the zika virus or progeny thereof is administered intravenously.

[0100] Embodiment 9 is the method of any one of embodiments 1-8, wherein the zika virus or progeny thereof is administered to the subject in a pharmaceutical composition comprising the zika virus or progeny thereof and a pharmaceutically acceptable carrier.

[0101] Embodiment 10 is the method of embodiment 9, wherein the pharmaceutical composition comprises a buffer, a salt, and an isotonic agent.

[0102] Embodiment 11 is the method of embodiment 10, wherein the buffer is a combination of potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4).

[0103] Embodiment 12 is the method of embodiment 10 or 11, wherein the salt is mono-sodium glutamate.

[0104] Embodiment 13 is the method of any one of embodiments 10-12, wherein the isotonic agent is a sugar.

[0105] Embodiment 14 is the method of embodiment 13, wherein the sugar is sucrose.

[0106] Embodiment 15 is the method of any one of embodiments 10-14, wherein the pharmaceutical composition comprises:(a) the zika virus or progeny thereof;(b) the buffer, wherein the buffer is a combination of potassium dihydrogen phosphate (KH2PO4) at a concentration of about 0.001M to about 0.01M and a dipotassium hydrogen phosphate (K2HPO4) at a concentration of about 0.001M to about 0.01M;(c) the salt is mono-sodium glutamate at a concentration of about 0.001M to about 0.01M; and(d) the isotonic agent is a sugar, wherein the sugar is sucrose at a concentration of about 0.05M to about 0.25M.

[0107] Embodiment 16 is the method of embodiment 15, wherein the pharmaceutical composition comprises:(a) the zika virus or progeny thereof;(b) KH2PO4 at a concentration of about 0.0038M and K2HPO4 at a concentration of about 0.0072M;(c) mono-sodium glutamate at a concentration of about 0.0054M; and(d) sucrose at a concentration of about 0.128M.

[0108] Embodiment 17 is the method of any one of embodiments 1-16, wherein the cancer is a solid cancer.

[0109] Embodiment 18 is the method of embodiment 17, wherein the solid cancer is selected from a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, metastatic melanoma, a breast cancer, an ovarian cancer,a cervical cancer, a head and neck cancer, a pancreatic cancer, a glioma, a glioblastoma, or other solid tumors.[001101 Embodiment 19 is the method of embodiment 18, wherein the solid cancer is a glioblastoma.

[0111] Embodiment 20 is the method of any one of embodiments 1-16, wherein the cancer is a hematologic malignancy.

[0112] Embodiment 21 is the method of embodiment 20, wherein the hematologic malignancy is selected from a lymphoma, a myeloma, a leukemia, and other hematologic malignancies.

[0113] Embodiment 22 is the method of any one of embodiments 1-21, wherein the zika virus or progeny thereof is administered with at least one other therapy.

[0114] Embodiment 23 is the method of embodiment 22, wherein the at least one other therapy is selected from a radiation therapy, an adjuvant temozolomide (TMZ) therapy, a carmustine (BCNU) therapy, a lomustine (CCNU) therapy, an immune checkpoint blockade therapy, a chimeric antigen receptor T cell (CAR-T) therapy, an oncolytic viral therapy, a vaccine therapy, a tumor-lysate loaded dendritic cell vaccine (DCVax-L) therapy, a focused ultrasound therapy, and / or combinations thereof.

[0115] Embodiment 24 is the method of embodiment 23, wherein the immune checkpoint blockade therapy comprises at least one agent that targets an immune checkpoint peptide, wherein the immune checkpoint peptide is selected from cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), PD-1 ligand 1 (PD-L1), indoleamine 2,3 dioxygenase 1 (IDO), cluster of differentiation 47 (CD47), or cluster of differentiation 73 (CD73).

[0116] Embodiment 25 is the method of embodiment 24, wherein the immune checkpoint blockade therapy comprises administration of atezolizumab, avelumab, cemiplimab, dostarlimab, durvalumab, nivolumab, ipilimumab, or pembrolizumab.

[0117] Embodiment 26 is a pharmaceutical composition comprising a zika virus and a pharmaceutically acceptable carrier, wherein the zika virus comprises a nucleotide sequence at least 99.6% identical with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2. In certain embodiments, the zika virus is at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to SEQ ID NO: 1. In certain embodiments the zika virus differs from SEQ ID NO: 1 by up to 44 nucleotides, up to 42 nucleotides, up to 40 nucleotides, up to 35 nucleotides, up to 25 nucleotides or up to 10 nucleotides. In certain embodiments, the zika virus is at least 99.6%, at least99.7%, at least 99.8%, at least 99.9% or 100% identical to SEQ ID NO: 2. In certain embodiments the zika virus differs from SEQ ID NO: 2 by up to 44 nucleotides, up to 42 nucleotides, up to 40 nucleotides, up to 35 nucleotides, up to 25 nucleotides or up to 10 nucleotides.

[0118] Embodiment 27 is the pharmaceutical composition of embodiment 26, wherein the zika virus comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2.

[0119] Embodiment 28 is the pharmaceutical composition of embodiment 26 or 27, wherein the pharmaceutical composition comprises a buffer, a salt, and an isotonic agent.

[0120] Embodiment 29 is the pharmaceutical composition of embodiment 28, wherein the buffer is a combination of potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4).

[0121] Embodiment 30 is the pharmaceutical composition of embodiment 28 or 29, wherein the salt is mono-sodium glutamate.

[0122] Embodiment 31 is the pharmaceutical composition of any one of embodiments 26-30, wherein the isotonic agent is a sugar.

[0123] Embodiment 32 is the pharmaceutical composition of embodiment 31, wherein the sugar is sucrose.

[0124] Embodiment 33 is the pharmaceutical composition of any one of embodiments 26-32, wherein the pharmaceutical composition comprises:(a) the zika virus or progeny thereof;(b) the buffer, wherein the buffer is a combination of potassium dihydrogen phosphate (KH2PO4) at a concentration of about 0.001M to about 0.01M and a dipotassium hydrogen phosphate (K2HPO4) at a concentration of about 0.001M to about 0.01M;(c) the salt is mono-sodium glutamate at a concentration of about 0.001M to about 0.01M; and(d) the isotonic agent is a sugar, wherein the sugar is sucrose at a concentration of about 0.05M to about 0.25M.

[0125] Embodiment 34 is the pharmaceutical composition of embodiment 33, wherein the pharmaceutical composition comprises:(a) the zika virus or progeny thereof;(b) KH2PO4 at a concentration of about 0.0038M and K2HPO4 at a concentration of about0.0072M;(c) mono-sodium glutamate at a concentration of about 0.0054M; and(d) sucrose at a concentration of about 0.128M.[001261 Embodiment 35 is the pharmaceutical composition of any one of embodiments 26-34, wherein the pharmaceutical composition further comprises at least one other therapy.

[0127] Embodiment 36 is the pharmaceutical composition of any one of embodiments 26-35, wherein the zika virus or progeny thereof is at a dose of about 104PFU, about 105PFU, about 105to about 106PFU, or about 105 7PFU.EXAMPLES

[0128] Methods

[0129] Cell lines and cultures

[0130] Glioblastoma tissues (based on WHO criteria) were obtained from excess tissue from patients at the Aurora St. Luke’s Medical Center with informed consent. Twelve established patient derived glioblastoma stem cell lines (GSCs; 8034, 8049, 7966, 8104, 8015, 8106, 7714, 7753, 8075, 8100, 7730, and 8136) were maintained in NeuroCult NS-A basal medium (Stemcell Technologies; Vancouver, BC, Canada), supplemented with B-27 without vitamin A, N-2, GlutaMAX and Pen / Strep (Thermo Fisher Scientific; Waltham, MA), bovine serum albumin (BSA) and heparin (Sigma-Aldrich; St. Louis, MO), human recombinant basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) (20 ng / ml each; PeproTech Inc.; Cranbury, NJ). Differentiated glioblastoma multiforme (GBM) cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) for 7-10 days before testing. All cell lines were routinely tested for mycoplasma contamination by using My coAlert Detection Kit (Lonza Inc.;Basel, Switzerland).

[0131] AXL knockout cell lines. Patient lines 8034, and 8049 were transfected with the AXL CRISPR / Cas9 KO Plasmid construct (Santa Cruz Biotechnology, SC-400393; Dallas, TX) via electroporation using the Neon Transfection kit (ThermoFisher, MPK10096). Electroporated cells were grown for 3 to 4 days before being single cell sorted via fluorescent activated cell sorting (FACS) (BD FACSAria III Cell Sorter) into 96-well plates. These single cell clones were then expanded, and AXL knockout was confirmed by western blotting against the wild-type parental lines. The following primary antibodies were used for western blot: AXL antibody (R&D Systems, 1 : 1000; Minneapolis, MN) and [3-actin antibody (Sigma-Aldrich, 1 : 1000).

[0132] Luciferase tagged cell lines.

[0133] The firefly luciferase lentivirus was purchased from BPS Bioscience (Cat# 79692) (BPS Bioscience; San Diego, CA). In brief, 8034 and 8049 cells were plated in laminin coated 6 well plates and infected with firefly luciferase lentivirus in the presence of 8 qg / ml polybrene at a multiplicity of infection (MOI) 1 for 3 days. After 3 days, successfully transduced cells were selected with puromycin for 5 days. After puromycin selection these stably transduced cell lines were confirmed by luciferase activity.

[0134] ZIKV strain preparation and titration:

[0135] ZIKV- SJRP / 2016 and ZIKV- Nicaragua / 2016 were propagated in Vero cells (ATCC, CCL-81). Vero cells were infected with Zika strains at multiplicity of infection of (MOI) 0.01. In brief, diluted virus in Opti-Pro SFM (Invitrogen; Carlsbad, CA) was added to Vero cells, rocked for 90 minutes at 37°C in a humidified incubator. After incubation, Opti-Pro SFM was added to the cells and incubated at 37°C in a humidified incubator with 5% CO2. At 6-day post-infection (for ZIKV- SJRP / 2016) and 4-day post-infection (for ZIKV- Nicaragua / 2016), cell supernatant from infected cells were harvested after adding IX SPG (Sucrose-phosphate-glutamate stabilizer) and purified by centrifugation at 1000X g for 20 minutes to remove cellular debris and flash freeze vials in dry ice for about 10 minutes. For mock infections, supernatant was collected from uninfected Vero cells and prepared by the same protocol used to make viral stock.

[0136] Viral titers for MR766 and ZIKVN (plaque forming units, PFU / mL) were calculated by the plaque-forming assay using Vero cells. The Vero cells (5xl03) were seeded in a 6-well plate and incubated at 37°C for 24 hours before infection. The next day, Vero cells were washed once with PBS, and serial dilutions of ZIKV strains were then added to the cells for 90 minutes at 37°C to initiate binding. After 90 minutes, 2 mb of an OptiMEM GlutaMAX media (Thermo Fisher) with 2% of FBS and 1% methylcellulose (Sigma, M0512 4000 cps) overlay was added to the wells and incubated at 37°C for five days. For plaque counting and MOI calculation, the cells were fixed with 4% formaldehyde and stained with 0.8% crystal violet in 50% ethanol. For ZIKVS, the immunostain procedure was used for plaque count. Initial steps were similar to ZIKVN until overlay incubation. In the immunostain procedure, cells were fixed with 80% methanol for 10 minutes, then antibody dilution buffer (5% skim milk in IxPBS) was added for 10 minutes. Flavivirus monoclonal antibody (4G2, 1:2000) was incubated for 2 hours and then washed with phosphate buffered saline (PBS). Antibody conjugate (peroxidase-labelled goat anti mouse IgG, Cat # 074-1806, 1 :2500) was addedand incubated for 2 hours, washed, and then TrueBlue substrate was added to develop. Peroxidase substrate was removed, and plaques were counted for MOI calculation.

[0137] ZIKV infection and cytotoxicity

[0138] Flow cytometry. Flow cytometry was used to determine the percentage of cells infected by ZIKV strains. At various time points, infected cells were washed, stained with viability dye eFluor 780 (ThermoFisher, 65-0865-14), fixed, and kept at 4°C until ready for flow analysis. Once ready for flow, infected cells were permeabilized for 20 minutes at 4°C, and then incubated at 4°C for 45 minutes with primary Flavi virus 4G2 antibody (Millipore MAB10216; Burlington, MA) followed by a secondary staining with phycoerythrin-conjugated goat anti-mouse IgG antibody. Acquisition was then performed on a BD LSR Fortessa HTS flow cytometer, collecting 20,000 events per run. Data were analyzed using FACSDiva and FlowJo v.10 software.

[0139] Immunofluorescence staining and microscopy. ZIKV infection was compared between 8034, 8049 parental and AXL-KO cell lines by Immunofluorescence staining. 2 x 105cells were infected at MOI 1 with ZIKV and cultured on Lab-Tek II 4- well chamber slides (Fisher Scientific) at indicated time points. Cells were washed with lx PBS, then fixed with 4% paraformaldehyde for 15 minutes, then permeabilized with permeabilization buffer (BD Biosciences) for 30 minutes at room temperature. Cells were blocked with 10% normal goat serum (Cell Signaling; Danvers, MA) for 1 hour and then incubated with flavivirus monoclonal antibody 4G2 (1 :500) for 2 hours at room temperature. After incubation, cells were washed three times with PBS, then incubated with Alexa Fluor 488-labeled anti-mouse antibody (Thermo Fisher, Al 1029) for 1 hour at room temperature. Cells were then washed and mounted with Fluoroshield mounting medium containing DAPI (Abeam, ab 104139; Cambridge, United Kingdom) for nuclei staining. All images were acquired with an Olympus 1X83 microscope, using CellSens Dimension software, and analyzed with ImageJ software.

[0140] RNA extraction and qRT-PCR. For viral RNA quantification, 8034, 8049 parental and AXL-KO cells were infected for the indicated lengths of time with ZIKV at MOI 1 PFU / cell. Total RNA was extracted from infected cells, using the RNeasy Mini Kit (Qiagen; Hilden, Germany). Isolated RNAs (1 pg total RNA) were then digested with 1 unit of DNase I (NEB; Ipswich, MA) at 37°C for 25 minutes to remove genomic DNA contamination before being processed for reverse transcription. Quantitative real time polymerase chain reaction (qRT-PCR) was performed using iTaq Universal SYBR Green One-Step Kit (Bio-Rad, 1725151) according to the manufacturer’sinstructions on a Roche LightCycler 480 instrument. The primers for viral RNA quantification targeted the nonstructural protein NS3 (Forward primer 5’ - CCA AC A AAC CTG GAG ATG AGT A -3’ (SEQ ID NO:3); Reverse primer 5’- GAG GCC ATC TTG GAG GTA AAT -3’ (SEQ ID NO:4)). The control primers were GAPDH (Forward primer 5’- GGA TTT GGT CGT ATT GGG - 3’ (SEQ ID NO:5); Reverse primer 5’- GGA AGA TGG TGA TGG GAT T -3’ (SEQ ID NO:6)). Relative expression quantification was performed based on the comparative CT Method (2-AACt), using GAPDH as endogenous reference control.

[0141] Annexin V assay. Cell death was measured using the fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit II (BD Biosciences) following the manufacturer's instructions. Cells were infected with ZIKV at varying MOIs and analyzed several days post-infection using BD LSR Fortessa HTS flow cytometer, collecting 10,000 events per run. Data analysis was performed using FlowJo software.

[0142] Glioblastoma organoids. Organoids were prepared by first suspending 1,000-5,000 patient derived glioblastoma stem cells into Matrigel (Corning; 354234; Corning, NY). From this suspension, 20 pL droplets were solidified on parafilm molds and cultured for 4 days without shaking on low-binding 6-well plates with the NeuroCult complete media. After 4 days, the GBM organoids were transferred to an orbital shaker inside of a tissue incubator, where they were further cultured in NeuroCult complete media for 8-10 weeks before characterization / experimentation.

[0143] Organoid Immunofluorescence:

[0144] Organoids were fixed in 4% paraformaldehyde (PF A) (Electron Microscopy Services) for 1 hour at room temperature (RT), washed 3 times in PBS, then cryopreserved in 30% sucrose overnight at 4°C. The following day, samples were embedded in optimum cutting temperature (O.C.T) medium (ThermoFisher) and 10 pm sections were cryosectioned onto charged slides.Sections were outlined with a hydrophobic pen and fixed in 4% PFA for 15 minutes at RT, then permeabilized for 15 minutes at RT with 1% Triton X-100. Slides were then blocked in a solution containing 10% goat serum and 1% Triton X-100 in PBS for 1 hour at RT and incubated in primary antibodies (primary antibody + 10% FBS in PBS) in a humidified chamber at 4°C overnight. The next day, after three washes with tris-buffered saline (TBST), slides were incubated in secondary antibodies (secondary + 10% FBS in PBS) for 2 hours at RT, washed with TBST, mounted with VectaShield mounting medium with DAPI (Abeam), and then sealed with cover glass.

[0145] Mice maintenance:

[0146] Fox N1 Nude homozygous female mice (Charles River Laboratories; Spencerville, OH), 42-56 days old, were received in the vivarium and acclimated for approximately one week prior to study start.

[0147] Patient derived xenografts:

[0148] Subcutaneous flank tumors. All animals received subcutaneous flank injections of GSCs (2.0xl06cells) in 100 pl Matrigel (Corning, 354234), under isoflurane anesthesia. When tumor size reached 200-250 mm3, approximately day 19-21 post inoculation, mice were randomly divided into groups: ZIKV MR766, ZIKVN, or saline placebo. Tumors were measured using calipers and volume determined using the LxW2x0.5 method. Euthanasia with CO2 or terminal anesthesia in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the euthanasia of animals, was performed using criteria of 20% weight loss, tumor ulceration, and / or when tumors crossed a size of 1.5 cm3. At euthanasia, tissues were harvested for immunohistochemistry, immunofluorescence, and quantitative reverse transcript! on-polymerase chain reaction (qRT-PCR).

[0149] Intracranial tumors. Single cell suspension of GSCs (2.0xl04cells in 5 pl) were implanted into the right cerebral hemisphere of 42-56 days old athymic nude female mice after mice were anesthetized with inhalation isoflurane anesthesia. Mice were mounted onto stereotactic apparatus and incisions were made on the top of the head and 1mm burr hole (2 mm posterior and 1.5 mm lateral to bregma) made to accommodate injection of GSCs. A 10 pL Hamilton syringe, loaded with GSCs was slowly lowered at a depth of 3.5 mm and withdrawn 0.5mm to depth of 2.5 mm. Injection was performed over a period of 5 minutes using the micro injector. The syringe was held in place for 5 minutes post injection before being withdrawn over a period of 5 minutes. Drill holes were sealed with sterile surgical bone wax before sealing the skin together with medical grade tissue adhesive. Mice were given a subcutaneous injection of analgesic, then placed in a warmed recovery cage until stable.

[0150] Bioluminescence imaging

[0151] In intracranial tumor, at day 9-17 after tumor implantation, mice with similar flux were randomized between groups. Using the same coordinates as for tumor implantation, mice were inoculated intratumorally with ZIKVN (5.7xl04) and ZIKVS (102, 103, 104, 3.7X105and 106PFU) or PBS, each in 10 pl.

[0152] Animals were observed daily, and weights and tumor measures were done three times per week with concurrent hydration assessment and observation of body condition.

[0153] Example 1 : ZIKV MR766 strain infected patient derived glioblastoma (GBM) stem cell lines.

[0154] The AXL expression of five patient derived GBM stem cell lines was determined by Western blot (FIG. 1A). The cell lines were then exposed to ZIKV MR766 strain at an MOI of 1. At six hours post infection, the amount of viral RNA as quantified by qRT-PCR varied considerably between the cell lines (FIG. IB). It was observed that more viral entry occurred in high AXL expressing patient derived cell lines. The infection rate for each cell line was determined by quantifying the percent of infected cells by flow cytometry at 24, 48, and 72 hours post infection (FIG. 1C). Cell lines 7730 and 7714 had the least amount of infected cells at 24 hours post infection. However, by 72 hours, all cell lines except 7730 reached nearly 100% infection. Finally, live virus, as quantified by plaque assay, recovered from the supernatant of infected cells increased over time and varied by cell line (FIG. ID). Again, infection in 7730 and 7714 cell lines lagged behind the 8034, 8049, and 7966 cell lines.

[0155] Example 2: AXL was required for ZIKV entry into GBM stem cell lines.

[0156] CRISPR was used to knockout AXL expression in the 8034 and 8049 cell lines. Knockout was confirmed by Western blot (FIG. 2A). Compared to their parental lines, 8034 AXLKOand 8049 AXLKOwere resistant to ZIKV MR766 infection. This was confirmed by flow cytometry (FIG. 2B, FIG. 2C), immunofluorescence imaging (FIG. 2D, FIG. 2E), qRT-PCR (FIG. 2F, FIG. 2G), and plaque assay (FIG. 2H, FIG. 21, and FIG. 2J).

[0157] Differentiated glioma cells (DGCs) were less permissive to ZIKV infection (FIG. 2). Immunofluorescence imaging (FIG. 2K, FIG. 2L) confirmed that DGCs have greater expression of GFAP than do their parental lines. Flow cytometry (FIG. 2M) demonstrated that there was a significantly lower infection rate in DGCs compared to their parental lines.

[0158] Example 3: AXL was required for ZIKVS and ZIKVN entry into GBM cell lines.

[0159] As with MR766, 8034 AXLKOand 8049 AXLKOwere resistant to ZIKVN and ZIKVS infection (FIG. 3A, FIG. 3B). In parental cell lines, the percent of infected cells is nearly 100% by days 3 or 4 post infection. By contrast, at 7 days, there is no discernible infection in the knockout cell lines. The cells were infected with ZIKV at an MOI of 1. Following exposure to ZIKVS and ZIKVN, cytotoxicity, as measured with the Annexin V flow cytometry assay, was abrogated in the AXL knockout cell lines (FIG. 3C).

[0160] Example 4: Virulence differed amongst ZIKV strains.

[0161] Using a panel of patient derived GBM stem cell lines, it was found that cell lines differed in their permissiveness to ZIKV infection and that the different ZIKV strains varied in their ability to infect the cell lines. The MR766 strain was the ancestral strain first isolated in the Zika forest of Uganda in 1947. Strains ZIKVN and ZIKVS are wild type strains isolated from a patient in Nicaragua (ZIKVN) and Brazil (ZIKVS) (FIG. 4). The strains infect a number of patient derived GBM cell lines with differing variability, with ZIKVN being more virulent.

[0162] Example 5: ZIKV treatment of immunocompromised mice bearing patient derived GBM tumors in their flanks led to prolonged survival.

[0163] The 8034 and 8049 cell lines were used to establish subcutaneous flank tumors. The volume of the tumors was recorded before and after mock, ZIKV MR766, or ZIKVN treatment (FIG. 5A, FIG. 5D). ZIKV treatment led to prompt, complete, and lasting response. Treatment with ZIKVN led to 100% survival with tumors from 8034 and 8049 cell lines (FIG. 5B, FIG. 5E). Interestingly, following a complete response 11 of 20 mice succumbed to weight loss in MR766 treatment. Six (6) of nine (9) mice in the 8034 group and five (5) of eleven (11) in the 8049 group treated with MR766 succumbed to weight loss after complete tumor response. This did not occur following ZIKVN treatment (FIG. 5C, FIG. 5F). There were no weight loss associated deaths in the ZIKVN treatment group. Because of the weight loss in MR766 treated group, the viral load in vital organs of mice with weight loss was evaluated using qRT-PCR (FIG. 5G). The relative expression of ZIKV RNA was not significantly different compared to mock infection in the organs tested.

[0164] Example 6: Treatment of immunocompromised mice bearing intracranial patient derived GBM tumors led to prolonged survival.

[0165] Luciferase tagged patient derived GBM tumors (cell line 8034) were established in mice. Around day 7, mice were randomized to mock treatment or treatment with ZIKVN or ZIKVS. The time course of response was monitored with luminescence imaging (FIG. 6A). Kaplan-Meier curves show a significant prolongation of survival following ZIKV treatment compared to mock treatment (FIG. 6B). In the ZIKVS group, 58% of mice survived to the end of the trial, 63% experienced a complete response, and there was no significant weight loss. In the ZIKVN group, 79% of mice experienced a complete response, however 6 of the 15 complete responders succumbed to weight loss. The change in luminescence (FIG. 6C), tumor area (FIG. 6D), and weight (FIG. 6E) were monitored over time.

[0166] Example 7: Determination of lowest effective dose of ZIKVS for intratumoral delivery.

[0167] Intracranial tumors were established with the luciferase tagged 8034 cell line. Mice were randomized to receive mock, or ZIKVS treatment at 102, 103, or 104PFU. At 87 days post injection, 80% of the 104group and 70% of the 102and 103groups survived (FIG. 7A). Tumor area and luminescence were followed over time (FIG. 7B, FIG. 7C). Interestingly, mice treated with placebo, or 103PFU experienced weight loss. Weight rebounded in the 103PFU treatment group (FIG. 7D).

[0168] Example 8: Intravenous ZIKVS delivery was also an effective treatment for intracranial GBM.

[0169] Once again patient derived GBM tumors (8034 cell line, luciferase tagged) were established in the brains of immunocompromised mice. Mice were randomized to receive placebo or intravenous ZIKVS, 106PFU. There were six complete responses, one partial response, and one tumor related death. At 80 days post treatment, 75% of the mice were alive and well (FIG. 8A). The change in tumor area (FIG. 8B), luminescence (FIG. 8C), and weight (FIG. 8D) were followed over time. Once again, treated mice experienced weight loss that recovered promptly.

[0170] Example 9: ZIKV MR766 treatment of GBM organoids leads to productive viral replication.

[0171] GBM organoids were established from 8049 and 8049 AXLKOcell lines. As seen using bright field imaging, the knockout derived cell lines have a ragged surface compared to the smooth surface of the parental organoids (FIG. 9A, FIG. 9B). Using immunofluorescence staining, it was found that 7-8 week old organoids contained Sox2 and Nestin expressing stem cells, oligodendrocyte lineage Olig2 expressing cells, and early neurons expressing 13-tubulin III (FIG. 9C). Parental but not AXLKOorganoids are permissive to ZIKV MR766 infection (FIG. 9D, FIG. 9E). Moreover, these data show that ZIKV MR766 was capable of infecting and replicating in a patient-derived organoid model.

[0172] Example 10: Melanoma cells lines were susceptible to ZIKV infection.

[0173] Patient derived metastatic melanoma cell lines (7895, 7993, 8131), commercially available metastatic melanoma cell lines (HTB63, HTB66, HTB69, HTB71, CRL7898 (ATCC; Manassas, VA)), and commercially available primary melanoma cell lines (HTB67, HTB72, CRL1619 (ATCC)) were infected with ZIKV strains (MR766, ZIKVN, ZIKVS) at an MOI of 1 for90 minutes and then washed. The percent of infected cells at 7 days post infection was determined by flow cytometry and summarized in FIG. 10.

[0174] Example 11: ZIKV infection of melanoma cell lines led to apoptosis of the melanoma cell lines.

[0175] HTB66 and HTB71 cell lines were exposed to ZIKV strains at an MOI of 1 for 90 minutes and then washed. Apoptosis was measured using the Annexin V flow cytometry assay at several days post infection. Compared to mock treated cells, ZIKV treatment resulted in death of significantly more cells (FIGs. 11 A-l IB).

[0176] Example 12: Breast cancer cell lines were susceptible to ZIKV infection.

[0177] A panel of commercially available breast cancer cell lines (MDA-231, CLR-2351, SKBR3, MDA-361, HTB21, HCC1937, HTB130, and HCC1428 (ATCC)) were screened for susceptibility to ZIKV infection. The cells were exposed to ZIKV strains, MOI 1, for 90 minutes and then washed. The percent of infected cells was determined by flow cytometry at 3 days post infection (FIG. 12).

[0178] Example 13: ZIKV infection of breast cancer cell lines led to apoptosis of the breast cancer cell lines.

[0179] Cell lines were exposed to ZIKV MR766 strain at an MOI of 1 for 90 minutes then washed. At 4 days post infection (dpi), except where noted, apoptosis was quantified using the Annexin V flow cytometry assay (FIG. 13).

[0180] Example 14: ZIKV infection led to tumor volume reduction in breast cancer mouse model system.

[0181] In this study, there were two groups of female FOX N 1 nude mice: unilateral tumor, mock treatment (N=9), and bilateral tumor, with only the left sided tumor treated with ZIKV MR766 (N=13) (FIG. 14A). For the tumors, the cell line MDA-MB-231 (2xl06cells in 100 p.L of 9.2% Matrigel) were injected into the mammary fat pads as described above. Tumors were treated with 5xl05ZIKV when they reached 125-150 mm3.

[0182] Tumor volumes were measured daily. Initially, both the treated tumor and the contralateral untreated tumor decreased in size. However, the untreated contralateral tumor began to increase in size and parallel, if not exceed, the growth rate for the mock treated tumors (FIG. 14B).

[0183] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.

Claims

CLAIMSIt is claimed:

1. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a zika virus, wherein the zika virus thereof comprises a nucleotide sequence at least 99.6% identical with the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2.

2. The method of claim 1, wherein the zika virus thereof comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2.

3. The method of claim 1 or 2, wherein the zika virus comprises the nucleotide sequence of SEQ ID NO: 1.

4. A method of treating a cancer in a subject in need thereof, the method comprising administering a Zika Virus Sao Jose do Rio Preto strain (ZIKVS) or a progeny thereof or a Zika Virus Nicaragua strain (ZIKVN) or a progeny thereof.

5. The method of claim 1, wherein the zika virus or progeny thereof is administered at a dose of about 102to about 107plaque forming units (PFU).

6. The method of claim 5, wherein the zika virus or progeny thereof is administered at a dose of about 104PFU, about 105PFU, about 105to about 106PFU, or about 105 7PFU.

7. The method of claim 1, wherein the zika virus or progeny thereof is administered intratumorally, intravenously, subcutaneously, intranasally, or intracranially.

8. The method of claim 7, wherein the zika virus or progeny thereof is administered intravenously.

9. The method of claim 1, wherein the zika virus or progeny thereof is administered to the subject in a pharmaceutical composition comprising the zika virus or progeny thereof and a pharmaceutically acceptable carrier.

10. The method of claim 9, wherein the pharmaceutical composition comprises a buffer, a salt, and an isotonic agent.

11. The method of claim 10, wherein the buffer is a combination of potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4).

12. The method of claim 10, wherein the salt is mono-sodium glutamate.

13. The method of claim 10, wherein the isotonic agent is a sugar.

14. The method of claim 13, wherein the sugar is sucrose.

15. The method of claim 10, wherein the pharmaceutical composition comprises:(a) the zika virus or progeny thereof;(b) the buffer, wherein the buffer is a combination of potassium dihydrogen phosphate (KH2PO4) at a concentration of about 0.001M to about 0.01M and a dipotassium hydrogen phosphate (K2HPO4) at a concentration of about 0.001M to about 0.01M;(c) the salt is mono-sodium glutamate at a concentration of about 0.001M to about 0.01M; and(d) the isotonic agent is a sugar, wherein the sugar is sucrose at a concentration of about 0.05M to about 0.25M.

16. The method of claim 15, wherein the pharmaceutical composition comprises:(a) the zika virus or progeny thereof;(b) KH2PO4 at a concentration of about 0.0038M and K2HPO4 at a concentration of about 0.0072M;(c) mono-sodium glutamate at a concentration of about 0.0054M; and(d) sucrose at a concentration of about 0.128M.

17. The method of claim 1, wherein the cancer is a solid cancer or a hematologic malignancy.

18. The method of claim 17, wherein the solid cancer is selected from a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, a glioma, a glioblastoma, or other solid tumors.

19. The method of claim 18, wherein the solid cancer is a glioblastoma.

20. The method of claim 17, wherein the hematologic malignancy is selected from a lympoma, a myeloma, a leukemia, or other hematologic malignancies.

21. The method of claim 1, wherein the zika vims or progeny thereof is administered with at least one other therapy.

22. The method of claim 21, wherein the at least one other therapy is selected from a radiation therapy, an adjuvant temozolomide (TMZ) therapy, a carmustine (BCNU) therapy, a lomustine (CCNU) therapy, an immune checkpoint blockade therapy, a chimeric antigen receptor T cell (CAR- T) therapy, an oncolytic viral therapy, a vaccine therapy, a tumor-lysate loaded dendritic cell vaccine (DCVax-L) therapy, a focused ultrasound therapy, and / or combinations thereof.

23. The method of claim 22, wherein the immune checkpoint blockade therapy comprises at least one agent that targets an immune checkpoint peptide, wherein the immune checkpoint peptide isselected from cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), PD-1 ligand 1 (PD-L1), indoleamine 2,3 dioxygenase 1 (IDO), cluster of differentiation 47 (CD47), or cluster of differentiation 73 (CD73).

24. The method of claim 23, wherein the immune checkpoint blockade therapy comprises administration of atezolizumab, avelumab, cemiplimab, dostarlimab, durvalumab, nivolumab, ipilimumab, or pembrolizumab.

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