ONCOLYTIC VIRUSES AND METHODS OF USE

PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial Number 62/446,271 filed January 13, 2017, the entirety of which is incorporated by reference herein.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 3, 2017, is named 50014-702_601_SL.TXT and is 44,653 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Cancers of the brain and nervous system are among the most difficult to treat, with treatment primarily consisting of surgical removal and radiation therapy, and limited chemotherapy options due to the difficulties of passing the brain-blood barrier. Unfortunately, new therapeutics have failed to improve overall survival or lead to a useful clinical response. Thus, there is an unmet need for a new therapeutic approach that can impact the course of the disease.

SUMMARY OF THE INVENTION

[0004] In one aspect, provided herein are viruses that address the need for a new therapeutic approach to cancer. Methods of use involve administering a virus to a patient having cancer such that the virus impairs cell division, impairs cell growth, and/or induces cell death of cancerous cells. In some cases, viruses having one or more anti -cancerous effects are oncolytic viruses. In order to minimize an effect on non-cancerous cells, one or more viruses are provided having tropism for cells that are undergoing division. An exemplary virus having such tropism is a Zika virus, or a recombinant virus derived from a Zika virus. Recent analysis suggests that Zika virus acts on dividing cells by diverting the phosphorylated TANK binding kinase 1 (pTBKl) away from its key role in mitosis. Accordingly, some methods provided herein exploit the tropism of viruses such as Zika virus for use in the treatment of cancer.

[0005] Further provided are compounds that mimic the mitotic impairment caused by viral infection and thus are useful in the treatment of cancer. Compounds useful for the treatment of cancer provided herein comprise modulators of TBKl, which include inhibitors of TBKl and agonists or antagonists of molecules involved in TBKl signaling. Non -limiting examples of compounds useful for the treatment of cancer include amlexanox, BX795, ΚΓΝ1408, and derivatives thereof.

[0006] In one aspect, provided herein is a method of treating cancer, comprising administering to a subject in need thereof a virus comprising a flavivirus or a portion of a flavivirus. In some embodiments, the flavivirus is within a phylogenetic group of mosquito-borne flaviviruses (MBFV). In some embodiments, the group of MBFV comprises viruses associated with Aedes spp. mosquitoes. In some embodiments, the flavivirus comprises Zika virus, spondweni virus, kedougous virus, or a combination thereof. In some embodiments, the virus comprises a nucleic acid sequence encoding for: a capsid protein of the flavivirus, membrane protein of the flavivirus, envelope protein of the flavivirus, nonstructural (NS) protein of the flavivirus, or a combination thereof. In some embodiments, the cancer is a brain cancer. In some embodiments, the brain cancer is an astrocytoma, oligodendroglioma, ependymoma, meningioma, schwannoma, craniopharyngioma, germinoma, pineocytoma, or a combination thereof. In some embodiments, the virus targets AXL receptor tyrosine kinase of a cell of the brain cancer. In some embodiments, the virus is engineered to comprise a suicide gene expressible under an inducible promoter. In some embodiments, the method further comprises administering to the subject an activator of the inducible promoter to express the suicide gene, thereby killing the virus. In some embodiments, the virus comprises a recombinant binding domain having specificity for a cancer cell biomarker. In some embodiments, the virus is engineered for selective proliferation in cancer cells. In some embodiments, the virus comprises a genetic element of a Zika virus genome that is not found in a dengue virus genome. In some embodiments, the genetic element is at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, or 200 nucleotides in length. In some embodiments, the genetic element encodes for a viral protein. In some embodiments, the genetic element is non-coding. In some embodiments, the method further comprises administering to the subject an antiviral agent configured to kill the virus.

[0007] In another aspect, provided is a method of treating a brain tumor in a subject in need thereof, the method comprising administering to the subject a virus having tropism for a brain tumor cell. In some embodiments, the brain tumor comprises an astrocytoma, oligodendroglioma, ependymoma, meningioma, schwannoma, craniopharyngioma, germinoma, pineocytoma, or a combination thereof. In some embodiments, the virus comprises or is engineered from a flavivirus or a portion of a flavivirus. In some embodiments, the virus comprises a Zika virus, a portion of a Zika virus, spondweni virus, a portion of a spondweni virus, kedougous virus, a portion of a kedouous virus, or a combination thereof. In some embodiments, the virus comprises a nucleic acid sequence encoding for a viral capsid protein, a viral membrane protein, a viral envelope protein, a viral nonstructural (NS) protein, or a combination thereof. In some embodiments, the virus comprises or is engineered from a virus of the Herpesviridae family, or a portion of a virus of the Herpesviridae family. In some embodiments, the virus comprises or is engineered from a virus of the Betaherpesvirinae subfamily, or a portion of a virus of the

Betaherpesvinnae subfamily. In some embodiments, the virus comprises or is engineered from a cytomegalovirus. In some embodiments, the virus is engineered to comprise a suicide gene expressible under an inducible promoter. In some embodiments, the method further comprises administering to the subject an activator of the promoter to express the suicide gene, thereby killing the virus. In some embodiments, the virus comprises a recombinant binding domain having specificity for a cancer cell biomarker. In some embodiments, the virus is engineered for selective proliferation a cell of the brain tumor. In some embodiments, the virus comprises a genetic element of a Zika virus genome that is not found in a dengue virus genome. In some embodiments, the genetic element is at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, or 200 nucleotides in length. In some embodiments, the genetic element encodes for a viral protein. In some embodiments, the genetic element is non-coding. In some embodiments, the method further comprises administering to the subject an antiviral agent configured to kill the virus.

[0008] In another aspect, provided herein is a method of treating cancer, comprising administering to a subject in need thereof an oncolytic virus comprising a flavivirus or a portion of the flavivirus. In some embodiments, the flavivirus is within a phylogenetic group of mosquito -borne flaviviruses (MBFV). In some embodiments, the group of MBFV comprises viruses associated with Aedes spp. mosquitoes, and the flavivirus is a virus associated with the Aedes spp. mosquito. In some embodiments, the flavivirus comprises Zika virus, spondweni virus, kedougous virus, or a combination thereof. In some

embodiments, the flavivirus or the portion of the flavivirus comprises a nucleic acid sequence encoding for a capsid protein of the flavivirus, membrane protein of the flavivirus, envelope protein of the flavivirus, nonstructural (NS) protein of the flavivirus, or a combination thereof In some embodiments, the nucleic acid sequence encodes for the capsid protein of the flavivirus, the capsid protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence 1-122 of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encodes for the membrane protein of the flavivirus, the membrane protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to the ammo acid sequence 216-290 of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encodes for the envelope protein of the flavivirus, the envelope protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence 795-1146 of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encodes for the NS protein of the flavivirus, the NS protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to one of a combination of amino acid sequences: 1147-1372, 1373-1502, 1503-2119, 2120-2246, 2247-2269 and 2521-3423 of SEQ ID NO: 2. In some embodiments, the NS protein comprises NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5, or a portion or combination thereof. In some embodiments, the flavivirus, or the portion of the flavivirus, provides a vehicle for delivering the oncolytic virus to the cancer cell.

[0009] In some embodiments, the oncolytic virus causes mitotic impairment, cancer cell death, or both mitotic impairment and cancer cell death. In some embodiments, the oncolytic virus causes preferential mitotic impairment to the cancer cell as compared to a non-cancer cell, adult stem cell, or post-mitotic cell. In some embodiments, the cancer cell is a cancer stem cell. In some embodiments, the cancer is a brain cancer and the cancer cell is a brain cancer cell. In some embodiments, the cancer cell is a cell of an astrocytoma, oligodendroglioma, ependymoma, meningioma, schwannoma, craniopharyngioma, germinoma, pineocytoma, or a combination thereof. In some embodiments, the oncolytic virus targets AXL of the brain cancer cell.

[0010] In some embodiments, the oncolytic virus is engineered to comprise a suicide gene expressible under an inducible promoter. In some embodiments, the method further comprises administering to the subject the promoter or an activator thereof to express the suicide gene, thereby killing the oncolytic virus. In some embodiments, the oncolytic virus comprises a recombinant binding domain having specificity for a cancer cell biomarker. In some embodiments, the oncolytic virus is engineered for selective proliferation in the cancer cell. In some embodiments, the oncolytic virus comprises a genetic element of a Zika virus genome that is not found in a dengue virus genome. In some embodiments, administering comprises intraperitoneal injection, intracranial injection, intratumoral injection, systemic administration, parenteral administration, intravenous injection, intrathecal administration, or intravenous administration. In some embodiments, the method further comprises administering to the subject an antiviral agent configured to kill the oncolytic virus. In some embodiments, the antiviral agent comprises a nucleoside analog. In some embodiments, the nucleoside analog comprises sofosbuvir, 2'-C-methyladenosine, or a combination thereof. In some embodiments, the subject receives radiation therapy. In some

embodiments, the subject receives immunotherapy. In some embodiments, the subject receives chemotherapy.

[0011] In another aspect, provided herein is a method of treating a brain tumor in a subject in need thereof, the method comprising administering to the subject an oncolytic virus having tropism for a brain tumor cell. In some embodiments, the brain tumor comprises an astrocytoma, oligodendroglioma, ependymoma, meningioma, schwannoma, craniopharyngioma, germinoma, pineocytoma, or a combination thereof. In some embodiments, the oncolytic virus comprises or is derived from a flavivirus or a portion of a flavivirus. In some embodiments, the flavivirus is within a phylogenetic group of mosquito-borne flaviviruses (MBFV). In some embodiments, the group of MBFV comprises viruses associated with Aedes spp. mosquitoes, and the flavivirus is a virus associated with the Aedes spp.

mosquito. In some embodiments, the oncolytic virus comprises Zika virus, spondweni virus, kedougous virus, or a combination thereof. In some embodiments, the oncolytic virus comprises a nucleic acid sequence encoding for a capsid protein, membrane protein, envelope protein, nonstructural (NS) protein, or a combination thereof. In some embodiments, the nucleic acid sequence encodes for the capsid protein, the capsid protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence 1-122 of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encodes for the membrane protein, the membrane protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence 216-290 of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encodes for the envelope protein, the envelope protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence 795-1 146 of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encodes for the NS protein, the NS protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to one of a combination of amino acid sequences: 1147-1372, 1373- 1502, 1503-21 19, 2120-2246, 2247-2269 and 2521-3423 of SEQ ID NO: 2. In some embodiments, the NS protein comprises NS 1, NS2A, NS2B, NS3, NS4A, NS4B, NS5, or a portion or combination thereof. In some embodiments, the oncolytic virus comprises or is derived from a virus of the Herpesviridae family, or a portion of a virus of the Herpesviridae family. In some embodiments, the oncolytic virus comprises or is derived from a virus of the Betaherpesvirinae subfamily, or a portion of a virus of the Betaherpesvirinae subfamily. In some embodiments, the oncolytic virus comprises or is derived from a cytomegalovirus. [0012] In some embodiments, the oncolytic vims is engineered to comprise a suicide gene expressible under an inducible promoter. In some embodiments, the method further comprises administering to the subject the promoter or an activator thereof to express the suicide gene, thereby killing the oncolytic virus. In some embodiments, the oncolytic virus comprises a recombinant binding domain having specificity for a cancer cell biomarker. In some embodiments, the oncolytic virus is engineered for selective proliferation in the cancer cell. In some embodiments, the oncolytic virus comprises a genetic element of a Zika virus genome that is not found in a dengue virus genome. In some embodiments, administering comprises intraperitoneal injection, intracranial injection, intratumoral injection, systemic administration, parenteral administration, intravenous injection, intrathecal administration, or intravenous administration. In some embodiments, the method further comprises administering to the subject an antiviral agent configured to kill the oncolytic virus. In some embodiments, the antiviral agent comprises a nucleoside analog. In some embodiments, the subject receives radiation therapy. In some embodiments, the subject receives immunotherapy. In some embodiments, the subject receives chemotherapy.

[0013] In another aspect, provided herein is a method of treating cancer comprising administering to a subject in need thereof a modulator of TBK1. In some embodiments, the modulator of TBK1 comprises BX795, KIN1408, amlexanox, or a salt, solvate or combination thereof.

[0014] In another aspect, provided herein is a method of treating cancer comprising administering to a subject in need thereof a compound of Formula (I)

provided that,

R _: is hydrogen, d-C ₆ alkyl, phenyl, -C0 ₂ H, -OH, d-Qalkoxy, -C0 ₂ C _r C ₆ alkyl, -CN, -NH(C=0) Q- C ₆ alkyl, or amino group which may be unsubstituted or substituted by up to two Ci-C ₆ alkyl groups;

m is 0, 1 or 2 and R ₂ is Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, Ci-C ₆ alkoxy, F, CI, Br, I, -N0 ₂ , -OH, -C0 ₂ H, butadienylene (-CH=^!H-CH=^H-) which forms a benzene ring with any adjacent carbon atoms, -CN, - C0 ₂ Ci-C ₆ alkyl, -CF ₃ , or amino group which is unsubstituted or substituted by at least one Ci-C ₆ alkyl; and R ₃ is -C0 ₂ H, -CN, -C0 ₂ d-C ₆ alkyl, -C(=0)NH ₂ , -C(=0)N(C ₁ -C ₆ alkyl) ₂ , -C(=0)NH(C ₁ -C ₆ alkyl).

[0015] In some embodiments, the compound of Formula (I) is Formula (la)

or a salt or solvate thereof.

[0016] In another aspect, provided herein is a method of treating cancer comprising administering to a subject in need thereof a compound of Formula (II)

provided that,

n is 2-6;

Ri is a 5 or 6 membered cyclic C ₃ -Ci ₀ alkyl nng or aromatic cyclic ring, wherein optionally one or more carbons in the ring structure is substituted by an 0, N or S;

R ₂ is a 5 or 6 membered cyclic alkyl ring or cyclic aromatic ring, wherein optionally one or more carbons in the ring structure is substituted by an 0, N or S, or is a linear or branched substituted or unsubstituted Ci-C ₆ alkyl, Ci-C ₆ alkoxy or C ₂ -C ₆ alkenyl, wherein when substituted the substituent group is a C(=0)NH ₂ ; -C0 ₂ H; -OH, -NH ₂ , -N0 ₂ ; and

R ₃ is a F, CI, Br, I, H, -CH ₃ , -CN, or -N0 ₂ .

[0017] In some embodiments, n is 2-4. In some embodiments, Ri is a 5 or 6 membered cyclic alkyl and optionally one carbon is substituted by an N. In some embodiments, Ri is a pyrrolidine group. In some embodiments, R ₂ is a 5 or 6 membered aromatic ring and optionally one carbon is substituted by S, or is a branched C ₂ -C ₄ alkyl substituted by -C0 ₂ H, or C(=0)NH ₂ . In some embodiments, R ₂ is a thiophene group, or is the group

[0018] In some embodiments, R is I or Br. In some embodiments, the compound of Formula (II) is Formula (Ila)

or a salt or solvate thereof.

[0019] In another aspect, provided herein is a method of treating cancer comprising administering to a subject in need thereof a compoun f Formula (III)

provided that,

V is -CR ² R ³ -, -C(=0)CR ² R ³ -, or -C(=0)-;

R ¹ is R ^a , OR ^a , or -NR ² R ³ ;

each R ^a is independently H, optionally substituted Ci-C ₆ alkyl, optionally substituted phenyl, optionally substituted naphthyl, or optionally substituted heteroaryl;

R ² and R ³ are each independently H, optionally substituted Ci-C ₆ alkyl, optionally substituted phenyl, optionally substituted naphthyl, or optionally substituted heteroaryl;

each R ⁴ is independently R ^a , -OR ^a , -C(=0)R ^a , -C(=0)NR ² R ³ , -NR ² R ³ , -NR ^a C(=0)R ^a , -SR ^a , -SOR ^a , -

S0 ₂ R ^a , -S0 ₂ NHR ^a , -S0 ₂ NR ² R ³ , -NR ^a COR ^a , halogen, tnhalomethyl, CN, or -N0 ₂ ;

W is S, O, or NR ⁵ ;

R ⁵ is R ^a , -C(=0)R ^a , or -S0 ₂ R ^a .

[0020] In some embodiments, V is -CHR ² -. In some embodiments, R ¹ is quinolinyl. In some embodiments, R ¹ is R ^a . In some embodiments, R ^a is H, optionally substituted Ci-C ₆ alkyl, optionally substituted phenyl, optionally substituted naphthyl, or optionally substituted heteroaryl. In some embodiments, R ² is H, optionally substituted Ci-C ₆ alkyl, optionally substituted phenyl, optionally substituted naphthyl, or optionally substituted heteroaryl. In some embodiments, each R ⁴ is independently H, -CH ₃ , -OCH ₃ , F, CI, Br, I, CN, or -N0 ₂ ; W is S. In some embodiments, the compound of Formula (III) is Formula (Ilia)

or a salt or solvate thereof.

[0021] In some embodiments, the cancer is a brain cancer. In some embodiments, the method further comprises administering an oncolytic virus to the subject. In some embodiments, the oncolytic virus is selected from a Zika virus or a virus engineered from a Zika virus. In some embodiments, the oncolytic virus is selected from a cytomegalovirus or a virus engineered from a cytomegalovirus.

DETAILED DESCRIPTION OF THE INVENTION

[0022] In one aspect, viruses are provided that impair cell division, cell growth, and/or induce cell death in cancerous cells, referred to in some embodiments as oncolytic viruses. A non-limiting example of an oncolytic virus is a virus having tropism for dividing cells, or a recombinant virus thereof, such as a Zika virus or cytomegalovirus. Another aspect of this disclosure relates to methods of cancer treatment comprising administering an oncolytic virus provided herein.

[0023] Before the present methods and compositions are described, it is to be understood that this disclosure is not limited to a particular method or composition described, and as such may vary. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications mentioned herein are incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0024] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0025] Where a range of values is provided, unless otherwise indicated, each intervening value to the tenth of the unit of the lower limit between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range is encompassed herein. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed herein, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0026] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a virus" includes a plurality of such virus and reference to "the compound" includes reference to one or more compounds and derivatives or analogs thereof known to those skilled in the art, and so forth. As used herein, "about" refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term "about" generally refers to a range of numerical values (e.g., +/-5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term "about" may include numerical values that are rounded to the nearest significant figure.

Oncolytic viruses

[0027] In one aspect, provided herein are oncolytic viruses that may have selectivity for cells undergoing cell division, such as cancerous cells, and as such are useful for killing said cancerous cells and/or inhibiting the growth of a tumor. Accordingly, an oncolytic virus provided herein is not limited to a virus having a lytic effect on cancerous cells, and instead encompasses any virus that acts on a cancer cell to inhibit cell growth, prevent cell growth, inhibit cell replication, prevent cell replication, cause cell death, or any combination thereof. The oncolytic virus may be a clinical isolate of a virus, or a clone or recombinant virus derived or engineered therefrom.

[0028] In another aspect, a composition is provided comprising one or more portions of a virus. For example, such portions of the virus include nucleic acids and proteins of the virus. Non-limiting examples of viral proteins include capsid proteins, membrane proteins, non-structural proteins, and envelope proteins. Non -limiting examples of viral nucleic acids include protein coding sequences and non-protein coding sequences.

[0029] In some embodiments, an oncolytic virus is a recombinant or engineered virus. A non-limiting example of an engineered virus is one comprising a nucleic acid sequence that is derived from a viral genome, for example, by mutation, insertion, and/or deletion of one or more nucleobases. An insertion includes a nucleic acid sequence encoding for one or more proteins. An insertion also includes a promoter, such as a promoter inducible in mammalian cells. A deletion includes removal of a coding sequence that allows for replication of the virus. Accordingly, in some cases an engineered virus indicates that the virus from which the engineered virus is derived has been modified by the introduction of a heterologous nucleic acid or protein and/or the alteration of a native nucleic acid sequence. As used herein, oncolytic viruses include both native and recombinant/engineered viruses, as well as viruses comprises portions of a native or recombinant/engineered virus.

[0030] One method of generating a recombinant virus involves introducing a viral genome or portion thereof into a cell in whole or in part, for example, in two or more fragments that assemble into the desired viral genome or portion thereof within the cell. A non-limiting method comprises cloning and amplifying nucleic acid fragments covering the genome of a virus, or portion thereof. Nucleic acid fragments or entire viral genomes may also be produced de novo. A promoter sequence, such as a cytomegalovirus (CMV) promoter may be inserted at the end of the first fragment. For viruses having a heterologous nucleic acid sequence, this sequence is fused into or adjoining one of the fragments. In one method to produce an oncolytic virus, amplified fragments are introduced into cells, for example, by electroporation, and the cells are then transferred into growth medium. Cell supernatants are then recovered and stored or used to infect cells from which clarified virus is obtainable from culture supernatants.

[0031] Any suitable method is useful for generating genetic modifications in a virus to generate an engineered virus, including mutagenesis, polymerase chain reaction, homologous recombination, or any other genetic engineering technique available in the art. Mutagenesis includes modification of a nucleotide sequence, a single gene, or blocks of genes, and involves removal, addition and/or substitution of a single or plurality of nucleotide bases. In some cases, genetic modification comprises use of genetic recombination techniques to delete or replace at least part of native viral sequence. For example, a polynucleotide replaces part or all of a region of native viral sequence of interest. Alternatively or additionally, a polynucleotide is inserted into the native viral sequence. This inserted polynucleotide may be functional and encode, e.g., a suicide protein, therapeutic agent, and/or reporter protein. Exemplary non-limiting polynucleotides encoding for reporter proteins include green fluorescent protein, enhanced green fluorescent protein, beta-galactosidase, luciferase, and HSV-tk.

[0032] In some embodiments, an oncolytic virus comprises one or more molecules that impart to the virus an enhanced level of tumor cell specificity. In this way, the oncolytic virus is targeted to specific tumor types using tumor cell-specific molecules or biomarkers.

[0033] In some embodiments, an oncolytic virus comprises a nucleic acid sequence encoding for a polypeptide that is heterologous to the virus, where the polypeptide is expressible under an inducible promoter. As such, the oncolytic virus may also be an expression vector from which the polypeptide may be expressed. For example, an engineered virus comprises a suicide gene expressible under an inducible promoter, such as CMV. Methods of cancer treatment involving such engineered virus optionally comprise inducing expression of the suicide gene to kill or otherwise prevent the virus from propagating. Accordingly, the viral infection may be controlled by a physician according to the needs of the patient.

[0034] In some embodiments, an oncolytic virus comprises a genetic modification that affects expression of a viral gene. For example, a mutation in a virulence gene that contributes to the pathogenicity of the virus to a host organism such that the expression of that gene is significantly decreased, or wherein the gene product is rendered nonfunctional, or its ability to function is significantly decreased. In some cases, the genetic modification compromises the ability of the virus to replicate, or to replicate in non-cancerous or non-dividing cells.

[0035] In some embodiments, the oncolytic virus is engineered to place at least one viral protein necessary for viral replication under the control of a tumor-specific promoter. In some embodiments, a gene that encodes a cytotoxic agent is placed under the control of a tumor-specific promoter. For example, cytotoxic agents include toxins, prodrugs, cytokines, and chemokines. [0036] As a non-limiting example, an oncolytic vims comprises a flavivirus or a portion of a flavivirus. In some cases, the flavivirus is within a phylogenetic group of mosquito-borne flaviviruses (MBFV). For example, the MBFV comprises viruses associated with Aedes spp. mosquitoes, and the flavivirus is a virus associated with the Aedes spp. mosquito. Examples of flaviviruses include Zika virus (ZIKV), spondweni virus, and kedougous virus. In some embodiments, an oncolytic virus comprises a nucleic acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% homologous or identical to a virus having a genome of SEQ ID NO: 1 as shown in Table 1. In some cases, an oncolytic virus encodes for a capsid protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% homologous or identical to a sequence selected from SEQ ID NO: 2 as shown in Table 1. In some cases, an oncolytic virus encodes for a membrane protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% homologous or identical to a sequence selected from SEQ ID NO: 2. In some cases, an oncolytic virus encodes for an envelope protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% homologous or identical to a sequence selected from SEQ ID NO: 2. In some cases, an oncolytic virus encodes for a non-structural protein comprising an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% homologous or identical to a sequence selected from SEQ ID NO: 2

[0037] In some cases, a portion of a virus, such as a flavivirus, includes at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200 nucleobases of the flavivirus. These nucleobases do not necessarily have 100% sequence identity to the flavivirus and may vary, e.g., the identity is at least about 80%, 85%, 90%, 95%, or 98%. In some cases a portion refers to a region of the virus that encodes for one or more proteins. Additionally or alternatively, a portion refers to a region of the virus that does not encode for a protein.

[0038] In one aspect, provided herein is a Zika virus and/or an engineered virus derived from a Zika virus, both of which are referred to herein as an oncolytic Zika virus. Oncolytic Zika viruses may be derived from any laboratory strain or clinical isolate (non-laboratory strain) of Zika virus. A number of Zika viruses are known in the art. In some embodiments, an oncolytic virus is an engineered virus derived from a Zika virus having a genome having SEQ ID NO: 1. In some cases, the engineered virus has at least about 50% sequence homology and less than about 100% sequence homology to the nucleic acid from which it was derived. The degree of homology or percent identity between a native and a modified or engineered sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the World Wide Web, such as provided by BLAST by the National Center for Biotechnology Information (NCBI).

[0039] Another non-limiting example of an oncolytic virus is one based on or derived from a virus of the Herpesviridae family, or a portion of Herpesviridae virus. The Herpesviridae family includes the Betaherpesvirinae subfamily, of which cytomegalovirus is a member. Accordingly, an oncolytic virus useful for the methods and compositions herein is a cytomegalovirus, or a virus derived therefrom. In some embodiments, an oncolytic virus comprises a nucleic acid sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% homologous or identical to a virus having a genome of a cytomegalovirus. Oncolytic cytomegalovimses may be derived from any laboratory strain or clinical isolate (non -laboratory strain). A number of cytomegaloviruses are known in the art. In some embodiments, an oncolytic virus is an engineered virus derived from a cytomegalovirus. In some cases, the engineered virus has at least about 50% sequence homology and less than about 100% sequence homology to the nucleic acid from which it was derived.

[0040] In another aspect, provided herein is a nucleic acid comprising the genome of an engineered virus. Another aspect relates to a host cell comprising the oncolytic virus or a nucleic acid comprising the genome of the oncolytic virus.

Compounds

[0041] In one aspect, a compound is provided for use in the treatment of cancer. The compound may be a modulator of TBK1 such that TBK1 is inhibited or induced to localize away from functioning during mitosis A first exemplary compound for the treatment of cancer has a structure of Formula (I):

Formula (I)

wherein Rj is hydrogen, d-Cgalkyl, phenyl, -C0 ₂ H, -OH, d-C ₆ alkoxy, -C0 ₂ C _! -C ₆ alkyl, -CN, -NH(C=0) Ci-C ₆ alkyl, or amino group which may be unsubstituted or substituted by up to two Ci-C ₆ alkyl groups, m is 0, 1 or 2 and R ₂ is C _r C ₆ alkyl, C ₂ -C ₆ alkenyl, d-Qalkoxy, F, CI, Br, I, -N0 ₂ , -OH, -C0 ₂ H,

butadienylene (-CH=CH-CH=CH-) which forms a benzene ring with any adjacent carbon atoms, -CN, - C0 ₂ Ci-C ₆ alkyl, -CF ₃ , or amino group which may be unsubstituted or substituted by at least one Ci- Cealkyl; and R ₃ is -C0 ₂ H, -CN, -C0 ₂ Ci-C ₆ alkyl, -C(=0)NH ₂ , -C(=0)N(d-C ₆ alkyl) ₂ , -C(=0)NH(Ci- dalkyl); or a salt or solvate thereof.

[0042] In some embodiments, the compound of Formula (I) is amlexanox, having Formula (la):

Formula (la)

[0043] A second exemplary compound for the treatment of cancer has a structure of Formula (II), or a salt or solvate thereof:

Formula (II)

wherein n is 2-6; R _t is a 5 or 6 membered cyclic C ₃ -Ci ₀ alkyl ring or aromatic cyclic ring, wherein optionally one or more carbons in the ring structure is substituted by an 0, N or S; R ₂ is a 5 or 6 membered cyclic alkyl ring or cyclic aromatic ring, wherein optionally one or more carbons in the ring structure is substituted by an O, N or S, or is a linear or branched substituted or unsubstituted Ci-C ₆ alkyl, Ci-C ₆ alkoxy or C ₂ -C ₆ alkenyl, wherein when substituted the substituent group may be a C(=0)NH ₂ ; - C0 ₂ H; -OH, -NH ₂ , -N0 ₂ , and R ₃ is a F, CI, Br, I, H, -CH ₃ , -CN, or -N0 ₂ . In some cases, n is 2-4. In other cases, n is 3.

[0044] In some cases, Ri is a 5 or 6 membered cyclic alkyl wherein optionally one carbon is substituted by an N. In some cases, Ri is a pyrrolidine group such as 1- pyrolidinyl. In some cases, R ₂ is a 5 or 6 membered aromatic ring wherein optionally one carbon is substituted by S, or is a branched C ₂ -C ₄ alkyl substituted by -C0 ₂ H, or C(=0) H ₂ . In some cases, R ₂ is a thiophene group such as 2-thienyl, or is the

group

[0045] In some cases, R ₃ is halo, such as I or Br.

[0046] In some embodiments, the compound of Formula (II) is BX795, having Formula (Ila):

Formula (Ila) [0047] A third exemplary compound for the treatment of cancer has a structure of Formula (III) :

Formula (III)

wherein,

V is -CR ² R ³ -, -C(=0)CR ² R ³ -, or -C(=0)-;

R ¹ is R ^a , OR ^a , or -NR ² R ³ ;

each R ^a is independently H, optionally substituted Ci-Qalkyl, optionally substituted phenyl,

optionally substituted naphthyl, or optionally substituted heteroaryl;

R ² and R ³ are each independently H, optionally substituted d-C ₆ alkyl, optionally substituted phenyl, optionally substituted naphthyl, or optionally substituted heteroaryl;

each R ⁴ is independently R ^a , -OR ^a , -C(=0)R ^a , -C(=0)NR ² R ³ , -NR ² R ³ , -NR ^a C(=0)R ^a , -SR ^a , -SOR ^a , - S0 ₂ R ^a , -S0 ₂ NHR ^a , -S0 ₂ NR R ³ , -NR ^a COR ^a , halogen, trihalomethyl, CN, or -N0 ₂ ;

W is S, O, or NR ⁵ ;

R ⁵ is R ^a , -C(=0)R ^a , or -S0 ₂ R ^a .

[0048] In some cases, heteroaryl is monocyclic or bicyclic, and is selected from the group consisting of pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, triazolyl, imidazolyl, pyrazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, oxadiazolyl, thiadiazolyl, furazanyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, indolizinyl, azaindolizinyl, indolyl, azaindolyl, indazolyl, azaindazolyl, benzimidazolyl,

azabenzimidazolyl, benzotriazolyl, azabenzotriazolyl, benzoxazolyl, azabenzoxazolyl, benzisoxazolyl, azabenzisoxazolyl, benzofuranyl, azabenzofuranyl, benzothienyl, azabenzothienyl, benzothiazolyl, azabenzothiazolyl, or purinyl.

[0049] Any optionally substituted group of Formula (III) is optionally substituted with one or more groups independently selected from H, F, CI, Br, I, -OH, -CN, d-C ₆ alkyl, d-C ₆ alkoxy, Ci-C ₆ fluoroalkyl, Ci-C ₆ fluoroalkoxy, Ci-C _fi difluoroalkyl, Ci-C ₆ difluoroalkoxy, Ci-C ₆ trifluoroalkyl, Ci-C ₆ trifluoroalkoxy, - C0 ₂ H, -C(=0)C ₁ -C ₆ alkyl, -C(=0)NH ₂ , -C(=0)N(C ₁ -C ₆ alkyl) ₂ , -C(=0)NH(C ₁ -C _fl alkyl), -NH ₂ , -NH(C Qalkyl), -N(d-C ₆ alkyl) ₂ , -N(C ₁ -C ₆ alkyl)C(=0)(C ₁ -C _f ,alkyl), -S(d-C ₆ arkyl), -SOid-dalkyl), -S0 ₂ (d- dalkyl), -S0 ₂ NH(C C ₆ alkyl), -S0 ₂ N(d-C ₆ alkyl) ₂ , and -N(C ₁ -C ₆ alkyl)C(=0)(C ₁ -C ₆ alkyl).

[0050] In some cases, V is -CHR ² -; R ¹ is R ^a ; R ^a is H, optionally substituted d-C ₆ alkyl, optionally substituted phenyl, optionally substituted naphthyl, or optionally substituted heteroaryl; R ² is H, optionally substituted Ci-C ₆ alkyl, optionally substituted phenyl, optionally substituted naphthyl, or optionally substituted heteroaryl; each R ⁴ is independently H, -CH ₃ , -OCH ₃ , F, CI, Br, I, CN, or -N0 ₂ ; W is S. [0051] In some cases, R ¹ is quinolinyl.

[0052] In some embodiments, the compound of Formula (III) is KIN1408 and has a structure of Formula (Ilia):

Formula (Ilia)

[0053] The compounds disclosed herein, including the compounds of Formula (I), (II) or (III), may be prepared by methods known in the field of synthetic chemistry. A compound of Formula (I), (II) or (III), or a pharmaceutically acceptable salt, solvate or prodrug thereof, may be formulated with a

pharmaceutically acceptable excipient in a pharmaceutical composition.

[0054] In the preceding description of compounds suitable for use in the methods described herein, definitions of referred-to standard chemistry terms may be found in reference works (if not otherwise defined herein), including Carey and Sundberg "Advanced Organic Chemistry 4th Ed." Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the ordinary skill of the art are employed. Unless specific definitions are provided, the nomenclature employed in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those known in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

Methods of use

[0055] Oncolytic viruses and compounds disclosed herein are useful for treating or preventing one or more diseases, conditions, and/or symptoms of cancer. Methods of use involve contacting a cancerous cell with an effective amount of an oncolytic virus, such as a Zika virus, and/or a compound, such as one having Formula (I), (II) or (III). The contacting may be carried out in vitro (e.g. , in biochemical and/or cellular assays), in vivo in a non-human animal, and in vivo in mammals, including humans. In some cases, contacting involves bringing a cancer cell and a composition comprising a virus and/or compound provided herein into sufficient proximity such that the composition exerts an effect on the cancer cell. Contacting includes physical interaction between the composition and a cancer cell, as well as interactions that do not require physical interaction. Contacting includes administering an effective amount of a composition to a subject comprising the cancer cell such that the composition impairs cancer cell growth, division and/or induces cancer cell death. In some embodiments, contacting by administration includes intravenous administration, intraperitoneal administration, intramuscular administration, intracoronary administration, intraarterial administration, subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation, intracerebral, nasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue or tumor of the subject.

[0056] Subjects include animals, such as humans, other higher primates, lower primates, and animals of veterinary importance, such as dogs, cats, horses, sheep, goats, and cattle and the like. Subjects also include animals for use in studies, for example, mice, rats and other rodents.

[0057] Provided is a method for treating cancer in a subject comprising administration of an effective amount of a pharmaceutical composition that includes an oncolytic virus provided herein, and/or at least one compound of Formula (I), (II) or (III) or a pharmaceutically acceptable salt, pharmaceutically acceptable prodrug, or pharmaceutically acceptable solvate thereof, to said subject. An effective amount or therapeutically effective amount of a composition is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in, the symptoms associated with a cancer. The amount of a composition administered to the subject may depend on the type and severity of the cancer and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. In some embodiments, an effective amount of an oncolytic virus is administered to a subject having cancer in an amount sufficient to induce oncolysis, the disruption or lysis of a cancer cell, slowing, inhibition and/or reduction in the growth or size of a tumor, and includes the eradication of the tumor in certain instances. In some embodiments, an effective amount of an oncolytic virus is administered to a subject having cancer in an amount sufficient to attenuate or halt division of a cancer cell.

[0058] Treatment of cancer includes amelioration, cure, and/or maintenance of a cure (i.e. the prevention or delay of relapse) and/or its associated symptoms. For example, a subject is successfully treated for a cancer if after receiving a therapeutic amount of the composition described herein, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of the cancer, e.g., reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; increase in length of remission, and/or relief to some extent, of one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. Treatment also includes preventing the cancer from becoming worse, slowing the rate of progression, and/or preventing the cancer from re-occurrence after initial elimination. A suitable dose and therapeutic regimen may vary depending upon the specific oncolytic virus used, the mode of delivery of the oncolytic virus, and whether it is used alone or in combination with one or more other oncolytic viruses or compounds.

[0059] In some embodiments, a method comprises administration of a virus and/or compound provided herein to a subject having a tumor, such that tumor cell growth or proliferation is inhibited. In some cases, tumor growth or proliferation is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or 100%, and includes inhibition of tumor cell division and/or induction of tumor cell death.

[0060] In some embodiments, a method comprises administration of a virus and/or compound provided herein to a subject having a tumor, such that tumor cell progression (e.g., tumorigenesis, tumor growth and proliferation, invasion and metastasis) is inhibited. In some cases, inhibiting tumor progression refers to inhibiting the development, growth, proliferation, or spreading of a tumor, including without limitation the following effects: inhibition of growth of cells in a tumor, (2) inhibition, to some extent, of tumor growth, including slowing down or complete growth arrest; (3) reduction in the number of tumor cells; (4) reduction in tumor size; (5) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into adjacent peripheral organs and/or tissues; (6) inhibition (i.e. reduction, slowing down or complete stopping) of metastasis; (7) increase in the length of survival of a patient or patient population following treatment for a tumor; and/or (8) decreased mortality of a patient or patient population at a given time point following treatment for a tumor. In some cases, tumor progression is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%.

[0061] In some embodiments, a therapeutic benefit is achieved after administration of a composition herein. A therapeutic benefit includes anything that promotes or enhances the well-being of the subject with respect to the medical treatment of his/her condition, which includes treatment of pre-cancer, cancer, and hyperproliferative diseases. For example, extension of the subject's life by any period of time, decrease or delay in the neoplastic development of the disease, decrease in hyperproliferation, reduction in tumor growth, delay of metastases, reduction in cancer cell or tumor cell proliferation rate, and a decrease in pain to the subject that can be attributed to the subject's condition.

[0062] Administration of a pharmaceutical composition to a subject is by means which the virus and/or compound of Formula (I), (II), or (III) contained therein will contact a target cell. The specific route will depend upon certain variables such as the cancer cell, and can be determined by the skilled practitioner. Suitable methods of administering a composition comprising a pharmaceutical composition of the present invention to a patient include any route of in vivo administration that is suitable for delivering a virus and/or compound to a patient. Exemplary methods of in vivo administration include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intracoronary administration, intraarterial administration (e.g., into a carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation (e.g., aerosol), intracerebral, nasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue. In an embodiment where the target cells are in or near a tumor, a preferred route of administration is by direct injection into the tumor or tissue surrounding the tumor.

[0063] Various routes of administration are contemplated for various tumor types. Where discrete tumor mass, or solid tumor, may be identified, a variety of direct, local and regional approaches may be taken. For example, the tumor is directly injected with the composition. A tumor bed may be treated prior to, during or after resection and/or other treatment(s). Following resection or other treatment(s), one generally will deliver the adenovirus by a catheter having access to the tumor or the residual tumor site following surgery. Methods of treating cancer include treatment of a tumor as well as treatment of the region near or around the tumor. This includes body cavities in which the tumor lies, as well as cells and tissue that are next to the tumor.

[0064] Combinations

[0065] In some embodiments, a pharmaceutical composition comprising an oncolytic virus and/or compound of Formula (I), (II), and/or (III) is administered in combination with another agent effective in the treatment of cancer ("anti -cancer agent"). An anti -cancer agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. Anti-cancer agents include biological agents, chemotherapy agents, and radiotherapy agents. The pharmaceutical composition provided herein and the anti-cancer agent may be administered simultaneously, e.g. as a combined preparation or as separate preparations one administered immediately after the other. Alternatively, they may be administered separately and sequentially, where one agent is administered and then the other administered later after a predetermined time interval. A pharmaceutical composition provided herein and an anti -cancer agent may be administered simultaneously, separately, or sequentially which may allow the two agents to be present in the tumor requiring treatment at the same time and thereby provide a combined therapeutic effect, which may be additive or synergistic.

[0066] It is contemplated that the pharmaceutical composition is useful in conjunction with surgery and/or chemotherapeutic, radiotherapeutic, immunotherapeutic or other biological intervention. Methods may comprise administering to the patient a second therapy, wherein the second therapy is anti- angiogenic therapy, chemotherapy, immunotherapy, surgery, radiotherapy, immunosuppresive agents, or gene therapy with a therapeutic polynucleotide. In some cases, the second therapy is administered to the patient before, at the same time, or after administration of the pharmaceutical composition provided herein.

[0067] Cancer

[0068] As used herein, cancer is a class of diseases characterized by uncontrolled cellular growth. Cancer includes all types of hyperproliferative growth, hyperplastic growth, neoplastic growth, cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.

[0069] In some embodiments, a composition provided herein (oncolytic virus and/or compound of Formula (I), (II), (III)), prevents and/or attenuates tumor cell growth when administered to a patient having cancer. In some embodiments, a composition herein prevents and/or attenuates tumor cell invasion when administered to a patient having cancer. In some embodiments, a composition herein prevents and/or attenuates tumor cell metastasis when administered to a patient having cancer. In some embodiments, a composition herein an oncolytic virus comprising or derived from a flavivirus, such as a Zika virus. In some embodiments, a composition herein an oncolytic virus comprising or derived from a Herpesviridae virus, such as a cytomegalovirus. In some embodiments, a composition herein is a compound having Formula (I), or a salt or solvate thereof. In some embodiments, a composition herein is a compound having Formula (II), or a salt or solvate thereof. In some embodiments, a composition herein is a compound having Formula (III), or a salt or solvate thereof.

[0070] Types of cancer include, but are not limited to, solid tumors (such as those of the bladder, bowel, brain, breast, endometrium, heart, kidney, lung, liver, uterus, lymphatic tissue (lymphoma), ovary, pancreas or other endocrine organ (thyroid), prostate, skin (melanoma or basal cell cancer)) and hematological tumors (such as the leukemias and lymphomas) at any stage of the disease, with or without metastases. In some embodiments, the cancer comprises a brain tumor. Non-limiting examples of tumors treatable with a composition provided herein include adenoma, angio-sarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hamartoma, hemangioendothelioma, hemangiosarcoma, hematoma, hepato-blastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and teratoma. Glioma refers to a tumor originating in the neuroglia of the brain or spinal cord. Gliomas are derived from the glial cell types such as astrocytes and oligodendrocytes, thus gliomas include astrocytomas and oligodendrogliomas, as well as anaplastic gliomas, glioblastomas, and ependymomas. Additional brain tumors include meningiomas, ependymomas, pineal region tumors, choroid plexus tumors, neuroepithelial tumors, embryonal tumors, peripheral neuroblastic tumors, tumors of cranial nerves, tumors of the hemopoietic system, germ cell tumors, and tumors of the sellar region.

[0071] Dosing and treatment regimens

[0072] Administration frequencies for a pharmaceutical composition comprising a virus and/or compound provided herein may vary based on the method being practiced, the physical characteristics of the subject, the severity of the cancer, cancer type, and the formulation and the means used to administer the composition. Non-limiting exemplary administration frequencies include 6, 5, 4, 3, 2 or once daily, every other day, every third day, every fourth day, every fifth day, every sixth day, once weekly, every eight days, every nine days, every ten days, bi-weekly, monthly, bi-monthly, every three months, every four months, every five months, and every six months. In certain aspects, the pharmaceutical composition is administered once daily. In some embodiments, the pharmaceutical composition is administered continuously.

[0073] The duration of treatment will be based on the condition being treated and may be determined by the attending physician. The duration of administration, in many instances, varies depending on a number of factors. Exemplary factors include, without limitation, patient response, severity of symptoms, and cancer type. Under some conditions, treatment is continued for a number of days, weeks, or months. Under other conditions, complete treatment is achieve through administering one, two or three dose of the pharmaceutical composition over the entire course of treatment. In certain aspects, complete treatment can be achieved using a single dose of the pharmaceutical composition. [0074] In certain embodiments wherein the patient's condition does not improve, upon the doctor's discretion the administration of the virus and/or compound of Formula (I), (II) or (III) is administered chronically, that is, for an extended period of time, including throughout the duration of the patient's life in order to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition.

[0075] In certain embodiments wherein a patient's status does improve, the dose of composition being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e. a "drug holiday"). In specific embodiments, the length of the drug holiday is between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days. The dose reduction during a drug holiday is, by way of example only, by 10%- 100%, including 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[0076] In certain embodiments the dose of the composition being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e. a "drug diversion"). In specific embodiments, the length of the drug diversion is between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days. The dose reduction during a drug diversion is, by way of example only, by 10%- 100%, including 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. After a suitable length of time, the normal dosing schedule is optionally reinstated.

[0077] In some embodiments, as a patient is started on a regimen of a virus and/or compound of Formula (I), (II) or (III), the patient is also weaned off (e.g., step-wise decrease in dose) a second treatment regimen.

[0078] In some embodiments, once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, in specific embodiments, the dosage or the frequency of administration, or both, is reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. In certain embodiments, however, the patient requires intermittent treatment on a long-term basis upon any recurrence of symptoms.

[0079] The amount of a given composition vanes depending upon factors such as the particular virus and/or compound, disease condition and its severity, the identity (e.g., weight, sex) of the subject in need of treatment, but can nevertheless be determined according to the particular circumstances surrounding the case. In some cases, doses employed for adult human treatment are typically in the range of 0.01 mg- 5000 mg per day. In some cases, doses employed for adult human treatment are from about 1 mg to about 1000 mg per day. In some embodiments, the desired dose is conveniently presented in a single dose or in divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[0080] In some embodiments, the daily dosages appropriate for a compound of Formula (I), (II) or (III) described herein are from about 0.01 to about 10 mg/kg per body weight. In some cases, an indicated daily dosage in a large mammal, including, but not limited to, humans, is in the range from about 0.5 mg to about 1000 mg, optionally administered in divided doses, including, but not limited to, up to four times a day. In some embodiments, the daily dosage is administered in extended release form. In certain embodiments, suitable unit dosage forms for oral administration comprise from about 1 to 500 mg compound. In other embodiments, the daily dosage or the amount of active in the dosage form are lower or higher than the ranges indicated herein, based on a number of variables in regard to an individual treatment regime. In various embodiments, the daily and unit dosages are altered depending on a number of variables including, but not limited to, the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0081] In some embodiments, a compound of Formula (I), (II), and/or (III) is administered an amount from about 25 mg to about 100 mg per dose. In some embodiments, a compound of Formula (I), (II), and/or (III) is administered an amount from about 100 mg to about 200 mg per dose. In some embodiments, a compound of Formula (I), (II), and/or (III) is administered an amount from about 200 mg to about 400 mg per dose. In some embodiments, a compound of Formula (I), (II), and/or (III) is administered an amount from about 400 mg to about 500 mg. In some embodiments, a compound of Formula (I), (II), and/or (III) is administered an amount from about 500 mg to about 1,500 mg.

[0082] In some embodiments, administration of a virus depending on the kind of virus and titer, and includes delivery of 1 to 100, 10 to 50, 100-1000, or up to 1 * 10 ⁴ , 1 * 10 ⁵ , 1 * 10 ⁶ , 1 * 10 ⁷ , 1 * 10 ⁸ , 1 * 10 ⁹ , 1 * 10 ¹⁰ , 1 * 10 ¹¹ , 1 * 10 ¹² or 1 * 10 ¹³ infectious particles to the patient in a pharmaceutically acceptable composition.

Pharmaceutical compositions and formulations

[0083] Provided herein are viruses and compounds having Formulas (I), (II) or (III), formulated into pharmaceutical compositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active agent into preparations that can be used pharmaceutically. A summary of pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's

Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and

Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins, 1999), herein incorporated by reference for such disclosure. For some virus delivery methods, a pharmaceutically acceptable vehicle is selected from known pharmaceutically acceptable vehicles for delivery, and should be one in which the virus is stable.

[0084] Further provided herein are pharmaceutical compositions that include a virus and/or a compound of Formula (I), (II) or (III), and another active agent; a pharmaceutically acceptable inactive ingredient; other medicinal or pharmaceutical agent; carrier; adjuvant; preserving, stabilizing, wetting or emulsifying agent; solution promoter; salt; buffer; excipients; binder; filling agent; suspending agent; flavoring agent; sweetening agents; disintegrating agent; dispersing agent; surfactants; lubricant; colorant; diluent; solubilizer; moistening agent; plasticizers; penetration enhancer; anti-foam agent; antioxidant;

preservative; or a combination thereof.

[0085] A composition as described herein, in various embodiments, comprises two or more

pharmaceutical compositions. In some embodiments, a pharmaceutical composition comprises one or more oncolytic viruses, for example, comprising or derived from a flavivirus, such as Zika virus. In some embodiments, a pharmaceutical composition comprises one or more compounds having Formulas (I), (II), and/or (III), or a pharmaceutically acceptable salt or solvate thereof. The two or more pharmaceutical compositions are administered together or separately.

[0086] In some embodiments, a pharmaceutical composition comprises at least one compound of Formula (I), (II) or (III) as an active agent in free-acid or free-base form, or in a pharmaceutically acceptable salt form.

[0087] In some embodiments, pharmaceutically acceptable salts are obtained by reacting a compound described herein with an acid to provide a "pharmaceutically acceptable acid addition salt." In some embodiments, the compound described herein (i.e. free base form) is basic and is reacted with an organic acid or an inorganic acid. Inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and metaphosphoric acid. Organic acids include, but are not limited to, 1 -hydroxy-2-naphthoic acid; 2,2-dichloroacetic acid; 2-hydroxyethanesulfonic acid; 2- oxoglutaric acid; 4-acetamidobenzoic acid; 4-aminosalicylic acid; acetic acid; adipic acid; ascorbic acid (L); aspartic acid (L); benzenesulfonic acid; benzoic acid; camphoric acid (+); camphor- 10-sulfonic acid (+); capric acid (decanoic acid); caproic acid (hexanoic acid); caprylic acid (octanoic acid); carbonic acid; cinnamic acid; citric acid; cyclamic acid; dodecylsulfuric acid; ethane- 1,2-disulfonic acid; ethanesulfonic acid; formic acid; fumaric acid; galactaric acid; gentisic acid; glucoheptonic acid (D); gluconic acid (D); glucuronic acid (D); glutamic acid; glutaric acid; glycerophosphoric acid; glycolic acid; hippuric acid; isobutyric acid; lactic acid (DL); lactobionic acid; lauric acid; maleic acid; malic acid (- L); malonic acid; mandelic acid (DL); methanesulfonic acid; monomethyl fumarate, naphthalene- 1,5-disulfonic acid; naphthalene-2-sulfonic acid; nicotinic acid; oleic acid; oxalic acid; palmitic acid; pamoic acid; phosphoric acid; proprionic acid; pyroglutamic acid (- L); salicylic acid; sebacic acid; stearic acid; succinic acid; sulfuric acid; tartaric acid (+ L); thiocyanic acid; toluenesulfomc acid (p); and undecylenic acid.

[0088] In some embodiments, pharmaceutically acceptable salts are obtained by reacting a compound described herein with a base to provide a "pharmaceutically acceptable base addition salt."

[0089] In some embodiments, the compound described herein is acidic and is reacted with a base. In such situations, an acidic proton of the compound described herein is replaced by a metal ion, e.g., lithium, sodium, potassium, magnesium, calcium, or an aluminum ion. In some cases, compounds described herein coordinate with an organic base, such as, but not limited to, ethanolamine,

diethanolamine, triethanolamine, tromethamine, meglumine, N-methylglucamine, dicyclohexylamine, tris(hydroxymethyl)methylamine. In other cases, compounds described herein form salts with amino acids such as, but not limited to, arginine, lysine, and the like. Acceptable inorganic bases used to form salts with compounds that include an acidic proton, include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium hydroxide, lithium hydroxide, and the like. In some embodiments, the compounds provided herein are prepared as a sodium salt, calcium salt, potassium salt, magnesium salt, meglumine salt, N-methylglucamine salt or ammonium salt.

[0090] It should be understood that a reference to a pharmaceutically acceptable salt includes the solvent addition forms. In some embodiments, solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and are formed during the process of isolating or purifying the compound with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of compounds described herein are conveniently prepared or formed during the processes described herein. In addition, the compounds provided herein optionally exist in unsolvated as well as solvated forms. The solvated forms of the compounds of Formula (I), (II) or (III) are also considered to be disclosed herein.

[0091] In addition, the methods and pharmaceutical compositions described herein include the use of N- oxides (if appropriate), crystalline forms, amorphous phases, as well as active metabolites of these compounds having the same type of activity.

[0092] In some embodiments, the compounds of Formula (I), (II) or (III) exist as tautomers. All tautomers are included within the scope of the compounds presented herein. As such, it is to be understood that a compound of the Formula (I), (II) or (III) or a salt thereof may exhibit the phenomenon of tautomerism whereby two chemical compounds that are capable of facile interconversion by exchanging a hydrogen atom between two atoms, to either of which it forms a covalent bond. Since the tautomeric compounds exist in mobile equilibrium with each other they may be regarded as different isomeric forms of the same compound. It is to be understood that the formulae drawings within this specification can represent only one of the possible tautomeric forms. However, it is also to be understood that the present disclosure encompasses any tautomeric form, and is not to be limited merely to any one tautomeric form utilized within the formulae drawings. The formulae drawings within this specification can represent only one of the possible tautomeric forms and it is to be understood that the specification encompasses all possible tautomeric forms of the compounds drawn not just those forms which it has been convenient to show graphically herein.

[0093] In some embodiments, compounds of Formula (I), (II) or (III) exist as enantiomers,

diastereomers, or other steroisomeric forms. The compounds disclosed herein include all enantiomeric, diastereomeric, and epimeric forms as well as mixtures thereof.

[0094] In some embodiments, compounds described herein are prepared as prodrugs. A prodrug refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because in some situations they are easier to administer than the parent drug. For example, in some cases a prodrug is bioavailable by oral administration whereas the parent is not. In some cases, the prodrug has improved solubility in pharmaceutical compositions over the parent drug. In a non-limiting examples, a prodrug of a compound described herein is administered as an ester (the prodrug) to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water-solubility is beneficial. A further example of a prodrug is a short peptide (polyaminoacid) bonded to an acid group where the peptide is metabolized to reveal the active moiety. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In certain embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound. Prodrugs metabolized in vivo to produce a compound of Formula (I), (II) or (III) as set forth herein are included within the scope of the claims. In some cases, a compounds described herein is a prodrug for another derivative or active compound.

[0095] In certain embodiments, compositions provided herein include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0096] In some embodiments, formulations described herein benefit from antioxidants, metal chelating agents, thiol containing compounds and other general stabilizing agents. Examples of such stabilizing agents, include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins,

(I) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0097] The pharmaceutical compositions described herein, which include a compound of Formula (I),

(II) or (III) are formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.

[0098] The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self -emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0099] In one aspect, a compound of Formula (I), (II) or (III) is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In some embodiments, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[00100] For intravenous injections or drips or infusions, compounds described herein are formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients.

[00101] Parenteral injections may involve bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical composition described herein may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In one aspect, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen -free water, before use.

[00102] For administration by inhalation, a compound of Formula (I), (II) or (III) is formulated for use as an aerosol, a mist or a powder. Pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound described herein and a suitable powder base such as lactose or starch.

[00103] Representative intranasal formulations are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. See, for example, Ansel, H. C. et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed. (1995). Preferably these compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients. The choice of suitable carriers is dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents are optionally present. Preferably, the nasal dosage form should be isotonic with nasal secretions.

[00104] Buccal formulations that include a compound of Formula (I), (II) or (III) are administered using a variety of formulations known in the art. In addition, the buccal dosage forms described herein can further include a bioerodible (hydrolysable) polymeric carrier that also serves to adhere the dosage form to the buccal mucosa. For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner.

[00105] For intravenous injections, a composition is optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. For other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients.

[00106] Parenteral injections optionally involve bolus injection or continuous infusion. Formulations for injection are optionally presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. In some embodiments, a pharmaceutical composition described herein is in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of an agent that modulates the activity of a carotid body in water soluble form. Additionally, suspensions of an agent that modulates the activity of a carotid body are optionally prepared as appropriate, e.g., oily injection suspensions.

[00107] Pharmaceutical preparations for oral use are obtained by mixing one or more solid excipient with one or more of the compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents are added, such as the cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. In some embodiments, dyestuffs or pigments are added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[00108] In some embodiments, pharmaceutical formulations of a compound of Formula (I), (II) or (III) are in the form of a capsules, including push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the compounds are dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In some embodiments, stabilizers are added. A capsule may be prepared, for example, by placing the bulk blend of the formulation of the compound described above, inside of a capsule. In some embodiments, the formulations (non-aqueous suspensions and solutions) are placed in a soft gelatin capsule. In other embodiments, the formulations are placed in standard gelatin capsules or non-gelatin capsules such as capsules comprising HPMC. In other embodiments, the formulation is placed in a sprinkle capsule, wherein the capsule is swallowed whole or the capsule is opened and the contents sprinkled on food prior to eating.

[00109] In some embodiments, a solid oral dosage forms are prepared by mixing a compound of Formula (I), (II) or (III) with one or more of the following: antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. In some cases, the solid dosage forms disclosed herein are in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder, a capsule, solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, beads, pellets, granules. In other embodiments, the pharmaceutical formulation is in the form of a powder. Compressed tablets are solid dosage forms prepared by compacting the bulk blend of the formulations described above. In various embodiments, tablets will include one or more flavoring agents. In some embodiments, the tablets will include a film surrounding the final compressed tablet. In some embodiments, the film coating can provide a delayed release of the compound of Formula (I), (II) or (III) from the formulation. In some embodiments, the film coating aids in patient compliance (e.g., Opadry ^® coatings or sugar coating). Film coatings including Opadry ^® typically range from about 1% to about 3% of the tablet weight. In some embodiments, solid dosage forms, e.g., tablets, effervescent tablets, and capsules, are prepared by mixing particles of a compound with one or more pharmaceutical excipients to form a bulk blend composition. The bulk blend is readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. In some embodiments, the individual unit dosages include film coatings. These formulations are manufactured by conventional formulation techniques.

[00110] Suitable carriers for use in the solid dosage forms described herein include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose, microcrystalline cellulose, lactose, mannitol and the like.

[00111] Suitable filling agents for use in the solid dosage forms described herein include, but are not limited to, lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, hydroxypropylmethycellulose (HPMC), hydroxypropylmethycellulose phthalate,

hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[00112] Suitable disintegrants for use in the solid dosage forms described herein include, but are not limited to, natural starch such as corn starch or potato starch, a pregelatinized starch, or sodium starch glycolate, a cellulose such as methylcrystalline cellulose, methylcellulose, microcrystalline cellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose, cross- linked carboxymethylcellulose, or cross-linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.

[00113] Binders impart cohesiveness to solid oral dosage form formulations: for powder filled capsule formulation, they aid in plug formation that can be filled into soft or hard shell capsules and for tablet formulation, they ensure the tablet remaining intact after compression and help assure blend uniformity prior to a compression or fill step. Materials suitable for use as binders in the solid dosage forms described herein include, but are not limited to, carboxymethylcellulose, methylcellulose,

hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxyethylcellulose, hydroxypropylcellulose, ethylcellulose, and microcrystalline cellulose, microcrystalline dextrose, amylose, magnesium aluminum silicate, polysaccharide acids, bentonites, gelatin,

polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone, starch, pregelatinized starch, tragacanth, dextrin, a sugar, such as sucrose, glucose, dextrose, molasses, mannitol, sorbitol, xylitol, lactose, a natural or synthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, starch, polyvinylpyrrolidone, larch arabogalactan, polyethylene glycol, waxes, sodium alginate, and the like.

[00114] In general, binder levels of 20-70% are used in powder-filled gelatin capsule formulations. Binder usage level in tablet formulations varies whether direct compression, wet granulation, roller compaction, or usage of other excipients such as fillers which itself can act as moderate binder. Binder levels of up to 70% in tablet formulations is common.

[00115] Suitable lubricants or glidants for use in the solid dosage forms described herein include, but are not limited to, stearic acid, calcium hydroxide, talc, corn starch, sodium stearyl fumerate, alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, magnesium stearate, zinc stearate, waxes, Stearowet ^® , boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol or a methoxypolyethylene glycol such as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium or sodium lauryl sulfate, and the like.

[00116] Suitable diluents for use in the solid dosage forms described herein include, but are not limited to, sugars (including lactose, sucrose, and dextrose), polysaccharides (including dextrates and maltodextrin), polyols (including mannitol, xylitol, and sorbitol), cyclodextrins and the like. [00117] Suitable wetting agents for use in the solid dosage forms described herein include, for example, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, quaternary ammonium compounds (e.g., Polyquat 10 ^® ), sodium oleate, sodium lauryl sulfate, magnesium stearate, sodium docusate, triacetin, vitamin E TPGS and the like.

[00118] Suitable surfactants for use in the solid dosage forms described herein include, for example, sodium lauryl sulfate, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic ^® (BASF), and the like.

[00119] Suitable suspending agents for use in the solid dosage forms described here include, but are not limited to, polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,

polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, vinyl pyrrolidone/vinyl acetate copolymer (S630), sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium

carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

[00120] Suitable antioxidants for use in the solid dosage forms described herein include, for example, e.g., butylated hydroxytoluene (BHT), sodium ascorbate, and tocopherol.

[00121] It should be appreciated that there is considerable overlap between additives used in the solid dosage forms described herein. Thus, the above-listed additives should be taken as merely exemplary, and not limiting, of the types of additives that can be included in solid dosage forms of the pharmaceutical compositions described herein. The amounts of such additives can be readily determined by one skilled in the art, according to the particular properties desired.

[00122] In some embodiments, the solid dosage forms described herein are formulated as enteric coated delayed release oral dosage forms, i.e., as an oral dosage form of a pharmaceutical composition as described herein which utilizes an enteric coating to affect release in the small intestine or large intestine. In one aspect, the enteric coated dosage form is a compressed or molded or extruded tablet/mold (coated or uncoated) containing granules, powder, pellets, beads or particles of the active ingredient and/or other composition components, which are themselves coated or uncoated. In one aspect, the enteric coated oral dosage form is in the form of a capsule containing pellets, beads or granules, which include a compound of Formula (I), (II) or (III), that are coated or uncoated. Any coatings should be applied to a sufficient thickness such that the entire coating does not dissolve in the gastrointestinal fluids at pH below about 5, but does dissolve at pH about 5 and above. Coatings include shellac, which dissolves in media of pH >7; acrylic polymers such as methacrylic acid copolymers and ammonium methacrylate copolymers; Eudragit series E, L, S, RL, RS and NE (Rohm Pharma) available as solubilized in organic solvent, aqueous dispersion, or dry powders; and Poly Vinyl Acetate Phthalate (PVAP) which dissolves in pH >5, and it is less permeable to water vapor and gastric fluids. The Eudragit series RL, NE, and RS are insoluble in the gastrointestinal tract but are permeable and are used primarily for colonic targeting. The Eudragit series E dissolve in the stomach. The Eudragit series L, L-30D and S are insoluble in stomach and dissolve in the intestine. Conventional coating techniques such as spray or pan coating are employed to apply coatings. The coating thickness must be sufficient to ensure that the oral dosage form remains intact until the desired site of topical delivery in the intestinal tract is reached.

[00123] In various embodiments, the particles of a compound of Formula (I), (II) or (III) and one or more excipients are dry blended and compressed into a mass, such as a tablet, having a hardness sufficient to provide a pharmaceutical composition that substantially disintegrates within less than about 30 minutes, less than about 35 minutes, less than about 40 minutes, less than about 45 minutes, less than about 50 minutes, less than about 55 minutes, or less than about 60 minutes, after oral administration, thereby releasing the formulation into the gastrointestinal fluid.

[00124] In other embodiments, a powder including a compound of Formula (I), (II) or (III) is formulated to include one or more pharmaceutical excipients and flavors. Such a powder is prepared, for example, by mixing the compound and optional pharmaceutical excipients to form a bulk blend composition.

Additional embodiments also include a suspending agent and/or a wetting agent. This bulk blend is uniformly subdivided into unit dosage packaging or multi -dosage packaging units.

[00125] In still other embodiments, effervescent powders are also prepared. Effervescent salts have been used to disperse medicines in water for oral administration.

[00126] In another aspect, dosage forms include microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulation material. Exemplary materials include, but are not limited to, pH modifiers, erosion facilitators, anti-foaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

Exemplary useful microencapsulation materials include, but are not limited to, hydroxypropyl cellulose ethers (HPC) such as Klucel® or Nisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC), hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC, Pharmacoat®, Metolose SR, Methocel®-E, Opadry YS, PrimaFlo, Benecel MP824, and Benecel MP843, methylcellulose polymers such as Methocel®-A, hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF-LG,HF-MS) and Metolose®, Ethylcelluloses (EC) and mixtures thereof such as E461, Ethocel®, Aqualon®-EC, Surelease®, Polyvinyl alcohol (PVA) such as Opadry AMB, hydroxyethylcelluloses such as Natrosol®, carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC) such as Aqualon®-CMC, polyvinyl alcohol and polyethylene glycol co-polymers such as Kollicoat IR®, monoglycerides (Myverol), triglycerides (KLX), polyethylene glycols, modified food starch, acrylic polymers and mixtures of acrylic polymers with cellulose ethers such as Eudragit® EPO, Eudragit® L30D-55, Eudragit® FS 30D

Eudragit® L100-55, Eudragit® L100, Eudragit® S100, Eudragit® RD100, Eudragit® E100, Eudragit® L12.5, Eudragit® S12.5, Eudragit® NE30D, and Eudragit® NE 40D, cellulose acetate phthalate, sepifilms such as mixtures of HPMC and stearic acid, cyclodextrins, and mixtures of these materials.

[00127] Liquid formulation dosage forms for oral administration are optionally aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to a virus and/or compound the liquid dosage forms optionally include additives, such as: (a) disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) at least one preservative, (e) viscosity enhancing agents, (f) at least one sweetening agent, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersions further includes a crystal-forming inhibitor.

[00128] In some embodiments, the pharmaceutical formulations described herein are self-emulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation. An advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or the aqueous phase is optionally added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient. Thus, the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides improvements in the bioavailability of hydrophobic active ingredients.

[00129] In some embodiments, pharmaceutical dosage forms are formulated to provide a controlled release of a virus and/or compound of Formula (I), (II) or (III). Controlled release refers to the release of the virus and/or compound from a dosage form in which it is incorporated according to a desired profile over an extended period of time. Controlled release profiles include, for example, sustained release, prolonged release, pulsatile release, and delayed release profiles. In contrast to immediate release compositions, controlled release compositions allow delivery of an agent to a subject over an extended period of time according to a predetermined profile. Such release rates can provide therapeutically effective levels of agent for an extended period of time and thereby provide a longer period of pharmacologic response while minimizing side effects as compared to conventional rapid release dosage forms. Such longer periods of response provide for many inherent benefits that are not achieved with the corresponding short acting, immediate release preparations.

[00130] In other embodiments, the formulations described herein are delivered using a pulsatile dosage form. A pulsatile dosage form is capable of providing one or more immediate release pulses at predetermined time points after a controlled lag time or at specific sites. In some embodiments, the pulsatile dosage form includes at least two groups of particles, (i.e. multiparticulate) each containing the formulation described herein. The first group of particles provides a substantially immediate dose of the compound of Formula (I), (II) or (III) upon ingestion by a mammal. The first group of particles can be either uncoated or include a coating and/or sealant. In one aspect, the second group of particles comprises coated particles. The coating on the second group of particles provides a delay of from about 2 hours to about 7 hours following ingestion before release of the second dose. Suitable coatings for pharmaceutical compositions are described herein or known in the art.

[00131] In some embodiments, pharmaceutical formulations are provided that include particles of a compound of Formula (I), (II) or (III) and at least one dispersing agent or suspending agent for oral administration to a subject. The formulations may be a powder and/or granules for suspension, and upon admixture with water, a substantially uniform suspension is obtained.

[00132] In some embodiments, liquid formulations are provided having inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers. Exemplary emulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, sodium lauryl sulfate, sodium doccusate, cholesterol, cholesterol esters, taurocholic acid, phosphotidylcholine, oils, such as cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, or mixtures of these substances, and the like.

[00133] In some embodiments, pharmaceutical compositions optionally include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[00134] Additionally, pharmaceutical compositions optionally include one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[00135] Other pharmaceutical compositions optionally include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridimum chloride.

[00136] In one embodiment, the aqueous suspensions and dispersions described herein remain in a homogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005 edition, chapter 905), for at least 4 hours. In one embodiment, an aqueous suspension is re-suspended into a homogenous suspension by physical agitation lasting less than 1 minute. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion. Examples of disintegrating agents for use in the aqueous suspensions and dispersions include, but are not limited to, a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch, or sodium starch glycolate; a cellulose such as methylcrystalline cellulose, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross- linked sodium carboxymethylcellulose, cross-linked carboxymethylcellulose, or cross-linked croscarmellose; a cross-linked starch such as sodium starch glycolate; a cross-linked polymer such as crospovidone; a cross-linked polyvinylpyrrolidone; alginate such as alginic acid or a salt of alginic acid such as sodium alginate; a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth; sodium starch glycolate; bentonite; a natural sponge; a surfactant; a resin such as a cation-exchange resin; citrus pulp; sodium lauryl sulfate; sodium lauryl sulfate in combination starch; and the like.

[00137] In some embodiments, the dispersing agents suitable for the aqueous suspensions and dispersions described herein include, for example, hydrophilic polymers, electrolytes, Tween ^® 60 or 80, PEG, polyvinylpyrrolidone, and the carbohydrate-based dispersing agents such as, for example,

hydroxypropylcellulose and hydroxypropyl cellulose ethers, hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ethers, carboxymethylcellulose sodium, methylcellulose,

hydroxyethylcellulose, hydroxypropylmethyl-cellulose phthalate, hydroxypropylmethyl-cellulose acetate stearate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), polyvinylpyrrolidone/vinyl acetate copolymer, 4-(l,l,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers; and poloxamines. In other embodiments, the dispersing agent is selected from a group not comprising one of the following agents: hydrophilic polymers, electrolytes; Tween ^® 60 or 80; PEG; polyvinylpyrrolidone (PVP);

hydroxypropylcellulose and hydroxypropyl cellulose ethers; hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ethers; carboxymethylcellulose sodium; methylcellulose;

hydroxyethylcellulose; hydroxypropylmethyl-cellulose phthalate; hydroxypropylmethyl-cellulose acetate stearate; non-crystalline cellulose; magnesium aluminum silicate; triethanolamine; polyvinyl alcohol (PVA); 4-(l,l,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde; poloxamers; or poloxamines.

[00138] Wetting agents suitable for the aqueous suspensions and dispersions described herein include, but are not limited to, cetyl alcohol, glycerol monostearate, polyoxyethylene sorbitan fatty acid esters (e.g., the commercially available Tweens ^® such as e.g., Tween 20 ^® and Tween 80 ^® , and polyethylene glycols, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, vitamin E TPGS, sodium taurocholate, simethicone, phosphotidylcholine and the like.

[00139] Suitable preservatives for the aqueous suspensions or dispersions described herein include, for example, potassium sorbate, parabens (e.g., methylparaben and propylparaben), benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl alcohol or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride. Preservatives, as used herein, are incorporated into the dosage form at a concentration sufficient to inhibit microbial growth.

[00140] Suitable viscosity enhancing agents for the aqueous suspensions or dispersions described herein include, but are not limited to, methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, Plasdon ^® S-630, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof. The concentration of the viscosity enhancing agent will depend upon the agent selected and the viscosity desired.

[00141] Examples of sweetening agents suitable for the aqueous suspensions or dispersions described herein include, for example, acacia syrup, acesulfame K, alitame, aspartame, chocolate, cinnamon, citrus, cocoa, cyclamate, dextrose, fructose, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup,

monoammonium glyrrhizinate (MagnaSweet ^® ), maltol, mannitol, menthol, neohesperidine DC, neotame, Prosweet ^® Powder, saccharin, sorbitol, stevia, sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfame potassium, mannitol, sucralose, tagatose, thaumatin, vanilla, xylitol, or any combination thereof.

Kits

[00142] In one aspect of the disclosure, provided herein are kits which include one or more reagents or devices for the performance of the methods disclosed herein. In some embodiments, the kit comprises a composition as described herein. In some embodiments, the kit comprises a means to administrate the composition. In some embodiments, one or more of the compositions of a kit comprises an oncolytic virus and/or one or more compounds of Formula (I), Formula (II), Formula (III), or salts, solvates, or combinations thereof.

[00143] In some embodiments, the kit comprises suitable instructions in order to perform the methods of the kit. The instructions may provide information of performing any of the methods disclosed herein, whether or not the methods may be performed using only the reagents provided in the kit. The kit and instructions may require additional reagents or systems.

[00144] For use in the therapeutic applications described herein, kits and articles of manufacture are also described herein. In some embodiments, such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) including one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. The container(s) optionally have a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprise a composition with an identifying description or label or instructions relating to its use in the methods described herein.

[00145] A kit will typically include one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and/or devices) desirable from a commercial and user standpoint for use of a compound described herein. Non-limiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes, carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[00146] In some embodiments, a label is on or associated with the container. A label can be on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. A label can be used to indicate that the contents are to be used for a specific therapeutic application. The label can also indicate directions for use of the contents, such as in the methods described herein.

[00147] In certain embodiments, a pharmaceutical composition comprising an oncolytic virus and/or compound described herein and optional additional active agent is presented in a pack or dispenser device which can contain one or more unit dosage forms. The pack can for example contain metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for

administration. The pack or dispenser can also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, can be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

EXAMPLES

Example 1: Tropism of Zika virus

[00148] A population of neuroepithelial stem (NES) cells derived from human neocortical (NCX) and spinal cord neuroepithelial stem (NES) cells served as an in vitro model for ZIKV -related

neuropathogenesis. Postmitotic neurons and NES cells were infected with Zika virus. The ability of Zika virus to infect postmitotic neurons was extremely low (-3.3%) in comparison to its ability to infect self- renewing NES cells (-90%).

Example 2: Cell culture assay

[00149] U87 human glioma cell line is obtained from American Type Culture Collection and maintained as recommended. Cells are incubated in a multi-well plate and infected with Zika virus at selected values of multiplicity of infection (MOI) in medium. The percentage of mitotic cells and viable cells is assessed. Example 3: Mouse model

[00150] Studies are performed in U-87 MG-based intracranial xenografts in nude mice. Treatments comprise intratumoral administration of Zika virus, a compound of Formula (I), a compound of Formula (II), or a compound of Formula (III). An exemplary dose of Zika virus is 10-1000 viral particles.

[00151] Primary studies are focused on survival. At death (cancer-induced or sacrificed), brain tissue is extracted. Tumors are examined using H&E staining and immunohistochemistry for viral proteins and angiogenesis. Example 4: Clinical Trail

[00152] A Phase 1, dose-escalating, two-part study of Zika virus for glioma is initiated. Primary objectives of the study are to determine the safety, tolerabihty, feasibility, and biological effect of injecting Zika virus into human brain tumors. To be eligible for the study, patients are required to have

histologically-proven, malignant glioma.

[00153] Group A is treated with direct intratumoral injection of Zika virus into an area of biopsy- confirmed glioma, and Group B is treated with injection of a divided dose of virus into the resection bed prior to and following glioma excision. The starting dose for both study groups is between 10 and 1000 viral particles, with a plan to dose escalate in half-log increments.

[00154] Patients are observed for 28 days following virus injection, and then follow-ups are scheduled to occur at monthly intervals for 4 months, every 2 months for 2 years, and every 4 months for life thereafter for both treatment groups. Patients are monitored for toxicity and symptoms, and evaluated using magnetic resonance imaging (MRI), spinal tap, and other tests as appropriate based on clinical standards of care for the duration of the study.

[00155] Example 5: Preparation of engineered oncolytic virus

[00156] The viral genome of Zika virus is assembled into Vero cells after electroporation of overlapping synthetic fragments encoding different parts of the viral genome. Briefly, the cDNA sequences of the fragments are cloned into a plasmid and amplified by PC using primers designed so that adjacent fragments to be assembled share sequence homology at their connecting ends. The first fragment comprises a cytomegalovirus (CMV) promoter upstream of the viral genome to allow for transcription initiation. Infectious virus is recovered by transfection of the assembled cDNA into Vero cells. The resultant viruses are plaque-purified.

[00157] For viruses having a heterologous nucleic acid sequence, this sequence is fused into or adjoining a region encoding for a structural protein of the virus in a viral fragment, for example, downstream or within the sequence encoding for the capsid protein. An exemplary heterologous nucleic acid is one encoding for a suicide gene.

[00158] Table 1. Sequences

SEQ Title Sequence

ID

NO

1 Zika virus AGTTGTTGATCTGTGTGAATCAGACTGCGACAGTTCGAGTTTGAAGCGA isolate AAGCTAGCAACAGTATCAACAGGTTTTATTTTGGATTTGGAAACGAGAG

ZIKV/H.s TTTCTGGTCATGAAAAACCCAAAAAAGAAATCCGGAGGATTCCGGATTG apiens/Bra TCAATATGCTAAAACGCGGAGTAGCCCGTGTGAGCCCCTTTGGGGGCTT zil/PE243/ GAAGAGGCTGCCAGCCGGACTTCTGCTGGGTCATGGGCCCATCAGGATG

2015,

complete GGGTCTCATCAATAGATGGGGTTCAGTGGGGAAAAAAGAGGCTATGGA genome AATAATAAAGAAGTTCAAGAAAGATCTGGCTGCCATGCTGAGAATAATC

AATGCTAGGAAGGAGAAGAAGAGACGGGGCGCAGATACTAGTGTCGGA

ATTGTTGGCCTCCTGCTGACCACAGCTATGGCAGCGGAGGTCACTAGAC

GTGGGAGTGCATACTATATGTACTTGGACAGAAACGATGCTGGGGAGGC

CATATCTTTTCCAACCACATTGGGGATGAATAAGTGTTATATACAGATC ATGGATCTTGGACACATGTGTGATGCCACCATGAGCTATGAATGCCCTA

TGCTGGATGAGGGGGTGGAACCAGATGACGTCGATTGTTGGTGCAACAC

GACGTCAACTTGGGTTGTGTACGGAACCTGCCATCACAAAAAAGGTGAA

GCACGGAGATCTAGAAGAGCTGTGACGCTCCCCTCCCATTCCACTAGGA

AGCTGCAAACGCGGTCGCAAACCTGGTTGGAATCAAGAGAATACACAA

AGCACTTGATTAGAGTCGAAAATTGGATATTCAGGAACCCTGGCTTCGC

GTTAGCAGCAGCTGCCATCGCTTGGCTTTTGGGAAGCTCAACGAGCCAA

AAAGTCATATACTTGGTCATGATACTGCTGATTGCCCCGGCATACAGCA

TCAGGTGCATAGGAGTCAGCAATAGGGACTTTGTGGAAGGTATGTCAGG

TGGGACTTGGGTTGATATTGTCTTGGAACATGGAGGTTGTGTCACCGTA

ATGGCACAGGACAAACCGACTGTCGACATAGAGCTGGTTACAACAACA

GTCAGCAACATGGCGGAGGTAAGATCCTACTGCTATGAGGCATCAATAT

CAGACATGGCTTCGGACAGCCGCTGCCCAACACAAGGTGAAGCCTACCT

TGACAAGCAATCAGACACTCAATATGTCTGCAAAAGAACGTTAGTGGAC

AGAGGCTGGGGAAATGGATGTGGACTTTTTGGCAAAGGGAGTCTGGTG

ACATGCGCTAAGTTTGCATGCTCCAAGAAAATGACCGGGAAGAGCATCC

AGCCAGAGAATCTGGAGTACCGGATAATGCTGTCAGTTCATGGCTCCCA

GCACAGTGGGATGATCGTTAATGACACAGGACATGAAACTGATGAGAA

TAGAGCGAAGGTTGAGATAACGCCCAATTCACCAAGAGCCGAAGCCAC

CCTGGGGGGTTTTGGAAGCCTAGGACTTGATTGTGAACCGAGGACAGGC

CTTGACTTTTCAGATTTGTATTACTTGACTATGAATAACAAGCACTGGTT

GGTTCACAAGGAGTGGTTCCACGACATTCCATTACCTTGGCACGCTGGG

GCAGACACCGGAACTCCACACTGGAACAACAAAGAAGCACTGGTAGAG

TTCAAGGACGCACATGCCAAAAGGCAAACTGTCGTGGTTCTAGGGAGTC

AAGAAGGAGCAGTTCACACGGCCCTTGCTGGAGCTCTGGAGGCTGAGAT

GGATGGTGCAAAGGGAAGGCTGTCCTCTGGCCACTTGAAATGTCGCCTG

AAAATGGATAAACTTAGATTGAAGGGCGTGTCATACTCCTTGTGTACCG

CAGCGTTCACATTCACCAAGATCCCGGCTGAAACACTGCACGGGACAGT

CACAGTGGAGGTACAGTACGCAGGGACAGATGGACCTTGCAAGGTTCC

AGCTCAGATGGCGGTGGACATGCAAACTCTGACCCCAGTTGGGAGGTTG

ATAACCGCTAACCCCGTAATCACTGAAAGCACTGAGAACTCTAAGATGA

TGCTGGAACTTGATCCACCATTTGGGGACTCTTACATTGTCATAGGAGTC

GGGGAGAAGAAGATCACCCACCACTGGCACAGGAGTGGCAGCACCATT

GGAAAAGCATTTGAAGCCACTGTGAGAGGTGCCAAGAGAATGGCAGTC

TTGGGAGACACAGCCTGGGACTTTGGATCAGTTGGAGGCGCTCTCAACT

GTTTGGAGGAATGTCCTGGTTCTCACAAATTCTCATTGGAACGTTGCTGA

TGTGGTTGGGTCTGAACACAAAGAATGGATCTATTTCCCTTATGTGCTTG

GCCTTAGGGGGAGTGTTGATCTTCTTATCCACAGCCGTCTCTGCTGATGT

GGGGTGCTCGGTGGACTTCTCAAAGAAGGAGACGAGATGCGGTACAGG

GGTGTTCGTCTATAACGACGTTGAAGCCTGGAGGGACAGGTACAAGTA

CCATCCTGACTCCCCCCGTAGATTGGCAGCAGCAGTCAAGCAAGCCTGG

GAAGATGGTATCTGCGGGATCTCCTCTGTTTCAAGAATGGAAAACATCA

TGTGGAGATCAGTAGAAGGGGAGCTCAACGCAATCCTGGAAGAGAATG

GAGTTCAACTGACGGTCGTTGTGGGATCTGTAAAAAACCCCATGTGGGG

AGGTCCACAGAGATTGCCCGTGCCTGTGAACGAGCTGCCCCACGGCTGG

AAGGCTTGGGGGAAATCGCACTTCGTCAGAGCAGCAAAGACAAATAAC

AGCTTTGTCGTGGATGGTGACACACTGAAGGAATGCCCACTCAAACATA

GAGCATGGAACAGCTTTCTTGTGGAGGATCATGGGTTCGGGGTATTTCA

CACTAGTGTCTGGCTCAAGGTTAGAGAAGATTATTCATTAGAGTGTGAT

CCAGCCGTTATTGGAACAGCTGTTAAGGGAAAGGAGGCTGTACACAGTG

ATCTAGGCTACTGGATTGAGAGTGAGAAGAATGACACATGGAGGCTGA

AGAGGGCCCATCTGATCGAGATGAAAACATGTGAATGGCCAAAGTCCC

ACACATTGTGGACAGATGGAATAGAAGAGAGTGATCTGATCATACCCA

AGTCTTTAGCTGGGCCACTCAGCCATCACAATACCAGAGAGGGCTACAG

GACCCAAATGAAAGGGCCATGGCACAGTGAAGAGCTTGAAATTCGGTTT

GAGGAATGCCCAGGCACTAAGGTCCACGTGGAGGAAACATGTGGAACA AGAGGACCATCTCTGAGATCAACCACTGCAAGCGGAAGGGTGATCGAG

GAATGGTGCTGCAGGGAGTGCACAATGCCCCCACTGTCGTTCCGGGCTA

AAGATGGCTGTTGGTATGGAATGGAGATAAGGCCCAGGAAAGAACCAG

AAAGCAACTTAGTAAGGTCAATGGTGACTGCAGGATCAACTGATCACAT

GGATCACTTCTCCCTTGGAGTGCTTGTGATTCTGCTCATGGTGCAGGAAG

GGCTGAAGAAGAGAATGACCACAAAGATCATCATAAGCACATCAATGG

CAGTGCTGGTAGCTATGATCCTGGGAGGATTTTCAATGAGTGACCTGGC

TAAGCTTGCAATTTTGATGGGTGCCACCTTCGCGGAAATGAACACTGGA

GGAGATGTAGCTCATCTGGCGCTGATAGCGGCATTCAAAGTCAGACCAG

CGTTGCTGGTATCTTTCATCTTCAGAGCTAATTGGACACCCCGTGAAAGC

ATGCTGCTGGCCTTGGCCTCGTGTCTTTTGCAAACTGCGATCTCCGCCTT

GGAAGGCGACCTGATGGTTCTCATCAATGGTTTTGCTTTGGCCTGGTTGG

CAATACGAGCGATGGTTGTTCCACGCACTGATAACATCACCTTGGCAAT

CCTGGCTGCTCTGACACCACTGGCCCGGGGCACACTGCTTGTGGCGTGG

AGAGCAGGCCTTGCTACTTGCGGGGGGTTTATGCTCCTCTCTCTGAAGG

GAAAAGGCAGTGTGAAGAAGAACTTACCATTTGTCATGGCCCTGGGACT

AACCGCTGTGAGGCTGGTCGACCCCATCAACGTGGTGGGGCTGCTGTTG

CTCACAAGGAGTGGGAAGCGGAGCTGGCCCCCTAGCGAAGTACTCACA

GCTGTTGGCCTGATATGCGCATTGGCTGGAGGGTTCGCCAAGGCAGATA

TAGAGATGGCTGGGCCCATGGCCGCGGTCGGTCTGCTAATTGTCAGTTA

CGTGGTCTCAGGAAAGAGTGTGGACATGTACATTGAAAGAGCAGGTGA

CATCACATGGGAAAAAGATGCGGAAGTCACTGGAAACAGTCCCCGGCT

CGATGTGGCGCTAGATGAGAGTGGTGATTTCTCCCTGGTGGAGGATGAC

GGTCCCCCCATGAGAGAGATCATACTCAAGGTGGTCCTGATGACCATCT

GTGGCATGAACCCAATAGCCATACCCTTTGCAGCTGGAGCGTGGTACGT

ATACGTGAAGACTGGAAAAAGGAGTGGTGCTCTATGGGATGTGCCTGCT

CCCAAGGAAGTAAAAAAGGGGGAGACCACAGATGGAGTGTACAGAGTA

ATGACTCGTAGACTGCTAGGTTCAACACAAGTTGGAGTGGGAGTTATGC

AAGAGGGGGTCTTTCACACTATGTGGCACGTCACAAAAGGATCCGCGCT

GAGAAGCGGTGAAGGGAGACTTGATCCATACTGGGGAGATGTCAAGCA

GGATCTGGTGTCATACTGTGGTCCATGGAAGCTAGATGCCGCCTGGGAC

GGGCACAGCGAGGTGCAGCTCTTGGCCGTGCCCCCCGGAGAGAGAGCG

AGGAACATCCAGACTCTGCCCGGAATATTTAAGACAAAGGATGGGGAC

ATTGGAGCGGTTGCGCTGGATTACCCAGCAGGAACTTCAGGATCTCCAA

TCCTAGACAAGTGTGGGAGAGTGATAGGACTTTATGGCAATGGGGTCGT

GATCAAAAATGGGAGTTATGTTAGTGCCATCACCCAAGGGAGGAGGGA

GGAAGAGACTCCTGTTGAGTGCTTCGAGCCTTCGATGCTGAAGAAGAAG

CAGCTAACTGTCTTAGACTTGCATCCTGGAGCTGGGAAAACCAGGAGAG

TTCTTCCCGAAATAGTCCGTGAAGCCATAAAAACAAGACTCCGTACTGT

GATCTTAGCTCCAACCAGGGTTGTCGCTGCTGAAATGGAGGAAGCCCT

TAGAGGGCTTCCAGTGCGTTATATGACAACAGCAGTCAATGTCACCCAC

TCTGGAACAGAAATCGTCGACTTAATGTGCCATGCCACCTTCACTTCAC

GTCTACTACAGCCAATCAGAGTCCCCAACTATAATCTGTATATTATGGAT

GAGGCCCACTTCACAGATCCCTCAAGTATAGCAGCAAGAGGATACATTT

CAACAAGGGTTGAGATGGGCGAGGCGGCTGCCATCTTCATGACCGCCAC

GCCACCAGGAACCCGTGACGCATTTCCGGACTCCAACTCACCAATTATG

GACACCGAAGTGGAAGTCCCAGAGAGAGCCTGGAGCTCAGGCTTTGATT

GGGTGACGGATTATTCTGGAAAAACAGTTTGGTTTGTTCCAAGCGTGAG

GAACGGCAATGAGATCGCAGCTTGTCTGACAAAGGCTGGAAAACGGGT

CATACAGCTCAGCAGAAAGACTTTTGAGACAGAGTTCCAGAAAACAAA

ACATCAAGAGTGGGACTTTGTCGTGACAACTGACATTTCAGAGATGGGC

GCCAACTTTAAAGCTGACCGTGTCATAGATTCCAGGAGATGCCTAAAGC

CGGTCATACTTGATGGCGAGAGAGTCATTCTGGCTGGACCCATGCCTGT

CACACATGCCAGCGCTGCCCAGAGGAGGGGGCGCATAGGCAGGAATCC

CAACAAACCTGGAGATGAGTATCTGTATGGAGGTGGGTGCGCAGAGAC

TGACGAAGACCATGCACACTGGCTTGAAGCAAGAATGCTCCTTGACAAT

ATTTACCTCCAAGATGGCCTCATAGCCTCGCTCTATCGACCTGAGGCCG ACAAAGTAGCAGCCATTGAGGGAGAGTTCAAGCTTAGGACGGAGCAAA

GGAAGACCTTTGTGGAACTCATGAAAAGAGGAGATCTTCCTGTTTGGCT

GGCCTATCAGGTTGCATCTGCCGGAATAACCTACACAGATAGAAGATGG

TGCTTTGATGGCACGACCAACAACACCATAATGGAAGACAGTGTGCCGG

CAGAGGTGTGGACCAGACACGGAGAGAAAAGAGTGCTCAAACCGAGGT

GGATGGACGCCAGAGTTTGTTCAGATCATGCGGCCCTGAAGTCATTCAA

GGAGTTTGCCGCTGGGAAAAGAGGAGCGGCTTTTGGAGTGATGGAAGC

CCTGGGAACACTGCCAGGACACATGACAGAGAGATTCCAGGAAGCCAT

TGACAACCTCGCTGTGCTCATGCGGGCAGAGACTGGAAGCAGGCCTTAC

AAAGCCGCGGCGGCCCAATTGCCGGAGACCCTAGAGACCATTATGCTTT

AACAAGGGCATAGGGAAGATGGGCTTTGGAATGGTGACTCTTGGGGCC

AGCGCATGGCTCATGTGGCTCTCGGAAATTGAGCCAGCCAGAATTGCAT

GTGTCCTCATTGTTGTGTTCCTATTGCTGGTGGTGCTCATACCTGAGCCA

GAAAAGCAAAGATCTCCCCAGGACAACCAAATGGCAATCATCATCATG

GTAGCAGTAGGTCTTCTGGGCTTGATTACCGCCAATGAACTCGGATGGT

TGGAGAGAACAAAGAGTGACCTAAGCCATCTAATGGGAAGGAGAGAGG

AGGGGGCAACCATGGGATTCTCAATGGACATTGACCTGCGGCCAGCCTC

AGCTTGGGCCATCTATGCTGCCTTGACAACTTTCATTACCCCAGCCGTCC

AACATGCAGTGACCACTTCATACAACAACTACTCCTTAATGGCGATGGC

CACGCAAGCTGGAGTGTTGTTTGGTATGGGCAAAGGGATGCCATTCTAC

GCATGGGACTTTGGAGTCCCGCTGCTAATGATAGGTTGCTACTCACAAT

TAACGCCCCTGACCCTAATAGTGGCCATCATTTTGCTCGTGGCGCACTAC

ATGTACTTGATCCCAGGGCTGCAGGCAGCAGCTGCGCGTGCTGCCCAGA

AGAGAACGGCAGCTGGCATCATGAAGAACCCTGTTGTGGATGGAATAG

TGGTGACTGACATTGACACAATGACAATTGACCCCCAAGTGGAGAAAA

AGATGGGACAGGTGCTACTCATGGCAGTAGCCGTCTCCAGCGCCATACT

GTCGCGGACCGCCTGGGGGTGGGGGGAGGCTGGGGCCCTGATCACAGC

CGCAACTTCCACTTTGTGGGAAGGCTCTCCGAACAAGTACTGGAACTCC

AGCTTCTCTAATCTACACAGTGACAAGAAACGCTGGCTTGGTCAAGAGA

CGTGGGGGTGGAACAGGAGAGACCCTGGGAGAGAAATGGAAGGCCCGC

TTGAACCAGATGTCGGCCCTGGAGTTCTACTCCTACAAAAAGTCAGGCA

TCACCGAGGTGTGCAGAGAAGAGGCCCGCCGCGCCCTCAAGGACGGTG

TGGCAACGGGAGGCCATGCTGTGTCCCGAGGAAGTGCAAAGCTGAGAT

GGTTGGTGGAGCGGGGATACCTGCAGCCCTATGGAAAGGTCATTGATCT

TGGATGTGGCAGAGGGGGCTGGAGTTACTACGCCGCCACCATCCGCAAA

GTTCAAGAAGTGAAAGGATACACAAAAGGAGGCCCTGGTCATGAAGAA

CCCGTGTTGGTGCAAAGCTATGGGTGGAACATAGTCCGTCTTAAGAG

TGGGGTGGACGTCTTTCATATGGCGGCTGAGCCGTGTGACACGTTGCTG

TGTGACATAGGTGAGTCATCATCTAGTCCTGAAGTGGAAGAAGCACGGA

CGCTCAGAGTCCTCTCCATGGTGGGGGATTGGCTTGAAAAAAGACCAGG

AGCCTTTTGTATAAAAGTGTTGTGCCCATACACCAGCACTATGATGGAA

ACCCTGGAGCGACTGCAGCGTAGGTATGGGGGAGGACTGGTCAGAGTG

CCACTCTCCCGCAACTCTACACATGAGATGTACTGGGTCTCTGGAGCGA

AAAGCAACACCATAAAAAGTGTGTCCACCACGAGCCAGCTCCTCTTGGG

GCGCATGGACGGGCCTAGGAGGCCAGTGAAATATGAGGAGGATGTGAA

TCTCGGCTCTGGCACGCGGGCTGTGGTAAGCTGCGCTGAAGCTCCCAAC

ATGAAGATCATTGGTAACCGCATTGAAAGGATCCGCAGTGAGCACGCG

GAAACGTGGTTCTTTGACGAGAACCACCCATATAGGACATGGGCTTACC

ATGGAAGCTATGAGGCCCCCACACAAGGGTCAGCGTCCTCTCTAATAAA

CGGGGTTGTCAGGCTCCTGTCAAAACCCTGGGATGTGGTGACTGGAGTC

ACAGGAATAGCCATGACCGACACCACACCGTATGGTCAGCAAAGAGTTT

TCAAGGAAAAAGTGGACACTAGGGTGCCAGACCCCCAAGAAGGTACTC

GTCAGGTTATGAGCATGGTCTCTTCCTGGTTGTGGAAAGAGCTAGGCAA

ACACAAACGGCCACGAGTCTGTACCAAAGAAGAGTTCATCAACAAGGT

TCGTAGCAATGCAGCATTAGGGGCAATATTTGAAGAGGAAAAAGAGTG GAAGACTGCAGTGGAAGCTGTGAACGATCCAAGGTTCTGGGCTCTAGTG

GACAAGGAAAGAGAGCACCACCTGAGAGGAGAGTGCCAGAGTTGTGTG

TACAACATGATGGGAAAAAGAGAAAAGAAACAAGGGGAATTTGGAAA

GGCCAAGGGCAGCCGCGCCATCTGGTATATGTGGCTAGGGGCTAGATTT

CTAGAGTTCGAAGCCCTTGGATTCTTGAACGAGGATCACTGGATGGGGA

GAGAGAACTCAGGAGGTGGTGTTGAAGGGCTGGGATTACAAAGACTCG

GATATGTCCTAGAAGAGATGAGTCGCATACCAGGAGGAAGGATGTATG

CAGATGACACTGCTGGCTGGGACACCCGCATCAGCAGGTTTGATCTGGA

GAATGAAGCTCTAATCACCAACCAAATGGAGAAAGGGCACAGGGCCTT

GGCATTGGCCATAATCAAGTACACATACCAAAACAAAGTGGTAAAGGT

CCTTAGACCAGCTGAAAAAGGGAAAACAGTTATGGACATTATTTCGAGA

CAAGACCAAAGGGGGAGCGGACAAGTTGTCACTTACGCTCTTAACACAT

TTACCAACCTAGTGGTGCAACTCATTCGGAATATGGAGGCTGAGGAAGT

CCTAGAGATGCAAGACTTGTGGCTGCTGCGGAGGTCAGAGAAAGTGAC

CAACTGGTTGCAGAGCAACGGATGGGATAGGCTCAAACGAATGGCAGT

CAGTGGAGATGATTGCGTTGTGAAGCCAATTGATGATAGGTTTGCACAT

GCCCTCAGGTTCTTGAATGATATGGGAAAAGTTAGGAAGGACACACAA

GAGTGGAAACCCTCAACTGGATGGGACAACTGGGAAGAAGTTCCGTTTT

GCTCCCACCACTTCAACAAGCTCCATCTCAAGGACGGGAGGTCCATTGT

GGTTCCCTGCCGCCACCAAGATGAACTGATTGGCCGGGCCCGCGTCTCT

CCAGGGGCGGGATGGAGCATCCGGGAGACTGCTTGCCTAGCAAAATCA

TATGCGCAAATGTGGCAGCTCCTTTATTTCCACAGAAGGGACCTCCGAC

TGATGGCCAATGCCATTTGTTCATCTGTGCCAGTTGACTGGGTTCCAACT

GGGAGAACTACCTGGTCAATCCATGGAAAGGGAGAATGGATGACCACT

GAAGACATGCTTGTGGTGTGGAACAGAGTGTGGATTGAGGAGAACGAC

CACATGGAAGACAAGACCCCAGTTACGAAATGGACAGACATTCCCTATT

TGGGAAAAAGGGAAGACTTGTGGTGTGGATCTCTCATAGGGCACAGAC

CGCGCACCACCTGGGCTGAGAACATTAAAAACACAGTCAACATGGTGC

GCAGGATCATAGGTGATGAAGAAAAGTACATGGACTACCTATCCACCCA

AGTTCGCTACTTGGGTGAAGAAGGGTCTACACCTGGAGTGCTGTAAGCA

CCAATCTTAATGTTGTCAGGCCTGCTAGTCAGCCACAGCTTGGGGAAAG

CTGTGCAGCCTGTGACCCCCCCAGGAGAAGCTGGGAAACCAAGCCTATA

GTCAGGCCGAGAACGCCATGGCACGGAAGAAGCCATGCTGCCTGTGAG

CCCCTCAGAGGACACTGAGTCAAAAAACCCCACGCGCTTGGAGGCGCA

GGATGGGAAAAGAAGGTGGCGACCTTCCCCACCCTTCAATCTGGGGCCT

GAACTGGAGATCAGCTGTGGATCTCCAGAAGAGGGACTAGTGGTTAGA

GGAGACCCCCCGGAAAACGCAAAACAGCATATTGACGCTGGGAAAGAC

CAGAGACTCCATGAGTTTCCACCACGCTGGCCGCCAGGCACAGATCGCC

GAATAGCGGCGGCCGGTGTGGGGAAATCCATGGGTCT

Zika virus MKNPKKKSGGFPJVNMLKRGVARVSPFGGLKRLPAGLLLGHGPIRMVLAIL isolate AFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKEKKR

ZIKV/H.s RGADTSVGIVGLLLTTAMAAEVTRRGSAYYMYLDRNDAGEAISFPTTLGM apiens/Bra NKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGT zil/PE243/ CHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFR

2015, NPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMS polyprotei GGTWVDIVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASIS n DMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVT

CAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAK

VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKE

WFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAV

HTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFT

KIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVIT

ESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGA

KRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIG

TLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSADVGCSVDFSKKETRCG

TGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGICGISSVSRMENI

MWRSVEGELNAILEENGVQLTVVVGSVKNPMWGGPQRLPVPVNELPHGW KAWGKSHFVRAAKTNNSFVVDGDTLKECPLKHRAW SFLVEDHGFGVFH

TSVWLKVREDYSLECDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKR

AHLIEMKTCEWPKSHTLWTDGIEESDLIIPKSLAGPLSHHNTREGYRTQMKG

PWHSEELEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTM

PPLSFRAKDGCWYGMEIRPRKEPESNLVRSMVTAGSTDHMDHFSLGVLVIL

LMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLAILMGATFAEM

NTGGDVAHLALIAAFKVRPALLVSFIFRANWTPRESMLLALASCLLQTAISA

LEGDLMVLINGFALAWLAIRAMWPRTDNITLAILAALTPLARGTLLVAWR

AGLATCGGFMLLSLKGKGSVKKNLPFVMALGLTAVRLVDPINVVGLLLLT

RSGKRSWPPSEVLTAVGLICALAGGFAKADIEMAGPMAAVGLLIVSYVVSG

KSVDMYIERAGDITWEKDAEVTGNSPRLDVALDESGDFSLVEDDGPPMREII

LKVVLMTICGMNPIAIPFAAGAWYVYVKTGKRSGALWDVPAPKEVKKGET

TDGVYRVMTRRLLGSTQVGVGVMQEGVFHTMWHVTKGSALRSGEGRLDP

YWGDVKQDLVSYCGPWKLDAAWDGHSEVQLLAVPPGERARNIQTLPGIFK

TKDGDIGAVALDYPAGTSGSPILDKCGRVIGLYGNGWIKNGSYVSAITQGR

REEETPVECFEPSMLKKKQLTVLDLHPGAGKTRRVLPEIVREAIKTRLRTVIL

APTRVVAAEMEEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTSRLLQ

PIRVPNYNLYIMDEAHFTDPSSIAARGYISTRVEMGEAAAIFMTATPPGTRD

AFPDSNSPIMDTEVEVPERAWSSGFDWVTDYSGKTVWFVPSVRNGNEIAAC

LTKAGKRVIQLSRKTFETEFQKTKHQEWDFVVTTDISEMGANFKADRVIDS

RRCLKPVILDGERVILAGPMPVTHASAAQRRGRIGRNPNKPGDEYLYGGGC

AETDEDHAHWLEARMLLDNIYLQDGLIASLYRPEADKVAAIEGEFKLRTEQ

RKTFVELMKRGDLPVWLAYQVASAGITYTDRRWCFDGTTNNTIMEDSVPA

EVWTRHGEKRVLKPRWMDARVCSDHAALKSFKEFAAGKRGAAFGVMEA

LGTLPGHMTERFQEAIDNLAVLMRAETGSRPYKAAAAQLPETLETIMLLGL

LGTVSLGIFFVLMRNKGIGKMGFGMVTLGASAWLMWLSEIEPARIACVLrV

VFLLLVVLIPEPEKQRSPQDNQMAIIIMVAVGLLGLITANELGWLERTKSDLS

HLMGRREEGATMGFSMDIDLRPASAWAIYAALTTFITPAVQHAVTTSYNNY

SLMAMATQAGVLFGMGKGMPFYAWDFGVPLLMIGCYSQLTPLTLIVAIILL

VAHYMYLIPGLQAAAARAAQKRTAAGIMKNPVVDGIWTDIDTMTIDPQV

EKKMGQVLLMAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYW

NSSTATSLCNIFRGSYLAGASLrYTVTRNAGLVKRRGGGTGETLGEKWKAR

LNQMSALEFYSYKKSGITEVCREEARRALKDGVATGGHAVSRGSAKLRWL

VERGYLQPYGKVIDLGCGRGGWSYYAATIRKVQEVKGYTKGGPGHEEPVL

VQSYGWNIVRLKSGVDVFHMAAEPCDTLLCDIGES SS SPEVEEARTLRVLS

MVGDWLEKRPGAFCIKVLCPYTSTMMETLERLQRRYGGGLVRVPLSRNST

HEMYWVSGAKSNTIKSVSTTSQLLLGRMDGPRRPVKYEEDVNLGSGTRAV

VSCAEAPNMKIIGNRIERIRSEHAETWFFDENHPYRTWAYHGSYEAPTQGSA

SSLINGVVRLLSKPWDVVTGVTGIAMTDTTPYGQQRVFKEKVDTRVPDPQE

GTRQVMSMVSSWLWKELGKHKRPRVCTKEEFINKVRSNAALGAIFEEEKE

WKTAVEAVNDPRFWALVDKEREHHLRGECQ S CV YNMMGKREKKQGEFG

KAKGSRAIWYMWLGARFLEFEALGFLNEDHWMGRENSGGGVEGLGLQRL

GYVLEEMSRIPGGRMYADDTAGWDTRISRFDLENEALITNQMEKGHRALA

LAIIKYTYQNKVVKVLRPAEKGKTVMDIISRQDQRGSGQVVTYALNTFTNL

VVQLIRNMEAEEVLEMQDLWLLRRSEKVTNWLQSNGWDRLKRMAVSGD

DCVVKPIDDRFAHALRFLNDMGKVRKDTQEWKPSTGWDNWEEVPFCSHH

FNKLHLKDGRSrVVPCRHQDELIGRARVSPGAGWSIRETACLAKSYAQMW

QLLYFHRRDLRLMANAICSSVPVDWVPTGRTTWSIHGKGEWMTTEDMLV

VWNRVWIEENDHMEDKTPVTKWTDIPYLGKREDLWCGSLIGHRPRTTWAE

NIKNTVNMVRRIIGDEEKYMDYLSTQVRYLGEEGSTPGVL

[00159] The preceding merely illustrates the principles of this disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of this disclosure and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present disclosure, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present disclosure is embodied by the appended claims.