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  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
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  • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/141—MicroRNAs, miRNAs
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/335—Modified T or U
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/353—Nature of the modification linked to the nucleic acid via an atom other than carbon
  • C12N2310/3533—Halogen

Definitions

• the present disclosure is generally directed to compositions and methods for treating cancer, and more particularly, to methods in which modified microRNAs alone or in conjunction with 5-fluorouracil are used in treating cancer, particularly colorectal, lung or pancreatic cancer.
• MicroRNAs are a class of highly conserved, non-coding small RNA molecules that mediate translation in a cell or organism by negatively regulating the expression of their target genes and thus causing translational arrest, mRNA cleavage or a combination thereof. See Bartel DP. Cell. (2009) 136(2):215-33. By targeting multiple transcripts, miRNAs regulate a wide range of biological processes, including apoptosis, differentiation and cell proliferation, thus aberrant microRNA function can lead to cancer (see Ambros V. Nature. (2004) 431(7006):350-5) and as such, miRNAs have recently been identified as as biomarkers, oncogenes or tumor suppressors. See, e.g., Croce, CM, Nat Rev Genet. (2009) 10:704-714).
• Colorectal cancer is the third most common malignancy and the second most common cancer-related cause of death in the United States. See, Hegde SR, et al., Expert review of gastroenterology & hepatology. (2008) 2(1): 135-49.
• pyrimidine antagonists such as fluoropyrimidine-based chemotherapeutic agents (e.g., 5-fluorouracil, S-l) are the gold standard for treating colorectal cancer.
• Pyrimidine antagonists block the synthesis of pyrimidine containing nucleotides (Cytosine and Thymine in DNA; Cytosine and Uracil in RNA).
• pyrimidine antagonists have similar structures when compared to endogenous nucleotides, they compete with the natural pyrimidines to inhibit crucial enzymatic activity involved in the replication process leading to the prevention of DNA and/or RNA synthesis and inhibition of cell division.
• Pancreatic cancer is a deadly cancer that is very difficult to treat. See Siegel, RL et al. CA Cancer J. Clin. (2015) 65: 5-29. Unique aspects of pancreatic cancer include a very low 5 year survivial rate of less than 7% (Id.), late presentation, early metastasis and a poor response to chemotherapy and radiation. See Maitra A and Hruban RH, Annu Rev. Pathol. (2008) 3:157-188. To date gemcitabine-based chemotherapy (?, 2'-difluoro
• 5-fluorouracil i.e., 5-FU, or more specifically, 5-fluoro-lH-pyrimidine-2,4-dione
• 5-FU is a well known pyrimidine antagonist that is used in many adjuvant chemotherapeutic medicants, such as Carac® cream, Efudex®, Fluoroplex®, and Adrucil®.
• chemotherapeutic medicants such as Carac® cream, Efudex®, Fluoroplex®, and Adrucil®.
• 5-FU targets a critical enzyme, thymidylate synthase (TYMS or TS), which catalyzes the methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) an essential step in DNA biosynthesis.
• TYMS or TS thymidylate synthase
• dUMP deoxyuridine monophosphate
• dTMP deoxythymidine monophosphate
• nucleic acid compositions i.e., a microRNA
• a 5-halouracil base have exceptional efficacy as anti-cancer agents.
• the data herein shows that contacting a cell with a modified microRNA composition of the present disclosure regulates cell cycle progression and reduced tumorigenesis by, for example, reducing cancer cell proliferation and increasing the efficacy of chemotherapeutic agents.
• the present disclosure is premised on the discovery that the incorporation of 5-halouracil bases within the nucleotide sequences of microRNAs increases microRNA efficacy as an anticancer therapeutic agent over the cancer therapeutic agents alone and/or the native microRNA.
• nucleic acid compositions that include a modified microRNA nucleotide sequence having at least one uracil base (U, U bases) that has been replaced by a 5-halouracil, such as 5-fluorouracil (5-FU) are described.
• the modified microRNA has more than one, or exactly one uracil that has been replaced by a 5-halouracil.
• the modified microRNA nucleotide sequence includes two, three, four, five, six, seven, eight or more uracil bases that have been replaced by a 5-halouracil.
• all of the uracil nucleotide bases of the modified mRNA have been replaced by a 5-halouracil.
• the 5-halouracil is, for example, 5-fluorouracil, 5- chlorouracil, 5-bromouracil, or 5-iodouracil. In specific embodiments, the 5-halouracil is 5- fluorouracil.
• the modified microRNA nucleotide sequence includes more than one 5-halouracil whereby each of the 5-halouracils are the same. In other embodiments, the modified microRNA nucleotide sequence includes more than one 5- halouracil whereby each of the 5-halouracils is different. In other embodiments, the modified microRNA nucleotide sequence includes more than two 5-halouracils, whereby the modified microRNA nucleotide sequence includes a combination of different 5-halouracils.
• a nucleic acid composition that contains a miR-129 nucleotide sequence that has been modified by replacing at least one of the uracil nucleotide bases with a 5-halouracil is provided. More specifically, the nucleic acid composition contains at least the following native miR-129 nucleotide sequence: wherein at least one, two, three, four, five, six, seven, eight or all of the uracil bases in the shown nucleic acid sequence or that may be covalently appended to the shown sequence, are replaced by a 5- halouracil.
• the modified microRNA has nucleic acid sequence consisting of
• U F is a halouracil, specifically 5-fluorouracil.
• a seed portion of the native miR-129 nucleotide sequence remains unmodified (i.e., does not include a 5-halouracil) while one or more (or all) of the remaining uracil nucleotide bases in the remainder of the modified miR-129 nucleotide sequence are replaced by an equivalent number of 5-halouracils.
• the modified miR-129 microRNA of the present disclosure has nucleic acid sequence consisting of whereby
• halouracil specifically 5-fiuorouracil.
• the 5-halouracil is, for example, 5-fiuorouracil, 5- chlorouracil, 5-bromouracil, or 5-iodouracil.
• the 5-halouracil is 5- fluorouracil.
• nucleic acid compositions that contain a miR-15a nucleotide sequence that has been modified by replacing at least one of the uracil nucleotide bases with a 5-halouracil, such as 5-fiuorouracil (5-FU) are provided.
• the nucleic acid composition contains at least the following native miR-15a nucleotide sequence: wherein at
• uracil nucleotide bases in the shown sequence are 5-halouracils.
• the modified miR-15a microRNA has nucleic acid sequence consisting of
• halouracil specifically 5-fluorouracil.
• a seed portion of the native miR-15a nucleotide sequence remains unmodified with a 5-halouracil, while one or more (or all) of the
• uracil bases in the remainder of the miR-15a nucleotide sequence are replaced by a 5-halouracil.
• the modified miR-129 microRNA has nucleic acid sequence consisting of
• U F is a halouracil, specifically 5-fluorouracil.
• the present disclosure is directed to nucleic acid compositions that include a miR-140 nucleotide sequence that has been modified.
• the native miR-140 nucleotide sequence has been modified by replacing at least one of the U bases with a 5-halouracil.
• precisely one of the U bases in the native miR-140 nucleic acid sequence sequence is a 5-halouracil.
• precisely or at least two U bases in the native miR-140 nucleotide sequence are replaced by 5- halouracils.
• precisely or at least three U bases in the miR- 140 nucleotide sequence are 5-halouracils.
• precisely or at least four U bases in the native miR-140 nucleotide sequence are 5-halouracils.
• precisely or at least five U bases in the miR-140 nucleotide sequence sequence are 5- halouracils.
• precisely or at least six U bases in the miR-140 nucleotide sequence are 5-halouracils.
• all of the U bases in the miR-140 nucleotide sequence, whether in the native and/or in an appended portion, are 5- halouracils.
• the modified microRNA nucleic acid composition of the present disclosure has a nucleotide sequence of
• the present disclosure is directed to nucleic acid compositions that include a modified native miR-192 or miR-215 nucleotide sequence that has been modified by replacing at least one of the uracil bases with a 5-halouracil.
• the modified miR-192 nucleotide sequence has been modified by replacing at least one of the U bases with a 5-fluorouracil.
• precisely one of the U bases in the modified miR- 192 nucleotide sequence is a 5-halouracil.
• precisely or at least two U bases in the modified miR-192 nucleotide sequence are 5-halouracils.
• precisely or at least three U bases in the modified miR-192 nucleotide sequence are 5-halouracils.
• precisely or at least four U bases in the modified miR-192 or miR-215 nucleotide sequence are 5-halouracils.
• all of the U bases in the modified miR-192 or miR-215 sequence, whether in the native and/or in an appended portion of the nucleic acid are 5-halouracils.
• the nucleic acid composition of the present disclosure has a modified miR-192 or modified miR-215 nucleotide sequence of
• halouracil specifically 5-fluorouracil.
• the present disclosure is directed to nucleic acid compositions that include a modified native miR-502 nucleotide sequence that has been modified by replacing uracil with 5-halouracil.
• the modified miR- 502 nucleotide sequence has been modified by replacing at least one of the U bases with a 5- fluorouracil.
• precisely one of the U bases in the miR-502 nucleotide sequence is a 5-halouracil.
• precisely or at least two U bases in the miR-502 nucleotide sequence are 5-halouracils.
• precisely or at least three U bases in the miR-502 nucleotide sequence are 5- halouracils.
• nucleotide sequence are 5-halouracils. In other embodiments, precisely or at least five U bases in the miR-502 nucleotide sequence are 5-halouracils. In other embodiments, precisely or at least six U bases in the modified miR-502 nucleotide sequence are 5-halouracils. In other embodiments, precisely or at least seven U bases in the miR-502 nucleotide sequence are 5-halouracils. In specific embodiments, all of the U bases in the miR-502 nucleotide sequence, whether in the native and/or in an appended portion, are 5-halouracils.
• the modified miR-502 nucleic acid composition of the present disclosure has a modified nucleotide sequence of
• the present disclosure is directed to nucleic acid compositions that include a modified miR-506 nucleotide sequence that includes a 5- halouracil.
• the modified miR-506 nucleotide sequence has been modified by replacing at least one of the U bases with a 5-halouracil, such as 5-fluorouracil.
• precisely one of the U bases in the native miR-506 nucleotide sequence is replaced by a 5-halouracil.
• precisely or at least two U bases in the modified miR-506 nucleotide sequence are 5-halouracils.
• precisely or at least three U bases in the modified miR-506 nucleotide sequence are 5-halouracils.
• precisely or at least four U bases in the modified miR-506 nucleotide sequence are 5-halouracils.
• precisely or at least five U bases in the modified miR-506 nucleotide sequence are 5-halouracils. In other embodiments, precisely or at least six U bases in the modified miR-506 nucleotide sequence are 5-halouracils. In other embodiments, precisely or at least seven U bases in the modified miR-506 nucleotide sequence are 5-halouracils. In specific embodiments, all of the U bases in the modified miR-506 nucleotide sequence, whether in the native and/or in an appended portion, are 5-halouracils.
• the miR-506 nucleic acid composition of the present disclosure has a modified microRNA nucleotide sequence of
• halouracil specifically 5-fluorouracil.
• the 5-halouracil is, for example, 5-fluorouracil, 5- chlorouracil, 5-bromouracil, or 5-iodouracil.
• the 5-halouracil is 5- fluorouracil, or a combination thereof.
• the present disclosure is also directed to formulations of a modified microRNA composition described herein or a formulation that includes combinations thereof.
• the formulations can include pharmaceutical preparations that comprise the above-described nucleic acid compositions and a pharmaceutically acceptable carrier.
• the present disclosure is directed to a method for treating cancer that includes administering to a subject an effective amount of one or more of nucleic acid compositions described herein.
• the nucleic acid compositions include a modified miR-129, miR-15a, miR-192, miR-215, miR-140, miR-502 or miR-506 nucleotide sequence wherein at least one, two, three, four, five, six or more of the uracil nucleotide bases in the native (unmodified) nucleotide sequence have been replaced by a 5-halouracil.
• the present methods include administering a nucleic acid composition of the present disclosure to a subject having cancer or a predisposition to cancer, whereby the nucleic acid composition is a modified miR-129 or a modified miR-15a nucleic acid.
• the modified microRNA administered has nucleic acid sequence selected from the group consisting of
• the subject is a mammal.
• the subject being treated is a human, dog, horse, pig, mouse, or rat.
• the subject is a human that has been diagnosed with cancer, or has been identified as having a predisposition to developing cancer.
• the cancer being treated can be, for example, colorectal, stomach, esophageal, lung, ovarian, pancreatic, or cervical cancer.
• the methods of the present disclosure treat a subject for colorectal cancer, pancreatic cancer or breast cancer.
• the data provided herein surprisingly shows an increased potency of the modified microRNAs described herein when compared to known anticancer agents, such as 5-FU alone in several different cancer models, including colorectal cancer, pancreatic cancer, and lung cancer.
• known anticancer agents such as 5-FU alone in several different cancer models, including colorectal cancer, pancreatic cancer, and lung cancer.
• the present disclosure provides the unexpected finding that the described modified nucleic acid compositions are substantially more potent in inhibiting cancer progression and tumorigenesis than 5-FU, miR-15a, miR-129, miR-140, miR-192, miR-215, miR-502 or miR-506 alone, or than a combination of 5-FU and corresponding native microRNAs.
• the present compositions and methods provide the additional benefit of permitting a lower dosing, which results in lower toxicity and fewer side effects.
• a further significant advantage exhibited by the described nucleic acid compositions is that the instant compositions have significantly improved efficacy compared to miR-140, miR-192, miR- 215, miR-502 or miR-506 sequences that have not been modified with a halouracil.
• the nucleic acid compositions disclosed herein represent a substantial advance in the treatment of cancer.
• FIGS. 1A-1H Chemical representation of exemplary modified microRNA nucleotide sequences of the present disclosure.
• A Chemical representation of miR-129 nucleotide sequence in which all U bases are replaced by a halouracil (i.e., U F ), as set forth in SEQ ID NO: 4.
• B Chemical representation of miR-129 in which only the non-seed portion of miR-129 has U bases replaced with halouracils), as set forth in SEQ ID NO: 5.
• C Chemical representation of miR-15a nucleotide sequence in which all U bases are replaced with a halouracil), as set forth in SEQ ID NO: 6.
• FIGS. 2A-C Exemplary modified miR-129 nucleic acids enter cancer cells and effectively reduce target protein expression.
• A Graph showing target (E2F3) specificity and the ability of a modified miR-129 (with all U bases replaced with 5-FU, 5-FU-miR-129) compared to that of control miRNA, and an unmodified miR-129 nucleic acid.
• B A quantitative Real-Time PCR analysis showing that miR-129 mimics enter cancer cells.
• FIG. 3 Graphs showing inhibition of colon cancer cell proliferation in 4 different colon cancer cell lines (HCT116, RKO, SW480 and SW620) by an exemplary modified miR-129 nucleic acid (mimic) having all U bases replaced by 5-FU (- - ⁇ - -), as compared to a non-specific (Negative control,- ⁇ -) control and ectopically expressed native miR-129 0).
• mimic modified miR-129 nucleic acid
• FIG. 4 Combination therapy with 5-FU and modified microRNA compositions of the present disclosure effectively inhibit cancer cell proliferation.
• FIGS. 5A-B Exemplary microRNA mimics induce apoptosis in colon cancer cells and cause cell cycle arrest.
• A Cell death was quantified by FITC-Annexin V apoptosis assay to show that modified miR-129 nucleic acid compositions of the present disclosure induce cancer cell apoptosis at significantly higher levels than negative controls, or ectopically expressed native miR-129 in several different colorectal cancer cell lines.
• FIG. 6 Modified MicroRNA nucleic acid compositions of the present disclosure eliminate chemotherapy resistant cancer stem cells. HCT116 derived colon cancer stem cells were treated with increasing concentrations of exemplary modified miR-129 nucleic acids of the present disclosure (O) or 5-FU ( ⁇ ). Results show that modified miR-129 nucleic acids killed 5-FU resistance cancer stem cells in a dose dependent manner.
• FIG. 7. In vivo systemic treatment with exemplary modified miR-129 nucleic acid compositions inhibits colon cancer metastasis without toxic side effects. A colon cancer metastasis mouse model was established via tail vein injection of metastatic human colon cancer cells.
• a modified miR-129 nucleic acid composition as set forth in SEQ ID NO: 4 was delivered by intravenous injection with a treatment frequency of one injection every other day for two weeks.
• the exemplary modified miR-129 nucleic acid was able to inhibit colon cancer metastasis (right panels) while negative control miRNA (left panels) had no effect. Mice treated with modified miR-129 nucleic acid did not exhibit any toxicity.
• FIGS. 8A-B Anti cancer activity of a second exemplary modified microRNA of the present disclosure.
• A Representative western blots comparing the ability of unmodified miR-15a (miR-15a) and a modified miR-15a nucleic acid composition (mimic-1) to modulate protein expression in colon cancer cells.
• Modified miR-15a as set forth in SEQ ID NO: 6 (mimic-1) retains the ability to regulate miR-15a targets (YAP1, BMI-1, DCLK1 and ECL2) and break downs TS-FdUMP in colorectal cancer cells.
• B Modified miR-15a (mimic-1) showed enhanced ability to inhibit colon cancer cell proliferation in three different colorectal cancer cell lines (HCT116, RKO, SW620) compared to unmodified miR-15a (miR-15a).
• FIG. 9 Graph showing cell cycle control for control (Negative), unmodified miR- 15a (miR-15a) and an exemplary modified miR-15a nucleic acid compositon as set forth in SEQ ID NO: 6 (mimic-1).
• FIG. 10 Modified miR-15a expression reduces the ability of cancer stem cells to induce cancer cell colony formation.
• expression of unmodified miR-15a inhibited cancer cell colony formation when compared to the ability of cancer stem cells provided with a non-specific control microRNA (Negative).
• Modified miR-15a is an effective anti cancer agent in vivo.
• a colon cancer metastasis mouse model was established via tail vein injection of metastatic human colon cancer cells.
• 40 ⁇ g of a modified miR- 15a nucleic acid composition as set forth in SEQ ID NO: 6 was delivered by intravenous injection with treatment frequency of one injection every other day for two weeks.
• the exemplary modified miR-15a nucleic acid (mimic) was able to inhibit colon cancer metastasis while negative control miRNA (negative) had no effect. Mice treated with modified miR-15a nucleic acid did not exhibit any toxicity.
• FIGS. 12 A-D Exemplary modified miR-15a and miR-129 mimics of the present disclosure exhibit enhanced ability to inhibit human breast cancer (A549;C,D) and pancreatic cancer (Pane- 1(A); AsPC-l(B)) cell proliferation compared to unmodified miR- 15a (miR-15a) or unmodified miR-129 (miR-129) or cells treated with negative controls.
• FIGS. 13 A-B Exemplary modified microRNAs of the present disclosure exhibit an enhanced ability to inhibit human colorectal cancer cell proliferation. Additional exemplary modified microRNAs were tested for their ability to inhibit colorectal cancer cell proliferation in HCT116 human colorectal cancer cells.
• An exemplary modified miR- 140 mimic as set forth in SEQ ID NO: 9 was administered to human colorectal cancer cells and revealed an increased ability to inhibit colorectal cancer cell proliferation when compared to negative control microRNAs.
• An exemplary modified miR-192 mimic as set forth in SEQ ID NO: 11 was administered to human colorectal cancer cells and revealed an increased ability to inhibit colorectal cancer cell proliferation when compared to negative control microRNAs.
• FIGS. 14A-D Exemplary modified microRNAs of the present disclosure exhibit an enhanced ability to inhibit human pancreatic and breast cancer cell proliferation.
• Additional exemplary modified microRNAs were tested for their ability to inhibit different types of human cancers by examining their affects on cancer cell proliferation.
• An exemplary modified miR-502 mimic as set forth in SEQ ID NO: 13 was administered to human pancreatic cancer cells (PANC1, A) and human breast cancer (A549, C) and revealed an increased ability to inhibit both types of cancer cell proliferation when compared to negative control microRNAs.
• Yet another exemplary modified microRNA, a miR-506 mimic as set forth in SEQ ID NO: 15 was administered to human pancreatic cancer cells (PANC1, B) and human breast cancer (A549, D) and revealed an increased ability to inhibit both types of cancer cell proliferation when compared to negative control microRNAs.
• the present disclosure provides nucleic acid compositions that incorporate one or more halouracil molecules.
• the present disclosure reveals that the replacement of uracil nucleotides within a microRNA oligonucleotide sequence with a 5 -halouracil increases the ability of the microRNA to inhibit cancer, development, progression and tumorigenesis.
• the present disclosure provides various nucleic acid (e.g., microRNA) compositions having S-halouracil molecules incorporated in their nucleic acid sequences and methods for using the same.
• the present disclosure further provides formulations, such as pharmaceutical compositions comprising the modified nucleic acid compositions, and methods for treating cancers that include administration of the same to a subject in need thereof.
• microRNA or “miRNA” or “miR” is used interchangeably to refer to small non-coding ribose nucleic acid (RNA) molecules that are capable of regulating the expression of genes through interacting with messenger RNA molecules (mRNA), DNA or proteins.
• RNA messenger RNA molecules
• mRNA messenger RNA molecules
• microRNAs are composed of nucleic acid sequences of about 19-25 nucleotides (bases) and are found in mammalian cells.
• modified microRNA refers to a microRNA that differs from the native or endogenous microRNA (unmodified microRNA) polynucleotide. More specifically, in the present disclosure a modified microRNA differs from the unaltered or unmodified microRNA nucleic acid sequence by one or more base. In some embodiments of the present disclosure, a modified microRNA of the present disclosure includes at least one uracil (U) nucleotide base replaced by a 5-halouracil.
• U uracil
• a modified microRNA includes an additional nucleotide (i.e., adenine (A), cytosine (C), uracil (U), and guanine (G)) and at least one uracil base that is substituted with a 5-halouracil.
• A adenine
• C cytosine
• U uracil
• G guanine
• nucleic acid compositions that include a modified microRNA nucleotide sequence having at least one uracil base (U, U bases) that has been replaced with a 5-halouracil, such as 5-fiuorouracil (5-FU) are described.
• a 5-halouracil such as 5-fiuorouracil (5-FU)
• the nucleic acid compositions of the present disclosure are useful, at least, in the treatment of cancer, particularly colorectal cancer, pancreatic cancer and breast cancer.
• the nucleic acid compositions contain a nucleotide sequence that has been modified by derivatizing at least one of the uracil nucleobases at the 5-position with a group that provides a similar effect as a halogen atom.
• the group providing the similar effect has a similar size in weight or spatial dimension to a halogen atom, e.g., a molecular weight of up to or less than 20, 30, 40, 50, 60, 70, 80, 90, or 80 g/mol.
• the group providing a similar effect as a halogen atom may be, for example, a methyl group, trihalomethyl (e.g., trifluoromethyl) group, pseudohalide (e.g., trifluoromethanesulfonate, cyano, or cyanate) or deuterium (D) atom.
• the group providing a similar effect as a halogen atom may be present in the absence of or in addition to a 5-halouracil base in the microRNA nucleotide sequence.
• the group providing a similar effect as a halogen atom may be located in the native (or seed) portion and/or in an appended portion of the microRNA nucleotide sequence, which will be readily identified by one of ordinary skill in the art.
• one or more (or all) of the above types of groups providing a similar effect as a halogen atom are excluded from the modified miRNA nucleotide sequence. When all such alternative groups are excluded, only one or more halogen atoms are present as substituents in the 5-position of one or more uracil groups in the microRNA nucleotide sequence.
• the modified microRNA has more than one, or exactly one uracil that has been replaced with a 5-halouracil.
• the modified microRNA nucleotide sequence includes three, four, five, six, seven, eight or more uracil bases that have been replaced with a 5- halouracil.
• all of the uracil nucleotide bases of the modified mRNA have been replaced by a 5-halouracil.
• the 5-halouracil is, for example, 5-fluorouracil, 5- chlorouracil, 5-bromouracil, or 5-iodouracil. In specific embodiments, the 5-halouracil is 5- fluorouracil
• C cytosine
• U uracil
• G guanine bases.
• the foregoing nucleotide sequence is herein referred to as an unmodified miR-129 (i.e., "native") sequence unless otherwise specified.
• MiR-129 may also be referred to in the field as hsa-miR-129 or hsa- miR-129-5p, with accession number(s) MI0000252 and MIMAT0000242.
• MiR-129 is well known and has been studied in detail. See, e.g., J. Wu et al., Cell Cycle, (2010) 9:9, 1809- 1818.
• the miR-129 sequence may be modified to produce a "miR-129 mimic", which has a sequence modified from the native sequence, but that retains the known function or activity of the native miR-129. Unless otherwise stated, all such modified miR-129 compositions are herein considered to be within the scope of the term "miR-129 mimic" as used herein.
• a particular modified miR-129 nucleic acid sequence (mimic) of interest contains two U bases (i.e., two U-containing nucleotides) covalently appended to an end of the miR- 129 native sequence, such as in CUUUUUGCGGUCUGGGCUUGC-UU [SEQ ID NO. 3].
• the two terminal U bases continue or extend the miR-129 native sequence from 21 nucleotide bases to 23 nucleotide bases.
• the miR-129 mimic contains no more than one, two, three, four, or five additional bases (i.e., as additional nucleotides) covalently appended to the miR-129 native sequence, wherein the additional bases are independently selected from C, U, G, and C, or the additional bases may be exclusively U.
• the miR-129 is used in single-strand form, but double-stranded versions are also considered herein.
• the present disclosure is directed to nucleic acid compositions that contain a miR-129 nucleotide sequence that has been modified by replacing at least one of the uracil nucleobases (i.e., U bases) with a 5-halouracil, i.e., wherein at least one of the U bases in the miR-129 sequence, whether in the native and/or in an appended portion, is a 5-halouracil.
• the 5-halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5- bromouracil, or 5-iodouracil.
• precisely one of the U bases in the miR-129 sequence is a 5-halouracil.
• precisely or at least two U bases in the miR-129 sequence are 5-halouracils.
• precisely or at least three U bases in the miR-129 sequence are 5-halouracils.
• precisely or at least four U bases in the miR-129 sequence are 5-halouracils.
• precisely or at least five U bases in the miR-129 sequence are 5-halouracils.
• all of the U bases in the miR-129 sequence, whether in the native and/or in an appended portion, are 5-halouracils.
• the nucleic composition of the present disclosure has a modified microRNA nucleotide sequence of
• U F is a halouracil, specifically 5-fluorouracil.
• the U bases that are replaced with 5-halouracils in the miR-129 sequence may be located in an unmodified part of the miR-129 sequence, as provided above, or, in the case of a miR-129 mimic, may be located in one or more U bases covalently appended to the native miR-129, as also provided above.
• a seed portion of the native miR- 129 nucleotide sequence, GUUUUUGC remains unmodified with a 5-halouracil while one or more (or all) of the remaining U bases in the remainder of the miR-129 nucleotide sequence are replaced with the equivalent number of 5-halouracils.
• the nucleic acid composition of the present disclosure has a modified microRNA nucleotide sequence of
• U F is a halouracil, specifically 5-fluorouracil.
• the nucleic acid composition contains a miR-129 nucleotide sequence that has been modified by derivatizing at least one of the uracil (U) nucleobases at the 5-position with a group that provides a similar effect as a halogen atom.
• the group providing the similar effect has a similar size in weight or spatial dimension to a halogen atom, e.g., a molecular weight of up to or less than 20, 30, 40, 50, 60, 70, 80, 90, or 80 g/mol.
• the group providing a similar effect as a halogen atom may be, for example, a methyl group, trihalomethyl (e.g., trifluoromethyl) group, pseudohalide (e.g., trifluoromethanesulfonate, cyano, or cyanate) or deuterium (D) atom.
• the group providing a similar effect as a halogen atom may be present in the absence of or in addition to a 5-halouracil base in the miR-129 nucleotide sequence.
• the group providing a similar effect as a halogen atom may be located in the native (or seed) portion and/or in an appended portion of the miR-129 nucleotide sequence.
• one or more (or all) of the above types of groups providing a similar effect as a halogen atom are excluded from the miR-129 nucleotide sequence.
• all such alternative groups are excluded, only one or more halogen atoms are present as substituents in the 5-position of one or more uracil groups in the miR-129 nucleotide sequence.
• the present disclosure is directed to nucleic acid compositions that include a miR-15a nucleotide sequence that has been modified.
• the miR-15a nucleotide sequence has been modified by replacing at least one of the U bases with a 5-halouracil.
• A adenine
• C cytosine
• U uracil
• G guanine bases.
• the foregoing nucleotide sequence is herein referred to as a miR-15a unmodified (i.e., "native") sequence unless otherwise specified.
• MiR-15a may also be referred to in the field as hsa- miR-15a or hsa-miR-15a-5p, with accession numbers) MI0000069.
• MiR-15a is well known and has been studied in detail, e.g., Xie T, et al. Clin Transl Oncol. (2015) 17(7):504- 10; and Acunzo M, and Croce CM, Clin. Chem. (2016) 62(4):655-6.
• miR-129 mimics methods for creating a miR-15a mimic are known by those of ordinary skill in the art. Unless otherwise stated, all such modified miR-15a forms are herein considered to be within the scope of the term "miR-15a mimic", as used herein.
• a modified miR-15a i.e., miR-15a mimic
• the miR-15a is used in single-strand form, but double-stranded versions are also considered herein.
• At least one of the U bases in the miR-15a sequence, whether in the native and/or in an appended portion, is a 5-halouracil.
• the 5-halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil.
• precisely one of the U bases in the miR-15a sequence is a 5-halouracil.
• precisely or at least two U bases in the miR-15a sequence are 5-halouracils.
• precisely or at least three U bases in the miR-15a oligonucleotide sequence are 5-halouracils.
• precisely or at least four U bases in the miR-15a sequence are 5-halouracils.
• precisely or at least five U bases in the miR-15a sequence are 5- halouracils.
• precisely or at least six U bases in the miR-15a sequence are 5-halouracils.
• all of the U bases in the miR-15a sequence, whether in the native and/or in an appended portion are 5-halouracils.
• the nucleic acid composition of the present disclosure has a modified microRNA nucleotide sequence of
• U F is a halouracil, specifically 5-fluorouracil.
• the U bases that are replaced with 5-halouracils in the miR-15a sequence may be located in an unmodified part of the miR-15a sequence, as provided above, or, in the case of a miR-15a mimic, may be located in one or more uracil bases that are appended to the native miR-15a, as also provided above.
• a seed portion of the native miR-15a nucleotide sequence, UAGCAGCA remains unmodified with a 5-halouracil while one or more (or all) of the remaining U bases in the remainder of the miR-15a nucleotide sequence (non-seed portion) are replaced with a 5-halouracil.
• the nucleic acid composition of the present disclosure has a modified miR-15a nucleotide sequence of
• the nucleic acid composition contains a miR-15a nucleotide sequence that has been modified by derivatizing at least one of the uracil (U) nucleobases at the 5-position with a group that provides a similar effect as a halogen atom.
• the group providing the similar effect has a similar size in weight or spatial dimension to a halogen atom, e.g., a molecular weight of up to or less than 20, 30, 40, 50, 60, 70, 80, 90, or 80 g/mol.
• the group providing a similar effect as a halogen atom may be, for example, a methyl group, trihalomethyl (e.g., trifluoromethyl) group, pseudohalide (e.g., trifluoromethanesulfonate, cyano, or cyanate) or deuterium (D) atom.
• the group providing a similar effect as a halogen atom may be present in the absence of or in addition to a 5-halouracil base in the miR-15a nucleotide sequence.
• the group providing a similar effect as a halogen atom may be located in the native (or seed) portion and/or in an appended portion of the miR-15a nucleotide sequence.
• one or more (or all) of the above types of groups providing a similar effect as a halogen atom are excluded from miR-15a nucleotide sequence.
• all such alternative groups are excluded, only one or more halogen atoms are present as substituents in the 5-position of one or more uracil groups in the miR-15a nucleotide sequence.
• the present disclosure is directed to nucleic acid compositions that include a miR-140 nucleotide sequence that has been modified.
• the miR-140 nucleotide sequence has been modified by replacing at least one of the U bases with a 5-halouracil.
• A adenine
• C cytosine
• U uracil
• G guanine bases.
• the foregoing nucleotide sequence is herein referred to as a miR-140 unmodified (i.e., "native") sequence unless otherwise specified.
• MiR-140 may also be referred by accession numbers) NT 010498 or by miRBase Accession MI0000456.
• MiR-140 is well known and has been studied in detail, e.g., Zhai, H. et al., Oncotarget. (2015) 6: 19735-46.
• methods for creating a miR-140 mimic are known by those of ordinary skill in the art. Unless otherwise stated, all such modified miR-140 forms are herein considered to be within the scope of the term "miR-140 mimic", as used herein.
• a modified miR-140 nucleic acid i.e., miR-140 mimic
• the miR-140 mimic is used in single-strand form, but double-stranded versions are also considered herein.
• At least one of the U bases in the miR-140 sequence, whether in the native and/or in an appended portion, is a 5-halouracil.
• the 5-halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil.
• precisely one of the U bases in the miR-140 mimic sequence is a 5-halouracil.
• precisely or at least two U bases in the miR-140 sequence are 5-halouracils.
• precisely or at least three U bases in the miR-140 oligonucleotide sequence are 5-halouracils.
• precisely or at least four U bases in the miR-140 sequence are 5-halouracils.
• precisely or at least five U bases in the miR-140 mimic sequence are 5-halouracils.
• precisely or at least six U bases in the miR-140 mimic sequence are 5-halouracils.
• all of the U bases in the miR- 140 sequence, whether in the native and/or in an appended portion are 5-halouracils.
• the nucleic acid composition of the present disclosure has a modified miR-140 nucleotide sequence of
• U F is a halouracil, specifically 5-fluorouracil.
• the U bases that are replaced with 5-halouracils in the miR-140 mimic sequence may be located in an unmodified part of the miR-140 sequence, as provided above, or may be located in one or more uracil bases that are appended to the native miR-140 sequence, as provided above.
• a seed portion of the native miR-140 nucleotide sequence remains unmodified with a 5-halouracil while one or more (or all) of the remaining U bases in the remainder of the miR-140 nucleotide sequence (non-seed portion) are replaced with a 5-halouracil.
• the present disclosure is directed to nucleic acid compositions that include a miR-192 nucleotide sequence that has been modified.
• the miR-192 nucleotide sequence has been modified by replacing at least one of the U bases with a 5-halouracil.
• miR- 192 is meant to be synonymous with the terms "microRNA-192", “miRNA-192” “microRNA-215", “miR-215" or “miRNA-215" and refers to an oligonucleotide having the following nucleotide sequence:
• MiR-192 unmodified (i.e., "native") sequence unless otherwise specified.
• MiR-192 may also be referred as hsa-mir-192, has-mir-215 or by miRBase Accession MI0000234, or MIMAT0000222.
• MiR-192 is well known and has been studied in detail, e.g., Song, B. et al., Clin. Cancer Res. (2008), 14: 8080-8086, and Song, B.
• miR-192 mimic As stated above for exemplary mimics miR-129, miR-140 and miR-lSa, methods for creating a miR-192 mimics are known by those of ordinary skill in the art. Unless otherwise stated, all such modified miR-192 nucleic acid forms are herein considered to be within the scope of the term "miR-192 mimic", as used herein.
• a modified miR-192 (i.e., miR-192 mimic) contains no more than one, two, three, four, or five additional nucleotides covalently appended to the miR-192 native sequence, wherein the additional bases are independently selected from C, U, G, and C, or the additional bases may be exclusively U.
• the miR-192 mimic is used in single- strand form, but double-stranded versions are also considered herein.
• At least one of the U bases in the miR- 192 or miR-21 S sequence, whether in the native and/or in an appended portion, is a 5-halouracil.
• the 5- halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5- iodouracil.
• precisely one of the U bases in the miR-192 mimic sequence is a 5-halouracil.
• precisely or at least two U bases in the miR-192 sequence are 5-halouracils.
• precisely or at least three U bases in the miR-192 oligonucleotide sequence are 5-halouracils.
• precisely or at least four U bases in the miR-192 sequence are 5-halouracils.
• all of the U bases in the miR-192 sequence, whether in the native and/or in an appended portion, are 5-halouracils.
• the nucleic acid composition of the present disclosure has a modified miR-192 nucleotide sequence of
• U F is a halouracil, specifically 5-fluorouracil.
• the U bases that are replaced with 5-halouracils in the miR-192 mimic sequence may be located in an unmodified part of the miR-192 sequence, as provided above, or may be located in one or more uracil bases that are appended to the native miR-192 sequence, as provided above.
• a seed portion of the native miR- 192 nucleotide sequence remains unmodified with a 5-halouracil while one or more (or all) of the remaining U bases in the remainder of the miR-192 nucleotide sequence (non-seed portion) are replaced with a 5-halouracil or combination thereof.
• the present disclosure is directed to nucleic acid compositions that include a miR-502 nucleotide sequence that has been modified.
• the miR-502 nucleotide sequence has been modified by replacing at least one of the U bases with a 5-halouracil.
• A adenine
• C cytosine
• U uracil
• G guanine bases.
• the foregoing nucleotide sequence is herein referred to as a miR-502 unmodified (i.e., "native") sequence unless otherwise specified.
• MiR-502 may also be referred as hsa-mir-502 or by miRBase Accession MI0003186, or MIMAT0002873.
• MiR-502 is well known and has been studied in detail, e.g., Zhai, H, et al., Oncogene. (2013), 32:12 pp. 1570-1579.
• methods for creating a miR-502 mimics are known by those of ordinary skill in the art.
• all such modified miR-502 nucleic acid forms are herein considered to be within the scope of the term "miR-502 mimic", as used herein.
• a modified miR-502 (i.e., miR-502 mimic) contains no more than one, two, three, four, or five additional nucleotides covalently appended to the miR-502 native sequence, wherein the additional bases are independently selected from C, U, G, and C, or the additional bases may be exclusively U.
• the miR-502 mimic is used in single- strand form, but double-stranded versions are also considered herein.
• at least one of the U bases in the miR-502 sequence, whether in the native and/or in an appended portion is a 5-halouracil.
• the 5-halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil.
• precisely one of the U bases in the miR-502 mimic sequence is a 5-halouracil.
• precisely or at least two U bases in the miR-502 sequence are 5-halouracils.
• precisely or at least three U bases in the miR-502 oligonucleotide sequence are 5-halouracils.
• precisely or at least four U bases in the miR-502 sequence are 5-halouracils.
• precisely or at least five U bases in the miR-502 sequence are 5- halouracils.
• precisely or at least six U bases in the miR-502 sequence are 5-halouracils.
• precisely or at least seven U bases in the miR-502 sequence are 5-halouracils.
• all of the U bases in the miR-502 sequence, whether in the native and/or in an appended portion, are 5-halouracils.
• the nucleic acid composition of the present disclosure has a modified miR-502 nucleotide sequence of
• the U bases that are replaced by 5-halouracils in the miR-502 mimic sequence may be located in an unmodified part of the miR-502 sequence, as provided above, or may be located in one or more uracil bases that are appended to the native miR-502 sequence, as provided above.
• a seed portion of the native miR-502 nucleotide sequence remains unmodified with a 5-halouracil while one or more (or all) of the remaining U bases in the remainder of the miR-502 nucleotide sequence (non-seed portion) are replaced by a 5- halouracil or combination thereof.
• the present disclosure is directed to nucleic acid compositions that include a miR-506 nucleotide sequence that has been modified.
• the miR-506 nucleotide sequence has been modified by replacing at least one of the U bases with a 5-halouracil.
• miR-506 is meant to be synonymous with the terms "microRNA-506” or “miRNA-506” and refers to an oligonucleotide having the following nucleotide sequence: U where it is
• MiR-506 unmodified (i.e., "native") sequence unless otherwise specified.
• MiR-506 may also be referred as hsa-mir-506 or by miRBase Accession MI0003193, or MIMAT0022701.
• MiR-506 is well known and has been studied in detail, e.g., Li, J, et al., Oncotarget. (2016), 7:38 pp. 62778-62788, and Li, J. et al., Oncogene. (2016) 35 pp. 5501-5514.
• miR-506 mimics As stated above for exemplary mimics miR- 129, miR-140, miR-502, miR-192 and miR-15a, methods for creating a miR-506 mimics are known by those of ordinary skill in the art. Unless otherwise stated, all such modified miR- 506 nucleic acid forms are herein considered to be within the scope of the term "miR-506 mimic", as used herein.
• a modified miR-506 (i.e., miR-506 mimic) contains no more than one, two, three, four, or five additional nucleotides covalently appended to the miR-506 native sequence, wherein the additional bases are independently selected from C, U, G, and C, or the additional bases may be exclusively U.
• the miR-506 mimic is used in single- strand form, but double-stranded versions are also considered herein.
• At least one of the U bases in the miR-506 sequence, whether in the native and/or in an appended portion, is a 5-halouracil.
• the 5-halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil.
• precisely one of the U bases in the miR-506 mimic sequence is a 5-halouracil.
• precisely or at least two U bases in the miR-506 sequence are 5-halouracils.
• precisely or at least three U bases in the miR-506 oligonucleotide sequence are 5-halouracils.
• precisely or at least four U bases in the miR-506 sequence are 5-halouracils.
• precisely or at least five U bases in the miR-506 sequence are 5- halouracils.
• precisely or at least six U bases in the miR-506 sequence are 5-halouracils.
• precisely or at least seven U bases in the miR-506 sequence are 5-halouracils.
• all of the U bases in the miR-506 sequence, whether in the native and/or in an appended portion, are 5-halouracils.
• the nucleic acid composition of the present disclosure has a modified miR-506 nucleotide sequence of
• U F is a halouracil, specifically 5-fluorouracil.
• the U bases that are replaced with 5-halouracils in the miR-506 mimic sequence may be located in an unmodified part of the miR-506 sequence, as provided above, or may be located in one or more uracil bases that are appended to the native miR-506 sequence, as provided above.
• a seed portion of the native miR-506 nucleotide sequence remains unmodified with a 5-halouracil while one or more (or all) of the remaining U bases in the remainder of the miR-506 nucleotide sequence (non-seed portion) are replaced with a 5-halouracil or combination thereof.
• modified microRNA nucleic acid compositions described herein can be synthesized using any of the well known methods for synthesizing nucleic acids.
• the nucleic acid compositions are produced by automated oligonucleotide synthesis, such as any of the well-known processes using phosphoramidite chemistry.
• a 5-halouracil nucleoside phosphoramidite can be included as a precursor base, along with the phosphoramidite derivatives of nucleosides containing natural bases (e.g., A, U, G, and C) to be included in the nucleic acid sequence.
• the nucleic acid compositions of the present disclosure may be produced biosynthetically, such as by using in vitro RNA transcription from plasmid, PCR fragment, or synthetic DNA templates, or by using recombinant (in vivo) RNA expression methods. See, e.g., C. M. Dunham et al., Nature Methods, (2007) 4(7), pp. 547- 548.
• microRNA sequence (e.g., miR-15a sequence, miR-140 sequence, miR-192 sequence, miR-502 sequence, miR-506 sequence or miR-129 sequence)may be further chemically modified such as by functionalizing with polyethylene glycol (PEG) or a hydrocarbon or a targeting agent, particularly a cancer cell targeting agent, such as folate, by techniques well known in the art.
• PEG polyethylene glycol
• a targeting agent particularly a cancer cell targeting agent, such as folate
• a reactive group e.g., amino, aldehyde, thiol, or carboxylate group
• a desired functional group may first be included in the oligonucleotide sequence.
• reactive or functional groups may be incorporated onto the as-produced nucleic acid sequence, reactive or functional groups can be more facilely included by using an automated oligonucleotide synthesis in which non-nucleoside phosphoramidites containing reactive groups or reactive precursor groups are included.
• the present disclosure is directed to formulations of the modified nucleic acid compositions described herein.
• the present nucleic acid compositions can be formulated for pharmaceutical uses.
• a formulation is a pharmaceutical composition containing a nucleic acid composition described herein and a pharmaceutically acceptable carrier.
• a formulation of the present disclosure comprises a modified miR-129 nucleic acid, a modified miR-15a nucleic acid, a modified miR-140 nucleic acid, a modified miR-192 nucleic acid, a modified miR-502, a modified miR-506 nucleic acid or a combination thereof and a pharmaceutically acceptable carrier.
• the modified microRNA nucleic acids set forth in the following nucleotide sequences can be formulated for pharmaceutical application and use;
• the term "pharmaceutically acceptable carrier” is used herein as synonymous with a pharmaceutically acceptable diluent, vehicle, or excipient.
• the nucleic acid composition may be dissolved or suspended (e.g., as an emulsion) in the pharmaceutically acceptable carrier.
• the pharmaceutically acceptable carrier can be any of those liquid or solid compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with tissues of a subject.
• the carrier should be "acceptable” in the sense of being not injurious to the subject it is being provided to and is compatible with the other ingredients of the formulation, i.e., does not alter their biological or chemical function.
• materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gelatin; talc; waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as ethylene glycol and propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents; water; isotonic saline; pH buffered solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
• sugars such as lactose, glucose and sucrose
• starches such as corn starch and potato starch
• the pharmaceutically acceptable carrier may also include a manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or stearic acid), a solvent, or encapsulating material. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
• a manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or stearic acid
• solvent e.g., a solvent, or encapsulating material.
• sweetening and/or flavoring and/or coloring agents may be added.
• suitable excipients can be found in standard pharmaceutical texts, e.g. in "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19 TH Ed. Mack Publishing Company, Easton, Pa., (1995).
• the pharmaceutically acceptable carrier may include diluents that increase the bulk of a solid pharmaceutical composition and make the pharmaceutical dosage form easier for the patient and caregiver to handle.
• Diluents for solid compositions include, for example, microcrystalline cellulose (e.g. Avicel ® ), microfine cellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, kaolin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, polymethacrylates (e.g. Eudragit ® ), potassium chloride, powdered cellulose, sodium chloride, sorbitol and talc.
• the nucleic acid compositions of the present disclosure may be formulated into compositions and dosage forms according to methods known in the art.
• the formulated compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, tablets, capsules, powders, granules, pastes for application to the tongue, aqueous or non-aqueous solutions or suspensions, drenches, or syrups; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin, lungs, or mucous membranes; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually or buccally; (6) ocularly; (7) transdermally; or (8) nasally.
• the formulations of the present disclosure include a solid pharmaceutical agent that is compacted into a dosage form, such as a tablet, may include excipients whose functions include helping to bind the active ingredient and other excipients together after compression.
• Binders for solid pharmaceutical compositions include acacia, alginic acid, carbomer (e.g. carbopol), carboxymethylcellulose sodium, dextrin, ethyl cellulose, gelatin, guar gum, hydrogenated vegetable oil, hydroxyethyl cellulose, hydroxypropyl cellulose (e.g. Klucel ® ), hydroxypropyl methyl cellulose (e.g. Methocel ® ), liquid glucose, magnesium aluminum silicate, maltodextrin, methylcellulose,
• polymethacrylates polymethacrylates, povidone (e.g. Kollidon ® , Plasdone ® ), pregelatinized starch, sodium alginate and starch.
• povidone e.g. Kollidon ® , Plasdone ®
• pregelatinized starch sodium alginate and starch.
• the dissolution rate of a compacted solid pharmaceutical composition in a subject's stomach may be increased by the addition of a disintegrant to the composition.
• Disintegrants include alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium (e.g. Ac-Di-Sol ® , Primellose ® ), colloidal silicon dioxide, croscarmellose sodium, crospovidone (e.g. Kollidon ® , Polyplasdone ® ), guar gum, magnesium aluminum silicate, methyl cellulose, microcrystalline cellulose, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate, sodium starch glycolate (e.g. Explotab**) and starch.
• alginic acid include alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium (e.g. Ac-Di-Sol ® , Primellose ® ), colloidal silicon dioxide, croscarmellose sodium, crospovidone (e.
• glidants can be added to formulations to improve the flowability of a non-compacted solid agent and to improve the accuracy of dosing.
• Excipients that may function as glidants include colloidal silicon dioxide, magnesium trisilicate, powdered cellulose, starch, talc and tribasic calcium phosphate.
• a dosage form such as a tablet is made by the compaction of a powdered composition
• the composition is subjected to pressure from a punch and dye.
• Some excipients and active ingredients have a tendency to adhere to the surfaces of the punch and dye, which can cause the product to have pitting and other surface irregularities.
• a lubricant can be added to the composition to reduce adhesion and ease the release of the product from the dye.
• Lubricants include magnesium stearate, calcium stearate, glyceryl monostearate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil, mineral oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, stearic acid, talc and zinc stearate.
• a formulated pharmaceutical composition for tableting or capsule filling can be prepared by wet granulation.
• wet granulation some or all of the active ingredients and excipients in powder form are blended and then further mixed in the presence of a liquid, typically water that causes the powders to clump into granules.
• the granulate is screened and/or milled, dried and then screened and/or milled to the desired particle size.
• the granulate may then be tableted, or other excipients may be added prior to tableting, such as a glidant and/or a lubricant.
• a tableting composition may be prepared conventionally by dry blending.
• the blended composition of the actives and excipients may be compacted into a slug or a sheet and then comminuted into compacted granules. The compacted granules may subsequently be compressed into a tablet.
• a blended composition may be compressed directly into a compacted dosage form using direct compression techniques. Direct compression produces a more uniform tablet without granules.
• Excipients that are particularly well suited for direct compression tableting include microcrystalline cellulose, spray dried lactose, dicalcium phosphate dihydrate and colloidal silica. The proper use of these and other excipients in direct compression tableting is known to those in the art with experience and skill in particular formulation challenges of direct compression tableting.
• a capsule filling may include any of the aforementioned blends and granulates that were described with reference to tableting; however, they are not subjected to a final tableting step
• liquid pharmaceutical compositions of the present disclosure the agent and any other solid excipients are dissolved or suspended in a liquid carrier such as water, water-for- injection, vegetable oil, alcohol, polyethylene glycol, propylene glycol or glycerin.
• a liquid carrier such as water, water-for- injection, vegetable oil, alcohol, polyethylene glycol, propylene glycol or glycerin.
• Liquid pharmaceutical compositions may contain emulsifying agents to disperse uniformly throughout the composition an active ingredient or other excipient that is not soluble in the liquid carrier.
• the liquid formulation may be used as an injectable, enteric, or emollient type of formulation.
• Emulsifying agents that may be useful in liquid compositions of the present invention include, for example, gelatin, egg yolk, casein, cholesterol, acacia, tragacanth, chondrus, pectin, methyl cellulose, carbomer, cetostearyl alcohol and cetyl alcohol.
• liquid pharmaceutical compositions of the present disclosure may also contain a viscosity enhancing agent to improve the mouth-feel of the product and/or coat the lining of the gastrointestinal tract.
• a viscosity enhancing agent include acacia, alginic acid bentonite, carbomer, carboxymethylcellulose calcium or sodium, cetostearyl alcohol, methyl cellulose, ethylcellulose, gelatin guar gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polyvinyl alcohol, povidone, propylene carbonate, propylene glycol alginate, sodium alginate, sodium starch glycolate, starch tragacanth and xanthan gum.
• the liquid composition of the present disclosure may also contain a buffer, such as gluconic acid, lactic acid, citric acid or acetic acid, sodium gluconate, sodium lactate, sodium citrate, or sodium acetate.
• Sweetening agents such as sorbitol, saccharin, sodium saccharin, sucrose, aspartame, fructose, mannitol and invert sugar, may be added to certain formulations of the present disclosure to improve the taste. Flavoring agents and flavor enhancers may make the dosage form more palatable to the patient. Common flavoring agents and flavor enhancers for pharmaceutical products that may be included in the composition of the present disclosure include maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol and tartaric acid.
• Preservatives and chelating agents such as alcohol, sodium benzoate, butylated hydroxy toluene, butylated hydroxyanisole and ethylenediamine tetraacetic acid, may be added at levels safe for ingestion to improve storage stability.
• Solid and liquid compositions may also be dyed using any pharmaceutically acceptable colorant to improve their appearance and/or facilitate patient identification of the product and unit dosage level.
• a dosage formulation of the present disclosure may be a capsule containing the composition, for example, a powdered or granulated solid composition of the disclosure, within either a hard or soft shell.
• the shell may be made from gelatin and optionally contain a plasticizer such as glycerin and sorbitol, and an opacifying agent or colorant.
• the modified microRNA nucleic acid compositions of the present disclosure and formulations thereof show unexpected and exceptional anticancer activity when compared to that exhibited by a native microRNA and/or a known cancer therapy (chemotherapy), such as 5-FU. Therefore, another aspect of the present disclosure provides a method for treating cancer in a mammal by administering to the mammal an effective amount of one or more of the modified microRNA nucleic acid compositions of the present disclosure, or formulations thereof.
• modified microRNA nucleic acids of the present disclosure i.e., modified miR-15a and modified MiR-129 suppress BCL2 expression and activity in the cancer cells of a subject, which results in an increased amount of available pro-apoptotic proteins which ultimately leads to increased cancer cell death.
• miR-129 for example, regulates apoptosis by directly targeting BCL2 as well as by impacting other critical cell death-related proteins.
• miR-129 reduces the expression, and thus activity of E2F3, a transcription factor protein that regulates cell cycle progression and reduces the expression or activity of thymidylate synthase (TS) protein levels, which results in increased cellular proliferation and increased efficacy of chemotherapeutic agents.
• TS thymidylate synthase
• microRNA's such as modified miR-506, miR-140, miR-192 and miR-502 also modulate cancer cell proliferation and cancer cell apoptosis, as shown in FIGS. 13A-B and 14A-D.
• FIGS. 7 and 11 show that intravenous treatment with two exemplary modified microRNA's of the present disclosure (e.g., modified miR-129 and modified miR- 15a) effectively treat colorectal cancer by inhibiting tumor growth and development.
• two exemplary modified microRNA's of the present disclosure e.g., modified miR-129 and modified miR- 15a
• the methods for treating cancer of the present disclosure include administering a nucleic acid composition of the present disclosure (e.g., a modified microRNA, such as modified miR-129 nucleic acid, a modified miR-15a nucleic acid, a modified miR-140 nucleic acid, a modified miR-192 nucleic acid, a modified miR-502, a modified miR-506 nucleic acid or a combination thereof) to a subject.
• a nucleic acid composition of the present disclosure e.g., a modified microRNA, such as modified miR-129 nucleic acid, a modified miR-15a nucleic acid, a modified miR-140 nucleic acid, a modified miR-192 nucleic acid, a modified miR-502, a modified miR-506 nucleic acid or a combination thereof.
• a nucleic acid composition of the present disclosure e.g., a modified microRNA, such as modified miR-129 nucleic acid, a modified miR-15a nu
• the nucleic acid composition can be administered as a formulation that includes a nucleic acid composition and a carrier.
• the nucleic acid composition of the present disclosure can be administered in the absence of a carrier (i.e., naked).
• the term "subject” as used herein refers to any mammal.
• the mammal can be any mammal, although the methods herein are more typically directed to humans.
• the phrase "subject in need thereof as used herein is included within the term subject and refers to any mammalian subject in need of a treatment, particularly cancer or has a medically determined elevated risk of a cancerous or pre-cancerous condition.
• the subject includes a human cancer patient.
• the subject has colorectal cancer or has a medically determined elevated risk of getting colorectal cancer.
• the subject has pancreatic cancer, or has a medically determined elevated risk of getting pancreatic cancer such as, for example, being diagnosed with chronic pancreatitis.
• a subject of the present disclosure has lung cancer, or has a medically determined elevated risk of getting lung cancer.
• treatment are synonomous with the term “to adminster an effective amount”. These terms shall mean the medical management of a subject with the intent to cure, ameliorate, stabilize, reduce one or more symptoms of or prevent a disease, pathological condition, or disorder such as cancer. These terms, are used interchangeably and include the active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
• treating includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the
• treatment while intended to cure, ameliorate, stabilize, or prevent a disease
• pathological condition, or disorder need not actually result in the cure, ameliorization, stabilization or prevention.
• the effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and/or quantitiative terms.
• characteristics or features of a disease, pathological condition, or disorder and/or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.
• the nucleic acid compositions of the present disclosure are used to treat cancer, such as colorectal cancer.
• cancer includes any disease caused by uncontrolled division and growth of abnormal cells, including, for example, the malignant and metastatic growth of tumors.
• cancer also includes pre-cancerous conditions or conditions characterized by an elevated risk of a cancerous or pre-cancerous condition.
• the treatment of cancer is herein also considered to include a method for the prevention of cancer or a method for preventing a pre-cancerous condition from transforming into a cancerous condition or into a completely non-cancerous condition.
• the cancer or pre-cancer can be located in any part of the body, including the internal organs and skin.
• Some examples of applicable body parts containing cancer cells include the colon, rectum (including anus), stomach, esophageal, digestive organs, lungs, pancreas, and liver.
• the cancer or neoplasm can also include the presence of one or more carcinomas, sarcomas, lymphomas, blastemas, or teratomas (germ cell tumors).
• the cancer may also be a form of leukemia.
• the nucleic acid compositions described herein are used to treat colorectal (i.e., colon or rectal), pancreatic or lung cancer in any of its stages, as further described below.
• colorectal i.e., colon or rectal
• pancreatic or lung cancer in any of its stages, as further described below.
• cancer spreads through a subject by invading the normal, non-cancerous tissue surrounding the tumor, via the lymph nodes and vessels, and by blood after the tumor invades the veins, capillaries and arteries of a subject.
• cancer cells break away from the primary tumor (“metastasize"), secondary tumors arise throughout an afflicted subject forming metastatic lesions.
• Stage 0 or carcinoma in situ abnormal potentially cancerous cells are found in the mucosa (innermost layer) of the colon wall.
• Stage I cancerous cells have formed in the mucosa of the colon wall and have spread to the submucosa (layer of tissue under the mucosa) and may have spread to the muscle layer of the colon wall.
• Stage ⁇ is composed of three subclasses: Stage IIA, wherein the cancerous tissue has spread through the muscle layer of the colon wall to the serosa (outermost layer) of the colon wall; Stage ⁇ , wherein the tumor has spread through the serosa of the colon wall but has not spread to nearby organs; and Stage IIC, wherein the cancer has spread through the serosa of the colon wall and invaded nearby organs.
• Stage ⁇ is also divided into three subclasses: Stage ⁇ , wherein the cancer may have spread through the mucosa of the colon wall to the submucosa and muscle layer, and has spread to one to three nearby lymph nodes or tissues near the lymph nodes; or the cancer has spread through the mucosa to the submucosa and four to six nearby lymph nodes; Stage MB, wherein the tumor has spread through the muscle layer of the colon wall to the serosa or has spread through the serosa but not to nearby organs and the cancer has spread to one to three nearby lymph nodes or to tissues near the lymph nodes; or has spread to the muscle layer or to the serosa, and to four to six nearby lymph nodes; or has spread through the mucosa to the submucosa and may have spread to the muscle layer and has spread to seven or more nearby lymph nodes.
• the tumor has spread through the serosa of the colon wall but not to nearby organs and the cancer has spread to four to six nearby lymph nodes; or the cancer has spread through the muscle layer to the serosa or has spread through the serosa but not to nearby organs and the cancer has spread to seven or more nearby lymph nodes; or the cancer has spread through the serosa to nearby organs and to one or more nearby lymph nodes or to tissues near the lymph nodes.
• Stage IV colon cancer is divided into two subclasses: Stage IV A, wherein the cancer has spread through the colon wall and into nearby organs and one organ that is not near the colon or to a distant lymph node; and Stage IVB, wherein the cancer has spread through the colon wall and into nearby organs and more than one organ that is not near the colon or into the lining of the abdominal wall.
• Stage A wherein the tumor is confined to the intestinal wall
• Stage B wherein the tumor exhibits invasion through the bowel but has not invaded the lymph nodes
• Stage C wherein cancerous cells or tissue is found within the lymph nodes of a subject
• Stage D wherein the tumor exhibits widespread metastases into several organs of the subject.
• Stage A colorectal cancer is identified as cancer that is only present in the mucosa of the intestine; Stage Bl whereby the tumor extends into muscularis basement but does not penetrate through it and the tumor has not metastasized into the lymph nodes, Stage B2 colorectal cancer is denoted by a tumor that penetrates through muscularis basement and the tumor has not metastasized into the lymph nodes; Stage CI is characterized by a tumor that extends into muscularis intestinal, but does not penetrate through it and the tumor has metastasized into the lymph nodes; Stage C2 colorectal cancer is classified as a tumor that penetrates through the muscularis basement where the tumor has metastasized into the lymph nodes; and Stage D describes a tumor that has metastasized throughout the organism or subject.
• the treatment methods of the present disclosure are more particularly directed to cancer subjects exhibiting reduced levels of miR-129 expression, miR-15a expression, miR-506 expression, miR-502, miR-140 or a combination thereof.
• miR-15a is down-regulated in cancers. See, for example, R I Aqeilan, et al., Cell Death and Differentiation (2010) 17, pp. 215-220.
• cancerous cells having reduced levels of miR-129 expression are resistant to 5- fluorouracil, as described, e.g., in U.S. Application Pub. No. 2016/0090636, the contents of which are incorporated by reference in their entirety.
• pancreatic cancer cells exhibit reduced levels of miR-506. See, e.g., Li, J, et al. Oncogene. 35 pp. 5501-5514.
• the microRNA mimics of the present disclosure are used to treat pancreatic cancer.
• Pancreatic cancer arises from precursor lesions called pancreatic intraepithelial neoplasia, or PanlNs. These lesions are typically located in the small ducts of the exocrine pancreas, and depending on the extent of cytologic atypia may be classified as low-grade dysplasia, moderate dysplasia or high-grade dysplasia lesions. Such lesions typically show that activating mutations in the KRAS gene present, along with certain inactivating mutations in CDKN2A, TP53 and SMAD4. Collectively, these genetic mutations lead to the formation of an infiltrating cancer.
• Pancreatic cancer is staged based on size of the primary tumor and whether it has grown outside of the pancreas into surrounding organs; whether the tumor has spread to the nearby lymph nodes, and whether it has metastasized to other organs of the body (e.g., liver, lungs, abdomen). This information is then combined and used to provide the specific stage, i.e., 0, 1 A, IB, 2A, 2B, 3 and 4.
• stage zero (0) the pancreatic tumor is confined to the top layers of pancreatic duct cells and has not invaded deeper tissues.
• the primary tumor has not spread outside of the pancreas such as in pancreatic carcinoma in situ or pancreatic intraepithelial neoplasia ⁇ .
• a stage 1 A pancreatic tumor is typically confined to the pancreas and is 2 cm across or smaller. Further a stage 1 A pancreatic tumor has not spread to nearby lymph nodes or distant sites.
• a stage IB pancreatic tumor confined to the pancreas and is larger than 2 cm across.
• a stage IB pancreatic tumor has not spread to nearby lymph nodes or distant sites.
• Stage 2A pancreatic tumors exhibit a tumor growing outside the pancreas but not into major blood vessels or nerves, but the cancer has not spread to nearby lymph nodes or distant sites.
• a subject exhibiting stage 2B pancreatic cancer presents a tumor is either confined to the pancreas or growing outside the pancreas but not into major blood vessels or nerves, but has spread to nearby lymph nodes.
• a subject exhibiting stage 3 pancreatic cancer presents a tumor that is growing outside the pancreas into major blood vessels or nerves, but has spread to distant sites.
• Stage 4 pancreatic cancer has metastasized to distant cites,
• the modified microRNA nucleic acid compositions of the present disclosure are used to treat lung cancer.
• the present methods include the treatment of non-small cell lung cancers, such as squamous cell carcinoma, adenocarcinoma, and large cell carcinoma.
• Lung cancer often arises from malignancies in the bronchi of the lungs and spreads to other parts of the body, such as lymph nodes.
• lymph nodes such as lymph nodes.
• small cell lung cancer a cancerous lesion is often found in once lung then spreads to the second lung, the fluid surrounding the lungs (pleura) or neighboring organs.
• Lung cancer is staged based on size of the primary tumor and whether it has grown outside of the lung into lymph nodes and whether it has metastasized to other organs of the body (e.g., bones, liver, breast, brain). This information is then combined and used to provide the specific stage, i.e., 0, 1, 2, 3 and 4.
• stage zero (0) i.e., carcinoma in situ
• the cancer is small in size and has not spread into deeper lung tissues or outside the lungs.
• Stage 1 lung cancer shows cancerous cells present in the underlying lung tissues, but the lymph nodes remain unaffected.
• Stage 2 lung cancer reveals that the cancer has spread to nearby lymph nodes or into the chest wall.
• Stage 3 lung cancer is classified by a continuous spread from the lungs to the lymph nodes or to nearby structures and organs, such as the heart, trachea and esophagus.
• Stage 4 lung cancer exhibits metastasized cancer throughout the body, which may affect the liver, bones or brain.
• nucleic acid compositions according to the present disclosure may be administered by any of the routes commonly known in the art. This includes, for example, (1) oral administration; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection; (3) topical administration; or (4) intravaginal or intrarectal administration; (5) sublingual or buccal administration; (6) ocular administration; (7) transdermal administration; (8) nasal administration; and (9) administration directly to the organ or cells in need thereof.
• routes commonly known in the art includes, for example, (1) oral administration; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection; (3) topical administration; or (4) intravaginal or intrarectal administration; (5) sublingual or buccal administration; (6) ocular administration; (7) transdermal administration; (8) nasal administration; and (9) administration directly to the organ or cells in need thereof.
• nucleic acid compositions of the present disclosure are administered depending on several factors, including the type and stage of the cancer, presence or absence of an auxiliary or adjuvant drug, and the subject's weight, age, health, and tolerance for the agent.
• the dosage may be, for example, about 2 mg/kg of body weight, about 5 mg/kg of body weight, about 10 mg/kg of body weight, about 15 mg/kg of body weight, about 20 mg/kg of body weight, about 25 mg/kg of body weight, about 30 mg/kg of body weight, about 40 mg/kg of body weight, about 50 mg/kg of body weight, about 60 mg/kg of body weight, about 70 mg/kg of body weight, about 80 mg/kg of body weight, about 90 mg/kg of body weight, about 100 mg/kg of body weight, about 125 mg/kg of body weight, about 150 mg/kg of body weight, about 175 mg/kg of body weight, about 200 mg/kg of body weight, about 250 mg/kg of body weight, about 300 mg/kg of body weight, about 350 mg/kg of body weight, about 400 mg/kg of body weight, about 500 mg/kg of body weight, about 600 mg/kg of body weight, about 700 mg/kg of body weight, about 800 mg/kg of body weight, about 900 mg
• the dosage may also be within a range bounded by any two of the foregoing values. Routine experimentation may be used to determine the appropriate dosage regimen for each patient by monitoring the compound's effect on the cancerous or pre-cancerous condition, or effect on microRNA expression level or activity (e.g., miR-15a, miR-129, miR-140, miR-192, miR-502, miR- 506), or effect on BCL2 level or activity, or effect on TS level or activity, or effect on E2F3 level or the disease pathology, all of which can be frequently and easily monitored according to methods known in the art.
• any of the above exemplary doses of nucleic acid can be administered once, twice, or multiple times per day.
• nucleic acid compositions described herein, and optionally, any additional chemotherapeutic agent for use with the current methods can be determined using pharmacological models well know in the art, such as cytotoxic assays, apoptosis staining assays, xenograft assays, and binding assays.
• nucleic acid compositions described herein may or may not also be coadministered with one or more chemotherapeutic agents, which may be auxiliary or adjuvant drugs different from a nucleic composition described herein.
• chemotherapy or the phrase a "chemotherapeutic agent” is an agent useful in the treatment of cancer.
• Chemotherapeutic agents useful in conjunction with the methods described herein include any agent that modulates BCL2, E2F3 or TS, either directly or indirectly.
• chemotherapeutic agents include: anti -metabolites such as methotrexate and fluoropyrimidine-based pyrimidine antagonist, 5-fiuorouracil (5-FU) (Carac® cream, Efudex®, Fluoroplex®, Adrucil®) and S-l; antifolates, including polyglutamatable antifolate compounds; raltitrexed (Tomudex®), GW1843 and pemetrexed (Alimta®) and non-polyglutamatable antifolate compounds; nolatrexed (Thymitaq®), plevitrexed, BGC945; folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; and purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, 5-
• the chemotherapeutic agent is a compound capable of inhibiting the expression or activity of genes, or gene products involved in signaling pathways implicated in aberrant cell proliferation or apoptosis, such as, for example, YAP1, BMI1, DCLK1, BCL2, thymidylate synthase or E2F3; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
• the chemotherapeutic agent is an anti-cancer drug, or a tissue sensitizer or other promoter for an anti-cancer drug.
• the co- drug may be another nucleic acid, or another miRNA, such as a microRNA mimic of the present disclosure, gemctiabine or free 5-FU.
• the other nucleic acid is a short hairpin RNA (shRNA), siRNA, or nucleic acid complementary to a portion of the BCL2 3'UTR.
• shRNA short hairpin RNA
• siRNA siRNA
• nucleic acid complementary to a portion of the BCL2 3'UTR is a short hairpin RNA (shRNA), siRNA, or nucleic acid complementary to a portion of the BCL2 3'UTR.
• the chemotherapy may be any of the following cancer drugs, such as one or more of methotrexate, doxorubicin, cyclophosphamide, cis-platin, oxaliplatin, bleomycine, vinblastine, gemcitabine, vincristine, epirubicin, folinic acid, paclitaxel, and docetaxel.
• the chemotherapeutic agent may be administered before, during, or after commencing therapy with the nucleic acid composition.
• the chemotherapeutic agent is a co-drug.
• E2F transcription factor 3 (RefSeq NG_029591.1, NM_001243076.2, NP 001230005.1) is a transcription factor that binds DNA and interacts with effector proteins, including but not limited to, retinoblastoma protein to regulate the expression of genes involved in cell cycle regulation. Therefore, any drug that inhibits the expression of E2F3 may be considered herein as a co-drug.
• B-cell lymphoma 2 (BCL2), (RefSeq NG_009361.1, NM_000633, NP_000624) including isoform a (NM_000633.2, NP_000624.2) and ⁇ NM 000657.2, NP_000648.2 thereof, are encoded by the Bcl-2 gene, which is a member of the BCL2 family of regulator proteins that regulate mitochondria regulated cell death via the intrinsic apoptosis pathway.
• BCL2 is an integral outer mitochondrial membrane protein that blocks the apoptotic death of cell cells by binding BAD and BAK proteins.
• Non-limiting examples of BCL2 inhibitors include anti sense oligonucleotides, such as Oblimersen (Genasense; Genta Inc.,), BID mimetic small molecule inhibitors including, ABT-737 (Abbott Laboratories, Inc.), ABT- 199 (Abbott Laboratories, Inc.), and Obatoclax (Cephalon Inc.). Any drug that inhibits the expression of BCL2 may be considered herein as a co-drug.
• Any drug that inhibits the expression of BCL2 may be considered herein as a co-drug.
• Thymidylate synthase (RefSeq: NG_028255.1, NM_001071.2, NP_001062.1) is a ubiquitous enzyme, which catalyses the essential methylation of dUMP to generate dTMP, one of the four bases which make up DNA.
• the reaction requires CH Hj-folate as a cofactor, both as a methyl group donor, and uniquely, as a reductant.
• the constant requirement for CH Hj-folate means that thymidylate synthase activity is strongly linked to the activity of the two enzymes responsible for replenishing the cellular folate pool:
• Thymidylate synthase is a homodimer of 30-35kDa subunits.
• the active site binds both the folate cofactor and the dUMP substrate simultaneously, with the dUMP covalently bonded to the enzyme via a nucleophilic cysteine residue (See, Carreras et al, Annu. Rev. Biochem., (1995) 64:721- 762).
• the thymidylate synthase reaction is a crucial part of the pyrimidine biosynthesis pathway which generates dCTP and dTTP for incorporation into DNA. This reaction is required for DNA replication and cell growth.
• Thymidylate synthase activity is therefore required by all rapidly dividing cells such as cancer cells. Due to its association with DNA synthesis, and therefore, cellular replication, thymidylate synthase has been the target for anti-cancer drugs for many years.
• Non-limiting examples of thymidylate synthase inhibitors include folate and dUMP analogs, such as 5-fluorouracil (5-FU). Any drug that inhibits the expression of thymidylate synthase may be considered herein as a co-drug.
• the administration of the nucleic acid composition described herein may be combined with one or more non-drug therapies, such as, for example, radiotherapy, and/or surgery.
• radiation therapy and/or administration of the chemotherapeutic agent in this case, the nucleic acid composition described herein, and optionally, any additional chemotherapeutic agent
• the chemotherapeutic agent may be given before surgery to, for example, shrink a tumor or stop the spread of the cancer before the surgery.
• radiation therapy and/or administration of the chemotherapeutic agent may be given after surgery to destroy any remaining cancer.
• [00163] modified miR-129 the 5-FU modified miR-129 molecules were synthesized by an automated oligonucleotide synthesis process and purified by HPLC. The two strands were annealed to make the mature modified 5-FU-miR-129. More specifically, a process referred to as "2 -ACE RNA synthesis" was used.
• the 2 -ACE RNA synthesis is based on a protecting group scheme in which a silylether is employed to protect the 5'-hydroxyl group in combination with an acid-labile orthoester protecting group on the 2'-hydroxy (2 -ACE). This combination of protecting groups is then used with standard phosphoramidite solid- phase synthesis technology. See, for example, S.A. Scaringe, F.E.
• modified miR-15a nucleic acid, modified miR-140 nucleic acid, modified miR-192 nucleic acid, modified miR-502, modified miR-506 nucleic acid or any other modified microRNAs that replace uracil with a 5-halouracil can be synthesized in the same manner as mi R- 129a.
• the human colon cancer cell lines HCT116, RKO, SW480, SW620, and the normal colon cell line CCD 841 CoN, pancreatic cancer cell lines ASPC-1, Panc-1, and lung cancer cell line A549 were obtained from the American Type Culture Collection (ATCC) and maintained in McCoy's 5A medium (HCT-116), DMEM (RKO, SW480, SW620) and MEM (CCD 841 CoN) (Thermo Fischer). Media was supplemented with 10% fetal bovine serum (Thermo Fischer).
• cells were cultured in DMEM7F12 supplemented with B27, 10 ng/mL bFGF, and 20 ng/mL EGF (Life Technologies) in ultra-low attachment flasks.
• the spheroid cells were maintained by collection through gentle centrifugation, dissociation to single cells and replating.
• mouse anti -human TS antibody (1:500), anti-a-tubulin (1:50000) (Santa Cruz Biotech Inc.), anti-GAPDH (1 : 100000) (Santa Cruz Biotech Inc.), ant-E2F3 (1 :500) (Santa Cruz Biotech Inc.),.
• Horseradish peroxidase-conjugated antibodies against mouse or rabbit (1:5000, Santa Cruz Biotech Inc.) were used as the secondary antibodies. Protein bands were visualized with autoradiography film using SuperSignal West Pico
• Cell proliferation assay Twenty-four hours after transfection, cells were seeded in 96-well plates at a density of 2000 cells per well. The cell proliferation assay was performed on days 1 to 5 by incubating 10 ul WST-1 (Roche Applied Science, Mannheim, Germany) in the culture medium for lh and reading the absorb an ce at 450 and 630nm. The cell proliferation rate was calculated by subtracting the absorbance at 450 nm from the absorbance at 630 nm. Experiments for the cell proliferation assay were performed at least three times. The O.D. was calculated by subtracting the absorbance at 630 nm from that at 450 nm. Proliferation experiments were performed three times.
• Cell cycle analysis Twenty-four hours after transfection, cells were harvested and resuspended at 0.5 to 1 x 10 6 cells/mL in modified Krishan's buffer supplemented with 0.02 mg/mL RNase H and 0.05 mg/mL propidium iodide. Stained cells were detected by flow cytometry and results were analyzed with Modfit LTTM software. The experiments for cell cycle analysis were performed at least three times.
• Apoptosis Assay To distinguish between early and late apoptosis, a fluorescein isothiocyanate (FITC)-Annexin assay was done (Becton Dickinson). HCT116, RKO, SW480 and SW620 cells were plated into 6 well plates (1 ⁇ 10 5 ) cells per well, after 24h, cells were transfected with 25 nM modified miRNAs using Oligofectamine.
• FITC fluorescein isothiocyanate
• [00173] 5-FU treatment and cytotoxicity assay Twenty-four hours after transfection, cancer cells were plated in 96-well plates at 2 x 10 3 cells per well in triplicates in 100 ⁇ . of medium. After 24 hours, fresh medium containing 2 ⁇ 5-FU alone, 50 nM native microRNA, 50 nM modified microRNA (e.g., modified miR-129), or a combination of 2 mM 5-FU with 50 nM a modified microRNA of the present disclosure e.g., modified miR- 129, were added, and cells were cultured for an additional 72 hours. Cell viability was measured using the WST-1 assay.
• 50 nM native microRNA e.g., 50 nM modified microRNA (e.g., modified miR-129)
• a modified microRNA of the present disclosure e.g., modified miR- 129
• Lentivirus production Briefly, 1.5 x 10 6 293T cells were plated in a 10-cm dish with 10 mL of DMEM + 10% FBS. Two days later, pEZX-MR03, a lentiviral plasmid, expressing miR-129 or hsa-miR-15a, was transfected with Lenti-Pac HIV expression packaging kit following the manufacturer's protocol. Forty-eight hours later, the virus was harvested and concentrated with Lenti-Pac lentivirus concentration solution. Then the titer of the virus (approximately 1011 virus particles/ml) was determined with Lenti-PacTM HIV qRT-PCR titration kit.
• serial dilution of the virus (0.1 ⁇ L, 0.5 ⁇ L, 2 ⁇ L,, 10 uL, 50 ⁇ ) was used to transduce 5 x 10 4 HCT116 CSC to determine the transduction efficiency.
• the lowest concentration (2 ⁇ .) to achieve 100% positive expression was used to infect the cells for mouse in vivo treatement experiments.
• Biosystems 7500 Real-Time PCR machine with miRNA-specific primers by TaqMan Gene Expression Assay (Applied Biosystems). Expression level of the exemplary miRs of the present disclosure was calculated by the AACT method based on the internal control RNU44, normalized to the control group and plotted as relative quantification.
• RNAs were extracted from cancer cells transfected with either exemplary microRNAs of the present disclosure or negative miRNA using TRIzol reagent (Thermo Fischer) in accordance with the manufacturer's protocol. RNAs were transcribed to first-strand cDNA using the RT2 First Strand Kit (Qiagen). Next, the cDNA is mixed with RT2 SYBR Green Mastermix (Qiagen), and this mixture is aliquoted into the wells of the Human Cancer Stem Cells RT2 Profiler PCR Array (Qiagen). Applied Biosystems 7500 Real-Time PCR machine was used for qRT-PCR (Applied Biosystems), and relative expression values were determined using the AACT method.
• TRIzol reagent Thermo Fischer
• Mouse subcutaneous tumor implantation model Two days before injection, HCT116 cancer stem cells were plated at 5 x 10 5 /well in a 6-well ultra low attachment plate. 20 ⁇ . of the virus or 100 pmole exemplary modified miR-129 or modified miR-15a were used to transduce or transfect cells. Forty-eight hours later, cells were collected and re- suspended at 10 6 /ml in DMEM/F12 knockout media with 30% matrigel. Ten-twelve week- old NOD/SCID mice (Jackson Laboratories, Bar Harbor, MA, USA) were used for tumor implantation. The mice were anesthetized by isoflurane inhalation.
• mice Two weeks after injection of colon cancer cells, mice were treated via tail vein injection with 40 ⁇ g of negative control or modified miR(s) packaged with in v/vo-jetPEI (Polyplus Transfection). Mice were treated every other day for 2 weeks (8 times). Following treatment, mice were screened using IVIS Spectrum In vivo Imaging System (IVIS) (PerkinElmer).
• IVIS IVIS Spectrum In vivo Imaging System
• RNA isolation For mouse xenografts, sectioned tissues were deparaffinized, hydrated, and digested with proteinase K, respectively. Subsequently, total RNA was isolated using the TRIzol ® reagent. Total RNA was also isolated from clinical specimens by the TRIzol ® -based approach.
• Example 2 Modified microRNAs of the present disclosure have anti-cancer activity.
• the modified miRNAs are more effective in inhibiting colon cancer, pancreatic cancer, and lung cancer cell proliferation than non-modified miRNA precursor.
• the modified miRNAs can be delivered into cancer cells without the transfection reagent (data not shown).
• the results show that cancer cell proliferation across several different colorectal cancer cell lines, pancreatic cancer cell lines, and lung cancer cell lines, is inhibited significantly when compared to cancer cells treated with control microRNAs.
• Modified miR-129 nucleic acids have anti-cancer activity.
• polynucleotides of the present disclosure were able to retain their target specificity to TS, BCL2 and E2F3 via.
• the results are shown in FIGS. 2 A and 2B, which shows the results for the modified miR-129 nucleic acid having all U bases were replaced with 5-FU, as obtained by two separate operators as set forth in SEQ ID. NO: 4.
• the exemplary miR-129 mimics were found to be more potent than unmodified (control) miR-129 in reducing the expression levels of TS, BCL2 and E2F3.
• modified miR-129 and 5-FU were compared using HCT-116 colon cancer cells.
• 50 nM (40-fold less than 5-FU) of modified miR-129 is unexpectedly much more potent than 2 ⁇ 5-FU in inhibiting tumor cell proliferation.
• Exemplary modified microRNAs of the present disclosure induce apoptosis in colon cancer cells.
• BCL2 being an important target of miR-129
• cell death was quantified using an apoptosis assay in HCT116, RKO, SW480, and SW620 colon cancer cells transfected with negative control miRNA, native miR-129 or an exemplary miR-129 mimic of SEQ ID NO: 4.
• results show that the miR-129 mimic was able to induce apoptosis by 2 to 30-fold in all 4 colon cancer cell lines via a fluorescence-activated cell sorting (FACS)-based FITC-Annexin assay compared to the native miR-129 and negative control miRNA (FIG. 5A).
• FACS fluorescence-activated cell sorting
• miR-129 mimic trigger Gl/S cell cycle check point control.
• Cell cycle analysis was performed using flow cytometry in HCT-116 cells treated with scramble control, miR-129 precursor, and an exemplary miR-129 mimic.
• FIG. SB cell cycle analysis revealed that the miR-129 mimic impacts colon cancer cell growth by inducing Gl arrest, and such impact is much more potent (more than two-fold) than native miR-129.
• miR-129 mimics eliminated chemotherapy resistant colon cancer stem cells.
• HCT116 derived colon cancer stem cells were treated with various concentrations of Mimic- 1 or 5-FU.
• the data shown in FIG. 6 reveal that exemplary microRNA mimics of the present disclosure are able to eliminate 5-FU resistant colon cancer stem cells by over 80% at 100 nM concentration, while a lethal dose of 5-FU at 100 ⁇ has minimal effect on tumor stem cell viability.
• polynucleotides of the present disclosure were able to inhibit cell proliferation of HCT116 colon cancer stem cells (FIG. 6). Such inhibitory effect by modified miR-129 was much more potent than native miR-129, as proliferation was nearly completely blocked with 25 nM miR-129 on day 6 (FIG. 6).
• modified miR-129 treated colon cancer stem cells and formed no visible spheres compared to cells treated with the native miR-129 or control miRNA (similar to those seen in FIG. 10).
• miR-129 mimics inhibit colon cancer metastasis in vivo.
• the therapeutic impact of modifiying miR-129 nucleic acids was evaluated using a colon cancer metastasis model. Two weeks after establishing metastasis, 40 ⁇ g of a miR-129 nucleic acid of SEQ ID NO; 4 was delivered by intravenous injection with treatment frequency of one injection every other day for two weeks.
• Example 3 Modified miR-lSa and anti-cancer activity thereof.
• Exemplary modified miR-15a compostions have anti-cancer activity.
• exemplary modified miR-lSa mimics in which all of the uracil bases (FIG. 1C) or only uracil bases that in the non-seed region (FIG. ID) of the miR-15a nucleic acid sequence were replaced with a 5-halouracil (i.e., 5-flurouracil) were synthesized as set forth above.
• Modified miR-15a has increased therapeutic efficacy in vitro. In order to determine whether the modified miR-15a compositions of the present disclosure are present disclosure.
• a WST-1 assay was used to assed cancer cell proliferation. As shown in FIG. 8B, six days after transfection, unmodified miR-15a had decreased cell proliferation by 53% compared to control. In the case of modified miR-15a, cell proliferation was decreased by 84%. Taken together, the experimental results show that modified miR-15a is more effective at decreasing cancer cell proliferation compared to the unmodified miR-lSa.
• Modified miR-15a nucleic acids were also analyzed for their ability to inhibit cell cycle progression in cancer cells.
• FIG. 9 shows that unmodified miR-15a induced cell cycle arrest and lead to about 3 -fold increase in the Gl/S ratio.
• FIG. 9 also shows that the exemplary modified miR-15a compositions of the present disclosure were more effective in stopping cell cycle progression when compared to their native counterpart. For example, a 7-fold increase in the Gl/S ratio was exhibited by cells expressing the exemplary modified miR-15a nucleic acids of the present disclosure when compared to the control. Therefore, modified miR-15a is more effective at inducing cell cycle arrest in colon cancer cells than unmodified miR-15a.
• Modified miR-15a inhibits ameer development and progression in vivo.
• halouracils e.g., 5-FU
• a miRNA nucleic acid sequence to enhance the chemotherapeutic function of the native microRNA molecule with or without the use of other chemotherapeutic agents.

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