KR20190086688A - 5-haloracil-modified microRNAs and their uses in cancer therapy - Google Patents

Abstract

The present invention provides a nucleic acid composition comprising at least one < RTI ID = 0.0 > halolacyl < / RTI > molecule. More specifically, the present invention reveals that the 5-haloracil replacement of uracil in the microRNA nucleotide sequence increases the ability of the microRNA to inhibit cancer progression and tumorigenesis. Thus, the present invention provides various nucleic acid (e. G., MicroRNA) compositions in which 5-halo lacyl molecules are incorporated into the nucleic acid sequence and methods of using the same. The present invention also provides pharmaceutical compositions (e. G., Formulations) comprising the modified nucleic acid compositions and methods of treating cancers such as colon, pancreatic, and lung cancer.

Description

5-haloracil-modified microRNAs and their uses in cancer therapy

Cross-reference to related application

This application claims the benefit of U.S. Provisional Application No. 62 / 464,491, filed February 28, 2017, which is incorporated herein by reference in its entirety, U.S. Provisional Application No. 62 / 422,298, filed November 15, 2016, and November 1, 2016 U.S. Provisional Application No. 62 / 415,740.

Government support

[0002] The present invention was made with government support under grant numbers HL127522 and CA197098 granted by the National Institutes of Health. The government has the right to the invention.

Includes reference to sequence list

[0003] A sequence listing of an ASCII text file named R8859_PCT_SequenceListing.txt of 7 KB created on October 30, 2017 and submitted to the US Patent and Trademark Office via EFS-Web is included herein by reference.

Field of the Invention

[0004] The present invention generally relates to compositions and methods for treating cancer, and more particularly, to a method of treating cancer in a cancer, particularly rectal, lung or pancreatic cancer, alone or in combination with 5-fluorouracil Lt; / RTI >

MicroRNAs (miRNAs, miRs) are highly conserved non-human genes that regulate translation in cells or organisms that negatively regulate the expression of their target gene, resulting in translation interruption, mRNA cleavage, or a combination thereof. Encryption is a kind of small RNA molecule. [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, and thus abnormal microRNA function can induce cancer (Ambros V. Nature. (2004) 431 (7006): 350-5). Thus, recently miRNA has been identified as a biomarker, oncogene or tumor suppressor. See, e.g., Croce, CM, Nat Rev Genet. (2009) 10: 704-714.

[0006] Colorectal cancer (CRC) is the third most common malignancy and the second most common cancer-related death in the United States. [Hegde SR, et al., Expert review of gastroenterology & hepatology. (2008) 2 (1): 135-49. There are a number of chemotherapeutic agents used to treat cancer, but pyrimidine antagonists such as fluoropyrimidine-based chemotherapeutic agents (e.g., 5-fluorouracil, S-1) (gold standard). Pyrimidine antagonists block the synthesis of pyrimidines (cytosine and thymine in DNA, cytosine and uracil in RNA) containing nucleotides. Since pyrimidine antagonists have a similar structure compared to endogenous nucleotides, they compete with natural pyrimidines to inhibit important enzymatic activities involved in the replication process, resulting in inhibition of DNA and / or RNA synthesis and inhibition of cell division.

[0007] Pancreatic cancer is a fatal cancer that is very difficult to treat. [Siegel, RL et al. CA Cancer J. Clin. (2015) 65: 5-29]. Unique features of pancreatic cancer include low 5-year survival rates of less than 7% (ld.), Delayed symptoms, early metastasis, chemotherapy, and poor response to radiotherapy. [Maitraa and Hruban RH, Annu Rev. Pathol. (2008) 3: 157-188. To date, gemcitabine chemotherapy (2 ', 2'-difluoro 2'-deoxycytidine) has been the standard for the treatment of pancreatic cancer, but the therapeutic intervention is limited due to drug resistance. [Oettle, H et al. JAMA (2013) 310: 1473-1481].

5-Fluorouracil (ie, 5-FU, or more specifically 5-fluoro-1H-pyrimidine-2,4-dione) is administered in the form of a large number of adjuvant chemotherapeutic agents, , Efudex (R), Fluoroplex (R) and Adrucil (R). (TYMS or TS), an important enzyme that catalyzes the methylation of deoxythymidine monophosphate (dTMP) of deoxyuridine monophosphate (dUMP), an essential step in DNA biosynthesis, as a target Is well known. [Danenberg P. V., Biochim. Biophys. Acta. (1977) 473 (2): 73-92). However, despite the steady improvement of 5-FU therapy, the patient response rate to 5-FU chemotherapy is still lukewarm due to the development of drug resistance. [Longley D. B, et al., Apoptosis, Cell Signaling, and Human Diseases, (2007) p. 263-78].

Nevertheless, existing cancer therapies are still at an early stage, and there are still many obstacles awaiting improvement or overcoming. For example, it is well known that 5-FU is quite effective in treating a variety of cancers, but 5-FU has substantial toxicity and can cause many side effects. With respect to miRNAs, this compound is known to cause enzymatic degradation upon administration, which results in poor stability. Tumor cells are also known to avoid apoptosis pathways by developing resistance to common therapeutic agents such as 5-FU and gemcitabine. [Gottesman M. M. et al., Nature Reviews Cancer, (2002) 2 (1): 48-58]. Thus, more effective, stable, less toxic drugs would be of considerable benefit for the treatment of cancer.

SUMMARY OF THE INVENTION

The present invention demonstrates that a nucleic acid composition comprising a 5-haloracil base (ie, microRNA) has excellent efficacy as an anti-cancer agent. Moreover, the data of the present invention further suggests that contacting the cell with a modified microRNA composition of the invention modulates progression of the cell cycle and reduction of tumorigenesis, for example, by reducing cancer cell proliferation and increasing the efficacy of the chemotherapeutic agent Show. The present invention presumes that the incorporation of the 5-halo lysyl base in the nucleotide sequence of the microRNA increases the microRNA efficiency as an anticancer agent rather than the cancer therapeutic alone and / or the original microRNA.

Thus, in one aspect of the present invention there is provided a modified microRNA nucleotide sequence having at least one uracil base (U, U base) replaced by a 5-halolactyl eg 5-fluorouracil (5-FU) ≪ / RTI > is described. In certain embodiments, the modified microRNA has at least one uracil or exactly one uracil substituted with 5-haloracil. In some embodiments, the modified microRNA nucleotide sequence comprises 2, 3, 4, 5, 6, 7, 8 or more uracil bases substituted with 5-haloracil. In another embodiment, all of the uracil nucleotide bases of the modified mRNA were replaced with 5-haloracil.

[0012] In some embodiments, the 5-haloracil is, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil or 5-iodouracil. In certain embodiments, the 5-haloracil is 5-fluorouracil.

[0013] In certain embodiments, the modified microRNA nucleotide sequence comprises one or more 5-Hollowacyls, whereby each of the 5-Hollowacyls is identical. In another embodiment, the modified microRNA nucleotide sequence comprises at least one 5-Hollow Lysyl, whereby each of the 5-Hollow Lysyl is different. In another embodiment, the modified microRNA nucleotide sequence comprises two or more 5-Hollowacyls, whereby the modified microRNA nucleotide sequence comprises a combination of different 5-Hollowacyls.

[0014] In one exemplary embodiment of the invention, a nucleic acid composition is provided that contains a modified miR-129 nucleotide sequence by replacing one or more uracil nucleotide bases with 5-haloracil. More specifically, the nucleic acid composition comprises at least the following native miR-129 nucleotide sequence:

CUUUUUGCGGUCUGGGCUUGC [SEQ ID NO. One]

Wherein at least one, two, three, four, five, six, seven, eight or all of the uracil bases that can be covalently bound to, or within the depicted nucleic acid sequence, - It is replaced by the Hollow Lassil.

[0015] In a specific embodiment of the present invention,

^{CU F U F U F U F} U F GCGGU F CU F GGGCU F U F GC [SEQ ID NO. 4], wherein U ^F is a halolacyl, specifically 5-fluorouracil.

[0016] In another embodiment, GUUUUUUC, the seed portion of the parent miR-129 nucleotide sequence, remains unmodified (ie, does not contain 5-halo lucyl), while the modified miR-129 nucleotide sequence One or more (or all) of the remaining uracil nucleotide bases in the remainder of the nucleotide sequence are replaced by the same number of 5-haloracils. In certain embodiments, the modified miR-129 microRNA of the present invention comprises,

CU ^F U ^{F F F} GGGCU CUUUUUGCGGU GC [SEQ ID NO. 5], whereby U ^F is a halolactyl, specifically 5-fluorouracil.

[0017] In some embodiments, the 5-haloracil is, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil or 5-iodouracil. In certain embodiments, the 5-haloracil is 5-fluorouracil.

[0018] In another embodiment of the present invention, a nucleic acid containing a modified miR-15a nucleotide sequence is obtained by replacing one or more of the uracil nucleotide bases with a 5-halolactyl such as 5-fluorouracil (5-FU) A composition is provided. Specifically, the nucleic acid composition comprises at least the following miR-15a nucleotide sequence:

UAGCAGCACAUAAUGGUUUGUG [SEQ ID NO. 2], wherein at least one, two, three, four, five, six or all of the uracil nucleotide bases that can be covalently bound to or in the depicted sequences, It is Rasile.

[0019] In a particular embodiment of the invention, the modified miR-15a microRNA comprises

^{F F} GGU AAU AGCAGCACAU ^{F F} U U U ^{F F F} GU G [SEQ ID NO. 6], wherein U ^F is a halolacyl, specifically 5-fluorouracil.

[0020] In another embodiment, the UAGCAGCA, which is the seed portion of the native miR-15a nucleotide sequence, remains unmodified to the 5-haloracil whereas the remainder of the miR-15a nucleotide sequence (Or all) of the remaining uracil bases are replaced by 5-haloracil.

[0021] In certain embodiments, the modified miR-129 microRNA comprises

GGU AAU ^{F F F F} U UAGCAGCACAU U ^{F F} GU G [SEQ ID NO. 7], wherein U ^F is a halolacyl, specifically 5-fluorouracil.

[0022] In another exemplary embodiment, the present invention relates to a nucleic acid composition comprising a modified miR-140 nucleotide sequence. In some embodiments, the native miR-140 nucleotide sequence has been modified by replacing one or more of the U bases with a 5-haloracil.

[0023] In one set of embodiments, exactly one of the U bases in the intrinsic miR-140 nucleic acid sequence is 5-Hollowacyl. In a second set of embodiments, exactly two or more U bases in the original miR-140 nucleotide sequence are replaced by 5-haloracil. In another set of embodiments, exactly three or more U bases in the miR-140 nucleotide sequence are 5-haloracil. In other embodiments, exactly four or more U bases in the native miR-140 nucleotide sequence are 5-halolactyl. In some embodiments, exactly five or more U bases in the miR-140 nucleotide sequence are 5-haloacyls. In yet another embodiment, exactly six or more U bases in the miR-140 nucleotide sequence are 5-haloracil. In certain embodiments, all of the U bases of the miR-140 nucleotide sequence are 5-halo lysyl, whether in its native portion and / or in the appended portion.

[0024] In one exemplary embodiment, the modified microRNA nucleic acid composition of the present invention comprises,

CAGU ^F GGUUUUACCCU ^F AUGGU ^F AG [SEQ ID NO. 9], wherein U ^F is a halocaryl, specifically 5-fluorouracil.

[0025] In another exemplary embodiment, the invention provides a nucleic acid composition comprising a modified native miR-192 or miR-215 nucleotide sequence modified by replacing one or more of the uracil bases with 5-haloracil . In some embodiments, the modified miR-192 nucleotide sequence has been modified by replacing one or more of the U bases with 5-fluorouracil.

[0026] In another set of embodiments, exactly one of the U bases in the modified miR-192 nucleotide sequence is 5-haloracil. In a second set of embodiments, exactly two or more U bases in the modified miR-192 nucleotide sequence are 5-haloacyls. In another set of embodiments, exactly three or more U bases in the modified miR-192 nucleotide sequence are 5-haloacyls. In another embodiment, exactly four or more U bases in the modified miR-192 or miR-215 nucleotide sequence are 5-haloracil. In certain embodiments, all of the U bases of the modified miR-192 or miR-215 sequence are 5-halo lysyl, whether in the intrinsic portion of the nucleic acid and / or in the appended portion.

[0027] In one exemplary embodiment, the nucleic acid composition of the present invention comprises,

GACCU AU ^{F F F F} GAAU CU U GACAGCC ^F [SEQ ID NO. 11] or a modified miR-215 nucleotide sequence, wherein U ^F is a halo lysyl, specifically 5-fluorouracil.

[0028] In another exemplary embodiment, the present invention relates to a nucleic acid composition comprising a modified, inherent miR-502 nucleotide sequence modified by replacing uracil with a 5-haloracil. In some embodiments, the modified miR-502 nucleotide sequence has been modified by replacing one or more of the U bases with 5-fluorouracil.

[0029] In another set of embodiments, exactly one of the U bases in the miR-502 nucleotide sequence is 5-halolactyl. In a second set of embodiments, exactly two or more U bases in the miR-502 nucleotide sequence are 5-halolactyl. In another set of embodiments, exactly three or more U bases in the miR-502 nucleotide sequence are 5-haloracil. In another embodiment, exactly four or more U bases in the miR-502 nucleotide sequence are 5-haloacyls. In another embodiment, exactly five or more U bases in the miR-502 nucleotide sequence are 5-haloracil. In another embodiment, exactly six or more U bases in the miR-502 nucleotide sequence are 5-haloacyls. In another embodiment, exactly seven or more U bases in the miR-502 nucleotide sequence are 5-haloracil. In certain embodiments, all of the U bases of the miR-502 nucleotide sequence are 5-halo lysyl, whether in the native portion and / or in the appended portion.

[0030] In one exemplary embodiment, the modified miR-502 nucleic acid composition of the present invention comprises

CCU U ^{F F F F} GCUAU AU CU GGGU ^{F F F} A GCU [SEQ ID NO. 13], wherein U ^F is a halo lysyl, specifically 5-fluorouracil.

[0031] In another exemplary embodiment, the present invention relates to a nucleic acid composition comprising a modified miR-506 nucleotide sequence comprising 5-haloracil. In some embodiments, the modified miR-506 nucleotide sequence has been modified by replacing one or more of the U bases with a 5-halolactyl, such as 5-fluorouracil.

[0032] In another set of embodiments, exactly one of the U bases in the native miR-506 nucleotide sequence is replaced by a 5-haloracil. In a second set of embodiments, exactly two or more of the U bases in the modified miR-506 nucleotide sequence are 5-haloracil. In another set of embodiments, exactly three or more U bases in the modified miR-506 nucleotide sequence are 5-haloucalyl. In another embodiment, exactly four or more U bases in the modified miR-506 nucleotide sequence are 5-haloracil. In another embodiment, exactly five or more U bases in the modified miR-506 nucleotide sequence are 5-haloacyls. In another embodiment, exactly six or more U bases in the modified miR-506 nucleotide sequence are 5-haloacyls. In another embodiment, exactly seven or more U bases in the modified miR-506 nucleotide sequence are 5-haloucalyl. In certain embodiments, all of the U bases of the modified miR-506 nucleotide sequence are 5-halo lysyl, whether in its native portion and / or in the appended portion.

[0033] In one exemplary embodiment, the miR-506 nucleic acid composition of the present invention,

AU ^{F F} U ^{F F} CAGGAAGGU U GU ^{F F} U U ^{F F} ACU AA [SEQ ID NO. 15], wherein U ^F is a haloracil, specifically 5-fluorouracil.

[0034] In some embodiments, the 5-haloracil is, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil. In certain embodiments, the 5-haloracil is 5-fluorouracil, or a combination thereof.

[0035] The present invention also relates to formulations comprising modified microRNA compositions described in the present invention or combinations thereof. In certain embodiments, the formulation may comprise a pharmaceutical formulation comprising the nucleic acid composition described above and a pharmaceutically acceptable carrier.

[0036] In another aspect, the invention is directed to a method of treating cancer comprising administering to a subject an effective amount of one or more nucleic acid compositions described herein. In certain embodiments of the method of the invention, the nucleic acid composition comprises a modified miR-129, miR-15a, miR-192, miR-215, miR-140, miR-502 or miR-506 nucleotide sequences 1, 2, 3, 4, 5, 6 or more uracil nucleotide bases of the native (unmodified) nucleotide sequence were replaced with 5-haloracil. In certain embodiments, the methods of the invention comprise administering a nucleic acid composition of the invention to a subject having or with cancer, wherein the nucleic acid composition comprises a modified miR-129 or a modified miR-15a It is a nucleic acid. In certain embodiments of the present invention, the modified microRNA, administered,

^{CU F U F U F U F} U F GCGGU F CU F GGGCU F U F GC [SEQ ID NO. 4],

CU ^F U ^{F F F} GGGCU CUUUUUGCGGU GC [SEQ ID NO. 5],

^{F F} GGU AAU AGCAGCACAU ^{F F} U U U ^{F F F} GU G [SEQ ID NO.6],

GGU AAU ^{F F F F} U UAGCAGCACAU U ^{F F} GU G [SEQ ID NO. 7],

CAGU ^F GGUUUUACCCU ^F AUGGU ^F AG [SEQ ID NO. 9],

GACCU AU ^{F F F F} GAAU CU U GACAGCC ^F [SEQ ID NO. 11],

CCU U ^{F F F F} GCUAU AU CU GGGU ^{F F F} A GCU [SEQ ID NO. 13], and

AU ^{F F} U ^{F F} CAGGAAGGU U GU ^{F F} U U ^{F F} ACU AA [SEQ ID NO. 15]. ≪ / RTI >

[0037] In certain embodiments, the subject is a mammal. In another embodiment, the subject to be treated is a human, dog, horse, pig, mouse or rat. In certain embodiments, the subject is a human who has been diagnosed with cancer or has been confirmed to have a predisposition to develop cancer. In some embodiments, the cancer to be treated may be, for example, colon cancer, gastric cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer or uterine cancer. In certain embodiments, the methods of the invention treat a subject for colon cancer, pancreatic cancer, or breast cancer.

[0038] The data provided in the present invention surprisingly indicate that the increased amount of modified microRNAs described in the present invention when compared to known anticancer agents such as 5-FU alone in several different cancer models, including colon cancer, pancreatic cancer and lung cancer Show efficacy. For example, the present invention contemplates that the modified nucleic acid compositions described above are more stable than 5-FU, miR-15a, miR-129, miR-140, miR-192, miR-215, miR- , Or is substantially more potent at inhibiting cancer progression and tumor formation than the combination of native microRNAs corresponding to 5-FU.

[0039] Thus, the compositions and methods of the present invention provide an additional advantage of allowing low doses, which results in lowering toxicity and reducing side effects. Another important advantage seen by the nucleic acid compositions described above is that the compositions have greatly improved efficacy compared to the miR-140, miR-192, miR-215, miR-502 or miR- It has. Thus, in light of at least the above-mentioned advantages, the nucleic acid compositions disclosed in the present invention exhibit substantial progress in cancer treatment.

Brief Description of Drawings

[0040] Figs. A chemical representation of an exemplary modified microRNA nucleotide sequence of the invention. (a) a chemical representation of the miR-129 nucleotide sequence in which all U bases have been replaced with a halo lysyl (i.e., UF), as described in SEQ ID NO: 4. (b) a chemical representation of miR-129 having a U base in which only the non-seed portion of miR-129 has been replaced with a halo lacyl, as described in SEQ ID NO: 5; (c) (D) a miR-15a nucleotide sequence having a U base in which only the non-seed portion of miR-15a has been replaced with a halo lysyl, as described in SEQ ID NO: 7; Chemical description of 15a. (e) a chemical representation of the miR-140 nucleotide sequence in which a specific three U base has been replaced by a halo lactate, as described in SEQ ID NO: 9. (f) a chemical representation of the miR-192 nucleotide sequence in which a particular 5 U base has been replaced by a halo lactate, as described in SEQ ID NO: 11. (g) a chemical representation of the miR-502 nucleotide sequence in which a specific seven U base has been replaced with a halo lysyl, as described in SEQ ID NO: 13. (h) a chemical representation of the miR-506 nucleotide sequence in which all (ie, eight) U bases have been replaced by a halo lactate, as described in SEQ ID NO:

2a-c. Exemplary modified miR-129 nucleic acids enter cancer cells and effectively reduce target protein expression. (E2F3) specificity and performance of variant miR-129 (all U-bases replaced by 5-FU, 5-FU-miR-129) compared to that of control miRNA and unmodified miR- Showing graph. (b) Quantitative real-time PCR analysis showing that miR-129 mimic enters tumor cells. (c) miR-129 mimetics enter cancer cells and degrade TS-FdUMP much better than 5-FU alone.

) 및 이상 발현된 (ectopically expressed) 본연의 miR-129 (○)에 비하여, 모든 U 염기가 5-FU로 대체된 예시적인 변형된 miR-129 핵산 (모방체)(

)에 의하여 4 가지 상이한 대장암 세포주 (HCT116, RKO, SW480 및 SW620)에서 대장암 세포 증식이 억제됨을 보여주는 그래프. [0042] Figure 3. Non-specific control (negative control,

) And an exemplary modified miR-129 nucleic acid (mimetic) in which all U bases are replaced with 5-FU, compared to the ectopically expressed intrinsic miR-129 (O)

(HCT116, RKO, SW480 and SW620) by four different colorectal cancer cell lines.

[0043] Figure 4. Combination therapy with 5-FU and the modified microRNA composition of the present invention effectively inhibits cancer cell proliferation. (NC), native miR-129, 5-FU, exemplary modified miR-129 nucleic acid mimetics (5-FU-miR-129) of the invention and 5-FU and exemplary miR -129 A graphical comparison of colon cancer cell proliferation for cancer cells treated with a combination of (5-FU-miR-129 + 5-FU).

5a-b. Exemplary microRNA mimetics induce apoptosis in colon cancer cells and cause cell cycle arrest. (a) Cell death was quantitated by FITC-Annexin V apoptosis assays to show that the modified miR-129 nucleic acid compositions of the present invention in a number of different colorectal cancer cell lines are significantly more potent than the negative control, or the over expressed miR-129, And induce apoptosis of cancer cells at a high level. (b) flow cytometry analysis to reveal that the modified miR-129 nucleic acid composition of the invention (mimetic-1) increases the G1 cell cycle arrest at significantly higher levels than the negative control or over expressed miR-129 .

[0045] Figure 6. The modified microRNA nucleic acid composition of the present invention removes chemotherapy-resistant cancer stem cells. HCT116-derived colon cancer stem cells were treated with increasing concentrations of the exemplary modified miR-129 nucleic acid (O) or 5-FU (●) of the present invention. The results show that the modified miR-129 nucleic acid killed 5-FU resistant cancer stem cells in a dose-dependent manner.

[0046] Figure 7. In vivo systemic treatment with an exemplary modified miR-129 nucleic acid composition inhibits colon cancer metastasis without toxic side effects. The colon cancer metastatic mouse model was established by intravenous injection of metastatic human colorectal cancer cells. Two weeks after establishing the metastasis, 40 [mu] g of the modified miR-129 nucleic acid composition as described in SEQ ID NO: 4 was delivered by intravenous injection at a treatment frequency of 1 injection every other day for 2 weeks. While the exemplary modified miR-129 nucleic acid (mimic) could inhibit colorectal metastasis (right panel), the negative control miRNA (left panel) was ineffective. Mice treated with the modified miR-129 nucleic acid showed no toxicity.

[0047] Figures 8a-b. An exemplary anticancer activity of the modified microRNA of the second invention. (a) Representative western blots comparing the ability of unmodified miR-15a (miR-15a) and modified miR-15a nucleic acid composition (mimic-1) to regulate protein expression in colon cancer cells. Modified miR-15a (mimetic-1) as described in SEQ ID NO: 6 is capable of modulating miR-15a targets (YAP1, BMI-1, DCLK1 and ECL2) and degrading TS-FdUMP in colon cancer cells . (b) The ability of the modified miR-15a (mimetic -1) to inhibit the growth of colorectal cancer cells in three different colorectal cancer cell lines (HCT116, RKO, SW620) as compared to the unmodified miR-15a .

[0048] Figure 9. Cell cycle (s) for the control (negative), unmodified miR-15a (miR-15a) and exemplary modified miR-15a nucleic acid composition (mimetic-1) as described in SEQ ID NO: Graph showing control. As shown by the increase in the G1 / S ratio exhibited by the colon cancer cells expressing the modified miR-15a compared to the cells and negative control cells that overexpressed miR-15a, the modified miR-15a nucleic acid Induced cell cycle arrest compared to unmodified miR-15a.

[0049] Figure 10. Modified miR-15a expression reduces the ability of cancer stem cells to induce cancer cell colony formation. In colon cancer stem cells, the expression of unmodified miR-15a (miR-15a) inhibited cancer cell colony formation when compared to the ability of cancer stem cells to be provided using nonspecific control microRNAs (negative). Treatment with the exemplary modified miR-15a (5-FU-miR-15a) of the present invention completely prevented cancer cell colony formation.

[0050] Figure 11. Modified miR-15a is an in vivo effective anti-cancer agent. A colon cancer metastatic mouse model was established by intravenous injection of metastatic human colorectal cancer cells. Two weeks after establishing the metastasis, 40 [mu] g of the modified miR-15a nucleic acid composition as described in SEQ ID NO: 6 was delivered by intravenous injection at a treatment frequency of 1 injection every other day for 2 weeks. Exemplary modified miR-15a nucleic acids (mimetics) were able to inhibit colorectal metastasis, whereas negative control miRNAs (negative) were ineffective. Mice treated with the modified miR-15a nucleic acid showed no toxicity.

12a-d. Exemplary modified miR-15a and miR-129 mimetics of the present invention can be used to inhibit the growth of human (i. E., MiR-15a) or miR- (A549; c, d) and pancreatic cancer cells (Panc-1 (a); AsPC-1 (b)).

[0052] Figures 13a-b. Exemplary modified microRNAs of the present invention exhibit enhanced ability to inhibit human colon cancer cell proliferation. Additional exemplary modified microRNAs were tested for their ability to inhibit colon cancer cell proliferation in HCT116 human colon cancer cells. (a) It has been found that the exemplary modified miR-140 mimic described in SEQ ID NO: 9 is administered to human colon cancer cells and has an increased ability to inhibit the growth of colon cancer cells when compared to negative control microRNAs . (b) It has been found that the exemplary modified miR-192 mimic described in SEQ ID NO: 11 is administered to human colon cancer cells and has an increased ability to inhibit the growth of colon cancer cells when compared to negative control microRNAs .

[0053] Figures 14a-d. Exemplary modified microRNAs of the present invention exhibit enhanced ability to inhibit human pancreatic cancer and breast cancer cell proliferation. Additional exemplary modified microRNAs were tested for their ability to inhibit various types of human cancers by examining their effect on cancer cell proliferation. The exemplary modified miR-502 mimetics described by SEQ ID NO: 13 were administered to human pancreatic cancer cells (PANC1, a) and human breast cancer (A549, c) and compared to negative control microRNAs, The ability to inhibit cancer cell proliferation is increased. Another exemplary modified microRNA, the miR-506 mimetic described by SEQ ID NO: 15, is administered to human pancreatic cancer cells (PANC1, b) and human breast cancer (A549, d) and compared with negative control microRNAs , The ability to inhibit both types of cancer cell proliferation was found to increase.

DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention provides a nucleic acid composition comprising at least one hollow lacyl molecule. Surprisingly, without being bound by any one particular theory, the present invention provides a method for inhibiting the cancer, onset, progression and tumorigenesis of said microRNA by replacing the uracil nucleotide in the microRNA oligonucleotide sequence with 5-haloracil . Thus, the present invention provides various nucleic acid (e. G., MicroRNA) compositions having 5-Hollow lysyl molecules incorporated into their nucleic acid sequences and methods for using them. The present invention also provides a pharmaceutical composition comprising a formulation, for example a modified nucleic acid composition, and a method of treating cancer comprising administration of said pharmaceutical composition to a subject in need thereof.

Nucleic acid composition

The term "microRNA" or "miRNA" or "miR" refers to small non-coding ribose nucleic acid (RNA) molecules capable of modulating gene expression through interaction with messenger RNA molecules (mRNA) Quot; is used interchangeably to refer to < / RTI > Typically, microRNAs are comprised of nucleic acid sequences of about 19-25 nucleotides (bases) and are found in mammalian cells.

The term "modified microRNA", "modified miRNA", "modified miR" or "mimic" refers to a microRNA that differs from native or endogenous microRNA (unmodified microRNA) polynucleotides And are used interchangeably herein. More specifically, in the present invention, the modified microRNA differs from the unmodified or unmodified microRNA nucleic acid sequence by one or more bases. In some embodiments of the invention, the modified microRNA of the invention comprises one or more uracil (U) nucleotide bases replaced by 5-haloracils. In another embodiment, the modified microRNA comprises at least one uracil base substituted with additional nucleotides (i.e., adenine (A), cytosine (C), uracil (U) and guanine (G) do.

[0057] In one aspect of the invention, a modified microRNA nucleotide sequence having at least one uracil base (U, U base) replaced by a 5-halolactyl, such as 5-fluorouracil (5-FU) ≪ / RTI > is described. As discussed further herein, the nucleic acid compositions of the present invention are useful in the treatment of at least cancers, particularly colon cancer, pancreatic cancer, and breast cancer.

[0058] In some embodiments, the nucleic acid composition contains a modified nucleotide sequence by derivatizing one or more uracil nucleobases using a group that provides a similar effect as a halogen atom at the 5-position. In some embodiments, the group providing such a similar effect has a scale similar to a halogen atom in the weight or spatial dimension, such as 20, 30, 40, 50, 60, 70, 80, 90, / mol. < / RTI > In certain embodiments, the group that provides an effect similar to a halogen atom includes, for example, a methyl group, a trihalomethyl group (e.g., trifluoromethyl group), a pseudohalide (e.g., trifluoromethanesulfonate, cyano or cyanate ) Or a deuterium (D) atom. Groups providing an effect similar to a halogen atom may be present in the microRNA nucleotide sequence in the absence or in addition of the 5-halo lysyl base.

[0059] Also, in other embodiments, a group that provides an effect similar to a halogen atom can be located in the native (or seed) portion and / or the ancillary portion of the microRNA nucleotide sequence, Will be readily identified by those skilled in the art. In some embodiments, one or more (or all) of the above types of groups that provide an effect similar to a halogen atom are excluded from the modified miRNA nucleotide sequence. When all these alternative groups are excluded, only one or more halogen atoms are present as substituents at the 5-position of the uracil group of one or more of the microRNA nucleotide sequences.

[0060] In certain embodiments, the modified microRNA has one or more, or exactly one uracil, replaced with a 5-haloracil.

[0061] In some embodiments, the modified microRNA nucleotide sequence comprises 3, 4, 5, 6, 7, 8 or more uracil bases substituted with 5-haloracil.

[0062] In another embodiment, all of the uracil nucleotide bases of the modified mRNA were replaced with 5-haloracil.

[0063] In some embodiments, the 5-haloracil is, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil or 5-iodouracil. In certain embodiments, the 5-haloracil is 5-fluorouracil.

The term "miR-129" as used in the present invention means a synonym with "microRNA-129" or "miRNA-129" and refers to an oligonucleotide having the following nucleotide sequence:

CUUUUUGCGGUCUGGGCUUGC [SEQ ID NO. One],

Where C = cytosine, U = uracil and G = guanine base. The nucleotide sequence refers to the unmodified (i.e., "native") miR-129 sequence in the present invention, unless otherwise specified. miR-129 can also be referred to in the art as accession numbers MI0000252 and MIMAT0000242, hsa-miR-129 or hsa-miR-129-5p. MiR-129 is well known and studied in detail. For example, [J. Wu et al., Cell Cycle , (2010) 9: 9, 1809-1818. As is well known in the art, the miR-129 sequence may be modified to produce a "miR-129 mimetic ", which has a sequence that is altered from its original sequence but has a known function or activity . Unless otherwise stated, all these modified miR-129 compositions are considered to be within the scope of the term "miR-129 mimetic" as used herein.

[0065] Certain modified miR-129 nucleic acid sequences (mimetics) of the target nucleic acid sequence are those sequences of miR-129, such as CUUUUUGCGGUCUGGGCUUGC-UU [SEQ ID NO. Containing two U-bases (i.e., two U-containing nucleotides) covalently attached to the terminus of the nucleotide sequence [3]. In this sequence, the two terminal U bases continue or extend the original miR-129 sequence from 21 nucleotides to 23 nucleotides. In general, the miR-129 mimetics contain no more than 1, 2, 3, 4 or 5 additional bases (i.e. as additional nucleotides) covalently attached to the parent sequence of miR-129, The base may be selected independently from C, U, G, and C, or the additional base may be exclusively U. Typically, the miR-129 is used in single-stranded form, but a double-stranded version is also contemplated in the present invention.

[0066] In one embodiment, the present invention relates to a nucleic acid composition comprising a modified miR-129 nucleotide sequence by replacing one or more of its uracil nucleobase (ie, U base) with a 5-haloracil , I.e. wherein at least one of the U bases in the miR-129 sequence is a 5-halo lysyl in its native and / or ancillary parts. The 5-haloracil can be, for example, 5-fluorouracil, 5-chloroauracyl, 5-bromouracil or 5-iodouracil.

[0067] In a first set of embodiments, exactly one of the U bases in the miR-129 sequence is 5-Hollowacyl. In a second set of embodiments, exactly two or more U bases in the miR-129 nucleotide sequence are 5-halolactyl. In a third set of embodiments, exactly three or more U bases in the miR-129 nucleotide sequence are 5-haloacyls. In a fourth embodiment, exactly four or more U bases in the miR-129 nucleotide sequence are 5-haloracil. In a fifth embodiment, exactly five or more U bases in the miR-129 nucleotide sequence are 5-haloracil. In the sixth embodiment, all of the U bases of the miR-129 nucleotide sequence are 5-halo lysyl, whether in its native part and / or in the appended part.

[0068] In certain embodiments, the nucleic acid composition of the present invention comprises a nucleotide sequence of SEQ ID NO. Having a micro-RNA nucleotide sequence variants of the ^{CU F U F U F U F} U F GCGGU F CU F GGGCU F U F GC described in 4, wherein U ^F is a hollow rasil uracil, and specifically 5-fluorouracil.

The U bases replaced with 5-haloracil in the miR-129 sequence may be located in the unmodified portion of the miR-129 sequence presented above, or in the case of the miR-129 mimic, As indicated, it may be located in one or more U bases that are covalently attached to the native miR-129. In another embodiment, GUUUUUGC, which is the seed portion of the native miR-129 nucleotide sequence, remains unmodified to the 5-haloracil while one of the U bases remaining in the remainder of the miR-129 nucleotide sequence (Or all) are replaced by an equivalent number of 5-haloracils.

[0070] For example, in certain embodiments, the nucleic acid composition of the present invention comprises SEQ ID NO. CUUUUUGCGGU CU ^{F F F} GGGCU gatneunde a modified micro-RNA nucleotide sequence of U ^F GC, whereby U ^F is described as a hollow rasil 5 is uracil, and specifically 5-fluorouracil.

[0071] In another embodiment, the nucleic acid composition comprises a modified, miR-129 nucleotide sequence modified by derivatizing one or more of the uracil (U) nucleobases at the 5-position to a group providing a halogen atom- do. In some embodiments, the group providing such a similar effect has a scale similar to a halogen atom in the weight or spatial dimension, such as 20, 30, 40, 50, 60, 70, 80, 90, / mol. < / RTI > Such a group providing an effect similar to a halogen atom may be, for example, a methyl group, a trihalomethyl group (e.g., trifluoromethyl group), a pseudohalide (e.g., trifluoromethanesulfonate, cyano or cyanate) ) Atoms. Such a group which provides an effect similar to a halogen atom may be present in the absence or addition of a 5-haloracil base in the miR-129 nucleotide sequence. In addition, the group that provides an effect similar to a halogen atom may be located in the native (or seed) and / or ancillary portions of the miR-129 nucleotide sequence. In some embodiments, one or more (or all) of the above types of groups providing an effect similar to a halogen atom are excluded from the miR-129 nucleotide sequence. When all such groups of alternatives are excluded, only one or more halogen atoms are present as substituents at the 5-position of one or more uracil groups in the miR-129 nucleotide sequence.

[0072] In another exemplary embodiment, the invention relates to a nucleic acid composition comprising a modified miR-15a nucleotide sequence. In some embodiments, the miR-15a nucleotide sequence has been modified by replacing one or more of the U bases with a 5-haloracil.

The term "miR-15a" used in the present invention means synonymous with "microRNA-15a" or "miRNA-15a" and refers to an oligonucleotide having the following nucleotide sequence:

UAGCAGCACAUAAUGGUUUGUG [SEQ ID NO. 2],

Where A = adenine, C = cytosine, U = uracil and G = guanine base. The nucleotide sequence refers to the unmodified (i.e., "native") miR-15a sequence in the present invention, unless otherwise specified. miR-15a can also be referred to in this field as accession number MI0000069, hsa-miR-15a or hsa-miR-15a-5p. MiR-15a is well known and studied in detail. See, 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]. Methods for generating miR-15a mimetics, as described above for miR-129 mimetics, are known to those skilled in the art. Unless otherwise stated, all such modified miR-15a forms are considered to be within the scope of the term "miR-15a mimetic" as used herein in the present invention.

In general, the modified miR-15a (ie, miR-15a mimetics) comprises no more than 1, 2, 3, 4 or 5 additional bases covalently attached to the original sequence of miR-15a , Said additional base being selected independently from C, U, G, and C, or said additional base may be exclusively U. Typically, the miR-15a is used in single-stranded form, but a double-stranded version is also contemplated in the present invention.

[0075] In some embodiments, at least one of the U bases in the miR-15a sequence is 5-haloulacyl, in its native and / or ancillary portions. The 5-haloracil can be, for example, 5-fluorouracil, 5-chloroauracyl, 5-bromouracil or 5-iodouracil.

[0076] In a first set of embodiments, exactly one of the U bases in the miR-15a sequence is 5-haloracil. In a second set of embodiments, exactly two or more U bases in the miR-15a sequence are 5-haloracil. In another set of embodiments, exactly three or more U bases in the miR-15a oligonucleotide sequence are 5-haloacyls. In another embodiment, exactly four or more U bases in the miR-15a sequence are 5-haloracil. In some embodiments, exactly five or more U bases in the miR-15a sequence are 5-haloracil. In yet another embodiment, exactly six or more U bases in the miR-15a sequence are 5-haloracil. In certain embodiments, all of the U bases of the miR-15a sequence are 5-halo lysyl, whether in its native portion and / or in the appended portion.

[0077] In one embodiment, the nucleic acid compositions of the present invention AGCAGCACAU ^F AAU ^F GGU ^{F F} U U U ^{F F F} GU G [SEQ ID NO. 6], wherein U ^F is a haloracil, specifically 5-fluorouracil.

The U bases replaced with 5-haloracil in the miR-15a sequence can be located in the unmodified portion of the miR-15a sequence presented above, or in the case of the miR-15a mimic, As indicated, it may be located in one or more u bases that are covalently attached to the native miR-15a.

[0079] In another embodiment, the UAGCAGCA, which is the seed portion of the native miR-15a nucleotide sequence, remains unmodified to the 5-haloracil while the U base remaining in the remainder of the miR-15a nucleotide sequence (Or all) are replaced by 5-haloracil.

[0080] In a particular embodiment, the nucleic acid compositions of the present invention ^{UAGCAGCACAU F AAU F GGU F U F} U F GU F G [SEQ ID NO. 7], wherein U ^F is a halo lysyl, in particular 5-fluorouracil.

In certain embodiments, the nucleic acid composition comprises a modified miR-15a nucleotide sequence modified by derivatizing at least one of the uracil (U) nucleobase at the 5-position to a group providing a halogen atom-like effect do. In some embodiments, the group providing such a similar effect has a scale similar to a halogen atom in the weight or spatial dimension, such as 20, 30, 40, 50, 60, 70, 80, 90, / mol. < / RTI > Such a group providing an effect similar to a halogen atom may be, for example, a methyl group, a trihalomethyl group (e.g., trifluoromethyl group), a pseudohalide (e.g., trifluoromethanesulfonate, cyano or cyanate) ) Atoms. Such a group providing an effect similar to a halogen atom may be present in the absence or addition of a 5-haloracil base in the miR-15a nucleotide sequence. In addition, the group that provides an effect similar to a halogen atom can be located in the native (or seed) portion and / or the ancillary portion of the miR-15a nucleotide sequence.

[0082] In some embodiments, one or more (or all) of the above types of groups providing an effect similar to a halogen atom are excluded from the miR-15a nucleotide sequence. Where all such groups of alternatives are excluded, only one or more halogen atoms are present as substituents at the 5-position of one or more uracil groups in the miR-15a nucleotide sequence.

[0083] In another exemplary embodiment, the present invention is directed to a nucleic acid composition comprising a modified miR-140 nucleotide sequence. In some embodiments, the miR-140 nucleotide sequence has been modified by replacing one or more of the U bases with a 5-haloracil.

As used herein, the term "miR-140" refers to an oligonucleotide having the following nucleotide sequence, which is synonymous with "microRNA-140" or "miRNA-140"

CAGUGGUUUUCCCCUAUGGUAG [SEQ ID NO. 8],

Where A = adenine, C = cytosine, U = uracil and G = guanine base. The nucleotide sequence refers to the unmodified (i.e., "native") miR-140 sequence in the present invention, unless otherwise specified. miR-140 can also be referred to as accession number NT_010498, or as miR base accession number MI0000456. MiR-140 is well known and studied in detail. See, e.g., Zhai, H. et al., Oncotarget . (2015) 6: 19735-46]. Methods for generating miR-140 mimetics, as described above for exemplary mimics miR-129 and miR-15a, are known to those of skill in the art. Unless otherwise stated, all these modified miR-140 forms are considered to be within the scope of the term "miR-140 mimetic" as used herein in the present invention.

In general, a modified miR-140 nucleic acid (ie, a miR-140 mimetic) can contain no more than 1, 2, 3, 4, or 5 additional bases covalently attached to the parent miR-140 sequence Wherein the additional base is selected independently from C, U, G and C, or the additional base may be exclusively U. < RTI ID = 0.0 > Typically, the miR-140 mimetics are used in single-stranded form, although double-stranded versions are also contemplated in the present invention.

[0086] In some embodiments, at least one of the U bases in the miR-140 sequence is 5-haloulacyl, in its native and / or ancillary portions. The 5-haloracil can be, for example, 5-fluorouracil, 5-chloroauracyl, 5-bromouracil or 5-iodouracil.

[0087] In a first set of embodiments, exactly one of the U bases in the miR-140 mimetic sequence is a 5-halolactyl. In a second set of embodiments, exactly two or more of the U bases in the miR-140 sequence are 5-haloracil. In another set of embodiments, exactly three or more U bases in the miR-140 oligonucleotide sequence are 5-haloracil. In another embodiment, exactly four or more U bases in the miR-140 sequence are 5-haloracil. In some embodiments, exactly five or more U bases in the miR-140 mimetic sequence are 5-halo lysyl. In yet another embodiment, exactly six or more U bases in the miR-140 mimetic sequence are 5-haloracil. In certain embodiments, all of the U bases of the miR-140 sequence are 5-halo lysyl, whether in the intrinsic portion and / or in the appended portion.

[0088] In one exemplary embodiment, the nucleic acid composition of the invention comprises CAGU ^F GGUUUUACCCU ^F AUGGU ^F AG [SEQ ID NO. 9], wherein U ^F is a halo lactyl, specifically 5-fluorouracil.

U bases that have been replaced with 5-haloracil in the miR-140 mimetic sequence can be located in the unmodified portion of the miR-140 sequence presented above, or, as indicated above, the native miR- Lt; RTI ID = 0.0 > U < / RTI >

[0090] In another embodiment, the seed portion of the native miR-140 nucleotide sequence remains unmodified to the 5-haloracil, while the remaining portion of the U bases remaining in the remainder of the miR-140 nucleotide sequence One or more (or all) is replaced by a 5-haloracil.

[0091] In another exemplary embodiment, the present invention relates to a nucleic acid composition comprising a modified miR-192 nucleotide sequence. In some embodiments, the miR-192 nucleotide sequence has been modified by replacing one or more of the U bases with a 5-haloracil.

The term "miR-192" as used in the present invention means a synonym with "microRNA-192", "miRNA-192", "microRNA-215", "miR215" Refers to oligonucleotides having the following nucleotide sequences:

CUGACCUAUGAAUUGACAGCC [SEQ ID NO. 10]

Where A = adenine, C = cytosine, U = uracil and G = guanine base. The nucleotide sequence refers to the unmodified (i.e., "native") miR-192 sequence in the present invention, unless otherwise specified. miR-192 may also be referred to as hsa-mir-192, has-mir-215, or miR base accession number MI0000234, or MIMAT0000222. MiR-192 is well known and studied in detail. See, e.g., Song, B. et al., Clin. Cancer Res . (2008), 14: 8080-8086, and Song, B. et al., Mol . Cancer . (2010), 9: 96 pp. 1476-4598]. Methods for generating miR-192 mimetics, as described above for the exemplary mimics miR-129, miR-140 and miR-15a, are known to those of skill in the art. Unless otherwise indicated, all such modified miR-192 nucleic acid forms are considered to be within the scope of the term "miR-192 mimic" as used herein in the present invention.

In general, a modified miR-192 (ie, a miR-192 mimetic) comprises 1, 2, 3, 4 or 5 additional bases covalently attached to the parent sequence of miR-192 , Said additional base being selected independently from C, U, G, and C, or said additional base may be exclusively U. Typically, the miR-192 mimetics are used in single-stranded form, although double-stranded versions are also contemplated in the present invention.

[0094] In some embodiments, at least one of the U bases in the miR-192 or miR-215 sequence is 5-haloracil in its native portion and / or an accessory portion. The 5-haloracil can be, for example, 5-fluorouracil, 5-chloroauracyl, 5-bromouracil or 5-iodouracil.

[0095] In another set of embodiments, exactly one of the U bases in the miR-192 mimetic sequence is a 5-halolactyl. In a second set of embodiments, exactly two or more of the U bases in the miR-192 sequence are 5-haloracil. In another set of embodiments, exactly three or more U bases in the miR-192 oligonucleotide sequence are 5-halolactyl. In another embodiment, exactly four or more U bases in the miR-192 sequence are 5-haloracil. In certain embodiments, all of the U bases of the miR-192 sequence are 5-halo lysyl, whether in its native portion and / or in the appended portion.

[0096] In one illustrative embodiment, the nucleic acid compositions of the invention ^{CU F GACCU F AU F GAAU F} U F GACAGCC [SEQ ID NO. 11], wherein U ^F is a halolacyl, specifically 5-fluorouracil.

The U bases replaced with 5-haloracil in the miR-192 mimetic sequence can be located in the unmodified portion of the miR-192 sequence presented above, or, as indicated above, the native miR- Lt; RTI ID = 0.0 > 192 < / RTI > sequence.

[0098] In another embodiment, the seed portion of the native miR-192 nucleotide sequence remains unmodified to the 5-haloracil, while the remaining portion of the U bases remaining in the remainder of the miR-192 nucleotide sequence One or more (or all) is replaced by a 5-haloracil or combinations thereof.

[0099] In another exemplary embodiment, the present invention relates to a nucleic acid composition comprising a modified miR-502 nucleotide sequence. In some embodiments, the miR-502 nucleotide sequence has been modified by replacing one or more of the U bases with a 5-haloracil.

The term "miR-502" as used in the present invention means a synonym with "microRNA-502" or "miRNA-502" and refers to an oligonucleotide having the following nucleotide sequence:

AUCCUUGCUAUCUGGGUGCUA [SEQ ID NO. 12],

Where A = adenine, C = cytosine, U = uracil and G = guanine base. The nucleotide sequence refers to the unmodified (i.e., "native") miR-502 sequence in the present invention, unless otherwise specified. miR-502 can also be referred to as hsa-mir-502, or as miR base accession number MI0003186, or MIMAT0002873. MiR-502 is well known and studied in detail. For example, [Zhai, H, et al., Oncogene . (2013), 32: 12 pp. 1570-1579]. Methods for generating miR-502 mimetics, as described above for the exemplary mimics miR-129, miR-140, miR-192 and miR-15a, are known to those of skill in the art. Unless otherwise stated, all such modified miR-502 nucleic acid forms are considered to be within the scope of the term "miR-502 mimetic" as used herein in the present invention.

In general, a modified miR-502 (ie, a miR-502 mimetic) comprises 1, 2, 3, 4 or 5 additional bases covalently attached to the parent sequence of miR-502 , Said additional base being selected independently from C, U, G, and C, or said additional base may be exclusively U. Typically, the miR-502 mimetics are used in single-stranded form, although double-stranded versions are also contemplated in the present invention.

[00102] In some embodiments, at least one of the U bases in the miR-502 sequence is 5-haloracil in its native portion and / or in the appendant portion. The 5-haloracil can be, for example, 5-fluorouracil, 5-chloroauracyl, 5-bromouracil or 5-iodouracil.

[00103] In another set of embodiments, exactly one of the U bases in the miR-502 mimetic sequence is 5-Hollowlaic. In a second set of embodiments, exactly two or more of the U bases in the miR-502 sequence are 5-haloracil. In another set of embodiments, exactly three or more U bases in the miR-502 oligonucleotide sequence are 5-halolactyl. In another embodiment, exactly four or more U bases in the miR-502 sequence are 5-halolactyl. In another embodiment, exactly five or more U bases in the miR-502 sequence are 5-haloracil. In another embodiment, exactly six or more U bases in the miR-502 sequence are 5-haloracil. In another embodiment, exactly seven or more U bases in the miR-502 sequence are 5-haloracil. In certain embodiments, all of the U bases of the miR-502 sequence are 5-halo lysyl, whether in the native portion and / or in the appended portion.

[00 104] In one illustrative embodiment, the nucleic acid compositions of the present invention CCU ^{F F} U ^F GCUAU ^F AU CU GGGU ^{F F F} A GCU [SEQ ID NO. 13], wherein U ^F is a halo lactyl, specifically 5-fluorouracil.

U bases replaced by 5-haloracil in the miR-502 mimic sequence can be located in the unmodified portion of the miR-502 sequence presented above or, as indicated above, Lt; RTI ID = 0.0 > U-bases < / RTI > covalently attached to the -502 sequence.

[00106] In another embodiment, the seed portion of the native miR-502 nucleotide sequence remains unmodified to the 5-haloracil, while the remaining portion of the U bases remaining in the remainder of the miR-502 nucleotide sequence One or more (or all) are replaced by 5-halo lysyl or a combination thereof.

[00107] In another exemplary embodiment, the invention relates to a nucleic acid composition comprising a modified miR-506 nucleotide sequence. In some embodiments, the miR-506 nucleotide sequence has been modified by replacing one or more of the U bases with a 5-haloracil.

The term "miR-506" as used in the present invention means a synonym with "microRNA-506" or "miRNA-506" and refers to an oligonucleotide having the following nucleotide sequence:

UAUUCAGGAAGGUGUUACUUAA [SEQ ID NO. 14],

Where A = adenine, C = cytosine, U = uracil and G = guanine base. The nucleotide sequence refers to the unmodified (i.e., "native") miR-506 sequence in the present invention, unless otherwise specified. miR-506 may also be referred to as hsa-mir-506, or as miR base accession number MI0003193, or MIMAT0022701. MiR-506 is well known and studied in detail. See, e.g., Li, J, et al., Oncotarget . (2016), 7:38 pp. 62778-62788, and Li, J. et al., Oncogene. (2016) 35 pp. 5501-5514]. Methods for generating miR-506 mimetics, as described above for the exemplary mimics miR-129, miR-140, miR-502, miR-192 and miR-15a, are well known to those skilled in the art will be. Unless otherwise stated, all such modified miR-506 nucleic acid forms are considered to be within the scope of the term "miR-506 mimetic" as used herein in the present invention.

Generally, a modified miR-506 (ie, a miR-506 mimetic) contains one, two, three, four, or five additional bases that are covalently attached to the original sequence of miR-506 , Said additional base being selected independently from C, U, G, and C, or said additional base may be exclusively U. Typically, the miR-506 mimetics are used in single-stranded form, although double-stranded versions are also contemplated in the present invention.

[00110] In some embodiments, at least one of the U bases in the miR-506 sequence is 5-haloracil in its native and / or ancillary portions. The 5-haloracil can be, for example, 5-fluorouracil, 5-chloroauracyl, 5-bromouracil or 5-iodouracil.

[00111] In yet another set of embodiments, exactly one of the U bases in the miR-506 mimetic sequence is 5-Hollowacil. In a second set of embodiments, exactly two or more U bases in the miR-506 sequence are 5-halolactyl. In another set of embodiments, exactly three or more U bases in the miR-506 oligonucleotide sequence are 5-haloracil. In other embodiments, exactly four or more U bases in the miR-506 sequence are 5-halolactyl. In another embodiment, exactly five or more U bases in the miR-506 sequence are 5-haloracil. In another embodiment, exactly six or more U bases in the miR-506 sequence are 5-haloracil. In other embodiments, exactly seven or more U bases in the miR-506 sequence are 5-haloracil. In certain embodiments, all of the U bases of the miR-506 sequence are 5-halo lysyl, whether in the intrinsic portion and / or in the appended portion.

[00 112] In one illustrative embodiment, the nucleic acid compositions of the present invention AU ^{F F} U ^F CAGGAAGGU ^F U ^F U ^F GU ACU ^F U ^F AA [SEQ ID NO. 15], wherein U ^F is a halo lacyl, specifically 5-fluorouracil.

U bases that have been replaced with 5-haloracil in the miR-506 mimetic sequence can be located in the unmodified portion of the miR-506 sequence presented above, or, as indicated above, the native miR- RTI ID = 0.0 > U < / RTI > bases that are covalently attached to the 506 sequence.

[00114] In another embodiment, the seed portion of the native miR-506 nucleotide sequence remains unmodified to the 5-haloracil, while the remaining portion of the U bases remaining in the remainder of the miR-506 nucleotide sequence One or more (or all) is replaced by a 5-haloracil or combinations thereof.

[00115] The modified microRNA nucleic acid compositions described in the present invention can be synthesized using any known method for synthesizing nucleic acids. In certain embodiments, the nucleic acid composition is prepared by any well known process using automated oligonucleotide synthesis, such as phosphoramidite chemistry. In order to introduce one or more 5-halo lysyl bases into a modified miR sequence (e.g., a miR-15a sequence, a miR-140 sequence, a miR-192 sequence, a miR-502 sequence, a miR-506 sequence, or a miR- 5-Hollow lysyl nucleoside phosphoramidite is included as a precursor base with a phosphoramidite derivative of a nucleoside containing natural bases (e.g., A, U, G and C) .

[00116] In some embodiments, the nucleic acid compositions of the present invention may be used biosynthetically, for example, by in vitro RNA transcription from plasmids, PCR fragments or synthetic DNA templates, or recombinant (in vivo) RNA expression Method. ≪ / RTI > See, for example, CM Dunham et al., Nature Methods , (2007) 4 (7), pp. 547-548]. The microRNA sequences (eg, miR-15a sequence, miR-140 sequence, miR-192 sequence, miR-502 sequence, miR-506 sequence or miR-129 sequence) can be obtained by techniques well known in the art, Can be further modified chemically by being functionalized using, for example, polyethylene glycol (PEG) or a hydrocarbon or targeting agent, especially a cancer cell targeting agent such as folic acid. To include these groups, a reactive group (e.g., amino, aldehyde, thiol or carboxylate group) that can be used to attach the desired functional group may first be included in the oligonucleotide sequence. Such reactive or functional groups may be incorporated into as-produced nucleic acid sequences, but the reactive or functional groups may be selected from the group consisting of non-nucleoside phosphoramidites containing reactive groups or reactive Can be included more easily by using automated oligonucleotide synthesis involving precursor groups.

Modified nucleic acid formulations

[00117] In another aspect, the invention is directed to formulations of modified nucleic acid compositions described herein. For example, the nucleic acid compositions of the present invention may be formulated for pharmaceutical use. In certain embodiments, the formulations are pharmaceutical compositions containing the nucleic acid compositions described herein and a pharmaceutically acceptable carrier. In other embodiments, formulations of the present invention comprise modified miR-129 nucleic acid, modified miR-15a nucleic acid, modified miR-140 nucleic acid, modified miR-192 nucleic acid, modified miR-502, Nucleic acid or a combination thereof and a pharmaceutically acceptable carrier. More specifically, the modified microRNA nucleic acid described by the nucleotide sequence below can be formulated for pharmaceutical use and use; ^{CU F U F U F U F} U F GCGGU F CU F GGGCU F U F GC [SEQ ID NO. 4], CUUUUUGCGGU CU ^{F F F} U ^F GGGCU GC [SEQ ID NO. 5], U ^{F F} AGCAGCACAU GGU AAU ^{F F} U U ^{F F F} GU G [SEQ ID NO.6], UAGCAGCACAU ^F GGU AAU ^{F F} U U ^{F F F} GU G [SEQ ID NO. 7], CAGU ^F GGUUUUACCCU ^F AUGGU ^F AG [SEQ ID NO. 9], CU ^F GACCU AU ^{F F F} U ^F GAAU GACAGCC [SEQ ID NO. 11], CCU AU ^{F F} U ^{F F} GCUAU CU GGGU ^{F F F} A GCU [SEQ ID NO. 13], and AU ^{F F} U ^F CAGGAAGGU ^F U ^F U ^F GU ACU ^F U ^F AA [SEQ ID NO. 15].

[00118] The term "pharmaceutically acceptable carrier" is used herein synonymously with pharmaceutically acceptable diluents, vehicles or excipients. Depending on the type of pharmaceutical composition and the intended mode of administration, the nucleic acid composition may be dissolved or suspended in the pharmaceutically acceptable carrier (e.g., as an emulsion). Pharmaceutically acceptable carriers may be liquid or solid compounds, materials, compositions and / or dosage forms suitable for use in contact with the tissue of a subject, within the scope of sound medical judgment. The carrier should be "acceptable" in the sense that it is not deleterious to the subject to which it is provided and should be compatible with the other ingredients of the formulation, i.e. not alter biological or chemical function.

[00119] Some of the materials capable of functioning as a pharmaceutically acceptable carrier include, but are not limited to: sugars such as lactose, glucose and sucrose; Starches such as corn starch and potato starch; Cellulose and derivatives thereof such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; gelatin; talc; Wax; 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; Buffers; water; Isotonic saline; pH buffer solution; And other non-toxic compatible materials used in pharmaceutical formulations. The pharmaceutically acceptable carrier may also comprise a manufacturing aid (e.g., a lubricant, talcum magnesium, calcium or zinc stearate or stearic acid), a solvent or an encapsulating material. If desired, certain sweetening and / or flavoring agents and / or coloring agents may be added. In another suitable excipient, standard pharmaceutical materials, such as [ "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19 th Ed. Mack Publishing Company, Easton, Pa., (1995).

[00120] In some embodiments, the pharmaceutically acceptable carrier may comprise a diluent which increases the volume of the solid pharmaceutical composition and makes the pharmaceutical dosage form easier for the patient and caregiver to handle. A diluent for the solid compositions, for example, microcrystalline cellulose (e.g., Avicel ^®), finely divided cellulose (microfine cellulose), lactose, starch, pregelatinized 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.

[00121] The nucleic acid compositions of the present invention may be formulated into compositions and dosage forms according to methods known in the art. In certain embodiments, the formulated compositions can be formulated specifically for administration in solid or liquid form, including those adapted for the following administrations: (1) oral administration such as 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 parenteral administration by subcutaneous, intramuscular or intravenous injection as a sterile solution or suspension; (3) topical application such as creams, ointments or sprays applied to topical applications such as skin, lungs or mucous membranes; Or (4) into the vagina or rectum as, for example, a pessary, cream or foam; (5) buccally or sublingually; (6) administration by eye; (7) Percutaneous administration; Or (8) administered intolerably.

[00122] In some embodiments, the formulations of the present invention comprise a solid pharmaceutical formulation compressed into a dosage form such as a tablet, which function helps to bind the active ingredient and other excipients together after compression, . ≪ / RTI > Binders for solid pharmaceutical compositions include, but are not limited to, acacia, alginic acid, carbomer (e.g., carbopol), carboxymethylcellulose sodium, dextrin, ethylcellulose, gelatin, guar gum, hydrogenated vegetable oil, hydroxyethylcellulose, hydroxypropylcellulose For example, Klucel ^®), hydroxypropyl methylcellulose (e.g., Methocel ^®), liquid glucose, magnesium aluminum silicate, maltodextrin, methylcellulose, polymethacrylates, povidone (Kollidon ^®, Plasdone ^®), arc Mars starch, sodium Alginate and starch.

[00123] The dissolution rate of the compressed solid pharmaceutical composition in the gastrointestinal tract of a subject can be increased by adding a disintegrant to the composition. Examples of disintegrants include alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium (e.g. Ac-Di-Sol ^® , Primellose ^® ), colloidal silicon dioxide, croscarmellose sodium, crospovidone (eg Kollidon ^® , Polyplasdone ^® ), guar gum, may be mentioned magnesium aluminum silicate, methylcellulose, microcrystalline cellulose, polacrilin potassium, powdered cellulose, starch arc Mars, see sodium carbonate, sodium starch glycolate (e.g., Explotab ^®) and starch.

[00124] Thus, in certain embodiments, a glidant may be added to the formulation to improve the flowability of the non-compressed solid formulation and to improve the accuracy of administration. Excipients that can serve as lubricants include colloidal silicon dioxide, magnesium trisilicate, powdered cellulose, starch, talc and tribasic calcium phosphate.

[00125] When a dosage form such as a tablet is prepared by compression of a powder composition, the composition is subjected to pressure from punches and dyes. Some excipients and active ingredients tend to adhere to the surface of the punches and dyes, which can cause pitting or other surface heterogeneity in the product. Lubricants can be added to the composition to reduce adhesion and facilitate release of product from the dye. Examples of lubricants include lubricants such as magnesium stearate, calcium stearate, glyceryl monostearate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil, mineral oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, Lactate, stearic acid, talc and zinc stearate.

[00126] Formulated pharmaceutical compositions for tabletting or capsule filling may be prepared by wet granulation. In wet granulation, some or all of the active ingredients and excipients in powder form are combined and then further mixed in the presence of liquid, typically water, which causes the powder to agglomerate into granules. The granules are screened and / or ground, dried and then screened and / or ground to the desired particle size. The granules may then be tableted, or other excipients such as lubricants and / or lubricants may be added prior to tableting. The tableting composition may be prepared conventionally by dry blending. For example, the combined composition of the active ingredient and the excipient may be compressed into a slug or sheet and then pulverized into compressed granules.

[00127] In another embodiment, as an alternative to dry granulation, the compounded composition may be compressed directly into a compressed dosage form using direct compression techniques. Direct compression produces more uniform tablets without granules. Particularly suitable excipients for direct compression tableting include microcrystalline cellulose, spray dried lactose, dicalcium phosphate dihydrate and colloidal silica. In direct compression tabletting, the proper use of these and other excipients is known to those skilled in the art, particularly with experience and knowledge of formulation attempts for direct compression tableting. Capsule filling may include any of the above-described formulations and granules described for tabletting; They do not undergo a final purification step.

[00128] In the liquid pharmaceutical compositions of the present invention, the medicament and any other solid excipient are dissolved or suspended in a liquid carrier, such as water, distilled water for injection, vegetable oil, alcohol, polyethylene glycol, propylene glycol or glycerin. The liquid pharmaceutical composition may contain an emulsifying agent to uniformly disperse the active ingredient or other excipient that is not soluble in the liquid carrier throughout the composition. The liquid formulation may be used as an injectable, enteric or softener type of formulation. Examples of emulsifiers that may be useful in the liquid composition of the present invention include gelatin, egg yolk, casein, cholesterol, acacia, tragacanth, chondrus, pectin, methylcellulose, carbomer, cetostearyl alcohol, Alcohol.

[00129] In some embodiments, the liquid pharmaceutical compositions of the present invention may also contain a viscosity enhancing agent to improve the oral sensation of the product and / or to coat the gastrointestinal lining. These preparations can be prepared by conventional means such as, for example, acacia, alginic acid bentonite, carbomer, carboxymethylcellulose calcium or sodium, cetostearyl alcohol, methylcellulose, ethylcellulose, gelatin guar gum, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, Dextrin, polyvinyl alcohol, povidone, propylene carbonate, propylene glycol alginate, sodium alginate, sodium starch glycolate, starch tragacanth and xanthan gum. In other embodiments, the liquid compositions of the present invention may also contain buffering agents such as, for example, gluconic acid, lactic acid, citric acid or acetic acid, sodium gluconate, sodium lactate, sodium citrate or sodium acetate.

[00130] Sweetening agents such as sorbitol, saccharin, sodium saccharin, sucrose, aspartame, fructose, mannitol, and phosgene may be added to certain formulations of the present invention to improve taste. Flavoring agents and flavor enhancers can make the dosage form feel more tasty to the patient. Common flavoring and flavor enhancers for pharmaceutical products that may be included in the compositions of the present invention include, but are not limited to, maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol and tartaric acid.

Preservatives and chelating agents such as alcohols, sodium benzoate, butylated hydroxytoluene, butylated hydroxyanisole and ethylenediaminetetraacetic acid can be added to safe levels to improve storage stability. The solid and liquid compositions may also be dyed using any pharmaceutically acceptable coloring agent to improve their appearance and / or facilitate patient identification for product and unit dosage levels.

[00132] Dosage forms of the present invention may be capsules containing such compositions, such as powdered or granulated solid compositions of the present invention, in a hard or soft shell. The shell may be made of gelatin and may optionally contain plasticizers such as glycerin and sorbitol, and opacifying agents or coloring agents.

How to treat cancer

[00133] As described above, the modified microRNA nucleic acid composition and its formulations of the present invention are useful in the treatment of unexpected (or unexpected) and / or unexpected Showing an exceptional anticancer activity. Therefore, another aspect of the present invention provides a method of treating cancer in a mammal by administering to the mammal an effective amount of at least one of the modified microRNA nucleic acid compositions of the invention or a formulation thereof.

[00134] As shown in Figures 2A and 8A, exemplary modified microRNA nucleic acids of the present invention, i.e., modified miR-15a and modified MiR-129, inhibit BCL2 expression and activity in cancer cells of a subject , Which results in increasing the amount of available pro-apoptotic proteins that ultimately increases amp eso killing. miR-129 directly targets BCL2, for example, and modulates apoptosis by affecting other important cell death-related proteins. 2A also shows that miR-129 decreases the expression of E2F3, a transcription factor protein that regulates cell cycle progression and decreases the expression or activity of the protein level of thymidylate synthetase (TS), thus reducing its activity , Which results in increased cell proliferation and increased efficacy of chemotherapeutic agents.

[00135] Other exemplary microRNAs such as the modified miR-506, miR-140, miR-192 and miR-502 may also be used to inhibit cancer cell proliferation and apoptosis of cancer cells as shown in Figures 13a-b and 14a- .

[00136] In fact, Figures 7 and 11 show that intravenous treatment with two exemplary modified microRNAs of the invention (e.g., modified miR-129 and modified miR-15a) inhibit tumor growth and development And that it effectively treats colon cancer.

[00137] Generally, methods of treating cancer of the present invention comprise administering a nucleic acid composition of the invention (eg, modified miR-129 nucleic acid, modified miR-15a nucleic acid, modified miR-140 nucleic acid, , Modified miR-502, modified miR-506 nucleic acid, or a combination thereof) to a subject. In certain embodiments, the nucleic acid composition can be administered as a formulation comprising the nucleic acid composition and the carrier. In other embodiments, the nucleic acid composition of the present invention may be administered in the absence of a carrier (i.e., in a naked state).

[00138] The term "subject" as used herein refers to any mammal. While the methods of the invention are generally directed to humans, the mammal may be any mammal. As used herein, the phrase "subject in need thereof " is encompassed within the term subject, and includes any mammal, particularly a mammal in need of treatment where the increased risk of cancer or a pre-cancerous disease is medically determined, It refers to the object. In certain embodiments, the subject comprises a human cancer patient. In some embodiments, the subject is at increased risk of developing colorectal cancer or medically determined colorectal cancer. In another embodiment, the subject is at increased risk of suffering from pancreatic cancer or medically determined pancreatic cancer, such as being diagnosed with, for example, chronic pancreatitis. In another embodiment, the subject is at increased risk of developing lung cancer or medically determined lung cancer.

[00139] The terms "treating", "treating" and "treating" are synonymous with the term "administering an effective amount". These terms refer to medical care having the purpose of treating, ameliorating, stabilizing, reducing, or preventing one or more symptoms of a disorder, such as a disease, condition, or cancer. These terms are used interchangeably and include active treatment, that is specifically indicated for the amelioration of a disease, pathological condition or disorder, as well as treatment of a causal treatment, i. E. The cause of the disease, pathologic condition or disorder Lt; RTI ID = 0.0 > and / or < / RTI > The treatment may also include palliative treatment, i.e., treatment designed for symptomatic relief rather than treatment of the relevant disease, pathological condition or disorder; Prophylactic treatment, i. E. Treatment directed at minimizing, partially or completely inhibiting the onset of the relevant disease, pathological condition or disorder; And treatments used to supplement adjunct therapy, i.e., another specific treatment directed to the improvement of the relevant disease, pathological condition or disorder. Treatment is intended to treat, ameliorate, stabilize or prevent a disease, pathological condition or disorder, but it is understood that there is no need to result in treatment, improvement, stabilization or prevention in practice. The effectiveness of the treatment may be measured or evaluated as described herein and as is known in the art, as appropriate to the relevant disease, pathological condition or disorder. Such measurements and evaluations may be made in terms of quality and / or in view. Thus, for example, the nature or characteristic of a disease, pathological condition or disorder and / or the symptoms of a disease, pathological condition or disorder can be reduced to any effect or to any amount.

[00140] In certain embodiments, the nucleic acid compositions of the present invention are used to treat cancer, such as colorectal cancer.

[00141] As used herein, the term "cancer" includes any disease caused by unregulated cleavage and growth of abnormal cells, including, for example, malignant and metastatic growth of tumors. The term "cancer " also encompasses a condition characterized by an elevated risk of pre-cancerous or cancerous or pre-cancerous conditions. Accordingly, the treatment of cancer in the present invention is also considered to include a method of preventing cancer or a method of preventing the precancerous state from being converted into a cancerous state or into a completely non-cancerous state. Cancer or precancerous (neoplastic conditions) can be located in any part of the body, including internal organs and skin. Examples of possible body parts that contain cancer cells include colon, rectum (including anal), stomach, esophagus, digestive tract, lung, pancreas and liver. The cancer or neoplasm may also include the presence of one or more carcinomas, sarcomas, lymphomas, osteosarcomas or teratomas (germ cell tumors). In some embodiments, the cancer may also be a form of leukemia.

[00142] In certain embodiments, the nucleic acid compositions described herein are used to treat colon cancer (ie, colorectal or rectal cancer), pancreatic cancer, or lung cancer at any stage as described below. As is well known, cancer spreads to the subject by invading the normal non-cancerous tissue surrounding the tumor through the lymph nodes and blood vessels, and after the tumor has invaded the subject's veins, capillaries and arteries. When cancer cells are isolated from primary tumors ("metaplasia"), secondary tumors develop through a disease entity that forms metastatic lesions.

[00143] For example, there are four stages of colon cancer, which are generally characterized by the degree of metastasis. In stage 0 or carcinoma in situ, abnormal potential cancerous cells are found in the mucosa (innermost layer) of the colon barrier. In step I, cancerous cells are formed in the mucosa of the fibrous barrier and spread to the submucosal layer (tissue layer beneath the mucosa) and to the muscle layer of the barrier wall. Phase II consists of three subclasses: stage IIA where the cancerous tissue spreads through the muscle layer of the barrier wall to the serosal layer (outermost layer) of the barrier wall; Stage IIB in which the tumor has spread through the serosal layer of the barrier but has not spread to nearby organs; And stage IIC where the cancer spreads through the serosal layer of the barrier wall and invades nearby organs. Phase III is also subdivided into three subclasses: cancer spreading through the mucosa of the barrier wall into the submucosal layer and muscle layer and spreading to 1-3 nearby lymph nodes or tissues near the lymph nodes; Or stage IIIA where cancer spreads through the mucosa into the submucosal layer and 4-6 nearby lymph nodes; The tumor spreads through the muscle layer of the barrier wall into the serous layer or through the serous layer but does not spread to nearby organs and the cancer spreads to 1-3 nearby lymph nodes or tissues near the lymph nodes; Or spread to muscle or mucosal layers and 4-6 nearby lymph nodes; Spreading through the mucosa into the submucosa, spreading to the muscle layer and spreading to more than 7 nearby lymph nodes IIIB. In stage IIIC colorectal cancer, the tumor spreads through the serous layer of the colon wall but does not spread to nearby organs; the cancer spreads to 4-6 nearby lymph nodes; Cancer spreads through the muscle layer to the serous layer or through the serous layer, but not to nearby organs, and cancer spreads to more than 7 nearby lymph nodes; Or cancer is spread through the serous layer to the nearby organs and to one or more nearby lymph nodes or tissues near the lymph nodes. Finally, stage IV colorectal cancer is divided into two subclasses: stage IVA spreading to one or more lymph nodes spreading through the colon barrier and not near the organs and colon; And step IVB spreading to one or more organs that spread through the colon barrier and not to the adjacent organs and the colon, or to the lining of the abdominal wall.

[00144] Another example of tumor staging is the Dukes classification system for colorectal cancer. Wherein the step comprises the steps A), < RTI ID = 0.0 > Step B where the tumor shows invasion through the intestine but does not involve the lymph node; Step C in which cancerous cells or tissues are found in the lymph nodes of the subject; And step D where the tumor exhibits extensive metastasis to various organs of the subject.

[00145] The Astler Coller classification system can also be used as an alternative. Wherein step A colorectal cancer is defined as cancer present only in the mucosa of the intestine; In stage B1, the tumor expanded into the muscularis propria but did not penetrate through it, and the tumor was not metastasized to the lymph nodes. Stage B2 colon cancer was defined as a tumor that penetrated through the muscle layer and the tumor did not metastasize to the lymph nodes ; Stage C1 is characterized by a tumor that extends into the muscle layer but does not penetrate through it and the tumor has not metastasized to the lymph nodes; Step C2 Colorectal cancer is classified as a tumor that has penetrated through the muscle layer where the tumor has metastasized to the lymph nodes; Step D describes a tumor that has metastasized through an individual or a subject.

[00146] In some embodiments, the method of treatment of the invention further comprises administering to a cancer subject exhibiting reduced levels of miR-129 expression, miR-15a expression, miR-506 expression, miR-502, miR-140, . In this regard, it is known that miR-15a is down regulated in cancer. See, for example, RI Aqeilan, et al., Cell Death and Differentiation (2010) 17, pp. 215-220]. Also, for example, cancerous cells with reduced miR-129 expression levels are known to be resistant to 5-fluorouracil, as described in U.S. Patent Application Publication No. 2016/0090636, the contents of which are incorporated herein by reference in its entirety. It is also known that pancreatic cancer cells show a decrease in miR-506 levels. See, e.g., Li, J, et al. Oncogene. 35 pp. 5501-5514].

[00147] In another example, the microRNA mimetics of the invention are used to treat pancreatic cancer. Pancreatic cancer occurs in precursor lesions called paninreatic intraepithelial neoplasia (PanINs). These lesions are usually located in small ducts of the exocrine pancreas and can be classified as low-grade dysplasia, intermediate dysplasia, or highly dysplastic lesions, depending on the degree of cell dysplasia. These lesions usually show the presence of an activating mutation in the KRAS gene, with specific inactivating mutations of CDKN2A, TP53 and SMAD4. Collectively, these genetic mutations lead to the formation of infiltrating cancers. Pancreatic cancer is the size of the primary tumor and whether it has grown outside the pancreas to the surrounding organs; It is stepped on the basis of whether the tumor has spread to nearby lymph nodes and whether it has metastasized to other organs of the body (e.g. liver, lung, abdomen). This information is then combined and used to present the specific steps: 0, 1A, 1B, 2A, 2B, 3 and 4. In stage 0, pancreatic tumors are confined to the top layer of pancreatic cells and do not invade deeper tissues. Primary tumors do not migrate outside the pancreas like pancreatic carcinoma in situ or pancreatic epithelial neoplasia III. Stage 1A Pancreatic tumors are usually limited to the pancreas and are 2 cm in size or less. Also, stage 1A pancreatic tumors do not spread to nearby lymph nodes or distant locations. Stage 1B pancreatic tumors confined to the pancreas are larger than 2 cm in size. Stage 1B Pancreatic tumors do not spread to nearby lymph nodes or distant locations. Step 2A Pancreatic tumors represent tumors that grow outside the pancreas but do not grow into the main blood vessels or nerves, and the cancer does not spread to nearby lymph nodes or distant locations. Stage 2B Patients with pancreatic cancer are spread to the nearby lymph nodes, with the tumor limited to the pancreas or to the outside of the pancreas but not to the main blood vessels or nerves. Step 3 An object representing pancreatic cancer is a tumor that grows outside the pancreas, grows into the main blood vessels or nerves, and spreads to distant locations. Stage 4 Pancreatic cancer is metastasized to distant locations, lymph nodes and organs.

[00148] In another example, the modified microRNA nucleic acid composition of the present invention is used to treat lung cancer. The method includes the treatment of non-small cell lung cancers such as squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Lung cancer often arises from malignant tumors of the bronchi of the lungs and spreads to other parts of the body, such as the lymph nodes. For example, in the case of small cell lung cancer, cancerous lesions are often found in the lungs and then spread to the second lung, lung (pleural) periphery, or neighboring organs. Lung cancer is graded on the basis of the size of the primary tumor and whether it has grown outside the lungs and grew to lymph nodes and whether it has metastasized to other organs of the body (eg, bone, liver, breast, brain). This information is then combined and used to suggest certain steps, namely 0, 1, 2, 3 and 4 steps. For stage 0, that is, for situ carcinoma, the cancer is small and does not spread to deeper lung tissue or lungs. Stage 1 Lung cancer shows cancerous cells present in the lower lung tissue, but the lymph nodes are not affected. Stage 2 Lung cancer shows that the cancer has spread to nearby lymph nodes or chest wall. Step 3 Lung cancer is classified by whether it has spread continuously from the lungs to the lymph nodes or to nearby structures and organs such as the heart, organs and esophagus. Step 4 Lung cancer represents cancer that has spread throughout the body, which can affect the liver, bone or brain.

[00149] The nucleic acid composition according to the present invention can be administered by any route commonly known in the art. This includes, for example, (1) oral administration; (2) parenteral administration, e. G., Subcutaneous, intramuscular or intravenous injection; (3) topical administration; Or (4) in vaginal or rectal administration; (5) sublingual or buccal administration; (6) ocular administration; (7) transdermal administration; (8) nasal administration; And (9) direct administration to an organ or cell in need thereof.

[00150] The amount (dose) of the nucleic acid composition of the present invention to be administered depends on several factors, such as the type and stage of cancer, the presence or absence of a prodrug or adjuvant drug, and the weight, The tolerance to the agent, and some other factors. Depending on these various factors, dosages may range from about 2 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 15 mg / kg body weight, about 20 mg / kg body weight, Kg, about 30 mg / kg body weight, about 40 mg / kg body weight, about 50 mg / kg body weight, about 60 mg / kg body weight, about 70 mg / kg body weight, about 80 mg / kg body weight, about 90 mg / body weight kg, about 100 mg / kg body weight, about 125 mg / kg body weight, about 150 mg / kg body weight, about 175 mg / kg body weight, about 200 mg / kg body weight, about 250 mg / kg body weight, about 300 mg / , About 350 mg / kg body weight, about 400 mg / kg body weight, about 500 mg / kg body weight, about 600 mg / kg body weight, about 700 mg / kg body weight, about 800 mg / kg body weight, about 900 mg / kg body weight, Or about 1000 mg / kg body weight, where the term "drug" is generally understood to be within ± 10%, 5%, 2% or 1% of the indicated value. The dose may also be within a range limited by any of the above values. Using conventional experimentation, the effect of a compound on a cancerous or pre-cancerous disease, or the effect of a microRNA (such as miR-15a, effect on expression level or activity, or on BCL 2 level or activity, or on TS level or activity, or on E2F3 level < RTI ID = 0.0 > Or by monitoring the effects on disease pathology, an appropriate dose regimen for each patient can be determined. Depending on the various factors described above, any of the exemplary nucleic acid dosages may be administered once, twice or multiply once per day.

[00151] The ability of the nucleic acid compositions described herein and optionally any additional chemotherapeutic formulations for use with the methods of the invention may be readily determined using pharmacological models well known in the art, including, for example, cytotoxicity assays, Colorimetric analysis, xenograft analysis, and binding analysis.

[00152] The nucleic acid composition described in the present invention may also be administered or not administered with one or more chemotherapeutic agents which may be a preliminary drug or an adjuvant drug different from the nucleic acid composition described in the present invention.

[00135] The phrases "chemotherapy" or "chemotherapeutic agent" used in the present invention are useful agents for the treatment of cancer. Useful chemotherapeutic agents in combination with the methods described herein include any agent that directly or indirectly modulates BCL2, E2F3, or TS. Examples of chemotherapeutic agents include: anti-metabolites such as methotrexate and fluoropyrimidine pyrimidine antagonists, 5-fluorouracil (5-FU) (Carac® cream, Efudex®, Fluoroplex®, Adrucil®) and S -One; Folic acid antifolates, such as polyglutamated antifolate compounds; Ralititrexid, GW1843 and pemetrexed (Alimta) and non-polyglutamated folic acid antagonist compounds; Thalitic (Thymitaq), Plebitectic, BGC945; Folic acid analogues such as denonfterin, methotrexate, proteopterin, trimethrexate; And purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogs such as ancitabine, azacytidine, 6-aza uridine, camouflur, cytarabine, dideoxyurilide, doxifluridine, enocitabine and phlox uridine. In certain embodiments of the present invention, the chemotherapeutic agent is a gene or a gene product associated with signal transduction pathways involved in abnormal cell proliferation or apoptosis, such as expression of YAP1, BMI1, DCLK1, BCL2, thymidyl synthetase or E2F3, or A compound capable of inhibiting activity; And a pharmaceutically acceptable salt, acid or derivative of any of the above compounds.

[00154] In some embodiments, the chemotherapeutic agent is an anti-cancer drug, or a tissue sensitizer or other promoter for an anti-cancer drug. In some embodiments, the co-drug may be another nucleic acid, or another miRNA, such as the microRNA mimetics of the invention, gemcitabine or free 5-FU.

[00155] In certain embodiments, the other nucleic acid is a nucleic acid complementary to a short hairpin RNA (shRNA), siRNA or BCL2 3'UTR portion.

[00156] In another embodiment, the chemotherapy comprises administering any one of the following cancer drugs: methotrexate, doxorubicin, cyclophosphamide, cis-platin, oxaliplatin, bleomycin, vinblastine, gemcitabine, Rubicin, polyphosphoric acid, paclitaxel, and coconut shell. The chemotherapeutic agent may be administered before, during or after the treatment with the nucleic acid composition.

[00157] In some embodiments, the chemotherapeutic agent is a co-drug.

E2F transcription factor 3, E2F3 (RefSeq NG_029591.1, NM_001243076.2, NP_00123305.1, NM_001243076.2, NP_001230005.1) is a transcription factor that binds to DNA and interacts with an effector protein, But are not limited to, retinoblastoma proteins that regulate the expression of genes involved in cell cycle regulation. Thus, any drug that inhibits the expression of E2F3 can be considered a co-drug in the present invention.

[B-cell lymphoma 2 (BCL2), (RefSeq NG_009361.1, NM_000633, NP_000624), such as its isoforms a (NM_000633.2, NP_000624.2) and β (NM_000657.2, NP_000648.2) 2 gene, which is a member of the BCL2 family of regulatory proteins that regulate mitochondrial-regulated apoptosis through an endogenous apoptosis pathway. BCL2 is a mitochondrial outer membrane protein that blocks apoptosis of cells by binding BAD and BAK proteins. Nonlimiting examples of BCL2 inhibitors include antisense oligonucleotides such as Oblimersen (Genasense; Genta Inc.), BH3 mimetic small molecule inhibitors such as ABT-737 (Abbott Laboratories, Inc.), ABT-199 (Abbott Laboratories, And Obatoclax (Cephalon Inc.). Any drug that inhibits the expression of BCL2 may be considered a co-drug in the present invention.

[00160] Timimidyl synthetase (RefSeq: NG_028255.1, NM_001071.2, NP_001062.1) is a ubiquitous enzyme and is an essential enzyme for producing dTMP, one of the four bases constituting DNA. Methylation. This reaction requires CH4-folic acid as a cofactor as both a methyl group leader and a unique reducing agent. A constant requirement for CH4-folate is that the activity of thymidylate synthetase is strongly related to the activity of two enzymes responsible for supplementing the cell's folate storage space: dihydrofolate reductase and serine transhydroxymethylase . Timimidyl synthetase is a homozygote of 30-35 kDa subunit. The active site binds simultaneously with both the folate promoter and the dUMP substrate, using dUMP covalently coupled to the enzyme through the nucleophilic cysteine residue. (See Carreras et al., Annu. Rev. Biochem., (1995) 64: 721-762). The thymidylate synthase reaction is an important part of the pyrimidine biosynthetic pathway that produces dCTP and dTTP for inclusion in DNA. This reaction is necessary for DNA replication and cell growth. Thus, thymidylate synthetase activity is required in all rapidly dividing cells such as cancer cells. Because of its DNA synthesis, and thus its association with cell replication, thymidyl synthetase has been the target of anticancer drugs for many years. Non-limiting examples of thymidylate synthetase inhibitors include folic acid and dUMP analogs such as 5-fluorouracil (5-FU). Any drug that inhibits the expression of thymidylate synthetase can be considered a co-drug in the present invention.

[00161] If desired, administration of the nucleic acid compositions described herein can be combined with one or more non-drug therapies, such as radiation therapy and / or surgery. As is well known in the art, the use of radiation therapy and / or chemotherapeutic agents (in this case, the nucleic acid composition described herein, and optionally, as needed, to reduce the tumor, Of an additional chemotherapeutic agent) may be given. As is also well known in the art, administration of radiation therapy and / or chemotherapeutic agents may be given after surgery to destroy any residual cancer.

[00162] The following examples are presented for purposes of illustration and to describe certain specific embodiments of the invention. However, the scope of the present invention is not limited by the embodiment described in the present invention.

Example

Example  1. Materials and Methods

[00163] Modified miR-129: The 5-FU modified miR-129 molecule was synthesized by an automated oligonucleotide synthesis process and purified by HPLC. Two strands were annealed to produce mature modified 5-FU-miR-129. More specifically, a method called "2'-ACE RNA synthesis" was used. 2'-ACE RNA synthesis is based on a protective group form in which a silyl ether is used to protect the 5'-hydroxyl group in combination with an acid-labile orthoester protecting group (2'-ACE) on 2'-hydroxy do. This combination of protection groups is then used in conjunction with standard phosphoramidite solid phase synthesis techniques. For example, the entire contents of each of these are expressly incorporated herein by reference [SA Scaringe, FE Wincott, and MH Caruthers, J. Am. Chem . Soc . , 120 (45), 11820-11821 (1998); International PCT Application WO / 1996/041809; MD Matteucci, MH Caruthers, J. Am. Chem . Soc . , 103, 3185-3191 (1981); [SL Beaucage, MH Caruthers, Tetrahedron Lett . 22, 1859-1862 (1981).

[00167] Exemplary modified miR-15a nucleic acids, modified miR-140 nucleic acids, modified miR-192 nucleic acids, modified miR-502, modified miR-506 nucleic acids or any Other modified microRNAs can be synthesized in the same manner as miR-129a.

[00165] Some exemplary structures of currently used protected and functionalized ribonucleoside phosphoramidites are shown below.

[00166] Cell culture . Human colon cancer cell line HCT116, RKO, SW480, SW620 and normal colon cell line CCD 841 CoN, pancreatic cancer cell line ASPC-1, Panc-1 and lung cancer cell line A549 were obtained from the American Type Culture Collection (ATCC) and cultured in McCoy's 5A medium ), DMEM (RKO, SW480, SW620) and MEM (CCD 841 CoN) (Thermo Fischer). The medium was supplemented with 10% fetal bovine serum (Thermo Fischer).

For transfection, 1 × 10 ⁵ cells were plated on 6-well plates and 24 h later, 100 nM of miR-15a precursor, nonspecific miRNA (Thermo Fischer) or modified miR-15a mimetics (Dharmacon). Ignored for illustration about the transfection, cells were plated per well (1x10 ⁵⁾ cells in a 6-well plate. After 24 hours, 100 pmol miRNA (control, miR-15a, mimic-1) was diluted in Optimem (Thermo Fischer) and added to the plate. After 24 hours, the medium was replaced. The medium was supplemented with 10% fetal bovine serum (Thermo Fischer). Briefly, cells were cultured in DMEM / F12 (Life Technologies) supplemented with B27, 10 ng / mL bFGF and 20 ng / mL EGF in an ultra-low attachment flask. The spheroid cells were maintained by weak centrifugation, dissociation into single cells, and collection through re-circulation.

[00 168] Western Immunoblot analysis : After 48 hours of transfection, an equal volume of protein (15 μg) extracted from cells lysed in RIPA buffer containing protease inhibitor (Sigma) was diluted with 10% -12% sodium dodecane And separated on silica gel-polyacrylamide gel. The primary antibodies used for the analysis were rabbit anti-YAP1 monoclonal antibody (1: 10000) (Cell Signaling Technologies), anti-DLCK1 (1: 500) (Abcam), anti- (1: 10000) (Cell Signaling Technologies), mouse anti-human TS antibody (1: 500), anti-alpha -tubulin (1: 50000) (Santa Cruz Biotech Inc.), anti-GAPDH (1: 100,000) (Santa Cruz Biotech Inc.), anti-E2F3 (1: 500) (Santa Cruz Biotech Inc.). As a secondary antibody, a mouse or rabbit horseradish peroxidase-conjugated antibody (1: 5000, Santa Cruz Biotech Inc.) was used. Protein bands were visualized with an autoradiographic film using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fischer). Western blot density was quantified using Image J software.

Cell proliferation assay : After 24 hours of transfection, cells were seeded into 96-well plates at a density of 2000 cells per well. Cell proliferation assays were performed on days 1 to 5 by culturing 10 μl WST-1 (Roche Applied Science, Mannheim, Germany) for 1 hour in culture medium and reading the absorbance at 450 and 630 nm. The cell growth rate was calculated by subtracting the absorbance at 450 nm from the absorbance at 630 nm. Experiments for cell proliferation assay were performed at least three times. The OD was calculated from the absorbance at 450 nm minus the absorbance at 630 nm. The proliferation experiments were performed three times.

[00170] Anchorage-independent proliferation was studied to determine cancer cell colony forming ability. By trypsinization and counting of cancer cells, it was 1 × 10 ⁵ per well illustration total cellular oligo using a peck vitamin transfected with a micro-RNA or miRNA negative control or miR inherent variation of 25 nM in in six-well plates, transfected Cells were counted again 6 hours after the selection. In a 35-mm dish, a total of 20,000 cells in 0.35% agar (Bacto Agar, Becton Dickinson) were plated onto 1 mL of a solidified 6% agar layer. Growth medium containing B27, 10 ng / mL bFGF and 20 ng / mL EGF was included in both layers. After two weeks of culture, colonies having a diameter of 50 mm or more were counted.

[00171] Cell cycle analysis : After 24 hours of transfection, cells were harvested and resuspended in modified Krishan buffer supplemented with 0.02 mg / mL RNase H and 0.05 mg / mL propidium iodide at 0.5-1 × 10 ⁶ cells / mL . The stained cells were detected by flow cytometry and the results were analyzed by Modfit LT ™ software. Experiments for cell cycle analysis were performed at least three times.

[00172] Analysis of apoptosis . Analysis of fluorescein isothiocyanate (FITC) -Annexin was performed to distinguish early and late apoptosis (Becton Dickinson). The HCT116, RKO, SW480 and SW620 cells per well (1 × 10 ⁵⁾ after the cells are plated on a 6-well plate, 24 hours, using Oligofectamine 25 nM cells with the modified miRNA were transfected. After 48 hours of transfection, cells were harvested and stained with propidium iodide and anti-annexin-V antibody according to the manufacturer's protocol (Annexin V-FITC Apoptosis Detection kit, Invitrogen, CA, USA) Cells were detected by flow cytometry.

[00173] 5-FU Treatment and Cytotoxicity Analysis : After 24 hours of transfection, cancer cells were plated in triplicates in 96-well plates in 2 × 10 ³ cells per well in 100 μl medium. After 24 hours, the modified microRNAs of the present invention of 2-μM 5-FU alone, 50 nM native microRNA, 50 nM modified microRNA (eg, modified miR-129), or 2 mM 5-FU and 50 nM A combination of microRNAs, such as modified miR-129, was added and the cells were incubated for an additional 72 hours. Cell viability was measured using WST-1 assay.

Lentivirus production : Briefly, 1.5 × 10 ⁶ 293T cells were plated in 10-cm dishes containing 10 mL DMEM + 10% FBS. Two days later, pEZX-MR03, a lentiviral plasmid expressing miR-129 or hsa-miR-15a, was transfected with the Lenti-Pac HIV expression packaging kit according to the manufacturer's protocol. After 48 hours, the virus was harvested and concentrated to a Lenti-Pac lentivirus concentration solution. The titer of the virus was then measured using a Lenti-Pac (TM) HIV qRT-PCR titration kit (approximately 1011 viral particles / ml). In addition, the virus was serially diluted (0.1 μL, 0.5 μL, 2 μL, 10 μL, and 50 μL) was used for transfection of 5 × 10 ⁴ HCT116 CSC and the transfection efficiency was determined. The cells were inoculated into mice for in vivo treatment experiments using the lowest concentration (2 [mu] L) to achieve 100% positive expression.

[00175] Real time qRT - PCR analysis of nucleic acid expression . Expression levels of microRNAs in cancer cells were quantitated. Briefly, primers specific for the subject microRNA and the internal control RNU44 gene were obtained from Ambion. cDNA synthesis was performed by High Capacity cDNA Synthesis Kit (Applied Biosystems) using miRNA specific primers. Real-time qRT-PCR was performed on an Applied Biosystems 7500 Real-Time PCR instrument using miRNA-specific primers by TaqMan Gene Expression Assay (Applied Biosystems). Exemplary miR expression levels of the present invention were calculated by the △ ΔCT method based on the internal control RNU44 and normalized to the control and plotted in relative quantities.

[00176] Human cancer stem cells Profiler : RNA was extracted from cancer cells transfected with the exemplary microRNA or negative miRNA of the present invention using a TRIzol reagent (Thermo Fischer) according to the manufacturer's protocol. RNA was transferred to the first strand cDNA using RT2 First Strand Kit (Qiagen). Next, the cDNA is mixed with RT2 SYBR Green Mastermix (Qiagen) and the mixture is divided into wells of Human Cancer Stem Cells RT2 Profiler PCR Array (Qiagen). Applied Biosystems 7500 Real-Time PCR instrument was used in qRT-PCR (Applied Biosystems) and relative expression values were determined using the △ ΔCT method.

[00177] Subcutaneous mouse tumor implantation model : Two days before injection, HCT116 cancer stem cells were plated at 5 × 10 ⁵ / well on a 6-well ultra-low attachment plate. Cells were transfected or transfected with 20 μL of virus or 100 pmoles of modified miR-129 or modified miR-15a. After 48 hours, the cells were harvested and resuspended to 10 ⁶ / ml in DMEM / F12 knockout medium containing 30% matrigel. NOD / SCID mice (Jackson Laboratories, Bar Harbor, MA, USA) at 10 to 12 weeks of age were used for tumor transplantation. Mice were anesthetized with isoflurane inhalation. 100 μL of cell suspension was subcutaneously injected on both sides of the waist. Tumor size was measured using a caliper (caliper), tumor volume was calculated using the formula of the ^2/2 V = length × width.

[00178] For in vivo miRNA delivery experiments, colon cancer cells expressing the lenti-luc reporter gene were generated by infecting parental HCT116 cells with recombinant lentivirus. Luciferase-expressing HCT116 cells (2.0x10 ⁶ cells per mouse) were suspended in 0.1 mL of PBS solution and injected through the tail vein of each mouse. Two weeks after colon cancer cells were injected, mice were treated with a modified negative miR (s) packaged in 40 μg of negative control or in vivo-jetPEI (Polyplus Transfection) via tail vein injection. Mice were treated twice a day (8 times) for 2 weeks. After treatment, mice were screened using the IVIS Spectrum In vivo Imaging System (IVIS) (PerkinElmer).

[00179] RNA Isolation : For mouse xenotransplantation, each of the slice tissues was deparaffinized, hydrated and digested with protease K. Total RNA was then isolated using TRIzol ^® reagent. Total RNA was also isolated from clinical specimens via the TRIzol ^® -based approach.

[00180] Statistical analysis . All experiments were repeated at least 3 times. All statistical analyzes were performed with SigmaPlot software. Statistical significance between the two groups was determined using the Student t-test (paired t -test for clinical samples and unpaired t-test for all other samples) . To compare two or more groups, one-way ANOVA followed by Bonferroni-Dunn test. Data were expressed as mean ± standard error of the mean (SEM). Statistical significance is described in the legend or marked with an asterisk (*). * = P &lt;0.05; ** = P &lt;0.01; *** = P < 0.001.

Example  2: Modified The microRNA  Have anticancer activity.

As shown in Figures 3, 8b, 12a-b, 13a-b and 14a-d, the modified miRNAs (modified miRs 129,15a, 192 (215), 140, 502 and 506) It is more effective at inhibiting the proliferation of colon, pancreatic, and lung cancer cells than the modified miRNA precursor. In addition, the modified miRNA can be transferred to cancer cells without a transfection reagent (data not shown). Notably, the results show that cancer cell proliferation across several different colon cancer cell lines, pancreatic cancer cell lines and lung cancer cell lines is significantly inhibited compared to cancer cells treated with control microRNAs.

Example  3: Modified miR -129 nucleic acid has anticancer activity.

[00182] In the following experiment, 5-FU was incorporated into miR-129. In one experiment, as shown in the structure provided in FIG. 1A, all U bases of miR-129 were replaced with 5-FU, wherein "U ^F " represents 5-fluorouracil or other 5-halo lacyl. In another experiment, all U bases except for the seed region of miR-129 were replaced with 5-FU as shown in the structure provided in FIG. 1b.

[00183] Target specificity analysis : The results of Western immunoblot experiments in colon cancer HCT-116 cells show that the exemplary modified miR-129 polynucleotides of the invention can retain their target specificity for TS, BCL2 and E2F3 . The results are shown in Figures 2a and 2b, which show that SEQ ID. The results for the modified miR-129 nucleic acid obtained by two separate operators as described in NO: 4, in which all U-bases have been replaced with 5-FU. More importantly, exemplary miR-129 mimetics were found to be more potent than unmodified (control) miR-129 in reducing the expression levels of TS, BCL2 and E2F3.

[00184] Enhancing the function of modified microRNAs of the present invention . The effect of an exemplary modified miR-129 on colon cancer cell proliferation was compared to native miR-129. The results show that 5-FU-miR-129 can completely inhibit HCT-116 tumor cell growth at a concentration of 50 nM. In addition, as shown in the results of Figure 3, 5-FU-miR-129 is much more potent than its parent miR-129, thereby providing a significantly higher inhibitory effect. This inhibition is specific because the scrambled control miR has no effect on cell proliferation.

[00185] Next, the effect of modified miR-129 and 5-FU on cell proliferation was compared using HCT-116 colon cancer cells. As shown by the results provided in FIG. 4, modified miR-129 at 50 nM (less than 40-fold less than 5-FU) is unexpectedly much more potent at suppressing tumor cell proliferation than 2 μM 5-FU.

[00186] An exemplary micro-modified RNA of the invention induces cell chair myeolsa in colon cancer cells. Using BCL2, an important target of miR-129, the effect of modified miR of the present invention on apoptosis was investigated. Specifically, apoptosis assays in HCT116, RKO, SW480 and SW620 colorectal cancer cells transfected with negative control miRNA, native miR-129 or exemplary miR-129 mimic of SEQ ID NO: 4, Were quantified. The results showed that miR-129 mimetics were detected in all four colorectal cancer cell lines by fluorescence-activated cell sorting (FACS) -based FITC-Annexin analysis at 2 to 30-fold compared to miR- Apoptosis can be induced (Fig. 5A).

[00187] The miR- 129 mimic triggers the G1 / S cell cycle checkpoint regulation . Cell cycle analysis was performed using flow cytometry in scrambled control, miR-129 precursor and HCT-116 cells treated with an exemplary miR-129 mimic. As shown in FIG. 5B, cell cycle analysis revealed that miR-129 mimics affect colon cancer cell growth by inducing a G1 arrest, and the effect is much more (more than two times) stronger than native miR-129 It turned out.

[00188] The miR- 129 mimic eliminates chemotherapy-resistant colon cancer stem cells . In order to determine the effect of certain exemplary modified microRNAs of the invention (i.e., miR-129 mimetics) on 5-FU resistant colon cancer stem cells, HCT116-derived colorectal stem cells were treated with various concentrations of mimetic-1 Or 5-FU. The data shown in Figure 6 show that the exemplary microRNA mimetics of the present invention can remove up to 80% of 5-FU resistant colon cancer stem cells at a concentration of 100 nM, while at a lethal dose of 5-FU 100 [ Indicating a minimal effect on cell viability.

[00189] Taken together, these results show that the exemplary modified microRNA polynucleotides of the present invention can inhibit cellular proliferation of HCT116 colon cancer stem cells (FIG. 6). This inhibitory effect of miR-129 was much more potent than the native miR-129, since the proliferation was almost completely blocked with 25 nM miR-129 at day 6 (Fig. 6). We also demonstrated the effect of cell therapy using modified miR-129 on non-adherent cell growth, using soft agar analysis. Modified miR-129 treated colon cancer stem cells and did not form visible sphere (similar to that seen in Figure 10) compared to cells treated with native miR-129 or control miRNA.

[00190] miR- 129 mimetics inhibit metastasis of colorectal cancer in vivo . The colon cancer metastasis model was used to evaluate the therapeutic effect of miR-129 nucleic acid modification. After two weeks of establishing the transfer, 40 [mu] g of miR-129 nucleic acid of SEQ ID NO: 4 was delivered by intravenous injection at a frequency of one injection every other day for 2 weeks.

[00191] The results shown in Figure 17 show no negative toxic side effects, but the modified microRNA-129 inhibits colorectal metastasis, while the negative control miRNA is ineffective.

Example  3. Modified miR -15a and its anticancer activity.

[00192] Exemplary modified miR- 15a compositions have anticancer activity . As shown in Fig. 1C and Fig. 1D, all uracil bases of the miR-15a nucleic acid sequence are replaced with 5-halo lacyl (i.e., 5-fluorouracil) An exemplary modified miR-15a mimic was synthesized as described above, wherein only the uracil base in the region was replaced with 5-haloucalyl (i.e., 5-fluorouracil) (Fig. 1d).

[00193] Three days after the transfection of the exemplary modified miR-15a described in FIG. 1c into HCT-116 colon cancer stem cells, proteins were collected and subjected to Western blotting to obtain the modified miR-15a Confirming that the nucleic acid composition retains the ability to regulate key miR-15a targets. As shown in FIG. 8A, miR-15a targets YAP1, BMI1, DCLK1, and BCL2 showed reduced protein levels upon transfection with unmodified miR-15a (native-miR15a) or modified miR- This indicated that the 5-halo lacyl modification did not inhibit the ability of miR-15a to regulate their targets in the cells.

[ 00194 ] Modified miR- 15a has increased therapeutic efficacy in vitro . To determine whether the modified miR-15a composition of the present invention showed increased efficacy in colon cancer cell lines as compared to unmodified miR-15a, HCT-116 colon cancer cells were treated with a negative control (non-specific oligosaccharide Nucleotides), unmodified miR-15a or an exemplary modified miR-15a composition.

[00195] WST-1 assay was used to assess cancer cell proliferation. As shown in Fig. 8B, after 6 days of transfection, unmodified miR-15a decreased cell proliferation by 53% as compared to the control group. In the case of modified miR-15a, cell proliferation was reduced by 84%. Taken together, the experimental results show that modified miR-15a is more effective at reducing cancer cell proliferation than unmodified miR-15a.

[00196] Modified miR-15a nucleic acids were also analyzed for their ability to inhibit cell cycle progression in cancer cells. Figure 9 shows that unmodified miR-15a induces cell cycle arrest and leads to approximately a 3-fold increase in G1 / S ratio. Figure 9 also shows that the exemplary modified miR-15a composition of the present invention was more effective at halting cell cycle progression as compared to the native counterpart. For example, a 7-fold increase in G1 / S ratio was seen by the cells expressing the exemplary modified miR-15a nucleic acid of the invention when compared to the control. Thus, modified miR-15a is more effective in inducing cell cycle arrest in colorectal cancer cells than unmodified miR-15a.

[00197] In the Matrigel matrix, the effect of the exemplary modified miR-15a composition on colony formation by colon cancer stem cells was also investigated. As shown in FIG. 10, although many colonies were formed by the cells transfected with the control miRNA (FIG. 10, negative), the cells transfected with the unmodified miR-15a produced almost no colonies (Fig. 10, miR-15a). In contrast, for cells transfected with modified miR-15a, no colonies were observed (Figure 10, 5-FU-miR-15a). These results indicate that the exemplary modified miR-15a compositions of the invention are indeed more potent inhibitors of cancer production and colon cancer progression.

[00198] Modified miR- 15a inhibits cancer development and in vivo progression . To further our understanding of miR-15a in colon CSCs, we established a mouse xenograft model containing colon cancer cells that had been transfected with modified miR-15a or negative control miRNA. Tumors were measured and harvested 8 weeks after injection. As shown in FIG. 11, there was a significant reduction in tumor size (> 25x) (n = 8) from tumors established from CSC expressing a modified miR-15a mimic.

[00199] The data presented in the present invention suggests that, in order to enhance the chemotherapeutic function of the original microRNA molecule, with or without other chemotherapeutic agents, the hormonal (eg, 5-FU) Supporting the feasibility of incorporating new variants.

                         SEQUENCE LISTING <110> The Research Foundation for the State University of New        York   <120> 5-HALOURACIL-MODIFIED MICRORNAS AND THEIR USE IN THE TREATMENT OF         CANCER <130> 34085 PCT <150> 62 / 464,491 <151> 2017-02-28 <150> 62 / 422,298 <151> 2016-11-15 <150> 62 / 415,740 <151> 2016-11-01 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 21 <212> DNA <213> Homo sapiens <400> 1 cuuuuugcgg ucugggcuug c 21 <210> 2 <211> 22 <212> DNA <213> Homo sapiens <400> 2 uagcagcaca uaaugguuug ug 22 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> modified microRNA129 with additional uracils on the 3 'end of the        nucleic acid sequence <220> <221> misc_feature &Lt; 222 > (22) <223> uracil nucleotides <400> 3 cuuuuugcgg ucugggcuug cuu 23 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> an exemplary modified microRNA-129 nucleic acid with all uracils        modified <220> <221> misc_feature <222> (2) (6) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (11) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (13) <223> halouracil nucleotides <220> <221> misc_feature <222> (18). (19) <223> halouracil nucleotides <400> 4 cuuuuugcgg ucugggcuug c 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> an exemplary modified microRNA-129 nucleic acid with all uracils        in non-seeded <220> <221> misc_feature &Lt; 222 > (11) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (13) <223> halouracil nucleotides <220> <221> misc_feature <222> (18). (19) <223> halouracil nucleotides <400> 5 cuuuuugcgg ucugggcuug c 21 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> an exemplary modified microRNA-15a nucleic acid with all uracils        modified <220> <221> misc_feature <222> (1) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (11) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (14) <223> halouracil nucleotides <220> <221> misc_feature <222> (17). (19) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (21) <223> halouracil nucleotides <400> 6 uagcagcaca uaaugguuug ug 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> an exemplary modified microRNA-15a nucleic acid with all uracils        in the non-seed portion of the nucleic acid sequence modified <220> <221> misc_feature &Lt; 222 > (11) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (14) <223> halouracil nucleotides <220> <221> misc_feature <222> (17). (19) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (21) <223> halouracil nucleotides <400> 7 uagcagcaca uaaugguuug ug 22 <210> 8 <211> 22 <212> DNA <213> Homo sapiens <400> 8 cagugguuuu acccuauggu ag 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> an exemplary modified microRNA-140 nucleic acid with a certain        uracils modified <220> <221> misc_feature <222> (4) (4) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (15) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (20) <223> halouracil nucleotides <400> 9 cagugguuuu acccuauggu ag 22 <210> 10 <211> 21 <212> DNA <213> Homo sapiens <400> 10 cugaccuaug aauugacagc c 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> an exemplary modified microRNA-192 nucleic acid with a certain        uracils modified <220> <221> misc_feature <222> (2) (2) <223> halouracil nucleotides <220> <221> misc_feature <222> (7) (7) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (9) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (13) <223> halouracil nucleotides <400> 11 cugaccuaug aauugacagc c 21 <210> 12 <211> 21 <212> DNA <213> Homo sapiens <400> 12 auccuugcua ucugggugcu a 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> an exemplary modified microRNA-502 nucleic acid with a certain        uracils modified <220> <221> misc_feature <222> (2) (2) <223> halouracil nucleotides <220> <221> misc_feature <222> (5) (6) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (11) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (13) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (17) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (20) <223> halouracil nucleotides <400> 13 auccuugcua ucugggugcu a 21 <210> 14 <211> 22 <212> DNA <213> Homo sapiens <400> 14 uauucaggaa gguguuacuu aa 22 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> an exemplary modified microRNA-506 nucleic acid with a certain        uracils modified <220> <221> misc_feature <222> (1) <223> halouracil nucleotides <220> <221> misc_feature <222> (3) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (13) <223> halouracil nucleotides <220> <221> misc_feature &Lt; 222 > (15) <223> halouracil nucleotides <220> <221> misc_feature <222> (19) <223> halouracil nucleotides <400> 15 uauucaggaa gguguuacuu aa 22

Claims (22)

A nucleic acid composition comprising a modified microRNA nucleotide sequence comprising at least one uracil nucleic acid, wherein the at least one uracil nucleic acid is 5-haloracil. The modified microRNA nucleotide sequence of claim 1, wherein the modified microRNA nucleotide sequence comprises a microRNA nucleotide sequence selected from the group consisting of miR-129, miR-15a, miR-140, miR-192, miR- &Lt; / RTI &gt; 3. The method of claim 2, wherein the modified microRNA nucleotide sequence comprises a nucleotide sequence selected from the group consisting of:
^{CU F U F U F U F} U F GCGGU F CU F GGGCU F U F GC [SEQ ID NO. 4],
CU ^F U ^{F F F} GGGCU CUUUUUGCGGU GC [SEQ ID NO. 5],
^{F F} GGU AAU AGCAGCACAU ^{F F} U U U ^{F F F} GU G [SEQ ID NO.6],
GGU AAU ^{F F F F} U UAGCAGCACAU U ^{F F} GU G [SEQ ID NO. 7],
CAGU ^F GGUUUUACCCU ^F AUGGU ^F AG [SEQ ID NO. 9],
GACCU AU ^{F F F F} GAAU CU U GACAGCC ^F [SEQ ID NO. 11],
CCU U ^{F F F F} GCUAU AU CU GGGU ^{F F F} A GCU [SEQ ID NO. 13], and
AU ^{F F} U ^{F F} CAGGAAGGU U GU ^{F F} U U ^{F F} ACU AA [SEQ ID NO. 15]. &Lt; / RTI &gt; 3. The method of claim 2, wherein the modified microRNA nucleotide sequence is SEQ ID NO. Lt; RTI ID = 0.0 &gt; miR-129 &lt; / RTI &gt;Lt; RTI ID = 0.0 &gt; miR-15a &lt; / RTI &gt; 5. The method of claim 4, wherein the microRNA nucleotide sequence is SEQ ID NO. 1, wherein at least one of the uracil nucleic acids is a 5-haloracil. 5. The method of claim 4, wherein the microRNA nucleotide sequence is SEQ ID NO. 2 miR-15a nucleotide sequence, wherein at least one of the uracil nucleic acids is 5-haloracil. 7. The composition of claim 1, wherein the 5-haloracil is 5-fluorouracil. 2. The composition of claim 1 wherein at least two of the uracil nucleic acids in the nucleotide sequence are 5-haloracil. 9. The composition of claim 8, wherein at least three of the uracil nucleic acids in the nucleotide sequence are 5-haloracil. 10. The composition of claim 9, wherein at least four of the uracil nucleic acids in the nucleotide sequence are 5-haloracil. 11. The composition of claim 10, wherein at least five of the uracil nucleic acids in the nucleotide sequence are 5-haloracil. 12. The composition of claim 11 wherein at least six of the uracil nucleic acids in the nucleotide sequence are 5-haloracil. 2. The composition of claim 1 wherein all uracil nucleic acid in the nucleotide sequence is 5-haloracil. 3. The composition of claim 2, wherein the 5-haloracil is 5-fluorouracil. A pharmaceutical composition comprising the nucleic acid composition of claim 1 and a pharmaceutically acceptable carrier. 15. The nucleic acid construct according to claim 14,
^{CU F U F U F U F} U F GCGGU F CU F GGGCU F U F GC [SEQ ID NO. 4],
CU ^F U ^{F F F} GGGCU CUUUUUGCGGU GC [SEQ ID NO. 5],
^{F F} GGU AAU AGCAGCACAU ^{F F} U U U ^{F F F} GU G [SEQ ID NO.6],
GGU AAU ^{F F F F} U UAGCAGCACAU U ^{F F} GU G [SEQ ID NO. 7],
CAGU ^F GGUUUUACCCU ^F AUGGU ^F AG [SEQ ID NO. 9],
GACCU AU ^{F F F F} GAAU CU U GACAGCC ^F [SEQ ID NO. 11],
CCU U ^{F F F F} GCUAU AU CU GGGU ^{F F F} A GCU [SEQ ID NO. 13], and
AU ^{F F} U ^{F F} CAGGAAGGU U GU ^{F F} U U ^{F F} ACU AA [SEQ ID NO. 15], wherein U ^F is 5-halouracil. 16. The pharmaceutical composition according to claim 15, wherein the 5-haloracil is 5-fluorouracil. A method of treating cancer comprising administering to a subject an effective amount of a nucleic acid composition according to claim 1, wherein the subject is diagnosed as having a cancer or having a cancer susceptibility, and the progression of the cancer is inhibited. 19. The method of claim 18, wherein the mammal is a human. 20. The method of claim 19, wherein the subject is caught in a cancer selected from the group consisting of colon cancer, pancreatic cancer, or lung cancer. 21. The method of claim 20, wherein the subject is caught in a colon cancer. 18. The nucleic acid construct according to claim 17,
^{CU F U F U F U F} U F GCGGU F CU F GGGCU F U F GC [SEQ ID NO. 4],
CU ^F U ^{F F F} GGGCU CUUUUUGCGGU GC [SEQ ID NO. 5],
^{F F} GGU AAU AGCAGCACAU ^{F F} U U U ^{F F F} GU G [SEQ ID NO.6],
GGU AAU ^{F F F F} U UAGCAGCACAU U ^{F F} GU G [SEQ ID NO. 7],
CAGU ^F GGUUUUACCCU ^F AUGGU ^F AG [SEQ ID NO. 9],
GACCU AU ^{F F F F} GAAU CU U GACAGCC ^F [SEQ ID NO. 11],
CCU U ^{F F F F} GCUAU AU CU GGGU ^{F F F} A GCU [SEQ ID NO. 13], and
AU ^{F F} U ^{F F} CAGGAAGGU U GU ^{F F} U U ^{F F} ACU AA [SEQ ID NO. 15], and the 5-haloracil is 5-fluorouracil.

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