JP2022173194A - 5-halouracil-modified micro rna and use thereof in treatment of cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide more efficacious, stable, and less toxic medications for the treatment of cancer.

SOLUTION: The present disclosure provides nucleic acid compositions that incorporate one or more halouracil molecules. More specifically, the present disclosure reveals that the replacement of uracil nucleotides within a microRNA nucleotide sequence with 5-halouracil increases the ability of the micro-RNA to inhibit cancer progression and tumorigenesis. As such, the present disclosure provides various nucleic acid (e.g., microRNA) compositions having 5-halouracil molecules incorporated in their nucleic acid sequences and methods for using the same. The present disclosure further provides pharmaceutical compositions (e.g., formulations) comprising the modified nucleic acid compositions, and methods for treating cancers, such as colorectal, pancreatic and lung cancer.

SELECTED DRAWING: Figure 1A

COPYRIGHT: (C)2023,JPO&INPIT

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is based on U.S. Provisional Patent Application No. 62/464,491 filed February 28, 2017; No. 62/415,740, filed on May 20, 2003, the entire contents of which are hereby incorporated by reference.

GOVERNMENT SUPPORT This disclosure was made with government support under grant numbers HL127522 and CA19709802 awarded by the National Institutes of Health. The Government has certain rights in this disclosure.

Incorporation by Reference in the Sequence Listing
The Sequence Listing in a 7KB ASCII text file named R8859_PCT_SequenceListing.txt, created October 30, 2017 and filed via EFS-Web with the United States Patent and Trademark Office, is hereby incorporated by reference. incorporated into the book.

FIELD OF THE DISCLOSURE The present disclosure relates generally to compositions and methods of treating cancer, and more particularly to the use of modified microRNAs alone or in combination with 5-fluorouracil to treat cancer, particularly colorectal, lung or pancreatic cancer. is directed to methods used in the treatment of cancer.

BACKGROUND MicroRNAs (miRNAs, miRs) are highly sensitive to mediate translation in cells or organisms by negatively regulating the expression of their target genes and thus causing translation arrest, mRNA cleavage or a combination thereof. It is a class of non-coding small RNA molecules conserved in . See Bartel DP. Cell. (2009) 136(2):215-33. By targeting multiple transcripts, miRNAs regulate a wide range of biological processes, including apoptosis, differentiation and cell proliferation, thus aberrant microRNA function can lead to cancer ( (2004) 431(7006):350-5) and recently miRNAs have themselves been identified as biomarkers, oncogenes or tumor suppressors. See, eg, Croce, CM, Nat Rev Genet. (2009) 10:704-714.

Colorectal cancer (CRC) is the third most common malignancy and the second most common cancer-related death in the United States. See Hegde SR et al., Expert review of gastroenterology & hepatology. (2008) 2(1):135-49. Although there are many chemotherapeutic agents used to treat cancer; pyrimidine antagonists, such as fluoropyrimidine-based chemotherapeutic agents (eg, 5-fluorouracil, S-1) treat colorectal cancer. is the gold standard for Pyrimidine antagonists block the synthesis of pyrimidine containing nucleotides (cytosine and thymine in DNA; cytosine and uracil in RNA). Pyrimidine antagonists have a similar structure when compared to endogenous nucleotides and they compete with natural pyrimidines to participate in replication processes resulting in prevention of DNA and/or RNA synthesis and inhibition of cell division. Inhibits critical enzymatic activity.

Pancreatic cancer is a deadly cancer that is very difficult to treat. See Siegel, RL et al., CA Cancer J. Clin. (2015) 65: 5-29. Unique aspects of pancreatic cancer include a very low 5-year survival rate of less than 7% (Id.), late presentation, early metastases and poor responsiveness to chemotherapy and radiation. See Maitra A and Hruban RH, Annu Rev. Pathol. (2008) 3:157-188. To date, gemcitabine-based chemotherapy (2',2'-difluoro-2'deoxycytidine) is the gold standard for the treatment of pancreatic cancer, but efficacy of therapeutic intervention is limited due to drug resistance. be. Oettle, H. et al., JAMA (2013) 310: 1473-1481.

5-fluorouracil (i.e., 5-FU, or more specifically 5-fluoro-1H-pyrimidine-2,4-dione) is used in many adjuvant chemotherapeutic medications, such as Carac® cream. , Efudex®, Fluoroplex®, and Adrucil® are well-known pyrimidine antagonists. Thymidylate synthase (TYMS or TS) has been established. Danenberg P.V., Biochim. Biophys. Acta. (1977) 473(2):73-92. However, despite steady improvements in 5-FU-based therapy, patient response rates to 5-FU-based chemotherapy remain modest due to the development of drug resistance. Longley D. B, et al., Apoptosis, Cell Signaling, and Human Diseases, (2007) p. 263-78.

Current cancer treatments are still in their early stages and still face many hurdles to be improved or overcome. For example, although 5-FU is highly effective in treating various cancers, it is well known that 5-FU is substantially toxic and can induce adverse side effects in the host. With respect to miRNAs, these compounds are known to be susceptible to enzymatic degradation and have poor stability when administered. Moreover, tumor cells are known to evade apoptotic pathways by developing resistance to common therapeutic agents such as 5-FU and gemcitabine. See Gottesman M. M. et al., Nature Reviews Cancer, (2002) 2(1):48-58. Therefore, there is a significant interest in more effective, stable and less toxic pharmaceuticals for the treatment of cancer.

U.S. Patent Application Publication No. 2016/0090636 International PCT application WO/1996/041809

Bartel DP. Cell. (2009) 136(2):215-33 Ambros V. Nature. (2004) 431(7006):350-5 Croce, CM, Nat Rev Genet. (2009) 10:704-714 Hegde SR et al., Expert review of gastroenterology & hepatology. (2008) 2(1):135-49 Siegel, RL et al., CA Cancer J. Clin. (2015) 65: 5-29 Maitra A and Hruban RH, Annu Rev. Pathol. (2008) 3:157-188 Oettle, H. et al., JAMA (2013) 310: 1473–1481 Danenberg P. V., Biochim. Biophys. Acta. (1977) 473(2):73-92. Longley D. B. et al., Apoptosis, Cell Signaling, and Human Diseases, (2007) pp. 263-78 Gottesman M. M. et al., Nature Reviews Cancer, (2002) 2(1):48-58 J. Wu et al., Cell Cycle, (2010) 9:9, 1809-1818. Xie T et al., Clin Transl Oncol. (2015) 17(7):504-10 Acunzo M, and Croce CM, Clin. Chem. (2016) 62(4):655-6 Zhai, H. et al., Oncotarget. (2015) 6: 19735–46. Song, B. et al., Clin. Cancer Res. (2008), 14: 8080-8086. Song, B. et al., Mol. Cancer. (2010), 9:96 pp. 1476-4598. Zhai, H. et al., Oncogene. (2013), 32:12 pp. 1570-1579. Li, J. et al., Oncotarget. (2016), 7:38 pp. 62778-62788 Li, J. et al., Oncogene. (2016) 35 pp. 5501-5514 C. M. Dunham et al., Nature Methods, (2007) 4(7), pp. 547-548 "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Ed. Mack Publishing Company, Easton, Pa., (1995) RI Aqeilan et al., Cell Death and Differentiation (2010) 17, pp. 215-220 Li, J et al., Oncogene. 35 pp. 5501-5514 Carreras et al, Annu. Rev. Biochem., (1995) 64:721-762. S.A. Scaringe, F.E. Wincott, and M.H. Caruthers, J. Am. Chem. Soc., 120 (45), 11820-11821 (1998) M.D. Matteucci, M.H. Caruthers, J. Am. Chem. Soc., 103, 3185-3191 (1981) S.L. Beaucage, M.H. Caruthers, Tetrahedron Lett. 22, 1859-1862 (1981)

SUMMARY OF THE DISCLOSURE The present disclosure demonstrates that nucleic acid compositions (ie, microRNAs) that incorporate a 5-halouracyl base have particular efficacy as anticancer agents. Moreover, the data herein demonstrate that contacting cells with the modified microRNA compositions of the present disclosure reduces cell cycle progression and tumorigenesis, such as by reducing cancer cell proliferation and increasing efficacy of chemotherapeutic agents. is controlled. The present disclosure states that the incorporation of 5-halouracyl bases within the nucleotide sequence of microRNAs increases the efficacy of microRNAs as anticancer therapeutics relative to cancer therapeutics alone and/or natural microRNAs. Assuming discovery.

Thus, in one aspect of the disclosure, a nucleic acid comprising a modified microRNA nucleotide sequence having at least one uracil base (U, U base) replaced by 5-halouracil, e.g., 5-fluorouracil (5-FU) Compositions are described. In certain embodiments, the modified microRNA has more than one or exactly one uracil replaced by 5-halouracil. In some embodiments, the modified microRNA nucleotide sequence includes 2, 3, 4, 5, 6, 7, 8 or more uracil bases replaced by 5-halouracil. In other embodiments, all uracil nucleotide bases of the modified mRNA are replaced by 5-halouracil.

In some embodiments, 5-halouracil is, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil. In certain embodiments, 5-halouracil is 5-fluorouracil.

In certain embodiments, the modified microRNA nucleotide sequence contains more than one 5-halouracil, wherein each 5-halouracil is the same. In other embodiments, the modified microRNA nucleotide sequence contains more than one 5-halouracil, wherein each 5-halouracil is different. In other embodiments, the modified microRNA nucleotide sequence comprises more than two 5-halouracils, wherein the modified microRNA nucleotide sequence comprises a combination of different 5-halouracils.

In an exemplary embodiment of the present disclosure, nucleic acid compositions containing miR-129 nucleotide sequences modified by replacing at least one uracil nucleotide base with 5-halouracil are provided. More specifically, the nucleic acid composition contains at least the following native miR-129 nucleotide sequences: CUUUUUGCGGUCUGGGCUUGC [SEQ ID NO: 1], where at least 1, 2, 3, 4, 5 in the nucleic acid sequences shown , 6, 7, 8 or all uracil bases or those that can be covalently attached to the sequences shown are replaced by 5-halouracil.

In certain embodiments of the disclosure, the modified microRNA has a nucleic acid sequence 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], wherein U ^F is halouracil, specifically 5-fluorouracil.

In other embodiments, the seed portion (GUUUUUGC) of the native miR-129 nucleotide sequence is left unmodified (i.e., free of 5-halouracil), while the remainder of the modified miR-129 nucleotide sequence contains residual uracil. One or more (or all) of the nucleotide bases are replaced by an equivalent number of 5-halouracyls. In certain embodiments, the modified miR-129 microRNA of the present disclosure has a nucleic acid sequence consisting of CUUUUUGCGGU ^F CU ^F GGGCU ^F U ^F GC [SEQ ID NO: 5], where U ^F is halouracil, specifically is 5-fluorouracil.

In some embodiments, 5-halouracil is, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil. In certain embodiments, 5-halouracil is 5-fluorouracil.

In another embodiment of the present disclosure, nucleic acid compositions are provided that contain a miR-15a nucleotide sequence that has been modified by replacing at least one uracil nucleotide base with 5-halouracil, such as 5-fluorouracil (5-FU). be done. Specifically, the nucleic acid composition contains at least the following native miR-15a nucleotide sequences: UAGCAGCACAUAAUGGUUUGUG [SEQ ID NO: 2], where at least 1, 2, 3, 4, 5, 6 in the sequence shown. Or all uracil nucleotide bases or those that can be covalently attached to the sequences shown are 5-halouracil.

In certain embodiments of the disclosure, the modified miR-15a microRNA has a nucleic acid sequence consisting of U ^F AGCAGCACAU ^F AAU ^F GGU ^F U ^F U ^F GU ^F G [SEQ ID NO: 6], wherein U ^F is halouracil, specifically 5-fluorouracil.

In other embodiments, the seed portion (UAGCAGCA) of the native miR-15a nucleotide sequence is left unmodified with 5-halouracil, while the remaining uracil bases in the remainder (non-seed portion) of the miR-15a nucleotide sequence are One or more (or all) are replaced by 5-halouracil.

In certain embodiments, the modified miR-129 microRNA has a nucleic acid sequence consisting of UAGCAGCACAU ^F AAU ^F GGU ^F U ^F U ^F GU ^F G [SEQ ID NO: 7], where U ^F is halouracil, specifically Specifically, it is 5-fluorouracil.

In another exemplary embodiment, the present disclosure is directed to nucleic acid compositions comprising modified miR-140 nucleotide sequences. In some embodiments, the native miR-140 nucleotide sequence is modified by replacing at least one U base with 5-halouracil.

In one set of embodiments, exactly one U base in the native miR-140 nucleic acid sequence is 5-halouracil. In a second set of embodiments, exactly or at least two U bases in the native miR-140 nucleotide sequence are replaced by 5-halouracil. In another set of embodiments exactly or at least 3 U bases in the miR-140 nucleotide sequence are 5-halouracil. In other embodiments exactly or at least 4 U bases in the native miR-140 nucleotide sequence are 5-halouracil. In some embodiments exactly or at least 5 U bases in the miR-140 nucleotide sequence are 5-halouracil. In still other embodiments, exactly or at least 6 U bases in the miR-140 nucleotide sequence are 5-halouracil. In certain embodiments, all U bases in the miR-140 nucleotide sequence, whether naturally occurring and/or in appendages, are 5-halouracyl.

In an exemplary embodiment, a modified microRNA nucleic acid composition of the disclosure has a nucleotide sequence of CAGU ^F GGUUUUACCCU ^F AUGGU ^F AG [SEQ ID NO: 9], where U ^F is halouracil, specifically , 5-fluorouracil.

In another exemplary embodiment, the present disclosure is directed to a nucleic acid composition comprising a modified native miR-192 or miR-215 nucleotide sequence that has been modified by replacing at least one uracil base with 5-halouracil. . In some embodiments, the modified miR-192 nucleotide sequence is modified by replacing at least one U base with 5-fluorouracil.

In another set of embodiments, exactly one U base in the modified miR-192 nucleotide sequence is 5-halouracil. In a second set of embodiments, exactly or at least two U bases in the modified miR-192 nucleotide sequence are 5-halouracyl. In another set of embodiments exactly or at least 3 U bases in the modified miR-192 nucleotide sequence are 5-halouracil. In other embodiments exactly or at least 4 U bases in the modified miR-192 or miR-215 nucleotide sequence are 5-halouracil. In certain embodiments, all U bases in the modified miR-192 or miR-215 sequence, whether naturally occurring and/or in the appendages of the nucleic acid, are 5-halouracyl.

In an exemplary embodiment, a nucleic acid composition of the disclosure has a modified miR-192 or modified miR-215 nucleotide sequence of CU ^F GACCU ^F AU ^F GAAU ^F U ^F GACAGCC [SEQ ID NO: 11], wherein UF is ^halouracil , specifically 5-fluorouracil.

In another exemplary embodiment, the disclosure is directed to a nucleic acid composition comprising a modified native miR-502 nucleotide sequence that has been modified by replacing uracil with 5-halouracil. In some embodiments, the modified miR-502 nucleotide sequence is modified by replacing at least one U base with 5-fluorouracil.

In another set of embodiments, exactly one U base in the miR-502 nucleotide sequence is 5-halouracil. In a second set of embodiments, exactly or at least two U bases in the miR-502 nucleotide sequence are 5-halouracil. In another set of embodiments, exactly or at least three U bases in the miR-502 nucleotide sequence are 5-halouracil. In other embodiments exactly or at least 4 U bases in the miR-502 nucleotide sequence are 5-halouracil. In other embodiments exactly or at least 5 U bases in the miR-502 nucleotide sequence are 5-halouracil. In other embodiments, exactly or at least 6 U bases in the modified miR-502 nucleotide sequence are 5-halouracil. In other embodiments, exactly or at least 7 U bases in the miR-502 nucleotide sequence are 5-halouracil. In certain embodiments, all U bases in the miR-502 nucleotide sequence, whether naturally occurring and/or in appendages, are 5-halouracyl.

In an exemplary embodiment, a modified miR-502 nucleic acid composition of the disclosure has a modified nucleotide sequence of AU ^F CCU ^F U ^F GCUAU ^F CU ^F GGGU ^F GCU ^F A [SEQ ID NO: 13], wherein UF is ^halouracil , specifically 5-fluorouracil.

In another exemplary embodiment, the present disclosure is directed to nucleic acid compositions comprising modified miR-506 nucleotide sequences that include 5-halouracyl. In some embodiments, the modified miR-506 nucleotide sequence is modified by replacing at least one U base with 5-halouracil, such as 5-fluorouracil.

In another set of embodiments, exactly one U base in the native miR-506 nucleotide sequence is replaced by 5-halouracil. In a second set of embodiments, exactly or at least two U bases in the modified miR-506 nucleotide sequence are 5-halouracyl. In another set of embodiments, exactly or at least 3 U bases in the modified miR-506 nucleotide sequence are 5-halouracil. In other embodiments exactly or at least 4 U bases in the modified miR-506 nucleotide sequence are 5-halouracil. In other embodiments exactly or at least 5 U bases in the modified miR-506 nucleotide sequence are 5-halouracil. In other embodiments exactly or at least 6 U bases in the modified miR-506 nucleotide sequence are 5-halouracil. In other embodiments, exactly or at least 7 U bases in the modified miR-506 nucleotide sequence are 5-halouracil. In certain embodiments, all U bases in the modified miR-506 nucleotide sequence, whether naturally occurring and/or in appendages, are 5-halouracyl.

In an exemplary embodiment, the miR-506 nucleic acid composition of the disclosure has a modified microRNA nucleotide sequence of U ^F AU ^F U ^F CAGGAAGGU ^F GU ^F U ^F ACU ^F U ^F AA [SEQ ID NO: 15], where U ^F is halouracil, specifically 5-fluorouracil.

In some embodiments, 5-halouracil is, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil. In certain embodiments, 5-halouracil is 5-fluorouracil, or a combination thereof.

The present disclosure also covers formulations comprising the modified microRNA compositions described herein or combinations thereof. In certain embodiments, formulations can include pharmaceutical formulations comprising the nucleic acid compositions described above and a pharmaceutically acceptable carrier.

In another aspect, the disclosure is directed to a method for treating cancer comprising administering to a subject an effective amount of one or more of the nucleic acid compositions described herein. In certain embodiments of the method, the nucleic acid composition comprises a modified miR-129, miR-15a, miR-192, miR-215, miR-140, miR-502 or miR-506 nucleotide sequence, wherein At least 1, 2, 3, 4, 5, 6 or more uracil nucleotide bases in the native (unmodified) nucleotide sequence are replaced by 5-halouracil. In certain embodiments, the method comprises administering to a subject having cancer or a predisposition to cancer a nucleic acid composition of the present disclosure, wherein the nucleic acid composition comprises modified miR-129 or miR-15a nucleic acid. In certain embodiments of the present disclosure, the modified microRNA administered is CU ^F U ^F U ^F U ^F U ^F GCGGU ^F CU ^F GGGCU ^F U ^F GC [SEQ ID NO: 4], CUUUUUGCGGU ^F CU ^F GGGCU ^F U ^F GC [SEQ ID NO: 5], U ^F AGCAGCACAU ^F AAU ^F GGU ^F U ^F U ^F GU ^F G [SEQ ID NO: 6], UAGCAGCACAU ^F AAU ^F GGU ^F U ^F U ^F GU ^F G [SEQ ID NO: 7], CAGU ^F GGUUUUACCCU ^F AUGGU ^F AG [SEQ ID NO: 9], CU ^F GACCU ^F AU ^F GAAU ^F U ^F GACAGCC [SEQ ID NO: 1], AU ^F CCU ^F U ^F GCUAU ^F CU ^F GGGU ^F GCU ^F A [SEQ ID NO: 13], and U ^F AU ^F U ^F CAGGAAGGU ^F GU ^F U ^F ACU ^F U ^F AA [SEQ ID NO: 15].

In certain embodiments, the subject is a mammal. In other embodiments, 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 identified as having a predisposition to develop cancer. In some embodiments, the cancer to be treated can be, for example, colorectal, gastric, esophageal, lung, ovarian, pancreatic, or cervical cancer. In certain embodiments, the methods of the present disclosure treat subjects with colorectal, pancreatic, or breast cancer.

Surprisingly, the data provided herein demonstrate that 5-FU alone in several different cancer models, including colorectal, pancreatic, and lung cancer, is superior to known anticancer agents such as Shows increased potency of the modified microRNAs described herein when compared. For example, the present disclosure provides that the modified nucleic acid compositions described are capable of Or 5-FU and the corresponding natural microRNA combination provide the unexpected finding that they are substantially more potent in inhibiting cancer progression and tumorigenesis.

As such, the present compositions and methods provide the added benefit of allowing lower dosing, resulting in lower toxicity and fewer side effects. A further significant advantage exhibited by the described nucleic acid compositions is that the ready-to-prepare compositions compared to non-halouracil-modified miR-140, miR-192, miR-215, miR-502 or miR-506 sequences. to have significantly improved efficacy as a Thus, at least in an interesting aspect, the nucleic acid compositions disclosed herein represent a substantial advance in the treatment of cancer.
Brief description of the drawing

)対照及び異所的に発現させた天然miR-129

と比較した、全てが5-FUによって置き換えられたU塩基を有する例示的な修飾miR-129核酸(模倣体)

による、4つの異なる結腸がん細胞株(HCT116、RKO、SW480及びSW620)における結腸がん細胞増殖の阻害を示すグラフである。
本開示の5-FU及び修飾マイクロRNA組成物での併用療法は、がん細胞増殖を効果的に阻害する。陰性対照(NC)、天然miR-129、5-FU、本開示の例示的な修飾miR-129核酸模倣体(5-FU-miR-129)、及び5-FU及び本開示の例示的なmiR-129模倣体の組合せ(5-FU-miR-129+5-FU)で処置されたがん細胞に関する結腸がん細胞増殖のグラフ比較。 例示的なマイクロRNA模倣体は、結腸がん細胞にアポトーシスを誘導し、細胞周期アレストの原因となる。(A)本開示の修飾miR-129核酸組成物が、陰性対照又はいくつかの異なる結腸直腸がん細胞株中で異所的に発現させた天然miR-129よりも有意に高いレベルでがん細胞アポトーシスを誘導することを示すため、FITC-アネキシンVアポトーシスアッセイによって細胞死が定量化された。(B)本開示の修飾miR-129核酸組成物(模倣体-1)が、陰性対照又は異所的に発現させた天然miR-129よりも有意に高いレベルでG1細胞周期アレストを増加させることを明らかにするため、フローサイトメトリーが行われた。 本開示の修飾マイクロRNA核酸組成物は、化学療法耐性がん幹細胞を排除する。HCT116由来結腸がん幹細胞を、漸増濃度の本開示の例示的な修飾miR-129核酸

又は5-FU

で処置した。結果は、修飾miR-129核酸が、用量依存的様式で5-FU耐性がん幹細胞を殺傷したことを示す。
例示的な修飾miR-129核酸組成物でのインビボ全身処置は、毒性副作用なく結腸がん転移を阻害する。結腸がん転移マウスモデルは、転移性ヒト結腸がん細胞の尾静脈注射を介して確立された。転移の確立の2週後、配列番号4に示される、40μgの修飾miR-129核酸組成物を、2週間隔日に1回の処置頻度の注射での静脈内注射で送達した。例示的な修飾miR-129核酸(模倣体)は、結腸がん転移(右のパネル)を阻害できたが、陰性対照miRNA(左のパネル)は、無効であった。修飾miR-129核酸で処置されたマウスは、毒性を呈さなかった。 本開示の第2の例示的な修飾マイクロRNAの抗がん活性。(A)結腸がん細胞におけるタンパク質発現をモジュレートする未修飾miR-15a(miR-15a)及び修飾miR-15a核酸組成物(模倣体-1)の能力を比較する代表的なウエスタンブロット。配列番号6(模倣体-1)に示される、修飾miR-15aは、miR-15a標的(YAP1、BMI-1、DCLK1及びECL2)を制御する能力を保持し、結腸直腸がん細胞においてTS-FdUMPを破壊する。 本開示の第2の例示的な修飾マイクロRNAの抗がん活性。(B)修飾miR-15a(模倣体-1)は、未修飾miR-15a(miR-15a)と比較した3種の異なる結腸直腸がん細胞株(HCT116、RKO、SW620)における結腸がん細胞増殖を阻害する能力の増強を示す。 対照(陰性)、未修飾miR-15a(miR-15a)及び配列番号6(模倣体-1)に示される例示的な修飾miR-15a核酸組成物に関する細胞周期制御を示すグラフである。修飾miR-15a核酸の投与は、天然miR-15aを異所的に発現する細胞及び陰性対照と比較した場合、修飾miR-15aを発現する結腸直腸がん細胞によって呈される増加したG1/S比によって示されるように、未修飾miR-15aと比較して細胞周期アレストを誘導する。 修飾miR-15a発現は、がん細胞コロニー形成を誘導するがん幹細胞の能力を低下させる。非特異的な対照マイクロRNA(陰性)を供給されたがん幹細胞の能力と比較した場合、結腸がん幹細胞において、未修飾miR-15a(miR-15a)の発現は、がん細胞コロニー形成を阻害した。本開示の例示的な修飾miR-15a(5-FU-miR-15a)での処置により、がん細胞コロニー形成が完全に防止された。 修飾miR-15aは、インビボで有効な抗がん剤である。結腸がん転移マウスモデルは、転移性ヒト結腸がん細胞の尾静脈注射を介して確立された。転移の確立の2週後、配列番号6に示される、40μgの修飾miR-15a核酸組成物を、2週間隔日に1回の処置頻度の注射での静脈内注射で送達した。例示的な修飾miR-15a核酸(模倣体)は、結腸がん転移を阻害できたが、陰性対照miRNA(陰性)は、無効であった。修飾miR-15a核酸で処置されたマウスは、毒性を呈さなかった。 本開示の例示的な修飾miR-15a及びmiR-129模倣体は、未修飾miR-15a(miR-15a)又は未修飾miR-129(miR-129)又は陰性対照で処置した細胞と比較して、膵がん(Panc-1(A);AsPC-1(B))細胞増殖を阻害する能力の増強を呈する。 本開示の例示的な修飾miR-15a及びmiR-129模倣体は、未修飾miR-15a(miR-15a)又は未修飾miR-129(miR-129)又は陰性対照で処置した細胞と比較して、ヒト乳がん(A549;C,D)細胞増殖を阻害する能力の増強を呈する。 本開示の例示的な修飾マイクロRNAは、ヒト結腸直腸がん細胞増殖を阻害する能力の増強を呈する。追加の例示的な修飾マイクロRNAを、HCT116ヒト結腸直腸がん細胞における結腸直腸がん細胞増殖を阻害する能力について試験した。(A)配列番号9に示される例示的な修飾miR-140模倣体を、ヒト結腸直腸がん細胞に投与したところ、陰性対照マイクロRNAと比較した場合に、結腸直腸がん細胞増殖を阻害する能力を増強することが明らかにされた。(B)配列番号11に示される例示的な修飾miR-192模倣体を、ヒト結腸直腸がん細胞に投与したところ、陰性対照マイクロRNAと比較した場合に、結腸直腸がん細胞増殖を阻害する能力を増強することが明らかにされた。 本開示の例示的な修飾マイクロRNAは、ヒト膵臓及び乳がん細胞増殖を阻害する能力の増強を呈する。追加の例示的な修飾マイクロRNAを、がん細胞増殖におけるそれらの影響を実験することによって、ヒトがんの異なるタイプを阻害する能力について試験した。配列番号13に示される例示的な修飾miR-502模倣体を、ヒト膵がん細胞(PANC1、A)に投与したところ、陰性対照マイクロRNAと比較した場合に、がん細胞増殖を阻害する能力を増強することが明らかにされた。更に別の例示的な修飾マイクロRNAである、配列番号15に示されるmiR-506模倣体を、ヒト膵がん細胞(PANC1、B)に投与したところ、陰性対照マイクロRNAと比較した場合に、がん細胞増殖を阻害する能力を増強することが明らかにされた。 本開示の例示的な修飾マイクロRNAは、ヒト膵臓及び乳がん細胞増殖を阻害する能力の増強を呈する。追加の例示的な修飾マイクロRNAを、がん細胞増殖におけるそれらの影響を実験することによって、ヒトがんの異なるタイプを阻害する能力について試験した。配列番号13に示される例示的な修飾miR-502模倣体を、ヒト乳がん(A549、C)に投与したところ、陰性対照マイクロRNAと比較した場合に、がん細胞増殖を阻害する能力を増強することが明らかにされた。更に別の例示的な修飾マイクロRNAである、配列番号15に示されるmiR-506模倣体を、ヒト乳がん(A549、D)に投与したところ、陰性対照マイクロRNAと比較した場合に、がん細胞増殖を阻害する能力を増強することが明らかにされた。 Chemical representations of exemplary modified microRNA nucleotide sequences of the present disclosure. (A) Chemical representation of the miR-129 nucleotide sequence shown in SEQ ID NO:4, with all U bases replaced by halouracil (ie, U ^F ). Chemical representations of exemplary modified microRNA nucleotide sequences of the present disclosure. (B) Chemical representation of miR-129, shown in SEQ ID NO:5, with only the non-seed portion of miR-129 having U bases replaced with halouracil. Chemical representations of exemplary modified microRNA nucleotide sequences of the present disclosure. (C) Chemical representation of the miR-15a nucleotide sequence shown in SEQ ID NO:6 with all U bases replaced with halouracil. Chemical representations of exemplary modified microRNA nucleotide sequences of the present disclosure. (D) Chemical representation of miR-15a, shown in SEQ ID NO:7, with only the non-seed portion of miR-15a having U bases replaced with halouracil. Chemical representations of exemplary modified microRNA nucleotide sequences of the present disclosure. (E) Chemical representation of the miR-140 nucleotide sequence shown in SEQ ID NO:9, where the U base at particular (3) is replaced by halouracil. Chemical representations of exemplary modified microRNA nucleotide sequences of the present disclosure. (F) Chemical representation of the miR-192 nucleotide sequence shown in SEQ ID NO: 11 in which the particular (5) U base is replaced by halouracil. Chemical representations of exemplary modified microRNA nucleotide sequences of the present disclosure. (G) Chemical representation of the miR-502 nucleotide sequence shown in SEQ ID NO: 13, in which the particular (7) U base is replaced by halouracil. Chemical representations of exemplary modified microRNA nucleotide sequences of the present disclosure. (H) Chemical representation of the miR-506 nucleotide sequence shown in SEQ ID NO: 15 with all (ie, 8) U bases replaced by halouracil. Exemplary modified miR-129 nucleic acids enter cancer cells and effectively reduce target protein expression. (A) Target (E2F3) specificity and potency of modified miR-129 (5-FU-miR-129 with all U bases replaced with 5-FU) compared to control miRNA and unmodified miR-129 nucleic acid. It is a graph showing. Exemplary modified miR-129 nucleic acids enter cancer cells and effectively reduce target protein expression. (B) Quantitative real-time PCR analysis showing that miR-129 mimics enter cancer cells. (C) MiR-129 mimics enter cancer cells and disrupt TS-FdUMP significantly better than 5-FU alone. non-specific (negative control,

) control and ectopically expressed native miR-129

Exemplary modified miR-129 nucleic acids with all U bases replaced by 5-FU (mimetics) compared to

is a graph showing the inhibition of colon cancer cell proliferation in four different colon cancer cell lines (HCT116, RKO, SW480 and SW620) by .
Combination therapy with 5-FU and modified microRNA compositions of the present disclosure effectively inhibits cancer cell proliferation. Negative control (NC), native miR-129, 5-FU, an exemplary modified miR-129 nucleic acid mimic of the disclosure (5-FU-miR-129), and 5-FU and an exemplary miR of the disclosure Graphical comparison of colon cancer cell proliferation for cancer cells treated with -129 mimetic combinations (5-FU-miR-129+5-FU). Exemplary microRNA mimics induce apoptosis in colon cancer cells and cause cell cycle arrest. (A) Modified miR-129 nucleic acid compositions of the present disclosure are associated with cancer at significantly higher levels than negative controls or native miR-129 ectopically expressed in several different colorectal cancer cell lines. To demonstrate that it induces cell apoptosis, cell death was quantified by the FITC-Annexin V apoptosis assay. (B) A modified miR-129 nucleic acid composition of the disclosure (Mimetic-1) increases G1 cell cycle arrest at significantly higher levels than the negative control or ectopically expressed native miR-129. To clarify, flow cytometry was performed. The modified microRNA nucleic acid compositions of the disclosure eliminate chemotherapy-resistant cancer stem cells. HCT116-derived colon cancer stem cells were treated with increasing concentrations of exemplary modified miR-129 nucleic acids of the present disclosure.

or 5-FU

treated with The results show that modified miR-129 nucleic acids killed 5-FU-resistant cancer stem cells in a dose-dependent manner.
In vivo systemic treatment with an exemplary modified miR-129 nucleic acid composition inhibits colon cancer metastasis without toxic side effects. A colon cancer metastasis mouse model was established via tail vein injection of metastatic human colon cancer cells. Two weeks after establishment of metastasis, 40 μg of the modified miR-129 nucleic acid composition shown in SEQ ID NO: 4 was delivered by intravenous injection with a treatment frequency of once every other day for two weeks. An exemplary modified miR-129 nucleic acid (mimetic) was able to inhibit colon cancer metastasis (right panel), whereas a negative control miRNA (left panel) was ineffective. Mice treated with modified miR-129 nucleic acids exhibited no toxicity. Anticancer Activity of a Second Exemplary Modified MicroRNA of the Disclosure. (A) Representative Western blot comparing the ability of unmodified miR-15a (miR-15a) and a modified miR-15a nucleic acid composition (mimic-1) to modulate protein expression in colon cancer cells. Modified miR-15a, shown in SEQ ID NO: 6 (Mimetic-1), retains the ability to regulate miR-15a targets (YAP1, BMI-1, DCLK1 and ECL2) and induces TS-1 in colorectal cancer cells. Destroy FdUMP. Anticancer Activity of a Second Exemplary Modified MicroRNA of the Disclosure. (B) Modified miR-15a (mimetic-1) colon cancer cells in three different colorectal cancer cell lines (HCT116, RKO, SW620) compared to unmodified miR-15a (miR-15a). Shows enhanced ability to inhibit proliferation. FIG. 10 is a graph showing cell cycle regulation for control (negative), unmodified miR-15a (miR-15a) and an exemplary modified miR-15a nucleic acid composition shown in SEQ ID NO: 6 (Mimetic-1). Administration of modified miR-15a nucleic acids resulted in increased G1/S exhibited by colorectal cancer cells expressing modified miR-15a when compared to cells ectopically expressing native miR-15a and negative controls. Induces cell cycle arrest compared to unmodified miR-15a, as indicated by the ratio. Modified miR-15a expression reduces the ability of cancer stem cells to induce cancer cell colony formation. In colon cancer stem cells, expression of unmodified miR-15a (miR-15a) reduced cancer cell colony formation when compared to the ability of cancer stem cells fed non-specific control microRNA (negative). inhibited. Treatment with an exemplary modified miR-15a (5-FU-miR-15a) of the disclosure completely prevented cancer cell colonization. Modified miR-15a is an effective anticancer agent in vivo. A colon cancer metastasis mouse model was established via tail vein injection of metastatic human colon cancer cells. Two weeks after establishment of the metastases, 40 μg of the modified miR-15a nucleic acid composition, shown in SEQ ID NO:6, was delivered by intravenous injection with a treatment frequency of one injection every other week for two weeks. An exemplary modified miR-15a nucleic acid (mimetic) was able to inhibit colon cancer metastasis, whereas a negative control miRNA (negative) was ineffective. Mice treated with modified miR-15a nucleic acids exhibited no toxicity. Exemplary modified miR-15a and miR-129 mimetics of the present disclosure are compared to cells treated with unmodified miR-15a (miR-15a) or unmodified miR-129 (miR-129) or negative control , exhibit enhanced ability to inhibit pancreatic cancer (Panc-1(A); AsPC-1(B)) cell proliferation. Exemplary modified miR-15a and miR-129 mimetics of the present disclosure are compared to cells treated with unmodified miR-15a (miR-15a) or unmodified miR-129 (miR-129) or negative control , exhibit enhanced ability to inhibit human breast cancer (A549; C,D) cell proliferation. Exemplary modified microRNAs of the present disclosure exhibit enhanced ability to inhibit human colorectal cancer cell proliferation. Additional exemplary modified microRNAs were tested for their ability to inhibit colorectal cancer cell proliferation in HCT116 human colorectal cancer cells. (A) An exemplary modified miR-140 mimetic set forth in SEQ ID NO: 9 is administered to human colorectal cancer cells and inhibits colorectal cancer cell proliferation when compared to negative control microRNAs. shown to enhance capacity. (B) An exemplary modified miR-192 mimic set forth in SEQ ID NO: 11 is administered to human colorectal cancer cells and inhibits colorectal cancer cell proliferation when compared to a negative control microRNA. shown to enhance capacity. Exemplary modified microRNAs of the present disclosure exhibit enhanced ability to inhibit human pancreatic and breast cancer cell proliferation. Additional exemplary modified microRNAs were tested for their ability to inhibit different types of human cancers by examining their effects on cancer cell proliferation. Ability of an exemplary modified miR-502 mimic set forth in SEQ ID NO: 13 to inhibit cancer cell proliferation when administered to human pancreatic cancer cells (PANC1, A) when compared to a negative control microRNA was shown to enhance the When administered to human pancreatic cancer cells (PANC1, B), yet another exemplary modified microRNA, the miR-506 mimetic set forth in SEQ ID NO: 15, when compared to the negative control microRNA: It has been shown to enhance the ability to inhibit cancer cell proliferation. Exemplary modified microRNAs of the present disclosure exhibit enhanced ability to inhibit human pancreatic and breast cancer cell proliferation. Additional exemplary modified microRNAs were tested for their ability to inhibit different types of human cancers by examining their effects on cancer cell proliferation. An exemplary modified miR-502 mimetic set forth in SEQ ID NO: 13 is administered to human breast cancer (A549, C) and enhances its ability to inhibit cancer cell proliferation when compared to negative control microRNAs It was revealed that Administration of yet another exemplary modified microRNA, the miR-506 mimetic set forth in SEQ ID NO: 15, to human breast cancer (A549, D) resulted in increased cancer cell It has been shown to enhance the ability to inhibit proliferation.

DETAILED DESCRIPTION OF THE DISCLOSURE The present disclosure provides nucleic acid compositions incorporating one or more halouracil molecules. While not wishing to be bound by any one particular theory, the present disclosure surprisingly suggests that replacement of uracil nucleotides within microRNA oligonucleotide sequences with 5-halouracil is associated with cancer development, cancer progression and It has been shown to increase the ability of tumorigenesis. As such, the present disclosure provides various nucleic acid (eg, microRNA) compositions having 5-halouracyl molecules incorporated into the nucleic acid sequence and methods of using the same. The disclosure further provides a formulation, such as a pharmaceutical composition, comprising the modified nucleic acid composition and a method of treating cancer comprising administering it to a subject in need thereof.

Nucleic acid composition.
The terms "microRNA" or "miRNA" or "miR" are used interchangeably and are small non-coding ribose nucleic acids capable of regulating gene expression by interacting with messenger RNA molecules (mRNA), DNA or proteins. (RNA) refers to a molecule. Typically, microRNAs consist of nucleic acid sequences of about 19-25 nucleotides (bases) and are found in mammalian cells.

The terms "modified microRNA", "modified miRNA", "modified miR" or "mimetic" are used interchangeably herein and refer to microRNAs that differ from natural or endogenous microRNA (unmodified microRNA) polynucleotides. refers to RNA. More specifically, in the present disclosure, modified microRNAs differ from unaltered or unmodified microRNA nucleic acid sequences by one or more bases. In some embodiments of the present disclosure, modified microRNAs of the present disclosure include at least one uracil (U) nucleotide base replaced by 5-halouracil. In other embodiments, the modified microRNA has additional nucleotides (i.e., adenine (A), cytosine (C), uracil (U), and guanine (G)) and at least one substituted with 5-halouracil. Contains uracil base.

In one aspect of the present disclosure, a nucleic acid composition comprising a modified microRNA nucleotide sequence having at least one uracil base (U, U base) replaced with 5-halouracil, such as 5-fluorouracil (5-FU). is described. As discussed further herein, the nucleic acid compositions of the present disclosure are useful, at least in the treatment of cancer, particularly colorectal, pancreatic and breast cancer.

In some embodiments, a nucleic acid composition contains a nucleotide sequence that has been modified by derivatizing at least one uracil nucleobase at position 5 with a group that provides an effect analogous to a halogen atom. In some embodiments, groups that provide similar effects have a mass or space relative to halogen atoms such as molecular weights up to or less than 20, 30, 40, 50, 60, 70, 80, 90, or 80 g/mol. have similar sizes in terms of volume. In certain embodiments, groups that provide similar effects to halogen atoms are, for example, methyl groups, trihalomethyl (e.g., trifluoromethyl) groups, pseudohalides (e.g., trifluoromethanesulfonate, cyano, or cyano acid) or deuterium (D) atoms. Groups that provide effects analogous to halogen atoms may be present in the absence or in addition to 5-halouracyl bases in microRNA nucleotide sequences.

Moreover, in other embodiments, groups that provide similar effects to halogen atoms are located in the natural (or seed) and/or appendages of microRNA nucleotide sequences, readily discernible by those skilled in the art. good too. In some embodiments, groups that provide similar effects to one or more (or all) of the above types of halogen atoms are omitted from the modified miRNA nucleotide sequence. When all such alternative groups are excluded, only one or more halogen atoms are present as substituents at position 5 of one or more uracil groups in the microRNA nucleotide sequence.

In certain embodiments, the modified microRNA has more than one or exactly one uracil replaced with 5-halouracil.

In some embodiments, the modified microRNA nucleotide sequence includes 3, 4, 5, 6, 7, 8 or more uracil bases replaced with 5-halouracil.

In other embodiments, all uracil nucleotide bases of the modified mRNA are replaced by 5-halouracil.

In some embodiments, 5-halouracil is, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil. In certain embodiments, 5-halouracil is 5-fluorouracil.

As used herein, the term "miR-129" is intended to be synonymous with the term "microRNA-129" or "miRNA-129" and refers to an oligonucleotide having the following nucleotide sequence: CUUUUUGCGGUCUGGGCUUGC[ SEQ ID NO: 1], where C=cytosine, U=uracil, and G=guanine bases. The foregoing nucleotide sequences are referred to herein as unmodified miR-129 (ie, "native") sequences, unless otherwise specified. MiR-129 may also be referred to in the art as hsa-miR-129 or hsa-miR-129-5p (accession numbers MI0000252 and MIMAT0000242). MiR-129 is well known and extensively studied. See, eg, J. Wu et al., Cell Cycle, (2010) 9:9, 1809-1818. As is well known in the art, miR-129 sequences may be modified to produce "miR-129 mimetics," which have sequences modified from the native sequence, but are -129 retains the known function or activity. Unless otherwise stated, all such modified miR-129 compositions are considered herein to be within the scope of the term "miR-129 mimetic" as used herein.

Certain modified miR-129 nucleic acid sequences (mimetics) of interest have two U bases (i.e., two U-containing nucleotides). In the above sequence, the two terminal U bases continue or extend the miR-129 native sequence from 21 nucleotide bases to 23 nucleotide bases. Generally, miR-129 mimetics contain no more than 1, 2, 3, 4, or 5 additional bases (i.e., additional nucleotides) covalently attached to the miR-129 native sequence, wherein , the additional bases may be independently selected from C, U, G, and C, or the additional bases may be exclusively U. Typically, miR-129 is used in single-chain form, although double-chain versions are also contemplated herein.

In one embodiment, the present disclosure is directed to a nucleic acid composition containing a miR-129 nucleotide sequence that has been modified by replacing at least one uracil nucleobase (i.e., U base) with a 5-halouracil, i.e., Here, at least one U base in the miR-129 sequence, whether naturally occurring and/or in the appendage, is 5-halouracil. 5-halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil.

In a first set of embodiments, exactly one U base in the miR-129 sequence is 5-halouracil. In a second set of embodiments, exactly or at least two U bases in the miR-129 sequence are 5-halouracil. In a third set of embodiments, exactly or at least three U bases in the miR-129 sequence are 5-halouracil. In a fourth set of embodiments exactly or at least 4 U bases in the miR-129 sequence are 5-halouracil. In a fifth set of embodiments exactly or at least 5 U bases in the miR-129 sequence are 5-halouracil. In a sixth set of embodiments, all U bases in the miR-129 sequence, whether naturally occurring and/or in appendages, are 5-halouracyl.

In certain embodiments, the nucleic acid composition of the disclosure has the modified microRNA nucleotide sequence of CU ^F U ^F U ^F U ^F U ^F GCGGU ^F CU ^F GGGCU ^F U ^F GC as set forth in SEQ ID NO: 4, wherein and U ^F is halouracil, specifically 5-fluorouracil.

The U base replaced by 5-halouracyl in the miR-129 sequence may be located in the unmodified portion of the miR-129 sequence, as provided above, or in the case of miR-129 mimetics, may be located at one or more U bases covalently attached to native miR-129, as provided above. In other embodiments, the seed portion (GUUUUUGC) of the native miR-129 nucleotide sequence is left unmodified with 5-halouracyl, while one or more of the U bases remaining in the remainder of the miR-129 nucleotide sequence (or all) are replaced with an equivalent number of 5-halouracils.

For example, in certain embodiments, a nucleic acid composition of the present disclosure has the modified microRNA nucleotide sequence of CUUUUUGCGGU ^F CU ^F GGGCU ^F U ^F GC set forth in SEQ ID NO: 5, where U ^F is halouracil, Specifically, it is 5-fluorouracil.

In an alternative embodiment, the nucleic acid composition comprises a miR-129 nucleotide sequence that has been modified by derivatizing at least one uracil (U) nucleobase with a group that provides an effect similar to a halogen atom at position 5. contains. In some embodiments, groups that provide similar effects have a mass or space relative to halogen atoms such as molecular weights up to or less than 20, 30, 40, 50, 60, 70, 80, 90, or 80 g/mol. have similar sizes in terms of volume. Groups that provide effects analogous to halogen atoms are, for example, methyl groups, trihalomethyl (e.g., trifluoromethyl) groups, pseudohalides (e.g., trifluoromethanesulfonate, cyano, or cyanate) or deuterium ( D) It may be an atom. Groups that provide effects analogous to halogen atoms may be present in the absence of or in addition to the 5-halouracyl base in the miR-129 nucleotide sequence. Moreover, groups that provide halogen-like effects may be located in the native (or seed) and/or appendages of the miR-129 nucleotide sequence. In some embodiments, groups that provide similar effects to one or more (or all) of the above types of halogen atoms are excluded from the miR-129 nucleotide sequence. When all such alternative groups are excluded, only one or more halogen atoms are present as substituents at position 5 of one or more uracil groups in the miR-129 nucleotide sequence.

In another exemplary embodiment, the present disclosure is directed to nucleic acid compositions comprising modified miR-15a nucleotide sequences. In some embodiments, the miR-15a nucleotide sequence is modified by replacing at least one U base with 5-halouracil.

As used herein, the term "miR-15a" is intended to be synonymous with the term "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 foregoing nucleotide sequences are referred to herein as miR-15a unmodified (ie, "native") sequences, unless otherwise specified. MiR-15a may also be referred to in the art as hsa-miR-15a or hsa-miR-15a-5p (accession number MI0000069). MiR-15a is well known and extensively studied, eg, 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. As described above for miR-129 mimetics, methods for making miR-15a mimetics are known to those skilled in the art. Unless otherwise stated, all such modified miR-15a forms are considered herein within the scope of the term "miR-15a mimetic" as used herein.

Generally, modified miR-15a (i.e., miR-15a mimics) contain no more than 1, 2, 3, 4, or 5 additional nucleotides covalently attached to the miR-15a native sequence; wherein the additional bases are independently selected from C, U, G and C or the additional bases may be exclusively U. Typically, miR-15a is used in single-chain form, although double-chain versions are also contemplated herein.

In some embodiments, at least one U base in the miR-15a sequence, whether naturally occurring and/or in an appendage, is 5-halouracyl. 5-halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil.

In one set of embodiments, exactly one U base in the miR-15a sequence is 5-halouracil. In a second set of embodiments exactly or at least two U bases in the miR-15a sequence are 5-halouracil. In another set of embodiments exactly or at least 3 U bases in the miR-15a oligonucleotide sequence are 5-halouracil. In other embodiments exactly or at least 4 U bases in the miR-15a sequence are 5-halouracil. In some embodiments exactly or at least 5 U bases in the miR-15a sequence are 5-halouracil. In still other embodiments, exactly or at least 6 U bases in the miR-15a sequence are 5-halouracil. In certain embodiments, all U bases in the miR-15a sequence, whether naturally occurring and/or in appendages, are 5-halouracyl.

In one embodiment, the nucleic acid composition of the present disclosure has a modified microRNA nucleotide sequence of U ^F AGCAGCACAU ^F AAU ^F GGU ^F U ^F U ^F GU ^F G [SEQ ID NO: 6], where U ^F is Halouracil, specifically 5-fluorouracil.

The U base replaced by 5-halouracyl in the miR-15a sequence may be located in the unmodified portion of the miR-15a sequence, as provided above, or, in the case of miR-15a mimics, similarly may be located at one or more uracil bases attached to native miR-15a, as provided above in .

In other embodiments, the seed portion (UAGCAGCA) of the native miR-15a nucleotide sequence is left unmodified with 5-halouracyl, while the remaining U bases in the remainder (non-seed portion) of the miR-15a nucleotide sequence are One or more (or all) are replaced with 5-halouracil.

In certain embodiments, the nucleic acid compositions of this disclosure have a modified miR-15a nucleotide sequence of UAGCAGCACAU ^F AAU ^F GGU ^F U ^F U ^F GU ^F G [SEQ ID NO: 7], where U ^F is Halouracil, specifically 5-fluorouracil.

In certain embodiments, the nucleic acid composition comprises a miR-15a nucleotide sequence that has been modified by derivatizing at least one uracil (U) nucleobase at position 5 with a group that provides an effect similar to a halogen atom. contains. In some embodiments, groups that provide similar effects have a mass or space relative to halogen atoms such as molecular weights up to or less than 20, 30, 40, 50, 60, 70, 80, 90, or 80 g/mol. have similar sizes in terms of volume. Groups that provide effects analogous to halogen atoms are, for example, methyl groups, trihalomethyl (e.g., trifluoromethyl) groups, pseudohalides (e.g., trifluoromethanesulfonate, cyano, or cyanate) or deuterium ( D) It may be an atom. Groups that provide effects analogous to halogen atoms may be present in the absence of or in addition to the 5-halouracyl base in the miR-15a nucleotide sequence. Moreover, groups that provide halogen-like effects may be located in the native (or seed) and/or appendages of the miR-15a nucleotide sequence.

In some embodiments, groups that provide similar effects to one or more (or all) of the above types of halogen atoms are excluded from the miR-15a nucleotide sequence. When all such alternative groups are excluded, only one or more halogen atoms are present as substituents at position 5 of one or more uracil groups in the miR-15a nucleotide sequence.

In another exemplary embodiment, the present disclosure is directed to nucleic acid compositions comprising modified miR-140 nucleotide sequences. In some embodiments, the miR-140 nucleotide sequence is modified by replacing at least one U base with 5-halouracil.

As used herein, the term "miR-140" is intended to be synonymous with the term "microRNA-140" or "miRNA-140" and refers to an oligonucleotide having the following nucleotide sequence: CAGUGGUUUUUACCCUAUGGUAG[ SEQ ID NO:8], where A=adenine, C=cytosine, U=uracil, and G=guanine base. The aforementioned nucleotide sequences are referred to herein as miR-140 unmodified (ie, "native") sequences, unless otherwise specified. MiR-140 may also be referred to by accession number NT_010498 or by miRBase accession number MI0000456. MiR-140 is well known and extensively studied, eg, Zhai, H. et al., Oncotarget. (2015) 6: 19735-46. Methods for generating miR-140 mimetics are known to those skilled in the art, as described above for the exemplary mimetics miR-129 and miR-15a. Unless otherwise stated, all such modified miR-140 forms are considered herein within the scope of the term "miR-140 mimetic" as used herein.

Generally, modified miR-140 nucleic acids (i.e., miR-140 mimetics) contain no more than 1, 2, 3, 4, or 5 additional nucleotides covalently attached to the miR-140 native sequence. , wherein the additional bases are independently selected from C, U, G, and C or the additional bases may be exclusively U. Typically, miR-140 mimics are used in single-chain form, although double-chain versions are also contemplated herein.

In some embodiments, at least one U base in the miR-140 sequence, whether naturally occurring and/or in an appendage, is 5-halouracyl. 5-halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil.

In one set of embodiments, exactly one U base in the miR-140 mimetic sequence is 5-halouracil. In a second set of embodiments, exactly or at least two U bases in the miR-140 sequence are 5-halouracil. In another set of embodiments, exactly or at least three U bases in the miR-140 oligonucleotide sequence are 5-halouracil. In other embodiments exactly or at least 4 U bases in the miR-140 sequence are 5-halouracil. In some embodiments exactly or at least 5 U bases in the miR-140 mimetic sequence are 5-halouracil. In still other embodiments, exactly or at least 6 U bases in the miR-140 mimetic sequence are 5-halouracil. In certain embodiments, all U bases in the miR-140 sequence, whether naturally occurring and/or in appendages, are 5-halouracyl.

In an exemplary embodiment, a nucleic acid composition of the disclosure has a modified miR-140 nucleotide sequence of CAGU ^F GGUUUUACCCU ^F AUGGU ^F AG [SEQ ID NO: 9], where U ^F is halouracil, specifically is 5-fluorouracil.

The U base replaced by 5-halouracyl in the miR-140 mimetic sequence may be located in the unmodified portion of the miR-140 sequence, as provided above, or It may be located at one or more uracil bases attached to native miR-140.

In other embodiments, the seed portion of the native miR-140 nucleotide sequence is left unmodified with 5-halouracyl, while one or more of the U bases remaining in the remainder (non-seed portion) of the miR-140 nucleotide sequence. (or all) are replaced with 5-halouracil.

In another exemplary embodiment, the disclosure is directed to nucleic acid compositions comprising modified miR-192 nucleotide sequences. In some embodiments, the miR-192 nucleotide sequence is modified by replacing at least one U base with 5-halouracil.

As used herein, the term "miR-192" is synonymous with the terms "microRNA-192," "miRNA-192," "microRNA-215," "miR-215," or "miRNA-215." is intended to refer to an oligonucleotide having the following nucleotide sequence: CUGACCUAUGAAUUGACAGCC [SEQ ID NO: 10], where A = adenine, C = cytosine, U = uracil, and G = guanine bases. The aforementioned nucleotide sequences are referred to herein as miR-192 unmodified (ie, "native") sequences, unless otherwise specified. MiR-192 may also be referred to as hsa-mir-192, has-mir-215 or by miRBase accession number MI0000234 or MIMAT0000222. MiR-192 is well known and extensively studied, eg, Song, B. et al., Clin. Cancer Res. (2008) 14: 8080-8086 and Song, B. et al., Mol. (2010), 9:96 pp. 1476-4598. As described above for the exemplary mimetics miR-129, miR-140 and miR-15a, methods for generating miR-192 mimetics are known to those skilled in the art. Unless otherwise stated, all such modified miR-192 nucleic acid forms are considered herein within the scope of the term "miR-192 mimetic" as used herein.

Generally, a modified miR-192 (i.e., a miR-192 mimetic) contains no more than 1, 2, 3, 4, or 5 additional nucleotides covalently attached to the miR-192 native sequence; wherein the additional bases are independently selected from C, U, G and C or the additional bases may be exclusively U. Typically, miR-192 mimetics are used in single-chain form, although double-chain versions are also contemplated herein.

In some embodiments, at least one U base in the miR-192 or miR-215 sequence, whether naturally occurring and/or in the appendage, is 5-halouracil. 5-halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil.

In another set of embodiments, exactly one U base in the miR-192 mimetic sequence is 5-halouracil. In a second set of embodiments, exactly or at least two U bases in the miR-192 sequence are 5-halouracil. In another set of embodiments, exactly or at least three U bases in the miR-192 oligonucleotide sequence are 5-halouracil. In other embodiments exactly or at least 4 U bases in the miR-192 sequence are 5-halouracil. In certain embodiments, all U bases in the miR-192 sequence, whether naturally occurring and/or in appendages, are 5-halouracyl.

In an exemplary embodiment, a nucleic acid composition of the disclosure has a modified miR-192 nucleotide sequence of CU ^F GACCU ^F AU ^F GAAU ^F U ^F GACAGCC [SEQ ID NO: 11], where U ^F is halouracil , specifically 5-fluorouracil.

The U base replaced by 5-halouracyl in the miR-192 mimetic sequence may be located in the unmodified portion of the miR-192 sequence, as provided above, or It may be located at one or more uracil bases attached to native miR-192.

In other embodiments, the seed portion of the native miR-192 nucleotide sequence is left unmodified with 5-halouracil, while one or more of the U bases remaining in the remainder (non-seed portion) of the miR-192 nucleotide sequence. (or all) are replaced with 5-halouracil or a combination thereof.

In another exemplary embodiment, the disclosure is directed to nucleic acid compositions comprising modified miR-502 nucleotide sequences. In some embodiments, the miR-502 nucleotide sequence is modified by replacing at least one U base with 5-halouracil.

As used herein, the term "miR-502" is intended to be synonymous with the term "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 aforementioned nucleotide sequences are referred to herein as miR-502 unmodified (ie, "native") sequences, unless otherwise specified. MiR-502 may also be referred to as hsa-mir-502 or MI0003186 or MIMAT0002873 by miRBase accession number. MiR-502 is well known and extensively studied, eg, Zhai, H. et al., Oncogene. (2013), 32:12 pp. 1570-1579. As described above for the exemplary mimetics miR-129, miR-140, miR-192 and miR-15a, methods for generating miR-502 mimetics are known to those skilled in the art. Unless otherwise stated, all such modified miR-502 nucleic acid forms are considered herein within the scope of the term "miR-502 mimetic" as used herein.

Generally, a modified miR-502 (i.e., miR-502 mimetic) contains no more than 1, 2, 3, 4, or 5 additional nucleotides covalently attached to the miR-502 native sequence; wherein the additional bases are independently selected from C, U, G and C or the additional bases may be exclusively U. Typically, miR-502 mimetics are used in single-chain form, although double-chain versions are also contemplated herein.

In some embodiments, at least one U base in the miR-502 sequence, whether naturally occurring and/or in an appendage, is 5-halouracil. 5-halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil.

In another set of embodiments, exactly one U base in the miR-502 mimetic sequence is 5-halouracil. In a second set of embodiments, exactly or at least two U bases in the miR-502 sequence are 5-halouracil. In another set of embodiments, exactly or at least three U bases in the miR-502 oligonucleotide sequence are 5-halouracil. In other embodiments exactly or at least 4 U bases in the miR-502 sequence are 5-halouracil. In other embodiments exactly or at least 5 U bases in the miR-502 sequence are 5-halouracil. In other embodiments, exactly or at least 6 U bases in the miR-502 sequence are 5-halouracil. In other embodiments, exactly or at least 7 U bases in the miR-502 sequence are 5-halouracil. In certain embodiments, all U bases in the miR-502 sequence, whether naturally occurring and/or in appendages, are 5-halouracyl.

In an exemplary embodiment, a nucleic acid composition of the disclosure has a modified miR-502 nucleotide sequence of AU ^F CCU ^F U ^F GCUAU ^F CU ^F GGGU ^F GCU ^F A [SEQ ID NO: 13], where U ^F is halouracil, specifically 5-fluorouracil.

The U base replaced by 5-halouracyl in the miR-502 mimetic sequence may be located in the unmodified portion of the miR-502 sequence, as provided above, or It may be located at one or more uracil bases attached to native miR-502.

In other embodiments, the seed portion of the native miR-502 nucleotide sequence is left unmodified with 5-halouracyl, while one or more of the U bases remaining in the remainder (non-seed portion) of the miR-502 nucleotide sequence. (or all) are replaced by 5-halouracil or a combination thereof.

In another exemplary embodiment, the present disclosure is directed to nucleic acid compositions comprising modified miR-506 nucleotide sequences. In some embodiments, the miR-506 nucleotide sequence is modified by replacing at least one U base with 5-halouracil.

As used herein, the term "miR-506" is intended to be synonymous with the term "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 foregoing nucleotide sequences are referred to herein as miR-506 unmodified (ie, "native") sequences, unless otherwise specified. MiR-506 may also be referred to as hsa-mir-506 or MI0003193 or MIMAT0022701 by miRBase accession number. MiR-506 is well known and extensively studied, see, for example, Li, J. et al., Oncotarget. (2016) 7:38 pp. 62778-62788 and Li, J. et al., Oncogene. 2016) 35 pp. 5501-5514. As described above for the exemplary mimetics miR-129, miR-140, miR-502, miR-192 and miR-15a, methods of making miR-506 mimetics are known to those skilled in the art. . Unless otherwise stated, all such modified miR-506 nucleic acid forms are considered herein within the scope of the term "miR-506 mimetic" as used herein.

Generally, a modified miR-506 (i.e., miR-506 mimetic) contains no more than 1, 2, 3, 4, or 5 additional nucleotides covalently attached to the miR-506 native sequence, wherein the additional bases are independently selected from C, U, G and C or the additional bases may be exclusively U. Typically, miR-506 mimics are used in single-chain form, although double-chain versions are also contemplated herein.

In some embodiments, at least one U base in the miR-506 sequence, whether naturally occurring and/or in an appendage, is 5-halouracyl. 5-halouracil can be, for example, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil.

In another set of embodiments, exactly one U base in the miR-506 mimetic sequence is 5-halouracil. In a second set of embodiments, exactly or at least two U bases in the miR-506 sequence are 5-halouracil. In another set of embodiments exactly or at least 3 U bases in the miR-506 oligonucleotide sequence are 5-halouracil. In other embodiments exactly or at least 4 U bases in the miR-506 sequence are 5-halouracil. In other embodiments exactly or at least 5 U bases in the miR-506 sequence are 5-halouracil. In other embodiments, exactly or at least 6 U bases in the miR-506 sequence are 5-halouracil. In other embodiments, exactly or at least 7 U bases in the miR-506 sequence are 5-halouracil. In certain embodiments, all U bases in the miR-506 sequence, whether naturally occurring and/or in appendages, are 5-halouracyl.

In an exemplary embodiment, a nucleic acid composition of the disclosure has a modified miR-506 nucleotide sequence of U ^F AU ^F U ^F CAGGAAGGU ^F GU ^F U ^F ACU ^F U ^F AA [SEQ ID NO: 15], wherein , U ^F is halouracil, specifically 5-fluorouracil.

The U base replaced by 5-halouracyl in the miR-506 mimetic sequence may be located in the unmodified portion of the miR-506 sequence, as provided above, or It may be located at one or more uracil bases attached to native miR-506.

In other embodiments, the seed portion of the native miR-506 nucleotide sequence is left unmodified with 5-halouracyl, while one or more of the U bases remaining in the remainder (non-seed portion) of the miR-506 nucleotide sequence. (or all) are replaced with 5-halouracil or a combination thereof.

The modified microRNA nucleic acid compositions described herein can be synthesized using any of the well-known methods for synthesizing nucleic acids. In certain embodiments, nucleic acid compositions are manufactured by any of the well-known processes using automated oligonucleotide synthesis, eg, phosphoramidite chemistry. To introduce one or more 5-halouracyl bases into modified miR sequences (e.g., miR-15a, miR-140, miR-192, miR-502, miR-506 or miR-129 sequences), 5-halouracyl nucleoside phosphoramidites can be included as precursor bases, along with phosphoramidite derivatives of nucleosides containing natural bases (eg, A, U, G, and C) included in nucleic acid sequences.

In some embodiments, the nucleic acid compositions of the present disclosure are synthesized biosynthetically, e.g., by using in vitro RNA transcription from plasmids, PCR fragments, or synthetic DNA templates, or using recombinant (in vivo) RNA expression methods. can be manufactured by See, for example, C. M. Dunham et al., Nature Methods, (2007) 4(7), pp. 547-548. A microRNA sequence (e.g., miR-15a sequence, miR-140 sequence, miR-192 sequence, miR-502 sequence, miR-506 sequence or miR-129 sequence) is isolated from polyethylene by techniques well known in the art, for example. It may be further chemically modified by functionalization with glycols (PEG) or carbohydrates or targeting agents, particularly cancer cell targeting agents such as folic acid. To include such groups, reactive groups (eg, amino, aldehyde, thiol, or carboxylate groups) that can be used to attach desired functional groups are first included in the oligonucleotide sequence. You can let Such reactive or functional groups may be incorporated into manufactured nucleic acid sequences, although reactive or functional groups include non-nucleoside phosphoramidite-containing reactive groups or reactive precursor groups, automated Inclusion may be easier by using oligonucleotide synthesis.

Modified Nucleic Acid Formulations In another aspect, the present disclosure is directed to formulations of the modified nucleic acid compositions described herein. For example, the nucleic acid compositions can be formulated for pharmaceutical use. In certain embodiments, the formulation is a pharmaceutical composition containing a nucleic acid composition described herein and a pharmaceutically acceptable carrier. In other embodiments, the formulations of the present disclosure contain modified miR-129 nucleic acids, modified miR-15a nucleic acids, modified miR-140 nucleic acids, modified miR-192 nucleic acids, modified miR-502, modified miR-506 nucleic acids or combinations thereof and including a pharmaceutically acceptable carrier. More specifically, the modified microRNA nucleic acids shown in the following nucleotide sequences can be formulated for pharmaceutical applications and uses; CU ^F U ^F U ^F U ^F U ^F GCGGU ^F CU ^F GGGCU ^F U ^F GC [SEQ ID NO: 4], CUUUUUGCGGU ^F CU ^F GGGCU ^F U ^F GC [SEQ ID NO: 5], U ^F AGCAGCACAU ^F AAU ^F GGU ^F U ^F U ^F GU ^F G [SEQ ID NO: 6], UAGCAGCACAU ^F AAU ^F GGU ^F U ^F U ^F GU ^F G [SEQ ID NO: 7], CAGU ^F GGUUUUACCCU ^F AUGGU ^F AG [SEQ ID NO: 9], CU ^F GACCU ^F AU ^F GAAU ^F U ^F GACAGCC [SEQ ID NO: 1], AU ^F CCU ^F U ^F GCUAU ^F CU ^F GGGU ^F GCU ^F A [SEQ ID NO: 13], and U ^F AU ^F U ^F CAGGAAGGU ^F GU ^F U ^F ACU ^F U ^F AA [SEQ ID NO: 15].

The term "pharmaceutically acceptable carrier" is used herein synonymously with pharmaceutically acceptable diluent, vehicle, or excipient. Depending on the type of pharmaceutical composition and the intended mode of administration, the nucleic acid composition can be dissolved or suspended (eg, as an emulsion) in the pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is any liquid or solid compound, material, composition and/or dosage form within the scope of sound medical judgment for use in contact with the tissue of interest. may be of A carrier should be "acceptable" in the sense of not being injurious to the subject to which it is provided, and being compatible with the other ingredients of the formulation, i.e., their biological or chemical do not change functionality.

Some non-limiting examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn and potato starch; cellulose and its derivatives such as carboxylate; waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as ethylene glycol and propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers; water; Contains compatible substances. Pharmaceutically acceptable carriers can also include manufacturing aids (eg, lubricants, magnesium talc, calcium or zinc stearate, or stearic acid), solvents, or encapsulating materials. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. Other suitable excipients are found in standard pharmaceutical texts such as "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Ed. Mack Publishing Company, Easton, Pa., (1995). sell.

In some embodiments, pharmaceutically acceptable carriers can include diluents, which increase the bulk of the solid pharmaceutical composition and make the pharmaceutical dosage form easier to handle for the patient and caregiver. Diluents for solid compositions include, for example, microcrystalline cellulose (eg Avicel®), microfine cellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates ( dextrate), dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, kaolin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, polymethacrylates (e.g. Eudragit®), potassium chloride, powder Includes modified cellulose, sodium chloride, sorbitol and talc.

Nucleic acid compositions of the disclosure can be formulated into compositions and dosage forms according to methods known in the art. In certain embodiments, formulated compositions may be specially formulated for administration in solid or liquid forms, including those adapted for: (1) tablets, capsules, powders, granules; oral administration such as preparations, pastes, aqueous or non-aqueous solutions or suspensions, drenches, or syrups for application to the tongue; (2) as sterile solutions or suspensions, such as subcutaneously; (3) topical application, such as as a cream, ointment, or spray applied to the skin, lungs, or mucous membranes; or (4) as a pessary, cream, or foam, etc. (5) sublingually or buccally; (6) ocularly; (7) transdermally; or (8) nasally.

In some embodiments, formulations of the present disclosure include solid pharmaceuticals that are compacted into a dosage form, such as a tablet, and are designed to help bind the active ingredient and other excipients together after compression. Excipients with the function of containing may be included. Binders for solid pharmaceutical compositions include acacia, alginic acid, carbomers (e.g. Carbopol), sodium carboxymethylcellulose, dextrin, ethylcellulose, gelatin, guar gum, hydrogenated vegetable oils, hydroxyethylcellulose, hydroxypropylcellulose (e.g. Klucel ®), hydroxypropylmethylcellulose (e.g. Methocel®), liquid glucose, magnesium aluminum silicate, maltodextrin, methylcellulose, polymethacrylates, povidone (e.g. Kollidon®, Plasdone®). ), pregelatinized starch, sodium alginate and starch.

The dissolution rate of a compacted solid pharmaceutical composition in the stomach of a subject can be increased by adding a disintegrant to the composition. Disintegrants include alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium (e.g. Ac-Di-Sol®, Primellose®), colloidal silicon dioxide, croscarmellose sodium, crospovidone (e.g. Kollidon (R), Polyplasdone (R)), guar gum, magnesium aluminum silicate, methylcellulose, microcrystalline cellulose, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate, sodium starch glycolate (e.g. Explotab ( ®)) and starch.

Thus, in certain embodiments, glidants may be added to the formulation to improve the flowability of non-compacted solid dosage forms and improve dosing accuracy. Excipients that can function as glidants include colloidal silicon dioxide, magnesium trisilicate, powdered cellulose, starch, talc and tribasic calcium phosphate.

When a dosage form such as a tablet is made from compaction of a powdered composition, the composition is subjected to pressure from punches and dye. Some excipients and active ingredients have a tendency to adhere to the surface of punches and pigments, which can cause the product to have pitting and other surface irregularities. Lubricants can be added to the composition to reduce adhesion and facilitate release of the product from the dye. Lubricants include magnesium stearate, calcium stearate, glyceryl monostearate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil, mineral oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, stearyl fumarate. Included are sodium stearyl fumarate, stearic acid, talc and zinc stearate.

A formulated pharmaceutical composition for tableting 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 blended and then further granulated in the presence of a liquid, typically water, which causes the powder to agglomerate into granules. mixed. The granules are screened and/or milled, dried and then screened and/or milled to the desired particle size. The granules may then be tableted, or other excipients may be added before tableting (eg, glidants and/or lubricants). A tableting composition may be prepared by conventional dry blending. For example, the mixed composition of active agent and excipients can be compacted into slugs or sheets and then ground into compacted granules. Compacted granules can subsequently be compressed into tablets.

In other embodiments, as an alternative to dry granulation, the blended composition can be directly compressed into a compacted dosage form using direct compression techniques. Direct compression produces a more uniform tablet without granules. Excipients that are particularly well suited for direct compression tableting include microcrystalline cellulose, spray dried lactose, dicalcium phosphate dihydrate and colloidal silica. The proper use of these and other excipients in direct compression tableting will be known to those skilled in the art, especially those with experience and skill in attempting to formulate direct compression tablets. Capsule filling may include any of the aforementioned mixtures and granules described with reference to tableting; however, they are not subjected to the final tableting step.

In liquid pharmaceutical compositions of this disclosure, the agent and any other solid excipients are dissolved or suspended in a liquid carrier such as water, water for injection, vegetable oil, alcohol, polyethylene glycol, propylene glycol or glycerin. Liquid pharmaceutical compositions may contain emulsifying agents to disperse uniformly throughout the composition an active ingredient or other excipient that is not soluble in the liquid carrier. Liquid formulations can be used as injectable, enteral, or emollient types of formulations. Emulsifiers that may be useful in the liquid compositions of the present invention include, for example, gelatin, egg yolk, casein, cholesterol, acacia, tragacanth, chondrus, pectin, methylcellulose, carbomer, cetostearyl alcohol and cetyl alcohol.

In some embodiments, liquid pharmaceutical compositions of the present disclosure also contain a viscosity enhancing agent that improves the texture of the product and/or the coating of the lining of the gastrointestinal tract. Such agents include acacia, bentonite alginate, carbomer, calcium or sodium carboxymethylcellulose, cetostearyl alcohol, methylcellulose, ethylcellulose, gelatin guar gum, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, maltodextrin, polyvinyl alcohol, Included are 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 disclosure also contain buffering agents such as gluconic acid, lactic acid, citric acid or acetic acid, sodium gluconate, sodium lactate, sodium citrate, or sodium acetate.

Sweeteners such as sorbitol, saccharin, sodium saccharin, sucrose, aspartame, fructose, mannitol and invert sugar may be added to certain formulations of the disclosure to improve palatability. Flavoring agents and taste enhancers can make the dosage form more palatable to the patient. Common flavorings and flavor enhancers for pharmaceutical products that may be included in the compositions of this disclosure include maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol and tartaric acid.

Preservatives and chelating agents such as alcohol, sodium benzoate, butylhydroxytoluene, butylhydroxyanisole and ethylenediaminetetraacetic acid may be added at levels safe for ingestion to improve shelf stability. Solid and liquid compositions may also be dyed using any pharmaceutically acceptable colorant to improve their appearance and/or facilitate patient identification of the product and unit dosage level.

A dosage form formulation of the present disclosure may be a capsule containing a composition, such as a powdered or granulated solid composition of the disclosure, within a hard or soft shell. The shell may be made of gelatin and optionally contain plasticizers such as glycerin and sorbitol, and opacifiers or coloring agents.

Methods for Treating Cancer As noted above, the modified microRNA nucleic acid compositions of the present disclosure and formulations thereof are directed to native microRNAs and/or known cancer treatments (chemotherapy), such as 5-FU. It exhibits unexpected and extraordinary anti-cancer activity when compared to activity. Accordingly, another aspect of the present disclosure provides a method for treating cancer in a mammal by administering to the mammal an effective amount of one or more modified microRNA nucleic acid compositions of the present disclosure or formulations thereof. offer.

As shown in FIGS. 2A and 8A, exemplary modified microRNA nucleic acids of the present disclosure, i.e., modified miR-15a and modified MiR-129, suppress BCL2 expression and activity in cancer cells of interest, increases the amount of available pro-apoptotic proteins, ultimately leading to increased cancer cell death. For example, miR-129 controls apoptosis by directly targeting BCL2 as well as by affecting other key cell death-associated proteins. Furthermore, FIG. 2A shows that miR-129 reduces the expression and thus activity of E2F3, a transcription factor protein that controls cell cycle progression, and reduces the expression or activity of thymidylate synthase (TS) protein levels, which increases cell proliferation and increases efficacy of chemotherapeutic agents.

Other exemplary microRNAs, such as modified miR-506, miR-140, miR-192 and miR-502, also have been shown to affect cancer cell proliferation and cancer cell growth, as shown in Figures 13A-B and 14A-D. Modulates cell apoptosis.

Indeed, Figures 7 and 11 demonstrate that intravenous treatment with two exemplary modified microRNAs of the present disclosure (e.g., modified miR-129 and modified miR-15a) inhibits tumor growth and development. to effectively treat colorectal cancer.

Generally, the methods for treating cancer of the present disclosure use the nucleic acid compositions (e.g., modified microRNAs, e.g., modified miR-129 nucleic acids, modified miR-15a nucleic acids, modified miR-140 nucleic acids, modified miR-140 nucleic acids, modified miR -192 nucleic acid, modified miR-502, modified miR-506 nucleic acid, or a combination thereof) to the subject. In certain embodiments, nucleic acid compositions may be administered as a formulation comprising the nucleic acid composition and a carrier. In other embodiments, the nucleic acid compositions of this disclosure can be administered in the absence of a carrier (ie, naked).

The term "subject" as used herein refers to any mammal. The mammal can be any mammal, but the methods herein are more typically directed to humans. As used herein, the phrase "subject in need thereof" is included within the term "subject" and specifically those in need of treatment for cancer or at elevated risk for cancerous or precancerous conditions. refers to a mammalian subject with a medically determined In certain embodiments, subjects include human cancer patients. In some embodiments, the subject has colorectal cancer or has been medically determined to be at an increased risk of developing colorectal cancer. In other embodiments, the subject has been medically determined to have an increased risk of developing pancreatic cancer, such as having pancreatic cancer or being diagnosed with chronic pancreatitis. In still other embodiments, the subject of this disclosure has lung cancer or has been medically determined to be at an increased risk of developing lung cancer.

The terms "treatment," "treat," and "treating" are synonymous with the term "to administer an effective amount." These terms refer to the medical management of a subject intended to cure, ameliorate, stabilize, reduce one or more symptoms, or prevent a disease, pathological condition, or disorder, such as cancer. do. These terms include active treatment, ie treatment specifically directed at amelioration of a disease, pathological condition or disorder, and causal treatment, ie elimination of the cause of the associated disease, pathological condition or disorder. It also includes treatments targeting In addition, treating is palliative treatment, i.e. treatment designed for the alleviation of symptoms rather than cure of a disease, pathological condition or disorder, and includes: prophylactic treatment; , i.e., treatment aimed at minimizing or partially or completely inhibiting the occurrence of the associated disease, pathological condition or disorder; and supportive treatment, i.e., associated disease, pathological condition or It is a treatment used in adjunctive alternative specific therapy aimed at ameliorating the disorder. Treatment is intended to cure, ameliorate, stabilize or prevent a disease, pathological condition or disorder, but it is understood that treatment may not actually cure, ameliorate, stabilize or prevent. be. The effect of treatment can be measured or assessed as known in the art, as described herein and as appropriate for the disease, pathological condition, or disorder. Such measurements and evaluations can be made in qualitative and/or quantitative terms. Thus, for example, a property or characteristic of a disease, pathological condition or disorder and/or a symptom of a disease, pathological condition or disorder may be reduced by any effect or amount.

In certain embodiments, the nucleic acid compositions of the disclosure are used to treat cancer, eg, colorectal cancer.

As used herein, the term "cancer" includes any disease caused by abnormal, uncontrolled cell division and growth, including, for example, malignant and metastatic growth of tumors. The term "cancer" also includes conditions characterized by precancerous conditions or an increased risk of cancer or precancerous conditions. Thus, treatment of cancer is herein also encompassed by methods for preventing cancer or for preventing a pre-cancerous condition from transforming into a cancerous state or an entirely non-cancerous state. considered in writing. Cancer or precancerous (neoplastic conditions) can be located in any part of the body, including internal organs and skin. Some examples of applicable body parts containing cancer cells include colon, rectum (including anus), stomach, esophagus, digestive tract, lung, pancreas, and liver. A cancer or neoplasia can also include the presence of one or more of cancer, sarcoma, lymphoma, blastoma, or teratoma (germ cell tumor). In some embodiments, the cancer may also be a form of leukemia.

In certain embodiments, the nucleic acid compositions described herein treat colorectal (i.e., colon or rectal), pancreatic or lung cancer, at any stage thereof, as further described below. used for As is well known, cancers invade normal, non-cancerous tissue surrounding tumors via lymph nodes and ducts, and tumors invade the veins, capillaries and arteries of the subject. Diffusion throughout the subject by later blood. When cancer cells detach (“metastasize”) from a primary tumor, a secondary tumor develops throughout the affected subject, forming a metastatic lesion.

For example, there are four stages of colorectal cancer that are generally characterized by the extent of metastasis. In stage 0, or carcinoma in situ, abnormal, potentially cancerous cells are found in the mucosa (innermost layer) of the colon wall. In Stage I, cancerous cells form in the mucosa of the colon wall and can spread into the submucosa (the layer of tissue under the mucosa) and into the muscle layer of the colon wall. Stage II consists of three subclasses: Stage IIA, cancerous tissue spreads through the muscular layer of the colon wall into the serosa (outermost layer) of the colon wall; It spreads through the serosa of the wall but not to adjacent organs; and in stage IIC, the cancer spreads through the serosa of the colon wall and invades adjacent organs. Stage III is also divided into three subclasses: In Stage IIIA, cancer can spread through the serosal membrane of the colon wall into the submucosa and muscle layers and into one to three adjacent lymph nodes or lymph nodes. spread to nearby tissues; or cancer spreads through the mucosa to the submucosa and 4 to 6 adjacent lymph nodes; in Stage IIIB, the tumor spreads through the muscle layer of the colon wall into the serosa but is adjacent through the serosa Does not spread to organs and cancer spreads to 1 to 3 adjacent lymph nodes or tissues near lymph nodes; or spreads to muscle layer or serosa and 4 to 6 adjacent lymph nodes; or submucosa through mucosa and may spread to the muscle layer and spread to 7 or more adjacent lymph nodes. In Stage IIIC colorectal cancer, the tumor spreads through the serosa of the colon wall to the serosa but not to adjacent organs and the cancer spreads to 4 to 6 adjacent lymph nodes; spread through the serosa or spread through the serosa but not to adjacent organs and cancer spreads to 7 or more adjacent lymph nodes; or cancer spreads through the serosa to adjacent organs and 1 or more adjacent lymph nodes Spread to tissues near nodes or lymph nodes. Finally, stage IV colon cancer is divided into two subclasses: in stage IVA, the cancer spreads through the colon wall to adjacent organs and in one organ or distant lymph nodes not near the colon. spread; and in stage IVB, cancer spreads through the colon wall to adjacent organs and to more than one organ or abdominal wall lining not near the colon.

Yet another example of tumor staging includes the Dukes classification system for colorectal cancer. Here the stage is identified as stage A, where the tumor is confined to the intestinal wall; in stage B, the tumor exhibits invasion through the intestine but does not invade the lymph nodes; Cancerous cells or tissue are found within the subject's lymph nodes; and in stage D, the tumor exhibits widespread metastasis to several organs of the subject.

The Astler Coller classification system can alternatively be used. Here, stage A colorectal cancer is identified as a cancer that resides only in the intestinal mucosa; in stage B1, the tumor spreads to the muscularis propria but does not penetrate through it and the tumor metastasizes to the lymph nodes. No, stage B2 colorectal cancer is represented by tumors that penetrate through the muscularis propria and the tumor does not metastasize to lymph nodes; characterized by nodal metastasis; Stage C2 colorectal cancer is classified as a tumor that penetrates through the muscularis propria, in which the tumor metastasizes to the lymph nodes; and Stage D is metastasis throughout the organism or subject. Describe the tumor that

In some embodiments, the treatment methods of the present disclosure more particularly exhibit reduced levels of miR-129 expression, miR-15a expression, miR-506 expression, miR-502, miR-140, or combinations thereof Intended for cancer patients. In this regard, miR-15a is known to be downregulated in cancer. See, eg, RI Aqeilan et al., Cell Death and Differentiation (2010) 17, pp. 215-220. In addition, cancerous cells with reduced levels of miR-129 expression, e.g., as described in US Patent Application Publication No. 2016/0090636, the contents of which are incorporated by reference in their entirety, 5 - Known to be resistant to fluorouracil. Additionally, pancreatic cancer cells are known to exhibit reduced levels of miR-506. See, eg, Li, J. et al., Oncogene. 35 pp. 5501-5514.

In yet another embodiment, the microRNA mimetics of the present disclosure are used to treat pancreatic cancer. Pancreatic cancer arises from precursor lesions called pancreatic intraepithelial neoplasia or PanINs. These lesions are typically located in the canaliculi of the exocrine pancreas and can be classified as low-, moderate-, or high-grade dysplastic lesions, depending on the degree of cytological atypicality. Such lesions typically indicate the presence of activating mutations in the KRAS gene along with specific inactivating mutations in CDKN2A, TP53 and SMAD4. Collectively, these genetic mutations lead to the formation of invasive cancers. Pancreatic cancer is determined by the size of the primary tumor and whether it has grown outside the pancreas into surrounding organs; whether the tumor has spread to nearby lymph nodes and whether it spreads to other organs in the body (e.g. liver, It is staged based on whether it has spread to the lungs, abdomen). This information is then used in combination to provide specific stages: 0, 1A, 1B, 2A, 2B, 3 and 4. For stage zero (0), pancreatic tumors are confined to the top layer of pancreatic ductal cells and do not invade deeper tissues. A primary tumor does not spread outside the pancreas (eg, pancreatic intraepithelial carcinoma or pancreatic intraepithelial neoplasia III). Stage 1A pancreatic tumors are typically confined to the pancreas and are 2 cm or smaller in diameter. Furthermore, stage 1A pancreatic tumors do not spread to adjacent lymph nodes or distant sites. Stage 1B pancreatic tumors are confined to the pancreas and are greater than 2 cm in diameter. Stage 1B pancreatic tumors do not spread to adjacent lymph nodes or distant sites. Stage 2A pancreatic tumors present tumors that grow outside the pancreas but do not grow into major blood vessels or nerves, but the cancer has not spread to nearby lymph nodes or distant sites. Subjects presenting with stage 2B pancreatic cancer present with a tumor that is confined to the pancreas or grows outside the pancreas but does not grow into major blood vessels or nerves and spreads to adjacent lymph nodes. Subjects who present with stage 3 pancreatic cancer present with tumors that grow outside the pancreas into major blood vessels or nerves but spread to distant sites. Stage 4 pancreatic cancer metastasizes to distant sites, lymph nodes and organs.

In another example, lung cancer is treated using the modified microRNA nucleic acid compositions of the present disclosure. The methods include treatment of non-small cell lung cancer, such as squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Lung cancer often forms malignant tumors in the bronchi of the lungs and spreads to other parts of the body (eg, lymph nodes). For example, in the case of small cell lung cancer, cancerous lesions are often found in one lung and then spread to a second lung, the fluid surrounding the lung (pleura) or adjacent organs. Lung cancer is staged based on the size of the primary tumor and whether it has grown outside the lungs into lymph nodes and whether it has metastasized to other organs of the body (e.g. bone, liver, breast, brain). divided. This information is then used in combination to provide the specific stages: 0, 1, 2, 3 and 4. For stage zero (0) (ie, carcinoma in situ), the cancer is small in size and does not spread to deeper lung tissue or outside the lung. Stage 1 lung cancer presents cancerous cells that are present in the underlying lung tissue but leave the lymph nodes unaffected. Stage 2 lung cancer reveals that the cancer has spread to nearby lymph nodes or to the chest wall. Stage 3 lung cancer is classified by continuous spread from the lung to lymph nodes or adjacent structures and organs such as the heart, trachea and esophagus. Stage 4 lung cancer presents as metastatic cancer throughout the body, which may affect the liver, bones or brain.

Nucleic acid compositions according to the disclosure may generally be administered by any route known in the art. (2) parenteral administration, such as by subcutaneous, intramuscular or intravenous injection; (3) topical administration; or (4) intravaginal or intrarectal administration; (5) (6) ocular administration; (7) transdermal administration; (8) nasal administration; and (9) administration directly to organs or cells in need thereof. is included.

The amount (dosage) of a nucleic acid composition of the present disclosure to be administered includes the type and stage of cancer, the presence or absence of supplemental or adjuvant drugs, and the subject's weight, age, health, and tolerance to the agent. Depends on several factors. Depending on these various factors, dosages may be, for example, 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, about 25 mg/kg body weight. , 30 mg/kg body weight, 40 mg/kg body weight, 50 mg/kg body weight, 60 mg/kg body weight, 70 mg/kg body weight, 80 mg/kg body weight, 90 mg/kg body weight, 100 mg/kg body weight, approximately 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/kg body weight, about 350 mg/kg body weight, about 400 mg/kg body weight, about 500 mg/kg body weight 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 "about" indicates It is generally understood to be within ±10%, 5%, 2% or 1% of the value. The dosage may also be within a range bounded by the two preceding values. Routine experimental methods can be used to determine the effects of compounds on cancerous or precancerous conditions, or microRNA expression levels or activity (e.g., miR-15a, miR-129, miR-140, miR-192, miR-192, miR-192, -502, miR-506), or the effect on BCL2 levels or activity, or the effect on TS levels or activity, or the effect on E2F3 levels or disease pathology to determine the appropriate dosing regimen for each patient may occur, all of which can be frequently and easily monitored according to methods known in the art. Depending on the various factors discussed above, any of the above exemplary doses of nucleic acid can be administered one, two, or more times per day.

The ability of the nucleic acid compositions and optionally any additional chemotherapeutic agents described herein to be used in current methods can be determined using pharmacological models well known in the art, such as cytotoxicity assays, Apoptosis staining assays, xenograft assays, and binding assays can be used to determine.

The nucleic acid compositions described herein may or may not be co-administered with one or more chemotherapeutic agents, which differs from the nucleic acid compositions described herein. It may be an auxiliary or adjuvant drug.

As used herein, "chemotherapy" or the phrase "chemotherapeutic agent" is an agent useful in the treatment of cancer. Chemotherapeutic agents useful in combination with the methods described herein include any agent that directly or indirectly modulates BCL2, E2F3 or TS. Examples of chemotherapeutic agents include: antimetabolites such as methotrexate and fluoropyrimidine-based pyrimidine antagonists, 5-fluorouracil (5-FU) (Carac® cream, Efudex®, Fluoroplex®, Adrucil®) and S-1; antifolates including polyglutamatable antifolate compounds; raltitrexed (Tomudex®), GW1843 and pemetrexed (Alimta®); Trademark)) and non-polyglutamated antifolate compounds; nolatrexed (Thymitaq®), plevitrexed, BGC945; folate analogs such as denopterin, methotrexate, pteropterin, trimetrexate; and purine analogs. , such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxyuridine. In certain embodiments of the present disclosure, chemotherapeutic agents are compounds capable of inhibiting the expression or activity of genes or gene products involved in signaling pathways involved in abnormal cell proliferation or apoptosis, e.g., YAP1, BMI1, DCLK1, BCL2, thymidylate synthase or E2F3; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In some embodiments, the chemotherapeutic agent is an anticancer drug, or a tissue sensitizer or other promoter for an anticancer drug. In some embodiments, the co-drug may be another nucleic acid, or another miRNA, such as a microRNA mimetic of the present disclosure, gemctiabine, or free 5-FU.

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

In other embodiments, chemotherapy is any of the following cancer drugs, e.g., one or more of methotrexate, doxorubicin, cyclophosphamide, cisplatin, oxaliplatin, bleomycin, vinblastine, gemcitabine, vincristine, epirubicin, folinic acid , paclitaxel, and docetaxel. A chemotherapeutic agent may be administered before, during, or after initiation of treatment with the nucleic acid composition.

In some embodiments, a chemotherapeutic agent is a co-drug.

E2F transcription factor 3, E2F3 (RefSeq NG_029591.1, NM_001243076.2, NP_001230005.1) binds DNA and regulates the expression of genes involved in cell cycle control, including but not limited to retinoblastoma proteins It is a transcription factor that interacts with effector proteins. Therefore, any drug that inhibits the expression of E2F3 can be considered a co-drug herein.

B-cell lymphoma 2 (BCL2), including isoform α (NM_000633.2, NP_000624.2) and isoform β (NM_000657.2, NP_000648.2), (RefSeq NG_009361.1, NM_000633, NP_000624), is associated with intrinsic apoptosis It is encoded by the Bcl-2 gene, a member of the BCL2 family of regulator proteins that regulates mitochondrial-controlled cell death via a pathway. BCL2 is a complex mitochondrial outer membrane protein that blocks apoptotic death of cells by binding BAD and BAK proteins. Non-limiting examples of BCL2 inhibitors include antisense oligonucleotides such as Oblimersen (Genasense; Genta Inc.,), ABT-737 (Abbott Laboratories, Inc.), ABT-199 (Abbott Laboratories, Inc.). , and BH3 mimetic small molecule inhibitors including Obatoclax (Cephalon Inc.). Any drug that inhibits expression of BCL2 may be considered a co-drug herein.

Thymidylate synthase (RefSeq: NG_028255.1, NM_001071.2, NP_001062.1) catalyzes the essential methylation of dUMP to produce dTMP, one of the four bases that make up DNA, a ubiquitous is the enzyme of The reaction requires CH 2 H4-folic acid as a cofactor (both as a methyl group donor and as a unique reductant). A constant requirement for CHH4-folate is that thymidylate synthase activity is strongly linked to the activity of dihydrofolate reductase and serine transhydroxymethylase, two enzymes responsible for replenishing the cellular folate pool. means Thymidylate synthase is a homodimer of 30-35 kDa subunits. The active site simultaneously binds both the folate cofactor and the dUMP substrate, and dUMP covalently binds to the enzyme through a nucleophilic cysteine residue (Carreras et al, Annu. Rev. Biochem., (1995). ) 64:721-762). The thymidylate synthase reaction is a critical part of the pyrimidine biosynthetic pathway, producing dCTP and dTTP for incorporation into DNA. This reaction is required for DNA replication and cell growth. Thymidylate synthase activity is therefore required for all rapidly dividing cells, such as cancer cells. Thymidylate synthase has been a long-standing target for anticancer drugs due to its association with DNA synthesis and thus cell replication. Non-limiting examples of thymidylate synthase inhibitors include folic acid and dUMP analogs such as 5-fluorouracil (5-FU). Any drug that inhibits the expression of thymidylate synthase can be considered a co-drug herein.

If desired, administration of the nucleic acid compositions described herein may be combined with one or more non-drug therapies such as radiation therapy and/or surgery. Radiotherapy and/or administration of chemotherapeutic agents (in this case, the nucleic acid compositions described herein and, optionally, any additional chemotherapeutic agents), as is well known in the art. may be given prior to surgery, eg, to shrink tumors or stop the spread of cancer prior to surgery. Radiation therapy and/or administration of chemotherapeutic agents can also be given after surgery to destroy any residual cancer, as is also well known in the art.

Examples are set forth below for purposes of illustration and to describe certain specific embodiments of the invention. However, the scope of the invention is not limited in any way by the examples presented herein.

(Example 1)
materials and methods.
Modified miR-129: 5-FU modified miR-129 molecules were synthesized by an automated oligonucleotide synthesis process and purified by HPLC. The two strands were annealed to create mature modified 5-FU-miR-129. More specifically, a process called "2'-ACE RNA synthesis" was used. 2'-ACE RNA synthesis is based on a protecting group scheme that uses a silyl ether to protect the 5'-hydroxyl group in combination with an acid-labile orthoester protecting group (2'-ACE) at the 2'-hydroxy. This combination of protecting groups is then used in standard phosphoramidite solid phase synthesis techniques. For example, SA Scaringe, FE Wincott, and MH Caruthers, J. Am. Chem. Soc., 120 (45), 11820-11821 (1998), the entire contents of each of which are expressly incorporated herein. International PCT Application WO/1996/041809; MD Matteucci, MH Caruthers, J. Am. Chem. Soc., 103, 3185-3191 (1981); SL Beaucage, MH Caruthers, Tetrahedron Lett. ).

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 that replace uracil with 5-halouracil are It can be synthesized in the same manner as miR-129a.

Some exemplary structures of currently used protected functionalized ribonucleoside phosphoramidites are shown below:

cell culture. Human colon cancer cell lines HCT116, RKO, SW480, SW620, and normal colon cell lines CCD 841 CoN, pancreatic cancer cell lines ASPC-1, Panc-1, and lung cancer cell line A549 were obtained from the American Type Culture Collection (ATCC). and maintained in McCoy's 5A medium (HCT-116), DMEM (RKO, SW480, SW620) and MEM (CCD 841 CoN) (Thermo Fischer). Medium was supplemented with 10% fetal bovine serum (Thermo Fischer).

For transfection, 1×10 ⁵ cells were placed in a 6-well plate and 100 nM miR-15a precursor, non-specific, was added 24 hours later using Oligofectamine (Thermo Fischer) according to the manufacturer's protocol. were transfected with either a targeted miRNA (Thermo Fischer) or a modified miR-15a mimetic (Dharmacon). For reagent-free transfections, cells were plated (1×10 ⁵ ) cells per well in 6-well plates. After 24 hours, 100 pmol miRNAs (control, miR-15a, mimetic-1) were diluted in Optimem (Thermo Fischer) and added to the plates. Medium was changed after 24 hours. Medium was supplemented with 10% fetal bovine serum (Thermo Fischer). Briefly, cells were cultured in DMEM/F12 supplemented with B27, 10 ng/mL bFGF, and 20 ng/mL EGF (Life Technologies) in ultra-low attachment flasks. Spheroid cells were collected by gentle centrifugation, dissociated into single cells and maintained by replating.

Western immunoblot analysis: 48 hours after transfection, equal amounts of protein (15 μg) extracted from cells lysed in RIPA buffer with protease inhibitors (Sigma) were added to 10% using standard procedures. Separated on ˜12% sodium dodecyl sulfate polyacrylamide gels. Primary antibodies used for analysis were rabbit anti-YAP1 monoclonal antibody (1:10000) (Cell Signaling Technologies), anti-DLCK1 (1:500) (Abcam), anti-BCL2 (1:500) (NeoMarkers), anti-BMI -1 (1:10000) (Cell Signaling Technologies), mouse anti-human TS antibody (1:500), anti-α-tubulin (1:50000) (Santa Cruz Biotech Inc.), anti-GAPDH (1:100000) (Santa Cruz Biotech Inc.), anti-E2F3 (1:500) (Santa Cruz Biotech Inc.). A horseradish peroxidase conjugated antibody (1:5000, Santa Cruz Biotech Inc.) to mouse or rabbit was used as secondary antibody. Protein bands were visualized on autoradiography film using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fischer). Western blot densities were quantified using Image J software.

Cell Proliferation Assay: Twenty-four hours after transfection, cells were seeded in 96-well plates at a density of 2000 cells per well. Cell proliferation assays were performed on days 1 to 5 by incubating 10 μl WST-1 (Roche Applied Science, Mannheim, Germany) in culture medium for 1 hour and reading the absorbance at 450 and 630 nm. Cell growth rate was calculated by subtracting absorbance at 450 nm from absorbance at 630 nm. Experiments on cell proliferation assays were performed at least three times. O.D. was calculated by subtracting the absorbance at 630 nm from the absorbance at 450 nm. Proliferation experiments were performed in triplicate.

Anchorage-independent growth was studied to determine cancer cell colony-forming ability. Cancer cells were trypsinized and counted, a total of 1×10 ⁵ cells per well were transfected with 25 nM modified microRNAs or native miRs or negative control miRNAs with oligofectamine in 6-well plates, Six hours after transfection, cells were recounted. A total of 20,000 cells in 0.35% agar (Bacto Agar; Becton Dickinson) were layered on top of a 1 mL layer of solidified 0.6% agar in a 35 mm dish. Both layers contained growth medium with B27, 10 ng/mL bFGF, and 20 ng/mL EGF. Colonies greater than 50 mm in diameter were counted after 2 weeks of incubation.

Cell Cycle Analysis: Twenty-four hours after transfection, cells were harvested and resuspended from 0.5 to ^1x106 cells/mL in modified Krishan's buffer supplemented with 0.02mg/mL RNaseH and 0.05mg/mL propidium iodide. . Stained cells were detected by flow cytometry and results were analyzed with Modfit LT™ software. Experiments on cell cycle analysis were performed at least three times.

Apoptosis assay. To distinguish between early and late apoptosis, a fluorescein isothiocyanate (FITC)-annexin assay (Becton Dickinson) was performed. HCT116, RKO, SW480 and SW620 cells were plated at (1×10 ⁵ ) cells per well in 6-well plates and 24 hours later cells were transfected with 25 nM modified miRNA using Oligofectamine. did. Forty-eight hours after transfection, cells were harvested and stained with propidium iodide and anti-Annexin V antibody (Annexin V-FITC Apoptosis Detection Kit, Invitrogen, Calif., USA) according to the manufacturer's protocol. Detected by flow cytometry.

5-FU treatment and cytotoxicity assay: Twenty-four hours after transfection, cancer cells were plated in triplicate at 2x10 ³ cells per well in 100 μL medium in 96-well plates. After 24 hours, 2 μM 5-FU alone, 50 nM native microRNA, 50 nM modified microRNA (e.g., modified miR-129), or 2 mM 5-FU in combination with 50 nM modified microRNA of the present disclosure, e.g., modified miR-129. Fresh medium containing was added and the cells were cultured for an additional 72 hours. Cell viability was measured using the WST-1 assay.

Lentivirus production: Briefly, 1.5×10 ⁶ 293T cells were plated in a 10 cm dish with 10 mL DMEM+10% FBS. Two days later, lentiviral plasmids pEZX-MR03 expressing miR-129 or hsa-miR-15a were transfected with the Lenti-Pac HIV Expression Packaging Kit according to the manufacturer's protocol. After 48 hours, virus was harvested and concentrated with Lenti-Pac Lentiviral Concentration Solution. Viral titers (approximately 10 11 viral particles/ml) were then determined with the Lenti-Pac™ HIV qRT-PCR titration kit. In addition, serial dilutions of virus (0.1 μL, 0.5 μL, 2 μL, 10 μL, 50 μL) were used to transduce 5×10 ⁴ HCT116 CSCs to determine transduction efficiency. The lowest concentration to achieve 100% positive expression (2 μL) was used to infect cells for mouse in vivo treatment experiments.

Real-time qRT-PCR analysis of nucleic acid expression. MicroRNA expression levels in cancer cells were quantified. Briefly, primers specific for the microRNA of interest and the internal control RNU44 gene were purchased from Ambion. cDNA synthesis was performed by High Capacity cDNA Synthesis Kit (Applied Biosystems) with miRNA-specific primers. Real-time qRT-PCR was performed on an Applied Biosystems 7500 Real-Time PCR instrument using miRNA-specific primers in TaqMan Gene Expression Assays (Applied Biosystems). Expression levels of exemplary miRs of the present disclosure were calculated by the ΔΔCT method based on the internal control RNU44 normalized to the control group and plotted as relative quantification.

Human Cancer Stem Cell Profiler: RNA is extracted from cancer cells transfected with either the exemplary microRNAs or negative miRNAs of this disclosure using TRIzol reagent (Thermo Fischer) according to the manufacturer's protocol. did. RNA was transcribed into first strand cDNA using the RT2 First Strand Kit (Qiagen). The cDNA was then mixed with the RT2 SYBR Green Mastermix (Qiagen) and this mixture was aliquoted into wells of the Human Cancer Stem Cells RT2 Profiler PCR Array (Qiagen). An Applied Biosystems 7500 Real-Time PCR instrument was used for qRT-PCR (Applied Biosystems) and relative expression values were determined using the ΔΔCT method.

Mouse Subcutaneous Tumor Implantation Model: Two days before injection, HCT116 cancer stem cells were plated at 5× ^{10 5} /well in 6-well ultra-low attachment plates. Cells were transduced or transfected with 20 μL of virus or 100 pmoles of exemplary modified miR-129 or modified miR-15a. After 48 hours, cells were harvested and resuspended at 10 ⁶ cells/ml in DMEM/F12 knockout medium with 30% matrigel. 10-12 week old NOD/SCID mice (Jackson Laboratories, Bar Harbor, Mass., USA) were used for tumor implantation. Mice were anesthetized with isoflurane inhalation. 100 μL of cell suspension was injected subcutaneously bilaterally in the lower dorsal region. Tumor size was measured using vernier calipers and tumor volume was calculated using the formula V=length x width2/ ² .

For in vivo miRNA delivery experiments, we generated colon cancer cells expressing the lenti-luc reporter gene by infecting parental HCT116 cells with recombinant lentivirus. Luciferase-expressing HCT116 cells (2.0×10 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 cell injection, mice were treated with tail vein injections of 40 μg negative control or modified miR packaged with in vivo-jetPEI (Polyplus Transfection). Mice were treated every other day for 2 weeks (8 times). After treatment, mice were screened using the IVIS spectral in vivo imaging system (IVIS) (PerkinElmer).

RNA isolation: For mouse xenografts, tissue sections were deparaffinized, hydrated and proteinase K digested, respectively. Total RNA was subsequently isolated using TRIzol® reagent. Total RNA was also isolated from clinical specimens by a TRIzol®-based approach.

statistical analysis. All experiments were repeated at least three times. All statistical analyzes were performed with SigmaPlot software. Statistical significance between two groups was determined using Student's t-test (paired t-test for clinical samples and unpaired t-test for all other samples). For comparisons of more than two groups, one-way ANOVA followed by Bonferroni-Dunn test was used. Data were expressed as mean±standard error of the mean (SEM). Statistical significance is either described in figure legends or indicated with an asterisk (*). *=P<0.05;**=P<0.01;***=P<0.001.

(Example 2)
Modified microRNAs of the present disclosure have anticancer activity.
As shown in Figures 3, 8B, 12A-B, 13A-B and 14A-D, modified miRNAs (modified miRs: 129, 15a, 192(215), 140, 502, and 506) It is more effective in inhibiting colon, pancreatic and lung cancer cell proliferation than unmodified miRNA precursors. Additionally, modified miRNAs can be delivered to cancer cells without transfection reagents (data not shown). Notably, the results demonstrated that across several different colorectal, pancreatic, and lung cancer cell lines, cancer cell proliferation was: It shows that it is significantly inhibited.

(Example 3)
Modified miR-129 nucleic acids have anticancer activity.
In the experiments below, 5-FU was incorporated into miR-129. In one experiment, all U bases in miR-129 were replaced with 5-FU, as shown in the structure provided in FIG. 1A, where "U ^F " is 5-fluorouracil or other represents the 5-halouracil of In another experiment, all U bases except the seed region of miR-129 were replaced with 5-FU, as shown in the structure provided in Figure 1B.

Analysis of Target Specificity: The results of Western immunoblot experiments in colon cancer HCT-116 cells demonstrate that exemplary modified miR-129 polynucleotides of the disclosure demonstrate their target specificity against TS, BCL2 and E2F3 via has been demonstrated to be able to hold The results are shown in Figures 2A and 2B, which are modified miR-129 nucleic acids with all U bases replaced with 5-FU, as obtained by two separate operators shown in SEQ ID NO:4. It shows the results for More significantly, exemplary miR-129 mimics were found to be more potent than unmodified (control) miR-129 in reducing expression levels of TS, BCL2 and E2F3. .

Functional enhancement of modified microRNAs of the present disclosure. The effect of exemplary modified miR-129s on colon cancer cell proliferation was compared to that of native miR-129. The results show that 5-FU-miR-129 at 50 nM concentration can completely suppress HCT-116 tumor cell growth. Moreover, as shown by the results in Figure 3, 5-FU-miR-129 is much more potent than native miR-129, thereby providing a significantly higher inhibitory effect. Such inhibition is specific as scrambled control miRs have no effect on cell proliferation.

Next, the efficacy 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 Figure 4, modified miR-129 at 50 nM (40-fold lower than 5-FU) was unexpectedly much more potent than 2 μM 5-FU in inhibiting tumor cell proliferation. be.

Exemplary modified microRNAs of the present disclosure induce apoptosis in colon cancer cells. Since BCL2 is an important target of miR-129, we investigated the effects of the modified miRs of the present disclosure on apoptosis. More specifically, cell death was induced by apoptosis in HCT116, RKO, SW480, and SW620 colon cancer cells transfected with a negative control miRNA, native miR-129, or an exemplary miR-129 mimic of SEQ ID NO:4. Quantitated using the assay. The results show that miR-129 mimics, by fluorescence-activated cell sorting (FACS)-based FITC-annexin assay, compared with native miR-129 and negative control miRNA in all four colon cancer cell lines: We show that apoptosis can be induced by 2- to 30-fold (Fig. 5A).

miR-129 mimics trigger G1/S cell cycle checkpoint regulation. Cell cycle analysis was performed on HCT-116 cells treated with scrambled control, miR-129 precursor, and exemplary miR-129 mimics using flow cytometry. As shown in Figure 5B, cell cycle analysis showed that miR-129 mimics affected colon cancer cell growth by inducing G1 arrest, and such effects were much more pronounced than native miR-129. It has been shown to be more potent (more than 2x).

miR-129 mimics eliminate chemotherapy-resistant colon cancer stem cells. To determine the effect of certain exemplary modified microRNAs of the present disclosure (i.e., miR-129 mimics) on 5-FU-resistant colon cancer stem cells, HCT116-derived colon cancer stem cells were treated with various concentrations of mimics. Treated with -1 or 5-FU. Data shown in FIG. 6 demonstrate that exemplary microRNA mimetics of the present disclosure can eliminate 5-FU-resistant colon cancer stem cells by more than 80% at 100 nM concentration, whereas a lethal dose of 5-FU at 100 μM Appears to have minimal effect on tumor stem cell viability.

Collectively, these results demonstrate that exemplary modified microRNA polynucleotides of the present disclosure were able to inhibit cell proliferation of HCT116 colon cancer stem cells (FIG. 6). The inhibitory effect by such modified miR-129 was much stronger than native miR-129, with proliferation almost completely blocked at 25 nM miR-129 at 6 days (Fig. 6). We used a soft agar assay to demonstrate the effect of treatment of cells with modified miR-129 on anchorage-independent cell growth. Modified miR-129-treated colon cancer stem cells did not form visible spheres compared to cells treated with native miR-129 or control miRNA (similar to those seen in Figure 10).

miR-129 mimetics inhibit colon cancer metastasis in vivo. The therapeutic impact of modifying miR-129 nucleic acids was evaluated using a colon cancer metastasis model. Two weeks after establishment of metastases, 40 μg of miR-129 nucleic acid of SEQ ID NO:4 was delivered by intravenous injection with a treatment frequency of once every other day for two weeks.

The results shown in Figure 7 demonstrate that modified microRNA-129 inhibits colon cancer metastasis, whereas the negative control miRNA is ineffective and does not exhibit toxic side effects.

(Example 3)
Modified miR-15a and its anticancer activity.
Exemplary modified miR-15a compositions have anti-cancer activity. As shown in Figures 1C and 1D, all uracil bases (Figure 1C) or only uracil bases in the non-seed region (Figure 1D) of the miR-15a nucleic acid sequence are 5-halouracil (i.e., 5-fluorouracil). An exemplary modified miR-15a was synthesized as shown above.

Three days after transfection of the exemplary modified miR-15a shown in FIG. 1C into HCT-116 colon cancer stem cells, proteins were harvested and Western blotting was performed to confirm , confirmed that they retained 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 either unmodified miR-15a (native-miR15a) or modified miR-15a compositions. , indicating that the 5-halouracil modification did not inhibit the ability of miR-15a to regulate its targets in those cells.

Modified miR-15a increased therapeutic efficacy in vitro. To determine whether modified miR-15a compositions of the present disclosure demonstrate increased potency in colon cancer cell lines compared to unmodified miR-15a, HCT-116 colon cancer cells were tested negatively. Transfected with control (non-specific oligonucleotide), unmodified miR-15a or the exemplary modified miR-15a composition shown in FIG. 1C.

Cancer cell proliferation was assessed using the WST-1 assay. As shown in Figure 8B, 6 days after transfection, unmodified miR-15a reduced cell proliferation by 53% compared to control. In the case of modified miR-15a cell proliferation was reduced by 84%. Collectively, the experimental results indicate that modified miR-15a is more effective in reducing cancer cell proliferation compared to unmodified miR-15a.

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, resulting in an approximately 3-fold increase in the G1/S ratio. FIG. 9 also shows that exemplary modified miR-15a compositions of this disclosure more effectively arrest cell cycle progression when compared to their natural counterparts. For example, a 7-fold increase in the G1/S ratio was exhibited by cells expressing exemplary modified miR-15a nucleic acids of the disclosure when compared to controls. Therefore, modified miR-15a is more effective than unmodified miR-15a in inducing cell cycle arrest in colon cancer cells.

The effect of exemplary modified miR-15a compositions on colonization by colon cancer stem cells in Matrigel matrix was also tested. As shown in Figure 10, many colonies were generated by cells transfected with control miRNA (Figure 10, negative), whereas only a few colonies were transfected with unmodified miR-15a. produced by cells (Fig. 10, miR-15a). In contrast, no colonies were observed in the case of cells transfected with modified miR-15a (Fig. 10, 5-FU-miR-15a). These results indicate that the exemplary modified miR-15a compositions of this disclosure are indeed more potent inhibitors of tumorigenesis and colorectal cancer progression.

Modified miR-15a inhibits cancer development and progression in vivo. Further to our understanding of miR-15a in colonic CSCs, a mouse xenograft model was established that included colorectal cancer cells pretransfected with either modified miR-15a or a negative control miRNA. was done. Eight weeks after injection, tumors were measured and harvested. A dramatic reduction in tumor size (>25x) for tumors established from CSCs expressing modified miR-15a mimics (n=8) is shown in FIG.

The data presented herein demonstrate that halouracil (e.g., 5-FU) is incorporated into miRNA nucleic acid sequences, resulting in chemotherapeutic effects of natural microRNA molecules in the presence or absence of the use of other chemotherapeutic agents. Supports the feasibility of novel modifications that enhance function.

Claims (15)

A double-stranded nucleic acid comprising a modified microRNA nucleotide sequence comprising a uracil nucleic acid, said uracil nucleic acid comprising a seed region of said modified microRNA nucleotide sequence that is complementary to a B-cell lymphoma 2 (BCL2) nucleotide sequence. All are 5-fluorouracil to combine the potency of the microRNA nucleotide sequence and 5-fluorouracil, and the modified microRNA nucleotide sequence is the miR-129 microRNA nucleotide sequence shown in SEQ ID NO: 1 or the miR-129 microRNA nucleotide sequence shown in SEQ ID NO: 2. A double-stranded nucleic acid that binds to said B-cell lymphoma 2 (BCL2) nucleotide sequence that is complementary to the miR-15a microRNA nucleotide sequence shown. A pharmaceutical composition comprising at least one double-stranded nucleic acid according to claim 1 . 3. The pharmaceutical composition of claim 2, wherein said modified microRNA nucleotide sequence comprises said miR-15a nucleotide sequence. 3. The pharmaceutical composition of claim 2, wherein said modified microRNA nucleotide sequence comprises said miR-129 nucleotide sequence. The double-stranded nucleic acid of claim 1, wherein said modified microRNA nucleotide sequence comprises said miR-129 nucleotide sequence. A method for treating breast cancer comprising administering to a subject an effective amount of a ^{modified microRNA having} the ^nucleotide sequence wherein progression of said breast cancer is inhibited by administration of said modified microRNA nucleic acid composition, and said breast cancer is triple-negative breast cancer. 7. The method of claim 6, wherein said subject is human. 8. The method of claim 7, wherein said modified microRNA nucleic acid binds to a complementary portion of the MDC1 3'UTR nucleotide sequence shown in SEQ ID NO:33. 8. The method of claim 7, wherein said modified microRNA nucleic acid composition is administered to said subject by injection. A method for treating breast cancer comprising administering to a subject an effective amount of a ^{modified microRNA consisting} of the ^nucleotide sequence and wherein progression of said breast cancer is inhibited by administration of said modified microRNA nucleic acid composition. 11. The method of claim 10, wherein said subject is human. 12. The method of claim 11, wherein said breast cancer is selected from the group of triple-negative breast cancer, ductal carcinoma and lobular carcinoma. 13. The method of claim 12, wherein said breast cancer is triple-negative breast cancer. 12. The method of claim 11, wherein said modified microRNA nucleic acid binds to a complementary portion of the MDC1 3'UTR nucleotide sequence shown in SEQ ID NO:33. 12. The method of claim 11, wherein said modified microRNA nucleic acid composition is administered to said subject by injection.

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