A computer readable file containing a sequence listing is being electronically co-filed herewith via EFS-Web. The computer readable file, submitted under 37 CFR § 1.831(a) and having the file name “41D9811.XML” was created on Jun. 15, 2023 and has a size of 11,884 bytes. The content of the computer readable file is hereby incorporated by reference in its entirety.
Acute myeloid leukemia (“AML”) presents a significant clinical challenge. AML cases, incidence, and deaths have been rising worldwide over the past 30 years and clinical outcomes remain poor. AML is most prevalent in elderly populations, with greater than 75% of cases occurring in patients over 65 years old. The 5-year overall survival rate for these patients is approximately 24% with median survival of 8.5 months. Traditional chemotherapeutic approaches based on anthracycline and cytarabine are somewhat effective for treating AML. However, elderly patients are not candidates for intensive chemotherapy.
Cytarabine (cytosine arabinoside or “ara-C”) is an antimetabolic agent that combines a cytosine base with an arabinose sugar. Cytarabine inhibits DNA synthesis in dividing cells resulting in cell death. It has been used as a chemotherapeutic for the treatment of various leukemias, e.g., AML, as well as lymphoma.
Aside from chemotherapy, cancer treatments based on micro-RNA (“miRNA”) are being developed. miRNAs are short non-coding RNAs with important roles in regulating gene expression. Individual miRNAs can inhibit the expression of many different target genes through imperfect base pairing to their 3′ untranslated region.
The importance of miRNAs in cancer was first identified in chronic lymphocytic leukemia, in which miRNA 15a (“miR-15a”) and miRNA 16 (“miR-16”) were found to have reduced expression. Since that discovery, miRNAs have been shown to play a significant role in many different cancer types.
There remains a need to develop new therapeutic approaches having reduced toxicity with the goal of extending survival for patients suffering from AML and other leukemias.
To meet the above need, a composition is provided that includes a miRNA mimic containing one or more cytosine nucleosides in which at least one cytosine nucleoside is replaced by cytosine arabinoside (“ara-C”).
Also provided is a method for killing a cancer cell by contacting it with a composition that includes a miRNA mimic containing ara-C and 5-fluorouracil (“5-FU”) in which the miRNA mimic is miR-15a, miR-16, miR-129, miR-194, miR-192, miR-139, miR-140, or miR-145.
Further, disclosed is a method for treating cancer by administering to a subject an effective amount of a composition containing a miRNA mimic having a guide strand and a passenger strand that each contains one or more cytosine nucleosides in which at least one cytosine nucleoside in the guide strand or at least one cytosine nucleoside in the passenger strand is replaced by ara-C.
The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description, from the drawings, and from the claims.
The description below refers to the accompanying drawings, of which:
As set out in the SUMMARY section above, a composition is disclosed that includes a miRNA mimic containing one or more cytosine nucleosides in which at least one cytosine nucleoside is replaced by ara-C. In the disclosed composition, the miRNA mimic can include a guide strand and a passenger strand. The ara-C can be present in the in the guide strand, in the passenger strand, or in both strands.
The composition can include a pharmaceutically acceptable excipient or carrier. The excipient can be a cationic polymer such as polyethyleneimine, and the carrier can be a lipid carrier, e.g., or a lipid nanoparticle. Additional examples of excipients and carriers are set forth in Ju et al., US Patent Application Publication 2019/0062754 (“Ju”), the contents of which are hereby incorporated by reference in its entirety.
In certain compositions, a single cytosine nucleoside in the guide strand, in the passenger strand, or in both strands is replaced by ara-C. In a particular composition, all of the cytosine nucleosides in the passenger strand are replaced with ara-C. In another composition, all of the cytosine nucleosides in the guide strand are replaced with cytosine arabinoside. Also within the scope of the invention is a composition in which all cytosine nucleosides in both the passenger strand and the guide strand are replaced by ara-C.
The compositions described above can include a guide strand that contains one or more uracil bases in which at least one uracil base is replaced by 5-halouracil. The 5-halouracil can be 5-bromouracil, 5-chlorouracil, 5-iodouracil, or 5-fluorouracil. In a particular composition the 5-halouracil is 5-fluorouracil (“5-FU”).
In certain compositions, a single uracil base in the guide strand is replaced with 5-FU. In other compositions, all of the uracil bases in the guide strand are replaced with 5-fluoruracil.
Any combination of ara-C- and 5-FU-modified guide strands and passenger strands can be included in the composition. See
In particular compositions, the miRNA mimic can be miR-15a, miR-16, miR-129, miR-194, miR-192, miR-139, miR-140, or miR-145. Each of these miRNA mimics can include a guide strand and a passenger strand modified with ara-C, 5-FU, or both as set forth, supra.
In an exemplary composition, the miRNA mimic is a miR-15a mimic that includes a guide strand consisting of the sequence UAGCAGCACAUAAUGGUUUGUG (SEQ ID NO: 1) and a passenger strand consisting of the sequence CAGGCCAUAUUGUGCUGCCUCA (SEQ ID NO: 2). In a specific composition, all three cytosine nucleosides in the guide strand and all seven cytosine nucleosides in the passenger strand are replaced with ara-C. In another specific composition, all three cytosine nucleosides in the guide strand and all seven cytosine nucleosides in the passenger strand are replaced with ara-C and all seven uracil bases in the guide strand are replaced with 5-FU.
Any of the above-described miRNA mimic compositions can be used in the method for killing a cancer cell set forth in the SUMMARY section. In this method, the miRNA mimic is miR-15a, miR-16, miR-129, miR-194, miR-192, miR-139, miR-140, or miR-145 in which the guide strand contains both ara-C and 5-FU and the passenger strand contains ara-C.
The miRNA mimic compositions can also be used in the method for treating cancer disclosed above. The method features administering the miRNA mimics described above to a subject suffering from cancer. The cancers that can be treated include, but are not limited to, leukemia, lymphoma, or multiple myeloma. For example, acute myeloid leukemia (“AML”) or acute lymphocytic leukemia (“ALL”) can be treated with the disclosed method.
Administration routes of the miRNA mimic compositions include, but are not limited to oral administration, parenteral administration (e.g., subcutaneous, intramuscular, intraperitoneal, or intravenous injection), and topical administration. Additional routes that can be used are set forth in Ju et al.
In the cancer-treating method, the miRNA mimics can be miR-15a, miR-16, miR-129, miR-194, miR-192, miR-139, miR-140, and miR-145 in which the guide strand contains both ara-C and 5-FU or solely 5-FU and the passenger strand contains ara-C.
The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.
The modified miR-15a mimic was synthesized with standard phosphoramidite chemistry as two separate RNA oligonucleotides, i.e., guide strand and passenger strand. The uracil bases were replaced with 5-FU by including in the reaction a 5-fluorouracil nucleoside phosphoramidite as a precursor, along with the phosphoramidite derivatives of nucleosides containing natural bases, e.g., A, G, and C). Similarly, an ara-C phosphoramidite was included during synthesis with natural A, U, and G to produce ara-C substituted miRNA molecules.
Different combinations of unmodified and modified guide and passenger strands were produced as shown in
Purified passenger and guide strands were annealed before use in transfection assays. Briefly, the passenger and guide strands were each dissolved in 100 mM acetic acid pH 3.8 to a concentration of 100 μM and mixed at a 1:1 volume ratio. The mixture was heated at 60° C. for 45 min. and then cooled at room temperature for 30 min. The annealed RNA was desalted by ethanol precipitation with 3M sodium acetate.
MV-4-11 (American Type Culture Collection; “ATCC”) AML cells were maintained in Iscove's Modified Dulbecco's Medium (IMDM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (“FBS”; VWR). REH ALL cells (ATCC) were maintained in RPMI-1640 Medium (Thermo Fisher Scientific) supplemented with 10% FBS.
For transfection with a vehicle, twenty-four hours before transfection, cells were plated in 6 well plates at 1×105 cells per well. Cells were transfected with negative control miRNA (Thermo Fisher Scientific), 5-FU miR-15a, or various versions of the ara-C modified miR-15a mimic, using Lipofectamine™ 2000 (Thermo Fisher Scientific) as directed by the manufacturer. MV-4-11 cells were transfected with 15 nM of miRNA and REH cells were transfected with 25 nM of miRNA. Twenty-four hours post-transfection, cells were replated at 2000 cells per well in 96 well plates.
For vehicle free transfection, cells were plated in 96 well plates at 2000 cells per well. Twenty-four hours later, miRNA was diluted in cell media and added to the 96 well plates. MV-4-11 cells were treated with 5 nM, 2.5 nM, and 1.25 nM miRNA. REH cells were treated with 10 nM, 5 nM, and 0.5 nM miRNA.
Cell numbers were measured on days 1, 3, and 6 post transfection for cells transfected with Lipofectamine 2000 and on day 6 post transfection for vehicle free transfection, using WST-1 dye (Roche). Briefly, cells were incubated with 10 μl of WST-1 dye per 100 μl of media for 1 hour (REH) or 2 hours (MV-4-11) at 37° C. and absorbance was read at 450 nm and 630 nm. The optical density (“OD”) was calculated by subtracting the absorbance at 630 nm from that at 450 nm. OD correlates with cell viability. In certain instances, OD was converted to percent viable cell relative to the negative control. The results are shown in
In MV-4-11 AML cells subjected to lipofection, both ara-C 5-FU miR-15a and passenger ara-C 5-FU miR-15a were more effective at inhibiting cell viability, as compared to both 5-FU miR-15a and ara-C miR-15a. See
When MV-4-11 cells were transfected vehicle free, only passenger ara-C 5-FU miR-15a was better than 5-FU miR-15a at inhibiting cell viability. See
The viability of REH ALL cells was decreased by all modified miRNAs introduced by lipofection, as compared to control miRNA. See
When REH cells were transfected without a vehicle, again all modified miRNAs tested inhibited cell viability. See
Wee1 is an important cell cycle regulator and a potential target for AML therapy. Wee1 is a direct target of miR-15a with two miR-15a binding sites in the 3′ UTR of Wee1. See
For transfection with a vehicle, twenty-four hours before transfection, cells were plated in 6 well plates at 1×105 cells per well. Cells were transfected with negative control miRNA (Thermo Fisher Scientific), 5-FU miR-15a, or various versions of the ara-C modified miR-15a mimic, using Lipofectamine™ 2000 (Thermo Fisher Scientific) as directed by the manufacturer. MV-4-11 cells were transfected with 15 nM of miRNA. Twenty-four hours post-transfection, RNA was isolated from cells using TRIzol™ (Thermo Fisher Scientific) reagent following the manufacturer's protocol. Wee1 expression was measured using qRT-PCR and normalized to GAPDH expression and calculated by the double delta threshold cycle (ΔΔCt) method. The results are shown in
miR-15a modified only with 5-FU on the guide strand was able to significantly suppress Wee1 gene mRNA expression, yet, miR-15a modified with ara-C alone on the guide strand did not. Compare 5-FU miR-15a to ara-C miR-15a in
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
The present application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/354,009, filed on Jun. 21, 2022. The content of the prior application is hereby incorporated by reference in its entirety.
Number | Date | Country | |
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63354009 | Jun 2022 | US |