Trimethylglycylglycerin compositions and their use in developing anti-cancer drugs and RNA vaccines

Abstract

This invention generally relates to a group of novel chemical compositions and their use for formulating RNA- and/or DNA-based medicine drugs/vaccines into stable compound complexes useful for both in-vitro and in-vivo delivery. Particularly, the present invention teaches the synthesis of a group of novel trimethylglycyl chemicals and their use for formulating cosmetic, therapeutic- and/or pharmaceutical-grade nucleic acid compositions, including but not limited microRNA precursors (pre-miRNA/miRNA), small hairpin RNAs (shRNA), short interfering RNAs (siRNA), ribozymes, antisense oligonucleotides, RNA-DNA hybrids and DNA-based vectors/vaccines, with or without modification, into delivery complexes, which can then be absorbed by cells in vivo, ex vivo and/or in vitro through an active mechanism of endocytosis via acetylcoline receptors for releasing the therapeutic and pharmaceutical effects of the formulated nucleic acid compositions.

Claims

1. A composition for formulating nucleic acid compositions with sugars and sugar alcohols into stable complexes for in-vitro, ex vivo and in-vivo delivery into mammalian cells, comprising: (a) at least a nucleic acid composition with at least a negative charge; and (b) at least a sugar or sugar alcohol composition modified by trimethylglycylation, wherein (a) and (b) are mixed together under a condition to form at least a delivery complex, wherein said delivery complex is delivered into mammalian cells via acetylcholine receptors.

2. The composition as defined in claim 1, wherein said nucleic acid composition is small hairpin RNAs.

3. The composition as defined in claim 1, wherein said sugar or sugar alcohol composition is glycerin (glycerol).

4. The composition as defined in claim 1, wherein said trimethylglycylation is a chemical reaction that replaces the hydroxyl (HO—) groups of a sugar alcohol or sugar with betaine's trimethylglycyl [(CH.sub.3).sub.3N.sup.+CH.sub.2COO—] groups and thus results in the formation of an ether (R—O—R) linkage between each OH-removed carbon of the sugar/sugar alcohol and the trimethylglycyl group.

5. The composition as defined in claim 1, wherein said sugar or sugar alcohol composition after modified by trimethylglycylation forms mono-trimethylglycylglycerins (mono-TMGG; C.sub.8H.sub.18O.sub.4N.sub.1 MW=190˜193g/mole), di-trimethylglycylglycerin (di-TMGG; C.sub.13H.sub.28O.sub.5N.sub.2 MW=290˜293g/mole), and/or tri-trimethylglycylglycerin (tri-TMGG; C.sub.18H.sub.38O.sub.6N.sub.3 MW=390˜393 g/mole).

6. The composition as defined in claim 1, wherein said sugar or sugar alcohol composition is capable of protecting said nucleic acid compositions from degradation.

7. The composition as defined in claim 1, wherein said sugar or sugar alcohol composition is capable of enhancing the delivery efficiency of said nucleic acid compositions into mammalian cells via acetylcholine receptors in vitro, ex vivo and in vivo.

8. The composition as defined in claim 1, wherein said condition is incubation at a temperature equal or larger than 75° C. and at a pressure equal or higher than 100 kPa.

9. The composition as defined in claim 8, wherein said condition is incubation at a temperature about 100° C. to 160° C. and at a pressure about 101 to 250 kPa.

10. The composition as defined in claim 1, wherein said delivery complex is formed by the ionic or electrostatic affinity occurring between the modified sugar or sugar alcohol and the nucleic acid composition.

11. The composition as defined in claim 1, wherein said delivery complex is useful for therapeutic applications.

12. The composition as defined in claim 1, wherein said modified sugar or sugar alcohol is capable of forming trimethylglycyl-glycylglycerin mixtures.

13. The composition as defined in claim 1, wherein said modified sugar or sugar alcohol is capable of forming polymers with glycylglycerins and amino acids.

14. The composition as defined in claim 1, wherein said modified sugar or sugar alcohol contains at least a trimethylglycyl [(CH.sub.3).sub.3N.sup.+CH.sub.2COO—] group that structurally resembles acetylcholine.

15. The composition as defined in claim 1, wherein said modified sugar or sugar alcohol is capable of interacting with acetylcholine receptors.

16. The composition as defined in claim 1, wherein said modified sugar or sugar alcohol is capable of being used to replace acetylcholine to stimulate or inhibit acetylcholine receptors.

17. The composition as defined in claim 1, wherein said modified sugar or sugar alcohol is useful for developing therapeutic applications and devices.

18. The composition as defined in claim 1, wherein said modified sugar or sugar alcohol is useful for formulating nucleic acid-based compositions into medicine drugs.

19. The composition as defined in claim 1, wherein said modified sugar or sugar alcohol is useful for formulating nucleic acid-based medicines for treating respiratory diseases and infections.

20. The composition as defined in claim 1, wherein said modified sugar or sugar alcohol is useful for formulating small hairpin RNAs into vaccines for treating viral infection.

21. The composition as defined in claim 20, wherein said formulated small hairpin RNAs vaccines induce RNAi effects.

22. The composition as defined in claim 1, wherein said nucleic acid composition induce immune responses to generate antibodies directed against viruses.

23. The composition as defined in claim 22, wherein said viruses are RNA viruses.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Referring particularly to the drawings for the purpose of illustration only and not limitation, there is illustrated:

(2) FIG. 1 shows the three major groups of glycylation-derived products, including mono-glycylglycerins (MGG; C5H11O4N1; each molecular weight around MW=149˜151 g/mole), di-glycylglycerins (DGG; C7H14O5N2; MW=206˜208 g/mole), and tri-glycylglycerin (TGG; C9H17O6N3; MW=263˜266 g/mole). MGG and DGG are partially glycylated products, while TGG is a completely (or fully) glycylated product. MGG and DGG can further form six- or five-member ring conformations similar to the chemical structures of six-carbon monosaccharides, in particular glucose, fructose and galactose, and thus are capable of being absorbed into cells via GLUT and SGLT carrier proteins.

(3) FIG. 2 shows the three major groups of trimethylglycylation-derived products, including mono-trimethylglycylglycerins (mono-TMGG; C8H18O4N1; each molecular weight around MW=190˜193 g/mole), di-trimethylglycylglycerins (di-TMGG; C13H28O5N2; molecular weight around MW=290˜293 g/mole) and tri-trimethylglycylglycerin (tri-TMGG; C18H38O6N3; MW=390˜393 g/mole). Mono- and di-TMGGs can further form six- and/or five-member ring conformations similar to the chemical structures of six-carbon monosaccharides; yet, all TMGGs preferably interact with acetylcholine receptors rather than GLUT/SGLT carrier proteins because the containing trimethylglycyl group highly resembles acetylcholine. This unique feature of TMGGs is completely different from glycylglycerins.

(4) FIG. 3 shows the chemical reaction of trimethylglycylation and the resulting mono-trimethylglycylglycerins (mono-TMGGs) thereof.

(5) FIG. 4 shows the chemical reaction of trimethylglycylation and the resulting di-trimethylglycylglycerins (di-TMGGs) thereof.

(6) FIG. 5 shows the chemical reaction of trimethylglycylation and the resulting tri-trimethylglycylglycerin (tri-TMGG) thereof.

(7) FIG. 6 shows the mixed reaction of trimethylglycylation and glycylation together, which mainly generates mono-trimethylglycyl-di-glycylglycerins (mono-TMG-DGGs; C12H25O6N3; molecular weight around MW=305˜308 g/mole) and their derivatives thereof.

(8) FIG. 7 shows the mixed reaction of glycylation first and then trimethylglycylation, of which the reaction mainly generates mono-trimethylglycyl-mono-glycylglycerins (mono-TMG-MGGs; C10H22O5N2; MW=248˜251 g/mole) thereof.

(9) FIG. 8 shows high performance liquid chromatography (HPLC) analyses of the results of trimethylglycylation between betaine (trimethylglycine, TMG) and glycerin (or called glycerol).

(10) FIG. 9 shows high performance liquid chromatography (HPLC) analyses of the results of trimethylglycylation among betaine (trimethylglycine, TMG), glycine and glycerin (or called glycerol).

(11) FIG. 10 shows the formation of TMGG-glycylglycerin polymers. The mixture reaction of mono-TMG-MGGs, mono-TMG-DGGs, and/or TMGG with other amino acids, particularly glycine, can form a variety of high-degree structural polymers through peptide bonding and/or electrostatic/ionic affinity.

(12) FIG. 11 shows the TMGG- and/or TMG-glycylglycerin-mediated nucleic acid-based drug delivery pathway and the following induced RNA interference (RNAi) effects thereof.

(13) FIG. 12 shows the formulation of anti-cancer drugs and/or RNA vaccines, using TMGG-, glycylglycerin- and/or TMGG-glycylglycerin-based delivery agents, and the related delivery and induced RNAi mechanisms thereof. In brief, these TMGG-, glycylglycerin- and/or TMGG-glycylglycerin-formulated nucleic acid-based drugs and/or vaccines are first delivered into target cells via either acetylcholine- or glucose/fructose-receptor-mediated endocytosis, depending on which delivery agents. After endocytosis, endosomes digest the delivery agents and release the anti-cancer drug and/or RNA vaccines, for example, such as pre-miRNAs/miRNAs and siRNAs. Intracellular Dicer RNases then further modify and protect the designed anti-cancer drugs and/or RNA vaccines to form mature miRNAs and/or siRNAs. Then, intracellular Argonuate (AGO) proteins, Dicer RNases and other RNAi-related proteins together form RNA-induced silencing complexes (RISC) around the mature miRNAs and/or siRNAs in order to elicit RNAi effects. As shown in FIG. 11, the induced RNAi effects then directly degrade and/or prevent the translation of the target RNAs, which contain complementary strand sequences to the mature miRNAs and/or siRNAs. The target RNAs include, but not limited to, viral genomes (viron; particularly RNA virus virons), oncogenes (particularly mutated cancer genes), and pathogenic genes that cause human diseases. It is further noticed that, since the RNAi effects only degrade or block a part of the target RNAs, the remaining target RNA fragments may still be translated into some small pieces of proteins, such as viral and/or cancer marker proteins, which are then excluded out of the cells for inducing immune responses and generating antibodies against the viruses and/or diseases (i.e. cancers).

(14) FIG. 13 shows the results of dose-dependent cancer therapy using formulated pre-miR-302 as an anti-cancer drug for treating normal lung epithelial cell line BEAS2B (top left), cancerous lung adenocarcinoma tissue cells isolated from a lung cancer patient CL1-0 (top middle), and lung adenocarcinoma tissue cells isolated from another cancer patient A549 (top right) as well as the dose-dependent therapeutic results of these lung cancers in response to pre-miR-302 treatments at two different concentrations of 50 (bottom left) and 100 (bottom right) microgram (μg)/mL, respectively.

(15) FIGS. 14A and 14B show the therapeutic potency of a formulated pre-miR-302 (F6) drug directed against the growth of malignant lung cancer cells in vitro, using a soft agar colony formation assay. FIG. 14A demonstrated the bar-chart results of the inhibitory effect of F6 on the colony number and size of human malignant lung cancer A 549 cell line. FIG. 14B showed the photos of the average cancer colony sizes before and after different F6 treatments, from left to right: control (original cancers treated with PBS), F5 (treated with the glycylglycerin-based formulation solution only), F6-25 (treated with 25 μg/mL F6), and F6-50 (treated with 50 μg/mL F6), respectively.

(16) FIG. 15 shows the mutation status of several driver genes in a variety of different human lung cancer cell lines and types, including the mutant types of EGFR, p53, and K-ras oncogenes (as shown on the panels to the left of the middle column of pictures of the different cancer cell colonies). The middle column of FIG. 15 shows the pictures of cancer colonies formed by original cancer cells derived from four different human lung cancer cell lines (types) without any treatment, whereas the panels to the right of the middle column of pictures display the inhibitory effect of one F6 treatment (50 μg/mL) on the colony formation of these different lung cancer types, of which the resulting drug potency is categorized into four groups: sensitive (reduced >50% in the average colony size), partial sensitive (reduced 25˜50%), partial resistant (reduced <25%), and resistant groups (no effect 0%).

(17) FIGS. 16A and 16B show the time schedule flowchart of treatment frequency (16A) and image taking frequency (16B) of the first animal trial experiments using a formulated pre-miR-302 drug, called F6, to treat highly malignant and metastatic human lung cancer implants in mice.

(18) FIGS. 17A, 17B and 17C show the therapeutic results of the first animal trial experiments using a formulated pre-miR-302 drug, called F6, to treat highly malignant and metastatic human lung cancer implants in mice. FIG. 17A demonstrated the numbers of lung cancer nodules found in different treatment and control groups of mice, and FIG. 17B showed the representative photo pictures of all lung cancer tissues found in both of the treatment and control groups, respectively. FIG. 17C showed the histological examination results of typical lung adenocarcinoma structures (circled and pointed by a black arrow).

(19) FIGS. 18A and 18B show the time schedule flowchart of treatment frequency (18A) and image taking frequency (18B) of the second animal trial experiments using a formulated pre-miR-302 (F6) drug to treat highly malignant and metastatic human NSCLC implants in mice.

(20) FIGS. 19A, 19B and 19C show the therapeutic results of the second animal trial experiments with a reduced frequency of drug treatments compared to that of the first animal trial, using a formulated pre-miR-302 (F6) drug, to treat highly malignant and metastatic human NSCLC in mice. FIG. 19A demonstrated the numbers of lung cancer nodules found in different treatment and control groups of mice, and FIG. 19B showed the representative photo pictures of all lung cancer tissues found in both of the treatment and control groups, respectively. FIG. 19C showed lymphocyte infiltration, a typical anti-cancer immune response in effect, in the implanted tumors/cancers after the F6 treatments (circled and pointed by a black arrow), of which the immune response may be likely induced by the RNAi-mediated mechanism as shown in FIG. 12.

EXAMPLES

(21) In the experimental disclosure which follows, the following abbreviations apply: M (molar); mM (millimolar); μm (micromolar); mol (moles); pmol (picomoles); gm (grams); mg (milligrams); μg (micrograms); ng (nanograms); L (liters); ml (milliliters); μl (microliters); ° C. (degrees Centigrade); RNA (ribonucleic acid); DNA (deoxyribonucleic acid); dNTP (deoxyribonucleotide triphosphate); PBS (phosphate buffered saline); NaCl (sodium chloride); HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid); HBS (HEPES buffered saline); SDS (sodium dodecyl sulfate); Tris-HCl (tris-hydroxymethylaminomethane-hydrochloride); ATCC (American Type Culture Collection, Rockville, Md.); hESC (human embryonic stem cells); and iPSC (induced pluripotent stem cells).

(22) 1. MicroRNA (miRNA) Production and Isolation and siRNA Synthesis

(23) Dicer-negative cells were acquired from Zymo Research (Irvine, Calif.), transduced with a pre-made miR-302 expression lentiviral vector pLenti-EF lalpha-RGFP-miR302 (Mello Biotech, Santa Fe Springs, Calif.), and maintained according to manufacturers' suggestions. MicroRNAs and microRNA precursors were isolated with a mirVana™ miRNA isolation kit (Ambion, Austin, Tex.), following the manufacturer's protocol. The isolated miRNAs (for example, pre-miR-302) were dissolved in autoclaved 1× Tris buffer at a concentration up to 5 mg/mL and stored at −80° C. till use. For stability tests with HPLC, a desired amount of the isolated RNAs was re-collected with an Amicon Ultra-0.5 mL 30K filter column (Millipore, Billerica, Mass.) and re-dissolved in autoclaved normal saline. For siR-302 preparation, synthetic miR-302 mimics were purchased from Sigma-Genosys (St. Louis, Mo.), containing two cyanine 5.5 (Cy5.5)-labeled RNA sequences: 5′-Cy5.5-UAAGUGCUUC CAUGUUUUAG UGU-3′ (SEQ.ID.NO.4) and 5′-Cy5.5-ACACUAAAAC AUGGAAGCAC UUA-3′ (SEQ.ID.NO.5). In experiments, siR-302 was formed by the hybrids of SEQ.ID.NO.4 and SEQ.ID.NO.5.

(24) 2. Trimethylglycylation of Sugar Alcohols and Formulation of miRNA/shRNA/siRNA

(25) Although the natural way of sugar/sugar alcohol glycylation is unclear, we have developed a chemical procedure to artificially make glycylated sugar alcohols and sugars. First, a pre-made base solution was prepared, containing 0˜5.0M, preferably 0.1˜2.0M glycerin (glycerol) and/or other substitutive sugar alcohols, such as xylitol and erythritol, and/or 0˜1.0M fructose/glucose (optional), and/or about 0.45%˜0.90% NaCl (w/v; optional) at around pH2.5˜pH8.0, depending on the source and amount of sugar alcohol(s) used. For activating trimethylglycylation, about 0.01˜10.0M, preferably 0.5˜5.0M, of USP-grade betaine (TMG) and 0˜2.0M glycine were added and mixed into the pre-made base solution, depending on the desired concentration and type(s) of final trimethylglycylated (TMG) and/or trimethylglycylated-(TMG)-glycylated-mixed sugar alcohol products. The final trimethylglycylated (TMG) sugar alcohol products may include mono-trimethyl-glycyl-glycerin (mono-TMGG; C.sub.8H.sub.18O.sub.4N.sub.1; FIG. 3), di-trimethyl-glycyl-glycerin (di-TMGG; C.sub.13H.sub.28O.sub.5N.sub.2; FIG. 4), and/or tri-trimethyl-glycyl-glycerin (tri-TMGG; C.sub.18H.sub.38O.sub.6N.sub.3; FIG. 5) as well as optional mono-trimethyl-glycyl-erythritol/xylitol, di-trimethyl-glycyl-erythritol/xylitol and/or tri-trimethyl-glycyl-erythritol/xylitol. Alternatively, the final products may also contain TMG-glycylated-mixed sugar alcohols, including but not limited to 2-trimethylglycyl-1,3-di-glycylglycerin (2-TMG-1,3-DGG), 1-TMG-2,3-DGG and/or 3-TMG-1,2-DGG as well as 2- or 3-mono-trimethylglycyl-1,4-di-glycylerythritol, 2-, 3- or 4-mono-trimethylglycyl-1,5-di-glycylxylitol, 2-/3-, 2-/4- or 3-/4-di-trimethylglycyl-1,5-di-glycyl-xylitol and some other mono- and/or di-trimethyl-glycyl-MGG/glycylerythritol/glycylxylitol compositions These different resulting products can be further separately purified and collected using HPLC. Then, this mixed solution was incubated under a dehydration condensation condition at ≥75° C. and ≥100 kPa, most preferably about between 100° C. and 160° C. at about between 101 kPa and 250 kPa (about 14˜37 psi or about 1˜25 bar). When the final volume of the mixed solution was reduced to about or over a half of the starting solution volume due to dehydration, the trimethylglycylation reaction was completed and the final products were ready to be further separately purified and used for formulating and delivering negatively charged materials, such as nucleic acid and similar chemical compositions. The principle of this novel formulation is based on the electrostatic affinity and/or ionic bonding between the trimethyl glycylated and/or the TMG-glycylated-mixed sugar alcohols and the nucleic acid compositions to form encapsulated delivery complexes, which can then be absorbed by cells through an active acetylcholine receptor-mediated endocytosis mechanism. For demonstration, a schematic trimethylglycylation reaction using glycerin as an example was shown in FIGS. 3, 4 and 5.

(26) 3. Human Cell Culture and Transfection

(27) Human lung epithelial cell line and lung cancer cell lines were purchased from ATCC and cultivated according to manufacturer's protocols at 37° C. under 5% CO.sub.2. Cells were passaged at about 50%-70% confluence by exposing the cells to trypsin/EDTA for about 1 min and then rinsing two times in HBSS containing trypsin inhibitor. The detached cells were replated at 1:5 dilutions in fresh EpiLife medium with HKGS supplements. For miRNA transfection, pri-/pre-miR-302 prepared from Example 1 was dissolved in 0.01˜5.0M, preferably 0.1˜2.0M, of TMGG solution at a desired concentration up to 5 mg/ml and then directly applied to cell culture medium based on the miRNA amount needed. For example, to deliver 200 pri-/pre-miR-302, we would need to add 40 μl of the TMGG-dissolved pri-/pre-miR-302 (at 5 mg/ml) into the cell culture medium and then mix well with the cells. Since TMGG is extremely safe and non-toxic, the tested cells could be cultivated in 0.01˜3.0M TMGG, preferably 0.1˜1.0M TMGG, with all necessary supplements and still not showing any adverse effect up to 48 hours.

(28) 4. High Performance Liquid Chromatography (HPLC) Analysis

(29) A reverse-phase HPLC method was developed for analyzing the purity and structural integrity of miR-302 and its precursors (i.e. pre-miR-302s). HPLC programs were run by an Ultimate 3000 HPLC machine (Thermo Scientific) with a DNA Pac PA-100 column (BioLC Semi-Prep 9×250 mm) at a flow rate of 3.6 ml/min. Starting buffer was 50 mM Tris-HCl (pH7.6) and mobile buffer was 50 mM Tris-HCl (pH7.6) with 500 mM sodium perchlorate. Signals of RNAs and DNAs were measured with an UV detector at 260 nm.

(30) 5. MicroRNA (miRNA) Microarray Analysis

(31) At about 70% confluency, small RNAs from each cell culture were isolated, respectively, using the mirVana™ miRNA isolation kit (Ambion). The purity and quantity of the isolated RNAs were assessed, using 1% formaldehyde-agarose gel electrophoresis and spectrophotometer measurement (Bio-Rad), and then immediately frozen in dry ice and submitted to LC Sciences (San Diego, Calif.) for miRNA microarray analyses. Each microarray chip was hybridized with a single sample labeled with either Cy3 or Cy5 or a pair of samples labeled with Cy3 and Cy5, respectively. Background subtraction and normalization were performed as manufacturer's suggestions. For a dual sample assay, a p-value calculation was performed and a list of differentially expressed transcripts more than 3-fold (yellow-red signals) was produced.

(32) 6. Formulation Using TMGG and/or TMG-Glycylglycerin Delivery Agents

(33) For enhancing the in-vitro and in-vivo delivery of DNA/RNA-based drugs and/or vaccines, the nucleic acid drug or vaccine compositions were first dissolved in a proper amount of autoclaved ddH2O, normal saline, Tris buffer or TE buffer at their highest possible soluble concentrations and then directly mixed with the pre-prepared TMGG and/or TMG-glycylglycerin delivery agents to reach a proper concentration for the use in treatments. Different treatments may require different concentrations of the TMGG and/or TMG-glycylglycerin-formulated nucleic acid drugs or vaccines for the best therapeutic results. For example, for testing toxicity, we injected 200 μg of synthetic siR-302 (from Example 1) dissolved in 200 μl of 0.1˜2.0M, preferably 0.5˜1.0M, TMGG or TMG-MGG/TMG-DGG solution into each of C57BL/6J strain mice via tail vein injection. Approximately 24 hours post-injection, we sacrificed two mice for observing the TMGG- and/or TMG-MGG-/TMG-DGG-delivered siR-302 distribution in vivo. As these siR-302 molecules were labeled with infra-red fluorescent dye Cy5.5, we could directly observe their in-vivo distribution using a bio-imaging system and/or their fluorescent signals in mouse tissue sections under a fluorescent microscope.

(34) 7. In Vitro Lung Cancer Sensitivity to Drug Tests

(35) TMGG-, glycylglycerin- and TMG-glycylglycerin-formulated nucleic acid drugs can elicit the same RNAi effects to silence pathogenic genes (such as oncogenes and viral genes), but through different carrier-protein-mediated endocytosis mechanisms. For lung cancer therapy, since lung cancer cells carry abundant GLUT and acetylcholine (particularly nicotinic) receptors, all three delivery agents will provide almost the same delivery results and RNAi effects on the treated cancer cells. For example, as shown in FIG. 13, the dose-dependent tumor suppression effect of a glycylglycerin-encapsulated pre-miR-302/shRNA drug (or called formula #6; F6) on the growth proliferation of different lung cancer cell types was tested, using various pre-miR-302 concentrations ranged from 0 to 200 μg/mL, preferably between 25˜100 μg/mL. In order to further test the F6 drug potency against the growth of malignant lung cancer cells, a soft agar colony formation assay was performed, as shown in FIGS. 14A and 14B. The results showed that both of the growth and colony formation abilities of a typical human malignant lung cancer cell line A549 were markedly inhibited after one F6 treatment (either 25 or 50 μg/mL), especially in the population of large size colonies (diameter ≥200 μm). In addition, FIG. 15 further demonstrated the mutation status of several driver oncogenes in a variety of different human lung cancer cell types, including the status of mutated EGFR, p53, and K-ras oncogenes. The middle column of FIG. 15 shows the pictures of cancer colonies formed by original cancer cells of four different human malignant/metastatic lung cancer cell lines (types) without any treatment, whereas the panels to the right of the middle column display the inhibitory effect of one F6 treatment (50 μg/mL) on the colony formation of these lung cancer types, of which the resulting drug potency in these cancer cell types was categorized into four groups: sensitive (reduced >50% in the average colony size), partial sensitive (reduced 25˜50%), partial resistant (reduced <25%), and resistant groups (0%).

(36) 8. In Vivo Lung Cancer Therapy Trials

(37) After understanding the tumor suppression potency of our formulated pre-miR-302 drug (F6) on different human lung cancer cell types, we further analyzed its in-vivo therapeutic potency using an orthotopic lung cancer mouse model. FIGS. 16A and 16B showed the time schedule flowchart of F6 treatment frequency and image taking frequency used in an in-vivo bio-imaging system (IVIS). For orthotopic tumor implantation assays, A549-Luci lung cancer cells (1*10.sup.5 cells in 20 μl PBS containing 10 ng Matrigel) were injected into the pleural cavity of 6-week-old NOD SCID mice (n=9 in treatment groups and n=3 in control group). A bio-imaging study with luciferase image observation indicated that these implanted mice developed many lung metastasis nodules four (4) weeks after implantation. After that, mice were treated with F6 twice per week via tail vein injection until sacrifice (FIG. 17A). On day-14 post-implantation, mice were divided into three groups: normal saline (NS), and treatments of either 50 or 100 μg/mL of F6, as shown in the imaging results of IVIS (FIG. 17B). The volume of F6 solution used was calculated based on the ratio of body weight and total blood volume in order to keep the same F6 concentration treated in the same group of tested mice. Luciferase signals were observed and measured once per week. In the end, mice were sacrificed 42 days after the first F6 treatment. Major organs such as lung, live, spleen, and kidneys were collected and fixed by 10% formalin, and then the resulting lung nodules were counted using gross and microscopic examination. The number of mice used for the experiments was based on the goal of having 98% power to detect a 2-fold between-group difference in nodule number at P<0.05.

(38) In animal trials using in-vivo orthotopic lung cancer assays (FIGS. 18A-18C), we injected lung cancer cells in the left pleural cavity of each tested mouse to observe the lung to lung metastases. As a result, the cancer nodules found in the right lobes indicated the metastasis of lung cancers from the primary cancer implant side in the left lobe. FIG. 18A demonstrated the numbers of lung cancer nodules in different experimental and control groups, and FIG. 18B showed the representative photo pictures. In FIG. 18A, the black bar illustrated the nodules found in left lobe, and the white bar showed the nodules found in right lobes. As a result shown in FIGS. 18A and 18B, the nodule numbers in both treatment groups (50 and 100 μg/ml) were significantly decreased in both left and right lobes of lung. Further histological examination (FIG. 18C) was also performed to observe typical lung adenocarcinoma structures (circled and pointed by a black arrow) in all groups

(39) In order to further evaluate the strong therapeutic effects of the F6 drug on metastatic lung adenocarcinoma, we reduced the treatment frequency of F6 solution in the in-vivo orthotopic lung cancer model (n=11 for both treatment groups and n=5 for control group). As shown in FIGS. 19A and 19B, in this repeated animal trials, mice were treated with F6 via tail vein injection twice per week during the week 3 and 4 and then once a week after week 5 until sacrifice. The applied dosage of F6 was calculated based on the ratio of body weight and total blood volume in order to keep the same F6 concentration treated in all tested mice. Luciferase signals were observed and measured once per week. In the end, mice were sacrificed on day-42 post-treatment. To assess the acute toxicity effects of the pre-miR-302 drug, the mice of one tested group were treated only four times of F6 during the week 3 and 4, which was labeled as the 50 (4) group (FIG. 19B). Furthermore, we also tested the toxicity of glycylglycerin only formula in this in-vivo mouse model to rule out any possible toxicity interference of the delivery formulation agent (F5), which actually presented neither toxicity nor any significant effect on the cancer cells.

(40) 9. Statistic Analysis

(41) Any change over 75% of signal intensity in the analyses of immunostaining, western blotting and northern blotting was considered as a positive result, which in turn is analyzed and presented as mean±SE. Statistical analysis of data was performed by one-way ANOVA. When main effects were significant, the Dunnett's post-hoc test was used to identify the groups that differed significantly from the controls. For pairwise comparison between two treatment groups, the two-tailed student t test was used. For experiments involving more than two treatment groups, ANOVA was performed followed by a post-hoc multiple range test. Probability values of p<0.05 was considered significant. All p values were determined from two-tailed tests.

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(43) TABLE-US-00001 SEQUENCE LISTING (1) GENERAL INFORMATION:     (iii) NUMBER OF SEQUENCES: 5 (2) INFORMATION FOR SEQ ID NO: 1:     (i) SEQUENCE CHARACTERISTICS:            (A) LENGTH: 23 base pairs            (B) TYPE: nucleic acid            (C) STRANDEDNESS: duplex            (D) TOPOLOGY: siRNA     (ii) MOLECULE TYPE: RNA            (A) DESCRIPTION: /desc = “synthetic”     (iii) HYPOTHETICAL: YES     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:     GUUGGUUGCC AUAACAAGUG UGC 23 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:            (A) LENGTH: 23 base pairs            (B) TYPE: nucleic acid            (C) STRANDEDNESS: duplex            (D) TOPOLOGY: siRNA     (ii) MOLECULE TYPE: RNA            (A) DESCRIPTION: /desc = “synthetic”     (iii) HYPOTHETICAL: YES     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:     GAUAAAGGAG UUGCACCAGG UAC 23 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:            (A) LENGTH: 23 base pairs            (B) TYPE: nucleic acid            (C) STRANDEDNESS: duplex            (D) TOPOLOGY: siRNA     (ii) MOLECULE TYPE: RNA            (A) DESCRIPTION: /desc = “synthetic”     (iii) HYPOTHETICAL: YES     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:     CUACCGAAGA GCUACCAGAC GAA 23 (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:            (A) LENGTH: 23 base pairs            (B) TYPE: nucleic acid            (C) STRANDEDNESS: single            (D) TOPOLOGY:     (ii) MOLECULE TYPE: RNA            (A) DESCRIPTION: /desc = “synthetic”     (iii) HYPOTHETICAL: YES     (iv) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:     UAAGUGCUUC CAUGUUUUAG UGU 23 (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:            (A) LENGTH: 23 base pairs            (B) TYPE: nucleic acid            (C) STRANDEDNESS: single            (D) TOPOLOGY:     (ii) MOLECULE TYPE: RNA            (A) DESCRIPTION: /desc = “synthetic”     (iii) HYPOTHETICAL: YES     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:     ACACUAAAAC AUGGAAGCAC UUA 23