Compositions and methods for treating cancer using modified sirna-gem agents

Abstract

The present invention provides a pharmaceutical composition comprising an RNAi agent comprising a gemcitabine ("GEM") modification, such as a siRNA molecule, in combination with a histidine-lysine copolymer carrier, intended for inhibiting the expression of a target gene involving a variety of cancers. The modifications to the siRNA backbone may include, but are not limited to, 2 '-methoxy, 2'-fluoro, and thiophosphate modifications Also provided are methods of treating a variety of cancers by administering such pharmaceutical compositions, including breast cancer, ovarian cancer, and pancreatic cancer in mammals.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 329,240, filed on April 8, 2022, the contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in XML format, and its entire contents are incorporated herein by reference. The sequence listing in XML format was created on April 7, 2023, named 4690_0032I_SL.xml, and is 178,201 bytes in size.

[0005] field

[0006] Modified RNAi agents or complexes containing gemcitabine for inhibiting the expression of selected target genes are provided. Methods for treating cancer are also provided, wherein pharmaceutical compositions containing these RNAi agents and complexes are used to treat various cancers.

[0007] background

[0008] RNAi agents (e.g., double-stranded siRNA molecules) containing gemcitabine ("GEM") have been previously reported in combination with histidine-lysine copolymer carriers for the treatment of cancer, see, e.g., WO2021 / 067825. The targeted gene is a gene that drives tumor development but is not important for normal cell function.

[0009] siRNA is a double-stranded RNA molecule consisting of a sense strand and a complementary antisense strand. These molecules may have modified backbones and may be blunt-ended, for example 19-29 bases per strand, or they may exhibit two base overhangs (usually dTdT).

[0010] Each strand of siRNA is typically prepared on a synthesizer by conjugating the next base in the desired sequence to the previous base attached to the oligonucleotide being synthesized. Amide chemistry and other synthetic methods are well known in the art. After synthesis, the two strands anneal to each other to form a double strand.

[0011] It has been demonstrated that siRNA directed against selected targets within cancer cells may be able to reduce the expression of proteins encoded by the silenced gene targets. Silencing these genes in turn can inhibit the growth of that cell. If the cell is a diseased cell (e.g., a cancer cell) that the siRNA can enter, the siRNA can act as a therapeutic agent. In addition, in some cases, it has been found that the use of specific therapies (small molecule inhibitors, monoclonal antibodies, etc.) that are currently the "gold standard" for treatment can enhance the efficacy by silencing genes in specific pathways.

[0012] GEM (2',2'-difluoro-2'-deoxycytidine) is a pyrimidine nucleoside analog that is taken up by nucleoside transporters when administered systemically, activated by triphosphorylation of deoxycytidine kinase, and then can be incorporated into RNA or DNA. It replaces nucleic acid cytidine during DNA replication (cell division) and can inhibit tumor growth; new nucleosides cannot attach to the nucleosides, leading to cell apoptosis (Damaraju et al., Oncogene 22:7524–7536 (2003)).

[0013] GEM is the mainstay of treatment for pancreatic cancer (Burris et al., J Clin Oncol 15:2403–2413 (1997)) but is also used to treat many other cancers, including cholangiocarcinoma (Jo and Song, "Chemotherapy of Cholangiocarcinoma: Current Management and Future Directions". Topics in the Surgery of the Biliary Tree", chapter 3; p35-52.

[0014] http: / / dx.doi.org / 10.5772 / intechopen.76134,SY(2018)), non-small cell lung cancer (Muggia et al., Expert Opinion on Investigational Drugs, 21:4, 403-408(2012)), ovarian cancer (Le et al., Gynaecol. Oncol. Res. Pract. 4:16(2017)) and breast cancer (Xie et al., al., Oncotarget, 9:7148-7161(2018)). Gemcitabine is taken up by nucleoside transporters, activated by deoxycytidine kinase, and incorporated into RNA and DNA. Its activity is enhanced by inhibition of ribonucleotide reductase and dCMP deaminase, while cytidine deaminase converts gemcitabine to its potentially inactive metabolite 2',2'-difluorodeoxyuridine, which in its nucleotide form may inhibit thymidylate synthase. Gemcitabine is administered systemically to patients by intravenous infusion. The standard dosing regimen for gemcitabine is weekly infusion over 30 minutes at a dose of 1000 mg / m 2, but its utility is limited because it is distributed not only in tumor cells but also in normal cells, thus exhibiting significant toxicity. In addition, in order to see efficacy against certain tumor types, the dose needs to be increased to very high levels, and the therapeutic index of the drug may be very limited.

[0015] GEMs are widely used in combination therapy, primarily with platinum analogs. Other alternatives to gemcitabine combination therapy for ovarian cancer include enhancing ribonucleotide reductase inhibition with triazepine or hydroxyurea. GEMs (like 5-FU and other nucleoside analogs) can be chemically synthesized by direct coupling to DNA or RNA bases, either by conventional synthesis (manually or using automated instrumentation).

[0016] Various approaches have been tried to overcome GEM toxicity. One of the most promising approaches is to use targeted delivery agents to selectively increase delivery to tumor cells while reducing delivery to normal cells.

[0017] Examples of target genes that enhance the effects of the compound gemcitabine include siRNAs targeting RAD17, CHK1, CHK2, ATR, ATM, etc. (to name a few) (references: Azorsa, J. Transl. Med. 7:43 (2009); Fredebohm (Journal of Cell Science 126:3380–3389 (2013), Plunkett et al., Semin Oncol 23:3–15 (1996)). As described above, these studies attempt to find other targets within cells that, when inhibited (by antagonists, such as small molecules or antibodies, etc.) or silenced (using siRNA or miRNA), result in a beneficial shift in the dose-response curve of the drug, allowing it to exhibit the same efficacy at a lower dose / concentration. This dose response can be observed using siRNAs targeting, for example, CHK1.

[0018] These siRNAs can be delivered to the tumor environment of animals / humans suffering from the disease using a variety of targeted or non-targeted delivery agents. These delivery vehicles can be composed of lipids, modified lipids, peptide delivery vehicles, etc., or even by attaching targeting ligands directly to modified (chemically stable) siRNA molecules, by modifying the backbone to prevent siRNA from being degraded by nucleases and other enzymes encountered in the circulation.

[0019] Recently, GalNAc-modified siRNAs have been used to facilitate the specific delivery of these siRNAs to hepatocytes within the liver.

[0020] The GalNAc moiety binds with very high affinity to the asialoglycoprotein receptor (ASGPR) which is specific and abundant on hepatocytes. ASGPR is thought to be internalized into the cell after binding, thus carrying the attached siRNA into the cell.

[0021] Other targeting ligands that can deliver payloads to specific cell types include GLP1 peptides (binding to the GLP1 receptor on pancreatic beta cells), RGD motifs (e.g., cRGD, iRGD, or peptides derived from foot-and-mouth disease virus that bind to the α5β3 integrin receptor (binding with nanomolar affinity to the α5β6 integrin receptor and almost micromolar affinity to the α5β3 receptor)), folate ligands (binding to the folate receptor), transferrin ligands that bind to the transferrin receptor, and targeting EGFR via the EGF receptor. Many other examples of targeting moieties show specificity for delivery to different cell types.

[0022] The disclosed embodiments provide pharmaceutical compositions and methods for treating cancerous tumors involving co-delivery of modified siRNA with GEMs to produce greater therapeutic benefit than administration of siRNA or GEMs alone or administration of unmodified siRNA with GEMs.

[0023] Summary

[0024] In the disclosed embodiments, a pharmaceutical composition or formulation is provided, which comprises a modified interfering ribonucleic acid (RNAi) molecule, which is combined with one or more gemcitabine molecules to replace the cytidine molecule. The composition also includes a histidine-lysine copolymer carrier, such as HKP or HKP (+H). The RNAi molecule can be siRNA, miRNA, shRNA or other oligonucleotides capable of silencing or inhibiting at least one target gene. Modifications of the main chain include 2'-OMe, 2'-fluorine and thiophosphate modifications, etc.

[0025] The composition may include one or more siRNA molecules identified in Figure 1 SEQ.ID Nos.1-10 and 13-14. In some embodiments, the composition may include any one of SEQ.ID Nos.1-10 in combination with at least SEQ.ID No.13 or SEQ.ID No.14. In some composition embodiments, lipofectamine (liposome) may be added. In certain other embodiments, the siRNA is a double-stranded siRNA with a modified backbone and at least one gemcitabine molecule.

[0026] Also provided are examples of methods for treating a variety of cancers in mammals. In one embodiment, a method for treating a subject with cancer is provided, and cancer growth is attenuated or inhibited when an effective amount of a pharmaceutical composition comprising a modified siRNA molecule is administered to the subject, wherein the modified siRNA molecule comprises gemcitabine and a histidine-lysine copolymer carrier, wherein the siRNA is at least one selected from the groups of SEQ.ID Nos.1-10 and 13-14. In some embodiments, the cancer to be treated is breast cancer; others are colon cancer or ovarian cancer. Still others are bladder cancer, lung cancer, pancreatic cancer, or gastric cancer. In some embodiments, the composition is administered intravenously; in other embodiments, it is administered intratumorally, and in some embodiments, the composition is administered by subcutaneous injection. In some embodiments for treating cancer, lipofectamine is included in the composition administered.

[0027] In certain embodiments, a composition comprising SEQ.ID No.9-10 and SEQ.ID No.8 is administered to a subject with cancer, and HKP(+H) is also included as a carrier. In certain embodiments involving this combination of siRNA sequences, the cancer being treated is breast cancer. In another treatment embodiment method, a composition comprising SEQ.ID No.9-10 and SEQ.ID No.13 is administered to a subject, and HKP(+H) is also included as a carrier. In certain embodiments involving this sequence combination, the cancer being treated is ovarian cancer. In some embodiments, some methods of cancer treatment may involve intravenous administration, while in other types of cancer, administration may involve in situ tumor administration. In certain embodiments of administering a pharmaceutical composition in situ tumor to treat a pancreatic cancer subject, the composition comprises SEQ.ID No.7-8. The expression of the target gene, tumor volume, or cancer cell level is reduced by more than 20%; in other embodiments, the expression of the target gene is reduced by more than 30% to 90%; in other treatment method embodiments, the expression of the targeted gene is reduced by more than 90%, and up to 100%. In certain treatment method embodiments, the subject is a mammal. In other embodiments, the subject is human. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 (a)-(b) shows (a) prChk1-GEM variant sequences and sense and antisense strands of non-silencing (NS) siRNA+GEM (SEQ.ID No. 1-12, respectively, in order of appearance); (b) sequences of some additional siRNA molecules (excluding GEM) used in the examples disclosed herein (SEQ.ID No. 13 to 16). Different fonts in the sequence represent the following: uppercase: 2'-methoxy; lowercase: 2'-fluoro; uppercase / underline: 2'-hydroxyl; Phos: phosphate; GEM: gemcitabine; and dT: deoxythymidine.

[0029] Figure 2 The effects of polyGEM variants on MiaPaCa-2 cell viability are shown, indicating that the IC 50 PNG6 (0.04 nM), STD1 (0.02 nM) and PNG7 (0.006 nM) showed increasingly significant effects, and all of these variant siRNAs were transfected by lipofectamine. (X±SD; n=4).

[0030] Figure 3 (a)-(c) shows the effect of two variants of PGN6 (DM and BH), with or without lipofectamine transfection, on cell viability when increasing siRNA levels in (a) BXPC3 (WT); (b) MiaPaCa (G12C); (c) Panc10.05 (G12D) pancreatic cancer cell lines.

[0031] Figure 4 (a)-(b) shows the effects of two variants of PGN6 (DM and BH), with and without lipofectamine ("lipo") transfection, on cell viability in (a) H358 (G12C) and (b) H1299 (WT) lung cancer cell lines.

[0032] Figure 5(a)-(b) shows the effect of these variants on cell viability in (a) HT29 (WT) and (b) LS180 (G12D / WT) colon cancer cell lines. Both variants showed potency and efficacy against pancreatic, lung, and colon cancer cells. In the absence of lipofectamine, PNG exerts its effect by releasing gemcitabine (GEM) from the structure. In the absence of lipofectamine, IC 50 The variation is usually 10 to 1000 times.

[0033] Figure 6(a)-(f) shows the efficacy of STD1, PGN6 and PGN7 in (a) Panc 10.05 (G12D); (b) MiaPaCa (G12C); (c) H1299 (WT); (d) H358 (G12C); (e) HT29 (WT); (f) LS180 (G12D / WT) pancreatic cancer, lung cancer and colon cancer cell lines, compared with non-silencing (NS) siRNA used as control.

[0034] Figure 7 (a)-(e) shows the effects of PGN7 and the combination of PGN7 and Wee1#7 siRNA on (a) Panc 10.05 (G12D); (b)

[0035] Efficacy of MiaPaCa(G12C); (c) H358(G12C); (d) H1299(WT); (e) LS180(G12D / WT) in pancreatic, lung and colon cancer cell lines.

[0036] Figure 8(a)-(e) show the efficacy of PGN7 variants and their combination with BCLxL in (a) Panc 10.05 (G12D); (b) MiaPaCa (G12C); (c) H358 (G12C); (d) H1299 (WT); (e) LS180 (G12D / WT) pancreatic cancer cell lines and lung cancer (but not colon cancer).

[0037] Figure 9(a)-(c) shows that PGN7+NS (i.e., combined with non-silencing siRNA), BCLxL#5+NS, and PGN7+

[0038] BCLxL#5 combination in (a) HT1376 (400 cells / well) and HTB-9 (800 cells / well) bladder cancer cell lines; (b) N87 gastric cancer cell line; (c) OVCAR-3 ovarian cancer cell line.

[0039] Figure 10 (a)-(d) show the efficacy of PGN7+NS (i.e., in combination with non-silencing siRNA), Wee1#7+NS, and PGN7+Wee1#7 combinations in (a) HT1376; (b) HTB-9 bladder cancer cell line; (c) N87 gastric cancer cell line; and (d) OVCAR-3 ovarian cancer cell line.

[0040] Figure 11(a)-(c) show the efficacy of PGN7+NS (i.e., combined with non-silencing siRNA), BCLxL+NS, Weel#7+NS, and PGN7+BCLxL#5 or PGN7+Wee1# in (a) HT1376; (b) HTB-9 bladder cancer cell line; (c) N87 gastric cancer cell line; and (d) OVCAR-3 ovarian cancer cell line.

[0041] Figure 12 (a)-(b) shows the effect of 2 mg / kg of PolyGEM variant PGN6 in HKP(+H) on (a) mean pancreatic tumor volume and (b) body weight of mice, compared to a control group receiving non-silencing (NS) siRNA and HKP(+H). Panel (a) shows treatment group (increase in mean tumor volume) and control group (stable volume) values ​​(mean ± SE). Panel (b) shows equivalent mean treatment group values ​​and control group values ​​(mean ± SD) over time.

[0042] Figure 13 (a)-(b) shows that the IC values ​​of PGN7 and BCLxL#5 were significantly higher in (a) triple-negative breast cancer (MDA-MB231) and (b) ovarian cancer (OVCAR3) cell lines than in (b) PGN7 or BCLxL#5 alone. 50 Significantly reduced. (a)IC 50 =1.1 nM; and BCLxL#5+NS, IC 50 =0.5nM. (b)IC 50 =0.06nM; compared with the effect of PGN7+NS, IC 50 =1.1 nM; and BCLxL#5+NS, IC 50 =0.5nM.

[0043] Figure 14 (a)-(c) shows the effects of STD1 and PGN7 alone on (a) tumor weight, (b) body weight, and (c) tumor volume when the treated tumor tissues were implanted in mice and grown for 3.5 weeks. Xenograft tumors from all treatment groups were harvested and weighed on day 25. There were no significant differences in the mean (±SE, n=8) tumor weights of STD 1, PGN 7, and the control group. Figure 14 (c) shows the growth results of xenograft tumors after NU / NU mice were subcutaneously implanted with MIA PaCa tumor cells and injected with STD1 and PGN7 twice a week in the single treatment group starting from day 0. Tumor volume was measured over time as shown in the figure (mean ± SE) and tested with ANOVA Tukey test. Only PGN7 significantly reduced tumor volume from day 14 (p<0.02).

[0044] Detailed description

[0045] The disclosed embodiments provide pharmaceutical compositions and methods of treatment using such compositions. Specifically, the disclosed embodiments provide interfering RNA (RNAi), such as modified siRNA comprising gemcitabine (GEM), which is used together with a histidine-lysine copolymer vector to silence the expression of one or more target genes.

[0046] In the disclosed embodiments, the modified siRNA or other nucleic acid molecules target and bind to complementary sequences on target genes to silence them and induce the desired therapeutic effect. The siRNA or other nucleic acid molecules are formulated into nanoparticle formulations together with a polypeptide polymer containing at least one histidine residue and at least one lysine residue. In some embodiments, more advantageously, the polypeptide polymer is HKP or HKP(+H). Chemical modification of siRNAs containing GEMs (e.g., those targeting Chk1) can improve serum stability compared to unmodified siRNAs containing GEMs.

[0047] One or more GEM molecules in each siRNA replace one or more cytosine moieties within a modified oligonucleotide (siRNA or other RNAi). The sequences of the sense and antisense strands of the prChk1-GEM variant and the non-silencing (NS) siRNA with GEM are shown in Figure 1(a).

[0048] Target selection for enhancing GEM activity

[0049] Constructs containing GEMs: Azorsa et al. (J. Transl. Med. 7: 43 (2009)) identified siRNAs against the gene CHK1, which showed an enhancement of the effect of gemcitabine in cultured pancreatic cancer cells. Subsequently, Fredebohm (Journal of Cell Science 126: 3380–3389, 2013) verified that CHK1 is a factor that enhances the effect of gemcitabine in pancreatic tumor cells, but also identified several other potential targets that can increase the activity of gemcitabine when silenced. RAD17 was also identified as one of these targets, and it was demonstrated that the effect of gemcitabine in reducing cell viability was significantly improved. The present disclosure again describes siRNA molecules against the CHK target, but the skilled person will recognize that the principles described herein can be applied to siRNA molecules containing GEMs against any target.

[0050] The study showed that even in the absence of gemcitabine, CHK1 silencing itself exhibits some toxicity in cells, which requires that siRNA targeting CHK1 must be used in a way that it is preferentially taken up by tumor cells rather than by normal dividing cells.

[0051] Polypeptide nanoparticles ("PNPs") prepared using histidine-lysine branched copolymers can simultaneously deliver multiple siRNAs to the same cell. These PNPs can also be used to deliver siRNAs to multiple tumor cell types when expressed as xenograft tumors in animal models.

[0052] As described below, siRNA molecules containing GEM portions can be prepared, which are linked to the ends and / or the interior of the siRNA sequence. These siRNAs containing GEMs still have the function of silencing the target gene, and enhance the efficacy of killing tumors by releasing GEMs into cells, where a synergistic effect can be seen between the gene silencing activity and the drug action, that is, the observed effect is greater than the effect achieved by administering siRNA and GEMs separately as separate components.

[0053] In order to determine whether GEM can be better processed by natural nucleotide separation, we took advantage of the fact that gemcitabine is a cytidine analog, so it can be directly incorporated into the siRNA sequence instead of cytidine (C). Gemcitabine amide can be used to form a polymer composed entirely of GEM parts. (Ma et al, Chem. Commun., 55: 6603-6606 (2019)) These polymers form nanogels and show certain activity in vitro and in vivo cancer cell models. Modified nucleosides incorporating amide functional groups can be used to introduce modified nucleosides (such as GEM) into one of the chains in the double strand during its synthesis. Since the antisense strand from the siRNA duplex must be annealed to the corresponding sequence in the sense strand in its siRNA region, and the antisense strand is integrated into the RISC complex and used to monitor the match with the mRNA sequence, the modified nucleosides are first attached to the sense strand of the siRNA to form an extension of the sense strand length relative to the antisense strand. Multiple (n) non-natural nucleobases can be incorporated into the termini of the strands such that delivery of a single siRNA molecule results in co-delivery of multiple (n) non-natural nucleosides.

[0054] Once in the cytoplasm, the antisense strand is incorporated into the RISC complex, and if a matching mRNA sequence is recognized, the mRNA is cleaved by the DICER enzyme. The sense strand is removed in the process and then cleaved within the cytoplasm. The unnatural nucleoside analog incorporated into the end of the sense strand will then be cleaved by endogenous nucleases. The released gemcitabine molecules (or similar structures with anticancer unnatural nucleoside analogs) subsequently inhibit the tumor cell replication machinery and, consistent with the reduced expression of the gene targeted by the siRNA, will exhibit a greater effect in reducing tumor cell growth than in the absence of these agents.

[0055] The chemically unmodified siRNA molecules incorporating GEM residues can be delivered in the form of suitable nanoparticle formulations to protect the siRNA from degradation in the blood circulation or tissues, tumor microenvironment, etc. Once entering the target tumor or tissue, the siRNA will be released into the tumor cells to produce its cytotoxic effect.

[0056] siRNA molecules synthesized using GEM amide can be delivered via a histidine-lysine polypeptide (copolymer) nanoparticle (HKP PNP) nanoparticle system. GEM can be included in a siRNA double-stranded sequence that targets a gene and then enhances the effect of gemcitabine in killing tumor cells - reducing the amount of drug required for treatment, achieving therapeutic effects and reducing the possibility of toxic effects caused by gemcitabine alone in patients. As described below, a previously disclosed siRNA sequence against CHK1 (Azorsa 2009, supra) has been shown to enhance the effect of gemcitabine when the siRNA sequence is administered separately from gemcitabine.

[0057] Methods well known in the art can be used, for example, by using chemical modifications such as 2'-OMe and / or 2'-F and / or thiophosphate modifications to stabilize siRNA to resist nuclease degradation. siRNA can be optionally combined with a targeting moiety (e.g., GalNac, RGD, folic acid, TFR, EGFR, peptide targeting ligands, aptamers or other carbohydrates, or even small molecules, antibodies or nanobodies). The targeting moiety has affinity for receptors or other targets on the cell surface, thereby enriching the uptake of these cells. As with unmodified siRNA, the SS and AS chains will separate in the cell, the AS chain will cause the target gene to be silenced, and the released SS-polyGEM structure will be degraded, releasing GEM, and will show a greater effect than any one alone when combined with the silenced gene.

[0058] Polygemcitabine can also be coupled into a single oligonucleotide with a sense strand and an antisense strand of equal length. After annealing, it forms a "polygemcitabine ring" connected between the two strands of double-stranded RNA. Compared with two strands, a single oligonucleotide sequence has a lower manufacturing cost and also has the potential to improve product stability, and each siRNA can deliver a larger number of GEM molecules, thereby improving the efficacy against cancer cells. In addition, in some cases, the two strands of RNA may not have to have the same length, as long as there are enough bases in the shorter strand to allow hybridization with the relevant homologous sequence in the longer strand. In these cases, the gemcitabine ring may be asymmetric between the two strands.

[0059] In another embodiment, these constructs are targeted for delivery to specific cell types by connecting a targeting ligand, which binds to a target present on a target cell with high affinity, but not on other non-target cells. For targeted delivery, multiple ligands can be connected to the gemcitabine ring region--multivalent targeting advantages (increasing binding affinity and possibly improving cell specificity) are provided by combining multiple ligands with its target simultaneously. The targeting ligand is conjugated to the gemcitabine in the ring region by a joint. This allows the conjugate of oligonucleotide and gemcitabine to be specifically delivered to the target cell or tissue, where they can then induce additional or synergistic or more extensive therapeutic effects. Ligands can also be added to the Gem ring in the asymmetric chain construct.

[0060] Figure 1(a) shows CHK1 siRNA molecules containing a GEM portion, as well as non-silencing (NS) siRNAs used in the examples of the present disclosure. Figure 1(b) shows additional siRNAs also used in these examples or discussed in the present disclosure. In the absence of a GEM, the SS and AS strands anneal to form siRNAs targeting CHK1.

[0061] Since gemcitabine is a cytidine analog, it can be used to replace the cytidine part in the RNA oligonucleotide sequence or the deoxycytidine part in the DNA oligonucleotide with gemcitabine. These GEMs will still hybridize with the counterparts on the second strand of the oligonucleotide, that is, the GEMs will hybridize with the "G" (guanosine / deoxyguanosine) on the opposite strand. Therefore, by replacing the GEMs into the sense strand or antisense strand of the siRNA or miRNA, or even the cytosine part in the single-stranded sequence (such as antisense oligonucleotide (ASO)), the effect of the siRNA sequence of the targeted gene is enhanced, and the gene will produce cell growth inhibition and can kill tumor cells when silenced. In the CHK1_AZ siRNA sequence that silences the CHK1 gene, replacing two cytosines in the sequence with GEMs can enhance the inhibitory activity of the individual siRNAs.

[0062] GEMs can also be added to the 3' or 5' end of the sense strand. They can also be added to the 3' or 5' end of the AS strand. The additions can independently contain 1, 2, 3, 4 or more GEM moieties.

[0063] Other nucleoside anticancer agents can be linked to the oligonucleotide backbone in place of gemcitabine. Such analogs can include cytarabine (ara-C), 5-FU, 5-deoxy-5-fluorouridine, fludarabine, capecitabine, cladribine, troxacitabine or clofarabine, azacitidine and 5-deoxyazacitidine, to name a few (see Damaraju, supra).

[0064] Many siRNA sequences can attach polynucleoside analogs on the sense strand or antisense strand. The example of such siRNA sequence is siRNA that silences the gene of the protein that the inhibitory mechanism of nucleoside analogs reacts against. For example, siRNA can be used to silence the enzyme (cytidine deaminase or 5'-nucleotidase) responsible for gemcitabine deamination and inactivation or the enzyme that is responsible for enhancing the release of nucleosides from cells--such as nucleoside transporter (see Damaraju above and references therein).

[0065] Other potential targets for use as siRNA sequences (linked to nucleoside analog chains) in these constructs include multidrug resistance (MDR) proteins such as P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1), encoded by the MDR1 and MRP1 genes, respectively. Expression of these proteins confers a resistance phenotype to a variety of drugs used in cancer chemotherapy. These transporters are broadly classified as ATP-binding cassette proteins (ABCs) and constitute a superfamily of proteins. Methods for silencing the MDR1 and MRP1 genes using siRNA have been described. See and Gündüz, Biomed Pharmacother. 65(2):85-9 (2011). Other examples of genes that can be silenced include methyltransferases, organic anion and cation transporters (OAT and OCT), oxidoreductase flavoproteins, and nucleoside transporters (NT), all of which are involved in intracellular drug transport and metabolism and may convey a drug-resistant phenotype to cancer cells.

[0066] Other gene targets for siRNA have been shown to enhance the effects of gemcitabine or other nucleoside analogs when silenced themselves. In addition to CHK1, such gene targets include ATR, RAD17, and WEE1. As other targets have been silenced and shown to enhance nucleoside activity, siRNAs directed against them can also be coupled to appropriate nucleoside analogs.

[0067] There are 2 C (cytosine) (on SS and AS strands) in the sequence used by Azorsa 2009 that can be replaced by Gem. Therefore, we made a construct in which only SS was modified and 2 Gems replaced Cs, and we made AS strand in the same way. We then annealed SS containing 2 GEMs and unmodified AS strand; and annealed AS strand containing one GEM modification and unmodified SS. We further annealed GEM-modified SSS and GEM-modified AS.

[0068] The siRNA sequence identified in Azorsa et al. 2009 (CHK1_AZ) is an example of an siRNA that can be used to prepare GEM-containing molecules. Using an oligonucleotide synthesizer, the following siRNA strands were made with or without GEM residues in place of the natural cytidine ("C") groups present in the sequence.

[0069] Synthesizing siRNA using GEM

[0070] CHK-Az siRNA is a 19 nucleotide long siRNA containing a 2 base (dTdT) overhang at the 3' end of each strand. When gemcitabine modifications were included within the sense strand, we inserted them between the sense strand end and the dTdT terminal group. The dTdT end may help stabilize the siRNA sequence against nuclease degradation, which in turn may affect the rate of gemcitabine release from the sense strand as it dissociates from the AS in the RISC complex during the period when the AS strand monitors the cognate mRNA sequence for cleavage and silencing.

[0071] Synthesized siRNA molecule analogs in which 2, 4 or 6 GEMs were appended to the 3' end of the SS, and several different siRNAs were also designed in which the C in the sequence could be modified by inserting a GEM in its place. The C was replaced with a Gem in one strand (sense or antisense) or in both strands (sense and antisense) to explore whether increasing the GEM content could improve efficacy or potency. Unmodified CHK1 siRNA, C1D-2A, C1D-2S and C1D-4 were transfected into the pancreatic cancer cell line BxPC3, which has been shown to be sensitive to CHK1+Gem (Azorsa 2009, supra). In previous work, a dose response of gemcitabine alone was performed, and a non-silencing (NS) siRNA (as a control) was transfected simultaneously.

[0072] It was previously observed that appending an extra GEM to the 3' end of the SS (usually before the dTdT terminal group) did not show any greater additive effect than adding 2 GEMs, nor did the potency and efficacy increase when 4 or 6 GEMs were added. It is suspected that nucleases present in the cell are unable to cleave the polyGEM sequence between the GEM motifs and are only able to cleave a single GEM from its previous unmodified nucleotide – regardless of the number of GEMs in the sequence, only 1 GEM will be released from the polyGEM sequence. Therefore, constructs with 4 or 6 GEMs at the 3' end of the SS had the same or slightly inferior potency compared to 2GEMs at this position. Logic for Improved Backbone and GEM Modifications in siRNAs Targeting CHK1

[0073] The 25-nucleotide-long (referred to as 25mer, the same below) siRNAs we designed are blunt-ended siRNAs, while the siRNA sequence used by Azorsa2009 is a 19mer siRNA with a 2-base (dTdT) overhang at the 3' end. When two GEMs were added between the last nucleotide of the siRNA SS and the dTdT overhang, we found that this sequence was much more effective against MiaPaca cells than against BxPC3 cells. The inclusion of dTdT at the end of the sequence may reduce the rate of release of GEMs from the SS in this construct when the SS is unwound from the AS chain and released into the cytoplasm. This may allow the AS sequence to induce gene silencing before the GEMs are released from the SS, and this may enhance the efficacy of this combination in MiaPaca cells compared to BxPC3 cells.

[0074] As further discussed in the Examples below, backbone-modified PGN7 siRNA (SEQ. ID No. 7-8) was expressed in MiaPaCa cells using lipofectamine (IC 50 0.006nM) transfection was more effective than other siRNA transfections using lipofectamine: PGN6 (IC 50 0.04nM)、STD1(IC 50 0.02 nM) and in contrast to GEM alone (IC 50 =8nM)( Figure 2 A-2B). When PGN6 siRNA was transfected with lipofectamine, it showed more effective results than PGN6 without lipofectamine not only in MiaPaCa (G12C) cells but also in BXPC3 (WT) cells and Panc10.05 (G12C) cell lines in pancreatic tissue (Figures 3A-3B). Similarly, in lung (Figures 4A-4B) and colon (Figures 5A-5B) tissues, PGN6 was clearly more effective in many cancer cell lines after lipofectamine transfection. In the absence of lipofectamine, PGN6 exerts its effect due to the release of GEM from the nucleic acid sequence. Compared with the presence of lipofectamine, IC 50 The changes in α- and β-catenin were typically in the range of 10- to 1000-fold (see Figures 3A-3B).

[0075] When STD1 (SEQ.ID No.3-4), PGN6 (SEQ.ID No.7-8) and PGN7 (SEQ.ID No.9-10) siRNAs were evaluated relative to non-silencing agents (as controls) in pancreatic (Figures 6(a)-(b)), lung (Figures 6(c)-(d)) and colon (Figures 6(e)-(f)) tissues, all three modified siRNAs were effective in reducing cell viability individually.

[0076] The combination of PGN7 with WEE1#7 or BCLxL#5 was unexpectedly and consistently effective in reducing IC5 in multiple tissues and cancer cell lines, with comparable or even better results than siRNA alone. siRNA PGN7 (SEQ.ID No.9-10) and WEE1#7 (SEQ.ID No.13) were evaluated individually and in combination in the same three tissues (Figure 7). In pancreatic, lung, and colon tissues, the combination of PGN7 and WEE1#7 was more effective against multiple cancer cell types than each siRNA alone. Similarly, in the same tissues and cancer cell types (Figure 8), as well as in three other tissues (bladder, stomach, and ovarian cancer tissues) (Figures 9-10), the same phenomenon was observed, that is, the combination of PGN7 and BCLxL#5 was more effective than either siRNA alone. When comparing the combination with PGN7 in the same study, each of PGN7+BCLxL#5 and PGN7+WEE1#7 was more effective than any single siRNA (Figures 11 and 13). In contrast, combinations of siRNAs containing Gem modifications with each siRNA targeting KRAS (SEQ.ID No.15) and MCL1 (SEQ ID No.16) were tested, but none showed efficacy (data not shown), emphasizing the specificity shown by the PGN7 (SEQ.ID No.9-10) / BCLxL#5 (SEQ.ID No.14) and PGN7 / Wee1#7 (SEQ.ID No.13) combinations in these examples.

[0077] In vivo studies to date have also yielded positive results, with PGN6 (SEQ.ID Nos.7-8) and PGN7 (SEQ.ID Nos.9-10) slowing the growth of pancreatic tumors in mice (Figures 12 and 14).

[0078] siRNA targeting BCLxL

[0079] Proteins in the BCL-2 family are regulators of the intrinsic apoptotic pathway. They contain one to four BCL-2 homology motifs (BH1–BH4) and can be divided into pro-apoptotic and anti-apoptotic proteins. The anti-apoptotic multidomain proteins (BH1–BH4) include BCL-2, BCLxL, BCL-w, BFL-1 / A1, and MCL-1, which function to counteract the pore-forming activity of the pro-apoptotic multidomain proteins BAX and BAK, which permeabilize the mitochondrial outer membrane. Following various stress signals, BH3-only proteins either neutralize anti-apoptotic proteins or directly activate effector proteins BAX and BAK, ultimately leading to apoptosis. Campbell and Tait, Open Biol. 8:180002 (2018); Quayle et al., Oncotarget 8:88670–88688 (2017). Cancer cells can evade apoptosis triggered by tumorigenesis or drug treatment by overexpressing BCL-2 anti-apoptotic proteins. Hanahan and Weinberg,Cell 144:646–

[0080] 674(2011). The siRNA sequence for targeting BCLxL#5 is shown in FIG1B , SEQ.ID No.14.

[0081] siRNA targeting WEE1

[0082] WEE1#7 is a tyrosine kinase in the Ser / Thr kinase family, which plays an important role in the time course of cell division and DNA replication, and is therefore considered a natural target for cancer therapy (Do, K., et al. Cell Cycle 12(19):3159-3164(2013)). The siRNA sequence for targeting WEE1 is shown in FIG1B , SEQ.ID No.13.

[0083] siRNA targeting KRAS

[0084] KRAS signaling protein plays an important role in transmitting instructions for cell growth and differentiation. KRAS gene mutations are often detected in pancreatic cancer and colon cancer. The siRNA sequence used to target KRAS#1 is shown in Figure 1B, SEQ.ID No.15.

[0085] siRNA targeting MCL1

[0086] Small molecule inhibitors developed against BCLxL (ABT-100 / ventoclax; 15Souers et al Nat. Med. 19:202-208 (2013)) and MCL1 (S63845; Li et al., Leukemia 33L:262-266 (2019)) have shown therapeutic benefits in treating multiple cancer types, including cervical cancer (Rahman et al, Biochem. Biophys. Reports, 22:100756 (2020)) and squamous cell carcinoma of the lung (Clare et al, Oncogene 37:4475-4488 (2018)) and head and neck cancer (Thomas et al., Oncotarget, 10:494-510 (2019)). It has also been shown that BCLxL and MCL1 siRNA can inhibit ovarian tumor growth (Brotin et al., Int. J. Cancer 126:885-895 (2010)). The siRNA sequence for targeting MCL1#4 is shown in FIG1B , SEQ. ID No. 16.

[0087] Improving cancer treatment options

[0088] Based on the data presented in the Examples, we have demonstrated that the efficacy of GEMs against multiple types of cancer cells is significantly improved by adding gemcitabine to one or more modified siRNAs. Incorporating multiple GEMs into the sense strand of an siRNA allows the GEMs to be delivered to the same cells where the siRNAs also act. Previous studies (Azorsa 2009) have shown that silencing CHK1 can enhance the effects of gemcitabine. Therefore, by adding GEMs to siRNA sequences that are capable of silencing CHK1, we expect that there will be some additive / synergistic effects in the interaction. Using histidine-lysine peptide nanoparticles (PNPs), we have demonstrated the ability to deliver siRNAs to many different tumor types in vitro and in vivo. Therefore, we are investigating the delivery of GEM CHK1 siRNA as a new therapeutic modality for treating cancer. In addition, peptide nanoparticles are able to deliver multiple siRNAs against multiple targets to the same cell at the same time, so we can also silence a second gene target that can further synergize with the first siRNA to maximize the effect of the target cell.

[0089] Choice of gemcitabine

[0090] The activity of gemcitabine is enhanced by inhibiting ribonucleotide reductase and dCMP deaminase.

[0091] Gemcitabine is used extensively in combination, primarily with platinum analogs, with other combinations being used less frequently or currently being explored.

[0092] The standard dosing of gemcitabine is 1000 mg / m2 weekly as a 30-minute infusion. 2 , but alternatives are being explored, such as prodrugs (e.g., CO-1.01, which bypasses transport defects), fixed-dose-rate infusions (10 mg / m 2 / min) and local routes of administration, such as 24-hour hepatic artery infusion, bladder instillation or intraperitoneal administration to treat advanced ovarian cancer. Other alternatives for gemcitabine combination therapy for ovarian cancer include combination with triazepine or hydroxyurea to enhance the inhibitory effect of ribonucleotide reductase. The effect of gemcitabine on signal transduction also provides a reasonable concept for its combination with signal transduction pathways.

[0093] Gemcitabine can be included as an amide during the synthesis of oligonucleotides and incorporated into the nucleotide sequence. We have previously demonstrated that the addition of GEM to siRNA allows for the co-delivery of siRNA and gemcitabine to the same cells. By selecting siRNAs against molecular targets that are expected to enhance the effect of gemcitabine in reducing tumor cell viability, we have seen significant improvements in efficacy that are not achievable with either agent alone.

[0094] Although we chose to use siRNA against CHK1, gemcitabine can be incorporated into other oligonucleotides with similar results. Examples of siRNAs that enhance gemcitabine activity also describe cMyc as a suitable gene target (Zhang et al., Zhang et Metastasis Rev 26:85–110 (2013)). Therefore, siRNAs against cMyc can also be directly incorporated into GEMs and used to treat cancers, such as NSCLC. In fact, oligonucleotides may not need to have a silencing effect at all, but only need to be used to deliver gemcitabine to exert anti-disease efficacy.

[0095] It is well known that chemical modification of bases within the oligonucleotide siRNA structure (e.g., 2'-OMe, 2'-F, phosphorothioate modification, etc.) can stabilize the oligonucleotides and make them resistant to nuclease attack when administered in vivo. Such chemically modified oligonucleotides can be directly coupled to targeting ligands, allowing direct delivery through binding to receptors on cancer cells.

[0096] Additionally, antisense oligonucleotides have been used to alter the expression of selected genes within cells, and these single-stranded oligonucleotides can also be modified by gemcitabine. These modifications (at the C base) or additions at the end of the sequence can be used to help alter the hybridization efficiency with a specific target and the co-delivery of gemcitabine.

[0097] Likewise, miRNAs (mimetics or inhibitors) have been described to have anticancer effects, and they can be modified by inserting gemcitabine into their sequence to deliver it to the same cells. In particular, miRNAs that can enhance the effects of gemcitabine will be of interest.

[0098] Finally, aptamers are RNA or DNA oligonucleotides that can be designed and manufactured to bind with high affinity to selected targets. In some cases, these can be receptors on cells, or specifically receptors that are upregulated on cancer cells. Gemcitabine can be appended or inserted into these sequences (instead of C bases) to allow high affinity binding to target receptors on the surface of cancer cells, and then the oligonucleotide is taken up into the cell, where it is released to exert its therapeutic effect.

[0099] Gemcitabine-modified oligonucleotides (DNA or RNA) may help improve cancer treatment options across multiple cancer types.

[0100] Gemcitabine can also act as a radiosensitizer (improve the efficacy of tumor radiotherapy). Therefore, it is feasible to deliver gemcitabine to specific cancer cells through the modification of the above-mentioned oligonucleotides. Once gemcitabine is delivered, the tumor is exposed to radiation and then kills the tumor cells. Sometimes, this radiation exposure can further produce an immune response to tumor-specific antigens released in the process.

[0101] Therefore, oligonucleotides containing gemcitabine or other non-nucleoside analogs can become a feasible treatment for cancer and other diseases for which non-nucleoside (or nucleotide) analogs are effective. Another example of using non-nucleoside analogs or nucleotide analogs is to treat viral diseases. For example, in viral diseases, oligonucleotides containing non-nucleoside (or nucleotide) analogs can be co-administered. These may include nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs), such as zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine and tenofovir, or non-nucleoside reverse transcriptase inhibitors (NNRTIs), such as nevirapine, delavirdine, efavirenz and etravirine.

[0102] RNAi

[0103] siRNA molecules can produce additive or synergistic effects in cells, depending on the composition and structure of the specific molecule. In certain embodiments, they produce synergistic effects that can enhance or maximize the effect, such as reducing tumor growth. Double-stranded RNA has been shown to silence gene expression through RNA interference (RNAi). RNAi modulation induced by short interfering RNA (siRNA) shows great potential for treating a variety of human diseases including cancer.

[0104] RNAi is a sequence-specific RNA degradation process that provides a relatively simple and straightforward method to knock down or silence theoretically any gene. In naturally occurring RNA interference, double-stranded (ds) RNA is cleaved by an endonuclease into siRNA molecules with 3' overhangs. These siRNAs are incorporated into a multicomponent ribonuclease called the RNA-induced silencing complex (RISC). One strand of the siRNA remains associated with the RISC and guides the complex to a cognate RNA whose sequence is complementary to the guide single-stranded siRNA (ss-siRNA) in the RISC. This siRNA-guided endonuclease digests the RNA, rendering it inactive. Studies have shown that using chemically synthesized 21-25 nucleotide ("nt" or "NT") siRNAs exhibit RNAi effects in mammalian cells, and that the thermodynamic stability of the siRNA hybrid (at the end or in the middle) plays a central role in determining the function of the molecule.

[0105] It is not currently possible to predict with high confidence which of the many candidate siRNA sequences that may target disease gene mRNA sequences will exhibit effective RNAi activity. Instead, specific candidate siRNA polynucleotide or oligonucleotide sequences must be generated and tested in mammalian cell culture to determine whether the expected interference with target gene expression occurs. The unique advantage of siRNA is that it can be combined with multiple siRNA duplexes to target multiple disease-causing genes in the same treatment, because all siRNA duplexes are chemically homogeneous, have the same source and the same manufacturing process.

[0106] As used herein, "siRNA molecules" are double-stranded oligonucleotides, i.e., short double-stranded polynucleotides, that interfere with the expression of a corresponding target RNA after the molecule is introduced into a cell. For example, it targets and binds to a complementary nucleotide sequence in a single-stranded ("ss") target RNA molecule (e.g., mRNA, microRNA (miRNA), or short hairpin RNA (shRNA)). The target RNA is then degraded by the cell. Such molecules are constructed by techniques known to those skilled in the art. Such techniques are described in U.S. Patent Nos. 5,898,031, 6,107,094, 6,506,559, 7,056,704, and European Patent Nos. 1214945 and 1230375. As is customary in the art, when a siRNA molecule is identified by a specific nucleotide sequence, the sequence refers to the sense strand of the double-stranded molecule.

[0107] The siRNA molecule can be made of naturally occurring ribonucleotides (i.e., ribonucleotides found in living cells), or one or more of the nucleotides can be chemically modified by techniques known in the art. In addition to modification at the level of one or more individual nucleotides, the backbone of the oligonucleotide can also be modified. Other modifications include the use of small molecules (e.g., sugar molecules), amino acid molecules, peptides, cholesterol, and other macromolecules to bind to the siRNA molecule.

[0108] In one embodiment, the molecule is an oligonucleotide having a length of about 19 to about 35 base pairs. In one aspect of this embodiment, the molecule is an oligonucleotide having a length of about 19 to about 27 base pairs. In another aspect, the molecule is an oligonucleotide having a length of about 21 to about 25 base pairs. In another embodiment, the siRNA molecule or other nucleic acid has a length of 20 to 30 base pairs; in another embodiment, the siRNA molecule or other nucleic acid has a length of 24 to 28 base pairs. The molecule can have a flat end at both ends, or have a sticky end at both ends, or each has one. As discussed in more detail below, the siRNA molecule can include one or more chemical modifications at the single nucleotide level or the oligonucleotide backbone level, or it may not have a modification. In an advantageous embodiment, the modified siRNA of the disclosed embodiment has a chain length of 25 nucleotides. In another embodiment, the modified siRNA has a chain length of 19 to 25 nucleotides. In some embodiments, the siRNA molecules can be asymmetric, where one strand is shorter than the other (usually 2 bases shorter, e.g., 21mer vs. 23mer or 19mer vs. 21mer or 23mer vs. 25mer). The strands can be modified by including a dTdT overhang group at the 3' end of the selected strand. In all of these aspects, the molecules can have blunt ends at both ends, or cohesive ends at both ends, or blunt ends at one end and cohesive ends at the other end.

[0109] In the compositions of the disclosed embodiments, the relative amounts of two different molecules and copolymers can vary. In one embodiment, the mass ratio of two different siRNA molecules is about 1: 1. In another embodiment, the mass ratio of these molecules to copolymers is about 1: 4, 1: 4.5 or 1: 5. Advantageously, the mass ratio of two different siRNA molecules is about 1: 1, and the mass ratio of these molecules to copolymers is about 1: 4, 1: 4.5 or 1: 5. By these ratios, the composition forms nanoparticles with an average diameter of about 150 nm.

[0110] In one embodiment, the siRNA molecule is selected from Figure 1. An example is PGN7, (SEQ.ID No.9-10), a modified siRNA-GEM RNAi agent: PGN7 sense strand: 5'-AAGAAAgAgau[GEM]uGuAu[GEM]AAu-3' (SEQ ID NO: 9); PGN7 antisense strand: 5'-AuUGAuACAGAuCuCuUuCuU[GEM][GEM]dTdT-3' (SEQ ID NO: 10).

[0111] The disclosed embodiments include a method for identifying desired siRNA molecules, the method comprising the following steps: (a) creating a set of siRNA molecules designed to target complementary nucleotide sequences in a target mRNA molecule, wherein the targeting strand of the siRNA molecule comprises various nucleotide sequences; (b) selecting siRNA molecules that exhibit the highest desired effect on the target mRNA molecule in vitro; (c) evaluating the selected siRNA molecules in an animal wound model; and (d) selecting siRNA molecules that exhibit the greatest silencing activity and therapeutic effect in the model.

[0112] Importantly, it is currently not possible to predict with high confidence which of the many candidate siRNA sequences that could potentially target the mRNA sequence of a disease gene will actually exhibit effective RNAi activity. Instead, a single specific candidate siRNA polynucleotide or oligonucleotide sequence must be generated and tested in mammalian cell culture, such as an in vitro organ culture assay, to determine whether the expected interference with target gene expression occurs. The unique advantage of siRNA makes it possible to combine multiple siRNA duplexes to target multiple disease-causing genes in the same treatment, as all siRNA duplexes are chemically homogeneous, having the same source and the same manufacturing process.

[0113] Advantageously, the siRNA molecules are evaluated in at least two animal models. In one method embodiment, the formulation of a pharmaceutical composition comprising an RNAi agent or complex comprises the steps of adding a pharmaceutically acceptable carrier to each siRNA molecule to form a pharmaceutical composition, and evaluating each pharmaceutical composition in an animal wound model or other model.

[0114] In another embodiment, siRNA sequences are prepared in such a way that each sequence can target and inhibit at least the same gene from humans and mice, or humans and non-human primates. On the one hand, siRNA molecules are combined with human mRNA molecules and homologous mouse mRNA molecules. That is, the protein with substantially the same structure or function of human and mouse mRNA molecules encoding. Other mammalian animal models of proteins with substantially the same structure or function of mRNA molecules encoding can be tested, and if feasible, it can also be used in the disclosed embodiments. The efficacy and toxic reactions observed in the mouse disease model can well understand the situation that will occur in humans. More importantly, the siRNA molecules tested in the mouse model are good candidates for human medicaments. The human / mouse homology design of siRNA drugs can eliminate the toxicity and side effects caused by species specificity observed in monoclonal antibody drugs.

[0115] In one embodiment, the disclosed embodiments provide a composition comprising two or more different siRNA molecules, which bind to mRNA encoding Chk proteins in mammalian cells. These molecules can bind to different nucleotide sequences in the target mRNA. The siRNA molecules can produce additional or synergistic effects in cells, depending on the composition and structure of the specific molecules. In an advantageous embodiment, they produce a synergistic effect. In some applications of these embodiments, siRNA molecules (e.g., SEQ.ID No.1-10) containing GEM or non-silencing nucleic acid (NS) molecules (SEQ.ID No.11-12) containing GEM are selected from those identified in Figure 1.

[0116] The siRNA molecules are combined with a pharmaceutically acceptable carrier to provide a pharmaceutical composition for administration to a mammal. In the disclosed embodiments, the patient or subject can be a mammal; the mammal can be an experimental animal, such as a dog, cat, pig, non-human primate or rodent, such as a mouse, rat or guinea pig. In certain advantageous embodiments, the mammal is a human.

[0117] Histidine-lysine copolymer.

[0118] The carrier is a histidine-lysine copolymer, which can form nanoparticles containing siRNA molecules, wherein the diameter of the nanoparticles is 100-400nm. In one aspect of this embodiment, the carrier is selected from a group including HKP species, H3K4b and PT73, which has a lysine backbone with four branches, and the branches contain multiple repeated histidine, lysine or asparagine, such as HKP (+H). When the HKP aqueous solution is mixed with siRNA at a N / P mass ratio of 4:1, the nanoparticles (average diameter of 100-200nm) will self-assemble. In another aspect of this embodiment, HKP has the following formula:

[0119] (R)K(R)-K(R)-(R)K(X), wherein R=KHHHKHHHKHHHKHHHK (SEQ ID NO: 17), or

[0120] R=KHHHKHHHNHHHNHHHN (SEQ ID NO: 18), X=C(O)NH2, K=lysine, H=histidine, N=asparagine.

[0121] In yet another aspect of this embodiment, HKP has the following formula:

[0122] (R)K(R)-K(R)-(R)K(X) (SEQ ID NO:21), wherein R=KHHHKHHHKHHHKHHHK (SEQ ID NO:17), X=C(O)NH2, K=lysine, H=histidine.

[0123] In yet another aspect of this embodiment, HKP has the following formula: (HKP(+H)), wherein the third repeat of the HHHK (SEQ ID NO: 19) motif has an additional H (located 6 characters from the right end)

[0124] (R)K(R)-K(R)-(R)K(X) (SEQ ID NO: 22), wherein R=KHHHKHHHKHHHHKHHHK (SEQ ID NO: 20), X=C(O)NH2, K=lysine, H=histidine.

[0125] A specific embodiment of the disclosed embodiment provides a method of administering a pharmaceutical composition to a human tissue, comprising injecting a therapeutically effective amount of a composition directly into a tumor tissue, the composition comprising a PGN7 siRNA molecule having a sense and antisense sequence as shown in FIG1: PGN7 sense strand: 5'-AAGAAAgAgau[GEM]uGuAu[GEM]AAu-3' and PGN7 antisense strand: 5'-AuUGAuACAGAuCuCuUuCuU[GEM][GEM]dTdT-3' (together, SEQ.IDNo.9-10) and a pharmaceutically acceptable histidine-lysine copolymer carrier. In one aspect of this embodiment, the histidine-lysine copolymer comprises a histidine-lysine copolymer species H3K4b or a histidine-lysine copolymer species PT73. In another aspect of this embodiment, the histidine-lysine copolymer has the following formula:

[0126] (R)K(R)-K(R)-(R)K(X) (SEQ ID NO: 21), wherein R=KHHHKHHHKHHHKHHHK (SEQ ID NO: 17), X=C(O)NH2, K=lysine, H=histidine, N=asparagine. In yet another aspect of this embodiment, the histidine-lysine copolymer has the formula:

[0127] (R)K(R)-K(R)-(R)K(X), wherein R=KHHHKHHHKHHHKHHHK (SEQ ID NO: 17), or

[0128] R=KHHHKHHHKHHHHKHHHK (SEQ ID NO: 20), X=C(O)NH2, K=lysine, H=histidine.

[0129] Another disclosed embodiment provides a method for treating cancer in a mammal, comprising injecting a therapeutically effective amount of a composition via intravenous or intratumoral route, the composition comprising a modified siRNA-GEM molecule PGN6 having the following sense and antisense sequences (SEQ.ID No.7-8) designated as the sense strand, respectively: 5'-AAGAAAgAgau[GEM]UGU AU [GEM] A AU–3'; and antisense strand: 5'-AuUGAuACAGAUCuCuuuucuu[GEM][GEM]dTdT-3', and a pharmaceutically acceptable histidine-lysine copolymer carrier. In one aspect of this embodiment, the histidine-lysine copolymer comprises a histidine-lysine copolymer species H3K4b or a histidine-lysine copolymer species PT73. In another aspect of this embodiment, the histidine-lysine copolymer has the following formula:

[0130] (R)K(R)-K(R)-(R)K(X) (SEQ ID NO:21), wherein R=KHHHKHHHKHHHKHHHK (SEQ ID NO:17), X=C(O)NH2, K=lysine, H=histidine, N=asparagine.

[0131] In yet another aspect of this embodiment, the histidine-lysine copolymer has the formula:

[0132] (R)K(R)-K(R)-(R)K(X), wherein R=KHHHKHHHKHHHKHHHK (SEQ ID NO:17), or having an extra H located 6 characters from the right end of the sequence: R=KHHHKHHHHHKHHHKHHHK (SEQ ID NO:20), X=C(O)NH2, K=lysine, H=histidine.

[0133] Other HKP copolymers suitable for use with the disclosed embodiments are provided, for example, in U.S. Patent Nos. 7,070,807; 7,163,695; 7,465,708; and 7,772,201.

[0134] Preparation of pharmaceutical compositions

[0135] In certain embodiments, the disclosed embodiments provide pharmaceutical compositions comprising (double-stranded, ds) RNAi agents of the disclosed embodiments. The dsRNA agent sample can be appropriately prepared and introduced into the cell environment by any means allowing a sufficient portion of the sample to enter the cell to induce gene silencing (if it occurs). Many dsRNA preparations are known in the art, and as long as the dsRNA enters the target cell to play a role, it can be used. See, for example, U.S. patent application Nos. 2004 / 0203145A1 and 2005 / 0054598A1. For example, the dsRNA agent of the disclosed embodiment can be formulated in a buffer solution such as phosphate buffered saline, liposomes, micellar structures and capsids. The dsRNA preparation containing cationic lipids can be used to promote the transfection of dsRNA preparations into cells. For example, many cationic lipids can be used, such as lipofectin (U.S. Patent No. 5,705,188), cationic glycerol derivatives and polycationic molecules, such as polylysine (disclosed PCT International Application WO 97 / 30731). Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, CO), or FuGene 6 (Roche), all of which can be used according to the manufacturer's instructions.

[0136] Such compositions generally comprise a nucleic acid molecule and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc., which are compatible with the drug. Supplementary active compounds may also be added to the composition.

[0137] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (water soluble) or dispersions and sterile powders (for the temporary preparation of sterile injection solutions or dispersions). For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL.TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is easy to inject. It should be stable under manufacturing and storage conditions, and must prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained by using a coating such as lecithin, maintaining the desired particle size in the case of dispersions, and using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0138] Sterile injection solutions can be prepared by incorporating the desired amount of active compound with one or a combination of the ingredients listed above (as needed) in a selected solvent, followed by filtration sterilization. Generally speaking, dispersions are prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, advantageous optional preparation methods are vacuum drying and freeze drying, which can obtain active ingredient powders and any other required ingredients from previously sterile filtered solutions.

[0139] Oral compositions typically include an inert diluent or edible carrier, such as HKP discussed below. For oral therapeutic administration, the active compound can be mixed with an excipient and used in the form of a tablet, pill, or capsule (e.g., a gelatin capsule). Oral compositions can also be prepared using a fluid carrier used as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, etc. may contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as mint, methyl salicylate, or orange flavoring.

[0140] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such preparations can be prepared using standard techniques. These materials can also be obtained from Alza Corporation and Nova Pharmaceuticals, Inc. commercially. Liposomal suspensions (including liposomes with targeted infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0141] The method by which dsRNA reagents are introduced into the cell environment will depend on the type of cell and the composition of its environment. For example, when cells are present in liquid, a favorable optional formulation is to use lipid formulations (such as lipofectamine transfection), and dsRNA reagents can be directly added to the liquid environment of the cell. Lipid formulations can be optionally administered to animals by intravenous, intramuscular or intraperitoneal injection, oral administration or by suction or other methods known in the art. When the preparation is suitable for administration to animals (such as mammals, more specifically humans), the preparation is also pharmaceutically acceptable. Pharmaceutically acceptable preparations for administering oligonucleotides are known and can be used. In some cases, it may be advantageous to prepare dsRNA reagents in buffer or saline solution and to inject the prepared dsRNA reagents directly into cells, as in oocyte research. It is also possible to directly inject dsRNA double-stranded reagents. For the suitable method for introducing dsRNA, see the patent application No. 2004 / 0203145A1 disclosed in the United States.

[0142] The modified siRNA molecules with GEM are mixed with a pharmaceutically acceptable carrier to provide a composition for use in a subject. Advantageously, the subject is a human being. In one embodiment, the composition includes a pharmaceutically acceptable carrier and at least three siRNA molecules, wherein each siRNA molecule is combined with an mRNA molecule, and the mRNA molecule encoding selects a gene from the group consisting of pro-inflammatory pathway genes, pro-angiogenic pathway genes, and pro-cell proliferation pathway genes. In another embodiment, each siRNA contains at least three siRNA double strands, which target at least three different gene sequences. Advantageously, each gene is selected from different approaches. The disclosed embodiments include pharmaceutically effective carriers for enhancing siRNA delivery to disease tissues and cells.

[0143] In various embodiments of the composition, the carrier comprises one or more components selected from the group consisting of saline solution, sugar solution, polymer, lipid, cream, gel and micellar material. Other components or carriers include: polycationic binders, cationic lipids, cationic micelles, cationic polypeptides, hydrophilic polymer graft polymers, non-natural cationic polymers, cationic polyacetals, hydrophilic polymer graft polyacetals, ligand functionalized cationic polymers and ligand functionalized hydrophilic polymer graft polymers, biodegradable polyesters such as polylactic acid (PLA), polyglycolic acid (PGA) and poly (lactic-glycolic acid) (PLGA), PEG-PEI (polyethylene glycol and polyethyleneimine), polyspermidine (spermidine) and polyamidoamine (PAMAM) dendrimers. Optionally, the carrier is a histidine-lysine copolymer, which is believed to form nanoparticles containing siRNA molecules, wherein the nanoparticles have a diameter of about 100 to 400 nm, or advantageously, a diameter of 80 to 200 nm; more advantageously, a diameter of 80 to 150 nm. In some embodiments, the siRNA molecule can be formulated for topical administration together with methylcellulose gel. In some advantageous embodiments, the nanoparticles having a size range of 80 to 150 or 80 to 200 nm and containing the siRNA molecule can be formulated as injection or infusion without methylcellulose gel. The method for preparing nanoparticles with methylcellulose gel is known in the art.

[0144] The phrase "pharmaceutically acceptable carrier" or "carrier" refers to a carrier for administering a therapeutic agent. Exemplary carriers include saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients, such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants (if present) are typically magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. The pharmaceutically acceptable carrier of the disclosed dsRNA composition may be a micellar structure, such as a liposome, a capsid, a capsid, a polymer nanocapsule, or a polymer microcapsule.

[0145] Polymer nanocapsules or microcapsules facilitate the transport and release of encapsulated or bound dsRNA into cells. They include polymeric and monomeric materials, particularly polybutylcyanoacrylate. A summary of materials and methods of manufacture has been published (see Kreuter, 1991). The polymeric materials formed from monomers and / or oligomeric precursors in the polymerization / nanoparticle generation step are known in the art per se, and the molecular weights and molecular weight distributions of polymeric materials that can be appropriately selected by a person skilled in the art of making nanoparticles are also known.

[0146] Modification and conjugation

[0147] The dsRNA agents of the disclosed embodiments may be conjugated (e.g., at the 5' or 3' end of the sense or antisense strand thereof) or not conjugated to another component (e.g., a non-nucleic acid component such as a peptide), an organic compound (e.g., a dye, cholesterol, etc.). Modifying the dsRNA agent in this manner can improve cellular uptake or enhance the cell targeting activity of the resulting dsRNA agent derivatives compared to the corresponding unconjugated dsRNA agent, can be used to track the dsRNA agent derivatives in cells or improve the stability of the dsRNA agent derivatives compared to the corresponding unconjugated dsRNA agent.

[0148] As used herein, the term "nucleic acid" refers to deoxyribonucleotides, ribonucleotides or modified nucleotides, and polymers thereof in single-stranded or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or bonds that are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties to the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphorodithioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, 2'-fluoro ribonucleotides, peptide nucleic acids (PNAs), and unlocked nucleic acids (UNAs); see, e.g., Jensen et al., Nucleic Acids Symposium Series 52: 133-4) and derivatives thereof.

[0149] As used herein, the art-recognized "nucleotide" is used to include those with natural bases (standard) and modified bases well known in the art. Such bases are usually located at the 1' position of the nucleotide sugar segment. Nucleotides are usually composed of a base, a sugar, and a phosphate group. Nucleotides can be unmodified or modified in the sugar, phosphate and / or base moieties (also interchangeably referred to as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and others, see, e.g., Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92 / 07065; Uhlman and Peyman et al., International PCT Publication No. WO 93 / 15187; summarized by Limbach et al., Nucleic Acids Res.22:2183, 1994. Some non-limiting examples of base modifications that can be introduced into nucleic acid molecules include hypoxanthine, purine, pyridine-4-1, pyridine-2-1, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydropyrimidine, naphthyl, aminophenyl, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkylpyrimidine (e.g., ribopyrimidine), 5-halopyrimidine (e.g., 5-bromopyrimidine) or 6-azapyrimidine or 6-alkylpyrimidine (e.g., 6-methyluracil), propyne, etc. (Burgin et al., Biochemistry 35:14090, 1996; Ullman and Payman, supra). The "modified base" referred to herein refers to a nucleotide base other than adenine, guanine, cytosine and uracil at the 1' position or its equivalent.

[0150] As used herein, "modified nucleotides" refer to nucleotides with one or more modifications to nucleosides, nucleobases, pentose rings or phosphate groups. For example, modified nucleotides do not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate and cytidine monophosphate and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate and deoxycytidine monophosphate. Modifications include those that occur naturally by modification of enzymes that modify nucleotides (such as methyltransferases). Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Synthetic or non-naturally occurring modifications in nucleotides include those with 2' modifications such as 2'-methoxy, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2--O-2'-bridge, 4'-(CH2)2--O-2'-bridge, 2'-LNA or other bicyclic or "bridged" nucleoside analogs, and 2'-O-(N-methylcarbamate) or base-containing analogs.

[0151] In the case of 2'-modified nucleotides described in the present disclosure, "amino" refers to 2'-NH2 or 2'-O-NH2, which can be modified or unmodified. For example, such modified groups are described by Eckstein et al. in U.S. Pat. No. 5,672,695 and Matulic-Adamic et al. in U.S. Pat. No. 6,248,878. The "modified nucleotides" of the disclosed embodiments may also include nucleotide analogs as described above.

[0152] With respect to the nucleic acid molecules of the present disclosure, modifications may be present on these agents in a pattern on one or both strands of a dsribonucleic acid (dsRNA). As used herein, an "alternating" position refers to a pattern in which every other nucleotide is a modified nucleotide, or an unmodified nucleotide (e.g., an unmodified ribonucleotide) is present between each modified nucleotide on a defined length of a dsRNA strand (e.g., 5'-MNMNMN3'; -3'-MNMNMN-5'; wherein M is a modified nucleotide and N is an unmodified nucleotide). According to the position numbering convention, the modification pattern starts at the first nucleotide position at the 5' or 3' end. The pattern of modified nucleotides at alternating positions may extend the full length of the strand, but in certain embodiments includes at least 4, 6, 8, 10, 12, 14 nucleotides, wherein at least 2, 3, 4, 5, 6, or 7 modified nucleotides are contained, respectively. "Alternating position pairs" refers to a pattern in which two consecutive modified nucleotides are separated by two consecutive unmodified nucleotides over a defined length of a dsRNA chain (e.g., 5'-MMNNMMNNMMNN-3'; 3'-MMNNMMNNMMNN-5'; wherein M is a modified nucleotide and N is an unmodified nucleotide). According to position numbering conventions such as those described herein, the modification pattern starts at the first nucleotide position at the 5' or 3' end. The pattern of modified nucleotides at alternating positions can run throughout the entire length of the chain, but advantageously includes at least 8, 12, 16, 20, 24, 28 nucleotides, containing at least 4, 6, 8, 10, 12 or 14 modified nucleotides, respectively. It should be emphasized that the above modification patterns are exemplary and are not intended to limit the scope of the disclosed embodiments.

[0153] As used herein, "loop" refers to a structure formed by a single strand of a nucleic acid in which complementary regions flanking a particular ss nucleotide region hybridize in a manner such that the ss nucleotide region between the complementary regions is excluded from duplex formation or Watson-Crick base pairing. A loop is a nucleotide region of arbitrary length. Examples of loops include unpaired nucleotides present in structures such as hairpins, stem loops, or extended loops.

[0154] In certain embodiments, the first and second oligonucleotide sequences of siRNA or other nucleic acids are present in a separate oligonucleotide chain that can and is usually chemically synthesized. In some embodiments, the length of both chains is 25 nucleotides, fully complementary and with flat ends. In certain embodiments of the disclosed embodiments, the modified siRNA is present in a separate RNA oligonucleotide (chain). In certain embodiments, the modified siRNA agent is composed of two oligonucleotide chains of different lengths, one of which has a flat end at the 3' end of the first chain (sense chain) and a 3' overhang (antisense chain) at the 3' end of the second chain. siRNA can also contain one or more deoxyribonucleic acid (DNA) base substitutions.

[0155] Suitable siRNA compositions containing two independent oligonucleotides can be chemically linked by chemical linking groups outside their annealing regions. Many suitable chemical linking groups are known in the art and can be used. Suitable groups will not block the endonuclease activity on the siRNA, and will not interfere with the directional destruction of the RNA transcribed from the target gene. Alternatively, two independent oligonucleotides can be connected by a third oligonucleotide so that a hairpin structure (shRNA) is produced when the two oligonucleotides constituting the siRNA composition are annealed. The hairpin structure will not block the endonuclease activity on the siRNA, nor will it interfere with the directional destruction of the target RNA.

[0156] The dsRNA molecules of the disclosed embodiments are added directly, or can be complexed with lipids (e.g., cationic lipids), packaged in liposomes, or otherwise delivered to target cells or tissues. Nucleic acids or nucleic acid complexes can be topically administered to relevant tissues in vitro or in vivo by direct skin application, transdermal application, or injection in vivo, whether incorporated into a biopolymer or not.

[0157] The dsRNA agent can be formulated as a pharmaceutical composition (preparation) comprising a pharmacologically effective amount of the dsRNA agent and a pharmaceutically acceptable carrier. The phrases "pharmacologically effective amount" and "therapeutically effective amount" or simply "effective amount" refer to the amount of RNA that effectively produces the expected pharmacological, therapeutic or preventive results. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition is reduced by at least 20%, then the therapeutically effective amount of the drug for treating the disease or condition is the amount required to reduce the parameter by at least 20%.

[0158] Based on the teachings contained herein, dosages, administration methods and administration times can be easily determined by those skilled in the art.

[0159] 剂量

[0160] As defined herein, the therapeutically effective amount (i.e., effective dose or therapeutically effective dose) of a nucleic acid molecule depends on the selected nucleic acid. For example, a single dose of dsRNA (or a construct encoding such dsRNA, for example) in the range of about 1 pg to up to 10 mg can be administered. In disclosed embodiments, 1, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg can be administered to one or more regions of a subject's body of 60 to 120 kg.

[0161] More specifically, the dosage administered according to the disclosed embodiments is between about 5 to about 170 μg, between about 10 to about 160 μg, between about 10 to about 130 μg, between about 10 to about 70 μg, between about 10 to about 40 μg, between about 20 to about 50 μg, between about 20 to about 30 μg, between about 30 to about 70 μg, between about 40 to about 80 μg, between about 60 to about 90 μg, between about 50 to about 100 μg, about 70 to about 100 μg, and about 80 to about 120 μg, at least once a week for 1 to 12 weeks.

[0162] Dosages and treatment cycles will vary. In some embodiments, a dose in the range of 60 to 150 μg is administered. Advantageously, the composition is administered subcutaneously or subcutaneously to multiple areas where remodeling of adipose tissue is desired, such as the submental or intra-mental adipose tissue. For example, each dose may be 60-150 μg / cm 2 , applied to several areas of a 60kg to 120kg patient. The skilled artisan will recognize that more or less than 60-150 μg / cm 2 Dosages, such as 10-300 μg / cm 2 .

[0163] The composition can be applied once or more per day to once or more per week to achieve the desired treatment length from one week to several months or one year or longer; in some embodiments, a dosage can be applied every other day. It will be appreciated by the technician that certain factors may affect the dosage and time required for the effective treatment of the subject, including but not limited to the severity of the disease or illness, previous treatment, the overall health and / or age of the subject, and other existing diseases. Treating the subject with a therapeutically effective amount of nucleic acid (e.g., dsRNA), protein, polypeptide or antibody can include a single treatment, or advantageously can include a series of treatments. In an advantageous embodiment, one or more dosages are applied weekly or half a week for a period of 1 and 6 to 12 weeks. In another embodiment, one or more dosages are applied every day.

[0164] In general, based on per kilogram body weight unit, the suitable dosage of dsRNA can be 1ng-2mg / kg body weight per day, week or month, but more likely in the following narrower range of about 0.01-20μg / kg body weight per day, week or month, or about 0.001-5μg / kg body weight per day, week or month, or about 1-500ng / kg body weight per day, week or month, or about 0.01-10μg / kg body weight per day, week or month, or about 0.10-5μg / kg body weight per day, week or month, or in the range of about 0.1-2.5μg / kg body weight per day, week or month. The pharmaceutical composition comprising dsRNA can be administered once a day. However, the therapeutic agent can also be included in the unit administration of two, three, four, five, six or more sub-doses administered at appropriate intervals within one day, one week or one month. In this case, the dsRNA contained in each sub-dose must be correspondingly smaller to reach the total dosage unit per day, week or month. The dosage unit can also be compounded into a single dose within a few days, for example, using a conventional sustained release formulation, which provides a continuous and consistent release of dsRNA within a few days. Sustained release formulations are well known in the art. In this embodiment, the dosage unit comprises a daily dose of a corresponding multiple. Regardless of the formulation, the pharmaceutical composition must contain the dsRNA of the amount that is enough to suppress the expression of the target gene in the treated animal or human. The composition can be formulated in such a way that the sum of multiple dsRNA units contains enough dosages together.

[0165] Depending on the specific target gene sequence and the dose of the delivered dsRNA agent material, the process may result in partial or complete loss of target gene function. A reduction or loss of at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of target cell expression (target gene expression or encoded polypeptide expression). Inhibition of target gene levels or expression refers to the absence (or significant reduction) of the target gene or the protein level encoded by the target gene. Specificity refers to the ability to inhibit the target gene without significantly affecting other genes of the cell. The consequences of inhibition can be confirmed by examining the external characteristics of the cell or organism or by biochemical techniques, such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, microarray gene expression monitoring, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blot, radioimmunoassay (RIA), other immunoassays and fluorescence activated cell analysis (FACS). The inhibition of target gene sequences by the dsRNA agents of the disclosed embodiments can also be measured based on the effect of administration of such dsRNA agents on the development / progression of target gene-related diseases or conditions in vivo or in vitro, for example, harmful adipose tissue remodeling, tumor formation, growth, metastasis, etc. due to obesity, overfeeding or metabolic disorders, whether in vivo or in vitro. Treatment with a pharmaceutical composition containing GEM-modified siRNA can result in a significant reduction in tumor volume or cancer cell levels, for example, up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more. The improvement can also be measured in logarithms, for example, by administering the modified dsRNA molecules (containing GEM) of the disclosed embodiments to cells, tissues or subjects, a 10-fold, 10 ... 5 times, 10 6 times, 10 7 For example, in some embodiments, the pharmaceutical composition (preparation) can be administered systemically by intratumoral, subcutaneous or intravenous injection to achieve a tumor volume reduction of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, treatment of a subject with a modified siRNA pharmaceutical composition comprising one or more GEMs results in a reduction in cancer progression of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%.

[0166] The data obtained from cell culture assays and animal studies (toxicity, therapeutic efficacy) can be used in formulating a range of dosages for use in humans and other mammals. Each dose of the compound is within the range of the ED that includes little or no toxicity. 50The dose may vary within this range depending upon the dosage form employed and the route of administration utilized. For compounds used in the methods of the disclosed embodiments, the therapeutically effective amount can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC determined in cell culture. 50 (ie, the concentration of the test compound which achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0167] Drug administration

[0168] The formulated compositions of the disclosed embodiments can be administered by means known in the art, for example, by parenteral routes, including intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including oral and sublingual) administration. In some embodiments, the pharmaceutical composition is administered by intravenous or parenteral infusion or injection. In one embodiment, the composition is administered by injection into a tissue (e.g., intratumor). In another embodiment, the composition is administered to a mammal by subcutaneous injection. In another embodiment, the composition is topically administered to a mammal. In certain advantageous embodiments, intravenous administration is an optional route. In other embodiments, intratumoral administration may be more effective. The pharmaceutical composition of a therapeutically effective amount is delivered to a tissue, which may include but is not limited to skin, liver, lung, kidney and heart tissue.

[0169] The preparation of the preparation should be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous injection, intradermal, subcutaneous, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, intratumoral, and rectal administration. Solutions or suspensions for parenteral, intratumoral, intradermal, subcutaneous, or subcutaneous applications may include the following ingredients: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates, and agents for adjusting tension, such as sodium chloride or glucose. The pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0170] For transmucosal or transdermal administration, penetrants suitable for the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents for transmucosal administration, bile salts and fusidic acid derivatives. Transmucosal administration can be accomplished by the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels or creams as known in the art.

[0171] siRNA preparations can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J. Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst. Pharm. 53(3), 325 (1996).

[0172] In addition, siRNA preparations can also be administered by methods suitable for nucleic acid reagents (e.g., DNA vaccines). These methods include gene guns, bio-injectors, and skin patches, as well as needle-free methods, such as the microparticle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, and the percutaneous needle-free vaccination of mammals with powdered vaccines disclosed in U.S. Patent No. 6,168,587. In addition, intranasal delivery is possible, especially as described in Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88 (2), 205-10. Liposomes (e.g., as described in U.S. Patent No. 6,472,375) and microencapsulation can also be used for delivery. DODAB can be used to improve absorption. Biodegradable targeted microparticle delivery systems (e.g., as described in U.S. Patent No. 6,471,996) can also be used.

[0173] Treating cancer

[0174] Typically, targeted delivery of siRNA focuses on silencing a single gene. However, cancer is polygenic and develops through the action of multiple genes. Unfortunately, when tumors become resistant to the inhibition of specific genes, they can continue to grow despite the silencing of a single target gene, because the selection of tumor cells does not depend on the progression of that gene. Therefore, either a very potent single siRNA molecule must be used, or, advantageously, the synergistic effect produced when at least two siRNA molecules are provided in a small molecule (e.g., a nanoparticle) can be exploited.

[0175] The siRNA molecules modified with GEM disclosed herein can be used to treat various types of cancer, such as pancreatic cancer, lung cancer, colon cancer, breast cancer, gastric cancer, ovarian cancer and bladder cancer. In one embodiment, a combination of Gem-modified siRNA and at least one other siRNA (with or without modification and with or without GEM) can be used for the additive or synergistic effect of treating cancer. In some embodiments, the administration is intravenous injection, while in other embodiments, the administration is intratumoral administration. In other cases, the route of administration will vary depending on the type of cancer and other factors. In certain embodiments, PGN6 or PGN7 combined with BCLxL or Wee1 or other siRNA can successfully reduce tumor growth, completely stop its growth, or even reduce the volume of existing tumors. In other embodiments, tumor volume and the expression level of one or more target genes can be reduced by 20% to 100%. In other embodiments, separate treatments can be used to achieve separate treatments for two or more types of cancers, each of which contains a single or combination of Gem-modified siRNA and histidine-lysine copolymer carriers to reduce the tumor volume caused by two or more types of cancers. Any number of embodiments comprising changes in modifications, siRNA sequences containing GEMs, histidine-lysine copolymer structures and other additives, routes of administration, dosages, and treatment regimens can be used to treat any of the various cancerous tumors disclosed herein.

[0176] The presently disclosed embodiments provide pharmaceutical compositions and methods of treatment for subjects with cancer. The disclosed embodiments can even address prophylactic administration to individuals at risk of developing or already having tumors, wherein the compositions can reduce the expression of selected genes that drive tumor growth but are not critical to normal cells.

[0177] "Treatment" or "treatment" as used herein is defined as the administration or administration of a therapeutic agent (e.g., a dsRNA agent or vector or a transgene encoding the same) to a patient, or the application or administration of a therapeutic agent to a tissue or cell line isolated from a patient, who has a disease or metabolic disorder, has characteristics of a disease or disorder, with the intent to cure, heal, alleviate, mitigate, alter, remedy, ameliorate, improve, or affect the disease or condition, symptoms of a disease or condition, or a predisposition to the disease.

[0178] In one aspect, the disclosed embodiments provide a method for preventing a disease or condition as described above in a subject by administering a therapeutic agent (e.g., a dsRNA agent or vector or a transgene encoding the same) to the subject (including, for example, preventing the initiation of a transformation event in the subject by inhibiting TGFβ1 and Cox-2 expression). For example, a subject at risk of a disease can be identified by one or a combination of the diagnostic or prognostic assays described herein. For example, administration of a prophylactic agent can be performed before cancer is detected in the subject or symptoms characteristic of the disease or condition are manifested, thereby preventing the disease or condition, or delaying its progression.

[0179] The dsRNA molecules (siRNA) can be used in combination with other therapies to treat, inhibit, reduce or prevent deleterious adipose remodeling in a subject or organism.

[0180] Another aspect of the disclosed embodiments relates to methods for therapeutic treatment of subjects, i.e., altering the onset of disease or disorder symptoms. These methods can be performed in vitro (e.g., by culturing cells with dsRNA agents), or alternatively in vivo (e.g., by administering dsRNA agents to subjects).

[0181] Regarding preventive and therapeutic treatment methods, such treatments can be specifically customized or modified based on knowledge in the field of pharmacogenomics. As used herein, "pharmacogenomics" refers to the application of genomic technologies such as gene sequencing, statistical genetics, and gene expression analysis in clinical development and the market. More specifically, the term refers to the study of how a patient's genes determine his or her response to a drug (e.g., the patient's "drug response phenotype" or "drug response genotype"). Therefore, another aspect of the disclosed embodiment provides a method for customizing an individual's preventive or therapeutic treatment using target TGFβ1 and Cox-2 genes or regulators based on the individual's drug response genotype. Pharmacogenomics allows clinicians or physicians to target preventive or therapeutic treatments for patients who benefit most from treatment and avoid treating patients who may experience toxic side effects associated with the drug.

[0182] Therapeutic agents can be tested in selected animal models. For example, the dsRNA agents described herein (or expression vectors or transgenics encoding the same) can be used in animal models to determine the efficacy, toxicity or side effects of treatment with the agents. Alternatively, agents (e.g., therapeutic agents) can be used in animal models to determine the mechanism of action of such agents.

[0183] The disclosed embodiments described and claimed are not limited to the scope of the specific advantageous embodiments cited herein, as these embodiments are intended to be illustrative rather than limiting. Any equivalent embodiments are intended to be within the scope of the present disclosure, and the disclosed embodiments are not mutually exclusive. Various modifications to the embodiments, in addition to the embodiments shown and described herein, will become apparent to those skilled in the art from the above description. These modifications are also intended to fall within the scope of the appended claims.

[0184] The terms and words used in the following description and claims are not limited to conventional definitions, but are used to enable the present disclosure to be clearly and consistently understood. Therefore, it should be apparent to those skilled in the art that the description of various embodiments is provided for illustrative purposes only and is not intended to limit the disclosure of the attached claims and their equivalents.

[0185] It is to be understood that the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise; for example, reference to "a dermatologically active compound" includes reference to one or more of such compounds.

[0186] Unless otherwise defined herein, all terms used have the same meaning as commonly understood by one of ordinary skill in the art. The terms used herein should be interpreted as having a meaning consistent with their meaning in the context of the relevant art.

[0187] As used herein, the terms "include," "comprising," or "comprises," in relation to any article, composition, preparation, apparatus, method, process, system, etc., defined or described elements, are intended to be inclusive or open-ended and include those specified elements or their equivalents. Additional elements may be included and still fall within the scope or definition of the defined item, composition, etc.

[0188] The terms "about" or "approximately" mean within what one of ordinary skill in the art would consider an acceptable error range for a particular value; this will depend in part on how the value is measured or determined according to the limitations of the measurement system.

[0189] "Co-administration" or "co-delivery" refers to the simultaneous administration of two pharmaceutical preparations in the blood or other fluids of a subject using the same or different administration routes. The pharmaceutical preparations can be administered simultaneously or sequentially in the same pharmaceutical carrier or in different pharmaceutical carriers.

[0190] The terms "subject," "patient," and "individual" are used interchangeably.

[0191] The following examples illustrate certain aspects of the disclosed embodiments and should not be construed as limiting the scope thereof.

[0192] example

[0193] Example 1: In vitro study evaluating the effects of various prChk1-PolyGEM variants on IC 50 Impact

[0194] MIA PaCa-2, MDA-MB 31, LS180, HT1376, HT29, H358, H1299, OVCAR-3, HTB-9, and Panc10.05 cells were obtained from ATCC (Rockville, MD). MiaPaCa cells were cultured in DMEM supplemented with 10% FBS. H358, H1299, HTB-9, N87, and Panc 10.05 cells were cultured in DMEM / 10% FBS; LS180 and HT1376 were cultured in EMEM / 10% FBS; HT29 cells were cultured in McCoy5A / 10% FBS; OVCAR-3 cells were incubated in RPMI / 20% FBS. All cells were maintained at 37°C with 5% CO2.

[0195] One day before transfection, cells were seeded into 384-well plates at an optimal density of 300 to 800 cells / well. The next day, ten 3-fold serial dilutions of single backbone modified siRNA or PGN6 / PGN7 siRNA and gemcitabine (GEM) mixed with siRNA were prepared: Wee1#7, KRAS#1, MCL1#3, or DM2-P47 siRNA was prepared in a 1:1 mix and mixed with diluted Lipofectamine RNAiMAX (Life Technologies, Carlsbad, CA). Within 20 minutes, 10 μl of the transfection mixture was added to the cells containing 40 μL of culture medium. The final concentration of siRNA or gemcitabine in the wells ranged from 100 nM to 0.002 nM.

[0196] 96 hours after adding GEM or transfection mixture, the number of viable cells was determined by using CellTiter-Glo(R)-2.0 reagent (Promega, Madison, WI) and detecting the luminescent signal using a Cytation 5 plate reader (BioTek Inc, Winooski, VT). The mean (±SD) of the treated group (n=4) was compared with the untreated control group and reported as relative cell viability.

[0197] Figure 1(a) shows the sequences of several prChk1-GEM variants and non-silencing siRNA+GEMs. Figure 1(b) shows some additional sequences of siRNAs used in these Examples. Figure 2The effect of PolyGEM variants on MiaPaCa-2 cell viability is shown, showing that PNG6 (0.0.4 nM), STD1 (0.02 nM), and PNG7 (0.006 nM) have a significant effect on the IC compared to gemcitabine (8 nM). 50 The effects were increasingly significant when they were co-transfected with lipofectamine respectively.

[0198] Example 2: Effects of PGN6 with and without lipofectamine transfection on the cell viability of pancreatic cancer cell lines, lung cancer cell lines and colon cancer cell lines.

[0199] Using the same experimental conditions as described in Example 1 above, the efficacy of PGN6 (SEQ.ID No.7-8) on cell viability with and without lipofentamine ("lipo") was evaluated in three types of cancer cell lines. Figure 3(a)-(c) shows the effect of PGN6 with or without lipo on cell viability in pancreatic cancer cell lines when increasing siRNA levels. Figure 4(a)-(b) similarly shows the effect of these variants on cell viability of lung cancer cell lines; Figure 5(a)-(b) shows the effect of these variants on cell viability of colon cancer cell lines. All data are shown as mean ± standard deviation. Both variants showed potency and efficacy against pancreatic, lung and colon cancer cells. In the absence of lipofectamine, the PNG6 product works by releasing gemcitabine (GEM) from the structure, IC 50 The variation is usually 10 to 1000 times.

[0200] Example 3: Effects of STD1, PGN6 and PGN7 variants on cell viability in pancreatic, lung and colon cancer cell lines.

[0201] Using the same experimental conditions as described in Example 1 above, the efficacy of STD1 (SEQ.ID Nos.1-2), PGN6 (SEQ.ID Nos.7-8) and PGN7 (SEQ.ID Nos.9-10) was evaluated in three types of cancer cell lines. Figure 6 (a)-(f) shows the efficacy of STD1, PGN6 and PGN7 siRNA in pancreatic cancer, lung cancer and colon cancer cell lines compared to non-silencing siRNA used as control. Data are shown as mean ± standard deviation.

[0202] Example 4: Effects of PGN7, WEE1 and combinations of PGN7 and WEE1 variants on cell viability in pancreatic, lung and colon cancer cell lines.

[0203] Using the same experimental conditions as described in Example 1 above, the efficacy of PGN7 (SEQ. ID No. 9-10) alone and in combination with WEE1#7 (SEQ. ID No. 13) was evaluated in three types of cancer cell lines. Figure 7 (a)-(e) shows the efficacy of PGN7 and the combination of PGN7 and WEE1#7 in pancreatic cancer cell lines, lung cancer cell lines, and colon cancer cell lines. The data are shown as mean ± standard deviation.

[0204] Example 5: Effects of PGN7, BCLxL, and combinations of PGN7 and BCLxL variants on cell viability in pancreatic, lung, and colon cancer cell lines.

[0205] The efficacy of PGN7 (SEQ. ID No. 9-10) and the combination of BCLxL#5 (SEQ. ID No. 14) and PGN7 was evaluated in three types of cancer cell lines using the same experimental conditions as described in Example 1 above. Figure 8 (a)-(e) shows the efficacy of PGN7 variants and their combination with BCLxL in pancreatic cancer cell lines and lung cancer cell lines, but not colon cancer cell lines. The data are shown as mean ± standard deviation.

[0206] Example 6: Effects of PGN7, BCLxL and PBH7+BCLxL combination variants on the viability of bladder, gastric and ovarian cancer cell lines.

[0207] Using the same or similar experimental conditions as described in Example 1 above, PGN7 (SEQ. ID No. 9-10) + NS (i.e., using non-silencing siRNA), BCLxL#5 (SEQ. ID No. 14) + NS and PGN7 + BCLxL#5 combinations showed significant efficacy in HT1376 (400 cells / well) and HTB-9 (800 cells / well) bladder cancer cell lines, N87 (600 cells / well) and OVCAR-3 (800 cells / well) cells [Figure 9 (a)-(d)]. Data are shown as mean ± standard deviation.

[0208] Example 7: Effects of PGN7, Wee1 or their combination on the viability of several bladder cancer, gastric cancer and ovarian cancer cell lines.

[0209] Using the same or similar experimental conditions as described in Example 1 above, the significant efficacy of PGN7 (SEQ.IDNo.9-10), Wee1#7 (SEQ.IDNo.13) or a combination thereof in bladder cancer cell lines HT1376 (400 cells / well) and HTB-9 (800 cells / well), gastric cancer cell line N87 (600 cells / well) and ovarian cancer cell line OVCAR-3 (800 cells / well) is shown in Figure 10 (a)-(d). Data are shown as mean ± standard deviation.

[0210] Example 8: Effects of PGN7, Wee1, BCLxL and their combination on the viability of bladder cancer, gastric cancer and ovarian cancer cell lines.

[0211] Using the same or similar experimental conditions as described in Example 1 above, the efficacy of PGN7 (SEQ.ID No.9-10), Wee1 (SEQ.ID No.13), BCLxL#5 (SEQ.ID No.14) or PGN7+Wee1 or PGN+BCLxL combination was evaluated in HT1376 (400 cells / well) and HTB-9 (800 cells / well) bladder cancer cell lines, N87 (600 cells / well) gastric cancer cell line and OVCAR-3 (800 cells / well) ovarian cancer cell line and is shown in Figure 11 (a)-(d). Data are shown as mean ± standard deviation.

[0212] Example 9: In vivo animal studies were used to evaluate the therapeutic efficacy of PGN6+DODAB siRNA on pancreatic tumor growth in the non-obese diabetic-severe combined immunodeficient mouse model (NOD / SCID) inoculated with the human pancreatic cancer cell line MIAPaCa-2.

[0213] Twenty NOD / SCID female mice, 6 to 9 weeks old, were purchased from Jackson Laboratory. Each mouse was marked for identification at the start of the study. A maximum of five mice were housed in a cage with standard corncob bedding for at least three days for stability; bedding was changed every two weeks. Fluorescent lighting was on a 12-hour cycle; temperature was maintained at 68-79 degrees Fahrenheit, and humidity was 30-70%. Mice were fed an 18% soy irradiated rodent diet (Envigo), and all animals had access to autoclaved acidified water (pH 2.5-3) ad libitum. Mice were observed daily for clinical signs of disease.

[0214] Tumor cell preparation: Cryogenic vials containing tumor cells were thawed and cultured according to the manufacturer's protocol. On the day of inoculation, cells were washed, counted, and resuspended in cold serum-free medium at a concentration of 3 million cells per 100 microliters. The matrigel-cell mixture was kept on ice during transportation to the animal room. Cell injection was prepared by drawing the matrigel-cell mixture into a frozen 1 mL smooth-tip syringe, which was placed on ice to prevent the matrigel from solidifying.

[0215] Tumor implantation: Animals were shaved and ear-tagged before inoculation. Each mouse was fixed and the injection site was disinfected with an alcohol swab, and then 100 mL of the cell suspension was injected subcutaneously into the flank of the mouse. The mice were injected with the cell suspension and left to rest for 7 days to observe the tumor growth.

[0216] Tumor Measurements: Monitor mice weekly for palpable tumors or changes in appearance or behavior. Once tumors are palpable, measure them weekly using calipers. Calculate tumor volume using the following formula:

[0217] (Longest diameter x Shortest diameter 2 ) / 2 = tumor volume

[0218] Once the tumor volume averages 150–250 mm 3 Tumors and body weights were measured at least twice weekly during the study. One individual was responsible for measuring all mice during the study.

[0219] Test Article Storage: The test article (PGN6 (SEQ.ID Nos.7-8) + DODAB siRNA, encapsulated by HKP (+H) copolymer carrier) used in the treatment group and the carrier (DODAB siRNA encapsulated by HKP (+H) copolymer carrier) used in the control group were stored at -20°C and were light-sensitive. The storage temperature after thawing and the storage temperature after formulation were both 4°C and the composition was light-sensitive. A new vial was provided for each injection. The remaining test article and dosing solution after the study were stored for subsequent studies.

[0220] Randomization and dose selection: Once mean tumor volume reaches 150-250mm 3Mice were randomly divided into a control group (1) and a treatment group (2) and injected intravenously with a single cell suspension (3 million cells in 0.1 mL 1:1 PBS:Matrigel). All mice were vaccinated within 24 hours of randomization. The treatment group (2) [i.e., HKP(+H) / (PGN6+DODAB siRNA)] was injected twice a week for three consecutive weeks via IV route at a dose of 2 mg / kg (dose volume: 4 mL / kg), while the control group mice were given (HKP(+H) / DODAB siRNA) at the same frequency, duration and volume, and the same IV administration method. The day of administration was defined as day 0, and the study was terminated four weeks after the first administration.

[0221] Body weight measurements and guidelines: All mice were weighed and tumor volumes were measured at least twice a week after randomization and initiation of treatment. A decrease in body weight of more than 20% from baseline was determined as a trigger for daily monitoring for signs of recovery for up to 72 hours; dosing holidays or nutritional supplements were provided based on animal health assessments; if no signs of recovery were observed, the animal was sacrificed for humane reasons as specified in the IACUC protocol.

[0222] Clinical observations: Clinical observations were performed twice weekly when tumors and body weights were measured and detailed in the study records. Mice were examined for tumor growth and adverse effects of treatment on behavior, such as mobility, food and water consumption, eye / hair matting, or other abnormalities. Any mouse showing signs of pain or distress was euthanized.

[0223] Moribund: Tumor size greater than 2000 mm 3 Mice were euthanized, as were any animals showing signs of pain or distress or that lost more than 20% of their body weight and showed no signs of recovery within 72 h.

[0224] Final Disposition: At the end of the study, all live animals were euthanized.

[0225] result

[0226] Figure 12(a): shows the mean (±SE) values ​​of pancreatic tumor volumes in NOD / SCID mice over a three-week treatment period for the control group (line showing increasing tumor growth over time) and the treated group (line showing little or no change over time), showing that PGN6 delivered by HKP(+H) had a significant effect in reducing or preventing tumor growth in treated animals.

[0227] Figure 12(b) shows the mean (±SD) body weight of NOD / SCID mice inoculated with human pancreatic cancer cells. Once the tumors were palpable, treatment (2 mg / kg twice a week) was started from day 0, with either the treated group (PGN6+non-silencing DODAB siRNA+HKP(+H) vector) or the control group (non-silencing DODAB siRNA+HKP(+H) vector) (n=10). In fact, during the treatment period (20 days), the mean body weight of the control and treated groups remained unchanged, while the tumor volume in the control group increased significantly (4-fold) during the same period, suggesting that the tumor weight had no significant effect on the body weight of the mice, or, if anything, the increase in tumor size / weight in the control group may have compensated for the loss of body weight in this group. In contrast, in the PGN6-treated group, body weight remained unchanged and the tumor did not grow.

[0228] Example 10: Effect of the combination of PGN7 and BCLxL on the viability of triple-negative breast cancer (MDA-MB231) and ovarian cancer (OVCAR3) cells

[0229] Using the same or similar experimental conditions as described in Example 1 above, (i) PGN7 (SEQ.ID No.9-10) plus non-signaling oligonucleotide ("NS"), (ii) BCLxL#5 (SEQ.ID No.14) + NS, and (iii) the effect of the combination of PGN7 and BCLxL#5 was evaluated in triple-negative breast cancer (MDA-MB231) and ovarian cancer (OVCAR3) cell lines, and the results are shown in Figure 13 (a)-(b). 400 cells were used per well for the MDA-MB231 cell line and 800 cells were used per well for the OVCAR3 cell line. Data are shown as mean ± standard deviation.

[0230] As shown in Figure 13 (a, MDA-MB231) and (b, OVCAR3), the significant synergistic effect of PGN7+BCLxL#5 (IC 50 (a): 0.06 nM; (b): 0.1 nM) were observed in both cell line types and compared with PGN7 (IC 50 (a): 1.1 nM; (b): 0.5 nM) or BCLxL#5 (IC 50 (a): 0.5 nM; (b): 2.7) compared.

[0231] Example 11: Effects of STD1 and PGN7 on xenograft tumor growth in NU / NU mice after subcutaneous implantation of MiaPaCa tumor cells

[0232] Twenty-four NU / NU nude mice were purchased from Charles River Laboratories (Frederick, MD) and housed in individual cages with standard bedding, lighting, and free access to food.

[0233] The human pancreatic cancer MiaPaCa-2 cell line was obtained from the American Type Culture Collection (ATCC, CRL-1420 TM ) and cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37°C, 5% CO2. Subculture was performed when the cell volume reached ~80-90% (approximately 3-5 days). The culture medium was changed every 3 to 4 days. All experiments were performed when the cells reached approximately 80-90% confluence.

[0234] On day 0, MiaPaCa tumor cells (2.5 x 10 6 The cells were injected subcutaneously into the left flank of each mouse. Body weight was measured daily using a digital scale and tumor volume was measured using a digital caliper. Tumor volume was measured using an external digital caliper and a modified ellipsoid formula: 1 / 2 (length x width) 2 ).

[0235] About five to ten days after subcutaneous implantation of MiaPaCa cells, when the tumor volume reached 100 mm 3 At the time of the experiment, mice were divided into three groups (n=8). Twice a week (Monday and Thursday) for three to four weeks, all three groups of mice received intratumoral injections of 80 μl each time. The control group mice were injected with non-silencing (NS) siRNA delivered with HKP(+H). One of the mice in the treatment group was injected with PGN7, i.e., GEM-modified SHK1(2.z)(SEQ.ID No.9-10) siRNA delivered with HKP(+H). The mice in the second treatment group were injected with STD1, i.e., GEM-modified CHK1(2.z)(SEQ.ID No.3-4) siRNA delivered with HKP(+H). All injections were performed under isoflurane anesthesia to minimize discomfort.

[0236] Tumor volumes (mm) were measured on days 0, 4, 7, 11, 14, 18, 21, and 25. 3 ) and body weight. Mean ± SE of each group (PGN7, STD1, and control) was statistically compared using analysis of variance (ANOVA) with Tukey's test. Mice were also observed for overgrowth, adverse responses to treatment, and other observable signs of poor health. Follow-up and data analysts were blinded to the treatments.

[0237] All mice were sacrificed and implanted xenografts removed on day 26 after the first treatment (the last treatment was performed on day 25). One-third of the xenografts were fixed in 0.9% formalin for histological analysis; one-third were frozen and one-third were stored in RNAlater stabilizing storage solution.

[0238] As shown in Figure 14(a), there was no significant difference in tumor weight (mean ± SE) between STD1, PGN7 and control groups (n = 8)

[0239] (p>0.05). Similarly, as shown in Figure 14(b), the mean (±SE) body weights of the two treatment groups and one control group did not differ over 25 days (p>0.05). PGN7 effectively attenuated the increase in mean (±SE) tumor volume over three and a half weeks compared to the control group (p<0.02) (Figure 14(c)).

Claims

1. A pharmaceutical composition comprising a modified interfering ribonucleic acid (RNAi) molecule combined with one or more gemcitabine molecules instead of cytidine molecules, and further comprising a histidine-lysine copolymer carrier, wherein the RNAi is selected from siRNA, miRNA, shRNA and mRNA. 2 . The pharmaceutical composition according to claim 1 , wherein the histidine-lysine copolymer carrier comprises HKP or HKP(+H).

3. The pharmaceutical composition according to any one of the preceding claims, wherein the modified RNAi is selected from 2'-methoxy modification, 2'-fluoro modification and phosphorothioate modification.

4. The pharmaceutical composition according to any one of the preceding claims, wherein the RNAi molecule comprises a molecule selected from SEQ.ID No. 1-2, SEQ.ID No. 3-4, SEQ.ID No. 5-6, SEQ.ID No. 7-8 and SEQ.ID No. 9-10.

5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition further comprises a combination of at least two RNAi molecules, SEQ.ID NO.13 or SEQ.ID NO.

14.

6. The pharmaceutical composition according to any one of the preceding claims, further comprising lipofectamine (a liposome).

7. A pharmaceutical composition according to any one of the preceding claims, wherein the RNAi molecule is a double-stranded siRNA comprising a gemcitabine modification, and wherein the siRNA comprises a sequence selected from SEQ.ID No.1-2, SEQ.ID NO.3-4, SEQ.ID NO.5-6, SEQ.ID NO.7-8 and SEQ.ID NO.9-10, And wherein the composition further comprises RNAi molecule SEQ.ID NO.13 or SEQ.ID NO.

14.

8. A method of treating a subject suffering from cancer, comprising: An effective amount of a pharmaceutical composition containing a gemcitabine-modified siRNA molecule is administered to a subject; wherein the composition further comprises a histidine-lysine copolymer carrier; and wherein the siRNA molecule also comprises at least one sequence selected from SEQ.ID No.1-2, SEQ.ID NO.3-4, SEQ.ID NO.5-6, SEQ.ID NO.7-8, SEQ.ID NO.9-10, SEQ.ID NO.13 and SEQ.ID NO.

14. Wherein treating the subject by administration of the pharmaceutical composition results in attenuation or inhibition of growth of the cancer.

9. The method of claim 8, wherein the cancer is selected from breast cancer, colon cancer, stomach cancer, pancreatic cancer, lung cancer, ovarian cancer, and bladder cancer.

10. The method of treatment according to claim 8 or 9, wherein administration of an effective amount of the pharmaceutical composition comprises a method selected from the group consisting of intravenous injection, intratumoral injection, and subcutaneous injection.

11. The method of treatment according to any one of claims 8 to 10, wherein liposomes are administered as a component of the pharmaceutical composition.

12. A method of treating a subject suffering from cancer, comprising: The subject is administered the pharmaceutical composition according to any one of claims 1-7.

13. The method of claim 12, wherein the cancer is selected from breast cancer, colon cancer, stomach cancer, pancreatic cancer, lung cancer, ovarian cancer, and bladder cancer.

14. A method for treating a subject having cancer, comprising administering to the subject an effective amount of a pharmaceutical composition comprising SEQ.ID.No.9-10 and SEQ.ID.No.14, wherein the composition further comprises HKP(+H), and wherein the pharmaceutical composition is administered by intravenous injection or intratumoral injection.

15. A method for treating a subject suffering from cancer, comprising administering to the subject an effective amount of a pharmaceutical composition comprising SEQ.ID.No.9-10 and SEQ.ID.No.13, wherein the composition further comprises HKP(+H), and wherein the pharmaceutical composition is administered by intravenous injection or intratumoral injection.

16. A method for treating a subject with pancreatic cancer, comprising administering to the subject an effective amount of a pharmaceutical composition comprising SEQ.ID.No.7-8, wherein the composition is administered by intratumoral injection.

17. The method according to any one of claims 8-16, wherein the amount of reduction in target gene expression is selected from the group consisting of 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more and up to 100%.

18. The method of any one of claims 8-17, wherein the amount of tumor volume reduction or cancer cell level reduction is selected from the group consisting of 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, and up to 100%.

19. The method of any one of claims 8-18, wherein the subject is a mammal.

20. The method of claim 19, wherein the subject is a human.

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