Polynucleotide for suppressing expression of transcript variant 1 of spindle microtubule assembly factor and use thereof

Abstract

The present disclosure relates to polynucleotides and methods for inhibiting the activity of development-related pathways, including the Wnt, Hedgehog, and Notch pathways, and the invasive capacity of malignant cells to treat primary or secondary solid tumors.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 414,461, filed October 8, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] The present disclosure relates to the field of cancer treatment. In particular, the present disclosure provides polynucleotides that inhibit the expression of assembly factor for spindle microtubules (ASPM) and their use in the treatment of cancer. [Background technology]

[0003] Developmental signaling pathways, including Wnt, Notch, and Hedgehog, regulate stem cell homeostasis in adult tissues and are often dysregulated during the malignant transformation process, resulting in tumor stem cell characteristics. Canonical Wnt signaling, such as β-catenin, is involved in epithelial tissue homeostasis by maintaining stem cell proliferation and migration, particularly in the intestine, mammary gland, and skin. Increasing evidence suggests that Wnt-regulated self-renewal processes in tissue progenitor and stem cells can be hijacked by cancer cells to promote malignant progression. Consistently, compelling data now support the role of the Wnt / β-catenin signaling pathway in sustaining the cancer stem cell (CSC) phenotype in solid tumors. Growing evidence suggests that Wnt signaling plays an essential role in the metastatic establishment of cancer cells, and these experimental studies highlight the interaction between microenvironmental factors and CSCs. For example, periostin, a stromal cell-derived extracellular matrix (ECM) protein, recruits Wnt ligands, thereby activating Wnt signaling in CSCs during breast cancer metastatic establishment.

[0004] Cancer invasiveness and distant metastasis are major causes of patient mortality, and understanding them is important for improving outcomes in patients with solid tumors. Invadopodia, or podosomes, are transient actin-based protrusions present in immune cells and certain cancer cells that mediate localized degradation of the extracellular matrix (ECM) through the localized proteolytic activity of proteases. In addition to directly degrading the ECM, invadopodia also initiate crosstalk with the ECM, exerting physical forces on the surrounding stroma and opening micron-sized channels to promote cancer cell invasiveness (Iizuka, S., Leon, RP, Gribbin, KP, Zhang, Y., Navarro, J., Smith, R., Devlin, K., Wang, LG, Gibbs, SL, Korkola, J. et al. (2020). Crosstalk between invadopodia and the extracellular matrix. Eur J Cell Biol 99, 151122). Invadopodia have been shown to exist in vivo and are therefore hypothesized to play a crucial role in the establishment of distant metastases by facilitating the processes of intravasation and extravasation. Consistent with these observations, a series of functional studies have demonstrated that specific variants of SRC, PDGFR-α, TKS-5, or MENA (MENA) INVWe have demonstrated that inhibiting invadopodia-regulating factors, including Twist1, can inhibit metastasis in various cancer models (Eckert, MA, Lwin, TM, Chang, AT, Kim, J., Danis, E., Ohno-Machado, L., and Yang, J. (2011). Twist1-induced invadopodia formation promotes tumor metastasis. Cancer Cell 19, 372-386; Weidmann, MD, Surve, CR, Eddy, RJ, Chen, X., Gertler, FB, Sharma, VP, and Condeelis, JS (2016). Mena (INV) dysregulates cortactin phosphorylation to promote invadopodium maturation. Sci Rep 6, 36142). Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need for drugs and methods for treating and preventing cancer and its metastasis. [Means for solving the problem]

[0006] The present disclosure relates to polynucleotides and methods for inhibiting the activity of development-related pathways, including the Wnt, Hedgehog, and Notch pathways, and the invasive capacity of malignant cells to treat primary or secondary solid tumors.

[0007] The present disclosure provides a polynucleotide comprising a nucleotide sequence complementary to the mRNA of the ASPM gene having the nucleotide sequence set forth in SEQ ID NO:1, or a nucleotide sequence comprising a contiguous segment having at least 70%, at least 80%, or at least 90% sequence identity to the nucleotide sequence complementary to SEQ ID NO:1.

[0008] In one embodiment, the polynucleotide comprises a nucleotide sequence complementary to an mRNA encoded by exon 18 of transcript variant 1 of the human ASPM gene having the nucleotide sequence set forth in SEQ ID NO:3, or a nucleotide sequence comprising a contiguous segment having at least 70%, at least 80%, or at least 90% sequence identity to the nucleotide sequence complementary to SEQ ID NO:3.

[0009] In some embodiments, the polynucleotides described herein are RNAi. Examples of RNAi include, but are not limited to, shRNA, siRNA, or dsRNA.

[0010] Certain embodiments of polynucleotides of the present disclosure are siRNA molecules, the siRNA molecule comprising: (a) a duplex region; and (b) zero or at least one overhang region, each overhang region containing six or fewer nucleotides; the duplex region consisting of a sense region and an antisense region, the sense region and the antisense region together forming the duplex region, the antisense region and the sense region each being 15 to 30 nucleotides in length; and the antisense region comprising a sequence that is the complement of a sequence selected from SEQ ID NOs: 3, 5, 7, or 9.

[0011] In some embodiments, the siRNA molecules described herein have antisense and sense regions that are 15-25, 15-20, or 15-18 bases in length, respectively.

[0012] In some embodiments, the siRNA molecule is a chemically synthesized double-stranded siRNA molecule, wherein (a) each strand of the double-stranded siRNA molecule is 15 to 30 nucleotides in length, and (b) one strand of the siRNA molecule comprises a sequence that is the complement of a sequence selected from SEQ ID NOs: 3, 5, 7, or 9.

[0013] In some embodiments, the siRNA molecules described herein comprise a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO:4 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO:5 by no more than 3 nucleotides.

[0014] In certain embodiments, the siRNA molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:5.

[0015] In some embodiments, the siRNA molecules described herein comprise a sense strand and an antisense strand that form separate double-stranded RNA duplexes, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO: 6 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO: 7 by no more than 3 nucleotides.

[0016] In certain embodiments, the siRNA molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:6 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:7.

[0017] In some embodiments, the siRNA molecules described herein comprise a sense strand and an antisense strand that form separate double-stranded RNA duplexes, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO: 8 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO: 9 by no more than 3 nucleotides.

[0018] In certain embodiments, the siRNA molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:8 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:9.

[0019] In some embodiments, the siRNA molecule has at least one overhang region or no overhang region.

[0020] In some embodiments, one or both of sense strand and antisense strand can be further modified as modified siRNA.The example of modified nucleotide includes but is not limited to 2'-O-methyl modified nucleotide, 2'-fluorophosphoramidite, 3'-terminal deoxy-thymine nucleotide, non-natural base that constitutes nucleotide, nucleotide that contains 5' phosphorothioate group, and terminal nucleotide that is linked with cholesteryl derivative and dodecanoic acid bisdecylamide group.

[0021] In some further embodiments, the modified siRNA comprises between 10% and about 30% of the nucleotides in the double-stranded region, comprises 2'-O-methyl (2'OMe) nucleotides, and comprises 2'OMe nucleotides in both strands of the modified siRNA.

[0022] The present disclosure provides a pool of siRNA molecules, comprising one or more of a first siRNA molecule or a modified siRNA molecule thereof, a second siRNA molecule or a modified siRNA molecule thereof, a third siRNA molecule or a modified siRNA molecule thereof, and a fourth siRNA molecule or a modified siRNA molecule thereof; the first siRNA molecule is a chemically synthesized double-stranded siRNA molecule, (a) each strand of the double-stranded siRNA molecule is 15 to 30 nucleotides in length, and (b) one strand of the siRNA molecule comprises a sequence that is a complementary sequence of a sequence selected from SEQ ID NO: 3; the second siRNA molecule is a chemically synthesized double-stranded siRNA molecule, (a) each strand of the double-stranded siRNA molecule is 15 to 30 nucleotides in length, and (b) one strand of the siRNA molecule comprises a sequence that is the complement of a sequence selected from SEQ ID NO: 5; the third siRNA molecule is a chemically synthesized double-stranded siRNA molecule, (a) each strand of the double-stranded siRNA molecule is 15 to 30 nucleotides in length, and (b) one strand of the siRNA molecule comprises a sequence that is the complement of a sequence selected from SEQ ID NO: 7; The fourth siRNA molecule is a chemically synthesized double-stranded siRNA molecule, (a) each strand of the double-stranded siRNA molecule is 15 to 30 nucleotides in length, and (b) one strand of the siRNA molecule comprises a sequence that is the complement of a sequence selected from SEQ ID NO:9.

[0023] In some embodiments, the siRNA molecules in the pool comprise one or more of a second siRNA molecule or a modified siRNA molecule thereof, a third siRNA molecule or a modified siRNA molecule thereof, and a fourth siRNA molecule or a modified siRNA molecule thereof.

[0024] In some embodiments, the siRNA molecules in the pool comprise a sense strand and an antisense strand that form separate double-stranded RNA duplexes, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO: 4, 6, or 8 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO: 3, 5, or 9 by no more than 3 nucleotides.

[0025] In a further embodiment, the siRNA molecules in the pool comprise a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:5.

[0026] In a further embodiment, the siRNA molecules in the pool comprise a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:6 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:7.

[0027] In a further embodiment, the siRNA molecules in the pool comprise a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:8 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:9.

[0028] The present disclosure provides pharmaceutical compositions comprising at least one polynucleotide described herein and a pharmaceutical carrier, diluent, and / or adjuvant.

[0029] The present disclosure provides nanoparticles comprising a polynucleotide described herein or any mixture thereof, and a lipid nanoparticle (LNP), liposome, micelle, virosome, or nucleic acid complex.

[0030] The present disclosure also provides a method of inhibiting malignant tumor growth, locoregional spread, and distant metastasis, and / or treating solid tumors and / or tumor metastases in a subject, comprising administering to the subject a polynucleotide described herein or any mixture thereof, a pool of siRNA molecules described herein, a pharmaceutical composition described herein, or a nanoparticle described herein.

[0031] In some embodiments, the human solid cancer is breast cancer, non-small cell lung carcinoma (NSCLC), PDAC, the sclerotic subtype of gastric adenocarcinoma, and the "stem / serrated / mesenchymal (SSM)" molecular subtype of colorectal cancer (CRC). [Brief explanation of the drawings]

[0032] [Figure 1](A) and (B) show that ASPM expression correlates with breast cancer metastasis. (A) shows that ASPM is the top-ranked Wnt-related factor associated with breast cancer metastasis in the Pawitan et al. dataset. (B) shows Kaplan-Meier survival curves comparing distant metastasis-free survival in breast cancer patients stratified according to ASPM expression levels queried from KM Plotter (http: / / kmplot.com / analysis / index.php?p=service) and SurvExpress (http: / / bioinformatica.mty.itesm.mx:8080 / Biomatec / SurvivaX.jsp). P values ​​are calculated using the log-rank test. [Figure 2] (A) and (B) show that genetic knockdown of ASPM expression reduced the invasive ability of breast cancer cells. ASPM-v1 expression was stably downregulated in HCC-1954 and MDA-MB-436 breast cancer cells using lentiviral-mediated knockdown. The ability of cancer cells with control or ASPM knockdown to invade recombinant basement membrane was examined using a modified Boyden chamber invasion assay. (A) shows a representative immunofluorescence image of invasive cells in which cell nuclei were stained with CYTOX-green. Scale bar = 100 μm. (B) shows the number of invasive cells in (A). Data are mean ± SEM. ***P<0.001. [Figure 3] (A) and (B) contain several panels showing the effect of gene knockdown (knockdown) of ASPM expression on distant metastasis in breast and pancreatic cancer. (A) shows representative bioluminescence (BLI) signals from metastatic lung tumors at the indicated time points (weeks, wk) after cell inoculation. (B) shows the BLI signal in (A), expressed as normalized photon counts, as a function of time. Data are shown as mean ± SEM (n = 5 mice per group). **P ≤ 0.01 compared to control IgG. [Figure 4](A) and (B) contain several panels related to elevated levels of ASPM expression in micrometastatic tumors from patient-derived xenograft (PDX) models of breast cancer progression. (A) shows a representative flow cytometry plot demonstrating the pattern of ASPM staining in CD298-positive cancer cells in primary tumors and lung micrometastases. (B) shows elevated ASPM expression in CD298-positive cancer cells in micrometastatic lesions from two PDX models (BR1282 and BR1474). Data are mean ± SEM. **P<0.01. [Figure 5] The predicted domain structure of human ASPM isoform 1 and isoform 2 proteins is shown. CH, calponin homology; ARM, armadillo; IQ, isoleucine and glutamine. The region encoded by exon 18 containing the 67 IQ domain is highlighted. [Figure 6A] (A)–(E) contain several panels related to the expression patterns of ASPM isoforms in normal and cancerous tissues. (A) shows the immunohistochemical (IHC) staining patterns of ASPM isoform 1 (ASPM-i1) and ASPM isoform 2 (ASPM-i2) in representative human breast cancer and normal breast tissues. Scale bar, 50 μm. [Figure 6B] (B) Bar graph showing the distribution of single-cell staining intensity (1+ to 3+) for ASPM-i1 and ASPM-i2. Data are mean ± SEM (n = 40). ***P < 0.001. [Figure 6C] (C) IHC staining pattern of ASPM-i1 at the invasive front of a representative human breast cancer tissue. Scale bar, 100 μm. [Figure 6D] (D) Bar graph showing the percentage of cancer cells with moderate (2+) to high (3+) ASPM-i1 staining intensity. Data are mean ± SEM (n = 46). ***P < 0.001. [Figure 6E] (E) shows the IHC staining patterns of ASPM-i1 and ASPM-i2 in representative normal, hepatitis, and cirrhotic liver tissues, as well as HCC tissues. Scale bar, 50 μm. [Figure 6F](F) Bar graph showing the distribution of single-cell staining intensity (1+ to 3+) of ASPM-i1 and ASPM-i2 in normal liver tissues (n = 24), hepatitis liver tissues (n = 10), cirrhosis liver tissues (n = 50), and HCC tissues (n = 111). Data are mean ± SEM. **P < 0.01, ***P < 0.001 vs. normal liver. [Figure 7] Figure 1 shows ASPM isoform 1 (ASPM-i1) immunoprecipitated with PAR-planar cell polarity proteins, including PAR-6α, PAR-6β, disheveled-2 (DVL2), CDC42, and SMURF1, in invasive breast cancer MDA-MB-436 cells. Non-invasive cancer cells are included as a control. [Figure 8] (A) and (B) show several panels related to the specific interaction of ASPM isoform 1 (ASPM-i1) with the PAR / PCP proteins DVL2, PAR6β, and CDC42 in cancer cell invadopodia. (A) Confocal images showing the colocalization of ASPM-i1 (red), DVL2, PAR6β, CDC42, or N-WASP (blue) with the invadopodia marker cortactin (green) in the invadopodia of breast cancer MDA-MB-436 cells. Scale, 10 μm. (B) Shows ASPM-i1 specifically immunoprecipitated with DVL2, PAR6β, CDC42, and N-WASP in the invadopodia of invasive MDA-MB-436 cells. Cortactin and TKS5 are included as invadopodia markers. [Figure 9](A)–(C) show several panels related to the effect of gene knockdown of ASPM variant 1 (ASPM-v1; encoding "ASPM isoform 1" or "ASPM-i1") expression on invadopodia formation and invasive ability of breast cancer cells. Breast cancer MDA-MB-436 cells were lentivirally transduced with small hairpin RNAs (shRNAs) specifically targeting ASPM gene transcript variant 1 (ASPM-v1 shRNA) to knockdown ASPM-v1 expression. (A) shows the effect of isoform-specific ASPM-v1 shRNAs, including ASPM.e18 shRNA #1 and ASPM.e18 shRNA #4, on the protein abundance levels of ASPM-i1 in MDA-MB-436 cells. (B) Knockdown of ASPM-v1 expression suppressed the recruitment of DVL2, PAR6β, CDC42, and membrane-type matrix metalloproteinase (MT1-MMP) to invadopodia of MDA-MB-436 cells. Immunoblots of the indicated proteins in invadopodia lysates from MDA-MB-436 cells lentivirally transduced with control shRNA or ASPM.e18 shRNA #4 are shown. (C) Knockdown of ASPM-v1 expression reduced the invasive ability of MDA-MB-436 cells as assessed using a modified Boyden chamber invasion assay. Left: Representative immunofluorescence images of invaded cells stained for cell nuclei with CYTOX-green. Scale bar, 50 μm. Right: Quantification of invaded cells. Data are mean ± SEM. ***P<0.001. [Figure 10]Genetic knockdown of ASPM variant 1 (ASPM-v1) expression affects multiple developmental and stemness-related pathways. Relative luciferase reporter activity of the indicated developmental and stemness-related signaling pathways in 293T cells in which ASPM-v1 was knocked down using lentiviral-mediated transduction of ASPM.18 shRNA#4 or in 293T cells infected with control shRNA lentivirus and transduced with the respective reporter constructs on the Cignal Finder Stem Cell & Differentiation 10-Pathway Reporter Array (n=3 per group). *P<0.05, **P<0.01, ***P<0.001 compared to control shRNA. [Figure 11] This panel contains two panels related to the selection of optimized siRNAs targeting ASPM variant 1 (ASPM-v1). Using qRT-PCR analysis, ASPM-v1 transcript levels are shown in MDA-MB-436 cells transduced with equal total amounts (50 nM for all siRNAs) of the top three siRNAs listed in step 4 or various combinations thereof (mean ± SEM, n=3). *P<0.5, **P<0.01, ***P<0.001 compared to NT siRNA. [Figure 12] (A) and (B) contain two panels related to the effects of a lipid nanoparticle (LNP)-formulated small interfering RNA (siRNA) mixture (LNP-siASPM-v1) specifically targeting the exon 18 mRNA region of ASPM variant 1. (A) shows that treatment of breast cancer MDA-MB-436 cells with increasing concentrations (0–50 nM) of LNP-ASPM-v1 siRNA dose-dependently reduced the transcript levels of ASPM-v1. (B) shows that treatment of MDA-MB-436 cells with LNP-siASPM-v1 dose-dependently reduced the protein abundance levels of ASPM isoform 1 (ASPM-i1), but not ASPM protein isoform 2 (ASPM-i2). NT siRNA, non-targeting siRNA. [Figure 13](A) and (B) show the effect of LNP-siASPM-v1 on invadopodia formation and invasive ability of cancer cells. (A) shows the inhibitory effect of LNP-formulated ASPM-v1-specific siRNA (LNP-siASPM-v1) on invadopodia formation of cancer cells. On the left, confocal images of cortactin+F-actin+ dots (yellow) representing cross sections of downward-protruding invadopodia in MDA-MB-436 cells treated with LNP-siASPM-v1 or LNP-nontargeting control siRNA (NT siRNA) (both at 100 nM for 72 hours) are shown. On the right, the number of cortactin+F-actin+ invadopodia per cell. Scale bar, 10 μm. Data are means ± SEM. ***P<0.001. (B) shows that treatment of MDA-MB-436 cells substantially inhibited their invasive ability when examined using a dual-chamber invasion assay. Representative immunofluorescence images of invaded cells, in which the cell nuclei were stained with CYTOX-green (green), are shown on the left. Scale bar = 500 μm. Right, number of invaded cells. Data are mean ± SEM. **P < 0.01. [Figure 14](A)–(C) contain several panels related to the effects of LNP-formulated ASPM variant 1-specific siRNA (LNP-siASPM-v1) on Wnt activity and the stem cell properties and tumorigenicity of hepatocellular carcinoma (HCC) cells. (A) shows the fold change in Wnt-specific luciferase expression in HuH-1 cells treated with LNP-siASPM-v1 or LNP-nontargeting control siRNA (NT siRNA) (both at 100 nM for 72 hours) followed by WNT3A (at 250 ng / ml for 16 hours). (B) shows the percentage of aldehyde dehydrogenase (ALDH)-positive cell population (representing cancer stemness in HCC) in HuH-1 cells treated with LNP-siASPM-v1 or LNP-nontargeting control siRNA, as in (A). Data are shown as mean ± SEM (n = 3). ***P ≤ 0.001 compared to LNP-NT siRNA in (A) and (B). (C) shows the inhibitory effect of LNP-siASPM-v1 on the tumorsphere-forming ability of HCC cells. HuH-1 cells were treated with LNP-siASPM-v1 or LNP NT siRNA as in (A). The cells were then cultured for 10 days in serum-free, nonadherent culture plates. Representative phase-contrast images of the resulting tumorspheres are shown. Scale bar, 50 μm. Right: Limiting dilution assay demonstrating the tumorsphere-forming efficacy of the cells. Data are mean ± SEM (n = 4 in each group). [Figure 15A] (A)–(D) show several panels related to the pharmacodynamic study of LNP-siASPM-v1, a LNP-formulated systemic siRNA targeting ASPM variant 1, in an orthotopic mouse model of triple-negative breast cancer (TNBC). (A) shows GFP- and firefly luciferase-expressing breast cancer MDA-MB-436 cells orthotopically injected into the mammary fat pad of immunodeficient NOD / SCID mice. Ten days after cell inoculation, when tumors became detectable by bioluminescence imaging (BLI), tumor-bearing mice received two intravenous injections of Cy5-labeled LNP-siASPM-v1 (100 μg [approximately 4 mg / kg] per mouse) every three days. At the indicated time points after the second injection, tumors were excised for cell dissociation, and GFP+ cancer cells were sorted for subsequent analysis. [Figure 15B] (B) Representative FACS plots showing the GFP and Cy5 staining patterns of the cancer cells described in (A), indicating the frequency of the Cy5+GFP+ cell population. Right, percentage of Cy5+ cancer cells at various time points after completion of treatment. T1 / 2, half-life. [Figure 15C] (C) Representative confocal images showing the accumulation of Cy5 (red)-labeled siRNA in the cytoplasm of breast tumor cells after intravenous LNP-ASPM-v1 siRNA treatment. The cytoplasm is shown by staining F-actin with phalloidin (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 5 μm. [Figure 15D] (D) Representative immunoblot showing the specific knockdown effect of LNP-siASPM-v1 systemic therapy on the protein abundance levels of ASPM isoform 1 (ASPM-i1) in treated tumors. Right: Relative protein abundance levels of ASPM-i1 in tumor-bearing mice treated with LNP-nontargeting control siRNA (NT siRNA) or LNP-siASPM-v1. Data are mean ± SEM (n = 3 in each group). *P < 0.05. [Figure 16](A)–(C) show several panels related to the anti-metastatic efficacy of ASPM variant 1 (ASPM-v1)-targeting siRNA therapy in an orthotopic mouse model of triple-negative breast cancer (TNBC). (A) shows TNBC MDA-MB-436 cells stably expressing firefly luciferase (FF-Luc) orthotopically injected into the mammary fat pad of immunodeficient NOD / SCID mice. Two weeks after cell inoculation, when tumors became detectable by BLI, tumor-bearing mice were injected intravenously (IV, 2 mg / kg or 4 mg / kg every 3 days, for a total of 6 injections) with LNP-siASPM-v1 or LNP-nontargeting control siRNA (NT siRNA). (B) shows representative BLI images of primary or metastatic tumors (lung fields) at the indicated time points after the initiation of treatment in (A). (C) shows the tumor volume in (B), quantified as BLI-normalized photon counts, as a function of time. Data are presented as mean ± SEM (n = 5 mice per group). **P ≤ 0.01, ***P ≤ 0.001 compared with LNP-NT siRNA. [Figure 17] (A)–(C) show several panels related to the anti-metastatic effect of ASPM variant 1 (ASPM-v1)-targeting siRNA systemic therapy in distant metastases of triple-negative breast cancer (TNBC). (A) Firefly luciferase (FF-Luc)-expressing MDA-MB-436 cells were injected via the tail vein into NOD / SCID mice. 24 h after cell inoculation, mice were intravenously injected with LNP-siASPM-v1 (100 μg [approximately 4 mg / kg] per mouse every 3 days, a total of 6 injections) or LNP-nontargeting control siRNA (NT siRNA), and the distribution of metastatic tumors was monitored by BLI. (B) shows representative BLI images of metastatic tumors at the indicated time points after cell inoculation. (C) shows the BLI signal in (B), expressed as normalized photon counts, as a function of time. Data are shown as mean ± SEM (n = 5 mice per group). **P ≤ 0.01 compared to LNP-NT siRNA. [Figure 18](A) and (B) show several panels related to the antitumor efficacy of intratumoral ASPM variant 1 (ASPM-v1)-targeted siRNA therapy in a mouse xenograft model of hepatocellular carcinoma (HCC). (A) HuH-1 cells stably expressing GFP and firefly luciferase (FF-Luc) were subcutaneously injected into the flanks of immunodeficient NOD / SCID mice. Tumor-bearing mice were administered LNP-siASPM-v1 or LNP-nontargeting control siRNA (NT siRNA, 0.8 mg / kg or 2 mg / kg oligonucleotide per intratumoral injection every 3 days, for a total of 3 injections) with or without concomitant intraperitoneal (IP) injections of sorafenib (15 mg / kg / day for 10 consecutive days) or vehicle. Representative bioluminescence (BLI) images of tumors at the indicated time points after cell inoculation are shown. (B) shows tumor volume, quantified as BLI-normalized photon counts, as a function of time in the tumor-bearing mice described in (A). Data are presented as mean ± SEM (n = 8 mice per group). *P < 0.05 compared to LNP-NT siRNA. Data are presented as mean ± SEM (n = 3 mice per group). [Figure 19] (A) and (B) show several panels related to the antitumor efficacy of ultrasound-guided intratumoral ASPM variant 1 (ASPM-v1)-targeted siRNA therapy in an orthotopic mouse xenograft model of hepatocellular carcinoma (HCC). (A) HuH-1 cells were injected into the left lobe of the liver of NOD / SCID mice. Two weeks after cell inoculation, tumor-bearing mice received intratumoral injections of LNP-siASPM-v1 or LNP-nontargeting control siRNA (NT siRNA, 0.8 mg / kg every 3 days, a total of 3 injections) under ultrasound guidance. (B) shows representative photographs of tumors in (A) at the end of the study. Right: Tumor volume plotted over time. Data are presented as mean ± SEM (n = 5 mice per group). **P ≤ 0.01 compared to LNP-NT siRNA. [Figure 20A](A)–(E) show several panels related to the antitumor efficacy of systemic ASPM variant 1 (ASPM-v1)-targeting siRNA in an orthotopic mouse model of hepatocellular carcinoma (HCC). (A) shows GFP- and FF-Luc-expressing HuH-1 cells injected into the left lobe of the liver of immunodeficient NOD / SCID mice. Two weeks after cell inoculation, tumor-bearing mice were then repeatedly intravenously injected with LNP-siASPM-v1 or LNP-nontargeting control siRNA (NT siRNA, 4 mg / kg every 3 days, a total of 6 injections). [Figure 20B] (B) shows the accumulation of siRNA in liver tumors of treated mice. Three days after two consecutive injections of LNP-siASPM-v1 (100 μg per mouse per injection), the tumors established in (A) were excised for cell dissociation, and the cells were subjected to FACS analysis. Representative FACS plots of Cy5 in GFP-positive tumor cells isolated from LNP-formulated and Cy5-labeled siASPM-v1 or cells from untreated tumors are shown. [Figure 20C] (C) shows representative BLI of tumors at the indicated time points after initiation of treatment in (A). [Figure 20D] (D) Tumor volume in (C) quantified as BLI-normalized photon counts as a function of time. Data are shown as mean ± SEM (n = 7 mice per group). *P < 0.05 compared to LNP-NT siRNA. [Figure 20E] (E) Percent survival as a function of time in the mice described in (D). Arrows: siRNA injection. [Figure 21](A) and (B) show several panels related to the gene silencing efficacy of unmodified and chemically modified siASPM-v1.7636 and siASPM-v1.4822 in cancer cells. (A) shows the ASPM variant 1 (ASPM-v1) transcript levels in breast cancer MDA-MB-436 cells transduced with siASPM-v1.7636, siASPM-v1.4822, or a 1:1 mixture thereof, with or without the chemical modifications listed in Table 5, analyzed using qRT-PCR analysis (mean ± SEM, n = 3). (B) shows the ASPM-v1 transcript levels in hepatocellular carcinoma HuH-1 cells transduced with unmodified or chemically modified siASPM-v1.7636, siASPM-v1.4822, or a 1:1 mixture thereof, with or without the chemical modifications listed in (A) (mean ± SEM, n = 3). ***P<0.001 vs. chemically modified non-targeting (NT) siRNA (NT siRNA). [Figure 22](A) and (B) show several panels related to the effect of siASPM-v1 active pharmaceutical ingredient (API) on invadopodia formation in cancer cells. (A) shows the inhibitory effect of siASPM-v1 API on invadopodia formation in HCC HuH-1 cells. On the left, confocal images of cortactin+F-actin+ dots (yellow) representing cross sections of downward-protruding invadopodia are shown in HuH-1 cells transduced with siASPM-v1 API or chemically modified non-targeting control siRNA (NT siRNA, both at 100 nM for 72 hours) using Lipofectamine LTX reagent. On the right, the number of cortactin+F-actin+ (representing invadopodia) per cell. Scale bar, 10 μm. Data are mean ± SEM. ***P<0.001. (B) shows the inhibitory effect of siASPM-v1 API on invadopodia formation in TNBC MDA-MB-436 cells. Confocal images of cortactin+F-actin+ dots (yellow) representing cross sections of downward-protruding invadopodia in HuH-1 cells transduced with siASPM-v1 API or non-targeting control siRNA (NT siRNA, both at 100 nM for 72 hours) using Lipofectamine LTX reagent are shown on the left. Right, number of invadopodia per cell. Scale bar, 10 μm. Data are mean ± SEM. ***P<0.001. [Figure 23] Figure 1 shows that transduction of siASPM-v1 active pharmaceutical ingredient (API) inhibited the invasive ability of hepatocellular carcinoma HuH-1 or breast cancer MDA-MB-436 cells. Representative immunofluorescence images of invasive cells, in which cell nuclei were stained with CYTOX-green, are shown on the left. Scale bar = 100 μm. Right, number of invasive cells. Data are mean ± SEM. ***P<0.001. [Figure 24](A) and (B) show several panels related to the effects of siASPM-v1 active pharmaceutical ingredient (API) on Wnt, Hedgehog, and Notch pathway activity in cancer cells. HuH-1 hepatocarcinoma cells (A) or MDA-MB-436 breast cancer cells (B) carrying a triple luciferase reporter for measuring Wnt, Hedgehog, and Notch pathway activity were transduced with 100 nM siASPM-v1 API or non-targeting control siRNA (NT siRNA) for 48 hours using Lipofectamine LTX reagent. Cells were then stimulated with SHH (3 μg / ml x 24 hours), WNT3A (250 ng / ml x 16 hours), JAG1-Fc (5 μg / ml x 24 hours), or vehicle for 24 hours, after which reporter activity was measured. **P<0.01, ***P<0.001 compared to control. [Figure 25] (A) and (B) show several panels related to the gene silencing efficacy of unmodified and chemically modified siASPM-v1.4360 and siASPM-v1.4822 in cancer cells. (A) shows ASPM-v1 transcript levels in HuH-1 hepatocarcinoma cells transduced with 100 nM (48 h) of siASPM-v1.4360 and siASPM-v1.4822, or a 1:1 mixture of these (referred to as "siASPM-v1 active pharmaceutical ingredient version 2" or "siASPM-v1 API_V2"), or their chemically modified versions (mean ± SEM, n = 3). siNT, non-targeting siRNA. (B) shows ASPM-v1 transcript levels in HCT-116 colorectal cancer cells transduced with unmodified or chemically modified siRNA (as in (A)) (mean ± SEM, n = 3). ***P<0.001 compared with siNT. [Figure 26](A) and (B) contain several panels related to the inhibitory effect of siASPM-v1 active pharmaceutical ingredient version 2 (API_V2) on invadopodia formation in cancer cells. (A) shows the inhibitory effect of siASPMv1 API_V2 on invadopodia formation in HuH-1 hepatocarcinoma cells. Confocal images of TKS5+Col1-3 / 4C+ dots (yellow) representing cross sections of downward-protruding invadopodia in HuH-1 cells transduced with siASPM-v1 API_V2 or chemically modified non-targeting control siRNA (m-siNT, both at 100 nM for 72 hours) are shown on the left. Right: The number of TKS5+Col1-3 / 4C+ (representing functional invadopodia) per cell. Scale bar, 10 μm. Data are mean ± SEM. ***P<0.001. (B) The inhibitory effect of siASPM-v1 API_V2 on invadopodia formation in HCT-116 colorectal cancer cells. Confocal images of TKS5+Col1-3 / 4C+ dots (yellow) representing cross sections of downward-protruding invadopodia in HCT-116 cells transduced with siASPM-v1 API_V2 or m-siNT (both at 100 nM for 72 hours) are shown on the left. Right: Number of invadopodia per cell. Scale bar, 10 μm. Data are mean ± SEM. ***P<0.001. [Figure 27] (A) and (B) contain several panels related to the inhibitory effect of siASPM-v1 active pharmaceutical ingredient version 2 (API_V2) on the invasive ability of cancer cells. (A) shows representative immunofluorescence images of invasive HhH-1 hepatocellular carcinoma cells or HCT-116 colorectal cancer cells treated with chemically modified non-targeting siRNA (m-siNT) or siASPM-v1 API_V2, where the cell nuclei were stained with CYTOX-green. Scale bar = 100 μm. (B) shows the number of invasive cells. Data are means ± SEM. ***P<0.001. [Figure 28]The inhibitory effects of siASPM-v1 active pharmaceutical ingredient version 2 (API_V2) on the Wnt, Hedgehog (Hh), and Notch pathways, as well as TEAD activity, in cancer cells are shown. HuH-1 hepatocellular carcinoma cells (left) or HCT-116 colorectal cells (right) were lentivirally infected with reporter constructs for Wnt, Hh, and Notch signaling pathways or TEAD transcriptional activity. Cells were then transduced with 100 nM chemically modified non-targeting siRNA (m-siNT) or siASPM-v1 API_V2 using Lipofectamine LTX reagent for 48 hours. Subsequently, cells were stimulated with SHH (3 μg / ml x 24 hours), WNT3A (250 ng / ml x 16 hours), or JAG-Fc (5 μg / ml x 24 hours), respectively, for 24 hours. **P<0.01, ***P<0.001 compared to m-siNT. [Figure 29] (A) and (B) contain several panels related to the inhibitory effect of siASPM-v1 active pharmaceutical ingredient version 2 (API_V2) on the tumorsphere-forming ability of cancer cells. (A) shows representative phase-contrast images of tumorspheres formed by HuH-1 hepatocellular carcinoma or HCT-116 colorectal cancer cells transduced with chemically modified non-targeting siRNA (m-siNT) or siASPM-v1 API_V2. Scale bar, 100 mm. (B) shows a limiting dilution assay demonstrating the tumorsphere-forming efficacy (1 / n) of HuH-1 or HCT-116 cells transduced with m-siNT or siASPM-v1 API_V2. n = 8 independent experiments. Maximum likelihood estimates with 95% confidence intervals. ***P < 0.001, likelihood ratio test and chi-square test. DETAILED DESCRIPTION OF THE INVENTION

[0033] Unless otherwise stated, the following terms and phrases have the meanings indicated below.

[0034] The terms "ribonucleotide" and "ribonucleic acid" (RNA) refer to modified or unmodified nucleotides or polynucleotides containing at least one ribonucleotide unit. A ribonucleotide unit contains a nitrogenous base attached to the 1'-position of the ribosyl moiety via an N-glycosidic bond, a hydroxyl group attached to the 2'-position of the ribosyl moiety, and a moiety that either allows or prevents linkage to another nucleotide.

[0035] As used herein, the term "interfering RNA" or "RNAi" or "interfering RNA sequence" refers to a double-stranded RNA (i.e., duplex RNA) that targets (i.e., silences, reduces, or inhibits) the expression of a target gene (i.e., by mediating the degradation of mRNA complementary to the sequence of the interfering RNA) when the interfering RNA is present in the same cell as the target gene. Thus, interfering RNA refers to a double-stranded RNA formed by two complementary strands or a single self-complementary strand. Specifically, RNAi molecules refer to shRNAs, siRNAs, or dsRNAs disclosed herein. Small hairpin RNAs (shRNAs) are RNA sequences that form a tight hairpin turn that can be used to suppress gene expression by RNA interference. shRNAs can be delivered to target cells using DNA plasmids, viral vectors, or bacterial vectors. Double-stranded RNAs (dsRNAs) include a wide range of viruses. Small interfering RNAs (siRNAs) are a class of double-stranded RNA molecules that contain a duplex of two separate strands and a single strand that can form a hairpin structure containing the duplex region. siRNAs are short (generally about 18-30 base pairs in length). siRNAs can be used to silence gene expression through RNA interference. Furthermore, siRNAs can vary in length and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNAs have unpaired overhanging bases at the 5' or 3' end of the sense and / or antisense strand.

[0036] As used herein, the term "complementary nucleotide sequence" refers to a complementary RNA that is complementary to a region of an mRNA transcript of a target mutant gene (i.e., the "corresponding nucleotide sequence" of the target gene).

[0037] As used herein, an excipient is a non-active ingredient in a pharmaceutical composition. Examples of excipients include fillers or diluents, surfactants, binders, glidants, lubricants, disintegrants, etc.

[0038] As used herein, the term "substantial identity" refers to a sequence that hybridizes to a reference sequence under stringent conditions or a sequence that has a specified percent identity over a specified region of the reference sequence.

[0039] As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence but not to other sequences, typically in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to nucleic acid hybridization can be found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assay" (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (when the target sequence is present in excess, 50% of the probes are occupied at equilibrium at the Tm). Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide. For selective or specific hybridization, a positive signal is at least two times background hybridization, preferably 10 times background hybridization.

[0040] As used herein, the term "substantially identical" or "substantial identity" in the context of two or more nucleic acids refers to two or more sequences or subsequences that are the same or have a specified percentage of identical nucleotides (i.e., at least about 60%, preferably 65%, 70%, 75%, preferably 80%, 85%, 90%, or 95% identity over the specified region) when compared and aligned for maximum correspondence over a comparison window or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also applies equally to the complement of a sequence, where indicated by context. Preferably, the substantial identity exists over a region that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides in length.

[0041] The term "transfection" refers to the process by which an agent is introduced into a cell.

[0042] The phrase "inhibiting the expression of a target gene" refers to the ability of the siRNA molecule of the present invention to suppress, reduce, or inhibit the expression of a target gene. To examine the degree of gene expression suppression (gene silencing), a test sample (e.g., a biological sample from an organism of interest that expresses the target gene, or a sample of cells in culture that express the target gene) is contacted with an siRNA that suppresses, reduces, or inhibits the expression of the target gene. The expression of the target gene in the test sample is compared with the expression of the target gene in a control sample that has not been contacted with the siRNA. The control sample is assigned a value of 100%. Suppression, reduction, or inhibition of the expression of a target gene is achieved when the value of the test sample relative to the control sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 10%. Suitable assays include, for example, techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, testing of protein or mRNA levels using enzymatic function, and phenotypic assays known to those of skill in the art.

[0043] The terms "treatment" and "treating" include therapeutic treatment of patients who already suffer from the condition, especially in its manifested form. Therapeutic treatment may be symptomatic treatment to alleviate the symptoms of a particular indication, or causal treatment to improve or partially improve the condition of the indication or to stop or slow the progression of the disease. Thus, the compositions and methods of the present invention may be used, for example, as therapeutic treatment over a period of time and for chronic therapy.

[0044] The terms "prophylactically treating," "preventatively treating," and "preventing" are used interchangeably and include treating patients who are at risk of developing the aforementioned conditions, thus reducing said risk.

[0045] ASPM has been identified as a key regulator of the Wnt signaling pathway. ASPM expression was found to be essential for cellular responsiveness to canonical Wnt ligands, such as Wnt-3a, in pancreatic and prostate cancer cells. Mechanistic studies revealed that ASPM interacts with upstream activators of β-catenin, including disheveled (Dvl)-2 or Dvl-3, as well as axin and protease-activated receptor-1 (PAR-1), and inhibits proteasome-dependent degradation of Dvl protein, thereby increasing the protein abundance of β-catenin and amplifying canonical Wnt signaling, which is crucial for its oncogenic effects. ASPM exerts its oncogenic and Wnt-activating effects primarily through protein stabilization of Dvl, along with the role of Dvl in both canonical and noncanonical Wnt signaling, suggesting that ASPM may also function as an activator of noncanonical Wnt signaling in adenocarcinoma cells or CSCs. ASPM and its binding partner DVL were found to be significantly upregulated in CSCs of various types of cancer cells, including pancreatic cancer, breast cancer, non-small cell lung cancer, as well as prostate cancer and hepatocellular carcinoma (HCC).

[0046] Several putative splicing variants of the ASPM transcript have been predicted to exist in normal and malignant human tissues, encoding protein isoforms consisting of 3477 amino acids (isoform 1), 1892 amino acids (isoform 2), 1389 amino acids (isoform 3), and 1062 amino acids (isoform 4), respectively (Kouprina, N., Pavlicek, A., Collins, N.K., Nakano, M., Noskov, V.N., Ohzeki, J., Mochida, G.H., Risinger, J.I., Goldsmith, P., Gunsior, M. et al. (2005). The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein. Hum Mol Genet 14, 2155-2165). The specific upregulation of ASPM isoform 1, together with its prognostic significance, makes this isoform an ideal and potentially safe therapeutic target in cancer.

[0047] In one aspect, the disclosure provides a polynucleotide comprising a nucleotide sequence complementary to the mRNA of the ASPM gene having the nucleotide sequence set forth in SEQ ID NO: 1; b) a nucleotide sequence comprising a contiguous segment having at least 70%, at least 80%, or at least 90% sequence identity to the polynucleotide described in a).

[0048] The polynucleotide of the present disclosure may be an RNAi molecule, such as an shRNA, siRNA, miRNA, dsRNA, or antisense oligonucleotide (ASO), or any derivative thereof, that is complementary to a coding or non-coding region of the mRNA of the ASPM gene (SEQ ID NO: 1), thereby inducing its specific degradation or reducing its amount.

[0049] The polynucleotides of the present disclosure can also be complementary to the mRNA encoded by exon 18 of the human ASPM gene as set forth in SEQ ID NO: 3, such that the polynucleotide induces degradation or reduces the amount of only ASPM transcript variant 1, but does not induce degradation or reduce the amount of other transcript variants.

[0050] In particular, polynucleotides of the present disclosure are siRNAs of about 15-60, 15-50, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30 or 15-25 (duplex) nucleotides in length, and preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each of the complementary sequences of a double-stranded siRNA is about 15-60, 15-50, 15-50, or 15-40 (duplex) nucleotides in length). The siRNA duplex may be about 15-60, 15-50, 15-50, 15-40, 15-30, or 15-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the siRNA duplex may be about 15-60, 15-50, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 20-24, 21-22, or 21-23 base pairs in length. The siRNA duplex may include a 3' overhang of about 1 to about 4 nucleotides, preferably about 2 to about 3 nucleotides, and a 5' phosphate terminus. Examples of siRNA include, but are not limited to, double-stranded polynucleotide molecules constructed from two separate oligonucleotides, one strand being a sense strand and the other being a complementary antisense strand; double-stranded polynucleotide molecules constructed from a single oligonucleotide, the sense and antisense regions being linked by a nucleic acid-based or non-nucleic acid-based linker; double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions; and circular single-stranded polynucleotide molecules having two or more loop structures and a stem with self-complementary sense and antisense regions, wherein the circular polynucleotide can be processed in vivo or in vitro to produce an active double-stranded siRNA molecule.

[0051] In one embodiment of the present disclosure, the siRNA may be double-stranded and may include at least one blunt end. For example, the siRNA may be an siRNA with blunt ends at both ends; an siRNA with one blunt end and a 2-nucleotide 5' overhang at the other end; an siRNA with one blunt end and a 2-nucleotide 3' overhang at the other end; and / or a combination thereof. Alternatively, in this embodiment, the siRNA introduced into a cell may be double-stranded and may include a 2-nucleotide 5' overhang at each end. In certain embodiments, the overhang may comprise from about 1 nucleotide to about 5 nucleotides. In another embodiment, the siRNA may be double-stranded and may include at least two overhangs. The overhang may comprise from about 1 nucleotide to about 5 nucleotides. In certain embodiments, each of the at least two overhangs comprises 2 nucleotides. In this embodiment of the invention, the siRNA may be an siRNA comprising a 2-nucleotide overhang at both 3' ends; an siRNA comprising a 2-nucleotide 3' overhang at one end and a 2-nucleotide 5' overhang at the other end; and / or a combination thereof.

[0052] siRNA can be modified to contain synthetic, naturally occurring, and non-naturally occurring backbone residues or linkages to form analogs that have the same binding properties as reference nucleic acids and are metabolized in the same way as reference nucleotides.SiRNA can be modified according to a process known in general knowledge, and modification includes the replacement or addition of one or more atoms or groups in one or more nucleotide bases.Some examples of the types of modification that can be used to construct modified nucleotides in base moieties include, but are not limited to, alkylation, halogenation, thiolation, amination, amidation, or acetylation, respectively or in combination.Certain examples include, for example, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 5-methylcytidine, 5-methyluridine, as well as other nucleotides with modifications at the 5-position, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deazaadenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, Included are 5-methyl-2-thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Further illustrative examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidites, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences of the particular nucleic acid sequence.Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more (or all) selected codons is substituted with mixed-base and / or deoxyinosine residues.

[0053] Chemical modification of siRNA involves attaching a conjugate to the siRNA molecule. The conjugate can be attached to the 5' and / or 3' end of the sense and / or antisense strand of the siRNA via a covalent bond, such as a biodegradable linker. The conjugate can also be attached to the siRNA via, for example, a carbamate group or other linking group. In certain cases, the conjugate is a molecule that facilitates the delivery of the siRNA into cells. Examples of conjugate molecules suitable for attachment to siRNA include, but are not limited to, steroids, such as cholesterol, glycols, such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs, and derivatives thereof), sugars (e.g., galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands of cell receptors that can mediate cellular uptake, and combinations thereof.

[0054] siRNA can be chemically synthesized or can be encoded by a plasmid (e.g., transcribed as a sequence that automatically folds into a double strand with a hairpin loop). siRNA can also be produced by cleaving longer dsRNA (e.g., dsRNA longer than about 25 nucleotides) with E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNA.

[0055] Substantial identity refers to a sequence that hybridizes to a reference sequence under stringent conditions, or a sequence that has a specified percent identity over a specified region of the reference sequence.

[0056] For sequence comparison, typically one sequence serves as a reference sequence to be compared with a test sequence.When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.Methods for aligning sequences for comparison are well known in the art.The optimal sequence alignment for comparison can be performed.

[0057] Preferred examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms. BLAST and BLAST 2.0 are used with the parameters described herein to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0058] Once potential siRNA sequence is identified, the sequence can be analyzed using various criteria known in the art.Those skilled in the art will understand that the sequence that has one or more of the above-mentioned characteristics can be selected as potential siRNA sequence for further analysis and testing.The siRNA sequence that is complementary to siRNA target site can also be designed.

[0059] The polynucleotides of the present disclosure are operably linked to regulatory sequences or gene promoters that direct expression of the polynucleotide to particular tissues and / or cell types. Such regulatory sequences are well known in the art and can be constructed by any of a variety of means known to those of ordinary skill in the art.

[0060] Methods suitable for use in the present disclosure to determine ASPM mRNA expression include assays selected from the group consisting of Northern blotting, reverse transcription polymerase chain reaction (PCR), RNase protection assay, cDNA or oligonucleotide microarray analysis, nucleotide sequencing, or probe-based digital mRNA profiling techniques, such as the NanoString nCounter gene expression system (Geiss, GK, Bumgarner, RE, Birditt, B., Dahl, T., Dowidar, N., Dunaway, DL, Fell, HP, Ferree, S., George, RD, Grogan, T. et al. (2008). Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 26, 317-325).

[0061] Methods suitable for use in the present disclosure to determine the expression of ASPM protein include assays selected from the group consisting of Western blotting, ELISA, immunoprecipitation, glutathione-S-transferase fusion protein pull-down, fluorescence anisotropy, fluorescence polarization, fluorescence resonance energy transfer, analytical ultracentrifugation, surface plasmon resonance, and isothermal titration calorimetry.

[0062] In one embodiment, the present disclosure provides a pool or kit of at least one siRNA, preferably in the form of a kit or therapeutic reagent, wherein one strand of each siRNA, i.e., the sense strand, contains a sequence substantially similar to a sequence in the target mRNA. The opposite strand, i.e., the antisense strand, preferably contains a sequence substantially complementary to the sequence of the target mRNA. More preferably, one strand of each siRNA contains a sequence identical to a sequence contained in the target mRNA. Most preferably, each siRNA is 15-25, 15-20, or 15-18 base pairs in length, and one strand of each siRNA is 100% complementary to a portion of the target mRNA. By using a pool or kit to increase the number of siRNAs targeting a specific target, it is possible to both increase the likelihood of including at least one siRNA with sufficient functionality and benefit from additive or synergistic effects. Furthermore, even if two or more siRNAs targeting a single gene do not have sufficient functionality alone, their combination may be sufficient to promote degradation of the target messenger RNA and successfully inhibit translation.

[0063] The siRNA duplexes in the above-mentioned siRNA pool or kit can correspond to overlapping sequences in specific mRNA or non-overlapping sequences of mRNA.However, preferably, they correspond to non-overlapping sequences.Furthermore, each siRNA can be selected randomly, or one or more siRNAs can be selected according to the criteria discussed above to maximize the efficacy of siRNA.

[0064] In some embodiments of the present disclosure, anti-tumor reagents or compositions can be delivered to cells, malignant tumors, or individuals by direct transfection or transfection and expression via an expression vector. Suitable expression vectors include mammalian expression vectors and viral vectors into which a polynucleotide encoding the sensitizing agent and an appropriate regulatory sequence, including a promoter, that causes the expression of the sensitizing agent in cells or malignant tumors is cloned. Suitable promoters can be constitutive or developmentally specific promoters. Transfection delivery can be achieved using liposomal transfection reagents known in the art (e.g., Xtreme Transfection Reagent, Roche, Alameda, CA; Lipofectamine formulation, Invitrogen, Carlsbad, CA). Cationic liposome-mediated delivery and direct delivery are efficient. Another possible delivery mode is targeting using an antibody against a cell surface marker of the target cell.

[0065] For transfection, the composition comprising one or more nucleic acid molecules (in vector or outside vector) can comprise a delivery vehicle, carrier and diluent and their salts, including liposomes for administration to a subject, and / or can be present in a pharmaceutically acceptable formulation.The delivery of siRNA molecules is also described in some US patent publications, including, for example, US Patent Application Publication No. 2006 / 0019912, US Patent Application Publication No. 2006 / 0014289, US Patent Application Publication No. 2005 / 0239687, US Patent Application Publication No. 2005 / 0222064 and US Patent Application Publication No. 2004 / 0204377, the disclosures of which are hereby incorporated by reference. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not limited to, encapsulation in liposomes, iontophoresis, electroporation, or incorporation into other vehicles, including biodegradable polymers, hydrogels, cyclodextrins (see, e.g., Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT Publication Nos. WO 03 / 47518 and WO 03 / 46185), poly(lactic-co-glycolic acid) (PLGA) and PLCA microspheres (see, e.g., U.S. Pat. No. 6,447,796 and U.S. Patent Application Publication No. 2002 / 130430), biodegradable nanocapsules, and bioadhesive microspheres, or protein vectors (O'Hare and Normand, International PCT Publication No. WO 00 / 53722). In another embodiment, the nucleic acid molecules of the present disclosure can also be formulated or complexed with polyethyleneimine and its derivatives, such as polyethyleneimine-polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.

[0066] Examples of liposome transfection reagents that can be used with the present invention include, for example, CellFectin; a 1:1.5 (M / M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoylphosphatidylethanolamine (DOPE) (GIBCO BRL); Cytofectin GSV (Glen Research), a 2:1 (M / M) liposome formulation of a cationic lipid and DOPE; DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammonium methylsulfate) (Boehringer Manheim); Lipofectamine (GIBCO BRL), a 3:1 (M / M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE; and (5) siPORT (Ambion); HiPerfect (Qiagen); X-treme GENE (Roche); RNAi Carrier (Epoch Biolabs) and TransPass (New England Biolabs).

[0067] Many types of human solid cancers, such as breast cancer, non-small cell lung carcinoma (NSCLC), PDAC, the sclerotic subtype of gastric adenocarcinoma, and the "stem cell-like / serrated / mesenchymal (SSM)" molecular subtype of colorectal cancer (CRC), are characterized by a prominent stromal reaction, termed the "desmoplastic response," which poses a major obstacle to the efficient delivery of cancer therapeutics to tumors (Isella, C., Terrasi, A., Bellomo, SE, Petti, C., Galatola, G., Muratore, A., Mellanno, A., Senetta, R., Cassenti, A., Sonetto, C. et al. (2015). Stromal contribution to the colorectal cancer transcriptome. Nat Genet 47, 312-319). Recently, two nanoparticle-formulated chemotherapy drugs, including albumin-bound paclitaxel (nab-paclitaxel) and liposome-encapsulated irinotecan, have been shown to prolong survival in patients with advanced PDAC. Both reagents were able to significantly increase the levels of chemotherapeutic agents in treated tumors (Wang-Gillam, A., Li, C.P., Bodoky, G., Dean, A., Shan, Y.S., Jameson, G., Macarulla, T., Lee, K.H., Cunningham, D., Blanc, J.F. et al. (2016). Nanoliposomal irinotecan with fluorouracil and folinic acid in metastatic pancreatic cancer after previous gemcitabine-based therapy (NAPOLI-1): a global, randomized, open-label, phase 3 trial. Lancet 387, 545-557), suggesting that nanoparticle formulations may be a clinically proven approach to improving the efficacy of desmoplastic cancer treatment.In liver disease, parenteral administration of transthyretin-specific lipid nanoparticle (LNP)-formulated siRNA (patisiran, Alnylam Pharmaceuticals, MA, USA) has been shown to reduce transthyretin produced by the liver of patients with hereditary transthyretin-mediated amyloidosis by up to 86.8%, thus becoming the first clinically approved RNAi drug (Adams, D., Gonzalez-Duarte, A., O'Riordan, WD, Yang, CC, Ueda, M., Kristen, AV, Tournev, I., Schmidt, HH, Coelho, T., Berk, JL et al. (2018). Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. N Engl J Med 379, 11-21). Furthermore, nanoparticle-delivered siRNA therapies, such as cyclodextrin polymer-based nanoparticles carrying siRNA targeting ribonucleotide reductase M2 (RRM2) and lipid nanoparticles carrying siRNA targeting VEGF-A and kinesin spindle protein (KSP), have shown promising pharmacodynamics and tolerability and antitumor efficacy in some treated patients in phase 1 clinical trials. Furthermore, systemic delivery of siRNA targeting tumor driver genes, such as BCR-ABL, also showed significant therapeutic efficacy in a mouse orthotopic model of hepatocellular carcinoma (HCC) (Tabernero, J., Shapiro, GI, LoRusso, PM, Cervantes, A., Schwartz, GK, Weiss, GJ, Paz-Ares, L., Cho, DC, Infante, JR, Alsina, M. et al. (2013). First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discov 3, 406-417).In contrast to microRNAs, siRNAs silence the expression of only one mRNA target; therefore, their genetic and biological effects are highly specific, with fewer off-target effects, as exemplified by the recent approval of liposomal siRNA targeting transthyretin as a first-in-class drug in patients with hereditary amyloidosis (Adams, D., Gonzalez-Duarte, A., O'Riordan, WD, Yang, CC, Ueda, M., Kristen, AV, Tournev, I., Schmidt, HH, Coelho, T., Berk, JL et al. (2018). Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. N Engl J Med 379, 11-21). In complex polygenic diseases, such as human malignancies, the implementation of siRNA-based therapies requires the identification of "driver" genes that function as key regulators of disease pathology.

[0068] Delivery of therapeutic genes to tumor cells or malignant tissues can be achieved by using nonviral vehicles, such as lipid-based and polymeric materials. For example, tumor-targeting immunoliposome nanocomplexes, termed scLs, in which a therapeutic molecular payload is encapsulated in cationic liposomes bearing anti-transferrin receptor (TfR) single-chain antibody fragments on their surface, have been designed to target tumor cells via TfR, which is highly expressed on the surface of tumor cells. A series of studies have demonstrated that scL nanocomplexes can specifically deliver various payloads, including plasmid DNA, siRNA, and small molecules, to both primary and metastatic tumor cells, as well as cancer stem cells, both in vitro and in vivo. Systemic administration of p53 plasmid DNA encapsulated in scL immunoliposomal nanoparticles, designated TfRscFv-Lip p53 or "SGT-53," has been shown to induce tumor-specific expression of exogenous p53, thereby improving the efficacy of chemotherapy and radiotherapy in various preclinical models of human tumors, including breast cancer, malignant glioma, head and neck cancer, and pancreatic cancer. Importantly, the clinical applicability of SGT-53 has been demonstrated in a recent phase I clinical trial, which successfully demonstrated that the tested therapeutic doses were well tolerated by study participants. Significantly, PCR analysis of tumor tissues revealed the unequivocal presence of the exogenous p53 transgene, providing strong support for scL nanoparticles as an effective systemic delivery vehicle for the effective delivery of therapeutic genes to human tumors.

[0069] Lipid nanoparticles (LNPs) suitable for in vivo delivery of oligonucleotides, such as siRNA, to liver or tumor tissue have been produced by AlCana ​​Technologies (Vancouver, BC) using a rational design approach (Jayaraman, M., Ansell, SM, Mui, BL, Tam, YK, Chen, J., Du, X., Butler, D., Eltepu, L., Matsuda, S., Narayanannair, JK et al. (2012). Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew Chem Int Ed Engl 51, 8529-8533). The LNP formulation consists of four lipid components, including DLin-MC3-DMA, an ionizable cationic amino lipid that complexes with siRNA, the amphiphilic phospholipid distearoylphosphatidylcholine (DSPC), cholesterol, and the coating lipid poly(ethylene glycol) lipid 1,2-dimyristoyl-rac-glycerol-methoxy(poly(ethylene glycol)) (DMG-PEG), mixed in a molar ratio of 50:10:38.5:1.5.The particle size of DLin-MC3-DMA LNPs ranges from 70 to 90 nm (Jayaraman, M., Ansell, SM, Mui, BL, Tam, YK, Chen, J., Du, X., Butler, D., Eltepu, L., Matsuda, S., Narayanannair, JK et al. (2012). Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew Chem Int Ed Engl 51, 8529-8533), which allows leakage of LNPs into tumor tissue through leaky endothelial fenestration (with estimated pore diameters of 380-780 nm), a phenomenon associated with prolonged circulation time and known as the "enhanced vascular permeability and retention (EPR)" effect (Agarwal, R. and Roy, K. (2013). Intracellular delivery of polymeric nanocarriers: a matter of size, shape, charge, elasticity and surface composition. Ther Deliv 4, 705-723).

[0070] DLin-MC3-DMA-based LNP formation was developed by Alnylam Pharmaceuticals and used in the first FDA-approved siRNA drug, patisiran (Onpattro). It has also been used for the systemic delivery of siRNA targeting tumor driver genes, such as BCR-ABL, VEGF-A, and kinesin spindle protein (KSP), which showed significant therapeutic efficacy in mouse models of chronic myeloid leukemia (CML) and orthotopic mouse models of hepatocellular carcinoma (HCC). Systemic delivery of encapsulated siRNA using DLin-MC3-DMA-based LNPs has been shown to be safe and generally well tolerated in a phase I clinical trial, with the exception of some infusion-related reactions and transient proinflammatory cytokine induction (Tabernero, J., Shapiro, GI, LoRusso, PM, Cervantes, A., Schwartz, GK, Weiss, GJ, Paz-Ares, L., Cho, DC, Infante, JR, Alsina, M. et al. (2013). First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discov 3, 406-417).

[0071] Recently, a next-generation LNP-based delivery vehicle has been described containing the highly biodegradable ionizable cationic lipid CL4H6, cholesterol, and 1,2-dimyristoyl-sn-glycero, methoxyethylene glycol 2000 ether (PEG-DMG2000) mixed in a 60:40:1 molar ratio (Sato, Y., Hashiba, K., Sasaki, K., Maeki, M., Tokeshi, M., and Harashima, H. (2019). Understanding structure-activity relationships of pH-sensitive cationic lipids facilitates the rational identification of promising lipid nanoparticles for delivering siRNAs in vivo. J Control Release 295, 140-152).CL4H6, a pH-sensitive cationic lipid systematically derivatized based on structure-activity relationship studies of its hydrophilic head group and hydrophobic tail, has higher in vivo gene silencing activity than the benchmark lipid DLin-MC3-DMA in a murine factor VII model (50% effective dose (ED50): 0.0025 mg / kg vs. 0.005 mg / kg) (Jayaraman, M., Ansell, SM, Mui, BL, Tam, YK, Chen, J., Du, X., Butler, D., Eltepu, L., Matsuda, S., Narayanannair, JK et al. (2012). Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew Chem Int Ed Engl 51, 8529-8533; Sato, Y., Hashiba, K., Sasaki, K., Maeki, M., Tokeshi, M., and Harashima, H. (2019). Understanding structure-activity relationships of pH-sensitive cationic lipids facilitates the rational identification of promising lipid nanoparticles for delivering siRNAs in vivo. J Control Release 295, 140-152).DLin-MC3-DMA, developed by Alnylam Pharmaceuticals and used in patisiran (Onpattro), the first FDA-approved siRNA drug, can cause liver toxicity and significant weight loss when administered to mice at high doses (>3 mg / kg) (Jayaraman, M., Ansell, SM, Mui, BL, Tam, YK, Chen, J., Du, X., Butler, D., Eltepu, L., Matsuda, S., Narayanannair, JK et al. (2012). Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew Chem Int Ed Engl 51, 8529-8533). In contrast, CL4H6 contains a biodegradable ester bond in its hydrophobic tail, and its levels rapidly decline in the liver or spleen after 24 hours. Due to the highly biodegradable nature of CL4H6, the potential for inducing liver or tissue toxicity is substantially reduced, especially in the repeated dosing schedules required for the treatment of cancer.

[0072] siRNA or oligonucleotides can be encapsulated in LNPs by mixing siRNA or oligonucleotides dissolved in 10 mM citrate buffer (pH 3.0) with LNPs in ethanol using a microfluidic device, such as the NanoAssemblr™ system (Precision Nanosystems, Vancouver, BC, Canada), at a flow rate of 0.5 ml / min and a flow ratio of 1:3 (lipid: siRNA = 0.125 ml / min: 0.375 ml / min). A syringe pump (Harvard Apparatus, MA, USA) can be used to control the flow rate. The resulting LNP / siRNA mixture solution is then dialyzed against phosphate-buffered saline using a Spectra / Por 4 dialysis membrane (Spectrum Laboratories, Rancho Dominguez, CA, USA). The LNP-encapsulated siRNA solution was then concentrated by ultrafiltration using an Amicon Ultra-15 unit (MWCO 50 kDa, Merch Millipore, Burlington, MA, USA). The size (number-weighted mean diameter) and zeta potential of the LNPs were measured using a Zetasizer Nano ZS ZEN3600 instrument (Malvern Instruments, Worcestershire, UK). The encapsulation efficiency and total concentration of siRNA were measured using Quanti-iT™ RiboGreen RNA reagent and kit (Invitrogen, Waltham, MA, USA).

[0073] Tumor targeting of lipid-based nanoparticles can also be achieved by using the CXCR-4 antagonist AMD-3100 as a targeting moiety. CXCR-4 is upregulated in hepatocellular carcinoma (HCC) after treatment with the targeting agent sorafenib. AMD-modified nanoparticles can efficiently deliver VEGF siRNA to HCC, synergizing with sorafenib to induce antiangiogenesis and suppress tumor growth and metastasis (Liu, JY, Chiang, T., Liu, CH, Chern, GG, Lin Ts, T., Gao, DY, and Chen, Y. (2015). Delivery of siRNA Using CXCR4-Targeted Nanoparticles Modulates Tumor Microenvironment and Achieves a Potent Antitumor Response in Liver Cancer. Mol Ther 23, 1772-1782). Interestingly, even in the absence of a specific tumor-targeting moiety, amphiphilic liposomes or neutral lipid emulsions, such as MaxSuppressor™ (BIOO Scientific, TX, USA) or SMARTICLES (Marina Biotech, WA, USA), have been found to be able to efficiently deliver RNAi agents to tumors. Most lipid-based delivery systems contain cationic lipids and have several drawbacks that may be due to their charge. In contrast, these neutral or amphoteric liposomes are neutral or anionic at neutral or higher pH and cationic at low pH. In biological fluids with a pH of 7 to 7.5, nanoparticles take on a slightly anionic character that may prevent unwanted interactions with the negatively charged cell membranes in endothelium and other tissues. For the same reason, these liposomes may be less toxic than those containing cationic lipids. Because the pH tends to be lower in tumor regions, the particles become cationic in these regions and may adhere to tumor cells.SMARTICLES formulations have been used to deliver tumor-suppressing miRNAs, such as miR-34a and let-7, to tumors in animal models of HCC, prostate cancer, and lung cancer, resulting in significant tumor regression and prolonged survival (Cortez, MA, Valdecanas, D., Niknam, S., Peltier, HJ, Diao, L., Giri, U., Komaki, R., Calin, GA, Gomez, DR, Chang, JY et al. (2015). In Vivo Delivery of miR-34a Sensitizes Lung Tumors to Radiation Through RAD51 Regulation. Mol Ther Nucleic Acids 4, e270).

[0074] Alternatively, combinatorial chemical synthesis was used to identify a low-molecular-weight polyamine and lipid compound, designated 7C1, that delivers siRNA to the lung endothelium without significant transfection of immune cells or hepatocytes. In a KrasG12Dp53flox / flox transgenic lung adenocarcinoma model, treatment of tumor-bearing mice with a microRNA34 mimetic and siRNA targeting the oncogene Kras using 7C1 nanoparticles reduced K-ras gene expression and MAPK signaling, inhibited tumor growth, and synergized with chemotherapy, prolonging survival.

[0075] The present disclosure also relates to pharmaceutical compositions comprising an anti-tumor reagent as defined herein, optionally in combination with a pharmaceutical carrier, diluent, and / or adjuvant. Any suitable pharmaceutically acceptable diluent, adjuvant, carrier, or excipient can be used in the compositions (see, for example, Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (ed.), Mack Publishing Company, April 1997). A preferred pharmaceutical form may be a combination with sterile saline, dextrose solution, or buffer solution, or other pharmaceutically acceptable sterile fluid. Alternatively, a solid carrier, such as microcarrier beads, may be used. Such compositions comprise an effective amount of an anti-tumor reagent sufficient to produce the desired therapeutic or prophylactic effect, and a pharmaceutically acceptable carrier or excipient. An "effective amount" includes a therapeutically effective amount or a prophylactically effective amount.

[0076] For gene therapy vectors, the dosage administered can depend to a large extent on the condition and size of the subject being treated, as well as the therapeutic formulation, frequency of treatment, and route of administration. Regimens for continuous therapy, including dose, formulation, and frequency, can depend on the initial response and clinical judgment. Parenteral routes of injection into blood vessels or the interstitial space of tissues may be preferred, although certain administrations may require other parenteral routes, such as inhalation of an aerosol formulation. In some protocols, a formulation containing the gene and gene delivery system in an aqueous carrier is injected into the tissue in an appropriate amount.

[0077] Sequence Listing SEQ ID NO: 1 sets forth the nucleotide sequence of the human ASPM transcript, transcript variant 1 (NCBI Reference Sequence: NM_018136.4). TIFF2025535734000002.tif175163TIFF2025535734000003.tif241163TIFF20255357340 00004.tif242163TIFF2025535734000005.tif241163TIFF2025535734000006.tif102162

[0078] SEQ ID NO: 2 sets forth the amino acid sequence of human ASPM gene isoform 1 (NCBI Reference Sequence: NP_060606.3). TIFF2025535734000007.tif105162TIFF2025535734000008.tif159162

[0079] SEQ ID NO: 3 sets forth the nucleotide sequence of the mRNA encoded by exon 18 of the human ASPM gene, corresponding to nucleotide 4323 to nucleotide 9077 of ASPM transcript variant 1 (NCBI Reference Sequence: NM_018136.4). TIFF2025535734000009.tif230162TIFF2025535734000010.tif208162

[0080] SEQ ID NO: 4 sets forth the nucleotide sequence of the sense strand of ASPM-v1 targeting siASPM-v1.4822, corresponding to nucleotide 4822 to nucleotide 4840 of ASPM transcript variant 1. GCGCACCAACUAUUUGCAG

[0081] SEQ ID NO:5 sets forth the nucleotide sequence of the antisense strand of ASPM-v1 targeting siASPM-v1.4822, which is complementary to the sense strand shown in SEQ ID NO:4. CUGCAAAUAGUUGGUGCGC

[0082] SEQ ID NO: 6 sets forth the nucleotide sequence of the sense strand of ASPM-v1 targeting siASPM-v1.7636, corresponding to nucleotide 7636 to nucleotide 7654 of ASPM transcript variant 1. CAGAAGACUGAUGGUAAAG

[0083] SEQ ID NO:7 sets forth the nucleotide sequence of the antisense strand of ASPM-v1 targeting siASPM-v1.7636, which is complementary to the sense strand shown in SEQ ID NO:6. CUUUACCAUCAGUCUUCUG

[0084] SEQ ID NO: 8 sets forth the nucleotide sequence of the sense strand of ASPM-v1 targeting siASPM-v1.4360, corresponding to nucleotide 4360 to nucleotide 4378 of ASPM transcript variant 1. CCUGCAAUCUAGGAUAAGA

[0085] SEQ ID NO:9 sets forth the nucleotide sequence of the antisense strand of ASPM-v1 targeting siASPM-v1.4360, which is complementary to the sense strand shown in SEQ ID NO:8. GGACGUUAGAUCCUAUUCU

[0086] [Example] The following examples are given for illustrative purposes only and are not intended to be limiting, unless otherwise specified. It should be understood by those skilled in the art that the techniques disclosed in the following examples are techniques discovered by the inventors to function well in the practice of the invention and are therefore believed to constitute preferred modes of its practice. It should be understood by those skilled in the art that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0087] [Example 1] Identification of ASPM as the top Wnt-related gene associated with cancer metastasis We focus on a list of 327 Wnt-related genes according to Gene Ontology terms. When ranked in descending order according to their prognostic significance (Cox regression P-value), the gene whose transcript levels best correlate with shorter recurrence-free survival (i.e., the highest probability of recurrence and / or metastasis) is ASPM (P<0.0001, Figure 1A). Consistently, in a large meta-analysis of patient cohorts, we found that ASPM was inversely correlated with distant metastasis-free survival (total n=2422, P<0.0001, Figure 1B).

[0088] Previous clinical correlation studies have raised the possibility that ASPM may play a functional role in breast cancer invasiveness and metastasis. Indeed, small hairpin RNA (shRNA)-mediated knockdown of ASPM expression in MDA-MB-436 and HCC-1954 breast cancer cells completely suppressed their ability to invade through recombinant basement membranes (Figure 2). To verify the functional importance of ASPM expression in vivo, human breast cancer MDA-MB-436 and MDA-MB-231 cells and pancreatic cancer AsPC-1 cells were lentivirally transduced with a GFP and firefly luciferase (FF-Luc) fusion vector (UBC-EGFP-T2A-Luc, System Biosciences), and GFP-positive cells were enriched by FACS. Control or ASPM-specific shRNA was transfected into MDA-MB-436-FF-Luc or MDA-MB-231-FF-Luc cells (10 cells) with lentivirus. 6 Cells (control knockdown or ASPM knockdown) were injected into immune-deficient NOD / SCID mice via the tail vein using a 27-gauge needle. The resulting lung metastatic tumors were visualized weekly by bioluminescence imaging (BLI) starting 3 weeks after cell inoculation, according to the manufacturer's recommendations (IVIS Imaging System, Caliper Life Sciences). As shown in Figure 3, control knockdown breast cancer cells developed metastatic lung tumors within 3 or 8 weeks after cell injection. In contrast, knockdown of ASPM expression almost completely suppressed distant metastasis. We further verified this finding in a pancreatic cancer metastasis model. Briefly, AsPC-1-FF-Luc cells were injected into the splenic medulla of NOD / SCID mice over a period of more than 1 minute, followed by splenectomy and ligation of the splenic vein. The development of liver and / or metastatic peritoneal tumors was monitored by BLI. Consistent with previous findings in breast cancer, control knockdown pancreatic cancer AsPC-1 cells rapidly spread to the liver and peritoneum 3 weeks after cell inoculation, whereas knockdown of ASPM expression substantially abolished the pro-metastatic ability of the cancer cells.

[0089] In line with the prevailing theory that metastatic colonization is initiated by rare tumor cells with unique properties (Lawson, DA, Bhakta, NR, Kessenbrock, K., Prummel, KD, Yu, Y., Takai, K., Zhou, A., Eyob, H., Balakrishnan, S., Wang, CY et al. (2015). Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells. Nature 526, 131-135), we sought to validate this finding by establishing patient-derived xenograft (PDX) models of breast cancer and analyzing ASPM expression in micrometastatic lesions. Briefly, we analyzed PDX breast tumors derived from two human TNBC tumors, NOD / Shi-scid / IL2Rγ. null Tumors (BR1282 and BR1474, CrownBio) were implanted into the mammary fat pad of (NOG) mice, and tumor growth was monitored by measuring the tumors weekly with a caliper. 3 When tumor size reached 100 μg / ml (at which point the development of pulmonary micrometastases had been confirmed in parallel experiments), lung tissue was harvested from tumor-bearing mice. The excised tissue was then digested, and cells were labeled with APC anti-CD298 (Lawson et al., 2015) (BioLegend 341706), a specific surface marker for human breast cancer cells. They were then fixed and analyzed by fluorescence-activated cell sorting (FACS) for subsequent labeling with PE anti-ASPM (Santa Cruz, sc-48883). Indeed, cancer cells isolated from the micrometastatic niche in the lungs of PDX-bearing mice expressed higher levels of ASPM compared with those in the parental tumor (Figure 4). Together, these in vitro and in vivo findings support the role of ASPM as an important and essential factor in cancer invasiveness and metastasis.

[0090] [Example 2] Specific upregulation of ASPM protein isoform 1 expression in malignant tumors Several splicing variants of the ASPM transcript are present in normal and malignant human tissues, encoding protein isoforms consisting of 3477 amino acids (isoform 1, ASPM-i1), 1892 amino acids (isoform 2, ASPM-i2), 1389 amino acids (isoform 2I), and 1062 amino acids (isoform 1V), respectively (Kouprina, N., Pavlicek, A., Collins, N.K., Nakano, M., Noskov, V.N., Ohzeki, J., Mochida, G.H., Risinger, J.I., Goldsmith, P., Gunsior, M. et al. (2005). The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein. Hum Mol Genet 14, 2155-2165). Importantly, the three shorter ASPM isoforms (isoforms 2–4) lack several functional domains of the full-length isoform 1 protein, such as the IQ (isoleucine and glutamine) motif and the calponin homology (CH) domain, raising the possibility that these ASPM isoforms may play distinct roles in normal and malignant cells. Notably, transcript variant 1 (ASPM-v1, NCBI RefSeq: NM_018136.4), encoding the full-length ASPM protein (ASPM isoform 1 or ASPM-i1, NCBI RefSeq: NP_060606.3), and variant 2 (NCBI RefSeq: NM_001206846), encoding a truncated protein lacking the IQ domain contained in exon 18 (ASPM-i1, NCBI RefSeq: NP_001193775.1), are the two major transcripts detected in pancreatic cancer cells (Hsu et al., 2019a).In several cancers, ASPM amplifies canonical Wnt signaling by positively regulating key upstream Wnt mediators, including disheveled (DVL) protein and β-catenin (Wang, WY, Hsu, CC, Wang, TY, Li, CR, Hou, YC, Chu, JM, Lee, CT, Liu, MS, Su, JJ, Jian, KY et al. (2013). A gene expression signature of epithelial tubulogenesis and a role for ASPM in pancreatic tumor progression. Gastroenterology 145, 1110-1120). Interestingly, in pancreatic cancer, using a rabbit polyclonal antibody generated using a peptide epitope located within a fragment encoded by exon 18 of the human ASPM gene (specific to ASPM-v1 and ASPM-i1), it was demonstrated that only ASPM-i1 associates with DVL2 and regulates its protein stability in cancer cells. In contrast, ASPM protein isoform 2 interacts with cyclin E, thereby regulating cell cycle progression in cancer cells. Therefore, ASPM-i1, but not ASPM isoform 2, contributes to Wnt activity and tumorigenicity in cancer cells, such as pancreatic cancer (Hsu, CC, Liao, WY, Chan, TS, Chen, WY, Lee, CT, Shan, YS, Huang, PJ, Hou, YC, Li, CR, and Tsai, KK (2019a). The differential distributions of ASPM isoforms and their roles in Wnt signaling, cell cycle progression, and pancreatic cancer prognosis. J Pathol 249, 498-508). These results suggest that different ASPM isoforms have distinct functions and pathogenic roles in cancer cells.

[0091] Previous studies have shown that ASPM-i1 is specifically expressed in the cytoplasm of pancreatic cancer cells, while ASPM-i2 is predominantly expressed in the cell nucleus (Hsu et al., 2019a). Importantly, immunohistochemistry (IHC) analysis of tissue microarrays (TMA) of human pancreatic or gastric cancer tissues revealed that ASPM-i1 expression was upregulated in tumor cells, whereas its staining was rarely positive in normal pancreatic duct epithelium (data not shown). To extend these observations to non-gastrointestinal (GI) tract malignancies, TMA analysis was performed on human breast cancer tissue and corresponding adjacent normal tissue (BR084b, US Biomax, Inc., Rockville, MD, USA). Tissue sections were deparaffinized, hydrated, and immersed in citrate buffer at pH 6.0 for epitope retrieval in a microwave oven. Endogenous peroxidase activity was quenched in 3% hydrogen peroxidase for 15 minutes, and then the slides were incubated with 10% normal horse serum to block nonspecific immunoreactivity. We generated rabbit polyclonal antibodies (1:1600) against immunogens specific for ASPM-i1 or ASPM-i2. Staining intensity was quantified at the single-cell level, with at least 300 tumor cells counted per tumor (three tissue sections per tumor; at least 100 tumor cells counted per section).

[0092] As shown in Figure 6A, a small subset of epithelial cells in normal human breast tissue (approximately 9.3%) exhibited weak (1+) ASPM-i1 staining intensity in the cytoplasm, whereas ASPM-i2 was expressed (≥1+) in either the cytoplasm (10.5%) or nucleus (9.3%) of a small proportion of breast epithelial cells. Consistent with previous findings in pancreatic cancer, ASPM-i1 expression was significantly upregulated in the cytoplasm of malignant cells in breast cancer tissue. In contrast, ASPM-i2 expression was primarily confined to the nucleus of breast cancer cells. Specifically, on average, approximately 21.1% of tumor cells exhibited weak (1+) ASPM-i1 staining, whereas within the same tumor, approximately 5.0% of tumor cells exhibited moderate (2+) staining and approximately 2.2% of tumor cells exhibited strong (3+) staining. In contrast, in the cytoplasm, an average of 9.4%, 0.3%, and 0.3% of tumor cells exhibited weak, moderate, and strong ASPM-i2 staining, respectively (Fig. 6B). To further examine the expression pattern of ASPM-i1 in breast cancer, we repeated the IHC staining in whole tumor tissue sections from three patients with breast cancer. Interestingly and importantly, we observed significant regional heterogeneity in the expression level of ASPM-i1 in breast cancer tissues, with the expression level being higher at the invasive front of the cancer than in other regions (Fig. 6C and 6D). This observation raised the possibility that ASPM-i1 may be associated with the invasiveness and / or metastasis of breast cancer.

[0093] After demonstrating the heterogeneity of ASPM isoform expression, we next sought to determine whether the ASPM expression pattern, particularly the specific upregulation of ASPM-i1 in the cytoplasm of cancer cells, could be generalized to other types of human cancer. Previously, ASPM transcript levels have been shown to be elevated in 66% of human HCC tissues and correlated with poor 5-year survival and early tumor recurrence in HCC patients (Lin, SY, Pan, HW, Liu, SH, Jeng, YM, Hu, FC, Peng, SY, Lai, PL, and Hsu, HC (2008). ASPM is a novel marker for vascular invasion, early recurrence, and poor prognosis of hepatocellular carcinoma. Clin Cancer Res 14, 4814-4820). To investigate the expression pattern of ASPM-i1 at the protein level in human normal and malignant liver tissues, we performed IHC staining of ASPM-i1 on two tissue microarrays (LVN241a and LV805b, US Biomax, Inc., Rockville, MD, USA) consisting of 24 normal liver tissues, 10 hepatitis liver tissues, 50 cirrhosis liver tissues, and 111 HCC tissues. Similar to the expression pattern in breast cancer tissues, ASPM-i1 was predominantly expressed (≥1+) in the cytoplasm of a subset of normal hepatocytes (average 28.3%) in normal liver tissues (Figure 6E). The percentage of epithelial cells with weak (1+) ASPM-i1 staining significantly increased in hepatitis, cirrhosis, and HCC tissues. Importantly, in HCC tissues, a significant number of epithelial cells showed moderate (2+, average 24.3%) or strong (3+, average 3.43%) ASPM-i1 staining intensity. In contrast, ASPM-i1 expression in the nuclei of normal, hepatitis, and cirrhotic liver tissues was negligible. In HCC tissues, only a small percentage of tumor cells showed weak (1+, average 13.5%) or moderate (2+, average 4.7%) nuclear staining of ASPM-i1.

[0094] In contrast to the ASPM-i1 staining pattern in liver tissue, the majority of hepatocytes in normal livers expressed ASPM-i2 (1+ or higher) in the cytoplasm (76.0%), and most hepatocytes also expressed ASPM-i2 in the cell nucleus (44.1%) (Figure 6E). Interestingly, ASPM-i2 expression was moderately upregulated in HCC tissues, with approximately two-thirds of tumor cells showing moderate to strong (2+ or higher) ASPM-i2 staining intensity in the cytoplasm (64.0%) or cell nucleus (67.5%) (Figure 6F).

[0095] Taken together, the very low expression frequency of ASPM-i1 in normal epithelial tissues, together with its specific upregulation in cancer cells, confirms that ASPM-i1 is a cancer cell-specific target with a favorable safety profile for the treatment of cancer.

[0096] [Example 3] ASPM isoform 1 (ASPM-i1) specifically promotes the assembly of PAR-planar cell polarity complexes and invadopodia formation in invasive cancer cells Given our previous findings regarding the critical role of ASPM in cancer cell invasiveness and metastasis, and the specific role of ASPM-i1 in Wnt activity and tumorigenesis, we sought to gain a deeper mechanistic understanding of how it regulates Wnt-related invasiveness in cancer cells. To this end, we isolated invasive breast cancer MDA-MB-436 cells using a modified Boyden chamber invasion assay (Transwell inserts, BD Biosciences). Transwell inserts were coated with a thin layer of growth factor-reduced reconstituted basement membrane (rBM, BD Biosciences) and cells were allowed to invade into the rBM for 12 hours. Cells that invaded the insert membrane and those that remained on the membrane were harvested. Next, we used high-throughput microwestern array analysis (Ciaccio, MF, Wagner, JP, Chuu, CP, Lauffenburger, DA, and Jones, RB (2010). Systems analysis of EGF receptor signaling dynamics with microwestern arrays. Nat Methods 7, 148-155) to screen a list of 107 annotated human Wnt proteins (http: / / web.stanford.edu / group / nusselab / cgi-bin / wnt / ) for interactions with ASPM-i1 in invasive breast cancer. Several members of the PAR-planar cell polarity (PCP) protein complex, including Par-6 family cell polarity regulator alpha (PAR6α), PAR6β, disheveled2 (DVL2), CDC42, and SMURF1, strongly interact with ASPM-i1 (Figure 7).

[0097] Because noncanonical Wnt-PCP signaling transmits tissue patterning information to individual cells and controls cell morphogenesis and the invasive behavior of malignant cells by regulating cell polarity and invadopodia (Luga, V., Zhang, L., Viloria-Petit, AM, Ogunjimi, AA, Inanlou, MR, Chiu, E., Buchanan, M., Hosein, AN, Basik, M., and Wrana, JL (2012). Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell 151, 1542-1556), we investigated the possibility that ASPM-i1 cooperates with PAR-PCP proteins to regulate invadopodia dynamics and thus cancer cell invasiveness. Therefore, we induced invadopodia formation in breast cancer MDA-MB-436 cells by plating them on a gelatin matrix (Sigma-Aldrich) as previously described (Eckert, MA, Lwin, TM, Chang, AT, Kim, J., Danis, E., Ohno-Machado, L., and Yang, J. (2011). Twist1-induced invadopodia formation promotes tumor metastasis. Cancer Cell 19, 372-386). Cells were plated on gelatin for more than 3 hours and then immunostained with anti-cortactin and Alexa Fluor 647 phalloidin (a stain for F-actin). The staining patterns were evaluated using confocal image analysis. Cortactin protrudes downward from the cells. + F-actin +These structures are thought to be invadopodia. As shown in Figure 8A, confocal imaging analysis revealed that ASPM-i1 colocalized with DVL2, PAR6β, CDC42, and cortactin in the invadopodia of invasive cancer cells. To confirm that ASPM-i1 specifically interacts with PAR / PCP proteins in invadopodia, we isolated invadopodia proteins from cells plated on a gelatin matrix for 3 hours to induce invadopodia formation. Cell bodies were sheared from the surface of the plate, leaving the invadopodia embedded in gelatin. The invadopodia protein and cell body protein fractions were then solubilized in IP buffer. For co-IP, cells were lysed in non-denaturing lysis buffer (1 mM PMSF, 1 mM Na3VO4, 1 μg / ml pepstatin, 20 mM NaF, phosphatase inhibitor cocktail, 0.5% NP-40, and 10% glycerol in PBS), and the lysate (1 mg) was cleared by incubation with a 50% Protein A-Sepharose bead slurry. Subsequently, 1 mL of the cleared lysate was incubated with antibody-conjugated 50% Protein A-Sepharose beads and 10 μL of 10% BSA overnight at 4 °C. The beads were washed three times with wash buffer (0.5% NP-40, 0.1% Triton X-100, 1 mM PMSF, and 1 mM Na3VO4 in PBS). Proteins were revealed after SDS-PAGE and immunoblotting with the indicated antibodies. Immunoblotting analysis of invadopodia proteins fractionated from invasive breast cancer MDA-MB-436 cells confirmed that ASPM interacted with DVL2, PAR6β, CDC42, and N-WASP in invadopodia, but not with other DVL isoforms or SMURF1 (Fig. 8B).

[0098] After demonstrating the specific interaction of ASPM-i1 with the PAR / PCP complex and its assembly in invadopodia of cancer cells, we next sought to deepen our understanding of its functional role in invadopodia formation and cancer invasiveness. To this end, we used BLOCK-iT™ RNAi Designer (Invitrogen) to synthesize four in silico predicted ASPM-v1 (SEQ ID NO: 3)-specific small hairpin RNA (shRNA) oligonucleotides whose 21-nucleotide target sequences were located within the mRNA region encoded by exon 18 (ASPM.e18) of the ASPM gene. We constructed lentiviral vectors expressing each of these ASPM-v1-targeting siRNAs by ligating the shRNA sequences containing both the sense and antisense strands separated by a 9-bp loop region for directional cloning into pGLV2-U6-Puro. We infected MDA-MB-436 cells with each of the lentiviral vectors and tested their efficacy in down-regulating the transcript and protein abundance levels of ASPM-i1. A non-targeting siRNA (NT siRNA, SHC002V, Sigma-Aldrich) was used as a control in subsequent experiments.

[0099] As shown in Figure 9A, transfection of breast cancer MDA-MB-436 cells with two of the ASPM.e18-specific shRNAs, including ASPM.e18 shRNA#1 and ASPM.e18 shRNA#4, dramatically downregulated the protein abundance of ASPM-i1 without affecting the protein abundance of ASPM isoform 2. Importantly, specific knockdown of ASPM-i1 expression using ASPM.e18.#4 shRNA significantly suppressed the recruitment of PAR-PCP proteins, including DVL2, PAR6β, and CDC42, as well as membrane-type matrix metalloproteinase (MT1-MMP; representing functional proteins on invadopodia), to invadopodia (Figure 9B), confirming the functional role of ASPM-i1 in the assembly of invadopodia-specific PAR6-PCP complexes. Complementing these molecular analyses, knockdown of ASPM-i1 significantly downregulated cortactin expression in cancer cells. + F-actin + It significantly suppressed the formation of invadopodia, thereby inhibiting their invasive potential (Figure 9C). Overall, these data suggest that ASPM-i1 regulates invadopodia formation and cancer invasiveness by promoting the assembly of a novel DVL2 / PAR6β / CDC42 / N-WASP protein complex in invadopodia.

[0100] [Example 4] ASPM-i1 regulates multiple development- and stemness-related pathways in cancer cells ASPM has been identified as a key regulator of the Wnt signaling pathway. ASPM expression was found to be essential for cellular responsiveness to canonical Wnt ligands, such as Wnt-3a, in pancreatic and prostate cancer cells. Mechanistic studies revealed that ASPM interacts with upstream activators of β-catenin, including disheveled 2 (DVL2) or DVL3, axin, and protease-activated receptor 1 (PAR1), inhibiting proteasome-dependent degradation of DVL protein, thereby increasing the protein abundance of β-catenin and amplifying canonical Wnt signaling, which is crucial for its oncogenic effects. Consistently, knockdown of ASPM expression resulted in a dramatic decrease in Wnt reporter activity in cancer cells, whereas increased DVL expression in these cells could restore their Wnt activity and stemness, suggesting that DVL functionally rescues ASPM deficiency and may function as a key regulator of Wnt signaling-associated cancer stemness.

[0101] The essential role of ASPM in neuronal development and its specific expression in normal or malignant stem cells, along with its pleiotropic roles in mitotic regulation, cell cycle, and developmental pathways, raises intriguing questions about its potential role in other developmental signaling pathways. To this end, we measured the activity of a panel of distinct developmental and stemness-related pathways using a commercial reporter array (Cignal Finder Stem Cell & Differentiation 10-Pathway Reporter Array, Qiagen, Germany). Briefly, highly transfectable human embryonic kidney 293T cells (HEK293T) were knocked down for ASPM-v1 expression using lentiviral-mediated shRNA with ASPM.e18.#4. Cells were transfected with reporter constructs in wells using Lipofectmine LTX reagent (ThermoFisher Scientific, Waltham, MA, USA), and then firefly and Renilla luciferase activities were measured in a SpectraMax® luminometer (Molecular Device, Waltham, MA, USA) using a dual-luciferase assay system (Promega, Madison, WI, USA). Results were expressed as the ratio of firefly luciferase (Fluc) activity to Renilla luciferase (Rluc) activity.

[0102] As shown in Figure 10, we demonstrated that Wnt signaling pathway activity was significantly reduced in HEK293T cells in which ASPM-v1 expression was knocked down. Notably, and importantly, the data revealed that knockdown of ASPM-v1 expression also reduced the activity of the Notch and Hedgehog (Hh) signaling pathways and the stemness-related transcription factors OCT4 and KLF4. Collectively, these data suggest that ASPM-v1 regulates not only Wnt signaling but also other development- and / or stemness-related pathways in cancer cells, highlighting its crucial role in cancer stemness and progression.

[0103] [Example 5] In silico and experimental screening of ASPM-v1-targeting siRNA To exploit ASPM-i1 as a therapeutic target for the treatment of both invadopodia-mediated cancer growth and cancer invasiveness, we attempted to develop siRNA therapies that specifically inhibit ASPM-i1 expression in cancer cells. To facilitate preclinical efficacy testing and primate toxicity studies of siRNA therapies targeting ASPM transcript variant 1 (ASPM-v1, NCBI RefSeq:NM_018136.4; encoding ASPM-i1), we searched for siRNA sequences that simultaneously target the mRNA regions encoded by exon 18 (specific to ASPM-v1; "ASPM-v1.e18") of both human and cynomolgus monkey (Macaca fascicularis) ASPM-v1. To design siRNAs targeting various regions in exon 18 of ASPM-v1 mRNA, we divided exon 18 of human ASPM-v1 equally into four segments of similar length and aligned them with the corresponding regions in the cynomolgus macaque gene using NCBI BLAST searches. For each of these segments, high identity (96%-98%) was observed between the human and primate sequences (Table 1).

[0104] [Table 1]

[0105] We then searched for various identical subsegments located within each of the four ASPM-v1.e18 segments (data not shown) and designed siRNAs for each of these subsegments using the Whitehead Institute's siRNA selection server (http: / / sirna.wi.mit.edu / home.php). We applied the following three selection criteria to select candidate siRNAs: (1) GC content of 40-60%, (2) a negative binding energy difference between the 5'-sense strand and the 5'-antisense strand (ΔΔG), and (3) an off-target score greater than 10. The off-target score of an siRNA was determined by subjecting the siRNA to a homology search against human mRNA sequences using NCBI BLAST, followed by extracting the number of mismatches in the non-seed, seed, and cleavage site regions of the siRNA for score calculation, as previously described (U.S. Patent No. 8,809,292 B2). and (4) the absence of known immunostimulatory motifs, including (5' to 3') "GUCCUUCAA" and "UGUGU" (Fedorov et al., 2006; Judge et al., 2005). Using these criteria, we identified 42 candidate siRNAs that met these selection criteria (Step 1). To validate the knockdown efficacy of ASPM-v1-targeting siRNA (siASPM-v1), we synthesized double-stranded siRNAs (Dharmacon) corresponding to the selected sequences and transfected each of them into highly transducible human embryonic kidney HEK293T cells using Lipofectamine LTX reagent (ThermoFisher Scientific). An siRNA (siASPM-v1.8602, 5'-GAGCUGCUAUCACUUUACAGC-3'), whose knockdown effect on ASPM-v1 expression has previously been demonstrated, was also synthesized and included as a positive control (Hsu et al., 2019b). A non-targeting control siRNA (5'-UGGUUUACAUGUCGACUAAUU-3', Dharmacon) was synthesized and included as a negative control.We measured ASPM-v1 transcript levels using quantitative real-time PCR (qRT-PCR) with the LightCycler FastStart DNA MASTERPLUS SYBR Green I Kit and the LightCycler System (Roche Diagnostics GmbH, Mannheim, Germany). Oligonucleotide primers were designed using Primer Bank (http: / / pga.mgh.harvard.edu / primerbank / index.html). All transduction experiments were performed in triplicate to allow for statistical analysis. We ranked 42 candidate siRNAs according to their respective gene silencing effects on ASPM-v1 transcript levels. Notably, not all of these in silico-designed siASPM-v1s were able to effectively knockdown ASPM-v1 expression; three of them had no effect on ASPM-v1 transcript levels after transduction into HEK293 cells. Using Student's t test, we identified a list of 30 siRNAs from these 42 candidate siRNAs that were able to significantly ( P value less than 0.05) reduce the expression of ASPM-v1 when transduced into HEK293K cells.

[0106] To verify the knockdown effects of the designed siRNAs, we synthesized double-stranded siRNAs (Dharmacon) corresponding to the selected sequences and transfected each of them into highly transducible human embryonic kidney HEK293T cells using Lipofectamine LTX reagent (ThermoFisher Scientific). An siRNA (siASPM-v1.8602, 5'-GAGCUGCUAUCACUUUACAGC-3') that has previously been shown to silence ASPM-v1 expression was also synthesized and included as a positive control (Hsu, CC, Liao, WY, Chan, TS, Chen, WY, Lee, CT, Shan, YS, Huang, PJ, Hou, YC, Li, CR, and Tsai, KK (2019b). The differential distributions of ASPM isoforms and their roles in Wnt signaling, cell cycle progression, and pancreatic cancer prognosis. J Pathol). A non-targeting control siRNA (NT siRNA, 5'-UGGUUUACAUGUCGACUAAUU-3', Dharmacon) was synthesized and included as a negative control. We measured ASPM-v1 transcript levels using quantitative real-time PCR (qRT-PCR) with the LightCycler FastStart DNA MASTERPLUS SYBR Green I Kit and the LightCycler System (Roche Diagnostics GmbH, Mannheim, Germany). Oligonucleotide primers were designed using Primer Bank (http: / / pga.mgh.harvard.edu / primerbank / index.html).We then ranked the 45 candidate siRNAs according to their respective knockdown effects on the ASPM-v1 transcript level and then selected the top two siRNA sequences from each of the four human-monkey homogenous fragments shown in Table 1 (Step 2). To further select the top siRNAs, we transduced the eight siRNAs selected from Step 2 into breast cancer MDA-MB-436 cells and ranked them according to their respective knockdown effects (Step 3). We then selected the top three siRNA sequences, including siASPM-v1.F2#1, siASPM-v1.F3#1, and siASPM-v1.F4#1, for subsequent analysis (Step 4).

[0107] To compare the efficacy of each of the three selected siRNAs or their combinations in knocking down ASPM-v1 expression, we transduced MDA-MB-436 cells with each of the three siRNAs or various combinations of them and analyzed the knockdown effect of each using qRT-PCR. Interestingly, we found that a 1:1 mixture of two of the siRNAs, including siASPM-v1.F3#1 and siASPM-v1.F4#1, and a single siRNA, siASPM-v1.F2#1, achieved the best gene silencing effect on ASPM-v1 among the various combinations (Figure 11). Notably, and importantly, a 1:1 mixture of siASPM-v1.F3#1 and siASPM-v1.F4#1 was chosen for subsequent development rather than a single siASPM-v1.F2#1 due to the following considerations: (1) the target mRNA sequences of siASPM-v1.F3#1 and siASPM-v1.F4#1 are located in different fragments in exon 18 of the ASPM gene (i.e., fragments 3 and 4, respectively); therefore, targeting them simultaneously using an siRNA mixture can avoid potential variations in knockdown efficacy caused by chromatin conformation; and (2) the siRNA mixture consists of half the amount of each siRNA as a single siRNA, thereby theoretically reducing potential toxicities, such as off-target silencing and immunostimulatory effects.

[0108] [Example 6] Biological effects of lipid nanoparticle-formulated ASPM-v1-targeting siRNA on cancer cells in vitro Many types of human solid cancers, such as breast cancer, non-small cell lung carcinoma, pancreatic ductal adenocarcinoma (PDAC), the scirrhous subtype of gastric adenocarcinoma, and the "stem cell-like / serrated / mesenchymal" molecular subtype of colorectal cancer, are characterized by a pronounced stromal reaction, termed the "desmoplastic response," which poses a major obstacle to the efficient delivery of cancer therapeutics to tumors. Recently, two nanoparticle-formulated chemotherapy agents, including albumin-bound paclitaxel (nab-paclitaxel) and liposome-encapsulated irinotecan, have been shown to prolong the survival of patients with advanced PDAC. Both agents significantly increased the levels of chemotherapy agents in treated tumors, suggesting that nanoparticle formulations may become a clinically proven approach to improve the efficacy of desmoplastic cancer treatment. In liver disease, parenteral administration of transthyretin-specific liposome-encapsulated siRNA (patisiran, Alnylam) has been shown to reduce transthyretin produced by the liver of patients with hereditary transthyretin-mediated amyloidosis by up to 86.8%, thus becoming the first clinically approved RNAi drug (Adams, D., Gonzalez-Duarte, A., O'Riordan, WD, Yang, CC, Ueda, M., Kristen, AV, Tournev, I., Schmidt, HH, Coelho, T., Berk, JL et al. (2018). Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. N Engl J Med 379, 11-21).Furthermore, nanoparticle-delivered siRNA therapies, such as cyclodextrin polymer-based nanoparticles bearing siRNA targeting ribonucleotide reductase M2 (RRM2) and lipid nanoparticles bearing siRNA targeting VEGF-A and kinesin spindle protein (KSP), have shown promising pharmacodynamics and tolerability and antitumor efficacy in some treated patients in phase 1 clinical trials (Davis, ME, Zuckerman, JE, Choi, CH, Seligson, D., Tolcher, A., Alabi, CA, Yen, Y., Heidel, JD, and Ribas, A. (2010). Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464, 1067-1070). Recently, intravenous injection of liposomal nanoparticle (LNP)-encapsulated small activating RNA (saRNA) designed to activate transcription of the CEBPA gene demonstrated an acceptable safety profile and potential synergistic efficacy with tyrosine kinase inhibitors in a phase Ib study in patients with advanced hepatocellular carcinoma (HCC) (Sarker, D., Sodergren, M., Plummer, ER, Basu, B., Meyer, T., Huang, KW, Evans, TRJ, Spalding, D., Ma, YT, Palmer, DH et al. (2020). First-in-human phase I trial of small activating RNA (saRNA) oligonucleotide MTL-CEBPA in combination with sorafenib in patients with advanced hepatocellular carcinoma (HCC). Journal of Clinical Oncology 38). Given these clinical advances and the important role of ASPM-i1 in invadopodia formation and cancer invasiveness, we attempted to develop a nanoparticle-formulated siRNA gene therapy targeting ASPM-v1 for the treatment of invasive and metastatic cancers.

[0109] Unmodified siRNAs are susceptible to degradation by serum exo- and endonucleases, resulting in a short serum half-life, and may induce immune responses via interferon and proinflammatory cytokines (Watts, JK, Deleaviey, GF, and Damha, MJ (2008). Chemically modified siRNA: tools and applications. Drug Discovery Today 13, 842-855). Therefore, several chemical modifications have been explored to improve siRNA stability and reduce its immunogenicity (Hassler, MR, Turanov, AA, Alterman, JF, Haraszti, RA, Coles, AH, Osborn, MF, Echeverria, D., Nikan, M., Salomon, WE, Roux, L. et al. (2018). Comparison of partially and fully chemically modified siRNA in conjugate-mediated delivery in vivo. Nucleic Acids Res 46, 2185-2196). To avoid potential immunogenicity of these siRNAs and increase their serum stability, we added 2'-O-methylation to the UA or CA site of the antisense strand and to all pyrimidines of the sense strand. This is because 2'-O-methylation of selected siRNAs is associated with less immune activation when administered systemically (Adami, RC, Seth, S., Harvie, P., Johns, R., Fam, R., Fosnaugh, K., Zhu, T., Farber, K., McCutcheon, M., Goodman, TT et al. (2011). An amino acid-based amphoteric liposomal delivery system for systemic administration of siRNA. Mol Ther 19, 1141-1151).To improve serum stability, we further added two deoxy-thymidine 3' overhangs with phosphorothioate linkages to both the sense and antisense strands.

[0110] Double-stranded partially chemically modified siASPM-v1.F3#1 and siASPM-v1.F4#1, and a similarly modified non-targeting double-stranded oligonucleotide (sense strand: 5'-UGGUUUACAUGUCGACUAA-3', non-targeting control siRNA) were synthesized by Dharmacon (Horizon Discovery Ltd., Waterbeach, UK). UU dinucleotides were added as overhangs to the 3' ends of both oligonucleotide strands. Each of them was formulated using a lipid nanoparticle (LNP)-based delivery vehicle containing DLin-MC3-DMA (MC3), an ionizable cationic amino lipid that complexes with siRNA, the amphiphilic phospholipid distearoylphosphatidylcholine (DSPC), cholesterol, and the coating lipid poly(ethylene glycol) lipid 1,2-dimyristoyl-rac-glycerol-methoxy(poly(ethylene glycol)) (DMG-PEG), mixed at a molar ratio of 50:10:38.5:1.5 (Adams, D., Gonzalez-Duarte, A., O'Riordan, WD, Yang, CC, Ueda, M., Kristen, AV, Tournev, I., Schmidt, HH, Coelho, T., Berk, JL et al. (2018). Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. N Engl J Med 379, 11-21). The rationale for selecting this MC3-based LNP as the delivery vehicle for our ASPM-targeted siRNA therapy is threefold: First, the same LNP formulation is used in patisiran (Onpattro), the first FDA-approved siRNA drug developed by Alnylam Pharmaceuticals.Second, this LNP formulation was shown to be effective in a mouse model of chronic myeloid leukemia (CML) (Jyotsana, N., Sharma, A., Chaturvedi, A., Budida, R., Scherr, M., Kuchenbauer, F., Lindner, R., Noyan, F., Suhs, KW, Stangel, M. et al. (2019). Lipid nanoparticle-mediated siRNA delivery for safe targeting of human CML in vivo. Ann Hematol 98, 1905-1918) or an orthotopic mouse model of hepatocellular carcinoma (HCC) (Tabernero, J., Shapiro, GI, LoRusso, PM, Cervantes, A., Schwartz, GK, Weiss, GJ, Paz-Ares, L., Cho, DC, Infante, JR, Alsina, M. et al. (2013). First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discov 3, 406-417) has been used for the systemic delivery of siRNA targeting tumor driver genes, such as BCR-ABL, VEGF-A, and kinesin spindle protein (KSP), which showed significant therapeutic efficacy. Third, this LNP-based systemic delivery of siRNA was shown to be safe and generally well-tolerated in a phase I clinical trial, with the exception of some infusion-related reactions and transient proinflammatory cytokine induction. The particle size of MC3-based LNPs ranges from 70 to 90 nm (Jayaraman et al., 2012), which allows leakage of LNPs into tumor tissue through leaky endothelial fenestration structures (with estimated pore diameters of 380 to 780 nm), a phenomenon associated with prolonged circulation time and known as the "enhanced vascular permeability and retention (EPR)" effect (Agarwal and Roy, 2013; Jain and Stylianopoulos, 2010).

[0111] We synthesized DLin-MC3-DMA and purchased DSPC (Avanti Polar Lipids, Alabaster, AL, USA), cholesterol (Sigma-Aldrich), and DMG-PEG (NOF America Corporation, White Plains, NY, USA). We encapsulated the above-mentioned mixture of siASPM-v1.F3.#1 and siASPM-v1.F4.#1 using an in-house designed microfluidic device with controlled mixing conditions of 0.5 ml / min flow rate and 1:3 flow ratio (lipid: siRNA = 0.125 ml / min: 0.375 ml / min). The size (number-weighted mean diameter) and zeta potential of LNPs were measured using a Zetasizer Nano ZS ZEN3600 instrument (Malvern Instruments, Worcestershire, UK). The encapsulation efficiency and total concentration of siRNA were measured using Quanti-iT™ RiboGreen RNA reagent and kit (Invitrogen, Waltham, MA, USA). The resulting 1:1 mixture of chemically modified siASPM-v1.F3#1 and siASPM-v1.F4#1 encapsulated in MC3-based LNPs was designated "LNP-siASPM-v1."

[0112] We first demonstrated that treatment of MDA-MB-436 cells with LNP-siASPM-v1 dose-dependently reduced the transcript level of ASPM-v1, with a 50% inhibitory concentration (IC50) of 0.43 nM (Figure 12A). Consistently, immunoblotting analysis confirmed that this treatment dose-dependently reduced the protein abundance level of ASPM-i1 (Figure 12B). In contrast, this treatment did not affect the transcript level of ASPM transcript variant 2 (data not shown) or the protein abundance level of ASPM-i2 (Figure 12B), reaffirming its specific knockdown effect on ASPM-v1 expression. Consistent with the knockdown effect on ASPM-i1, treatment of cancer cells with siASPM-v1 significantly reduced the expression of cortactin. + F-actin +It significantly inhibited the formation of invadopodia (Fig. 13A) and their invasive ability (Fig. 13B).

[0113] Previous studies have shown that ASPM, specifically ASPM-i1, contributes to canonical Wnt activity, stemness, and tumorigenesis in various types of cancer, such as pancreatic cancer, prostate cancer, and hepatocellular carcinoma (HCC) (Hsu, CC, Liao, WY, Chan, TS, Chen, WY, Lee, CT, Shan, YS, Huang, PJ, Hou, YC, Li, CR, and Tsai, KK (2019a). The differential distributions of ASPM isoforms and their roles in Wnt signaling, cell cycle progression, and pancreatic cancer prognosis. J Pathol 249, 498-508). Therefore, we attempted to evaluate the effects of siRNA targeting ASPM-i1 on Wnt activity and stemness in cancer cells. To this end, we treated HCC HuH-1 cells with LNP-siASPM-v1 and demonstrated that it could substantially reduce WNT3A-stimulated Wnt-specific TEF / LEF reporter activity in HuH-1 cells (Figure 14A). Consistently, it also reduced the number of aldehyde dehydrogenase (ALDH)-positive cells, which are known to contain enriched stem cell-like cells in HCC (Figure 14B). At the functional level, transduction of LNP-ASPM-v1 siRNA into HuH-1 cells could substantially inhibit their ability to form tumorspheres in serum-free and ultra-low-adhesion culture conditions (Figure 14C). These findings collectively support the Wnt and stemness-inhibitory function of LNP-formulated ASPM-v1-targeting siRNA.

[0114] [Example 7] Pharmacodynamic study of lipid nanoparticle-formulated ASPM-v1-targeted siRNA therapy To confirm the pharmacodynamic activity of LNP-siASPM-v1 systemic therapy in treated tumor cells, triple-negative breast cancer (TNBC) MDA-MB-436 cells were lentivirally transduced with a GFP and firefly luciferase (FF-Luc) fusion vector (UBC-EGFP-T2A-Luc, System Biosciences), and GFP-positive cells were sorted using a BD Influx™ cell sorter (BD Biosciences). The cells were then orthotopically injected into the mammary fat pad of immunodeficient NOD / SCID mice. Ten days after cell inoculation, when tumors became detectable by bioluminescence imaging (BLI), tumor-bearing mice were intravenously injected with LNP-siASPM-v1 at a dose of 100 μg per mouse (approximately 4 mg / kg). Three days later, this treatment was repeated, and tumors were excised 24 hours after the second injection. Tumors were enzymatically dissociated into single cells and analyzed using a FACSAria™ III cell sorter (BD Biosciences) for subsequent analysis. + Cancer cells were sorted (Figure 15A).

[0115] As shown in Figure 15B, the majority (average 88.7%) of GFP isolated from LNP-siASPM-v1 treated mice + Tumor cells were Cy5-positive 24 hours after completion of treatment, confirming their successful transduction with siRNA. The percentage of cells transduced with siRNA gradually decreased over time, with an estimated half-life of 4.8 days.

[0116] Next, to confirm the accumulation of LNP-siASPM-v1 in tumor cells, tumor tissues excised from siRNA-treated mice were rapidly frozen in liquid nitrogen and sectioned for immunofluorescence staining using phalloidin (a stain for actin filaments) to clarify cell contours. As shown in Figure 15C, confocal images revealed that systemically administered siRNA was successfully delivered to the cytoplasm of tumor cells in the orthotopic TNBC model.

[0117] Finally, to confirm the knockdown effect of LNP-siASPM-v1 systemic therapy on the protein abundance level of ASPM-i1 in treated tumor cells, we analyzed freshly sorted GFP-positive cells from treated tumors. + Protein lysates were collected from tumor cells and blotted with an ASPM-i1-specific rabbit polyclonal antibody (Hsu et al., 2019a). As expected, siRNA therapy substantially reduced the protein abundance of ASPM-i1 compared to untreated tumors (Figure 15D), confirming that siASPM-v1 systemic therapy specifically inhibits ASPM-i1 expression in treated tumor cells.

[0118] [Example 8] Antitumor and anti-metastatic effects of siRNA therapy targeting ASPM-v1 in triple-negative breast cancer (TNBC) After demonstrating the expected inhibitory effect of LNP-ASPM-v1 siRNA on the invadopodia formation and invasive ability of cancer cells, we next attempted to investigate its therapeutic effect in vivo. Using an orthotopic breast cancer progression model, we explored whether systemic LNP-siASPM-v1 could exert anti-metastatic efficacy. The GFP- and FF-Luc-expressing TNBC MDA-MB-436 cells (10 6LNP-siASPM-v1 (100 μg [approximately 4 mg / kg] per mouse) was orthotopically injected into the mammary fat pad of NOD / SCID mice. Ten days after cell inoculation, when tumors became detectable by BLI, tumor-bearing mice were repeatedly injected intravenously (IV, 100 μg [approximately 4 mg / kg] per mouse every 3 days, a total of 10 times) with LNP-siASPM-v1 or LNP-nontargeting control siRNA (NT siRNA). The resulting primary or metastatic lung tumors were visualized weekly by bioluminescence imaging (BLI) according to the manufacturer's recommendations (IVIS Imaging System, Caliper Life Sciences) (Figure 16A). The results showed that IV LNP-siASPM-v1 therapy at different dose levels (2 mg / kg or 4 mg / kg per injection) significantly reduced primary tumor growth in a dose-dependent manner, with a tumor control rate of 81.2% at the high dose level (Figure 16B). As expected, gene systemic therapy could completely prevent the appearance of distant metastatic tumors in the lungs (FIG. 16C), highlighting the potent antitumor and antimetastatic efficacy of ASPM-v1-targeted siRNA therapy.

[0119] Despite advances in adjuvant chemotherapy, approximately 40% of breast cancer patients who undergo surgical removal of the primary tumor will experience a recurrence and ultimately die from metastatic disease (Weigelt, B., Peterse, JL, and van't Veer, LJ (2005). Breast cancer metastasis: markers and models. Nat Rev Cancer 5, 591-602). Current adjuvant chemotherapy increases the 15-year survival rate of breast cancer patients by only 3%-10%, highlighting the urgent need to develop anti-metastatic therapies that are safer and more effective than chemotherapy. Along these lines, the ability of nanoparticles to improve tissue penetration and tumor uptake of cargo drugs, along with the potential to integrate multiple therapeutic functions into a single platform, positions nanoparticle therapeutics as a highly promising strategy for the treatment of metastatic cancer (Schroeder, A., Heller, DA, Winslow, MM, Dahlman, JE, Pratt, GW, Langer, R., Jacks, T., and Anderson, DG (2011). Treating metastatic cancer with nanotechnology. Nat Rev Cancer 12, 39-50). Because ASPM-v1-targeted siRNA therapy exhibits dramatic anti-metastatic efficacy in an orthotopic model of breast cancer, we hypothesized that this nanogene therapy may also be highly useful in preventing metastatic disease. To address this possibility, we injected MDA-MB-436 cells into NOD / SCID mice via the tail vein. Twenty-four hours after cell inoculation, mice were intravenously injected with LNP-siASPM-v1 (4 mg / kg per mouse every 3 days, a total of 6 injections) (Figure 17A). Indeed, this therapy completely prevented the appearance of distant metastases, particularly in the lungs (Figures 17B and 17C), confirming the therapeutic potential of ASPM-v1-targeted siRNA therapy in the adjuvant setting.

[0120] [Example 9] Antitumor efficacy of siRNA therapy targeting ASPM-v1 in hepatocellular carcinoma (HCC) Our previous findings demonstrated that ASPM-v1-targeted siRNA therapy inhibits invadopodia formation and invasiveness of cancer cells, as well as their Wnt activity and stem cell properties (Figure 14). Consistent with this, intratumoral (IT) injection of LNP-siASPM-v1 was able to shrink primary tumor masses in the orthotopic breast cancer model described above. Given these findings, the recent FDA approval of liposome-encapsulated siRNA therapy (patisiran, Alnylam) for transthyretin-mediated amyloidosis, a liver disease, and the important role of the ASPM gene in HCC tumorigenesis and progression, we sought to evaluate the therapeutic potential of ASPM-v1-targeted siRNA therapy in the treatment of localized and / or advanced HCC. Therefore, we administered LNP-siASPM-v1 to established HCC via IT injection into a subcutaneous xenograft mouse model with or without simultaneous oral administration of sorafenib, a multikinase inhibitor that also has Wnt inhibitory effects and is routinely used for the first-line treatment of advanced HCC (Lachenmayer, A., Alsinet, C., Savic, R., Cabellos, L., Toffanin, S., Hoshida, Y., Villanueva, A., Minguez, B., Newell, P., Tsai, HW et al. (2012). Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib. Clin Cancer Res 18, 4997-5007). In this model, HuH-1 cells were lentivirally transduced with GFP and firefly luciferase fusion vectors, and GFP-positive cells were selected as described above. 100 μl of cells (1 × 10 6 8-week-old NOD / SCID mice were inoculated with 1000 cells (1:1 Matrigel:cells) subcutaneously into the flank or orthotopically into the left liver lobe, and tumor mass and distribution were assessed by BLI.

[0121] As shown in Figure 18A, intratumoral injection of LNP-siASPM-v1 (0.8 mg / kg or 2 mg / kg every 3 days for a total of 3 doses) significantly suppressed tumor growth in a dose-dependent manner. Remarkably, this treatment dramatically reduced primary tumor growth, with an average tumor control rate of 92.8% at the 2 mg / kg dose level. Although LNP-siASPM-v1 therapy at the 0.8 mg / kg dose level did not exhibit significant antitumor efficacy, it was able to significantly enhance the antitumor efficacy of sorafenib, a standard HCC therapeutic agent, suggesting potential synergistic effects between LNP-formulated ASPM-v1-targeting siRNA and sorafenib in the treatment of HCC (Figure 18B).

[0122] Next, we attempted to further extend our findings to a more clinically relevant context. Because image-guided local treatments, such as ablation and transcatheter tumor therapy, are widely accepted treatment options for patients with early-stage HCC, we performed ultrasound-guided injection of LNP-formulated ASPM-v1-targeting siRNA into an orthotopic mouse model of HCC (Figure 19A). Human HCC HuH-1 cells were injected into the left lobe of the liver of NOD / SCID mice under ultrasound guidance. Three weeks after cell inoculation, LNP-siASPM-v1 was directly injected into the tumor under ultrasound guidance. Notably, injection of a low dose level (0.8 mg / kg) of LNP-siASPM-v1 into orthotopic HuH-1 tumors was able to substantially suppress their growth (Figure 19B), confirming that intratumoral ASPM-v1-targeting siRNA therapy is a clinically feasible means of treating patients with early-stage HCC who present with accessible lesions.

[0123] Systemic administration of multikinase inhibitors or immune checkpoint inhibitors is a treatment option for patients with advanced HCC, but is associated with very limited survival benefit or response rates. Therefore, we investigated whether systemic administration of LNP-formulated ASPM-v1-targeting siRNA also exerts antitumor efficacy in an orthotopic model of HCC. Briefly, 100 μl of FF-Luc-expressing HuH-1 cells (1 × 10 68-week-old NOD / SCID mice were inoculated with LNP-siASPM-v1 (100 μg [approximately 4 mg / kg] per mouse every 3 days, a total of 6 doses) or LNP-nontargeting control siRNA via tail vein injection (Figure 20A). As shown in Figure 20B, systemically administered LNP-siASPM-v1 was able to efficiently transduce tumor cells in orthotopically established HCC. Importantly, LNP-siASPM-v1 systemic therapy significantly suppressed tumor progression with a mean tumor control rate of 57.5% (Figures 20C and 20D). Accordingly, mice receiving the therapy survived significantly longer than mice receiving the control treatment (Figure 20E).

[0124] [Example 10] siRNA combinations effectively down-regulate ASPM-v1 expression in various types of human cancer cells Our previous findings in Example 5 showed that the gene expression silencing effect of ASPM-v1-specific siRNA varied greatly between different types of malignant cells, such as HEK293T cells and breast cancer MDA-MB-436 cells (Figure 11). Because we attempted to select siRNAs that could effectively suppress ASPM-v1 expression in various types of cancer, we again attempted to select siRNAs that could achieve the best knockdown efficacy across a variety of malignant cells.

[0125] To this end, we first selected 10 siRNAs from the 30 siRNAs described in Example 5 that were able to achieve >80% knockdown of ASPM-v1 expression in HEK293T cells. We then obtained synthetic double-stranded siRNAs (Dharmacon) and transduced each of them into HEK293T cells, MDA-MB-436 TNBC cells, and HuH-1 HCC cells using Lipofectamine LTX reagent (ThermoFisher Scientific). An siRNA (siASPM-v1.8602, 5'-GAGCUGCUAUCACUUUACAGC-3') previously shown to knockdown ASPM-v1 expression was included as a positive control (Hsu et al., 2019b), and a non-targeting control siRNA (NT siRNA, 5'-UGGUUUACAUGUCGACUAAUU-3', Dharmacon) was included as a negative control. We measured ASPM-v1 transcript levels using quantitative real-time PCR (qRT-PCR) with the LightCycler FastStart DNA MASTERPLUS SYBR Green I Kit and the LightCycler System (Roche Diagnostics GmbH, Mannheim, Germany). Oligonucleotide primers were designed using Primer Bank (http: / / pga.mgh.harvard.edu / primerbank / index.html), including the forward primer: GCG AAG AGT CTT AGC ACA G and the reverse primer: GTG GAA TAT CTT CTC CAA TAT CCC. We averaged the knockdown efficacy of each of the 13 siRNAs in each strain and ranked their performance accordingly, thereby identifying the top siRNAs that could consistently achieve >80% knockdown efficacy in all three strains.

[0126] In our efforts to develop an active pharmaceutical ingredient (API) for siRNA therapeutics according to a preferred embodiment of the present invention, we reasoned that (1) siRNAs targeting various fragments in exon 18 of ASPM-v1 mRNA could avoid potential variations in knockdown efficacy caused by chromatin conformation, and (2) siRNA mixtures consisting of half the amount of each siRNA compared with single siRNAs could theoretically reduce potential toxicities, such as off-target silencing and immunostimulatory effects. Therefore, we selected siASPM-v1.4822 (sense strand: SEQ ID NO: 4, antisense strand: SEQ ID NO: 5), which targets fragment 1 in exon 18 (ASPM-v1.e18.F1), and siASPM-v1.7636 (sense strand: SEQ ID NO: 6, antisense strand: SEQ ID NO: 7), which targets fragment 3 in exon 18 (ASPM-v1.e18.F3). The inventors envisioned using their 1:1 combination as the API for ASPM-v1-targeted siRNA therapeutics for further development.

[0127] To avoid potential immunogenicity and enhance serum stability of these siRNAs, we added 2'-O-methylation to the UA or CA sites of the antisense strand and to all pyrimidines of the sense strand. This is because 2'-O-methylation of siRNA formulated using LNPs is associated with less immune activation when administered systemically (Adami, RC, Seth, S., Harvie, P., Johns, R., Fam, R., Fosnaugh, K., Zhu, T., Farber, K., McCutcheon, M., Goodman, TT et al. (2011). An amino acid-based amphoteric liposomal delivery system for systemic administration of siRNA. Mol Ther 19, 1141-1151). To improve serum stability, we further added two deoxythymidine 3' overhangs with phosphorothioate linkages to both the sense and antisense strands (Table 2).

[0128] [Table 2]

[0129] To compare and validate the knockdown efficacy of the two newly selected siRNAs and their mixtures, we transfected MDA-MB-436 cells with siASPM-v1.7636, siASPM-v1.4822, or a 1:1 mixture of these siRNAs, with or without chemical modifications, or a non-targeting control siRNA (NT siRNA) for 48 hours using Lipofectamine LTX reagent. Afterwards, RNA was isolated and the transcript levels of ASPM-v1 were measured using qRT-PCR. As shown in Figure 21A, all tested siRNAs, except for unmodified siASPM-v1.7636, achieved comparable knockdown efficacy of over 70% for all sequences tested. Chemically modified siASPM-v1.7636 and siASPM-v1.4822 achieved knockdown efficacies comparable to those of unmodified siRNAs, except for chemically modified siASPM-v1.7636, which achieved a knockdown efficacy greater than that of unmodified siASPM-v1.7636. Using the same transfection method, we also verified the knockdown efficacy of chemically modified siRNAs in HuH-01 cells. As shown in Figure 21B, all tested sequences achieved knockdown efficacies greater than 75%, except for siASPM-v1.4822 (mean knockdown efficacy = 68.6%). Overall, these data confirmed that transfection of these siRNAs achieved sufficient knockdown efficacy in both TNBC and HCC cells, and that their performance was not affected by the introduction of chemical modifications.

[0130] After confirming the gene silencing efficacy of a 1:1 mixture of siASPM-v1.7636 and siASPM-v1.4822 (hereinafter referred to as siASPM-v1 API), we used it as the API for ASPM-v1-targeting siRNA therapeutics as a preferred embodiment of the present invention. To verify the inhibitory effect of siASPM-v1 API on cancer cell invadopodia formation, invasion, and developmental pathway activity, we transduced HuH-01 or MDA-MD-436 cells with 100 nM siASPM-v1 API or chemically modified non-targeting control siRNA (NT siRNA) using Lipofectamine LTX reagent for 48 hours, and then plated the cells on a gelatin matrix (Sigma-Aldrich, G1393) as previously described (Eckert et al., 2011). Cells were plated on gelatin for 6 hours or more and then immunostained with anti-cortactin (4F11, Abcam, Cambridge, UK) or Alexa Fluor 647 phalloidin (a stain for F-actin, Invitrogen). Staining patterns were assessed using confocal image analysis with a Leica TCS SP5 confocal microscope system (Leica Microsystems GmbH, Wetzlar, Germany). Cortactin as seen under a confocal microscope. + F-actin + The dots represent cross sections of invadopodia protruding downward from the cell body. As shown in Figure 22, pretreatment of both HhH-1 and MDA-MB-436 cells with siASPM-v1 API significantly reduced the number of invadopodia by an average of 64.8% (HuH-1) to 63.7% (MDA-MB-436) compared to cells treated with NT siRNA.

[0131] After confirming the inhibitory effect of siASPM-v1 API on invadopodia formation in HCC and TNBC cells, we investigated whether it could effectively inhibit the invasive ability of cancer cells. HuH-1 or MDA-MB-436 cells were transduced with 100 nM siASPM-v1 API using Lipofectamine LTX reagent for 48 hours. The cells were then seeded onto Transwell inserts (BD Biosciences, San Jose, CA) with a thin layer of type I collagen (BD Biosciences) in the presence of 10% FBS and allowed to invade the collagen for 12 hours. Cells that invaded the insert membrane were fixed, stained with SYTOX Green (Invitrogen), and counted using a fluorescent microscope. As shown in Figure 23, consistent with an inhibitory effect on invadopodia, transduction of cells with siASPM-v1 API was able to substantially reduce the invasive capacity of both HuH-1 cells (89.2% on average) and MDA-MB-436 cells (75.3% on average) compared to cells treated with NT siRNA.

[0132] As described in our previous findings in Example 4, ASPM-i1 contributes to the activity of multiple developmental signaling pathways, including Wnt, Hedgehog, and Notch, in cancer cells. To confirm that inhibiting the function of ASPM-i1 by transducing cells with siASPM-v1 API can suppress the activity of these developmental signaling pathways in cancer cells, we infected HuH-1 or MDA-MB-436 cells with a lentiviral vector (pMuLE_EXPR_CMV-eGFP_TOP-NLuc1.1_12GLI-FLuc_CBF-GLuc, Addgene plasmid #113862) carrying a triple luciferase reporter of Wnt, Hedgehog (Hh), and Notch (Maier et al., 2019). In separate experiments, to further stimulate the activity of these pathways, cells were treated with recombinant human WMT3A (250 ng / ml for 16 hours, R&D Systems, Minneapolis, MN), recombinant human Sonic hedgehog (SHH, 3 μg / ml for 24 hours, Sigma-Aldrich) (Liu et al., 2006), recombinant human JAG1-Fc (5 μg / ml for 24 hours, Sigma-Aldrich, Steinheim, Germany) (Dees et al., 2011), or vehicle. Wnt, Hedgehog, and Notch reporter activity in unstimulated or stimulated cells was then measured using the Nano-Glo® Luciferase Assay System, ONE-Glo® Luciferase Assay System, and Renilla-Glo® Luciferase Assay System (Promega, Madison, WI), respectively.

[0133] As shown in Figure 24, transduction of HuH-1 cells with siASPM-v1 API significantly reduced Wnt, Hedgehog, and Notch reporter activity, which was more pronounced in ligand-stimulated cells. Similarly, transduction of siASPM-v1 API also significantly inhibited Wnt / Hedgehog / Notch reporter activity in both unstimulated and ligand-stimulated MDA-MB-436 cells. Overall, these data confirmed that inhibition of ASPM-v1 expression by siASPM-v1 API can indeed effectively inhibit these oncogenic pathways in different types of cancer cells.

[0134] The present inventors expanded the above-described siRNA selection strategy to include additional higher mammalian species in the design of ASPM-v1-targeting siRNAs. We selected the dog (Canis familiaris) because its ASPM gene shares the second highest percent identity with the human gene (82.69%), second only to the monkey orthologue (99.52%). We aligned the exon 18 sequences of the human, dog, and monkey ASPM genes using NCBI BLAST search, thereby identifying 10 identical subsegments with conserved sequences longer than 25 nucleotides. Notably, the sequences of these subsegments were identical among the human, dog, and cynomolgus monkey genes, allowing for flexible selection of relevant species for subsequent toxicity testing and preclinical studies. We designed and selected candidate siRNAs using the criteria described above.

[0135] To compare the gene silencing efficacy of candidate ASPM-v1-targeting siRNAs (siASPM-v1), we obtained synthetic double-stranded siRNAs (Dharmacon) and transfected them into HuH-1 hepatocellular carcinoma (HCC) cells or HCT-116 colorectal cancer (CRC) cells using Lipofectamine LTX reagent (ThermoFisher Scientific). We measured ASPMv1 transcript levels using qRT-PCR as described above. We ranked their performance and identified the top two siRNAs, siASPM-v1.4360 (sense strand: SEQ ID NO: 8, antisense strand: SEQ ID NO: 9) and siASPM-v1.4822 (sense strand: SEQ ID NO: 4, antisense strand: SEQ ID NO: 5), which consistently achieved >75% knockdown efficacy in both HuH-1 and HCT-116 cells. We demonstrated that transduction of two siASPM-v1 constructs into canine A-72 fibroblasts (Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan, #60480) resulted in sufficient (>75%) KD of ASPM-v1 expression. To exploit the potential utility of selected siASPM-v1 constructs as active pharmaceutical ingredients (APIs) for ASPM-v1-targeting siRNA drugs against extrahepatic cancers, we tested their gene silencing efficacy against ASPM-v1 in MDA-MB-436 triple-negative breast cancer (TNBC) cells and NCI-H209 and NCI-H146 small cell lung cancer (SCLC) cells (American Type Culture Collection, Manassas, VA). We transduced each cell line with candidate siASPM-v1 using Lipofectamine LTX reagent (ThermoFisher Scientific) and measured ASPM-v1 transcript levels using qRT-PCR as described above.We demonstrated that the top two siASPM-v1s selected from HCC and CRC cells, including siASPM-v1.4360 and siASPM-v1.4822, performed best in MDA-MB-436 TNBC cells, with knockdown efficacies of 96.71% and 83.95%, respectively. Similarly, transduction of the two siASPM-v1s into NCI-H209 and NCI-H146 SCLC cells also achieved excellent gene silencing effects against ASPM-v1, achieving knockdown efficacies of 84.49% and 86.31%, respectively, in NCI-H209 cells and 81.37% and 83.77%, respectively, in NCI-H146 cells. These highly consistent and robust data support the inclusion of these two siASPM-v1s for the subsequent development of ASPM-v1-targeting siRNA therapeutics in different cancers.

[0136] siASPM-v1.4360 and siASPM-v1.4822 target different mRNA subsegments in exon 18. Therefore, we designated their 1:1 combination as another version of the API of LNP-encapsulated siASPM-v1 (siASPM-v1 API_V2). Furthermore, to avoid potential immunogenicity of these siRNAs and increase their serum stability, we added 2'-O-methylation modifications to the UA or CA sites of the antisense strand and all pyrimidines of the sense strand. This is because 2'-O-methylation of siRNA formulated using LNPs is associated with less immune activation when administered systemically (Adami, RC, Seth, S., Harvie, P., Johns, R., Fam, R., Fosnaugh, K., Zhu, T., Farber, K., McCutcheon, M., Goodman, TT et al. (2011). An amino acid-based amphoteric liposomal delivery system for systemic administration of siRNA. Mol Ther 19, 1141-1151). To improve serum stability, we added two deoxythymidine 3' overhangs with phosphorothioate linkages to the sense and antisense strands. The chemically modified siRNAs were named "siASPM-v1.m4360-4378" and "siASPM-v1.m4822-4840," respectively, where "m" stands for "modified" (Table 3).

[0137] [Table 3]

[0138] To compare and verify gene silencing efficacy, we used Lipofectamine LTX reagent to transduce Huh-1 and HCT-116 cells with siASPM-v1.m4360, siASPM-v1.m4822, a 1:1 mixture of them (siASPM-v1 API_V2), or non-targeting siRNA (siNT) with or without chemical modifications as shown in Tables 2 and 3 for 48 hours. RNA was then isolated, and ASPM-v1 transcript levels were measured using qRT-PCR. As shown in Figure 25A, all tested siRNAs achieved approximately 70% gene silencing efficacy against ASPM-v1 in HuH-1 HCC cells. Notably, chemically modified siRNAs achieved slightly better knockdown efficacy (approximately 75%) than unmodified siRNAs. Similarly, in HCT-116 CRC cells (Figure 25B), all tested sequences achieved an average knockdown efficacy of 82.26%. These data demonstrated that siASPM-v1.m4360-4378, siASPM-v1.m4822-4840, or their 1:1 mixture (i.e., siASPM-v1 API_V2) all achieved sufficient gene silencing effects against ASPM-v1 in both HCC and CRC cells, and that their performance was not affected by the introduction of chemical modifications.

[0139] Next, we functionally characterized siASPM-v1 API_V2 in terms of its effectiveness in inhibiting invadopodia formation, invasion, and development-related pathway activity in cancer cells. First, we transduced HuH-1 or HCT-116 cells with 100 nM siASPM-v1 API_V2 or chemically modified non-targeting siRNA (m-siNT) using Lipofectamine LTX reagent for 48 hours, and then plated the cells on a gelatin matrix (Sigma-Aldrich, G1393). Cells were seeded on gelatin for 12 hours and then immunostained with the invadopodia marker TKS5 (anti-TKS5, 1:200, clone 13H6.3, Merck, Burlington MA) or anti-Col1-3 / 4C (type I collagen cleavage site, 1:25, ImmunoGlobe Antikoerpertechnik GmbH, Himmelstadt, Germany). Staining patterns were assessed using confocal image analysis with a Leica TCS SP5 confocal microscope system (Leica Microsystems GmbH, Wetzlar, Germany). TKS5 as seen under a confocal microscope. + Col1-3 / 4C + The dots represent cross sections of functional invadopodia, which protrude downward from the cell body and degrade the surrounding collagen 1 matrix. As shown in Figure 26, pretreatment of both HhH-1 and HCT-116 cells with siASPM-v1 API_V2 significantly reduced the number of invadopodia by an average of 90.69% in HuH-1 cells or 81.31% in HCT-116 cells compared to cells treated with siNT.

[0140] After confirming the inhibitory effect of siASPM-v1 API_V2 on invadopodia formation in cancer cells, we next investigated whether it could effectively inhibit the invasive ability of cancer cells. To this end, HuH-1 or HCT-116 cells were transduced with 100 nM siASPM-v1 API_V2 using Lipofectamine LTX reagent for 48 hours. The cells were then seeded onto Transwell inserts (BD Biosciences, San Jose, CA) with a thin layer of type I collagen (BD Biosciences) in the presence of 10% FBS and allowed to invade into the collagen for 12 hours. Cells that invaded the insert membrane were fixed, stained with SYTOX Green (Invitrogen), and counted using a fluorescent microscope. As shown in Figure 27, consistent with an inhibitory effect on invadopodia, transduction of cells with siASPM-v1 API_V2 was able to substantially reduce the invasive ability of HuH-1 cells by an average of 69.1% and that of HCT-116 cells by an average of 77.7% compared to cells treated with m-siNT.

[0141] ASPM-iI has been shown to critically regulate the activity of multiple developmental and oncogenic signaling pathways, including the Wnt, Hedgehog (Hh), and Notch pathways. We recently demonstrated that ASPM-iI also enhances the stability of Yes-associated protein (YAP) and PDZ-binding motif (TAZ), both coactivators of TEAD transcription factors, in cancer cells. To confirm that inhibiting the function of ASPM-i1 by transducing cells with siASPM-v1 API_V2 could suppress the activity of these aforementioned development- and stemness-related pathways in cancer cells, we infected HuH-1 or HCT-116 cells with a lentiviral vector carrying a triple luciferase reporter of Wnt, Hh, and Notch (pMuLE_EXPR_CMV-eGFP_TOP-NLuc1.1_12GLI-FLuc_CBF-GLuc, Addgene plasmid #113862) (Maier et al., 2019) or a lentiviral vector carrying the Hippo pathway transcription factor TEAD (BPS Bioscience #79833, San Diego, CA). To stimulate the activity of the Wnt, Hh, or Notch pathways, cells were treated with recombinant human WNT3A (250 ng / ml for 16 hours, R&D Systems, Minneapolis, MN), recombinant human sonic hedgehog (SHH, 3 μg / ml for 24 hours, Sigma-Aldrich), or recombinant human JAG1-Fc (5 μg / ml for 24 hours, Sigma-Aldrich, Steinheim, Germany), respectively. Wnt, Hh, Notch, and TEAD reporter activities in unstimulated or stimulated cells were then measured using the Nano-Glo® Luciferase Assay System (Wnt reporter), the ONE-Glo® Luciferase Assay System (Hh and TEAD reporters), and the Renilla-Glo® Luciferase Assay System (Notch reporter) (Promega, Madison, WI).As shown in Figure 28, transduction of HuH-1 cells with siASPM-v1 API_V2 significantly reduced the reporter activity of all tested pathways, including Wnt, Hh, Notch, and TEAD, confirming that inhibition of ASPM-v1 expression by siASPM-v1 API_V2 can effectively inhibit multiple oncogenesis- and development-related pathways in cancer cells.

[0142] The ability to form large three-dimensional sphere-like structures, or "tumorspheres," under serum-free and anchorage-independent conditions reflects the tumorigenic potential of cancer cells. Given that knockdown of ASPM-v1 expression significantly inhibited the transcriptional activity of developmental and stemness-related Wnt, Hh, and Notch pathways, as well as YAP / TEAD, we investigated whether treatment of cancer cells with siASPM-v1 API_V2 could inhibit their tumorsphere-forming ability. HuH-1 hepatocellular carcinoma cells or HCT-116 colorectal cancer cells were transduced with 100 nM siASPM-v1 API_V2 or m-siNT using Lipofectamine LTX reagent for 48 hours, after which the cells were plated at limiting dilutions (10,000, 1,000, 100, and 10 cells per well) into 24-well nonadherent culture plates. After 10 days, the presence of tumorspheres was assessed. Data from the limiting dilution assay were analyzed and plotted using ELDA software (http: / / bioinf.wehi.edu.au / software / elda / index.html). As shown in Figure 29, transduction of siASPM-v1 API_V2 into HhH-1 or HCT-116 cells significantly inhibited the tumorsphere-forming ability of HuH-1 or HCT-116 cells, highlighting its therapeutic potential in hepatocellular carcinoma and colorectal cancer.

Claims

1. 1) a polynucleotide comprising a nucleotide sequence complementary to the mRNA of the ASPM gene having the nucleotide sequence shown in SEQ ID NO: 1, or 2) a nucleotide sequence comprising a contiguous segment having at least 70%, at least 80%, or at least 90% sequence identity to the nucleotide sequence complementary to SEQ ID NO:

1.

2. The polynucleotide of claim 1, comprising a nucleotide sequence complementary to an mRNA encoded by exon 18 of transcript variant 1 of the human ASPM gene having the nucleotide sequence set forth in SEQ ID NO: 3, or a nucleotide sequence comprising a contiguous segment having at least 70%, at least 80%, or at least 90% sequence identity to the nucleotide sequence complementary to SEQ ID NO:

3.

3. 3. The polynucleotide of claim 1, which is an shRNA, an siRNA, or a dsRNA.

4. 4. The polynucleotide of claim 1, wherein the polynucleotide is an siRNA molecule, the siRNA molecule comprising (a) a double-stranded region and (b) zero or at least one overhanging region, each overhanging region containing six or fewer nucleotides, the double-stranded region consisting of a sense region and an antisense region, the sense region and the antisense region together forming the double-stranded region, the antisense region and the sense region each being 15 to 30 nucleotides in length, and the antisense region comprising a sequence that is the complement of a sequence selected from SEQ ID NO:

3.

5. The polynucleotide according to any one of claims 1 to 4, wherein the siRNA molecule has an antisense region and a sense region, each of which is 15 to 25 bases in length.

6. The polynucleotide of any one of claims 1 to 5, wherein the siRNA molecule is a chemically synthesized double-stranded siRNA molecule, (a) each strand of the double-stranded siRNA molecule is 15 to 30 nucleotides in length, and (b) one strand of the siRNA molecule comprises a sequence that is the complement of a sequence selected from SEQ ID NOs: 3, 5, 7, or 9.

7. The polynucleotide of any one of claims 1 to 6, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO: 4 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO: 5 by no more than 3 nucleotides.

8. The polynucleotide of any one of claims 1 to 7, wherein the siRNA molecule comprises a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 4, and the antisense strand comprising the nucleotide sequence set forth in SEQ ID NO:

5.

9. The polynucleotide of any one of claims 1 to 8, wherein the siRNA molecule described herein comprises a sense strand and an antisense strand that form separate double-stranded RNA duplexes, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO: 6 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from SEQ ID NO: 7 by no more than 3 nucleotides.

10. The polynucleotide of any one of claims 1 to 9, wherein the siRNA molecule comprises a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 6, and the antisense strand comprising the nucleotide sequence set forth in SEQ ID NO:

7.

11. The polynucleotide of any one of claims 1 to 10, wherein the siRNA molecule described herein comprises a sense strand and an antisense strand that form separate double-stranded RNA duplexes, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from SEQ ID NO: 8 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from SEQ ID NO: 9 by no more than 3 nucleotides.

12. The polynucleotide of any one of claims 1 to 11, wherein the siRNA molecule comprises a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 8, and the antisense strand comprising the nucleotide sequence set forth in SEQ ID NO:

9.

13. The polynucleotide of any one of claims 1 to 12, wherein the siRNA molecule has at least one overhang region or no overhang region.

14. The polynucleotide according to any one of claims 1 to 13, wherein one or both of the sense strand and the antisense strand may be further modified to form a modified siRNA.

15. 15. The polynucleotide of any one of claims 1 to 14, wherein the modified nucleotides include 2'-O-methyl modified nucleotides, 2'-fluorophosphoramidites, 3'-terminal deoxythymine nucleotides, non-natural bases that make up nucleotides, nucleotides containing 5' phosphorothioate groups, and terminal nucleotides linked with cholesteryl derivatives and dodecanoic acid bisdecylamide groups.

16. 16. The polynucleotide of any one of claims 1 to 15, wherein the modified siRNA comprises 10% to about 30% of nucleotides in the double-stranded region, comprises 2'-O-methyl (2'OMe) nucleotides, and comprises 2'OMe nucleotides in both strands of the modified siRNA.

17. a pool of siRNA molecules, comprising one or more of a first siRNA molecule or a modified siRNA molecule thereof, a second siRNA molecule or a modified siRNA molecule thereof, a third siRNA molecule or a modified siRNA molecule thereof, and a fourth siRNA molecule or a modified siRNA molecule thereof; the first siRNA molecule is a chemically synthesized double-stranded siRNA molecule, (a) each strand of the double-stranded siRNA molecule is 15 to 30 nucleotides in length, and (b) one strand of the siRNA molecule comprises a sequence that is the complement of a sequence selected from SEQ ID NO: 3; the second siRNA molecule is a chemically synthesized double-stranded siRNA molecule, (a) each strand of the double-stranded siRNA molecule is 15 to 30 nucleotides in length, and (b) one strand of the siRNA molecule comprises a sequence that is the complement of a sequence selected from SEQ ID NO: 5; the third siRNA molecule is a chemically synthesized double-stranded siRNA molecule, (a) each strand of the double-stranded siRNA molecule is 15 to 30 nucleotides in length, and (b) one strand of the siRNA molecule comprises a sequence that is the complement of a sequence selected from SEQ ID NO: 7; The fourth siRNA molecule is a chemically synthesized double-stranded siRNA molecule, (a) each strand of the double-stranded siRNA molecule is 15 to 30 nucleotides in length, and (b) one strand of the siRNA molecule comprises a sequence that is the complement of a sequence selected from SEQ ID NO:

9.

18. 18. The pool of claim 17, comprising one or more of a second siRNA molecule or a modified siRNA molecule thereof, a third siRNA molecule or a modified siRNA molecule thereof, and a fourth siRNA molecule or a modified siRNA molecule thereof.

19. the pool comprises one or more of a second siRNA molecule or a modified siRNA molecule thereof, a third siRNA molecule or a modified siRNA molecule thereof, and a fourth siRNA molecule or a modified siRNA molecule thereof; the second siRNA molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 4 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 5; the third siRNA molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 6 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 7; The pool of claim 17, wherein the fourth siRNA molecule comprises a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 8, and the antisense strand comprising the nucleotide sequence set forth in SEQ ID NO:

9.

20. 17. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 16 and a pharmaceutical carrier, diluent and / or adjuvant.

21. 17. A nanoparticle comprising the polynucleotide of any one of claims 1 to 16, and a lipid nanoparticle (LNP), a liposome, a micelle, a virosome, a nucleic acid complex, and any mixture thereof.

22. A method for inhibiting the growth, local-regional spread, and distant metastasis of malignant tumors and / or treating solid tumors and / or tumor metastases in a subject, the method comprising administering to the subject a polynucleotide described in any one of claims 1 to 16, a pool described in claim 17 or 18, a pharmaceutical composition described in claim 20, or a nanoparticle described in claim 21.

23. 23. The method of claim 22, wherein the solid cancer is hepatocellular carcinoma, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, and lung cancer.

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