SiRNA for inhibiting AKT1 gene expression and application thereof

Abstract

The present disclosure provides a double-stranded RNA (dsRNA) inhibitor for inhibiting expression of human serine / threonine kinase 1 (AKT1), a lipid composition comprising the dsRNA inhibitor, and uses thereof. The present disclosure also provides the use of a dsRNA inhibitor or a lipid composition comprising the dsRNA inhibitor in combination with abiraterone for the treatment of cancer.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of medicine, in particular to double-stranded RNA (dsRNA) inhibitors, lipid compositions thereof and uses thereof. BACKGROUND

[0002] Serine / threonine kinase Akt (also known as protein kinase B) plays a very important role in the classical signaling pathway PI3K-AKT-mTOR. Akt signaling is a central pathway that regulates metabolism, proliferation, cell survival and angiogenesis in response to various extracellular signals. Current research has found that the AKT family has three highly homologous subtypes, namely AKT1 (PKBα), AKT2 (PKBβ) and AKT3 (PKBγ). The three subtypes of AKT are composed of an N-terminal pleckstrin homology (PH) domain separated by a 39-amino acid hinge region, a large central kinase domain, and a C-terminal regulatory domain (RD). Among them, the PH domain has about 60% homology, while the homology of the kinase domain is more than 85%. Each AKT member has a wide variety of downstream effects in different environments under the stimulation of various extracellular stimuli. Among them, AKT1 and AKT2 are more widely distributed in various cells. AKT1 is involved in cell survival pathways by inhibiting the process of apoptosis, can prevent apoptosis and promote cell survival, and is considered a major factor in many types of cancer. In various cancerous tissues, the expression of AKT is significantly increased. In more than 40% of human liver cancer cases, the Akt signaling pathway is activated, and the expression of PTEN (a negative regulator of Akt) is inhibited; studies have shown that the level of pAKT (phosphorylated AKT) protein in non-small cell lung cancer cells is related to the occurrence and progression of lung cancer, and the expression level in lung cancer cells is significantly higher than that in normal tissues, and is related to lymph node metastasis and differentiation degree; colorectal cancer and prostate cancer are more common malignant tumors, and studies have found that there is overexpressed AKT protein in these two cancer tissues, which is positively correlated with the degree of cancer progression and can significantly promote the invasion and metastasis of cancer cells. The role of AKT in tumor cell growth may be related to its ability to promote angiogenesis. The PI3K / Akt signaling pathway is one of the main mitogenic signaling pathways, which can be activated by receptor tyrosine kinases, causing downstream protein mTOR phosphorylation, and then regulating the synthesis of various proteins related to angiogenesis, thereby achieving the purpose of regulating angiogenesis. Abundant blood vessels can provide nutrients for tumor cells, leading to an accelerated growth rate of tumors, and also cause increased vascular permeability, promoting cell migration, i.e. tumor cell metastasis.

[0003] Intrinsic or acquired drug resistance has been the main reason for the failure of cancer drug therapy and ultimately leads to treatment failure. Therefore, with the continuous exploration of the underlying mechanism, finding more promising drug targets is becoming the best treatment for cancer prevention and treatment. The abnormal activation of the PI3K / AKT pathway and the upstream or downstream target transduction play a crucial role as an important signaling pathway responsible for drug resistance of various tumor formation. Given the key role of Akt in cancer development, progression, and even subsequent treatment, the development of drugs targeting Akt has always been a research hotspot. Early small molecule inhibitors can be divided into PH domain inhibitors, allosteric inhibitors and ATP competitive inhibitors according to the structure of Akt and the binding site of the inhibitor. The PH domain of Akt binds to PIP3, and the PH domain inhibitor binds to the PH domain, which can prevent the translocation of Akt to the cell membrane and the phosphorylation of PDK1 and mTOR2; allosteric inhibitors do not directly act on the ATP binding site or the PH domain, but bind to a unique hydrophobic region at the junction of the PH domain and the kinase domain, stabilize the Akt "PH-in" conformation, block the phospholipid binding site, and block the activation of Akt; ATP competitive inhibitors can bind to the ATP site of the kinase domain, showing ATP competitive inhibition. PH domain inhibitors and allosteric inhibitors have failed to achieve the desired effect in clinical research, while ATP competitive inhibitors with more drug potential are difficult to find specific kinase inhibitors due to the high structural similarity of the ATP binding sites of the AGC kinase family, and the sequence homology of the kinase domains of the three subtypes of Akt is more than 80%. The ATP competitive inhibitors currently in the clinical stage cannot distinguish between the different subtypes of Akt, for example, the new drug Akt inhibitor Capivasertib of AstraZeneca can target and inhibit three subtypes of Akt (Akt1 / 2 / 3), and the development of ATP competitive inhibitors that selectively inhibit specific subtypes of Akt is also a goal to be achieved in the future. SUMMARY

[0004] The present disclosure provides double-stranded RNA (such as siRNA) capable of specifically targeting and silencing the corresponding gene of human serine / threonine kinase 1 (AKT1), which reduces the expression level of AKT1 protein. Using the siRNA of the present disclosure can achieve precise targeting of Akt1, exclude interference from different subtypes, and specifically inhibit the expression of Akt1 in tumor cells. In some embodiments, the gene silencing efficiency can be improved and the drug efficacy can be improved by methods such as sequence screening, optimization, etc. of siRNA for inhibiting AKT1 protein expression. The present disclosure provides siRNA of the present disclosure encapsulated by nano-lipid particles (LNP), which on the one hand reduces the risk of degradation of siRNA in vivo, and on the other hand can improve the delivery efficiency, further improve the siRNA silencing efficiency, and achieve the purpose of inhibiting tumor growth.

[0005] In a first aspect, the present disclosure provides a double-stranded RNA (dsRNA) inhibitor for inhibiting expression of human AKT1, wherein the dsRNA inhibitor comprises a sense strand and an antisense strand, the sense strand and the antisense strand form a double-stranded region, and the antisense strand comprises a sequence complementary to a human AKT1 mRNA sequence. In some preferred embodiments, the dsRNA inhibitor is a small interfering RNA (siRNA). In some preferred embodiments, the AKT1 mRNA sequence is set forth in NCBI Gene ID: 207.

[0006] In a second aspect, the present disclosure provides a lipid composition comprising an active ingredient and a lipid component, wherein the active ingredient comprises the dsRNA inhibitor of the first aspect of the present disclosure. In some preferred embodiments, the lipid composition of the present disclosure is selected from one or more of the following lipid compositions: a cationic polymer, a nano-lipid particle (LNP), a cationic liposome, a QTsome, a lipopolyplex, a microparticle, a microsphere, a nanoemulsion. A QTsome is a lipid nanoparticle coexisting with a Quaternary and Tertiary cationic phospholipid.

[0007] In a third aspect, the present disclosure provides a pharmaceutical composition comprising the dsRNA inhibitor of the first aspect of the present disclosure or the lipid composition of the second aspect of the present disclosure, and one or more pharmaceutically acceptable carriers or excipients.

[0008] In a fourth aspect, the present disclosure provides a vector comprising a nucleotide sequence encoding the dsRNA inhibitor of the first aspect of the present disclosure.

[0009] In a fifth aspect, the present disclosure provides a host cell comprising the dsRNA inhibitor of the first aspect of the present disclosure, the lipid composition of the second aspect of the present disclosure, or the vector of the fourth aspect of the present disclosure.

[0010] In a sixth aspect, the present disclosure provides a method of treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the dsRNA inhibitor of the first aspect of the present disclosure, the lipid composition of the second aspect of the present disclosure, or the pharmaceutical composition of the third aspect of the present disclosure.

[0011] In a seventh aspect, the present disclosure provides use of the dsRNA inhibitor of the first aspect, the lipid composition of the second aspect, or the pharmaceutical composition of the third aspect in the manufacture of a medicament for treating or preventing a disease.

[0012] In an eighth aspect, the present disclosure provides a method of reducing AKT1 expression, comprising administering to a sample or subject a dsRNA inhibitor of the first aspect of the present disclosure, a lipid composition of the second aspect of the present disclosure, or a pharmaceutical composition of the third aspect of the present disclosure.

[0013] In a ninth aspect, the present disclosure provides a kit comprising a dsRNA inhibitor, a lipid composition, or a pharmaceutical composition provided herein, and a second therapeutic agent.

[0014] In a tenth aspect, the present disclosure provides a method of preparing a lipid composition described herein, comprising the following steps (a)-(d): (a) taking an active component, and formulating the active component with a buffer to obtain an active component buffer; (b) taking a lipid component, and formulating the lipid component with an organic solvent to obtain a lipid solution; (c) mixing the lipid solution and the active component buffer at a mass ratio of the lipid component to the active component of (8-12): 1; and (d) removing the organic solvent, diluting, and adjusting the pH to 6.0-8.0. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A Western-blot in vitro test of the efficiency of siAktl in knocking down the target gene Aktl in different cells is provided in Example 1.

[0016] Figure 2 A column chart of the efficiency of siAktl in knocking down Aktl in different tumor cells in Example 1 is provided.

[0017] Figure 3 A line chart of the average body weight of mice over time after the mice were administered physiological saline, a blank control, and siAktl in Example 2 is provided.

[0018] Figure 4 A line chart of the tumor growth inhibition rate of mice over time after the mice were administered a blank control and siAktl in Example 2 is provided.

[0019] Figure 5 A schematic diagram of the administration regimen of mice in Example 3 is provided.

[0020] Figure 6 A line chart of the average body weight of mice over time after the mice were administered physiological saline, LNP-encapsulated siAktl, abiraterone, LNP-encapsulated siAKTl, and abiraterone in Example 3 is provided.

[0021] Figure 7 A line chart of the tumor growth inhibition rate of mice over time after the mice were administered LNP-encapsulated siAktl, abiraterone, LNP-encapsulated siAKTl, and abiraterone in Example 3 is provided. DETAILED DESCRIPTION

[0022] Unless otherwise stated, the terminology used herein has the common meaning understood by one of ordinary skill in the art. It may vary for those skilled in the art depending on the desired properties and effects sought through this application, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or as understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the experimental procedures in organic chemistry, medicinal chemistry, and biology described herein are well-known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Where multiple definitions exist for terms used herein, the definitions in this section shall prevail unless otherwise stated.

[0023] Unless otherwise stated, all figures used in this specification and claims to indicate content, concentration, proportion, weight, particle size, percentage, technical effect, etc., shall in any event be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. “About” or “approximately” can be understood to be within a range of plus or minus 5% to 10% of the indicated values.

[0024] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0025] As used herein, the terms “include,” “contain,” or “comprising” mean that in addition to having the listed elements, other elements are not excluded.

[0026] As used herein, the term "chain containing a sequence" refers to an oligonucleotide comprising a single nucleotide chain, the nucleotide chain being a sequence referred to using standard nucleotide nomenclature.

[0027] The letters “G”, “C”, “A”, “T”, and “U”, whether modified or unmodified, typically represent nucleotides containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively. The letter “dT”, whether modified or unmodified, typically represents a nucleotide containing deoxythymine as a base.

[0028] As used herein, "functional variant" refers to a variant of the indicated nucleotide sequence, but which retains substantially the same function as the original nucleotide sequence. For example, a sense strand variant of an siRNA provided herein binds in complementary association with the original antisense strand and / or antisense strand variant; an antisense strand variant of an siRNA provided herein binds in complementary association with the target gene (mRNA of Aktl); and / or a sense strand variant of an siRNA provided herein, or the original sense strand, forms a double-stranded RNA with an siRNA antisense strand variant or original antisense strand, and inhibits expression of Aktl protein. Those skilled in the art will appreciate that guanine, cytosine, adenine, thymine and uracil can be replaced by other molecules without materially altering the molecule. For example, a nucleotide with an inosine base can base pair with a nucleotide containing adenine, cytosine or uracil, but is not limited thereto. Thus, in the dsRNA nucleotide sequences of the disclosure, a nucleotide containing uracil, guanine or adenine can be replaced by a nucleotide containing inosine, and the like. As another example, adenine and cytosine at any position in an oligonucleotide can be replaced by guanine and uracil, respectively, to form a G-U Wobble base pair with a target mRNA.

[0029] As used herein, "short interfering RNA (siRNA)" comprises a sense strand and an antisense strand, in some cases with characteristic overhangs (Bernstein et al. (2001) Nature 409:363). siRNA is incorporated into an RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA duplex, allowing the antisense strand to recognize a target by complementarity (Nykanen et al. (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in RISC cleave the target mRNA, inducing silencing (Elbashir et al. (2001) Genes Dev. 15:188).

[0030] The term "double-stranded RNA (dsRNA)" refers to a complex of ribonucleic acid molecules having a double-stranded structure, consisting of two antiparallel and substantially complementary nucleic acid strands, having "sense" and "antisense" orientation with respect to a target RNA (e.g., Aktl mRNA), the substantially complementary nucleic acid strands being referred to as the sense strand (also referred to as the positive strand) and the antisense strand, respectively. In some embodiments of the disclosure, double-stranded RNA (e.g., siRNA) triggers degradation of a target RNA (e.g., Aktl mRNA). Generally, a dsRNA molecule can include ribonucleotides, but as detailed herein, either or both strands can also include one or more non-ribonucleotides, such as deoxynucleotides, modified nucleotides. Further, as used herein, "double-stranded RNA" can include modified ribonucleotides.

[0031] As used herein, the term "modified nucleotide" refers to a nucleotide having a modified sugar, a modified internucleotide linkage, or a modified nucleobase. Thus, the term "modified nucleotide" includes substitution, addition, or removal of an internucleotide linkage, sugar group, or nucleobase, e.g., substitution, addition, or removal of a functional group or atom. Modifications suitable for the RNA of the present disclosure include all types of modifications disclosed herein or known in the art.

[0032] As described herein, unless otherwise indicated, the listed nucleotide sequences are in the 5' to 3' direction, left to right.

[0033] dsRNA inhibitor

[0034] The present disclosure provides a dsRNA inhibitor for inhibiting the expression of AKT1, wherein the dsRNA inhibitor comprises a sense strand and an antisense strand, the sense strand and the antisense strand form a double-stranded region, and the antisense strand comprises a sequence complementary to an AKT1 nucleic acid sequence (e.g., mRNA sequence). In some preferred embodiments, the dsRNA inhibitor is an siRNA. In some preferred embodiments, the AKT1 nucleic acid sequence is set forth in NCBI Gene ID: 207.

[0035] In some embodiments, the dsRNA inhibitor provided by the present disclosure comprises a sequence of any one of the following (a)-(c) groups:

[0036] (a) the antisense strand of the dsRNA inhibitor comprises a modified or unmodified nucleotide sequence as set forth in UCACACCACCUGACCAAGA (SEQ ID NO: 1) or a modified or unmodified functional variant thereof having 1, 2, or 3 different nucleotides from the nucleotide sequence set forth in UCACACCACCUGACCAAGA;

[0037] (b) the sense strand of the dsRNA inhibitor comprises a modified or unmodified nucleotide sequence as set forth in UCUUGGUCAGGUGGUGUGA (SEQ ID NO: 2) or a modified or unmodified functional variant thereof having 1, 2, or 3 different nucleotides from the nucleotide sequence set forth in UCUUGGUCAGGUGGUGUGA;

[0038] (c) the antisense strand of the dsRNA inhibitor comprises a modified or unmodified sequence of nucleotides as shown in UCACACCACCUGACCAAGA or a modified or unmodified functional variant thereof having 1, 2, or 3 different nucleotides from the sequence of nucleotides as shown in UCACACCACCUGACCAAGA, and the sense strand of the dsRNA inhibitor comprises a modified or unmodified sequence of nucleotides as shown in UCUUGGUCAGGUGGUGUGA or a modified or unmodified functional variant thereof having 1, 2, or 3 different nucleotides from the sequence of nucleotides as shown in UCUUGGUCAGGUGGUGUGA.

[0039] In some embodiments, the modification of the antisense strand and / or the sense strand comprised in the dsRNA inhibitor provided by the present disclosure comprises one or more of the following: replacing P=O linkage between all or part of two adjacent nucleotides in the sequence with P=S linkage, replacing all or part of deoxyribonucleosides in the sequence with ribonucleosides, replacing all or part of ribonucleosides in the sequence with deoxyribonucleosides, replacing the group at 2' position in all or part of the sugar ring of nucleotides in the sequence with methoxy, methoxyethyl, amino, alkyl, or halogen, bridging between the 2nd and 4th carbon atoms of all or part of the sugar ring of nucleotides in the sequence to form a locked nucleic acid or (S)-cEt-BNA, comprising one or more abasic nucleotides, morpholino nucleotides, phosphoramidate nucleotides, and / or nucleotides comprising non-natural bases in the sequence, comprising at least one of phosphothioate, alkylphosphonate, alkylphosphonate, alkenylphosphonate, vinylphosphonate RNA end modification at the 5' end of the RNA strand. In some preferred embodiments, the sense strand of the dsRNA inhibitor provided by the present disclosure comprises U*C*UUGGUCAGGUGGUGUGA (SEQ ID NO: 3), and / or the antisense strand comprises U*C*ACACCACCUGACCAAGA (SEQ ID NO: 4), wherein * represents a phosphorothioate linkage between its adjacent two nucleotides.

[0040] In some embodiments, to improve the stability of siRNA in cells, the phosphorothioate backbone modification can comprise: replacing P=O linkage between part or all of the nucleotides with P=S linkage in the direction from 5' end to 3' end.

[0041] In some embodiments, to improve the stability of siRNA, to enhance the recognition of the antisense strand by the RNA-induced silencing complex and to reduce off-target rate, the modification of the nucleotide sequence of siRNA can comprise: methoxy modification at the 2' position of the sugar ring of nucleotides in the direction from 5' end to 3' end.

[0042] In some embodiments, the dsRNA inhibitor provided by the present disclosure comprises 1, 2, or 3 mismatched base pairs between the antisense strand and the sense strand.

[0043] In some embodiments, the dsRNA inhibitors provided by the present disclosure comprise a nucleotide overhang at either or both ends (e.g., 5' end and / or 3' end) of at least one of the RNA single strands (e.g., the sense strand and / or the antisense strand). In some preferred embodiments, the dsRNA inhibitors provided by the present disclosure comprise a nucleotide overhang at either or both ends of both of the RNA strands (e.g., the sense strand and the antisense strand). In some preferred embodiments, the dsRNA inhibitors provided by the present disclosure comprise a nucleotide overhang at the 3' end of the sense strand and a nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the nucleotide overhang comprises 2 to 5 nucleotides. In some preferred embodiments, the nucleotide overhang comprises at least one of dTdT, UU, UC, CU. In some embodiments, the modification of the nucleotide sequence can comprise a deoxyribonucleotide dTdT overhang at the 3' end of the sense strand and the antisense strand. In some embodiments, the modification of the nucleotide sequence can comprise a deoxyribonucleotide UU overhang at the 3' end of the sense strand and the antisense strand. In some embodiments, the antisense strand in the dsRNA inhibitors provided by the present disclosure comprises UCACACCACCUGACCAAGA(dT)(dT) (SEQ ID NO: 5) and / or the sense strand comprises UCUUGGUCAGGUGGUGUGA(dT)(dT) (SEQ ID NO: 6). In some embodiments, the antisense strand in the dsRNA inhibitors provided by the present disclosure comprises U*C*UUGGUCAGGUGGUGUGA*(dT)*(dT) (SEQ ID NO: 7), and / or the antisense strand comprises U*C*ACACCACCUGACCAAGA*(dT)*(dT) (SEQ ID NO: 8), wherein * represents a phosphorothioate bond between its adjacent two nucleotides.

[0044] The siRNAs provided by the present disclosure can specifically target and silence the corresponding genes, and reduce the expression level of the corresponding proteins. The use of the siRNAs provided by the present disclosure can achieve precise targeting of Akt1, exclude the interference of different subtypes, specifically inhibit the expression of Akt1 in tumor cells, and improve the gene silencing efficiency and drug efficacy through sequence screening, optimization, etc.

[0045] Lipid composition

[0046] The present disclosure provides a lipid composition comprising an active component and a lipid component, wherein the active component comprises a dsRNA inhibitor provided by the present disclosure. In some embodiments, the lipid composition of the present disclosure is selected from one or more of the following lipid compositions: a cationic polymer, a nano-lipid particle (LNP), a cationic liposome, a QTsome, a lipopolyplex, a microparticle, a microsphere, a nanoemulsion. In some embodiments, the dsRNA inhibitor provided by the present disclosure is encapsulated by a QTsome. In some embodiments, the dsRNA inhibitor provided by the present disclosure is encapsulated by an LNP. In some embodiments, the lipid component of the lipid composition of the present disclosure comprises one or more of an ionizable lipid, a neutral phospholipid, a cholesterol, and a PEG lipid. In some preferred embodiments, the mass ratio of the lipid component to the active component in the lipid composition provided by the present disclosure is (8-12): 1, for example, 10: 1.

[0047] The ionizable lipid includes, but is not limited to, MC3, A066, SM-102, DODMA (1,2- dioleyl-3-dimethylamino-propane, CAS: 104162-47-2), ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA, DLinDMA class, DLin-K-DMA class. The neutral phospholipid includes, but is not limited to, 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), dioleoyl L-a-phosphatidyl ethanolamine (DOPE, CAS: 4004-05-1), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC, CAS: 4235-95-4), 1-hexadecan 2-(cis-9-octadecenoyl)-sn-glycero-3-phosphocholine (POPC), egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC, CAS: 816-94-4), HPSC. The PEG lipid includes, but is not limited to, DSG-PEG, DMG-PEG.

[0048] In some embodiments, the ionizable lipid of the present disclosure is selected from at least one of DODMA, MC3, SM-102, A066, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA, DLinDMA class, DLin-K-DMA class, the neutral phospholipid is selected from at least one of DPPC, DSPC, HPSC, EPC, DOPC, DOPE, POPC, and the PEG lipid is selected from at least one of DSG-PEG, DMG-PEG. In some preferred embodiments, the lipid component of the lipid composition of the present disclosure comprises MC3, DSPC, cholesterol, and PEG-DSG.

[0049] In some embodiments, the molar ratio of ionizable lipid, neutral phospholipid, cholesterol, and PEG lipid in the lipid composition of the present disclosure is (10-60):(10-60):(15-50):(0-10), preferably the molar ratio of ionizable lipid, neutral phospholipid, cholesterol, and PEG lipid is 45:27.5:20:7.5.

[0050] The lipid composition provided by the present disclosure reduces the risk of degradation of siRNA in vivo on the one hand, and improves the delivery efficiency on the other hand, further enhances the silencing efficiency of siRNA, and achieves the purpose of inhibiting tumor growth.

[0051] Pharmaceutical composition

[0052] The present disclosure provides a pharmaceutical composition comprising the dsRNA inhibitor or the lipid composition of the present disclosure, and one or more pharmaceutically acceptable carriers or excipients.

[0053] In some embodiments, the pharmaceutical composition of the present disclosure further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from one or more of nucleic acid drugs, small molecule drugs, polypeptides, proteins, antibodies, chemical drugs, chemotherapy drugs. In some embodiments, the second therapeutic agent is selected from one or more of androgen inhibitors, for example CYP17 enzyme inhibitors, such as abiraterone. In some embodiments, the second therapeutic agent is selected from one or more of polyinosinic acid-polycytidylic acid (Poly(I:C)), STING agonists, imiquimod, resiquimod, gardiquimod, sorafenib, sunitinib, erlotinib, doxorubicin, paclitaxel, gemcitabine, radiotherapy. In some embodiments, the second therapeutic agent comprises abiraterone and prednisone.

[0054] In some embodiments, the antibody includes, but is not limited to, an antibody with anti-tumor effect. The antibody with anti-tumor effect includes, but is not limited to, an antibody targeting PD-1, such as nivolumab, pembrolizumab, cemiplimab, toripalimab, sintilimab, camrelizumab, etc.; an antibody targeting PD-L1, such as atezolizumab, avelumab, and durvalumab, etc.; an antibody targeting CTLA4, such as ipilimumab, tremelimumab, etc. In some embodiments, the antibody includes, but is not limited to, an anti-tumor associated antigen (TAA) antibody. The TAA includes, but is not limited to, GPC, CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, EGFR, Ep-CAM, EphA3, Her2, Her3, ROR2, PSMA, STEAP1, FGFR2, TROP2, B7-H3, B7-H4, B7-H6, FOLR1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, SSX-2, fibronectin, MART-2, PDL-1, PD-1, CTLA4, VEGFR, CLAUDIN, etc.

[0055] In some embodiments, the chemotherapeutic drug includes, but is not limited to, doxorubicin, paclitaxel, gemcitabine, radiotherapy.

[0056] In some embodiments, the small molecule drug or nucleic acid drug includes, but is not limited to, a pattern recognition receptor agonist, an anthracycline, a camptothecin, an immunogenic cell death (ICD) inducer, a tyrosine kinase inhibitor, a taxane, a Bruton's tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 / PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, an AKT1 inhibitor, an EGFR inhibitor, a VEGF inhibitor, a PARP inhibitor Her2, LAG-3, TNFR2 inhibitor, a corticosteroid drug (such as prednisone), etc.

[0057] In some embodiments, the anthracycline includes, but is not limited to, doxorubicin, epirubicin, pirarubicin, daunorubicin, aclarubicin, idarubicin, amrubicin, etc.

[0058] In some embodiments, the camptothecin class of compounds includes, but is not limited to, topotecan, irinotecan, belotecan, exatecan (DX-8951), lurtotecan, sinotecan, rubitecan (9-NC), 9-aminocamptothecin (9-AC), gimatecan, karenitecin, DB-67, and the like.

[0059] In some embodiments, the tyrosine kinase inhibitor includes, but is not limited to, sorafenib, sunitinib, gefitinib, erlotinib, lapatinib, afatinib, dacomitinib, vandetanib, neratinib, pelitinib (EKB-569), canertinib (CI-1033), osimertinib, rociletinib (CO-1686, Clovis Oncology), olmutinib (HM61713, Hanmi Pharmaceutical), naquotinib (ASP8273, AstraZeneca), tesevatinib (XL647 / KD019, Kadmon Corporation), nazartinib (EGF816, Novartis), and PF-06747775 (Pfizer), and the like.

[0060] In some embodiments, the immunogenic cell death inducer includes, but is not limited to, a DNA damaging agent, bleomycin, and the like.

[0061] In some embodiments, the taxane class of compounds includes, but is not limited to, paclitaxel, docetaxel, and the like.

[0062] In some embodiments, the pattern recognition receptor agonist includes, but is not limited to, Toll-like receptors, RIG-I-like receptors, Nod-like receptors, AIM2-like receptors, and C-type lectin receptors, and cGas and other intracellular DNA sensor agonists.

[0063] In some embodiments, the dsRNA inhibitor, lipid composition, or pharmaceutical composition provided by the present disclosure is administered by one or more of the following routes: intratumoral injection, intravenous injection, subcutaneous injection, intramuscular injection, intra-arterial injection, intraperitoneal injection, intracerebral injection, intracavity perfusion (e.g., intravesical perfusion, gavage), interventional therapy, oral, transdermal, transpulmonary, ocular, and topical administration.

[0064] As used herein, the term "pharmaceutical composition" refers to a mixture of the dsRNA inhibitor or lipid composition provided by the disclosure with other chemical components, such as vehicles, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the dsRNA inhibitor or lipid composition to an organism. Multiple ways exist in the art for administering a dsRNA inhibitor, lipid composition, or pharmaceutical composition provided in the disclosure, including, but not limited to, intratumoral injection, intravenous injection, subcutaneous injection, intramuscular injection, arterial injection, intracavitary infusion (e.g., intravesical infusion, gavage), intraperitoneal injection, intracerebral injection, such as intrathecal injection, interventional therapy, oral, transdermal, transpulmonary, ocular, and topical administration.

[0065] As used herein, the pharmaceutical composition can be configured in a dosage form suitable for administration to a subject by the desired route of delivery. The dosage forms include, but are not limited to, tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalers, creams, ointments, lotions, pastes, sprays, lyophilized, injectables, and gels, among others.

[0066] The term "pharmaceutically acceptable carrier" includes a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the dsRNA inhibitor or lipid composition within an subject to an subject such that it can perform its intended function. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laureate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethylene glycol; phosphate buffer solutions; diluents; granulating agents; lubricants; binders; disintegrating agents; wetting agents; emulsifying agents; colorants; demulcents; coatings; sweetening agents; flavoring agents; perfuming agents; preservatives; antioxidants; plasticizers; jelling agents; thickening agents; stiffening agents; texturizers; suspending agents; surface active agents; humectants; carriers; stabilizers; and other nontoxic compatible substances used in pharmaceutical formulations, or any combination thereof.

[0067] Vectors, host cells

[0068] The present disclosure provides vectors comprising a nucleotide sequence encoding a dsRNA inhibitor of the present disclosure.

[0069] As used herein, the term "vector" refers to a nucleic acid molecule that carries genetic material into another cell and replicates and / or expresses it therein. In certain embodiments, the vector is an expression vector. In certain embodiments, the vector is a viral vector, such as a lentiviral vector. In certain embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.

[0070] The present disclosure provides host cells comprising a vector, a dsRNA inhibitor, or a lipid composition of the present disclosure.

[0071] As used herein, the host cell can be an animal cell (e.g., a mammalian cell (such as a mouse cell, a rat cell, a monkey cell, a dog cell, a human cell, etc.)), a plant cell, a yeast cell, and / or a bacterial cell. The host cell can be an in vivo cell and / or an in vitro cell. In some embodiments, the host cell is a cancer cell, such as a human liver cancer cell (e.g., Hep G2, Hep 3B, SNU 387), a mouse liver cancer cell (e.g., Hepal-6), a mouse prostate cancer cell (e.g., LNCaP, PC-3). In some embodiments, the host cell is a tissue cell (e.g., a liver cell, a heart cell, a spleen cell, a lung cell, a kidney cell, etc.). In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0072] Medical uses and methods

[0073] The present disclosure provides methods of treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a dsRNA inhibitor, a lipid composition, or a pharmaceutical composition of the present disclosure.

[0074] In some embodiments, the dsRNA inhibitor, the lipid composition, or the pharmaceutical composition provided by the present disclosure is administered by one or more of the following routes: intratumoral injection, intravenous injection, subcutaneous injection, intramuscular injection, intraarterial injection, intraperitoneal injection, intracerebral injection, intracavity perfusion (e.g., intravesical perfusion, gavage), interventional therapy, oral, transdermal, transpulmonary, ocular, and topical administration.

[0075] In some embodiments, the dsRNA inhibitor, the lipid composition or the pharmaceutical composition of the present disclosure is co-administered with other therapeutic agents. In some embodiments, the dsRNA inhibitor, the lipid composition or the pharmaceutical composition of the present disclosure is administered prior to administration of other therapeutic agents. In some embodiments, the dsRNA inhibitor, the lipid composition or the pharmaceutical composition of the present disclosure is administered after administration of other therapeutic agents. In some embodiments, the dsRNA inhibitor, the lipid composition or the pharmaceutical composition of the present disclosure is administered concurrently with other therapeutic agents. In some embodiments, the dsRNA inhibitor, the lipid composition or the pharmaceutical composition of the present disclosure is administered by the same or different route as other therapeutic agents.

[0076] In some embodiments, the other therapeutic agent is selected from one or more of nucleic acid drugs, small molecule drugs, polypeptides, proteins, antibodies, chemical drugs, chemotherapy drugs. In some embodiments, the other therapeutic agent is selected from one or more of nucleic acid drugs, small molecule drugs, polypeptides, proteins, antibodies, chemical drugs, chemotherapy drugs. In some embodiments, the other therapeutic agent is selected from one or more of androgen inhibitors, for example CYP17 enzyme inhibitors, such as abiraterone. In some embodiments, the other therapeutic agent is selected from one or more of polyinosinic acid: polycytidylic acid (Poly(I:C)), STING agonists, imiquimod, resiquimod, gardiquimod, sorafenib, sunitinib, erlotinib, doxorubicin, paclitaxel, gemcitabine, radiotherapy. In some embodiments, the other therapeutic agent comprises abiraterone and prednisone.

[0077] The present disclosure provides use of the dsRNA inhibitor, the lipid composition or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating or preventing a disease.

[0078] As used herein, the disease includes, but is not limited to, AKT1 expression related diseases, angiogenesis related diseases, for example solid tumors. Preferably, the solid tumor is selected from one or more of head and neck cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, thyroid cancer, lung cancer, gastric cancer, liver cancer, pancreatic cancer, spleen cancer, non-small cell lung cancer, colorectal cancer, colon cancer, intestinal cancer, kidney cancer, brain tumor, glioma, bladder cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, uterine cancer, bone cancer, osteosarcoma, chondrosarcoma, liposarcoma, neuroblastoma, synovial sarcoma, astrocytoma, glioblastoma multiforme, anaplastic astrocytoma, peritoneal cavity cancer, soft tissue sarcoma, sarcoma, rhabdomyosarcoma, advanced mucinous disease, melanoma, skin cancer.

[0079] The present disclosure provides methods of reducing AKT1 expression comprising administering to a sample or subject a dsRNA inhibitor, lipid composition, or pharmaceutical composition of the present disclosure. In some embodiments, the methods of reducing AKT1 expression provided by the present disclosure can be in vivo or in vitro methods. In some embodiments, the methods of reducing AKT1 expression of the present disclosure are in vitro methods comprising administering to an in vitro sample a dsRNA inhibitor, lipid composition, or pharmaceutical composition of the present disclosure. In some embodiments, the methods of reducing AKT1 expression of the present disclosure are in vitro, non-therapeutic methods comprising administering to an in vitro sample a dsRNA inhibitor, lipid composition, or pharmaceutical composition of the present disclosure.

[0080] As used herein, the term "subject" includes an animal, such as a vertebrate, preferably a mammal, such as a dog, cat, pig, cow, sheep, horse, rodent (e.g., mouse, rat, or guinea pig), or primate (e.g., gorilla, chimpanzee, and human).

[0081] As used herein, the term "treatment" refers to alleviating or ameliorating a disease or disorder (i.e., slowing or arresting the development of a disease or at least one of its clinical symptoms); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder.

[0082] As used herein, the term "therapeutically effective dose" refers to an amount that results in a benefit or treatment of a disease as compared to a corresponding subject that does not receive the amount, but that is low enough to avoid serious side effects, within the scope of sound medical judgment. The therapeutically effective dose of a dsRNA inhibitor, lipid composition, or pharmaceutical composition described herein will vary with the dsRNA inhibitor, lipid composition, or pharmaceutical composition chosen; the route of administration; the severity of the disease being treated; the age, size, body weight, and physical condition of the patient being treated; the medical history of the patient being treated; the duration of treatment; the nature of concurrent therapy; the desired therapeutic effect; and like factors, but can nevertheless be determined in a routine manner by one of ordinary skill in the art.

[0083] As used herein, the term "prevention" includes providing prophylaxis against the occurrence or recurrence of a disease in an individual who can be predisposed to the disease but has not yet been diagnosed with the disease.

[0084] As used herein, the term "prophylactically effective dose" refers to an amount that can prevent a disease as compared to a corresponding subject that does not receive the amount, but that is low enough to avoid serious side effects, within the scope of sound medical judgment. The prophylactically effective dose described herein will vary with the lipid composition or pharmaceutical composition chosen; the route of administration; the severity of the disease being prevented; the age, size, body weight, and physical condition of the subject; the desired prophylactic effect; and like factors, but can nevertheless be determined in a routine manner by one of ordinary skill in the art.

[0085] As used herein, “sample” includes, but is not limited to, a bodily fluid sample of a subject, such as a blood sample, a saliva sample; a tissue sample; a lesion sample, a tumor sample, an organ sample, a heart sample, a liver sample, a spleen sample, a lung sample, a kidney sample, a bone sample, a muscle sample, a soft tissue sample. A “sample” can also be a cell, including but not limited to, an animal cell, a plant cell, a fungal cell, a eukaryotic cell, a prokaryotic cell, and the like.

[0086] Kit

[0087] The present disclosure provides a kit comprising a dsRNA inhibitor of the present disclosure, a lipid composition or a pharmaceutical composition, and a second therapeutic agent. In some embodiments, the kit of the present disclosure has multiple chambers, wherein one chamber comprises a dsRNA inhibitor of the present disclosure, a lipid composition or a pharmaceutical composition, and another chamber comprises a second therapeutic agent. In some embodiments, the kit of the present disclosure further comprises an instruction or a manufacturer’s guide.

[0088] In some embodiments, the second therapeutic agent is selected from one or more of a nucleic acid drug, a small molecule drug, a polypeptide, a protein, an antibody, a chemical drug, a chemotherapy drug. In some embodiments, the second therapeutic agent is selected from one or more of an androgen inhibitor, for example, a CYP17 enzyme inhibitor, such as abiraterone. In some embodiments, the second therapeutic agent is selected from one or more of a polyinosinic acid: polycytidylic acid (Poly(I:C)), a STING agonist, imiquimod, resiquimod, gardiquimod, sorafenib, sunitinib, erlotinib, doxorubicin, paclitaxel, gemcitabine, a radiotherapy. In some embodiments, the second therapeutic agent comprises abiraterone and prednisone.

[0089] In some embodiments, the antibody includes, but is not limited to, an antibody with anti-tumor effect. The antibody with anti-tumor effect includes, but is not limited to, an antibody targeting PD-1, such as nivolumab, pembrolizumab, cemiplimab, toripalimab, sintilimab, camrelizumab, etc.; an antibody targeting PD-L1, such as atezolizumab, avelumab, and durvalumab, etc.; an antibody targeting CTLA4, such as ipilimumab, tremelimumab, etc. In some embodiments, the antibody includes, but is not limited to, an anti-tumor associated antigen (TAA) antibody. The TAA includes, but is not limited to, GPC, CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, EGFR, Ep-CAM, EphA3, Her2, Her3, ROR2, PSMA, STEAP1, FGFR2, TROP2, B7-H3, B7-H4, B7-H6, FOLR1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, SSX-2, fibronectin, MART-2, PDL-1, PD-1, CTLA4, VEGFR, CLAUDIN, etc.

[0090] In some embodiments, the chemotherapy drug includes, but is not limited to, doxorubicin, paclitaxel, gemcitabine, radiotherapy.

[0091] In some embodiments, the small molecule drug or nucleic acid drug includes, but is not limited to, a pattern recognition receptor agonist, an anthracycline, a camptothecin, an immunogenic cell death (ICD) inducer, a tyrosine kinase inhibitor, a taxane, a Bruton's tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 / PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, an AKT1 inhibitor, an EGFR inhibitor, a VEGF inhibitor, a PARP inhibitor Her2, LAG-3, TNFR2 inhibitor, a corticosteroid drug (such as prednisone), etc.

[0092] In some embodiments, the anthracycline includes, but is not limited to, doxorubicin, epirubicin, pirarubicin, daunorubicin, aclarubicin, idarubicin, amrubicin, etc.

[0093] In some embodiments, the camptothecin class of compounds includes, but is not limited to, topotecan, irinotecan, belotecan, exatecan (DX-8951), lurtotecan, sinotecan, rubitecan (9-NC), 9-aminocamptothecin (9-AC), gimatecan, karenitecin, DB-67, and the like.

[0094] In some embodiments, the tyrosine kinase inhibitor includes, but is not limited to, sorafenib, sunitinib, gefitinib, erlotinib, lapatinib, afatinib, dacomitinib, vandetanib, neratinib, pelitinib (EKB-569), canertinib (CI-1033), osimertinib, rociletinib (CO-1686, Clovis Oncology), olmutinib (HM61713, Hanmi Pharmaceutical), naquotinib (ASP8273, AstraZeneca), tesevatinib (XL647 / KD019, Kadmon Corporation), nazartinib (EGF816, Novartis), and PF-06747775 (Pfizer), and the like.

[0095] In some embodiments, the immunogenic cell death inducer includes, but is not limited to, a DNA damaging agent, bleomycin, and the like.

[0096] In some embodiments, the paclitaxel class of compounds includes, but is not limited to, paclitaxel, docetaxel, and the like.

[0097] In some embodiments, the pattern recognition receptor agonist includes, but is not limited to, Toll-like receptors, RIG-I-like receptors, Nod-like receptors, AIM2-like receptors, and C-type lectin receptors, and cGas and other intracellular DNA sensor agonists.

[0098] Method of preparation

[0099] The present disclosure provides a method for preparing the lipid composition of the present disclosure, comprising the following steps (a)-(d): (a) taking the active ingredient, and preparing an active ingredient buffer with a buffer solution; (b) taking the lipid ingredient, and preparing a lipid solution with an organic solvent; (c) mixing the lipid solution and the active ingredient buffer at a mass ratio of lipid ingredient to active ingredient of (8-12): 1; (d) removing the organic solvent, diluting, and adjusting the pH to 6.0-8.0.

[0100] In some embodiments, the active component is selected from the dsRNA inhibitors provided by the present disclosure. In some embodiments, the organic solvent in the lipid solution is removed by methods such as ultrafiltration and / or dialysis. In some embodiments, the active component is formulated into an active component-containing solution using any one of the commonly used buffers such as sodium acetate buffer, citric acid buffer, Tris buffer, PBS buffer, etc. with pH ranging from 3.0 to 6.0. In some embodiments, the organic solvent is ethanol. In some embodiments, the active component-containing solution and the lipid solution are preheated to a temperature ranging from 20°C to 30°C, for example 25°C, before mixing. In some embodiments, the organic solvent in the lipid solution is removed by methods such as dialysis, ultrafiltration, tangential flow filtration, etc.

[0101] Examples

[0102] Exemplary embodiments of the present application are described herein with reference to the accompanying drawings, which are meant to be exemplary in nature, and are in no way intended as limiting the scope of the present application. The scope of the present application is defined only by the appended claims. Therefore, it should be understood by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope of the present application. Also, for the sake of brevity and clarity, descriptions of well-known functions and constructions are omitted herein.

[0103] The experimental methods in the following examples are routine methods, and are performed according to the techniques or conditions described in the literature in the art or according to the instructions of the products, unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0104] Example 1. Detection of Aktl protein expression level after transfection of different tumor cells

[0105] The siRNA against Aktl, i.e. siAktl, is designed according to the sequence of Aktl (NCBI Gene ID: 207) and the basic principles of siRNA design, and the sequence of the sense strand of siAktl used in the examples of the present disclosure is U*C*UUGGUCAGGUGGUGUGA*(dT)*(dT), and the sequence of the antisense strand is U*C*ACACCACCUGACCAAGA*(dT)*(dT), wherein * represents that the phosphodiester bond between the two adjacent nucleotides is a phosphorothioate bond.

[0106] Freeze four kinds of tumor cells Hep G2 (human hepatoma cells), Hep 3B (human hepatoma cells), SNU 387 (human hepatoma cells), Hepa1-6 (mouse hepatoma cells) are taken out, placed in a 37°C water bath pot to melt, Hep G2 and Hep 3B are added to 5mL of MEM (Gibco, 11095080) + 10% FBS (Gibco, 100991148) + 1% non-essential amino acids (Beyotime, C0332-100ml) + 1% sodium pyruvate (Beyotime, C0331-100ml), SNU 387 is added to 5mL of RPMI 1640 (gibco, 21870092) + 10% FBS, Hepa1-6 is added to 5mL of DMEM (Gibco, 11965092) + 10% FBS, after centrifugation (1100rpm, 5min) the supernatant is discarded, and the corresponding medium is added to resuspend and transfer to the culture dish, and placed in a 37°C, 5% CO2 incubator for culture.

[0107] When the above four kinds of cells grow well and are in the logarithmic growth phase, the supernatant is discarded, rinsed with PBS (Gibco, 10010023) once, and then 1mL of 0.25% trypsin-EDTA (Solarbio, T1300) is added for digestion, 5 minutes later, the corresponding medium containing serum is added to terminate digestion, the cells are blown evenly after centrifugation (1100rpm, 5min), the supernatant is discarded, and the cells are resuspended to 2*10 5 / mL, inoculated into a 12-well plate at a density of 2*10 5 / well (i.e. 1mL of cell suspension is added to each well), and cultured overnight before the next step of transfection. The sample siAkt1 is transfected, the transfection reagent is lipo3000 (Invitrogen, 92008), and the transfection concentration is 200nM. After 4 hours of transfection, replace with fresh corresponding medium, and then continue to culture for 20 hours. The blank control group is only given the same dose of lipo3000 transfection reagent.

[0108] The transfected cells are added with 100μL of RIPA lysis buffer (Beyotime, P0013B) + 1% PMSF (Beyotime, ST506) per well on ice for 20 minutes, blown evenly, collected into an EP tube, centrifuged at 12000rpm for 10 minutes, and the supernatant is taken to determine the protein concentration by BCA method (Beyotime, P0009), 5* loading buffer (YEASEN, 20317ES05) is added to prepare the protein sample, and finally the metal bath is incubated at 100°C for 15 minutes.

[0109] Subsequent analysis was performed using 12% SDS-PAGE. Prepared protein samples were added to each well at a rate of 10 μg. The stacking gel voltage was adjusted to 70 V, and the separating gel voltage to 120 V. Electrophoresis was stopped when the bands reached the bottom of the SDS-PAGE gel. After electrophoresis, the gel was transferred using a conventional wet transfer method (constant current 200 mA, 90 min). Blocking buffer (TBST + 5% skim milk powder) was then added for blocking, and the mixture was incubated on a shaker at room temperature for 1 hour. Akt1 antibody (CST, 75692) and GAPDH-HRP antibody (Proteintech, HRP-60004) were diluted with blocking buffer and added to the mixture for incubation on a shaker at room temperature for 2 hours. Wash three times with TBST, each time for 10 minutes on a shaker at room temperature. Then, add diluted secondary antibody HRP-conjugated affinipure goat anti-rabbit IgG (H+L) (Proteintech, SA00001-2) to the Akt1 antibody-incubated sample and incubate on a shaker at room temperature for 1 hour. Repeat the TBST wash three times for 10 minutes each time. Then add a 1:1 mixture of chemiluminescent substrate (Thermo, 34577) for development and incubate at room temperature for about 1 minute before detection.

[0110] GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is widely distributed in cells of various tissues, has a highly conserved sequence, and is a housekeeping gene. It is expressed at high levels in almost all tissues with a constant expression level, and was therefore chosen as an internal control gene to ensure consistent sample size. Figure 1 As shown, the expression level of GAPDH was basically the same in different cells; in Hep G2 and Hep 3B cells, siAkt1 could effectively knock down Akt1 expression compared with the blank control; the expression level of Akt1 was slightly reduced in SNU 387 cells; in mouse Hepa1-6 cells, the expression level of Akt1 did not change significantly compared with the blank control, which also shows that this sequence has specific targeting of human Akt1.

[0111] Use ImageJ software to... Figure 1 Grayscale analysis was performed. First, the IntDen values ​​of all stripes were subtracted from the background. Then, the Akt1 knockdown efficiency was calculated using the following formula:

[0112]

[0113] like Figure 2 As shown, the knockdown efficiency of siAkt1 varies in different cell lines, with the highest knockdown efficiency of the target gene in Hep 3B cells.

[0114] Example 2. Drug inhibition of LNCaP tumor growth in mouse prostate cancer

[0115] Take siAkt1 samples and prepare a siAkt1 sodium acetate solution with pH=5.0, a siAkt1 concentration of 0.25 mg / mL, and a sodium acetate concentration of 25 mM using sodium acetate buffer (pH=5.0). Take MC3, DSPC, cholesterol, and PEG-DSG in a molar ratio of 45:27.5:20:7.5 and add anhydrous ethanol to prepare a lipid ethanol solution with a total lipid concentration of 7.5 mg / mL. Take 0.75 mL of the siAkt1 sodium acetate solution and 0.25 mL of the lipid ethanol solution respectively. The solution was preheated in a 25°C water bath, then the lipid ethanol solution was injected into the siAkt1 sodium acetate solution, magnetically stirred until homogeneous, and incubated for 1 minute. It was then diluted 4 times with 25 mM sodium acetate buffer at pH 5.0 to obtain a siAkt1-encapsulated nanoliposome solution. Subsequently, the solution was centrifuged using a 100 kDa ultrafiltration tube to remove ethanol, and the concentration was adjusted to a suitable level with 25 mM Tris buffer. The pH was then adjusted to 7.0 with 0.1 M sodium hydroxide solution to finally obtain the siAkt1-encapsulated LNP suspension.

[0116] Forty 6-7 week old NCG mice were used for a one-week acclimatization period. LNCaP tumor cell lines (human prostate cancer cells) were revived and passaged. Once the cells were in good condition, they were injected with 1×10⁻⁶ cells / mL. 7 / cells were subcutaneously inoculated into mice, and tumor growth was observed regularly until the tumors grew to an average volume of 163 mm². 3 Mice were randomly assigned to three groups based on tumor size and body weight: a saline group, a negative control group (scramble), and an experimental group (siAkt1), with seven mice in each group. The negative control group received siRNA that did not target any mRNA. Its sense strand sequence was: ACGCGUAACGCGGGAAUUU*(dT)*(dT) (SEQ ID NO:9), and its antisense strand sequence was: AAAUUCCCGCGUUACGCGU*(dT)*(dT) (SEQ ID NO:10), where * represents a phosphate thioester bond between two adjacent nucleotides. Each mouse received 4 mg of the drug / kg body weight intravenously (adjustments could be made based on experimental results), administered every three days for a total of five doses. The first dose was given on the day of grouping, designated as Day 0. During the administration period, tumor growth and proliferation were observed and recorded in each group, including mouse body weight and tumor volume. The tumor growth inhibition rate (%) was calculated using the following formula. At the end of the experiment (Day 20 after grouping), the tumors were removed, their volume measured, and photographed.

[0117] Tumor growth inhibition rate (%) = [1 - tumor volume (experimental group) / tumor volume (saline group)] * 100%

[0118] As shown in Figure 3 , the experimental group (siAktl) had a significant decrease in body weight within 3 days of the first administration (dose 4 mg / kg), so the dose was reduced to 2 mg / kg for the second and third administrations, and the dose was restored to 4 mg / kg after the mice adapted. It can be seen that the body weight of the mice returned to normal after the initial fluctuation, indicating that the dose did not affect the health of the mice. As shown in Figure 4 , compared with the saline group, the negative control group slightly inhibited tumor growth, with a maximum tumor growth inhibition rate of 28.41%, while the tumor growth inhibition rate of the experimental group (siAktl) was significantly higher than that of the negative control group, with a maximum tumor growth inhibition rate of 41.88%, and was superior to the negative control group at different doses.

[0119] Example 3. Inhibition of prostate cancer PC-3 tumor growth in mice by siAktl combined with Abiraterone

[0120] The siAktl used in this example is LNP-encapsulated siAktl. The preparation process of the LNP suspension loaded with siAktl is as described in Example 2.

[0121] Thirty 6-8-week-old B-NDG mice were used for adaptive feeding, and the PC-3 tumor cell line was recovered and subcultured. When the cells grew well, 2 x 10 6 cells were inoculated subcutaneously on the right side of the back of the mice, and the tumor growth was observed regularly. When the tumor grew to an average volume of 100-150 mm 3 , the mice were randomly grouped according to the tumor size and body weight for administration.

[0122] The mice were divided into four groups, G1 (saline), G2 (siAktl), G3 (Abiraterone), and G4 (siAktl + Abiraterone), with 7 mice in each group. The administration frequency and concentration are shown in Figure 5 . During the experiment, the mice were administered on Day 0, Day 3, Day 6, Day 9, Day 12, Day 19, and Day 26 after grouping. Abiraterone is a pregnenolone analogue that can effectively and irreversibly inhibit CYP17 enzyme in the human body, thus inhibiting the synthesis of androgens from the source and inhibiting the development of androgen-dependent prostate cancer. During the administration period, the growth and proliferation of tumors in each group of mice were observed and recorded, including the body weight and tumor volume of the mice, and the tumor growth inhibition rate (%) was calculated according to the above formula.

[0123] like Figure 6 As shown, mice experienced a significant decrease in body weight after an initial dose of 198 mg / kg of Abiraterone. However, after halving the Abiraterone dose to 96 mg / kg, the mice's body weight slowly recovered, indicating that the current dosage does not affect the mice's health. Figure 7 It can be seen that the tumor growth inhibition rate of the G4 (siAkt1+Abiraterone) group was the highest, while the tumor growth inhibition rate of the G2 (siAkt1) group was slightly lower than that of the combination group. However, both groups were significantly better than the G3 (Abiraterone) group, indicating that siAkt1 itself can inhibit tumor cell growth, and its inhibitory ability is significantly higher than that of Abiraterone. After being used in combination with Abiraterone, the tumor inhibition ability increased slightly. Moreover, even after increasing the dosing interval in the last two administrations, it still showed a significant ability to inhibit tumor cell growth.

[0124] The above descriptions are merely some embodiments of this disclosure. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of this disclosure, and all such modifications and improvements fall within the scope of protection of this disclosure.

Claims

1. A double-stranded RNA (dsRNA) inhibitor for inhibiting the expression of human serine / threonine kinase 1 (AKT1), wherein the dsRNA inhibitor comprises a sense strand and an antisense strand forming a duplex region, and the antisense strand comprises a sequence complementary to a human AKT1 mRNA sequence, preferably the dsRNA inhibitor is a small interfering RNA (siRNA).

2. The dsRNA inhibitor of claim 1, comprising a sequence of any one of the following (a)-(c) groups: (a) the antisense strand comprises a modified or unmodified nucleotide sequence as set forth in UCACACCACCUGACCAAGA or a modified or unmodified functional variant thereof having 1, 2, or 3 different nucleotides from the nucleotide sequence as set forth in UCACACCACCUGACCAAGA; (b) the sense strand comprises a modified or unmodified nucleotide sequence as set forth in UCUUGGUCAGGUGGUGUGA or a modified or unmodified functional variant thereof having 1, 2, or 3 different nucleotides from the nucleotide sequence as set forth in UCUUGGUCAGGUGGUGUGA; (c) the antisense strand comprises a modified or unmodified nucleotide sequence as set forth in UCACACCACCUGACCAAGA or a modified or unmodified functional variant thereof having 1, 2, or 3 different nucleotides from the nucleotide sequence as set forth in UCACACCACCUGACCAAGA, and the sense strand comprises a modified or unmodified nucleotide sequence as set forth in UCUUGGUCAGGUGGUGUGA or a modified or unmodified functional variant thereof having 1, 2, or 3 different nucleotides from the nucleotide sequence as set forth in UCUUGGUCAGGUGGUGUGA.

3. The dsRNA inhibitor of claim 2, wherein the modification comprises one or more of the following: replacing P=O linkage between all or part of two adjacent nucleotides in the sequence with P=S linkage, replacing all or part of deoxyribonucleosides in the sequence with ribonucleosides, replacing all or part of ribonucleosides in the sequence with deoxyribonucleosides, replacing the group at 2’ position in all or part of nucleotide sugar rings in the sequence with methoxy, methoxyethyl, amino, alkyl, or halogen, bridging between the 2nd and 4th carbon atoms of all or part of nucleotide sugar rings in the sequence to form a locked nucleic acid or (S)-cEt-BNA, including one or more abasic nucleotides, morpholino nucleotides, phosphoramidate nucleotides, and / or nucleotides comprising non-natural bases in the sequence, RNA end modification at the 5’ end of the RNA strand comprising at least one of phosphotriester, alkylphosphonate, alkylidene phosphonate, alkenyl phosphonate, vinyl phosphonate; preferably, the sense strand of the dsRNA inhibitor comprises U*C*UUGGUCAGGUGGUGUGA, and / or the antisense strand comprises U*C*ACACCACCUGACCAAGA, wherein * represents a phosphorothioate linkage between its adjacent two nucleotides.

4. The dsRNA inhibitor of any one of claims 1-3, wherein the anti-sense strand and the sense strand comprise 1, 2, or 3 mismatched base pairs.

5. The dsRNA inhibitor of any one of claims 1-4, wherein the dsRNA inhibitor comprises a nucleotide overhang on either or both ends of at least one of the RNA single strands, preferably, the dsRNA inhibitor comprises a nucleotide overhang on either or both ends of both of the RNA strands.

6. The dsRNA inhibitor of claim 5, wherein the nucleotide overhang comprises 2 to 5 nucleotides, preferably, the nucleotide overhang comprises at least one of dTdT, UU, UC, CU.

7. The dsRNA inhibitor of claim 6, wherein the anti-sense strand comprises UCACACCACCUGACCAAGA(dT)(dT) and / or the sense strand comprises UCUUGGUCAGGUGGUGUGA(dT)(dT).

8. The dsRNA inhibitor of claim 7, wherein the sense strand comprises U*C*UUGGUCAGGUGGUGUGA*(dT)*(dT), and / or the anti-sense strand comprises U*C*ACACCACCUGACCAAGA*(dT)*(dT), wherein * represents a phosphorothioate linkage between its adjacent two nucleotides.

9. A lipid composition comprising an active component and a lipid component, wherein the active component comprises the dsRNA inhibitor of any one of claims 1-8.

10. The lipid composition of claim 9, which is selected from one or more of the following lipid compositions: a cationic polymer, a nano-lipid particle (LNP), a cationic liposome, a QTsome, a lipopolyplex, a microparticle, a microsphere, a nanoemulsion.

11. The lipid composition of claim 9, wherein the lipid component comprises one or more of an ionizable lipid, a neutral phospholipid, a cholesterol, and a PEG lipid, preferably, the mass ratio of the lipid component to the active component is (8-12):

1.

12. The lipid composition of claim 11, wherein the ionizable lipid is selected from at least one of DODMA, MC3, SM-102, A066, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA, DLinDMA class, DLin-K-DMA class, the neutral phospholipid is selected from at least one of DPPC, DSPC, HPSC, EPC, DOPC, DOPE, POPC, the PEG lipid is selected from at least one of DSG-PEG, DMG-PEG, preferably, the lipid component comprises MC3, DSPC, cholesterol, and PEG-DSG.

13. The lipid composition of claim 12, wherein the molar ratio of the ionizable lipid, neutral phospholipid, cholesterol, and PEG lipid is (10-60):(10-60):(15-50):(0-10), preferably, the molar ratio of the ionizable lipid, neutral phospholipid, cholesterol, and PEG lipid is 45:27.5:20:7.

5.

14. The lipid composition of claim 13, wherein the lipid component comprises MC3, DSPC, cholesterol, and PEG-DSG, and the molar ratio of MC3, DSPC, cholesterol, and PEG-DSG is 45:27.5:20:7.

5.

15. A pharmaceutical composition comprising the dsRNA inhibitor of any one of claims 1-8 or the lipid composition of any one of claims 9-14, and one or more pharmaceutically acceptable carriers or excipients.

16. The pharmaceutical composition of claim 15, further comprising a second therapeutic agent, preferably, the second therapeutic agent is selected from one or more of a nucleic acid drug, a small molecule drug, a polypeptide, a protein, an antibody, a chemical drug, a chemotherapy drug, more preferably, the second therapeutic agent is selected from Abiraterone.

17. A kit comprising the dsRNA inhibitor of any one of claims 1-8, the lipid composition of any one of claims 9-14 or the pharmaceutical composition of claim 15, and a second therapeutic agent.

18. A vector comprising a nucleotide sequence encoding the dsRNA inhibitor of any one of claims 1-8.

19. A host cell comprising the dsRNA inhibitor of any one of claims 1-8, the lipid composition of any one of claims 9-14 or the vector of claim 17.

20. Use of the dsRNA inhibitor of any one of claims 1-8, the lipid composition of any one of claims 9-14 or the pharmaceutical composition of claim 15 or 16 in the manufacture of a medicament for treating or preventing a disease.

21. A method of reducing AKT1 expression, comprising administering to a sample or a subject the dsRNA inhibitor of any one of claims 1-8, the lipid composition of any one of claims 9-14 or the pharmaceutical composition of claim 15 or 16.

22. The use of claim 19 or the method of claim 20, wherein the dsRNA inhibitor, lipid composition or pharmaceutical composition is co-administered with another therapeutic agent, preferably, the another therapeutic agent is selected from one or more of a nucleic acid drug, a small molecule drug, a polypeptide, a protein, an antibody, a chemical drug, a chemotherapy drug, more preferably, the another therapeutic agent is selected from Abiraterone.

23. The use of claim 19 or the method of claim 20, wherein the dsRNA inhibitor, lipid composition or pharmaceutical composition is administered by one or more of the following routes: intratumoral injection, intravenous injection, subcutaneous injection, intramuscular injection, intraarterial injection, intraperitoneal injection, intracerebral injection, intracavity perfusion, intravesical perfusion, interventional therapy, oral, transdermal, transpulmonary, ocular and topical administration.

24. The use of claim 19, wherein the disease comprises an AKTl expression related disease, an angiogenesis related disease, for example a solid tumor, preferably the solid tumor is selected from one or more of head and neck cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, thyroid cancer, lung cancer, gastric cancer, liver cancer, pancreatic cancer, spleen cancer, non-small cell lung cancer, colorectal cancer, colon cancer, intestinal cancer, kidney cancer, brain tumor, glioma, bladder cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, uterine cancer, bone cancer, osteosarcoma, chondrosarcoma, liposarcoma, neuroblastoma, synovial sarcoma, astrocytoma, glioblastoma multiforme, anaplastic astrocytoma, peritoneal cavity cancer, soft tissue sarcoma, sarcoma, rhabdomyosarcoma, advanced mucinous disease, melanoma, skin cancer.

25. A method for preparing the lipid composition of any one of claims 9-14, comprising the following steps (a)-(d): (a) taking the active ingredient, formulating with buffer to active ingredient buffer; (b) taking the lipid ingredient, formulating with organic solvent to lipid solution; (c) mixing the lipid solution and the active ingredient buffer with a mass ratio of lipid ingredient to active ingredient of (8-12): 1; (d) removing the organic solvent, diluting and adjusting the pH to 6.0-8.0.

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