WO2023040828A1 - 靶向FAP阳性细胞的siRNA缀合物及其药物组合物和应用 - Google Patents

靶向FAP阳性细胞的siRNA缀合物及其药物组合物和应用 Download PDF

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WO2023040828A1
WO2023040828A1 PCT/CN2022/118435 CN2022118435W WO2023040828A1 WO 2023040828 A1 WO2023040828 A1 WO 2023040828A1 CN 2022118435 W CN2022118435 W CN 2022118435W WO 2023040828 A1 WO2023040828 A1 WO 2023040828A1
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sirna
fap
cancer
sirna conjugate
conjugate according
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French (fr)
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苏金
魏新茹
杨鹏辉
叶婷
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菲柏生物医学技术（广州）有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the present disclosure relates to a small nucleic acid drug, in particular to an siRNA conjugate targeting fibroblast activation protein (FAP), its preparation method, pharmaceutical composition and use.
  • FAP fibroblast activation protein
  • FAP fibroblast activation protein
  • FAP is highly upregulated in a variety of cancers, expressed in more than 90% of epithelial malignancies, and is often used as a marker of tumor-promoting stromal growth.
  • Extracellular Matrix ECM
  • IPF Idiopathic pulmonary fibrosis
  • fibroblasts may induce inflammation and tissue damage in a series of autoimmune diseases, such as rheumatoid arthritis, bone erosion and destruction of joint cartilage.
  • the researchers analyzed synovial samples from rheumatoid arthritis and found that high expression of the protein FAP in these tissues directly correlates with the progression of the disease.
  • Studies in mouse models of arthritis have found that high levels of FAP are associated with the severity of the ankle bulge and that cells expressing the protein migrate into bone and cartilage, a prerequisite for cell destruction.
  • FAP-expressing fibroblasts two types of fibroblasts in joints were identified by mass cytometry, one in the synovium in the lining layer (LL) region and the other in the synovium.
  • SL sub-lining layer
  • the researchers found that when arthritis develops, the levels of all cell types increase in the joints of mice, and that the number of fibroblasts in the SL area correlates with the severity of inflammation, while the number of fibroblasts in the LL area correlates with the severity of the cartilage.
  • the results suggest that different types of fibroblasts may play key roles in different aspects of rheumatoid arthritis.
  • a siRNA conjugate targeting FAP-positive cells which includes: (i) a ligand portion targeting FAP, (ii) an siRNA portion that inhibits the expression of survival genes in FAP-positive cells, And (iii) a linker moiety between the ligand moiety and the siRNA moiety, wherein the FAP-targeting ligand moiety comprises a structure of the following formula (I):
  • X is Its left side is connected to formula (I), and its right side is connected to linker part;
  • R 1 and R 2 are the same or different, and are each independently selected from hydrogen, halogen and C 1 -C 4 alkyl, preferably halogen, more preferably fluorine;
  • R 3 is C 1 -C 4 alkyl, nitrile or isonitrile, preferably nitrile (-CN);
  • R4 is H or -CH3 ;
  • R 5 and R 6 are the same or different, and are each independently selected from hydrogen, halogen and C 1 -C 4 alkyl, preferably hydrogen;
  • R 7 , R 8 and R 9 are the same or different, and are each independently selected from hydrogen, methoxy, halogen, CF 3 and C 1 -C 4 alkyl, preferably hydrogen.
  • the ligand moiety targeting FAP comprises the following structural formula:
  • the ligand moiety targeting FAP comprises the following structure:
  • the siRNA portion is directed against one of the following survival genes: polr2a, polr2b, dkc1, cenpe, eif-3b.
  • the siRNA portion is directed at one of the following survival genes: survivin, phb2, c-myc and col1a1.
  • said FAP positive cells are activated fibroblasts.
  • said activated fibroblasts are present in tumor stroma, arthritic or fibrotic tissue.
  • the tumor is selected from basal cell carcinoma, squamous cell carcinoma, skin cancer, oral cancer, melanoma, esophageal cancer, adenocarcinoma, gastric cancer, colorectal cancer, rectal cancer, pancreatic cancer, hepatocellular Carcinoma, lung, mesothelioma, cholangiocarcinoma, liver, bladder, breast, kidney, prostate, cervix, ovary, glioma, thyroid, parathyroid, sarcoma, myeloma, and astrocytoma tumor.
  • the tumor is selected from lung cancer, cholangiocarcinoma, liver cancer, glioma, ovarian cancer, breast cancer and pancreatic cancer.
  • the fibrotic tissue is pulmonary fibrotic tissue or liver cirrhosis tissue.
  • the arthritis is rheumatoid arthritis.
  • the pulmonary fibrosis tissue is derived from one or more of idiopathic fibrosis (IPF), silicosis, and usual interstitial pneumonia (UIP).
  • IPF idiopathic fibrosis
  • UIP usual interstitial pneumonia
  • the siRNA moiety comprises complementary sense and antisense strands, and the FAP-targeting ligand moiety is linked to the 3' end of the sense strand.
  • the linker moiety comprises an amide bond.
  • the linker moiety has the following structural formula:
  • m and p are each independently an integer from 0 to 10.
  • m and p are each independently an integer of 1 to 5 or 1 to 3.
  • the siRNA conjugate has the following structural formula:
  • the sense strand is shown in SEQ ID NO: 9
  • the antisense strand is shown in SEQ ID NO: 10
  • the siRNA conjugate inhibits the expression of the eif-3b gene.
  • compositions which comprises any siRNA conjugate described in the present invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated as an injection.
  • Another aspect of the present invention provides the application of any one of the siRNA conjugates described above in the preparation of a drug for inhibiting the proliferation or survival of FAP-positive cells.
  • Another aspect of the present invention provides a method for inhibiting the proliferation or survival of FAP-positive cells, the method comprising contacting the FAP-positive cells with any of the siRNA conjugates of the present invention, so that the siRNA conjugates The substance is taken up by the FAP-positive cells, thereby inhibiting the proliferation or survival of the FAP-positive cells.
  • Another aspect of the present invention provides a method of treating a subject for a disease mediated by FAP positive cells, the method comprising administering to the subject a therapeutically effective amount of any one of the siRNA conjugates of the present invention or its pharmaceutical composition.
  • the disease mediated by FAP positive cells is a tumor, a fibrotic disease or arthritis.
  • the tumor is selected from basal cell carcinoma, squamous cell carcinoma, skin cancer, oral cancer, melanoma, esophageal cancer, adenocarcinoma, gastric cancer, colorectal cancer, rectal cancer, pancreatic cancer, hepatocellular Carcinoma, lung, mesothelioma, cholangiocarcinoma, liver, bladder, breast, kidney, prostate, cervix, ovary, glioma, thyroid, parathyroid, sarcoma, myeloma, and astrocytoma tumor.
  • the tumor is selected from lung cancer, cholangiocarcinoma, liver cancer, glioma, ovarian cancer, breast cancer and pancreatic cancer.
  • the fibrotic disease is pulmonary fibrosis or liver cirrhosis.
  • said fibrotic disease is idiopathic pulmonary fibrosis (IPF).
  • the disease mediated by FAP positive cells is rheumatoid arthritis (RA).
  • Figure 1 Flow cytometric detection of hFAP positive rate in cell lines stably expressing hFAP.
  • Figure 2 A synthetic route diagram showing siRNA conjugates.
  • Figure 3 Actin mRNA inhibition caused by administration of an exemplary siRNA conjugate reagent targeting Actin at a concentration of 50 nM in 293T and 293T-hFAP cells.
  • FIG. 4 GFP mRNA suppression caused by administration of an exemplary siRNA conjugate targeting GFP at a concentration of 50 nM to A549-GFP, A549-GFP-hFAP cells.
  • FIG. 5 Inhibition of GAPDH mRNA by administration of an exemplary siRNA conjugate targeting GAPDH at a single tail vein dose of 3 mg/kg to A549, A549-hFAP nude mice.
  • An siRNA conjugate reagent targeting the FAP ligand-free moiety of GAPDH was used as a negative control.
  • FIG. 6 GFP mRNA inhibition by an exemplary siRNA conjugate targeting GFP given a single tail vein dose of 3 mg/kg to A549-GFP, A549-GFP-hFAP nude mice.
  • the siRNA conjugate of the present invention targeting MYC was used as a negative control.
  • siRNA conjugates of the invention targeting the cell survival-related gene eIF-3 ⁇ lead to inhibition of the expression of eIF-3 ⁇ mRNA.
  • FIG. 8 The expression of FAP in different fibrosis patients.
  • B Western blot Detection of FAP expression in healthy donors, IPF, Silicosis lung tissue;
  • D Western blot detection of the expression of FAP in the lung tissue of healthy donors, UIP, and NSIP.
  • shRNAs (survivin, eIF-3B, PHB2, POLR2B, DKC1) targeting cell survival-related genes inhibit the proliferation of IPF patient fibroblasts in vitro.
  • shRNA (survivin, eIF-3B, PHB2, POLR2B, DKC1) targeting genes related to cell survival inhibits fibrosis of IPF patient fibroblasts in vitro.
  • IPF patient fibroblasts mixed with matrigel form microvessels one week after subcutaneous injection.
  • FIG. 21 Subcutaneous injection of FAPI-sieIF-3B effectively inhibits fibrosis in IPF fibroblasts in the long term.
  • siRNA is a double-stranded RNA of 20 to 25 nucleotides in length, comprising two antiparallel and substantially complementary nucleic acid strands. The two nucleic acid strands are named sense and antisense relative to the target RNA. siRNAs in part trigger the degradation of target RNA (eg, mRNA) through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).
  • target RNA eg, mRNA
  • RNA interference post-transcriptional gene silencing mechanism
  • each strand of the siRNA moiety typically includes one or more non-ribonucleotides, e.g., deoxyribonucleosides acids and/or modified nucleotides.
  • siRNA moieties can include ribonucleotides with chemical modifications, and can also include substantial modifications at multiple nucleotides. These modifications may include all types of modifications disclosed herein or known in the art. Any such modifications are intended to be included within the definition of "siRNA”.
  • RNA complementary means that in a double-stranded nucleic acid molecule, the bases of one strand are each paired with the bases of the other strand in a complementary manner.
  • the purine base adenine (A) is always paired with the pyrimidine base uracil (U); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G).
  • Each base pair consists of a purine and a pyrimidine.
  • mismatch in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary form.
  • substantially complementary means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “substantially complementary” means that there are no more than 3 base mismatches between the two nucleotide sequences. 1 base mismatch; “completely complementary” means that there is no base mismatch between two nucleotide sequences.
  • inhibitor is used interchangeably with “reduce”, “silence”, “downregulate”, “suppress” and other similar terms, and includes any level of inhibition.
  • the phrase “inhibiting the expression of a gene” includes inhibiting the gene in any species (such as a mouse gene, a rat gene, a monkey gene, or a human gene) as well as variants of that gene (such as a naturally occurring variant) or mutant expression.
  • the gene may be a wild-type gene, a mutated gene or, in the case of a genetically manipulated cell, population of cells or organism, a transgene.
  • “Inhibiting the expression of a gene” or similar terms include any level of inhibition of a gene, such as at least partial inhibition of gene expression, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, At least about 97%, at least about 98%, or at least about 99%.
  • at least partial inhibition of gene expression such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least
  • the expression of the gene can be assessed based on the levels of any variable associated with the expression of the gene, such as mRNA levels, encoded protein levels, and the like.
  • the degree of inhibition is assessed by the reduction in the absolute or relative level of one or more of these variables compared to control levels.
  • the control level can be any type of control level utilized in the art, such as a pre-dose baseline level or from a similar untreated or control-treated subject, cell, or sample, such as a buffer-only control or an inert control. determined level.
  • FAP-positive cells refers to cells with increased or overexpressed FAP expression on the cell surface compared to normal cells.
  • FAP is usually absent or lowly expressed in normal tissues, and activated fibroblasts upregulate the expression of FAP in conditions associated with wound healing, rheumatoid arthritis, cancer, and fibrosis, among others.
  • said FAP positive cells mediate one or more diseases or conditions in the subject.
  • Typical examples of FAP-positive cells are activated fibroblasts located in fibrotic tissue, joint synovium or tumor stroma. Activated fibroblasts may have different nomenclature in different tissues and pathological states.
  • activated fibroblasts in the tumor stroma are also referred to as FAP-positive tumor cells (eg, FAP-positive lung cancer cells, FAP-positive colorectal cancer cells, etc.).
  • FAP-positive tumor cells eg, FAP-positive lung cancer cells, FAP-positive colorectal cancer cells, etc.
  • the activated fibroblasts are called fibroblast-like synoviocytes or rheumatoid arthritis fibroblast-like synoviocytes (HFLS-RA).
  • HFLS-RA rheumatoid arthritis fibroblast-like synoviocytes
  • Whether the cell surface is positive for FAP can be determined by techniques known in the art (eg, immunohistochemistry, staining).
  • Reagents eg, fluorescently labeled FAP antibodies
  • instruments eg, flow cytometry
  • FAP positive cells includes any disease or condition associated with FAP positive cells.
  • the disease can be caused by overexpression of the FAP protein.
  • a "FAP-positive cell-mediated disease” preferably refers to a disease in which a significant expression of FAP is detected in an individual suffering from the disease. As noted above, most normal adult tissues show little or no detectable FAP expression, and in humans the plasma concentration of FAP measured by ELISA in healthy individuals is approximately 100 ng/mL or 0.6 nmol/L.
  • FAP-positive cell-mediated disease refers to an individual with the disease having a FAP plasma concentration of at least about 1 ⁇ g/mL, at least about 2 ⁇ g/mL, at least about 3 ⁇ g/mL, at least about 4 ⁇ g/mL, at least about 5 ⁇ g/mL, at least about 6 ⁇ g/mL, at least about 7 ⁇ g/mL, at least about 8 ⁇ g/mL, at least about 9 ⁇ g/mL, at least about 10 ⁇ g/mL, at least about 15 ⁇ g/mL, at least about 20 ⁇ g/mL, at least about 25 ⁇ g/mL mL, at least about 50 ⁇ g/mL, at least about 100 ⁇ g/mL or higher.
  • Typical examples of diseases mediated by FAP positive cells are tumors, fibrotic diseases and rheumatoid arthritis.
  • the tumor is selected from basal cell carcinoma, squamous cell carcinoma, skin cancer, oral cancer, melanoma, esophageal cancer, adenocarcinoma, gastric cancer, colorectal cancer, rectal cancer, pancreatic cancer, hepatocellular Carcinoma, lung, mesothelioma, cholangiocarcinoma, liver, bladder, breast, kidney, prostate, cervix, ovary, glioma, thyroid, parathyroid, sarcoma, myeloma, and astrocytoma tumor.
  • the tumor is selected from lung cancer, cholangiocarcinoma, liver cancer, glioma, ovarian cancer, breast cancer and pancreatic cancer.
  • the fibrotic disease is pulmonary fibrosis or liver cirrhosis.
  • said fibrotic disease is idiopathic pulmonary fibrosis (IPF).
  • the disease mediated by FAP positive cells is rheumatoid arthritis (RA).
  • the fibrotic disease is one or more of idiopathic fibrosis (IPF), silicosis, and usual interstitial pneumonia (UIP).
  • survival genes is used interchangeably with “essential genes” and refers to genes that are necessary for the survival or reproduction of a particular cell, a defect in the function of one of these genes causing the cell to appear Lethal phenotype with inability to survive or reproduce.
  • Typical survival genes are those encoding key proteases in the transcription, replication and expression of genetic material, including but not limited to POLR2A (DNA-directed RNA polymerase II subunit RPB1, the largest subunit of RNA polymerase II, which can synthesize Pre-mRNA and many functional non-coding RNAs form the polymerase active center together with the second largest subunit), POLR2B (DNA-directed RNA polymerase II subunit RPB2, the second largest subunit of RNA polymerase II, and the first The large subunits together form the polymerase active center), DKC1 (H/ACA ribonucleoprotein complex subunit DKC1, ribonucleoprotein complex subunit), CENPE (Centromere-associated protein E, mitosis-related protein E), eIF-3b (eukaryotic translation initiation factor 3 subunit b, eukaryotic transcription initiation factor 3 subunit b), COL1A1 (type I collagen ⁇ 1 chain gene), survivin, PHB2 (pro
  • the term "survival genes in FAP-positive cells” refers to genes necessary for the survival or propagation of FAP-positive cells, particularly genes necessary for the survival or propagation of fibroblasts (preferably activated).
  • Methods for determining whether a specific gene in a human cell is an essential gene are known in the art, for example, see Wang T et al. Identification and characterization of essential genes in the human genome. Science 27 Nov 2015: Vol.350, Issue 6264 , pp.1096-1101 or Vincent A.Blomen et al., Gene essentiality and synthetic lethality in haploid human cells. Science 27 Nov 2015: Vol.350, Issue 6264, pp.1092-1096.
  • siRNA conjugates refers to the covalent linkage between two or more chemical moieties each having a specific function; the compound formed.
  • siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA.
  • the siRNA conjugate of the present disclosure may also be simply referred to as "conjugate”.
  • siRNA conjugates should be understood as a general term for siRNA conjugates, which roughly include a targeting ligand part, an siRNA part and a linker part connecting the two, and the three are covalently bound together by a chemical reaction to form a " siRNA conjugates" specific compounds.
  • contacting a cell with an siRNA conjugate includes contacting a cell by any possible means, including contacting the cell with the siRNA conjugate in vitro or contacting the cell with the siRNA conjugate in vivo.
  • Contacting a cell in vitro can be performed, for example, by incubating the cell with the siRNA conjugate.
  • Contacting a cell in vivo can be performed, for example, by injecting the siRNA conjugate into or near the tissue in which the cell is located, or by injecting the siRNA conjugate into another area (e.g., by intravenous or subcutaneous injection), such that the agent subsequently Reach the tissue where the cells to be contacted are located.
  • terapéuticaally effective amount is intended to include an amount of siRNA conjugate sufficient to effect treatment of a disease mediated by FAP positive cells (eg, by attenuating, ameliorating or maintaining the disease or symptom) when administered to a patient to treat the disease mediated by FAP positive cells.
  • the "therapeutically effective amount” can be based on the siRNA conjugate itself, the route of administration, the mode of administration, the type of disease and its severity, medical history, age, body weight, family history, genetic makeup, stage of pathological process, previous or concomitant The type of treatment, if any, and other individual characteristics of the patient being treated will vary.
  • patient or “subject” includes human or non-human animals, preferably mammals, such as monkeys. Most preferably, the subject or patient is a human.
  • siRNA moieties that inhibit the expression of survival genes in FAP-positive cells
  • the "siRNA part that inhibits the expression of survival genes in FAP-positive cells” is a double-stranded RNA molecule that, after being taken up by target cells, specifically targets Degradation of mRNA to specific survival genes, thereby inhibiting the expression of survival genes in FAP-positive cells.
  • the siRNA part has a sense strand and an antisense strand with the same or different lengths, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/ 25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the sense and antisense strands of the siRNA moiety have a length ratio of 19/21
  • the sense strand further comprises nucleotide sequence III
  • the antisense strand further comprises nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each 1-4 nuclei Nucleotide
  • the nucleotide sequence III and the nucleotide sequence IV are equal in length and substantially reverse complementary or completely reverse complementary
  • the nucleotide sequence III is connected to 5 of the nucleotide sequence I ' end
  • the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence
  • the second nucleotide sequence refers to the nucleotides of the target mRNA fragment A nucleotide sequence that is adjacent to the 5' end of the sequence and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides, according to the direction from the 5' end to the 3' end, the nucleoside
  • the base composition of the acid sequence III is CU, and the base composition of the nucleotide sequence IV is AG; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequence III and IV Both are 3 nucleotides, according to the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is UCU, and the base composition of the nucleotide sequence IV is AGA
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, and according to the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is Cu , the base composition of the nucleotide sequence IV is AG; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and nucleotide sequence IV are completely reverse complementary, therefore, given the base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.
  • siRNA design sites that provide online design services (e.g. DSIR: http://biodev.extra.cea.fr/DSIR/DSIR.html or siDirect version 2.0: http://sidirect2.rnai.jp/) . These design sites can simultaneously obtain multiple representative siRNA sequences for different fragments of a target gene based on its mRNA. These representative siRNAs can be ranked based on a number of options (eg, thermodynamics, higher order structure, etc.). Candidate siRNA sequences can be further screened (manually or automatically) to exclude sequences containing SNP sites or binding non-ORF segments.
  • Candidate siRNA sequences can also be analyzed by off-target prediction software for siRNA sequences with potential high off-target (eg http://rnai.cs.unm.edu/rnai/off-target).
  • siRNA optionally follows general design guidelines. For example, find a 21 nt sequence starting with an AA dinucleotide in the targeted mRNA sequence. Starting from the AUG start codon of the transcript, AA dinucleotide sequences were scanned. Record each AA and the next 19 nucleotides 3' adjacent as potential siRNA targeting sites. This selection of siRNA targeting sites is due to the fact that siRNAs with a UU dinucleotide overhang at the 3' end were found to be most effective. This approach is also consistent with the use of RNA pol III to transcribe hairpin siRNA, because RNA pol III terminates transcription at 4-6 nucleotide poly(T), resulting in RNA with a short poly(U) tail.
  • siRNAs with additional 3'-terminal dinucleotide overhangs have been shown to be effective in inducing RNAi.
  • This targeting site selection can also be modified to design siRNAs with other dinucleotide overhangs if desired, although it is generally recommended to avoid selection of GG overhangs because siRNAs may be blocked by RNases at single-stranded G residues. cut.
  • Another guideline is to choose 2-4 targeting sequences. Typically more than half of the randomly designed siRNAs resulted in at least a 50% reduction in the RNA level of the targeted mRNA, and about a quarter of the siRNAs resulted in a 75-95% reduction.
  • Select the target site for the sequence identified in the first step according to the following guidelines: the study found that siRNA with a GC content of 30-50% was more active than siRNA with a higher G/C content. Since 4-6 nucleotide poly(T) will become the termination signal of RNA pol III, if it is necessary to use RNA pol III to initiate expression, it is necessary to avoid more than 4 T or A consecutively appearing in the target sequence.
  • siRNA targeting sites are usually selected at different positions in the gene sequence. No correlation was observed between the location of the targeting site on the mRNA and siRNA potency. Compare potential target sites with corresponding genomic databases (human, mouse, rat, etc.), if the target sequence has more than 16-17 contiguous base pair homology to other gene coding sequences, it cannot Choose such a sequence.
  • BLAST is recommended (visit NCBI server: www.ncbi.nlm.nih.gov/BLAST).
  • a complete siRNA experiment should include a series of controls to ensure data validity.
  • Two exemplary controls are: (1) Negative control siRNA, which has the same nucleotide composition as siRNA, but has no obvious sequence homology with the genome. To design a negative control siRNA, the nucleotide sequence of a gene-specific siRNA can be scrambled and searched to ensure that it does not share homology to any gene. (2) Other siRNA sequences targeting the same mRNA. Perhaps the best way to ensure confidence in RNAi data is to run experiments with two or more different siRNAs for the same gene, one individual siRNA at a time. Before these experiments, each siRNA needs to be tested to ensure that they reduce the expression level of the target gene to a similar level.
  • siRNA sequences can also be chemically modified in various ways to enhance their stability, prolong half-life, and/or enhance efficacy (eg, silencing efficiency).
  • Representative chemical modifications include 4-thio modification to increase the stability of siRNA; LNA (locked nucleic acid) combined with siRNA to increase stability; modification of the phosphate backbone; and modification of the 2' pentose ring.
  • LNA locked nucleic acid
  • siRNA sequences can also be chemically modified in various ways to enhance their stability, prolong half-life, and/or enhance efficacy (eg, silencing efficiency).
  • Representative chemical modifications include 4-thio modification to increase the stability of siRNA; LNA (locked nucleic acid) combined with siRNA to increase stability; modification of the phosphate backbone; and modification of the 2' pentose ring.
  • LNA locked nucleic acid
  • the siRNA sequence of the siRNA moiety targets a survival gene of FAP positive cells.
  • the siRNA sequence targets one or more fragments of one or more of the polr2a, polr2b, dkcl, cenpe, eif-3b genes.
  • after the siRNA sequence is delivered to the cell it binds to the target gene through the RNAi pathway, resulting in the inhibition of the expression of the target gene.
  • inhibition of expression of a target gene results in apoptosis, death, and/or inhibition of proliferation of FAP-positive cells.
  • apoptosis, death and/or inhibition of proliferation of FAP-positive cells mediates treatment or cure of the disease.
  • Suitable survival genes include, but are not limited to, one or more of the polr2a, polr2b, dkcl, cenpe, eif-3b genes.
  • Other suitable survival genes include, but are not limited to, one or more of the survivin, phb2, c-myc, and col1a1 genes. siRNA sequences targeting survival genes can be obtained and tested as described above.
  • the siRNA portion comprises a sense strand and an antisense strand, wherein the sense strand is shown in SEQ ID NO: 9, the antisense strand is shown in SEQ ID NO: 10, and the siRNA is conjugated with The compound inhibits the expression of eif-3b gene.
  • the siRNA portion comprises a sense strand and an antisense strand, wherein the sense strand is shown in SEQ ID NO: 11, the antisense strand is shown in SEQ ID NO: 12, and the siRNA is conjugated with The compound inhibits the expression of survivin gene.
  • the siRNA portion comprises a sense strand and an antisense strand, wherein the sense strand is shown in SEQ ID NO: 13, the antisense strand is shown in SEQ ID NO: 14, and the siRNA is conjugated with The compound inhibits the expression of the col1a1 gene.
  • the siRNA portion comprises a sense strand and an antisense strand, wherein the sense strand is shown in SEQ ID NO: 15, the antisense strand is shown in SEQ ID NO: 16, and the siRNA is conjugated with Compound inhibits the expression of phb2 gene.
  • the siRNA portion comprises a sense strand and an antisense strand, wherein the sense strand is shown in SEQ ID NO: 17, the antisense strand is shown in SEQ ID NO: 18, and the siRNA is conjugated with Compound inhibits the expression of polr2b gene.
  • the siRNA portion comprises a sense strand and an antisense strand, wherein the sense strand is shown in SEQ ID NO: 19, the antisense strand is shown in SEQ ID NO: 20, and the siRNA is conjugated with The compound inhibits the expression of dkc1 gene.
  • the siRNA provided in the present disclosure can be obtained by conventional siRNA preparation methods in the art (such as solid-phase synthesis and liquid-phase synthesis). Among them, solid-phase synthesis has commercialized customized services.
  • the modified nucleotide group can be introduced into the siRNA described in the present disclosure by using the correspondingly modified nucleoside monomer, the method for preparing the correspondingly modified nucleoside monomer and the introduction of the modified nucleotide group Methods of siRNA are also well known to those skilled in the art.
  • the FAP-targeting ligand moiety constitutes the targeting moiety of the siRNA conjugates of the present disclosure, which is used to recognize and bind to FAP-positive cells, thereby delivering the loaded siRNA moiety to the target cells.
  • the ligand moiety targeting FAP is a small molecule compound moiety, preferably a targeting ligand having a binding affinity to FAP of about 1 nM to about 20 nM.
  • the FAP-targeting ligand moiety has a molecular weight below 10,000.
  • the ligand targeting FAP is a small molecule inhibitor of FAP.
  • the ligand targeting FAP comprises the structure of formula (I):
  • X is Its left side is connected to formula (I), and its right side is connected to linker part;
  • R 1 and R 2 are the same or different, and are each independently selected from hydrogen, halogen and C 1 -C 4 alkyl, preferably halogen, more preferably fluorine;
  • R 3 is C 1 -C 4 alkyl, nitrile or iso Nitrile, preferably nitrile;
  • R 4 is H or -CH 3 ;
  • R 5 and R 6 are the same or different, and each independently selected from hydrogen, halogen and C 1 -C 4 alkyl, preferably hydrogen;
  • R 7 , R 8 and R 9 are the same or different, and are each independently selected from hydrogen, methoxy, halogen, CF 3 and C 1 -C 4 alkyl, preferably hydrogen.
  • the ligand moiety targeting FAP comprises the following structural formula:
  • the ligand moiety targeting FAP comprises the following structure:
  • More ligand molecules targeting FAP can be found in Chinese Invention Patent Application Publication CN110291401A or CN111699181A, the contents of which are incorporated herein in their entirety by reference.
  • the siRNA conjugates of the invention may comprise more than one FAP-targeting ligand molecule, for example 2 to 5 ligand molecules, preferably 3 ligand molecules.
  • Each FAP-targeting ligand molecule can be the same or different, preferably the same.
  • the siRNA conjugate of the present invention comprises three ligands as shown in the following structural formula:
  • the siRNA conjugate of the present invention contains more than one ligand molecule
  • multiple ligand molecules can be covalently bound to the same atom (eg, C atom) to form a multi-ligand structure.
  • the multi-ligand structure is believed to increase the ability and efficiency of the siRNA conjugates to recognize and bind to FAP-positive cells.
  • the molecule forming the ligand moiety targeting FAP may have a carboxyl group (-COOH) to facilitate amide bond formation through the primary amine group of the linker moiety (see below for details).
  • carboxyl group (-COOH)
  • NHS or sulfo-NHS to activate carboxyl groups is known, which facilitates the reaction of carboxyl groups with terminal primary amine groups to form amide bonds. Therefore, in some embodiments of the present invention, the FAP ligand having a carboxyl group is reacted with EDC/NHS or EDC/sulfo-NHS system to generate FAP ligand-NHS.
  • the ligand moiety targeting FAP is also labeled FAPI-.
  • FAPI-NHS can be purchased from, for example, Wuxi Jiehua Pharmaceutical Technology Co., Ltd., or can also refer to CN110291401A or CN111699181A to prepare FAPI-NHS.
  • a linker moiety is interposed between the siRNA moiety and the ligand moiety, which form covalent linkages with the siRNA moiety and the ligand moiety, respectively, thereby forming the siRNA conjugate molecule.
  • the linker moiety has a first linker moiety to which the siRNA moiety is attached and a second linker group to which the FAP-targeting ligand moiety is attached.
  • the first linking group and the second linking group of the linker moiety are linked by one or more methylene groups.
  • the first linking group is a hydroxyl group.
  • the second linking group is a primary amine group.
  • the first linking group is a hydroxyl group and the second linking group is a primary amine group.
  • a linker moiety can be first ligated to an siRNA moiety to form a linker-siRNA moiety.
  • a linker of the following structural formula can be used to connect to the 3' end of the sense strand, wherein p is an integer from 0 to 10:
  • the resulting linker-siRNA moiety was modified with a free primary amine group at the 3' end to facilitate attachment to the FAP ligand moiety.
  • an exemplary structure of a linker-siRNA is as follows:
  • the linker has a reactive group other than the primary amine group to chemically react with a corresponding reactive group of the FAP ligand moiety to form a covalent bond.
  • exemplary other linker structures include, but are not limited to, those used in antibody drug conjugates (ADCs), cleavable or non-cleavable linker structures.
  • the resulting linker-siRNA moiety is then covalently linked to the FAP ligand moiety via a second linking group.
  • the FAP ligand moiety has a carboxyl group that can react with a primary amine group of a second linking group that is the linker moiety to form an amide bond to covalently link the FAP ligand moiety to the linker-siRNA moiety,
  • the use of NHS or sulfo-NHS to activate carboxyl groups is known, which facilitates the reaction of carboxyl groups with terminal primary amine groups to form amide bonds.
  • the FAP ligand having a carboxyl group is reacted with the EDC/NHS or EDC/sulfo-NHS system to generate the FAP ligand-NHS, and then the FAP ligand-NHS is linked with the above-mentioned sub-siRNA reaction to generate siRNA conjugates of the present invention.
  • said linker moiety comprises an amide bond.
  • the linker moiety has the following structural formula:
  • m and p are each independently an integer from 0 to 10.
  • m and p are each independently an integer of 1 to 5 or 1 to 3.
  • the linker moiety can be attached to a phosphate group, a 3'-position hydroxyl group, or a base of a nucleotide. In some embodiments, the linker moiety can be attached to the hydroxyl group at the 3'-position, and in this case, the nucleotides are connected by 2'-5' phosphodiester bonds.
  • the linker part is usually connected to the phosphate group of the nucleotide; when the linker part is connected to the internal sequence of the siRNA, the linker part is usually connected to the ribose sugar ring or base.
  • the siRNA and the linker moiety may be linked by acid-labile or reducible chemical bonds, which can be degraded in the acidic environment of endosomes, thereby leaving the siRNA in a free state.
  • the linker moiety can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on the activity of the siRNA.
  • the structural demarcation of the three parts of the siRNA conjugates can vary.
  • the structural boundary between the ligand portion and the linker portion of FAP can vary.
  • the structural boundaries of the linker moiety and the siRNA moiety may vary. That is, for one or more groups or groups of groups in the linking region, it can be used as a part of the FAP ligand part, it can also be part of the linker part, or it can be part of the linker part, or it can be part of the linker part.
  • the linker moiety in the siRNA conjugate comprises an amide bond
  • the m methylene moieties on the left side of the amide bond of the linker portion can be derived from a FAP-targeting ligand.
  • Body part m is as defined above. That is, by definition, the part to the left of the amide bond in the linker can also be classified as part of the FAP-targeting ligand, thereby constituting a part of the FAP-targeting ligand.
  • all methylene groups to the left of the amide bond in the linker can be considered part of the ligand moiety targeting FAP.
  • siRNA conjugates are formed by covalently bonding a FAP-targeting ligand moiety to an siRNA moiety via a linker moiety.
  • the siRNA conjugate targeting FAP-positive cells comprises (i) a ligand moiety targeting FAP, (ii) an siRNA moiety that inhibits expression of survival genes in FAP-positive cells, and (iii) a moiety located at the A linker moiety between the ligand moiety and the siRNA moiety.
  • each part is as defined above respectively.
  • the FAP-targeting ligand moiety exerts the targeting effect of the conjugate, which directs the conjugate to bind to cells positive for FAP expression on the surface, such as cancer cells or joint synovial fibroblasts.
  • the molecular imaging diagnostic technology developed with FAP as a target has been applied in the field of nuclear medicine.
  • 68 Ga-FAPI PET/CT imaging has been used in the diagnosis of different types of cancer patients such as pancreatic cancer, breast cancer, liver cancer, head and neck cancer, and thyroid cancer. Examples of the present disclosure demonstrate that conjugates with a ligand moiety targeting FAP are able to specifically bind cells positive for FAP expression.
  • siRNA moiety that inhibits the expression of survival genes in FAP-positive cells exerts the pharmacodynamic effect of the conjugate.
  • siRNA drugs mediate the silencing effect of target genes through the RNAi mechanism.
  • the first siRNA drug, Onpattro (Patisiran, Alnylam), was approved by the FDA for the treatment of patients with polyneuropathy caused by hATTR.
  • siRNA drugs there are currently 14 small nucleic acid drugs on the global market, including 4 siRNA drugs, all of which were developed or co-developed by Alnylam for the treatment of familial amyloid polyneuropathy (Onpattro), acute hepatic porphyria (Givlaari), Primary hyperoxaluria type 1 (Oxlumo) and adult hypercholesterolemia and mixed dyslipidemia (Inclisiran).
  • siRNA partially targets the survival genes of FAP-expressing positive cells to inhibit the expression of the survival genes, which ultimately leads to the death or inhibition of proliferation of the target cells.
  • the embodiments of the present disclosure confirm that the FAP ligand can effectively carry siRNA to target FAP positive cells and inhibit the expression of target genes in cells.
  • the conjugate of the present invention is labeled as FAPI-gene siRNA, FAPI-si gene (such as FAPI-siSurvivin) or FAPI-gene RNAi reagent, for example, FAPI-GFP siRNA means that the target gene is GFP with FAP Double-stranded siRNA targeting ligand, FAPI-eIF-3 ⁇ siRNA means double-stranded siRNA with FAP targeting ligand whose target gene is eIF-3 ⁇ , FAPI-siSurvivin means double-stranded siRNA with FAP targeting ligand whose target gene is Survivin Stranded siRNA, sigene (eg siSurvivin) means naked siRNA without FAP targeting ligand.
  • FAPI-GFP siRNA means that the target gene is GFP with FAP Double-stranded siRNA targeting ligand
  • FAPI-eIF-3 ⁇ siRNA means double-stranded siRNA with FAP targeting ligand whose target gene is eIF-3 ⁇
  • the structural formula of the siRNA conjugate targeting FAP-positive cells is as follows:
  • the sense strand represents the sense strand of the siRNA molecule
  • the antisense strand represents the antisense strand of the siRNA molecule
  • the present disclosure provides a pharmaceutical composition containing the above-mentioned siRNA conjugate as an active ingredient and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition containing the above-mentioned siRNA conjugate as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition there is no special requirement on the content of the siRNA conjugate and the pharmaceutically acceptable carrier, which can be the conventional content of each component.
  • the weight ratio of the siRNA conjugate to the pharmaceutically acceptable carrier may be 1:(1-500), and in some embodiments, the above weight ratio is 1:(1-50).
  • the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds routinely used in the art.
  • the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent and an osmotic pressure regulator.
  • the pH buffer can be a tris hydrochloride buffer with a pH value of 7.5-8.5 and/or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5 buffer.
  • the protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator can be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmol/kg (mOsm/kg). According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.
  • the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder injection, which is mixed with a liquid excipient during administration to prepare a liquid preparation.
  • the liquid preparation can be used for subcutaneous, intramuscular or intravenous injection administration, but can also be administered by spraying to the lungs or to other organs and tissues (such as the liver) through the lungs by spraying.
  • the pharmaceutical composition is for intravenous administration.
  • the pharmaceutical composition may be in the form of a liposomal formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid, and/or a pegylated Lipid.
  • the present disclosure provides a use of an siRNA conjugate of the present disclosure in the manufacture of a medicament for the treatment of a disease mediated by FAP positive cells.
  • the present disclosure provides a method of treating a disease mediated by FAP positive cells, the method comprising administering to a subject in need thereof an effective amount of an siRNA conjugate of the present disclosure.
  • an siRNA conjugate of the present disclosure By administering the siRNA conjugate of the present disclosure to a subject in need, the purpose of treating diseases mediated by FAP-positive cells can be achieved through the mechanism of RNA interference. Therefore, the siRNA conjugates of the present disclosure can be used to treat diseases mediated by FAP-positive cells, or to prepare medicaments for treating diseases mediated by FAP-positive cells.
  • administration/administration refers to the placement of an siRNA conjugate of the present disclosure by a method or approach that at least partially localizes the siRNA conjugate of the present disclosure at a desired site to produce a desired effect. into the subject's body.
  • Routes of administration suitable for the methods of the present disclosure include topical and systemic administration. In general, local administration results in delivery of more siRNA conjugates to a specific site as compared to the subject's systemic circulation; whereas systemic administration results in delivery of the siRNA conjugates of the present disclosure to the subject's systemic circulation.
  • Administration to a subject may be by any suitable route known in the art, including, but not limited to: oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal Drugs, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual).
  • Dosing frequency can be one or more times per day, every week, every two weeks, every three weeks, every month or year.
  • the dosage of the siRNA conjugates described in the present disclosure can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially the age, body weight and sex of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining LD50 (the lethal dose that causes 50% of the population to die) and ED50 (the dose that can cause 50% of the maximum response in quantitative response, and in qualitative response). The middle response refers to the dose that can cause a positive reaction in 50% of the test subjects).
  • a range of dosage for use in humans can be derived based on the data obtained from cell culture assays and animal studies.
  • the amount of siRNA when administering the siRNA conjugates of the present disclosure, for example, for C57BL/6N mice, male or female, 6-12 weeks old, weighing 18-25 g, for the siRNA conjugates, the amount of siRNA, the siRNA
  • the dosage may be 0.001-100 mg/kg body weight, in some embodiments 0.01-50 mg/kg body weight, in some embodiments 0.05-20 mg/kg body weight, in other embodiments 0.1-15 mg/kg body weight, in other embodiments In some embodiments, 0.1-10 mg/kg body weight.
  • Example 1 Construction of a cell line stably expressing hFAP
  • hFAP human FAP
  • NM_004460.5 human FAP mRNA sequence
  • NCBI NCBI
  • construct hFAP lentiviral overexpression vector and package the virus in vitro after sequencing verification.
  • flow cytometry was used to detect hFAP in 293T and A549 cells The positive rate was used to confirm the successful establishment of 293T-hFAP, A549-hFAP, and A549-GFP-hFAP cell lines.
  • siRNA sequences targeting mRNAs of different target genes through online design tools (see Table 1), synthesize sense and antisense strands by solid-phase synthesis, and perform RPC reverse purification to obtain siRNA sequences after annealing.
  • the sense strand of the siRNA sequence is covalently linked to a linker so that the 3' end of the sense strand is modified with NH 2 .
  • siRNA interference hFAP stably transfected cell lines Take 293T and 293T-hFAP cells, A549-GFP and A549-GFP-hFAP cells in the logarithmic growth phase, adjust the cell density by counting cells, and spread them at a density of 2 ⁇ 10 5 cells/well In a 24-well plate, 500 ⁇ l of complete medium was added to each well, and cultured overnight at 37° C. in an atmosphere of 5% CO 2 for interference experiments the next day.
  • RNA extraction cells were harvested and dissolved in 400 ⁇ l lysate, and the lysate was pipetted 5-10 times with a pipette gun to break the DNA. Transfer the lysate to a gDNA filter column, centrifuge at 14,000 ⁇ g for 2 min, add an equal volume of 70% ethanol to the filtrate, and pipette 3-5 times.
  • RNA concentration and purity were determined using a Thermo NanoDrop 2000.
  • cDNA synthesis was performed using TAKARA PrimeScript RT Master Mix Kit (RR036a): Add 4 ⁇ l 5 ⁇ PrimeScript RT Master Mix, 1 ⁇ g total RNA to RNase Free dH 2 O to make the total volume 20 ⁇ l. Use the Q1000+ fluorescent quantitative PCR system to generate cDNA through the following steps: 37°C, 15min; 85°C, 5s, 4°C hold.
  • hFAP overexpression animal model subcutaneously inject 2 ⁇ 10 6 A549-hFAP cell suspension (left side) into nude mice, and subcutaneously inject A549 cells into the right side as a negative control; 2 ⁇ 10 6 A549-GFP-hFAP cell suspension per mouse was subcutaneously injected (left side) into nude mice, and A549-GFP cells were subcutaneously injected into the right side as a negative control. After 21 days of feeding, they were used for interference experiments in vivo.
  • FAPI-GAPDH siRNA (50nM) in vivo can down-regulate the expression of GAPDH in A549-hFAP cells; FAPI-GFP siRNA (50nM) in vivo can down-regulate the expression of GFP in A549-GFP-hFAP cells Express.
  • Example 5 In vivo inhibitory effect of siRNA conjugates targeting cell survival-related gene eIF-3 ⁇ fruit
  • A549-hFAP cell suspensions were subcutaneously injected into nude mice, fed for 21 days, and then used for interference experiments in vivo.
  • a single dose of eIF-3 ⁇ RNAi reagent (FAPI-eIF-3 ⁇ ) was given to the mice in the experimental group through the tail vein, and the mice in the control group were injected with an equal volume of PBS. After 72 hours, the tumors of the nude mice in the experimental group and the control group were taken to extract total RNA. And carry out real-time PCR experiment, the specific operation refers to embodiment 3.
  • Example 6FAP is specifically highly expressed in the lung tissue of IPF patients
  • Tissue blocks from 13 healthy donors and 34 patients with pulmonary fibrosis (IPF: 8 cases; UIP: 6 cases, NSIP: 8 cases, silicosis: 12 cases) were collected, and the presence of FAP in healthy donor lungs was determined by qPCR and Western blot. It is hardly expressed in IPF, silicosis and the usual interstitial pneumonia (UIP), which is mainly characterized by fibroblast foci (including fibrous matrix and activated fibroblasts) ( Figure 8) , especially highly expressed in lesions of IPF patients.
  • IPF interstitial pneumonia
  • the purified IPF fibroblasts were induced with 10 ng/ml TGF- ⁇ 1 for 48 hours to further activate and overexpress hFAP ( FIG. 9 ). Untreated cells were used as the control group.
  • FAPI-siRNA FAPI is prepared as shown in Figure 2, siRNA sequence is shown in Table 1, the same below), control siRNA (this embodiment involves survivin, eIF-3B, COL1A1, PHB2 , C-MYC, 5 genes) were incubated with activated FAP+IPF fibroblasts at a concentration of 100nM for 24h, the RNA of the above cells was extracted, and the knockdown effect of the target gene was verified by qPCR ( Figure 10- Figure 14).
  • FAPI-coupled cell survival-related gene siRNA can interfere with the endocytosis of siRNA into the cytoplasm through the combination of FAP and receptor-mediated endocytosis.
  • FAPI-siSurvivin, FAPI-sieIF-3B, FAPI-siCOL1A1, FAPI-siPHB2, and FAPI-siC-MYC had the strongest interference effects compared with the other three groups.
  • Example 8 ShRNA targeting cell survival-related genes inhibits fibrosis in IPF patients in vitro Effects on cell proliferation and fibrosis
  • interfering with the expression of survivin and eIF-3B alone, or jointly interfering with the expression of survivin+eIF-3B, or interfering with survivin+eIF-3B+PHB2 can reduce the fibrosis index ⁇ -sMA (smooth muscle muscle Protein), Collagen 1 (collagen), Fn (Fibronectin, fibronectin), the above results show that interference (survivin, eIF-3B, PHB2, POLR2B, DKC1) and other genes can inhibit the proliferation of fibroblasts, and can also inhibit fibrosis.
  • ⁇ -sMA smooth muscle muscle Protein
  • Collagen 1 Collagen
  • Fn Fibronectin, fibronectin
  • the fibroblasts of 2* 106 IPF patients induced by TGF- ⁇ 1 were evenly mixed with 500ul Corning Matrigel matrix, and injected subcutaneously into immunodeficient mice. After 7 days, FAPI-sieIF-3B was injected subcutaneously, and the control group ( con, injection of sieIF-3B naked siRNA), the doses were 3mg/kg and 9mg/kg respectively, and 14 days after subcutaneous injection, fibroblasts were taken out to verify the interference effect of eIF-3B gene, and it was found that subcutaneous injection (FAPI-coupled purpose gene) method can also effectively inhibit the expression of the target gene.

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Abstract

提供了靶向FAP阳性细胞的siRNA缀合物，其包括：靶向FAP的配体部分、抑制FAP阳性细胞中的存活基因的表达的siRNA部分、以及位于所述配体部分和所述siRNA部分之间的连接子部分。还提供了制备该siRNA缀合物的方法、其药物组合物及用途。

Description

靶向FAP阳性细胞的siRNA缀合物及其药物组合物和应用 技术领域
本公开涉及小核酸药物，具体涉及靶向成纤维细胞活化蛋白(Fibroblast activation protein,FAP)的siRNA缀合物、其制备方法、药物组合物及用途。
背景技术
在与伤口愈合、癌症和纤维化相关的条件下，活化的成纤维细胞上调表达成纤维细胞活化蛋白(FAP)。FAP在多种癌症中高度上调，表达于90％以上的上皮恶性肿瘤，通常作为促肿瘤基质生长的标志物。肿瘤生长环境中存在着大量肿瘤因子，这些肿瘤因子可以诱导FAP上调，上调的FAP可以激活生长因子，促进血管生成、肿瘤生长及浸润；同时，由于FAP具有胶原酶活性，可以水解细胞外基质(Extracellular Matrix,ECM)中的胶原，从而破坏ECM，促进肿瘤的侵袭和转移。因此，FAP可以直接促进肿瘤细胞的增殖、迁移和侵袭；与FAP高表达细胞共培养也能促进肿瘤、内皮细胞和免疫细胞的增殖、活化和侵袭。
在病理情况下，正常的组织修复反应逃脱了内稳态调节机制并演变为不受控制的纤维化过程，其特征在于细胞外基质的进行性过量产生，这破坏了正常器官的结构并最终导致器官衰竭。在生长因子的刺激下，成纤维细胞可以分化为肌成纤维细胞，继而过度合成胶原蛋白和其它细胞外基质蛋白。与非致病性成纤维细胞不同，活化的肌成纤维细胞会表达成纤维细胞活化蛋白(FAP)。特发性肺纤维化(IPF)是一种慢性进行性致死疾病，其病理学特点为肺实质破坏、肺泡上皮细胞表型显著变化、细胞外基质的沉积以及成纤维细胞异常增殖与积累。目前，FDA在2014年批准了两种药物用于治疗IPF：吡非尼酮和尼达尼布。然而，这两种药物在IPF患者中均显示出有限且不一致的疗效。
研究发现，成纤维细胞或会在一系列自身免疫性疾病中诱发炎症及组织损伤，比如风湿性关节炎、骨质侵蚀和关节软骨的破坏。研究人员对来自风湿性关节炎的滑膜样本进行分析，发现这些组织中蛋白质FAP的高表达与疾病的进展直接相关。通过对关节炎小鼠模型进行研究，发现高水平的FAP与踝关节隆起部位的严重性有关，而且表达该蛋白的细胞能迁移到骨质和软骨中，而这 是细胞破坏的前提。为了分析表达FAP的成纤维细胞的特性，利用大量细胞计数法鉴别出关节中的两类成纤维细胞，其中一种位于内衬(LL，lining layer)区域的滑膜中，而另一种则位于次内衬层(SL，sub-lining layer)中。研究者发现，当关节炎发生时，小鼠关节中所有类型的细胞水平都会增加，而且SL区域中成纤维细胞的数量与炎症的严重性有关，而LL区域中成纤维细胞的数量则与软骨损伤有关，结果表明，不同类型的成纤维细胞或在风湿性关节炎的不同方面扮演着关键角色。为了调查相关研究结果是否能够应用于人类疾病的研究，对来自风湿性关节炎或骨关节炎患者机体滑膜中的细胞样本进行分离分析，发现相比骨关节炎患者而言，风湿性关节炎患者机体中存在大量表达FAP和Thy-1的成纤维细胞群体。
对于癌症、风湿性关节炎和纤维化疾病的治疗的临床需求仍未被满足，寻找更多或更加有效的替代性治疗方案仍然是研究者们的普遍追求。
发明内容
本申请的一个方面提供一种靶向FAP阳性细胞的siRNA缀合物，其包括：(i)靶向FAP的配体部分、(ii)抑制FAP阳性细胞中的存活基因的表达的siRNA部分、以及(iii)位于所述配体部分和所述siRNA部分之间的连接子部分，其中所述靶向FAP的配体部分包含下式(I)的结构：
Figure PCTCN2022118435-appb-000001
其中：
X是
Figure PCTCN2022118435-appb-000002
其左侧连接 至式(I)，其右侧连接至连接子部分；
R 1与R 2相同或不同，并且各自独立地选自氢、卤素和C 1-C 4烷基，优选为卤素，更优选为氟；
R 3是C 1-C 4烷基、腈或异腈，优选为腈(-CN)；
R 4是H或-CH 3；
R 5与R 6相同或不同，并且各自独立地选自氢、卤素和C 1-C 4烷基，优选为氢；
R 7、R 8和R 9相同或不同，并且各自独立地选自氢、甲氧基、卤素、CF 3和C 1-C 4烷基，优选为氢。
在优选的实施方式中，所述靶向FAP的配体部分包含以下结构式：
Figure PCTCN2022118435-appb-000003
Figure PCTCN2022118435-appb-000004
其右侧连接至连接子部分。
在优选的实施方式中，所述靶向FAP的配体部分包含以下结构：
Figure PCTCN2022118435-appb-000005
其右侧连接至连接子部分。
在优选的实施方式中，所述siRNA部分是针对选自以下存活基因中的一个：polr2a、polr2b、dkc1、cenpe、eif-3b。
在优选的实施方式中，所述siRNA部分是针对选自以下存活基因中的一个：survivin,phb2,c-myc以及col1a1.
在优选的实施方式中，所述FAP阳性细胞是活化的成纤维细胞。在优选的实施方式中，所述活化的成纤维细胞存在于肿瘤基质、关节炎或纤维化组织中。在优选的实施方式中，所述肿瘤选自基底细胞癌、鳞状细胞癌、皮肤癌、口腔癌、黑色素瘤、食道癌、腺癌、胃癌、结直肠癌、直肠癌、胰腺癌、肝细胞癌、肺癌、间皮瘤、胆管癌、肝癌、膀胱癌、乳腺癌、肾癌、前列腺癌、宫颈癌、卵巢癌、胶质瘤、甲状腺癌、副甲状腺瘤、肉瘤、骨髓瘤和星形细胞瘤。在优选的实施方式中，所述肿瘤选自肺癌、胆管癌、肝癌、胶质瘤、卵巢癌、乳腺癌和胰腺癌。在优选的实施方式中，所述纤维化组织是肺纤维化组织或肝硬化组织。在优选的实施方式中，所述关节炎是类风湿性关节炎。
在优选的实施方式中，其中所述肺纤维化组织来源于特发性纤维化(IPF)、矽肺、寻常型间质性肺炎(UIP)中的一种或多种。
在优选的实施方式中，所述siRNA部分包含互补的正义链和反义链，所述靶向FAP的配体部分连接至所述正义链的3’端。
在优选的实施方式中，所述连接子部分包含酰胺键。在优选的实施方式中，所述连接子部分具有以下结构式：
Figure PCTCN2022118435-appb-000006
其左侧连接至所述靶向FAP的配体部分，右侧连接至所述siRNA部分，并且其中m和p各自独立地为0至10的整数。在优选的实施方式中，m和p各自独立地为1至5或1至3的整数。
在优选的实施方式中，所述siRNA缀合物具有以下结构式：
Figure PCTCN2022118435-appb-000007
在优选的实施方式中，其中所述正义链如SEQ ID NO:9所示，所述反义链如SEQ ID NO:10所示，所述siRNA缀合物抑制eif-3b基因的表达。
本发明的另一个方面提供一种药物组合物，其包含本发明所述的任一siRNA缀合物以及药学上可接受的载体。在优选的实施方式中，所述药物组合物被配制成注射液。
本发明的另一个方面提供上述任一所述的siRNA缀合物在制备抑制FAP阳性细胞的增殖或存活的药物中的应用。
本发明的另一个方面提供一种抑制FAP阳性细胞的增殖或存活的方法，所述方法包括使所述FAP阳性细胞与本发明任一所述的siRNA缀合物接触，使得所述siRNA缀合物被所述FAP阳性细胞摄取，从而抑制FAP阳性细胞的增殖或存活。
本发明的另一个方面提供一种治疗对象的由FAP阳性细胞介导的疾病的方法，所述方法包括向所述对象施用治疗有效量的本发明的任一所述的siRNA缀合物或其药物组合物。在优选的实施方式中，所述由FAP阳性细胞介导的疾病是肿瘤、纤维化疾病或关节炎。在优选的实施方式中，所述肿瘤选自基底细胞癌、鳞状细胞癌、皮肤癌、口腔癌、黑色素瘤、食道癌、腺癌、胃癌、结直肠癌、直肠癌、胰腺癌、肝细胞癌、肺癌、间皮瘤、胆管癌、肝癌、膀胱癌、乳腺癌、肾癌、前列腺癌、宫颈癌、卵巢癌、胶质瘤、甲状腺癌、副甲状腺瘤、肉瘤、骨髓瘤和星形细胞瘤。在优选的实施方式中，所述肿瘤选自肺癌、胆管癌、肝癌、胶质瘤、卵巢癌、乳腺癌和胰腺癌。在优选的实施方式中，所述纤维化疾病是肺纤维化或肝硬化。在优选的实施方式中，所述纤维化疾病是特发性肺纤维化(IPF)。在优选的实施方式中，所述由FAP阳性细胞介导的疾病是类风湿性关节炎(RA)。
附图说明
图1.稳定表达hFAP细胞株hFAP阳性率流式检测图。
图2.显示siRNA缀合物的合成路线图。
图3.给予293T、293T-hFAP细胞50nM浓度的一种靶向Actin的示例性siRNA缀合物试剂导致的Actin mRNA抑制。
图4.给予A549-GFP、A549-GFP-hFAP细胞50nM浓度的一种靶向GFP的示例性siRNA缀合物导致的GFP mRNA抑制。
图5.给予A549、A549-hFAP裸鼠一个3mg/kg的单个尾静脉剂量的一种靶向GAPDH的示例性siRNA缀合物导致的GAPDH mRNA抑制。以靶向GAPDH的无FAP配体部分的siRNA缀合物试剂为阴性对照。
图6.给予A549-GFP、A549-GFP-hFAP裸鼠一个3mg/kg的单个尾静脉剂量的一种靶向GFP的示例性siRNA缀合物导致的GFP mRNA抑制。以靶向MYC的本发明的siRNA缀合物为阴性对照。
图7.靶向细胞存活相关基因eIF-3β的本发明的siRNA缀合物导致eIF-3β的mRNA的表达抑制。
图8.FAP在不同纤维化病人中的表达情况。A.来自健康供体(n＝6)、特发性肺纤维化(IPF，n＝8)和矽肺(Silicosis，n＝12)患者的肺组织中FAP的mRNA相对表达量；B.Western blot检测健康供者、IPF、Silicosis肺组织中FAP的表达量；C.来自另一批健康供者(n＝7)、寻常型间质性肺炎(UIP，n＝6)、特异性间质性肺炎(NSIP，n＝8)患者的肺组织中FAP的mRNA相对表达量；D.Western blot检测健康供者、UIP、NSIP肺组织中FAP的表达量。
图9.TGF-β刺激成纤维细胞后，流式细胞术检测到FAP表达上调。
图10.FAPI-siSurvivin在体外抑制FAP+的IPF成纤维细胞中Survivin的表达。
图11.FAPI-sieIF-3B在体外抑制FAP+的IPF成纤维细胞中eIF-3B的表达。
图12.FAPI-siCOL1A1在体外抑制FAP+的IPF成纤维细胞中COL1A1的表达。
图13.FAPI-siPHB2在体外抑制FAP+的IPF成纤维细胞中PHB2的表达。
图14.FAPI-siC-MYC在体外抑制FAP+的IPF成纤维细胞中C-MYC的表达。
图15.靶向细胞存活相关基因的shRNA(survivin、eIF-3B、PHB2、POLR2B、DKC1)在体外抑制IPF病人成纤维细胞增殖。
图16.靶向细胞存活相关基因的shRNA(survivin、eIF-3B、PHB2、POLR2B、DKC1)在体外抑制IPF病人成纤维细胞的纤维化。
图17.IPF病人成纤维细胞与基质胶混合后皮下注射一周后可形成微血管。
图18.FAPI-siSurvivin在体内可以有效抑制IPF成纤维细胞的纤维化。
图19.FAPI-sieIF-3B在小鼠体内可以有效抑制IPF成纤维细胞的纤维化。
图20.FAPI-siCOL1A1在小鼠体内可以有效抑制IPF成纤维细胞的纤维化。
图21.FAPI-sieIF-3B皮下注射可长期有效抑制IPF成纤维细胞的纤维化。
*，P<0.05，**，P<0.01,***，P<0.001。
具体实施方式
定义
术语“siRNA”是长度为20到25个核苷酸的双链RNA，包含两条反向平行的和基本上互补的核酸链。相对于靶RNA，这两条核酸链被命名为正义链和反义链。siRNA部分通过转录后基因沉默机制(在此称为RNA干扰或RNAi)触发靶RNA(例如mRNA)的降解。通常，siRNA部分的每条链的大部分的核苷酸是核糖核苷酸，但是两条链的每一条或两者还可以包括一个或多个非核糖核苷酸，例如，脱氧核糖核苷酸和/或修饰的核苷酸。另外，siRNA部分可以包括具有化学修饰的核糖核苷酸，也可以包括在多个核苷酸处的实质性修饰。这些修饰可以包括在此披露的或在本领域中已知的所有类型的修饰。任何这样的修饰均意在被包括在“siRNA”的定义范围内。
术语“互补”是指在双链核酸分子中，一条链的碱基各自与另一条链上的碱基以互补的方式相配对。在RNA中，嘌呤碱基腺嘌呤(A)始终与嘧啶碱基尿嘧啶(U)相配对；嘌呤碱基鸟嘌呤(C)始终与嘧啶碱基胞嘧啶(G)相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸尿嘧啶配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，并且从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。术语“基本上互补”是指所涉及的两段核苷酸序列之间存在不多于3个的碱基错配；“实质上互补”是指两段核苷酸序列之间存在不多于1个的碱基错配；“完全互补”是指两段核苷酸序列之间不存在碱基错配。
术语“抑制”，可以与“减少”、“沉默”、“下调”、“压制”和其他类似术语交替使用，并且包括任何水平的抑制。如在此使用的，短语“抑制...基因的表达”包括抑制任何物种的该基因(如小鼠基因、大鼠基因、猴基因或人基因)以及该基因的变体(例如天然存在的变体)或突变体的表达。因此，该基因可以是野生型基因、突变基因或在遗传操作的细胞、细胞群组或生物体的情形下的转基因。“抑制...基因的表达”或类似术语包括任何水平的基因的抑制，例如至少部分抑制基因的表达，如抑制至少约5％、至少约10％、至少约15％、至少约20％、 至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、或至少约99％。基于与该基因表达相关的任何变量水平，例如mRNA水平、编码蛋白的水平等可以评估该基因的表达情况。将这些变量中的一个或多个与对照水平相比，通过其绝对或相对水平的减少来评估抑制程度。对照水平可以是本领域中利用的任何类型的对照水平，例如给药前基线水平或从类似的未经处理或经对照(例如仅缓冲液对照或惰性剂对照)处理的对象、细胞、或样品确定的水平。
术语“FAP阳性细胞”是指与正常细胞相比细胞表面的FAP表达升高或过表达的细胞。FAP在正常组织中通常不表达或低表达，在与伤口愈合、类风湿性关节炎、癌症和纤维化等相关的条件下，活化的成纤维细胞上调表达FAP。在优选的实施方式中，所述FAP阳性细胞介导对象中的一种或多种疾病或症状。FAP阳性细胞的典型例子是位于纤维化组织、关节滑膜或肿瘤基质中的活化的成纤维细胞。活化的成纤维细胞在不同组织和病理状态下可能具有不同的命名。例如，在某些情况下，肿瘤基质中的活化的成纤维细胞也被称为FAP阳性肿瘤细胞(例如FAP阳性肺癌细胞、FAP阳性结直肠癌细胞等)。在类风湿性关节炎中，活化的成纤维细胞称为成纤维样滑膜细胞或类风湿关节炎成纤维样滑膜细胞(HFLS-RA)。通过本领域的已知技术(例如免疫组化、染色)可确定细胞表面是否呈FAP阳性。用于检测FAP阳性的试剂(如荧光标记的FAP抗体)和仪器(如流式细胞仪)在本领域是容易获得的。
术语“FAP阳性细胞介导的疾病”包括任何与FAP阳性细胞相关的疾病或症状。例如，这种疾病可以由FAP蛋白的过量表达引起。“FAP阳性细胞介导的疾病”优选地是指患有该疾病的个体体内检测到显著的FAP表达的疾病。如上所述，大多数正常的成人组织显示很少或没有可检测到的FAP表达，在人类中通过ELISA在健康个体中测得的FAP血浆浓度约为100ng/mL或0.6nmol/L。因此，“FAP阳性细胞介导的疾病”是指患有该疾病的个体体内FAP血浆浓度为至少约1μg/mL、至少约2μg/mL、至少约3μg/mL、至少约4μg/mL、至少约 5μg/mL、至少约6μg/mL、至少约7μg/mL、至少约8μg/mL、至少约9μg/mL、至少约10μg/mL、至少约15μg/mL、至少约20μg/mL、至少约25μg/mL、至少约50μg/mL、至少约100μg/mL或更高。
典型的FAP阳性细胞介导的疾病的例子是肿瘤、纤维化疾病和类风湿性关节炎。在优选的实施方式中，所述肿瘤选自基底细胞癌、鳞状细胞癌、皮肤癌、口腔癌、黑色素瘤、食道癌、腺癌、胃癌、结直肠癌、直肠癌、胰腺癌、肝细胞癌、肺癌、间皮瘤、胆管癌、肝癌、膀胱癌、乳腺癌、肾癌、前列腺癌、宫颈癌、卵巢癌、胶质瘤、甲状腺癌、副甲状腺瘤、肉瘤、骨髓瘤和星形细胞瘤。在优选的实施方式中，所述肿瘤选自肺癌、胆管癌、肝癌、胶质瘤、卵巢癌、乳腺癌和胰腺癌。在优选的实施方式中，所述纤维化疾病是肺纤维化或肝硬化。在优选的实施方式中，所述纤维化疾病是特发性肺纤维化(IPF)。在优选的实施方式中，所述由FAP阳性细胞介导的疾病是类风湿性关节炎(RA)。在优选的实施方式中，所述纤维化疾病是特发性纤维化(IPF)、矽肺、寻常型间质性肺炎(UIP)中的一种或多种。
术语“存活基因(survival genes)”可与“必需基因(essential genes)”互换使用，是指对于特定细胞的生存或繁殖所必需的基因，这些基因中的一个的功能缺陷会致使该细胞出现致死表型而无法存活或繁殖。典型的存活基因是编码那些在遗传物质的转录、复制和表达过程中的关键蛋白酶的基因，包括但不限于POLR2A(DNA-directed RNA polymerase II subunit RPB1，RNA聚合酶II的最大亚基，可合成mRNA前体和许多功能性非编码RNA，与第二大亚基一起形成聚合酶活性中心)、POLR2B(DNA-directed RNA polymerase II subunit RPB2,RNA聚合酶II的第二大亚基，与第一大亚基一起形成聚合酶活性中心)、DKC1(H/ACA ribonucleoprotein complex subunit DKC1，核糖核蛋白复合物亚基)、CENPE(Centromere-associated protein E，有丝分裂相关蛋白E)、eIF-3b(eukaryotic translation initiation factor 3subunit b，真核转录起始因子3亚基b)、COL1A1(I型胶原α1链基因)、survivin、PHB2(prohibitin 2)等等。相应地，术语“FAP阳性细胞中的存活基因”是指对于FAP阳性细胞的生存或繁殖所必需的基因，特别是指对于成纤维细胞(优选是活化的)的生存或繁殖所必需的基因。确定人类细胞中特定基因是否是必须基因的方法在本领域是已知的，例如可参 见Wang T et al.Identification and characterization of essential genes in the human genome.Science 27 Nov 2015:Vol.350,Issue 6264,pp.1096-1101或Vincent A.Blomen et al.,Gene essentiality and synthetic lethality in haploid human cells.Science 27 Nov 2015:Vol.350,Issue 6264,pp.1092-1096。
术语“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“siRNA缀合物”表示一个或多个具有特定功能的化学部分与siRNA共价连接至而形成的化合物。在下文中，有时也将本公开的siRNA缀合物简称为“缀合物”。在本文中，siRNA缀合物应理解为siRNA缀合物的总称，其大致包括靶向配体部分、siRNA部分和连接两者的连接子部分，三者通过化学反应共价结合在一起形成“siRNA缀合物”的特定化合物。
短语“使细胞与siRNA缀合物接触”或类似表述包括通过任何可能手段使细胞接触，包括使细胞在体外与该siRNA缀合物接触或使细胞在体内与siRNA缀合物接触。在体外接触细胞可通过例如用该siRNA缀合物孵育该细胞来进行。体内接触细胞可以通过以下进行，例如通过将siRNA缀合物注入该细胞所在的组织或其附近，或通过将siRNA缀合物注入另一个区域(例如通过静脉注射或皮下注射)，使得该试剂随后到达待接触的细胞所在的组织。
术语“治疗有效量”旨在包括当施用至患者以治疗FAP阳性细胞介导的疾病时足以实现该疾病的治疗(例如通过削弱、改善或维持该疾病或症状)的siRNA缀合物的量。该“治疗有效量”可以根据该siRNA缀合物本身、给药途径、给药方式、疾病类型及其严重程度、病史、年龄、体重、家族史、遗传组成、病理过程的阶段、先前或伴随治疗(如有)的类型以及待治疗的患者的其他个体特征而变化。
术语“患者”或“对象”包括人类或者非人类动物，优选哺乳动物，例如猴。最优选地，该对象或患者是人。
抑制FAP阳性细胞中的存活基因的表达的siRNA部分
作为本公开的siRNA缀合物的药效部分，“抑制FAP阳性细胞中的存活基因的表达的siRNA部分”是一条双链RNA分子，其在被靶细胞摄取后，通过RNAi机制途径特异性靶向特定存活基因的mRNA的降解，从而抑制FAP阳性 细胞中的存活基因的表达。
该siRNA部分具有正义链和反义链，长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸。这样，本公开提供的siRNA正义链和反义链的长度比可以是19/19、19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25或23/26。在一些实施方案中，所述siRNA部分的正义链和反义链的长度比为19/21、21/23或23/25。
在一些实施方案中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV长度各自为1-4个核苷酸；所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或者完全反向互补；所述核苷酸序列III连接在所述核苷酸序列I的5'末端，所述核苷酸序列IV连接在所述核苷酸序列II的3'末端。在一些实施方案中，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA片段的核苷酸序列的5'末端相邻，且长度与所述核苷酸序列IV相同的核苷酸序列。
在一些实施方案中，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为U，核苷酸序列IV的碱基为A；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CU，核苷酸序列IV的碱基组成为AG；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UCU，核苷酸序列IV的碱基组成为AGA；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UUCU，核苷酸序列IV的碱基组成为AGAA；此时，正义链和反义链的长度比为23/23。在一些实施方案中，所述核苷酸序列III和核苷酸序列IV的长度为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CU，核苷酸序列IV的碱基组 成为AG；此时，正义链和反义链的长度比为21/21。在一些实施方案中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。
针对靶基因设计siRNA的方法是本领域的常规技术。例如，有多个siRNA设计网站可以提供在线设计服务(例如DSIR:http://biodev.extra.cea.fr/DSIR/DSIR.html或siDirect version 2.0:http://sidirect2.rnai.jp/)。这些设计网站可基于靶基因的mRNA同时获得针对该靶基因的不同片段的多个代表性siRNA序列。这些代表性siRNA可基于多个选项(例如热力学、高级结构等)排序。候选siRNA序列可进一步被筛选(人工或自动)，以排除含有SNP位点或结合非开放阅读框片段的序列。在一些实施方式中，选择在不同在线设计网站中的重合或重叠结果作为候选siRNA序列。候选siRNA序列还可以通过脱靶(off-target)预测软件来分析具有潜在高脱靶性的siRNA序列(如http://rnai.cs.unm.edu/rnai/off-target)。
siRNA的设计可选地遵循一般设计准则。例如，在靶向mRNA序列中找到一段21nt的以AA二核苷酸起始的序列。从转录本的AUG起始密码子开始，扫描AA二核苷酸序列。记录下每一个AA及之后3’相邻的19个核苷酸作为潜在的siRNA靶向位点。这种选择siRNA靶向位点的方式是因为研究发现3’端带有UU二核苷酸悬臂的siRNA最为有效。该方式同样与使用RNA pol III来转录发卡siRNA相符，因为RNA pol III会在4-6个核苷酸poly(T)处终止转录，从而得到带有短poly(U)尾的RNA。带有其他3’端终止双核苷酸突出的siRNA已被证明可以有效诱导RNAi。若需要，还可以对这种靶向位点选择方式进行修改，以设计带有其他双核苷酸突出的siRNA，不过一般建议避免选择GG突出末端，因为siRNA在单链G残基处可能被RNase剪切。
另一个准则是选择2-4个靶向序列。通常超过一半的随机设计siRNA可以使得靶向的mRNA在RNA水平上至少降低50％，大约四分之一的siRNA则会得到75-95％的降低。按照如下指南对第一步鉴定得到的序列进行靶向位点选择：研究发现GC含量在30-50％的siRNA比G/C含量更高的siRNA活性高。由于4-6个核苷酸poly(T)会成为RNA pol III的终止信号，如果需要用RNA pol III启动来进行表达，要避免4个以上T或A连续出现在目标序列中。由于mRNA 的部分区域可能具有较复杂的结构或与调控蛋白质相结合，通常会在基因序列的不同位置来选择siRNA靶向位点。未观察到mRNA上靶向位点的位置与siRNA效能之间的相关性。将潜在的靶向位点与相应的基因组数据库(人、小鼠、大鼠等)进行对比，如果靶向序列与其他基因编码序列具有超过16-17个连续碱基对同源性，就不能选择这样的序列。建议使用BLAST(请访问NCBI服务器：www.ncbi.nlm.nih.gov/BLAST)。
完整的siRNA实验应当包含一系列对照，以确保数据的有效性。示例性的两种对照为：(1)阴性对照siRNA，其与siRNA具有相同的核苷酸成分，但与基因组无明显的序列同源性。要设计阴性对照siRNA，可以将基因特异性siRNA的核苷酸序列打乱，然后进行搜索以确保其与任意基因均不具有同源性。(2)靶向相同mRNA的其他siRNA序列。确保RNAi数据可信度的最佳方法可能就是每次使用一条单独siRNA，对同一条基因使用两条或更多不同siRNA进行实验。在这些实验之前，需要对每条siRNA进行测试以确保它们能够将目标基因的表达水平降到比较接近的水平。
siRNA序列还可通过各种化学修饰以增强其稳定性、延长半衰期和/或增强疗效(例如沉默效率)。代表性的化学修饰包括4位的硫代修饰以增加siRNA的稳定性；LNA(locked nucleic acid)与siRNA结合增加稳定性；磷酸骨架的修饰；以及2’戊糖环的修饰。各种修饰方法是本领域已知的，并且可以从商业上获得。
在一些实施方式中，所述siRNA部分的siRNA序列靶向FAP阳性细胞的存活基因。例如，所述siRNA序列靶向polr2a、polr2b、dkc1、cenpe、eif-3b基因中的一个或多个的一个或多个片段。在一些实施方式中，当siRNA序列被递送至细胞后，经RNAi途径与靶基因结合，导致靶基因的表达的抑制。在一些实施方式中，靶基因的表达抑制导致FAP阳性细胞的凋亡、死亡和/或增殖抑制。在一些实施方式中，FAP阳性细胞的凋亡、死亡和/或增殖抑制介导疾病的治疗或治愈。合适的存活基因包括但不限于polr2a、polr2b、dkc1、cenpe、eif-3b基因中的一个或多个。其他合适的存活基因包括但不限于survivin,phb2,c-myc以及col1a1基因中的一个或多个。靶向存活基因的siRNA序列可通过如上所述的方法获得并测试。
在一些实施方式中，所述siRNA部分包含正义链和反义链,其中所述正义链如SEQ ID NO:9所示，所述反义链如SEQ ID NO:10所示，所述siRNA缀合物抑制eif-3b基因的表达。
在一些实施方式中，所述siRNA部分包含正义链和反义链,其中所述正义链如SEQ ID NO:11所示，所述反义链如SEQ ID NO:12所示，所述siRNA缀合物抑制survivin基因的表达。
在一些实施方式中，所述siRNA部分包含正义链和反义链,其中所述正义链如SEQ ID NO:13所示，所述反义链如SEQ ID NO:14所示，所述siRNA缀合物抑制col1a1基因的表达。
在一些实施方式中，所述siRNA部分包含正义链和反义链,其中所述正义链如SEQ ID NO:15所示，所述反义链如SEQ ID NO:16所示，所述siRNA缀合物抑制phb2基因的表达。
在一些实施方式中，所述siRNA部分包含正义链和反义链,其中所述正义链如SEQ ID NO:17所示，所述反义链如SEQ ID NO:18所示，所述siRNA缀合物抑制polr2b基因的表达。
在一些实施方式中，所述siRNA部分包含正义链和反义链,其中所述正义链如SEQ ID NO:19所示，所述反义链如SEQ ID NO:20所示，所述siRNA缀合物抑制dkc1基因的表达。
本公开提供的siRNA可以通过本领域常规的siRNA制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。
靶向FAP的配体部分
靶向FAP的配体部分构成本公开的siRNA缀合物的靶向部分，其用于识别、结合FAP阳性细胞，从而将负载的siRNA部分递送至靶细胞。在本公开中，靶向FAP的配体部分是小分子化合物部分，优选是与FAP具有约1nM至约20nM的结合亲和力的靶向配体。在优选的实施方式中，靶向FAP的配体部分是分子量低于10,000。在一些实施方式中，靶向FAP的配体是FAP小分子抑 制剂。
在一些实施方式中，靶向FAP的配体包含下式(I)的结构：
Figure PCTCN2022118435-appb-000008
其中：
X是
Figure PCTCN2022118435-appb-000009
其左侧连接至式(I)，其右侧连接至连接子部分；
R 1与R 2相同或不同，并且各自独立地选自氢、卤素和C 1-C 4烷基，优选为卤素，更优选为氟；R 3是C 1-C 4烷基、腈或异腈，优选为腈；R 4是H或-CH 3；R 5与R 6相同或不同，并且各自独立地选自氢、卤素和C 1-C 4烷基，优选为氢；R 7、R 8和R 9相同或不同，并且各自独立地选自氢、甲氧基、卤素、CF 3和C 1-C 4烷基，优选为氢。
在优选的实施方式中，所述靶向FAP的配体部分包含以下结构式：
Figure PCTCN2022118435-appb-000010
Figure PCTCN2022118435-appb-000011
Figure PCTCN2022118435-appb-000012
其右侧连接至连接子部分。
在优选的实施方式中，所述靶向FAP的配体部分包含以下结构：
Figure PCTCN2022118435-appb-000013
其右侧连接至连接子部分。
更多的靶向FAP的配体分子可见于中国发明专利申请公布CN110291401A或CN111699181A，通过引用将其内容整体合并至本文中。
在一些实施方式中，本发明的siRNA缀合物可包含不止一个靶向FAP的 配体分子，例如2至5个配体分子，优选为3个配体分子。每个靶向FAP的配体分子可以相同或不同，优选相同。在一个实施方式中，本发明的siRNA缀合物包含3个如以下结构式所示的配体：
Figure PCTCN2022118435-appb-000014
当本发明的siRNA缀合物包含不止1个配体分子时，多个配体分子可以共价结合于同一个原子(例如C原子)，以形成多配体结构。多配体结构被认为可增加siRNA缀合物识别和结合FAP阳性细胞的能力和效率。
在一些实施方式中，形成靶向FAP的配体部分的分子可具有羧基(-COOH)，以利于通过连接子部分的伯胺基(详见下文)形成酰胺键。NHS或sulfo-NHS在活化羧基的用途方面是已知的，其有助于将羧基与末端伯胺基反应生成酰胺键。因此，在本发明的一些实施方式中，将具有羧基的FAP配体与EDC/NHS或EDC/sulfo-NHS体系反应，以生成FAP配体-NHS。在本公开中，靶向FAP的配体部分也被标记为FAPI-。
一个示例性的FAP配体-NHS的结构如下所示：
Figure PCTCN2022118435-appb-000015
FAPI-NHS可购自例如无锡捷化医药科技有限公司，或者也可参见 CN110291401A或CN111699181A制备FAPI-NHS。
连接子部分
在本公开的siRNA缀合物中，连接子部分介于siRNA部分和配体部分之间，其分别与siRNA部分和配体部分形成共价连接，从而形成siRNA缀合物分子。
在本公开中，连接子部分具有连接siRNA部分的第一连接基团和具有连接靶向FAP的配体部分的第二连接基团。在一些实施方式中，连接子部分的第一连接基团和的第二连接基团通过一个或多个亚甲基相连。在一些实施方式中，第一连接基团是羟基。在一些实施方式中，第二连接基团是伯胺基。在一些实施方式中，第一连接基团是羟基并且第二连接基团是伯胺基。
在一些实施方式中，连接子部分可与siRNA部分首先连接以形成连接子-siRNA部分。在一个实施例中，例如可以使用以下结构式的连接子连接至正义链的3’端，其中p为0至10的整数：
Figure PCTCN2022118435-appb-000016
所形成的连接子-siRNA部分在3’端修饰有游离的伯胺基以便于与FAP配体部分连接。
在一个实施例中，连接子-siRNA的示例性结构如下：
Figure PCTCN2022118435-appb-000017
在其他的实施方式中，连接子具有不同于伯胺基的其他反应性基团，从而与FAP配体部分的相应反应性基团发生化学反应以形成共价键合。示例性的其他连接子结构包括但不限于那些用于抗体药物偶联物(ADC)的可裂解或不可裂解接头结构。
在一些实施方式中，所得到的连接子-siRNA部分再与FAP配体部分通过第二连接基团共价连接。在一些实施方式中，FAP配体部分具有羧基，因而可与作为连接子部分的第二连接基团的伯胺基反应形成酰胺键而将FAP配体部分与连接子-siRNA部分共价连接，从而形成本公开的缀合物。NHS或sulfo-NHS在活化羧基的用途方面是已知的，其有助于将羧基与末端伯胺基反应生成酰胺键。在本发明的一些实施方式中，将具有羧基的FAP配体与EDC/NHS或EDC/sulfo-NHS体系反应，以生成FAP配体-NHS，再将FAP配体-NHS与以上所述的连接子-siRNA反应，以生成本发明的siRNA缀合物。
在优选的实施方式中，在所述siRNA缀合物中，所述连接子部分包含酰胺键。在优选的实施方式中，在所述siRNA缀合物中，所述连接子部分具有以下结构式：
Figure PCTCN2022118435-appb-000018
其左侧连接至所述靶向FAP的配体部分，右侧连接至所述siRNA部分，并且其中m和p各自独立地为0至10的整数。在优选的实施方式中，m和p各自独立地为1至5或1至3的整数。
在一些实施方案中，所述连接子部分可以连接在核苷酸的磷酸基团、3'-位羟基或者碱基上。在一些实施方案中，所述连接子部分可以连接在3'-位羟基上，此时核苷酸之间采用2'-5'磷酸二酯键连接。当连接子部分连接在siRNA链的末端时，所述连接子部分通常连接在核苷酸的磷酸基团上；当连接子部分连接在siRNA的内部序列时，所述连接子部分通常连接在核糖糖环或者碱基上。在一些实施方案中，所述siRNA与连接子部分间可以通过酸不稳定的或可还原的化学键相连，在细胞内涵体的酸性环境下，这些化学键可降解，从而使siRNA成为自由状态。对于不可降解的缀合方式，连接子部分可连接在siRNA的正义链，从而尽量降低缀合对siRNA活性的影响。
在本公开中，所述siRNA缀合物的三个部分在结构上的划分界限是可变 化的。例如FAP配体部分与连接子部分在结构上的界限是可变化的。可选地或并且，连接子部分与siRNA部分在结构上的界限是可变化的。也就是说，对于处于连接区域的一个或多个基团或基团集合，其可作为FAP配体部分的一部分，也可以作为连接子部分的一部分，或者其可作为连接子部分的一部分，也可以作为siRNA部分的一部分(例如siRNA修饰部分)。例如，在一些实施方式中，所述siRNA缀合物中的所述连接子部分包含酰胺键，所述连接子部分的酰胺键左侧的m个亚甲基部分可来自于靶向FAP的配体部分(m为如上定义)。即，在定义上，连接子中酰胺键左侧的部分也可以被划归为靶向FAP的配体部分，从而构成靶向FAP的配体的一部分。例如连接子中酰胺键左侧的所有亚甲基可被认为是靶向FAP的配体部分的一部分。
靶向FAP阳性细胞的siRNA缀合物
在本公开中，siRNA缀合物是通过将靶向FAP的配体部分与siRNA部分通过连接子部分共价结合形成的。因此，所述靶向FAP阳性细胞的siRNA缀合物包含(i)靶向FAP的配体部分、(ii)抑制FAP阳性细胞中的存活基因的表达的siRNA部分、以及(iii)位于所述配体部分和所述siRNA部分之间的连接子部分。在本发明的一些实施方式中，所述各个部分如以上所分别定义。
在本公开中，靶向FAP的配体部分发挥所述缀合物的靶向作用，其引导缀合物结合至表面呈FAP表达阳性的细胞，例如癌细胞或关节滑膜成纤维细胞。以FAP作为靶标开发的分子成像诊断技术已在核医学领域应用。例如 68Ga-FAPI PET/CT显像已用于胰腺癌、乳腺癌、肝癌、头颈癌、甲状腺癌等不同类型的癌症患者的诊断。本公开的实施例证实，具有靶向FAP的配体部分的缀合物能够特异性结合FAP表达阳性的细胞。
在本公开中，抑制FAP阳性细胞中的存活基因的表达的siRNA部分发挥缀合物的药效作用。siRNA药物通过RNAi机制介导靶基因的沉默效应。首款siRNA药物Onpattro(Patisiran，Alnylam)被FDA批准用于治疗由hATTR引起的多发性神经病变患者。目前全球上市的小核酸药物共有14款，包括4款siRNA药物，均由Alnylam公司开发或合作开发，用于治疗家族性淀粉样多发性神经病变(Onpattro)、急性肝卟啉症(Givlaari)、原发性高草酸尿症1型(Oxlumo)和成人高胆固醇血症及混合型血脂异常(Inclisiran)。在本公开中， siRNA部分靶向FAP表达阳性细胞的存活基因，以抑制存活基因的表达，从而最终导致靶细胞的死亡或增殖抑制。本公开的实施例证实，FAP配体能够有效携带siRNA靶向FAP表达阳性细胞，并抑制细胞内靶基因的表达。
在一些实施方式中，本发明的缀合物被标记为FAPI-基因siRNA、FAPI-si基因(如FAPI-siSurvivin)或FAPI-基因RNAi试剂，例如FAPI-GFP siRNA表示靶基因为GFP的具有FAP靶向配体的双链siRNA，FAPI-eIF-3βsiRNA表示靶基因为eIF-3β的具有FAP靶向配体的双链siRNA，FAPI-siSurvivin表示靶基因为Survivin的具有FAP靶向配体的双链siRNA，si基因(如siSurvivin)表示不含FAP靶向配体的裸siRNA。
在一个实施方式中，所述靶向FAP阳性细胞的siRNA缀合物的结构式如下所示：
Figure PCTCN2022118435-appb-000019
其中sense strand代表siRNA分子的正义链，antisense strand代表siRNA分子的反义链。
药物组合物
本公开提供了一种药物组合物，所述药物组合物含有如上所述的siRNA缀合物作为活性成分和药学上可接受的载体。在所述药物组合物中，对siRNA缀合物和药学上可接受的载体的含量没有特别要求，可以是各组分常规的含量。在一些实施方案中，siRNA缀合物与药学上可接受的载体的重量比可以为1:(1-500)，在一些的实施方案中，上述重量比为1:(1-50)。在一些实施方案中，所述药物组合物中，还可以包含药学上可接受的其它辅料，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括pH缓冲液、保护剂和渗透压调节剂中的至少一种。所述pH缓冲液 可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，例如可以为pH值5.5-8.5的磷酸盐缓冲液。所述保护剂可以为肌醇、山梨醇、蔗糖、海藻糖、甘露糖、麦芽糖、乳糖和葡萄糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％。所述渗透压调节剂可以为氯化钠和/或氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克(mOsm/kg)。根据所需渗透压，本领域技术人员可以容易地确定所述渗透压调节剂的含量。
在一些实施方案中，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉或静脉注射给药，也可以但不限于通过喷雾给药到肺脏或通过喷雾经肺脏给药到其它脏器组织(如肝脏)。在一些实施方案中，所述药物组合物用于静脉注射给药。在一些实施方案中，所述药物组合物可以为脂质体制剂的形式。在一些实施方案中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化脂质。
方法和应用
在一些实施方案中，本公开提供了本公开的siRNA缀合物在制备用于治疗FAP阳性细胞介导的疾病的药物中的用途。
在一些实施方案中，本公开提供了一种治疗FAP阳性细胞介导的疾病的方法，该方法包括将有效量的本公开的siRNA缀合物给予有需要的对象。通过将本公开的siRNA缀合物给予有需要的对象，可以通过RNA干扰的机制达到治疗FAP阳性细胞介导的疾病的目的。因此，本公开的siRNA缀合物可用于治疗FAP阳性细胞介导的疾病，或用于制备用于治疗FAP阳性细胞介导的疾病的药物。
本文所使用的术语“给药/给予”是指通过使得至少部分地将本公开的siRNA缀合物定位于期望的位点以产生期望效果的方法或途径，将本公开的siRNA缀合物放置入受试者体内。适于本公开方法的给药途径包括局部给药和全身给药。一般而言，局部给药导致与受试者体循环相比将更多siRNA缀合物递送至特定位点；而全身给药导致将本公开的siRNA缀合物递送至受试者的体 循环。
可通过本领域已知的任何合适途径向受试者给药，所述途径包括但不仅限于：口服或胃肠外途径，如静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月或每年一次或多次。
本公开所述的siRNA缀合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是受试者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD50(使50％的群体死亡的致死剂量)和ED50(在量反应中指能引起50％最大反应强度的剂量，在质反应中指能引起50％实验对象出现阳性反应时的剂量)。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。
在给予本公开所述siRNA缀合物时，例如，对于雄性或雌性、6-12周龄、体重18-25g的C57BL/6N小鼠，对于siRNA缀合物，以siRNA的量计，其siRNA用量可以为0.001-100mg/kg体重，在一些实施方案中为0.01-50mg/kg体重，在一些实施方案中为0.05-20mg/kg体重，另一些实施方案中为0.1-15mg/kg体重，另一些实施方案中为0.1-10mg/kg体重。
实施例
实施例1.稳定表达hFAP细胞株构建
根据NCBI查找、确认人FAP(hFAP)的mRNA序列(NM_004460.5)，构建hFAP慢病毒过表达载体，经测序验证后，体外包装病毒。取对数生长期293T、A549细胞，细胞计数调整细胞密度，分别以3×10 5个/孔的密度铺于6孔板中，每孔加入2ml完全培养基，在37℃、5％CO 2的氛围下培养过夜用于次日病毒感染实验(次日细胞密度达到60％-70％)。弃去6孔板中培养基，PBS漂洗一次，加入4ml上述步骤包装得到的过表达hFAP慢病毒或过表达hFAP-GFP慢病毒，进行病毒感染。缓慢、均匀滴加病毒液至细胞中，在37℃、5％CO 2的氛围下培养48h后更换新的完全培养基。待细胞长满后进行细胞传代，新的培养基中加入2μg/ml嘌呤霉素进行药筛，经过3-4代的持续性药物筛选后，用流式细胞仪检测293T、A549细胞中的hFAP阳性率，以确定293T-hFAP、A549-hFAP、 A549-GFP-hFAP细胞系构建成功。
结果：流式细胞术结果展示于图1中，可见A549-hFAP、A549-GFP-hFAP、293T-hFAP稳转细胞系构建成功。
实施例2.siRNA缀合物的制备
siRNA的设计与合成：通过在线设计工具设计针对不同靶基因的mRNA的siRNA序列(见表1)，通过固相合成法合成正义链和反义链，退火后进行RPC反向纯化得到siRNA序列。siRNA序列的有义链与连接子共价连接，使有义链的3’端修饰有NH 2。
siRNA缀合物的制备：本文实施例中所用各FAPI-siRNA(以例如FAPI-si基因或FAPI-基因siRNA的形式表示)均按图2显示的siRNA缀合物的制备流程制备，其中FAPI-NHS来自无锡捷化医药科技有限公司( 1HNMR(400MHz,CDCl 3)δ8.80(d,J＝4.0Hz,1H),8.06(d,J＝8.0Hz,1H),7.70(brs,1H),7.50-7.51(m,1H),7.43-7.47(m,1H),7.07(brs,1H),5.03-5.05(m,1H),4.41-4.47(m,1H),4.21-4.30(m,3H),3.98-4.07(m,2H),2.79-2.94(m,8H),2.31-2.36(m,2H).MS(ESI-API):544[M+1] +。
表1.合成的靶向不同基因的siRNA的序列
Figure PCTCN2022118435-appb-000020
Figure PCTCN2022118435-appb-000021
实施例3.体外用FAPI-Actin/GFP siRNA抑制Actin/GFP
siRNA干扰hFAP稳转细胞株：取对数生长期的293T和293T-hFAP细胞、A549-GFP和A549-GFP-hFAP细胞，细胞计数调整细胞密度，分别以2×10 5个/孔的密度铺于24孔板中，每孔加入500μl完全培养基，在37℃、5％CO 2的氛围下培养过夜用于次日干扰实验。弃去24孔板中培养基，更换为OPTI-MEM培养基，并加入50nM FAPI-Actin siRNA(293T和293T-hFAP细胞)或者FAPI-GFP siRNA(A549-GFP和A549-GFP-hFAP细胞)，阴性对照只需加入等体积OPTI-MEM培养基。在37℃、5％CO 2的氛围下培养24h后检测mRNA表达。
使用Magen HiPure Total RNA Mini Kit试剂盒(R4111-03)进行总RNA提取：将细胞收获，并且溶解于400μl裂解液中，用移液枪吹打裂解液5-10次以便打断DNA。将裂解液转移至gDNA过滤柱中，14000×g离心2min，加入等倍体积70％乙醇至滤液中，吹打3-5次。将前步所得混合液加入HiPure RNA纯化柱中，12000×g离心1min，弃滤液，Buffer RW1洗涤一次，Buffer RW2洗涤2次后，加入40μl洗脱液，室温静置2min后，12000×g离心1min收集总RNA，使用Thermo NanoDrop 2000测定RNA浓度和纯度。
使用TAKARA PrimeScript RT Master Mix试剂盒(RR036a)进行cDNA合成：加入4μl 5×PrimeScript RT Master Mix、1μg总RNA至RNase Free dH 2O中，使总体积为20μl。使用Q1000+型荧光定量PCR系统通过以下步骤产生cDNA：37℃，15min；85℃，5s，4℃保持。
使用YEASEN Hieff
Figure PCTCN2022118435-appb-000022
qPCR SYBR Green Master Mix(抗体法，No Rox)试剂盒进行实时PCR测定：加入3μl RNase Free dH 2O、6μl Hieff
Figure PCTCN2022118435-appb-000023
qPCR SYBR Green Master Mix、0.5μl Actin/GFP Forward Primer(10μM)、0.5μl Actin/GFP Reverse Primer(10μM)至2μl稀释500倍后的cDNA中。使用Q1000+型荧光定量PCR系统进行实时PCR，将实时数据使用ΔΔCt方法分 析。
结果展示于图3和图4中，由图可见，体外用FAPI-Actin siRNA(50nM)能够下调293T-hFAP细胞中Actin的表达；体外用FAPI-GFP siRNA(50nM)能够下调A549-GFP-hFAP细胞中GFP的表达。
实施例4.体内用FAPI-GAPDH/GFP siRNA抑制GAPDH/GFP
过表达hFAP动物模型构建：将2×10 6个/只的A549-hFAP细胞悬液皮下注射(左侧)于裸鼠体内，A549细胞作为阴性对照皮下注射于右侧；将2×10 6个/只的A549-GFP-hFAP细胞悬液皮下注射(左侧)于裸鼠体内，A549-GFP细胞作为阴性对照皮下注射于右侧。饲养21天后用于体内干扰实验。
过表达hFAP裸鼠中的GAPDH/GFP mRNA的沉默：将FAPI-GAPDH siRNA试剂或GAPDH-siRNA试剂(阴性对照)以3mg/kg的一个单个尾静脉剂量给予饲养21天后的A549、A549-hFAP裸鼠体内；将FAPI-GFP siRNA试剂或FAPI-MYC-siRNA试剂(阴性对照)以3mg/kg的一个单个尾静脉剂量给予饲养21天后的A549-GFP、A549-GFP-hFAP裸鼠体内。饲养3天后，取裸鼠肿瘤提取总RNA，并进行实时PCR实验，具体操作参照前述实施例。
结果展示于图5和图6中，体内用FAPI-GAPDH siRNA(50nM)能够下调A549-hFAP细胞中GAPDH的表达；体内用FAPI-GFP siRNA(50nM)能够下调A549-GFP-hFAP细胞中GFP的表达。
实施例5.靶向细胞存活相关基因eIF-3β的siRNA缀合物的体内抑制效 果
将2×10 6个/只的A549-hFAP细胞悬液皮下注射裸鼠体内，饲养21天后用于体内干扰实验。将单一剂量的eIF-3βRNAi试剂(FAPI-eIF-3β)尾静脉给予实验组小鼠，对照组小鼠注射等体积的PBS，72小时后，取实验组以及对照组裸鼠肿瘤提取总RNA，并进行实时PCR实验，具体操作参照实施例3。
结果展示于图7中，可见体内用FAPI-eIF-3βsiRNA(3mg/kg)能够下调A549-hFAP细胞中eIF-3β的表达。
实施例6.FAP在IPF病人肺组织中特异性高表达
收集13例健康供者以及34例肺纤维化患者(IPF：8例；UIP：6例，NSIP：8例，矽肺：12例)的组织块，通过qPCR、Western blot确定FAP在健 康供体肺中几乎不表达，在IPF、矽肺和以成纤维细胞灶(包括纤维基质和活化成纤维细胞)为主要特征的寻常型间质性肺炎(usual interstitial pneumonitis，UIP)中显著高表达(图8)，尤其是在IPF患者病灶中高表达。
实施例7.FAPI偶联细胞存活相关基因在体外抑制IPF病人成纤维细胞 的效果
无菌选取绿豆大小的纤维化病灶部位的组织在1.5ml EP管剪碎5min，使之呈碎末状，转移至10cm细胞培养皿中，加入5ml含20％FBS 2％双抗的DMEM培养基，置于培养箱培养，2～3天换液，等原代成纤维细胞从组织碎块中爬出。将纯化的IPF成纤维细胞用10ng/ml的TGF-β1诱导IPF成纤维细胞48h，使其进一步活化并过表达hFAP(图9)，未处理细胞作为对照组。不加任何转染试剂，将FAPI-siRNA(FAPI按图2所示制备，siRNA序列如表1中所示，下同)、control siRNA(本实施例共涉及survivin、eIF-3B、COL1A1、PHB2、C-MYC，5个基因)分别以100nM的浓度与活化的FAP+IPF成纤维细胞孵育24h，提取上述细胞的RNA，以qPCR法验证目的基因的敲降效果(图10-图14)。
结果显示：FAPI偶联的细胞存活相关基因siRNA可以通过与FAP的结合，通过受体介导的内吞作用将siRNA内吞到细胞质中发挥干扰效果。在上述5个基因中，FAPI-siSurvivin、FAPI-sieIF-3B、FAPI-siCOL1A1、FAPI-siPHB2、FAPI-siC-MYC相较其他三组的干扰效果最强。
实施例8.靶向细胞存活相关基因的shRNA在体外抑制IPF病人成纤维 细胞增殖以及纤维化的效果
构建相应存活基因siRNA对应的shRNA质粒(survivin、eIF-3B、PHB2、POLR2B、DKC1)，包装慢病毒，感染IPF病人的成纤维细胞，通过CCK8增殖检测试剂盒检测，结果表明干扰上述基因的表达能明显抑制成纤维细胞的增殖(图15)。除此之外，单独干扰survivin、eIF-3B的表达、或是联合干扰survivin+eIF-3B的表达，或是干扰survivin+eIF-3B+PHB2都可以降低纤维化指标α-sMA(平滑肌肌动蛋白)、Collagen 1(胶原蛋白)、Fn(Fibronectin，纤连蛋白)，上述结果说明，干扰(survivin、eIF-3B、PHB2、POLR2B、DKC1)等基因可以抑制成纤维细胞的增殖，也可抑制纤维化。
实施例9.FAPI偶联靶向细胞存活相关基因的siRNA在体内抑制IPF病 人成纤维细胞增殖以及纤维化的效果
TGF-β1诱导后的2*10 6的IPF病人的成纤维细胞与500ul康宁Matrigel基质混合均匀后，皮下注射到免疫缺陷小鼠的皮下，7天后微血管会形成(图16)，尾静脉注射FAPI-siRNA，以及对照组不含FAPI的裸siRNA，注射剂量为3mg/kg。注射72小时后将其取出，一部分进行RNA提取，通过QRT-PCR检测干扰基因的下调情况(图17)；一部分固定做切片后进行胶原COL1A1的免疫组化，马松染色，天狼星红染色等，查看纤维化指标是否下降。
本次体内实验使用的是FAPI-siSurvivin(图18)、FAPI-sieIF-3B(图19)、FAPI-siCOL1A1(图20)，对照组是siSurvivin、sieIF-3B、siCOL1A1裸的siRNA。
马松染色结果表明，与注射siSurvivin裸的siRNA相比，FAPI-siSurvivin实验组中存活细胞较少，且纤维化指标胶原纤维(波浪线)含量也减少了；与注射sieIF-3B裸的siRNA相比，FAPI-sieIF-3B实验组中存活细胞较少，且纤维化指标胶原纤维(波浪线)含量也减少了。
免疫组化结果显示，与注射siCOL1A1裸的siRNA相比，FAPI-siCOL1A1实验组中存活细胞也较少，靶向胶原蛋白的含量降低。
此外，TGF-β1诱导后的2*10 6的IPF病人的成纤维细胞与500ul康宁Matrigel基质混合均匀后，皮下注射到免疫缺陷小鼠皮下，7天后皮下注射FAPI-sieIF-3B，对照组(con，注射sieIF-3B裸的siRNA)，剂量分别为3mg/kg、9mg/kg，皮下注射14天后，取出成纤维细胞，验证eIF-3B基因的干扰效果，发现皮下注射(FAPI-偶联目的基因)方式也可有效抑制目的基因的表达。

Claims (24)

  1. 靶向FAP阳性细胞的siRNA缀合物，其包括：(i)靶向FAP的配体部分、(ii)抑制FAP阳性细胞中的存活基因的表达的siRNA部分、以及(iii)位于所述配体部分和所述siRNA部分之间的连接子部分，其中所述靶向FAP的配体部分包含下式(I)的结构：
    Figure PCTCN2022118435-appb-100001
    其中：
    X是
    Figure PCTCN2022118435-appb-100002
    其左侧连接至式(I)，其右侧连接至连接子部分；
    R 1与R 2相同或不同，并且各自独立地选自氢、卤素和C 1-C 4烷基；
    R 3是C 1-C 4烷基、腈或异腈；
    R 4是H或-CH 3；
    R 5与R 6相同或不同，并且各自独立地选自氢、卤素和C 1-C 4烷基；
    R 7、R 8和R 9相同或不同，并且各自独立地选自氢、甲氧基、卤素、CF 3和C 1-C 4烷基。
  2. 根据权利要求1所述的siRNA缀合物，其中式(I)的结构中的R 1和R 2各自为卤素。
  3. 根据权利要求2所述的siRNA缀合物，其中所述卤素为氟。
  4. 根据权利要求1至3任一项所述的siRNA缀合物，其中式(I)的结构中的R 3是腈。
  5. 根据权利要求1至4任一项所述的siRNA缀合物，其中式(I)的结构中的R 5至R 9独立地为氢。
  6. 根据权利要求1所述的siRNA缀合物，其中所述靶向FAP的配体部分包含以下结构式：
    Figure PCTCN2022118435-appb-100003
    Figure PCTCN2022118435-appb-100004
    其右侧连接至连接子部分。
  7. 根据权利要求1所述的siRNA缀合物，其中所述靶向FAP的配体部分包含以下结构：
    Figure PCTCN2022118435-appb-100005
    其右侧连接至连接子部分。
  8. 根据权利要求1至7任一项所述的siRNA缀合物，其中所述siRNA部分是针对选自以下存活基因中的一个：polr2a、polr2b、dkc1、cenpe、eif-3b。
  9. 根据权利要求1至7任一项所述的siRNA缀合物，其中所述FAP阳性细胞是活化的成纤维细胞。
  10. 根据权利要求9所述的siRNA缀合物，其中所述活化的成纤维细胞存在于肿瘤基质、关节炎或纤维化组织中。
  11. 根据权利要求10所述的siRNA缀合物，其中所述肿瘤选自基底细胞癌、鳞状细胞癌、皮肤癌、口腔癌、黑色素瘤、食道癌、腺癌、胃癌、结直肠癌、直肠癌、胰腺癌、肝细胞癌、肺癌、间皮瘤、胆管癌、肝癌、膀胱癌、乳腺癌、肾癌、前列腺癌、宫颈癌、卵巢癌、胶质瘤、甲状腺癌、副甲状腺瘤、肉瘤、骨髓瘤和星形细胞瘤。
  12. 根据权利要求10所述的siRNA缀合物，其中所述肿瘤选自肺癌、胆管癌、肝癌、胶质瘤、卵巢癌、乳腺癌和胰腺癌。
  13. 根据权利要求10所述的siRNA缀合物，其中所述纤维化组织是肺纤维化组织或肝硬化组织。
  14. 根据权利要求10所述的siRNA缀合物，其中所述关节炎是类风湿性关节炎。
  15. 根据权利要求1至7任一项所述的siRNA缀合物，其中所述siRNA部分包含互补的正义链和反义链，所述靶向FAP的配体部分连接至所述正义链的3’端。
  16. 根据权利要求1至7任一项所述的siRNA缀合物，其中所述连接子部分包含酰胺键。
  17. 根据权利要求16所述的siRNA缀合物，其中所述连接子部分具有以下结构式：
    Figure PCTCN2022118435-appb-100006
    其左侧连接至所述靶向FAP的配体部分，右侧连接至所述siRNA部分，并且其中m和p各自独立地为0至10的整数。
  18. 根据权利要求1所述的siRNA缀合物，其中所述siRNA缀合物具有以下结构式：
    Figure PCTCN2022118435-appb-100007
  19. 根据权利要求15所述的siRNA缀合物，其中所述正义链如SEQ ID NO:9所示，所述反义链如SEQ ID NO:10所示，所述siRNA缀合物抑制eif-3b基因的表达。
  20. 根据权利要求1至7任一项所述的siRNA缀合物，其中所述siRNA部分是针对选自以下存活基因中的一种：survivin,phb2,c-myc以及col1a1。
  21. 根据权利要求13所述的siRNA缀合物，其中所述肺纤维化组织来源于特发性纤维化(IPF)、矽肺、寻常型间质性肺炎(UIP)中的一种或多种。
  22. 一种药物组合物，其包含权利要求1至19任一项所述的siRNA缀合物以及药学上可接受的载体。
  23. 根据权利要求22所述的药物组合物，其中所述药物组合物被配制成注射液。
  24. 权利要求1至19任一项所述的siRNA缀合物在制备抑制FAP阳性细胞的增殖或存活的药物中的应用。
PCT/CN2022/118435 2021-09-14 2022-09-13 靶向FAP阳性细胞的siRNA缀合物及其药物组合物和应用 WO2023040828A1 (zh)

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