SiRNA for inhibiting melanocortin receptor 4 (MC4R) gene expression, medicine and application thereof

Abstract

The invention provides siRNA for inhibiting melanocortin receptor 4 (MC4R) gene expression in human cells, a polypeptide oligonucleotide conjugate and application of the polypeptide oligonucleotide conjugate, the siRNA comprises a positive-sense strand and a corresponding antisense strand, and the nucleotide composition of the siRNA is each group of corresponding positive-sense strand and corresponding complementary antisense strand in a table 1, or a sequence having a difference of no more than 3 nucleotides from its sense strand or antisense strand. According to the invention, a plurality of siRNAs which can be used for inhibiting MC4R expression are screened, and on the basis, proper modification is carried out to improve the silence capability of a target. And moreover, a unique delivery system is adopted to enable the siRNA to be delivered into the brain to play a role, so that the siRNA is expected to be applied to energy metabolism disorder diseases, such as cachexia, sarcopenia, emaciation syndrome and anorexia nervosa, which are clinically related to MC4R target spots, and other consumptive diseases caused by chronic inflammation, cancer and immune disorder, such as cachexia, sarcopenia, emaciation syndrome and anorexia nervosa.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to siRNA, drugs and applications thereof for inhibiting melanocortin receptor 4 (MC4R) gene expression. Background Art

[0002] Cachexia is a systemic wasting disease characterized by progressive weight loss, muscle atrophy, and loss of adipose tissue. It is considered a late-stage consequence of disease and can occur in patients with a variety of infectious diseases, such as HIV / AIDS, malaria, and tuberculosis; many chronic conditions, such as cancer, chronic heart failure (CHF), chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), cystic fibrosis, cirrhosis, Crohn's disease, rheumatoid arthritis, stroke, and neurodegenerative diseases; and major trauma and sepsis (Farkas, von Haehling et al. 2013, Ferrer, Anthony et al. 2023). Cachexia is most common in patients with cancer, especially those with advanced cancer, and contributes to at least 20% of cancer deaths (Muscaritoli, Bossola et al. 2006). Cachexia increases mortality from chronic diseases, with approximately 30% of deaths occurring in patients with cachexia (Farkas, von Haehling et al. 2013). There is currently no effective treatment for cachexia. In addition to treating the cause, it also includes a variety of intervention measures, such as nutritional therapy, exercise, and psychosocial intervention (Borg, Anker et al. 2015, Arends, Strasser et al. 2021, Setiawan, Sari et al. 2023).

[0003] The neuroendocrine system plays a crucial role in the development and progression of cachexia. The hypothalamus, pituitary gland, and adrenal glands can also regulate the progression of cancer cachexia by controlling appetite (Setiawan, Sari et al. 2023). The hypothalamus is a key regulator of energy homeostasis, specifically fine-tuning energy balance by transmitting signals to coordinate food intake and thereby inhibit energy expenditure (Laviano, Seelaender et al. 2012). Melanocortin receptor 4 (MC4R) signaling plays a crucial role in the paraventricular nucleus of the hypothalamus (PVH) and is a key regulator of appetite and food intake (Tao 2010). MC4R regulates food intake and energy expenditure. Loss-of-function mutations in the MC4R gene can lead to monogenic obesity, the most common form of monogenic obesity. Gain-of-function mutations or abnormal activation of the MC4R receptor can inhibit feeding behavior and increase energy expenditure, leading to emaciation (Tao 2005).

[0004] Compared to traditional small molecule drugs, small interfering RNA (siRNA) drugs utilize the cell's endogenous system to specifically degrade the targeted target gene, offering promising clinical therapeutic prospects. Mechanistically, exogenously introduced double-stranded siRNAs can be recognized by the cellular endoribonuclease Dicer and cleaved into a single-stranded guide strand and its complementary strand by the RNA-induced silencing complex (RISC). The guide stranded siRNA then binds to the Argonate 2 protein (AGO2) and directs it to the target RNA, where AGO2 mediates the degradation of the target RNA. In addition to degrading RNAs in the cytoplasm, siRNAs can also promote chromosome remodeling and histone modification in the nucleus, leading to transcriptional silencing (Matzke et al. Nature reviews. Genetics vol. 6, 1 (2005): 24-35.). The delivery of siRNA drugs is an important guarantee for RNAi therapy. The most mature siRNA intrahepatic delivery system currently uses N-oligosaccharide acetylgalactosamine (GalNAc) to specifically bind to the asialoglycoprotein receptor (ASGPR) specifically expressed by hepatocytes, thereby achieving active delivery to liver cells. This is an important breakthrough in the field of RNA therapy (Zhang L et al,. Front Pharmacol. 2022; 13: 1090237.). In addition to liver tissue, researchers have found that adenovirus targeting the mouse brain containing BACE1 siRNA shows the potential to improve the pathological state of AD (Zhou Y et al,. Sci Adv. 2020; 6 (41): eabc7031.). However, effective, safe and comprehensive siRNA extrahepatic delivery systems still face huge challenges, especially in brain tissue where the blood-brain barrier exists.

[0005] Regarding the development of RNAi therapeutic drugs targeting the central nervous system, researchers have established a system of chemically modified siRNAs linked to fatty alcohol 2'-O-hexadecyl (C16). Injecting C16-siRNA targeting APP into the brain ventricles of Alzheimer's mice can reduce amyloid β deposition, inflammation, and behavioral defects. Similarly, after administering C16-siRNA to the central nervous system of non-human primates, extensive siRNA distribution and RNAi activity were achieved, with target gene silencing greater than 75% for at least 3 months. This provides an important technical platform for extrahepatic targeted therapy of RNAi drugs (Brown KM et al,. Nat Biotechnol. 2022; 40(10): 1500-1508.). In addition, utilizing target cell surface receptor proteins to achieve transcellular transport of drugs across the blood-brain barrier is also an important idea for drug delivery to the central nervous system. Among them, low-density lipoprotein receptor-related protein (LRP1) is widely expressed in neurons and brain endothelial cells. It is a multifunctional endocytic receptor that participates in many biological processes, including the transport of metabolites (Aβ peptide) across the blood-brain barrier. Therefore, it is a very attractive target for drug delivery to the central nervous system (Ulery PG et al,. J Biol Chem. 2000; 275(10): 7410-7415.). LRP1 has multiple ligands, reflecting its multiple physiological functions. In recent years, researchers have developed a transport peptide called Angiopep-2 (ANG), which can be used to transport drugs coupled to ANG to brain tissue by utilizing its interaction with the LRP1 receptor. Among them, ANG-mediated paclitaxel delivery is the first example, and clinical trials have been conducted (Régina A et al,. Br J Pharmacol. 2008; 155(2): 185-197.).

[0006] Sandwich enzyme-linked immunosorbent assay and fluorescence resonance energy transfer experiments showed that WT MC4R (Melanocortin-4 Receptor) has a dimer (Biebermann, Krude et al. 2003). Bioluminescence resonance energy transfer also showed that MC4R constitutively dimerizes and is not affected by ligand binding (Nickolls and Maki 2006). MC4R receptors can be activated by partial agonists and internalized. For example, long-term administration of MTII and other agonists has been shown to induce a rapid response (Pierroz, Ziotopoulou et al. 2002, Kumar, Sutton et al. 2009). In heterologous cells expressing MC4R, peptide agonist-activated receptors can be internalized through clathrin-coated pits (Shinyama, Masuzaki et al. 2003, Mohammad, Baldini et al. 2006). MC4R receptors can also be internalized by a natural antagonist, AgRP, also known as an inverse agonist: AgRP induces MC4R binding to β-arrestin and subsequent internalization (Breit, Wolff et al. 2006). Chronic stimulation of the MC4R receptor leads to its translocation from endosomes to lysosomes, where it is subsequently degraded (Gao, Lei et al. 2003). Summary of the Invention

[0007] Based on this, the purpose of the present invention is to provide an siRNA or a pharmaceutically acceptable salt thereof, a drug and its use for inhibiting the expression of melanocortin receptor 4 (MC4R) gene, which has the advantages of good biological activity and low toxicity.

[0008] The first aspect of the present invention provides an siRNA or a pharmaceutically acceptable salt thereof for inhibiting the expression of the melanocortin receptor 4 (MC4R) gene in human cells, wherein the siRNA comprises a sense strand and a corresponding complementary antisense strand, the sense strand comprising at least 15, 16 or 17 consecutive nucleotides, which differs from any of the sense strand sequences shown in Table 1 by no more than 3 nucleotides; and the antisense strand comprises at least 15, 16 or 17 consecutive nucleotides, which differs from any of the antisense strand nucleotide sequences shown in Table 1 by no more than 3 nucleotides.

[0009] In some embodiments, the base composition of the siRNA is each corresponding sense strand and corresponding complementary antisense strand in Table 1.

[0010] The present invention also provides a method for modifying the multi-stranded siRNA, which is used to improve the stability and activity of the siRNA in vivo and in vitro and to reduce non-target activity.

[0011] In some embodiments, the sense strand comprises no more than 3, 2, 1 or 0 unmodified nucleotides, the modified nucleotides in the sense strand comprise 2'-O-methyl modified nucleotides, 2'-deoxy nucleotides, 2'-fluoro modified nucleotides, inverted abasic residues, LNA, respectively, and the sense strand contains 0, or 1, or 2, or 3 phosphorothioate bonds at the 3' or 5'-end; and wherein the antisense strand comprises no more than 3, 2, 1 or 0 unmodified nucleotides, the modified nucleotides in the antisense strand comprise 2'-O-methyl modified nucleotides, 2'-deoxy nucleotides, 2'-fluoro modified nucleotides, 5'-(E)-vinylphosphonic acid nucleotides and analogs thereof, open nucleic acid nucleotides (UNA) or isoglycerol nucleotides (isoGNA), respectively, and the antisense strand contains 0, or 1, or 2, or 3 phosphorothioate bonds at the 3' or 5'-end.

[0012] In some embodiments, the sense strand contains two phosphorothioate bonds at the 5'-terminus and 0-2 phosphorothioate bonds at the 3'-terminus; and the antisense strand contains two phosphorothioate bonds at both the 5'-terminus and the 3'-terminus.

[0013] In some embodiments, the sense strand comprises at least 15, 16, 17, 18 or 19 consecutive nucleotides that differ by no more than 3 nucleotides from any of the sense strand sequences shown in Table 2; and wherein the antisense strand comprises at least 15, 16, 17, 18 or 19 consecutive nucleotides that differ by no more than 3 nucleotides from any of the antisense strand nucleotide sequences shown in Table 3.

[0014] In some embodiments, the double-stranded siRNA is selected from any modified double-stranded siRNA in Table 2, or a sequence that differs from its sense strand or antisense strand by no more than 3 nucleotides.

[0015] The second aspect of the present invention provides a biological agent or a pharmaceutical preparation for inhibiting MC4R expression, which comprises any

[0016] The siRNA or a pharmaceutically acceptable salt thereof, and a targeted delivery vector connected to or encapsulated with the siRNA, wherein the targeted delivery vector transports the siRNA or a pharmaceutically acceptable salt thereof to the central nervous system.

[0017] In some embodiments, the targeted delivery vector comprises a targeting ligand and a linker connecting the targeting ligand and the siRNA.

[0018] The targeting ligand is composed of a linker, and the targeting ligand is a polypeptide targeting low-density lipoprotein receptor-related protein 1 (LRP1) or a polypeptide targeting transferrin receptor (TfR) or a polypeptide targeting melanocortin receptor (MCR).

[0019] In some embodiments, the ligand targeting LRP1 is ANG2, and the amino acid sequence from N-terminus to C-terminus is as follows: Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr.

[0020] In some embodiments, the structure of the ligand ANG2 after being linked to the siRNA is as follows:

[0021]

[0022] Wherein X and Y are O or CH2; R is O or S; p and q are integers of 0-4, and k is 1, 2, 4 or 8.

[0023] Preferably, in some embodiments, the structure of the ligand ANG2 after being linked to the siRNA is as follows:

[0024]

[0025] BiAng2(K10N3)-siRNA,

[0026]

[0027] Bi-Sym-Ang2(K10N3)-siRNA,

[0028]

[0029] BiAng2(K10N3)-siRNA-2,

[0030]

[0031] Bi-Sym-Ang2(K10N3)-siRNA-2,

[0032]

[0033] BiAng2(K10N3)-Q22-siRNA.

[0034] In some embodiments, there are multiple ligands targeting melanocortin receptors, and the unmodified ligands of the melanocortin receptors are selected from any of the following amino acid sequences: Nle-c[Asp-Pro-D-Nal-Arg-Trp-Lys]-D-Val-D-Pro-Gly; Arg-c[Cys-D-Ala-His-D-Phe-Arg-Trp-Cys]; Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val; Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]; Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr; Tyr-Val-Met-Gly-His-Phe-Arg-D-Trp-Asp-Arg-Phe-Gly ;Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-D-Trp-Gly-Lys-Pro-Val ;Tyr-Val-Met-Gly-His-D-Phe-Arg-D-Trp-Asp-Arg-Phe-Gly ;Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp(SCF3)-Gly-Lys-Pro-Val.

[0035] In some embodiments, the modified ligand polypeptide is selected from the following structures: Ac-Nle-c[Asp-Pro-D-Nal-Arg-Trp-Lys]-D-Val-D-Pro-Gly-Azk-NH2 (referred to herein as P1); Ac-Azk-Arg-c[Cys-D-Ala-His-D-Phe-Arg-Trp-Cys]-NH2 (referred to herein as P2); Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-Azk-NH2 (referred to herein as P3); Ac-Nle-c[Asp-H is-DPhe-Arg-Trp-Lys]-Azk-NH2 (referred to herein as P4); Ac-Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-Azk-NH2 (referred to herein as P5); Tyr-Val-Met-Gly-His-Phe-Arg-D-Trp-Asp-Arg-Phe-Gly-Azk-NH2 (referred to herein as P6); Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-D-Trp-Gly-Lys-Pro-Val-Azk-NH2 (referred to herein as P7); Tyr-Val-Met-Gly-His-D-Phe-Arg-D-Trp-Asp-Arg-Phe-Gly-Azk-NH2 (referred to herein as P8); Ac-Azk-AEEA-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 (referred to herein as P3-2); 6-Azido-hexanoic-AEEA-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 (referred to herein as P3-3); A c-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys(AEEA-6-Azido-hexanoic)-Pro-Val-NH2 (referred to herein as P3-4); Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp(SCF3)-Gly-Lys-Pro-Val-Azk-NH2 (referred to herein as P3-5); wherein, Trp(SCF3) represents, Nle represents L-norleucine, Dpr represents L-2,3-diaminopropionic acid, and D-Nal represents 3-(2-naphthyl)-D-alanine.

[0036] P3 and P3-5 are preferred.

[0037] In some embodiments, the specific chemical structure of the above polypeptide is as follows:

[0038]

[0039]

[0040] In some embodiments, the structure of the ligand and the siRNA after being connected via a linker group is as follows:

[0041]

[0042]

[0043] Wherein X and Y are O or CH2 atoms; R is O or S; p and q are integers of 0-4; k is 1, 2 or 8; 2; Ac is acetyl.

[0044] Further preferred structures are as follows:

[0045]

[0046]

[0047] Wherein R is O or S, and Peptide is the ligand targeting the melanocortin receptor. A further preferred structure is as follows:

[0048]

[0049]

[0050] Wherein, AEEA represents 2-(2-(2-aminoethoxy)ethoxy)acetic acid, Trp(SCF3) represents Nle represents L-norleucine, Dpr represents L-2,3-diaminopropionic acid, and D-Nal represents 3-(2-naphthyl)-D-alanine.

[0051] In some embodiments, the structure of the ligand after being linked to the siRNA is as follows:

[0052]

[0053]

[0054]

[0055] The third aspect of the present invention provides the use of any of the above-mentioned siRNAs or pharmaceutically acceptable salts thereof, or any of the above-mentioned delivery vector-siRNAs in the preparation of biological preparations or pharmaceutical preparations that inhibit MC4R expression, or in the preparation of drugs that inhibit melanocortin receptor 4 (MC4R) gene expression.

[0056] A fourth aspect of the present invention is to provide a method for inhibiting MC4R expression, the method comprising:

[0057] (a) contacting a cell with any of the multi-targeting siRNA agents or the biological or pharmaceutical agents described above; and

[0058] (b) maintaining the cells produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcripts expressed by MC4R, thereby simultaneously inhibiting the expression of MC4R in the cells.

[0059] In some embodiments, the cell is within a subject.

[0060] In some of these embodiments, the subject is a human.

[0061] In some embodiments, MC4R expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0062] The siRNA is used in the preparation of drugs for preventing or treating diseases mediated by the MC4R gene. The diseases mediated by the MC4R gene include, but are not limited to, diseases related to energy metabolism disorders, such as cachexia, sarcopenia, wasting syndrome, anorexia nervosa, etc., and wasting diseases caused by chronic inflammation, cancer, immune disorders, and other factors.

[0063] Through extensive research on siRNA sequences and extensive experimentation, the present invention screened multiple siRNA agents capable of inhibiting MC4R expression. Based on these, appropriate modifications were made to enhance target silencing. Furthermore, a unique delivery system was employed to enable siRNA delivery to the brain for effective action. This approach is expected to be clinically applicable to treating energy metabolism disorders related to the MC4R target and other wasting diseases caused by other factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 It was shown that BiP3-8022 and BiP3-5-8022 had a significant effect of increasing body weight. DETAILED DESCRIPTION

[0065] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0066] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0068] The abbreviations and structures of nucleotide monomers used in nucleic acid sequence representation are shown in the following table:

[0069]

[0070]

[0071] The specific structure is as follows:

[0072]

[0073]

[0074] In the above and below text, unless otherwise specified, in the polypeptide sequence, G or Gly represents glycine, A or Ala represents alanine (wherein D-Ala represents D-configuration alanine), V or Val represents valine (wherein D-Val represents D-configuration valine), L or Leu represents leucine, I or Ile represents isoleucine, P or Pro represents proline (wherein D-Pro represents D-configuration proline), F or Phe represents phenylalanine (wherein D-Phe represents D-configuration phenylalanine ), Y or Tyr represents tyrosine, W or Trp represents tryptophan (wherein D-Trp represents tryptophan in the D configuration), S or Ser represents serine, T or Thr represents threonine, C or Cys represents cysteine, M or Met represents methionine, N or Asn represents asparagine, Q or Gln represents glutamine, D or Asp represents aspartic acid, E or Glu represents glutamic acid, K or Lys represents lysine, R or Arg represents arginine, and H or His represents histidine. In particular, Nle represents L-norleucine, D-Nal represents 3-(1-naphthyl)-L-alanine, Azk represents (2S)-6-azidohexanoic acid, and Dpr represents L-2,3-diaminopropionic acid.

[0075] According to existing technologies, those skilled in the art know that the pharmaceutically acceptable salt of the siRNA may be a sodium salt or a potassium salt. For example, a sodium salt of the siRNA is produced during purification.

[0076] The present invention is further described in detail below with reference to specific embodiments.

[0077] Example 1 Synthesis of siRNA

[0078] Oligonucleotides were synthesized using a solid-phase oligonucleotide synthesis protocol on a 12-channel nucleic acid synthesizer from Beijing Qingke Biotechnology Co., Ltd. 0.2-1 μmol of oligonucleotides were synthesized. GalNAc-containing siRNA sequences were synthesized on a prepacked CPG column coupled to GalNAc. Aminolysis reagent was added to the synthesized oligonucleotides and incubated at 45-80°C to separate the oligonucleotides from the solid support and free them. The crude oligonucleotides were then precipitated with ethanol, and the supernatant discarded. This process was repeated twice to obtain the crude oligonucleotides, which were then resuspended in DEPC-free water. The crude oligonucleotides were purified by ion-pairing HPLC, and the collected product was dried to a powder in a vacuum centrifuge. The purified product was dissolved in DEPC-free water and analyzed by time-of-flight LC-MS. The oligonucleotide concentration was determined, and the required volumes of equimolar amounts of the sense and antisense strands were calculated. The equimolar amounts of the sense and antisense strands were mixed and annealed to form a duplex by heating at 95°C for 5 minutes and then cooling to room temperature.

[0079] Table 1: Sense and antisense strand sequences of unmodified siRNA targeting MC4R.

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Table 2: Describes the modified siRNA double-stranded sequences

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] Example 2: Construction of hMC4R-293Ta stable cell line using liposome transfection method for in vitro screening of siRNA in a stable cell line overexpressing human MC4R:

[0099] a. The hMC4R ("h" stands for Homo sapiens) CDS region sequence was customized at Qingke Biotechnology and ligated to the lentiviral vector pLEX-MCS via PCR.

[0100] b. The lentiviral plasmid pLEX-MCS linked to the hMC4R CDS region was transfected into 293T cells to produce lentivirus;

[0101] c. 293Ta cells were infected with lentivirus and screened using medium without IL-3 or medium containing puromycin to obtain hMC4R stably transfected cell lines.

[0102] Cell culture and 96-well plate transfection: In vitro experiments were performed in hMC4R-293Ta stable cell lines using MEM + 10% FBS + 1X penicillin-streptomycin + 1X non-essential amino acids. When the cell confluence area reached 80%, the cells were trypsinized and the cell density was determined using a Scepter automated cell counter (Millipore, #PHCC00000). Simultaneously, siRNA, Opti-MEM, and INTERFERin (Polyplus transfection) were mixed in a 96-well plate and incubated at room temperature for 10 minutes. Then, complete medium containing h / r / mMC4R-293Ta cells was added to each well, and the 96-well plate was incubated in a 37°C, 5% CO2 incubator for 24 hours.

[0103] Unmodified siRNA was used in screening assays at a final concentration of 1 nM.

[0104] 96-well plate RNA extraction and reverse transcription: Dynabeads mRNA DI RECT kit (Ambion) was used to extract mRNA from 96-well plate cells. The culture medium in the 96-well plate was aspirated, washed once with DPBS, and 50-300 μl of cell lysis buffer was added to each well, followed by 20-100 μl of Beads, shake on the oscillator, place the 96-well plate on the magnetic separation rack, aspirate the lysate in the well, add 50-300μl of washing buffer A to each well, place it on the magnetic separation rack after blowing, aspirate the washing buffer A, then blow up the beads with washing buffer B, transfer them to a new 96-well plate, place it on the magnetic separation rack, aspirate the washing buffer B, and then blow up the beads with buffer B and transfer them to a 96-well PCR plate. At the same time, prepare the reversal reagent, place the 96-well PCR plate on the magnetic separation rack, aspirate the washing buffer B, add 20μl of reversal reagent to each well, seal the plate with a sealing film, and incubate at 25℃ for 10 minutes on a PCR instrument, then incubate at 37℃ for two hours, then at 85℃ for 5 minutes, cool to 4℃, and the reverse transcription is completed.

[0105] Real-time fluorescence quantitative PCR: After reverse transcription, place the 96-well plate on a magnetic separation rack until the beads are adsorbed to the bottom. Aspirate the reverse transcription reagent, add the prepared QPCR system to the 96-well PCR plate, seal the plate with a sealing film, and perform PCR on the StepOnePlus real-time PCR system (Applied Biosystems).

[0106] The data were analyzed using the ΔΔCt method and normalized using cells transfected with a negative control sequence.

[0107] The negative control AD-1955 sequence is: CUUACGCUGAGUACUUCGAdTdT (SEQ ID NO. 335)

[0108] UCGAAGUACUCAGCGUAAGdTdT (SEQ ID NO. 336).

[0109] Table 3 shows the experimental results of screening modified siRNA double strands in hMC4R-293Ta cells at a concentration of 10 nM by liposome transfection

[0110]

[0111]

[0112] Example 3: In vitro activity test method for verifying related sequences in primary mouse liver cells of the constructed AVV8-hMC4R virus

[0113] In vitro screening was performed in primary hepatocytes from mice infected with AAV8 virus expressing human MC4R (hMC4R).

[0114] Construction of mice infected with AAV8 virus stably expressing hMC4R: 1x10 11 Mice were infected with ultrapurified recombinant AAV8 hMC4R viral particles to obtain transgenic mice stably expressing hMC4R.

[0115] Mouse primary hepatocyte extraction: Mouse hepatocytes were extracted by collagenase digestion via inferior vena cava perfusion. Viable primary hepatocytes were filtered through a tissue cell strainer (BIOLOGIX, 15-1070) and resuspended in DMEM medium supplemented with 10% FBS and 1X penicillin-streptomycin. Cell density was determined using a Scepter automated cell counter.

[0116] The methods of siRNA transfection, RNA extraction, reverse transcription, and real-time fluorescence quantitative PCR were as described above. The free uptake method for delivering siRNA did not require the addition of INTERFERin.

[0117] Table 4 shows the experimental results of screening modified siRNA duplexes in AVV8-hMC4R primary liver cells at a concentration of 10 nM by lipofectamine transfection.

[0118]

[0119]

[0120]

[0121] Table 5 shows the experimental results of free uptake of modified siRNA duplexes at 1 nM concentration in AVV8-hMC4R primary liver cells

[0122]

[0123]

[0124] Table 6 shows the experimental results of free uptake of modified siRNA duplexes in AVV8-hMC4R primary liver cells

[0125]

[0126] Example 4 In vivo activity of modified siRNA duplexes in AVV8-hMCR mice

[0127] The effects of the in vitro preferred sequences on hMC4R expression in the liver were assessed in mice expressing hMC4R.

[0128] Experimental methods:

[0129] 1. Adenoviral Integration of hMC4R

[0130] Pass 1-10x10 11 Purified recombinant AAV8 viral particles were used to infect mice to obtain transgenic mice stably expressing hMC4R.

[0131] 2. Medication

[0132] Fourteen days after virus injection, mice were divided into groups of 4. siRNA was dissolved in saline and injected subcutaneously at a dose of 1 mg / kg.

[0133] 3. Liver sampling and testing

[0134] Liver samples were harvested at different time points after siRNA injection. RNA was extracted from the liver tissue using the TRI REAGENT (MRC, Catalog No. TR118) and reverse transcribed into cDNA using the PrimeScript RTregent Kit (Takara, Catalog No. RR047A). The prepared QPCR system was added to a 96-well PCR plate, sealed with a film, and QPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) to measure hMC4R expression.

[0135] Table 7 In vivo activity of preferred sequences in AVV8-hMC4R mice

[0136]

[0137]

[0138] NA means not detected.

[0139] Example 5 Synthesis of Polypeptide Oligonucleotide Conjugates (siRNA Agents)

[0140] 1) Synthesis of polypeptides:

[0141] General synthesis of linear peptides: Linear peptides were synthesized on a PurePep Chorus 4-channel peptide synthesizer at a 0.2 mmol scale using conventional solid-phase peptide synthesis methods, such as those described in Adam G et al., Standard practices for Fmoc-based solid-phase peptide synthesis in the Nowick laboratory. The conditions of the main process are as follows: (1) Resin swelling: Place Fmoc-Tyr-Wang resin or Rink-Amide-MBHA-Resin resin in DMF and swell at room temperature for 30 minutes; (2) Deprotection: Treat the resin with a DMF solution containing 20% ​​piperidine at 50°C for 2 minutes; (3) Amino acid condensation: Use 5eq (1eq of resin) Fmoc amino acid, HCTU as a condensing agent (5eq), DIPEA (5eq) as a base, and react at 50°C for 10 minutes; (4) N-terminal acetylation: 5% N-acetylimidazole, react at room temperature for 1 hour; (5) TFA cleavage of resin: After the synthesis is completed, treat the resin with a cleavage reagent containing TFA (TFA / Tips / H2O=95:2.5:2.5; TFA / Tips / EDT / H2O=92.5:2.5:2.5:2.5) for more than 2 hours, filter and discard the resin, and collect the filtrate. (6) Precipitation and purification: Add pre-cooled anhydrous ether to the filtrate and centrifuge to obtain a white precipitate. Wash the precipitate three times with anhydrous ether and dry under reduced pressure at room temperature to obtain a crude polypeptide product as an off-white powder. Dissolve the crude polypeptide in acetonitrile / water and purify it by high performance liquid chromatography.

[0142] General synthesis method of cyclized peptides: On a PurePep Chorus 4-channel peptide synthesizer, peptide synthesis was performed on a 0.2 mmol scale according to the general synthesis method for linear peptides described above to obtain the corresponding linear crude peptide. HBTU (3.0 eq), HOBT (3.0 eq), and DIEA (6.0 eq) were added to the linear crude peptide in sequence, and DMF was used as the solvent. The reaction was allowed to proceed at room temperature for 1 hour. After the reaction was complete as monitored by LC-MS, the target cyclized peptide was purified by HPLC using an acetonitrile / water system to obtain the target cyclized peptide.

[0143] In this example, the following polypeptides were synthesized according to the above method:

[0144] P1 cyclized peptide

[0145]

[0146] 34 mg of the target cyclized polypeptide P1 was obtained (theoretical molecular weight: 1441.7, measured: 1441.9 [M+H] + ).

[0147] P2 cyclized peptide

[0148]

[0149] 40 mg of the target cyclized polypeptide P2 (theoretical molecular weight: 1271.5, measured: 1273 [M+H] + ) was obtained.

[0150] P3 peptide

[0151]

[0152] 28 mg of the target polypeptide P3 (theoretical molecular weight: 1801, measured: 901.3 [M+2H]2+) was obtained.

[0153] P4 cyclized peptide

[0154]

[0155] 36 mg of the target cyclized polypeptide P4 (theoretical molecular weight: 1178.4, measured: 1178.5 [M+H]) was obtained. + ).

[0156] P5 cyclized peptide

[0157]

[0158] 24 mg of the target cyclized polypeptide P5 (theoretical molecular weight: 1505.7, measured: 1506.15 [M+H]) was obtained. + ).

[0159] P6 polypeptide

[0160]

[0161] 38 mg of target polypeptide P6 (theoretical molecular weight: 1724, measured: 1725.3 [M+H]) was obtained. + ).

[0162] P7 polypeptide

[0163]

[0164] 31 mg of target peptide P7 (theoretical molecular weight: 1801, measured: 901.6 [M+2H]) was obtained. 2+ ).

[0165] P8 polypeptide

[0166]

[0167] 16 mg of target polypeptide P8 (theoretical molecular weight: 1724, measured: 1725.7 [M+H] + ).

[0168] P9 polypeptide

[0169]

[0170] 43 mg of target polypeptide P9 (theoretical molecular weight: 2455. Measured: 819.50 [M+3H] 3+ ).

[0171] P10 peptide

[0172]

[0173] 28 mg of target polypeptide P10 (theoretical molecular weight: 2679. Measured: 894.6 [M+3H] 3+ ).

[0174] P3-2 polypeptide

[0175]

[0176] 59 mg of the target polypeptide P3-2 (theoretical molecular weight: 1946.21, measured: 974.13 [M+2H] 2+ ).

[0177] P3-3 polypeptide

[0178]

[0179] 72 mg of the target polypeptide P3-3 (theoretical molecular weight: 1889.15, measured: 945.38 [M+2H] 2+ ).

[0180] P3-4 polypeptide

[0181]

[0182] 63 mg of target polypeptide P3-4 (theoretical molecular weight: 1931.19, measured: 966.32 [M+2H]) was obtained. 2+ ).

[0183] P3-5 polypeptide

[0184]

[0185] 72 mg of the target polypeptide P3-5 (theoretical molecular weight: 1901.11, measured: 951.36 [M+2H] 2+ ).

[0186] Table 8 shows the relevant peptide sequences

[0187]

[0188]

[0189] Wherein, AEEA represents 2-(2-(2-aminoethoxy)ethoxy)acetic acid, Trp(SCF3) represents Nle represents L-norleucine, Dpr represents L-2,3-diaminopropionic acid, D-Nal represents 3-(2-naphthyl)-D-alanine, and AZK represents 6-azido-norleucine.

[0190] 2) Synthesis of B04

[0191]

[0192] Synthesis of compound B04-1

[0193] Fluorenylmethyloxycarbonyl-6-aminohexanoic acid (10.00 g, 86.84 mmol), DIEA (22.45 g, 173.68 mmol), and HBTU (36.23 g, 95.53 mmol) were dissolved in DMF (100 ml) and stirred at room temperature for 5 min. Compound 17 (38.25 g, 91.18 mmol) was then dissolved in DMF (50 mL) and added to the reaction solution, which was stirred at room temperature for 2 h. The reaction mixture was added to saturated NaHCO₃ and extracted with DCM. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (DCM / MeOH = 100:1, 2% TEA) to obtain 54.3 g of a colorless oil, with a yield of 85.2%. LCMS: 754.92, found: 777.11 [M+Na] + .

[0194] Synthesis of compound B04-2

[0195] Compound B04-1 (8.00 g, 10.6 mmol) was dissolved in EA (80 ml), and diethylamine (38.80 g) was added. The reaction system was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure and separated by column chromatography (DCM / MeOH = 20:1, 0.5% TEA → 10:1, 0.1% TEA) to give 4.2 g of the product as a white foamy solid, with a yield of 74.4%. LCMS: 532.68, found 533.65 [M+H] + .

[0196] Synthesis of compound B04-3

[0197] Compound B04-2 (3.00 g, 5.63 mmol) was dissolved in MeOH (30 ml), and ethyl trifluoroacetate (2.00 g, 14.08 mmol) was added. The system was stirred at room temperature overnight. The reaction solution was concentrated and separated by column chromatography (DCM / MeOH = 30:1, 0.1% TEA) to obtain 2.49 g of the product as a white foamy solid, with a yield of 70.3%. LCMS: 628.69, found 650.72 [M+Na] + .

[0198] Synthesis of compound B04

[0199] Compound B04-3 (2.00 g, 3.18 mmol) and DIPEA (1.23 g, 9.54 mmol) were dissolved in anhydrous DCM (10 ml). Cyanoethyl N,N-diisopropylchlorophosphoramidite (1.13 g, 4.77 mmol) was added, and the system was stirred at room temperature for 1 h. The reaction mixture was concentrated at 30°C and separated by column chromatography (PE, 2% TEA → PE / EA = 4:1, 2% TEA) to give 1.37 g of the product as an off-white foamy solid in a 52.0% yield. LCMS: 828.91, found 830.21 [M+H] + .

[0200] 3) Synthesis of B05

[0201]

[0202] Synthesis of compound B05-1

[0203] Compound 3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)propanoic acid (4 g, 11.3 mmol), DIEA (3.6 g, 27.9 mmol), and HATU (4.3 g, 11.3 mmol) were dissolved in DMF (40 ml) and stirred at room temperature for 5 min. Compound 17 (3.93 g, 9.37 mmol) was then dissolved in DMF (10 ml) and added to the above reaction solution, and the system was stirred at room temperature for 1 h. The reaction mixture was added to saturated NaHCO3 and extracted with DCM. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (DCM / MeOH = 50:1, 0.5% TEA) to obtain 6.5 g of a colorless oil with a yield of 76.4%. LCMS: 756, found 779 [M+23] + .

[0204] Synthesis of compound B05-2

[0205] Compound B05-1 (6.5 g, 8.60 mmol) was dissolved in EA (76 ml), and diethylamine (12.6 g) was added. The reaction system was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure and separated by column chromatography (DCM / MeOH = 20:1, 0.5% TEA → 10:1, 0.1% TEA) to obtain 3.1 g of the product as a white foamy solid, with a yield of 67.5%. LCMS: 534, found 535 [M+H] + 1H NMR (500 MHz, Chloroform-d) δ 7.36 (d, J = 6.3 Hz, 2H), 7.33-7.13 (m, 7H), 6.81 (dt, J = 10.8, 6.3 Hz, 4H), 4.57-4.24 (m, 2H), 4.17-3.91 (m, 1H), 3.78 (t, J = 5.4 Hz, 8H), 3.63 (h, J = 5.9, 5.0 Hz, 1H), 3.54-3.33 (m, 3H), 3.17 (dt, J = 21.6, 5.2 Hz, 1H), 2.88-2.64 (m, 3H), 2.47 (s, 3H), 2.29-1.96 (m, 3H). Synthesis of compound B05-3

[0206] Compound B05-2 (2.5 g, 4.67 mmol) was dissolved in ACN (30 ml), and ethyl trifluoroacetate (1.65 g, 11.6 mmol) was added. The system was stirred at room temperature overnight. The reaction solution was concentrated and separated by column chromatography (DCM / MeOH = 30:1, 0.1% TEA) to obtain 2.8 g of the product as a white foamy solid, with a yield of 95.2%. LCMS: 630, found 629 [MH] - .

[0207] 1 H NMR(500MHz,Chloroform-d)δ7.99(d,J=74.9Hz,1H),7.29(dd,J=8.0,6.3Hz,2H),7.21(d,J=10.1Hz,2H),7.18-7.15(m,4H),7 .12(q,J=7.3,6.1Hz,1H),6.78-6.69(m,4H),4.56(p,J=4.3Hz,1H),4.46-4.31(m,1H),3.71(d,J=4.9Hz,7H),3.69-3.58(m,2H ),3.52(dtt,J=14.6,9.8,4.1Hz,3H),3.43(dt,J=8.5,4.4Hz,2H),3.33(ddd,J=17.5,11.1,4.7Hz,1H),3.14-3.06(m,1H),2.5 6-2.45(m,1H),2.40-2.29(m,1H),2.21(dt,J=13.3,5.7Hz,1H),2.06(q,J=9.3,7.4Hz,1H),1.92(ddd,J=13.2,8.7,4.0Hz,1H).

[0208] Synthesis of compound B05

[0209] Compound B05-3 (2.6 g, 4.13 mmol) and DIPEA (1.3 g, 10.1 mmol) were dissolved in anhydrous DCM (25 ml). Cyanoethyl N,N-diisopropylchlorophosphoramidite (1.1 ml, 4.95 mmol) was added, and the system was stirred at room temperature for 1 hour. The reaction mixture was concentrated at 30°C and separated by column chromatography (PE, 2% TEA → PE / EA = 4:1, 2% TEA) to give 0.9 g of the product as an off-white foamy solid in a 26.3% yield. LCMS: 830, found 746 [M-83-H]-, 693 [M-136-H] - .

[0210] 1H NMR(500MHz,Chloroform-d)δ8.18-7.95(m,1H),7.69-7.42(m,1H),7.36-7.24(m,2H),7.12(q,J=7.3 ,6.5Hz,1H),6.82-6.69(m,4H),4.74-4.60(m,1H),4.31(d,J=7.9Hz,1H),4.18-3.97(m,1H),3.88-3. 80(m,1H),3.77-3.63(m,10H),3.59-3.39(m,8H),2.52(dt,J=12.8,6.3Hz,3H),2.42(dddd,J=21.8,1 6.1,10.5,5.4Hz,1H),2.32-2.09(m,2H),2.03(dt,J=14.4,6.3Hz,1H),1.09(dt,J=13.5,6.5Hz,11H).

[0211] 4) Synthesis of B08

[0212]

[0213] Synthesis of compound B08-1

[0214] Compound [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (10 g, 26.0 mmol), DIEA (6.7 g, 52.0 mmol), and HBTU (10.8 g, 28.6 mmol) were dissolved in DMF (100 ml) and stirred at room temperature for 5 min. Compound 17 (11.4 g, 27.3 mmol) was then dissolved in DMF (20 mL) and added to the reaction solution, which was stirred at room temperature for 2 h. The reaction mixture was added to saturated NaHCO₃ and extracted with DCM. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (DCM / MeOH = 100:1, 2% TEA) to obtain 17.4 g of a colorless oil, with a yield of 85.2%. LCMS: 786.9, found: 808.9 [M+Na] + .

[0215] Synthesis of compound B08-2

[0216] Compound B05-1 (8.0 g, 10.2 mmol) was dissolved in EA (76 ml), and diethylamine (12.6 g) was added. The reaction system was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure and separated by column chromatography (DCM / MeOH = 20:1, 0.5% TEA → 10:1, 0.1% TEA) to obtain 3.8 g of the product as a white foamy solid, with a yield of 66.2%. LCMS: 564.7, found 565.8 [M+H]+ .

[0217] Synthesis of compound B08-3

[0218] Compound B05-2 (2.0 g, 3.5 mmol) was dissolved in ACN (35 ml), and ethyl trifluoroacetate (1.3 g, 8.8 mmol) was added. The system was stirred at room temperature overnight. The reaction solution was concentrated and separated by column chromatography (DCM / MeOH = 30:1, 0.1% TEA) to obtain 1.87 g of the product as a white foamy solid, with a yield of 79.9%. LCMS: 660.7, found 682.7 [M+Na] + .

[0219] 4. Synthesis of B08

[0220] B05-3 (1.5 g, 2.3 mmol) and DIPEA (0.9 g, 6.8 mmol) were dissolved in anhydrous DCM (10 ml). Cyanoethyl N,N-diisopropylchlorophosphoramidite (0.6 g, 2.7 mmol) was added, and the system was stirred at room temperature for 1 h. The reaction mixture was concentrated at 30°C and separated by column chromatography (PE, 2% TEA → PE / EA = 4:1, 2% TEA) to give 0.8 g of the product as an off-white foamy solid in a 38.8% yield. LCMS: 860.9, found 861.9 [M+H]

[0221] 5) Synthesis of the dipeptide linker BPL-2

[0222]

[0223] Step 1. Preparation of (S)-22-(2,2-dimethyl-4,13-dioxo-3,8-11-trioxa-5,14-diazaoctadec-18-yl)-2,2-dimethyl-4,3,20-trioxa-3,8,11-trioxa-5,14,21-triazatricosan-23-oic acid methyl ester (Compound B1): (6-aminohexanoyl)-L-lysine methyl ester (3.0 g, 10.9 mmol, 1.0 eq), 2,2-dimethyl-4-oxo-3,8-11-trioxa-5,14-diazaoctadec-18-yl)-2,2-dimethyl-4,3,20-trioxa-3,8,11-trioxa-5,14,21-triazatricosan-23-oic acid methyl ester were added at room temperature. A mixture of 3,8,11-trioxo-5-azatridecane-13-oic acid (6.35 g, 24.2 mmol, 2.2 eq.) and N,N-diisopropylethylamine (5.7 ml, 32.9 mmol, 3.0 eq.) in dichloromethane was stirred uniformly, and then 1-hydroxybenzotriazole (3.7 g, 27.5 mmol, 2.5 eq.) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.24 g, 27.5 mmol, 2.5 eq.) were added and reacted for 1 hour. The plate was stirred (DCM:MeOH=20:1, R f=0.3) and LCMS monitoring, the reaction was complete, and dichloromethane was added to dilute the reaction solution and extract. The organic layer was washed twice with saturated sodium bicarbonate and then washed twice with saturated ammonium chloride, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane:methanol) to obtain the product (S) 22-(2,2-dimethyl-4,13-dioxo-3,8-11-trioxa-5,14-diazaoctadecane-18-yl)-2,2-dimethyl-4,3,20-trioxa-3,8,11-trioxa-5,14,21-triazatricosane-23-oic acid methyl ester (3.9 g, yield: 46.8%), MS (ES) + ):m / z=764(M+H) + .

[0224] Step 2. Preparation of methyl-N2-(6-(2-(2-(2-(2-aminoethoxy)ethoxy)acetylamino)hexanoyl)-N6-(2-(2-(2-aminoethoxy)ethoxy)acetyl)-L-lysine ester (Compound B2): Methyl-22-(2,2-dimethyl-4,13-dioxo-3,8-11-trioxa-5,14-diazaoctadec-18-yl)-2,2-dimethyl-4,3,20-trioxa-3,8,11-trioxa-5,14,21-triazatricosan-23-oate (3.9 g, 5.11 mmol, 1.0 eq.) and hydrogen chloride were added at room temperature. The mixture was stirred with a dioxane solution (40 mL, 2 mol / L) for 1.5 hours. The reaction was complete under LCMS monitoring. The reaction solution was concentrated under reduced pressure, and water (30 mL) was added. Sodium hydroxide solution was added to adjust the pH to approximately 7-8. The aqueous phase was concentrated under reduced pressure using an oil pump. The resulting solid was washed with a solution of dichloromethane / methanol = 10 / 1, stirred, and filtered. The filtrate was concentrated under reduced pressure to afford methyl N2-(6-(2-(2-(2-(2-aminoethoxy)ethoxy)acetylamino)hexanoyl)-N6-(2-(2-(2-aminoethoxy)ethoxy)acetyl)-L-lysine ester (2.9 g, yield: 85.8%) as a pale yellow oil. MS (ES) + ):m / z=564(M+H) + .

[0225] Step 3. Preparation of (S)-2-(6,15-dioxo-8,11-dioxo-5,14-diazaicos-20-yn-1-yl)-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25-ynoic acid methyl ester (Compound B3): Methyl N2-(6-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)acetylamino)hexanoyl)-N6-(2-(2-(2-aminoethoxy)ethoxy)acetyl)-L-lysine ester (440 mg, A mixture of dichloromethane (0.691 mmol, 1.0 equivalent), 6-heptynoic acid (178 mg, 1.41 mmol, 2.04 equivalent), N,N-diisopropylethylamine (263 μl, 1.52 mmol, 2.2 equivalent) was stirred evenly, and then 1-hydroxybenzotriazole (205 mg, 1.52 mmol, 2.2 equivalent) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (290 mg, 1.52 mmol, 2.2 equivalent) were added and reacted for 1 hour. Spot plate (DCM:MeOH=20:1, R f =0.2) and LCMS monitoring, the reaction was complete, and dichloromethane was added to dilute the reaction solution and extract. The organic layer was washed twice with saturated sodium bicarbonate and then twice with saturated ammonium chloride, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane:methanol) to obtain the product (S)-2-(6,15-dioxo-8,11-dioxo-5,14-diazaicos-20-yn-1-yl)-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25-ynoic acid methyl ester (310 mg, yield: 57.6%). MS (ES + ):m / z=780(M+H) + .

[0226] Step 4. Preparation of (S)-2-(6,15-dioxo-8,11-dioxo-5,14-diazaicos-20-yn-1-yl)-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25ynoic acid (Compound B4): Methyl 1-hydroxy-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25-ynoate (310 mg, 0.398 mmol, 1.0 eq) was added to a mixture of tetrahydrofuran, methanol, and water (6 mL). A solution of lithium hydroxide (20.1 mg, 0.478 mmol, 1.2 eq) in water (1 mL) was then added and stirred at room temperature for 1 hour. The reaction was complete as monitored by LCMS. The reaction mixture was concentrated under reduced pressure, water (25 mL) was added, and the pH was adjusted to approximately 3-4 with 1 mol / L dilute hydrochloric acid. The mixture was diluted with water (20 mL) and extracted with dichloromethane (25 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure to give a white solid (S) 2-(6,15-dioxo-8,11-dioxo-5,14-diazaicos-20-yn-1-yl)-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25-ynoic acid (290 mg, yield: 95.4%). 1 HNMR (500MHz, CDCl3) δ6.90 (s, 2H), 6.83 (d, J = 8.3Hz, 1H), 6.55 (s, 2H), 4.15-4.1 0(m,1H),3.69-3.62(m,4H),3.55(s,8H),3.50(t,J=4.8Hz,10H),3.41-3.35(m,10 H),2.45-2.36(m,4H),2.32(t,J=6.8Hz,2H),2.24(t,J=6.9Hz,2H),2.15(dt,J=7. 1,6.2Hz,7H),1.89(dd,J=15.2,8.1Hz,4H),1.72-1.64(m,4H),1.52-1.46(m,4H). MS(ES + ):m / z=766(M+H) + .

[0227] Step 5. Preparation of BPL-2: Compound B4 (290 mg, 0.38 mmol) was dissolved in 4 mL of dichloromethane, and N-hydroxysuccinimide (52 mg, 0.46 mmol, 1.2 eq.) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (88 mg, 0.46 mmol, 1.2 eq.) were added in sequence. The mixture was stirred at room temperature for 1.5 hours. The reaction was completed under LCMS monitoring. Dichloromethane was added to dilute the reaction solution and extracted. The organic layer was washed twice with saturated sodium bicarbonate and then washed twice with saturated ammonium chloride, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. BPL-2 (310 mg, yield: 94.5%) was obtained as a white viscous solid and used directly in the next step without purification. MS (ES) + ):m / z=864(M+H) + .

[0228] 6) Synthesis of solid phase carrier Q22-CPG

[0229]

[0230] Step 1. A mixture of B4 (5.0 g, 6.53 mmol, 1.0 eq.), Q22-1 (2.26 g, 6.86 mmol, 1.05 eq.), DIEA (1.68 g, 13.06 mmol, 2.0 eq.), and HATU (3.72 g, 9.8 mmol, 1.5 eq.) in DMF (10 mL) was stirred at room temperature for 2 hours. The reaction was diluted with ethyl acetate, washed twice with water, saturated NaCO, and NHCl solutions, and then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to afford 6.14 g of crude Q22-2. MS (ES+): m / z = 1079.46 (M+H)+.

[0231] Step 2. Dissolve the crude product Q22-2 (6.14 g) in 5 mL of dichloromethane. Add 5 mL of trifluoroacetic acid with stirring, and stir at room temperature for 1 hour. The reaction solution is concentrated under reduced pressure, and the residue is diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to afford 5.53 g of crude product Q22-3. MS (ES+): m / z = 1023.25 (M+H)+

[0232] Step 3. A mixture of crude Q22-3 (5.53 g), B08-2 (3.2 g, 5.67 mmol), DIEA (1.4 g, 10.8 mmol), and HATU (3.07 g, 8.1 mmol) in DMF (10 mL) was stirred at room temperature for 2 hours. The reaction was diluted with ethyl acetate, washed twice with water, twice with saturated NaCO solution, and then with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by RP-HPLC to afford the desired product Q22-4 as a white oil (5.1 g, 49.7% yield over three steps). 1HNMR(500MHz,DMSO-d6)δ7.90-7.77(m,5H),7.61(t,J=5.9Hz,2H),7.35-7.26 (m,4H),7.21-7.17(m,4H),6.91-6.84(m,4H),4.97(s,1H),4.42-4.36(m,1H),4 .27(d,J=14.0Hz,1H),4.19(td,J=8.6,5.1Hz,2H),4.11(t,J=13.4Hz,2H),4.0 6(s,1H),3.85(d,J=1.7Hz,4H),3.73(s,7H),3.62-3.46(m,17H),3.46-3.35(m, 9H),3.19(p,J=7.5,6.6Hz,9H),3.10-3.01(m,5H),2.90(d,J=12.7Hz,1H),2.7 2(d,J=2.6Hz,2H),2.54(s,1H),2.43-2.30(m,1H),2.18-2.04(m,11H),2.01(dd ,J=12.5,5.9Hz,1H),1.84(ddd,J=12.9,8.3,4.4Hz,1H),1.64(s,2H),1.56(p,J =7.5Hz, 5H), 1.43 (ddq, J = 24.8, 17.3, 10.1, 8.8Hz, 14H), 1.22 (q, J = 7.5Hz, 4H). MS(ES+): m / z=1267.30[(M-303)+H]+.

[0233] Step 4. Dissolve Q22-4 (5.1 g, 3.25 mmol, 1 eq.) in dichloromethane (20 mL). Succinic anhydride (1.95 g, 19.5 mmol, 6.0 eq.), triethylamine (3.94 g, 39 mmol, 12.0 eq.), and 4-dimethylaminopyridine (39 mg, 0.325 mmol, 0.1 eq.) were added sequentially with stirring. The reaction mixture was allowed to react overnight at room temperature. The reaction mixture was diluted with dichloromethane and washed three times with 10% sodium bicarbonate solution and once with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated to yield a brown oil. NMR(500MHz, CDCl3)δ7.57(dt,J=20.0,5.0Hz,1H),7.35(d,J=10.0Hz,2H),7.29-7.19(m,6 H),7.22-7.17(m,3H),7.08-6.90(m,4H),6.69-6.59(m,1H),6.50-6.39(m,1H),5.46-5.38( m,1H),4.58-4.43(m,2H),4.33-4.01(m,4H),3.98(d,J=5.0Hz,4H),3.79-3.78(m,7H),3.67 -3.60(m,16H),3.57(q,J=5.0Hz,8H),3.52-3.49(m,2H),3.48-3.43(m,5H),3.43-3.38(m,2 H),3.38-3.31(m,1H),3.27(dq,J=10.0,5.0Hz,4H),3.16-3.11(m,1H),3.06(s,1H),2.58-2 .50(m,5H),2.46-2.36(m,1H),2.31(dd,J=10.0,5.0Hz,1H),2.26-2.17(m,10H),2.11(td,J =10.0,5.0Hz,1H),1.98(t,J=5.0Hz,2H),1.84(dt,J=10.0,5.0Hz,2H),1.75(dt,J=15.0,5. 0Hz,6H),1.65(dt,J=15.0,5.0Hz,4H),1.55(dq,J=15.0,5.0Hz,9H),1.39-1.33(m,4H).13C NMR (126MHz, CDCl3) δ174.77,174.60,173.25,173.11,173.02,172.93,172.20,169.98,169.87,168 .55,168.04,167.58,167.51,158.66,158.47,144.85,135.86,129.95,128.00,127.84,126.84,113.27, 113.13, 86.06, 84.18, 84.16, 73.19, 70.96, 70.92, 70.61, 70.54, 70.12, 70.04, 70.01, 70.00, 68.69, 55.22, 52.78, 52.19, 45.02, 39.44, 39.26, 39.07, 38.69, 38.57, 36.24, 35.90, 35.86, 32.87, 32.48, 31.22, 30.57, 29.32, 29.14, 28.72, 27.97, 26.51, 25.30, 25.25, 24.79, 22.73, 18.20, 9.05. MS (ES+): m / z = 1367.35 [(M-303)+H]+.

[0234] The above oil (5.26 g) was dissolved in DMF, DIEA (1.63 g, 12.6 mmol), HATU (1.44 g, 3.78 mmol) and HOBt (510 mg, 3.78 mmol) were added with stirring, the mixture was stirred at room temperature for 5 minutes, 5.3 g of controlled-pore glass support (CPG, substitution rate 1.2 mmol / g) was added to the reaction mixture. The system was stirred at room temperature overnight, filtered, the filter cake was washed with dichloromethane, acetonitrile and dichloromethane in turn, and dried under vacuum for 1 hour. The above dried solid phase carrier was placed in pyridine / acetic anhydride (20 mL / 7 mL) and shaken at room temperature for 3 hours, filtered, the filter cake was washed with dichloromethane, acetonitrile and dichloromethane in turn, and dried under vacuum for 1 hour to obtain a light yellow solid phase carrier Q22-CPG (10.2 g).

[0235] 7) Synthesis of polypeptide oligonucleotide conjugates:

[0236] The synthesis and annealing of siRNA were carried out according to the following conventional method:

[0237] On the Agilent MerMade-12 channel nucleic acid synthesizer, 10-40 μmol of oligonucleotide synthesis was carried out using a solid phase oligonucleotide synthesis protocol. An aminolysis reagent was added to the synthesized oligonucleotide, and the oligonucleotide was separated from the solid phase carrier to free the oligonucleotide. Then the crude oligonucleotide was precipitated with ethanol, the supernatant was discarded by high-speed centrifugation twice to obtain the crude oligonucleotide, and the precipitate was resuspended in DEPC water. The crude oligonucleotide was purified by ion pair HPLC method, and the collected product was dried to powder in a vacuum centrifugal dryer. The product obtained after purification was dissolved in DEPC water, and analyzed by TOF LC-MS.

[0238] After obtaining the purified peptide-sense strand conjugate, the nucleotide concentration was determined by dissolving it in DEPC-treated water. The volumes required for equimolar amounts of the sense and antisense strands were calculated. The equimolar amounts of the peptide, sense, and antisense strands were then mixed and heated at 75°C for 5 minutes, followed by annealing with natural cooling to room temperature to produce peptide-siRNA duplexes. After cooling to room temperature, the annealed product was diluted with 10 volumes of DEPC-treated water and transferred to a Millipore 15mL ultrafiltration centrifuge tube (molecular cutoff 3 kDa) for ultrafiltration. This step removes residual chemicals (such as n-hexylamine) from the peptide and nucleic acid preparation process. The ultrafiltration tube was centrifuged at 4000 RPM for approximately 40 minutes. Once the product volume remained at approximately 1 mL, the product was collected and washed with 3 mL of DEPC-treated water. The ultrafiltration product was sterilized by filtration through a 0.22 μm filter membrane and then dried to a powder in a vacuum centrifuge. After dissolution with the appropriate solvent, it could be used for subsequent in vitro and in vivo experiments.

[0239] The peptide oligonucleotide conjugates P1-8001, P2-8001, P3-8001, P4-8001, P5-8001, P6-8001, P7-8001, and P8-8001 have the same nucleotide sequence and linker except for the peptide portion. The synthesis steps are taken as an example for P3-8001:

[0240]

[0241] 1. Using conventional nucleotide monomers and the special monomer B04, a standard solid-phase oligonucleotide procedure was performed for synthesis and purification to obtain the 5'-end B04-modified oligonucleotide sense strand 8001SM1-B04 (33 mg, 4.60 μmol) (molecular weight: 7171, measured: 7171).

[0242] 2. Synthesis of siRNA-BCN: 8001SM1-B04 (33 mg, 4.60 μmol) was dissolved in 30 mL of phosphate buffer (0.1 M, pH 7.4), endo-BCN-PEG2-NHS ester (144 mg, 320 μmol) was dissolved in 15 mL of DMF, the DMF solution of endo-BCN-PEG2-NHS ester was mixed with the phosphate buffer of 8001SM1-B04, and the mixture was shaken at 40 °C for 30 min. After the reaction was completed by LC-MS monitoring, 4.5 mL of 3 M NaCl solution and 150 mL of pre-cooled ethanol were added to the reaction solution, which was shaken thoroughly and then centrifuged at 4 °C and 15000 rpm for 3 min. The supernatant was discarded, and the precipitate was dissolved in about 3 mL of DEPC water. Purification was performed by HPLC, and the white powder solid 8001SM1-B04-BCN (22.4 mg, 2.98 μmol) was obtained by lyophilization (molecular weight: 7506, measured: 7506).

[0243] 3. 8001SM1-B04-BCN (6 mg, 0.80 μmol) was dissolved in 2.5 mL of phosphate buffer (0.1 M, pH 7.4), and the polypeptide P3 (1.8 mg, 1.0 μmol) was dissolved in 1 mL of DMF. The phosphate buffer of 8001SM1-B04-BCN was mixed with the DMF solution of P3, and the mixture was shaken at 40 °C for 2 h under N2 protection. After the reaction was completed by LC-MS monitoring, 0.3 mL of 3 M NaCl solution and 10 mL of pre-cooled ethanol were added to the reaction solution, which was shaken thoroughly and then centrifuged at 4 °C and 15000 rpm for 3 min. The supernatant was discarded, and the precipitate was dissolved in about 2 mL of DEPC water. Purification was performed by HPLC, and the white powder solid P3-8001SM1 (2.8 mg, 0.30 μmol) was obtained by lyophilization (molecular weight: 9308, measured: 9308).

[0244] 4. P3-8001SM1 was mixed with 8001AM2 to obtain P3-8001.2-1, which was abbreviated as P3-8001.

[0245] BiP3-8022 is a siRNA with two identical polypeptides at one end, and the synthesis steps are as follows:

[0246]

[0247] 1. The 3'-end amino-containing oligonucleotide sense strand 8022SM2-B08 (molecular weight: 7012, measured: 7012) was synthesized and purified by a standard solid-phase oligonucleotide procedure using conventional nucleotide monomers and a special monomer B08.

[0248] 2. Dissolve 8022SM2-B08 (160 mg, 23 μmol) in 20 mL of phosphate buffer (0.1 M, pH 7.4), dissolve BPL-2 (569 mg, 660 μmol) in 10 mL of DMF, mix the above BPL-2 DMF solution with the 8022SM2-B08 phosphate buffer, shake the reaction at room temperature for 60 min. After the reaction is complete, monitor by LC-MS, add 3 mL of 3 M NaCl solution and 90 mL of pre-cooled ethanol to the reaction, shake well and centrifuge for 3 min (4 °C, 15000 rpm), discard the supernatant, dissolve the precipitate with about 5 mL of DEPC water, purify by HPLC, and lyophilize to obtain white powder solid 8022SM2-B08-BPL-2 (120 mg, 14.8 μmol) (molecular weight: 8084, measured: 8084).

[0249] 3. Dissolve 8022SM2-B08-BPL-2 (20 mg, 2.5 μmol) in 10 mL of phosphate buffer (0.1 M, pH 7.4), dissolve the polypeptide P3 (22 mg, 12 μmol) in 10 mL of DMF, mix the above P3 DMF solution with the 8022SM2-B08-BPL-2 phosphate buffer, add CuSO4 pentahydrate, tris-(3-hydroxypropyl triazolylmethyl) amine (THPTA), and sodium L-ascorbate (NaVc) under N2 protection, react at 40 °C for 2 h, after the reaction is complete, monitor by LC-MS, add 0.3 mL of 3 M NaCl solution and 90 mL of pre-cooled ethanol to the reaction, shake well and centrifuge for 3 min (4 °C, 15000 rpm), discard the supernatant, dissolve the precipitate with about 2 mL of DEPC water, purify by HPLC, and lyophilize to obtain white powder solid BiP3-8022SM2 (8.2 mg, 0.70 μmol) (molecular weight: 11688, measured: 11688).

[0250] 4. Mix BiP3-8022SM2 with 8022AM4, anneal to obtain BiP3-8022.4-2, which is referred to as BiP3-8022 in the present application. BiP3-8022-2 is a siRNA with two identical polypeptides attached to one end, and the synthesis steps are as follows:

[0251]

[0252] 1. Synthesis of 8022SM3-Q22: Use the solid support Q22-CPG to synthesize and purify the 3' end of the oligonucleotide sense strand 8022S3-Q22 containing a double alkyne group modification by following the standard solid-phase oligonucleotide procedure of Example 7 (molecular weight: 8316.95, measured: 8315.86)

[0253] 2. Synthesis of BiP3-Q22-8022SM3: 8022SM3-Q22 (5 mg, 0.6 μΜ) was dissolved in 3 mL of phosphate buffer (0.1 M, pH 7.4), P3 (5 mg, 2.7 μmol) was dissolved in 3 mL of DMF, the phosphate buffer of 8022SM3-Q22 and the DMF solution of P3 were mixed, CuSO4.5H2O (10.6 mg, 42.4 μmol), tris-(3-hydroxypropyl triazolylmethyl) amine (THPTA, 6 mg, 13.8 μmol) and sodium L-ascorbate (NaVc, 17 mg, 85.8 μmol) were added under N2 protection, the reaction was carried out at 40 °C for 1 h, after the reaction was completed by LC-MS monitoring, 0.6 mL of 3 M NaCl solution and 20 mL of pre-cooled ethanol were added to the reaction solution, after shaking thoroughly, centrifugation was carried out for 3 min (4 °C, 15000 rpm), the supernatant was discarded, about 2 mL of DEPC water was added to dissolve the precipitate, HPLC was used for purification, and freeze-drying was carried out to obtain white solid powder BiP3-Q22-8022SM3 (3 mg, 0.25 μmol, yield: 41.7 %) (molecular weight: 11919.05, measured: 11918.23).

[0254] 4. BiP3-Q22-8022SM3 was mixed with 8022AM4, and annealing was carried out according to the method in Example 7 to obtain BiP3-Q22-8022.4-3, which is referred to herein as BiP3-8022-2.

[0255] DP3-8070 is a siRNA to which one kind of the same polypeptide is connected at each of the 2 ends, and the synthesis steps are as follows:

[0256]

[0257] 1. A 5' end and 3' end containing one B05 modified oligonucleotide sense strand B05-8070SM1-B05 (34 mg, 5.0 μmol) (molecular weight: 6785, measured: 6785) was synthesized and purified by using conventional nucleotide monomers and special monomer B05 according to the standard solid-phase oligonucleotide procedure.

[0258] 2. Synthesis of siRNA-BCN: B05-8070SM1-B05 (34 mg, 5.0 μmol) was dissolved in 30 mL of phosphate buffer (0.1 M, pH 7.4), and endo-BCN-PEG2-NHS ester (288 mg, 640 μmol) was dissolved in 15 mL of DMF. The DMF solution of endo-BCN-PEG2-NHS ester and the phosphate buffer solution of B05-8070SM1-B05 were mixed and shaken at 40°C for 30 min. After the reaction was complete as monitored by LC-MS, 4.5 mL of 3 M NaCl solution and 150 mL of pre-cooled ethanol were added to the reaction solution, which was thoroughly shaken and centrifuged for 3 min (4°C, 15,000 rpm). The supernatant was discarded, and about 3 mL of DEPC water was added to dissolve the precipitate. The precipitate was purified by HPLC and freeze-dried to obtain a white powder solid BCN-B05-8070SM1-B05-BCN (22.3 mg, 3.00 μmol) (molecular weight: 7456, measured: 7456).

[0259] 3. BCN-B05-8070SM1-B05-BCN (6 mg, 0.80 μmol) was dissolved in 2.5 mL of phosphate buffer (0.1 M, pH 7.4), and polypeptide P3 (3.6 mg, 2.0 μmol) was dissolved in 1 mL of DMF. The phosphate buffer of BCN-B05-8070SM1-B05-BCN and the DMF solution of P3 were mixed, and the mixture was shaken for 2 hours under N2 protection. After the reaction was complete, 0.3 mL of 3 M NaCl solution and 10 mL of pre-cooled ethanol were added to the reaction solution. After sufficient shaking, the mixture was centrifuged for 3 minutes (4°C, 15000 rpm). The supernatant was discarded, and about 2 mL of DEPC water was added to dissolve the precipitate. The precipitate was purified by HPLC and lyophilized to obtain a white powder solid DP3-8070SM1 (3.4 mg, 0.31 μmol) (molecular weight: 11060, measured: 11060).

[0260] 4. DP3-8070SM1 and 8070AM1 were mixed and annealed to obtain DP3-8070.1-1, abbreviated as DP3-8070.

[0261] The names and sequences of the biological agent-polypeptide oligonucleotide conjugates (siRNA agents) synthesized by the above method are shown in Table 14 below, wherein the abbreviations of the siRNA agents, such as P1 or BiP3, represent the corresponding targeting ligand and carrier, and the synthesized oligonucleotide sequences are all siRNA sequences targeting MC4R.

[0262] Table 9 Names and sequences of polypeptide oligonucleotide conjugates

[0263]

[0264]

[0265] The specific structure of the polypeptide oligonucleotide conjugate is as follows:

[0266]

[0267]

[0268] Example 6 In vitro activity of polypeptide oligonucleotide conjugates in hMC4R-293Ta stable cell lines

[0269] Cell culture and 96-well plate free uptake transfection: In vitro experiments were performed in hMC4R-293Ta stable cell lines using MEM + 10% FBS + 1X penicillin-streptomycin + 1X non-essential amino acids. When the cell confluence area reached 80%, the cells were trypsinized and the cell density was determined using a Scepter automated cell counter (Millipore, #PHCC00000). Then, the corresponding concentrations of siRNA and complete culture medium containing hMC4R-293Ta cells were added to 96-well plates and incubated in a 37°C, 5% CO2 incubator for 24 or 48 hours.

[0270] 96-well plate RNA extraction and reverse transcription: Dynabeads mRNADIRECT kit (Ambion) was used to extract mRNA from 96-well plate cells. The culture medium in the 96-well plate was aspirated, washed once with DPBS, and 50-300 μl of cell lysis buffer was added to each well, followed by 20-100 μl of Beads, shake on the oscillator, place the 96-well plate on the magnetic separation rack, aspirate the lysate in the well, add 50-300μl of washing buffer A to each well, place it on the magnetic separation rack after blowing, aspirate the washing buffer A, then blow up the beads with washing buffer B, transfer them to a new 96-well plate, place it on the magnetic separation rack, aspirate the washing buffer B, and then blow up the beads with buffer B and transfer them to a 96-well PCR plate. At the same time, prepare the reversal reagent, place the 96-well PCR plate on the magnetic separation rack, aspirate the washing buffer B, add 20μl of reversal reagent to each well, seal the plate with a sealing film, and incubate at 25℃ for 10 minutes on a PCR instrument, then incubate at 37℃ for two hours, then at 85℃ for 5 minutes, cool to 4℃, and the reverse transcription is completed.

[0271] Real-time fluorescence quantitative PCR: After reverse transcription, place the 96-well plate on a magnetic separation rack until the beads are adsorbed to the bottom. Aspirate the reverse transcription reagent, add the prepared QPCR system to the 96-well PCR plate, seal the plate with a sealing film, and perform PCR on a StepOnePlus real-time PCR system (applied biosystems).

[0272] The data were analyzed using the ΔΔCt method and normalized using cells transfected with a negative control sequence.

[0273] The negative control AD-1955 sequence is: CUUACGCUGAGUACUUCGAdTdT (SEQ ID NO. 335)

[0274] UCGAAGUACUCAGCGUAAGdTdT (SEQ ID NO. 336).

[0275] Table 10 shows the in vitro activity of the polypeptide oligonucleotide conjugates in hMC4R-293Ta stable cells

[0276]

[0277] The data showed that naked-chain siRNA 8001 had only low target activity when freely taken up, even at a high concentration of 1000nM. The experimental data of the P2-8001 and P3-8001 groups indicated that the delivery vectors of ligands P2 and P3 can deliver siRNA in human cells.

[0278] Table 11 In vitro activity of polypeptide oligonucleotide conjugates in hMC4R-293Ta stable cell lines

[0279]

[0280]

[0281] The in vitro delivery activity of the polypeptide oligonucleotide conjugate (siRNA agent) showed that the delivery vectors with ligands P2, P3, and P4 can successfully deliver the connected siRNA into cells through the human MC4R receptor and exhibit significant target knockdown activity. When the delivery vector with the ligand P3 transports the siRNA, the activity of the siRNA agent is better.

[0282] Example 7 In vitro activity of polypeptide oligonucleotide conjugates in primary liver cells of mice infected with AAV8-hMC4R virus. Experimental method: In vitro screening was performed in primary liver cells of mice infected with AAV8 virus expressing human MC4R (hMC4R).

[0283] Construction of mice infected with AAV8 virus stably expressing hMC4R: 1x10 11 Mice were infected with ultrapurified recombinant AAV8 hMC4R viral particles to obtain transgenic mice stably expressing hMC4R.

[0284] Mouse primary hepatocyte extraction: Mouse hepatocytes were extracted by collagenase digestion via inferior vena cava perfusion. Viable primary hepatocytes were filtered through a tissue cell strainer (BIOLOGIX, 15-1070) and resuspended in DMEM medium supplemented with 10% FBS and 1X penicillin-streptomycin. Cell density was determined using a Scepter automated cell counter.

[0285] The methods of siRNA transfection, RNA extraction, reverse transcription, and real-time fluorescence quantitative PCR were the same as those described above. siRNA was delivered by free uptake without the addition of INTERFERin.

[0286] Table 12 shows the in vitro activity of polypeptide oligonucleotide conjugates in AVV8-hMC4R primary liver cells

[0287]

[0288] The data showed that BiP3-8022-2 had high activity in primary liver cells of AVV8-hMC4R virus-infected mice and had a certain dose-dependency.

[0289] Example 8 In vitro activity of polypeptide oligonucleotide conjugates in stable mMC4R-293Ta cells

[0290] The experimental methods refer to the previous article.

[0291] Table 13 shows the in vitro activity of the polypeptide oligonucleotide conjugates in the stable mMC4R-293Ta cell line. The in vitro delivery activity of the polypeptide oligonucleotide conjugate showed that when the ligand was P3, the linked siRNA could be successfully delivered into cells through the mouse MC4R receptor, and MC4R could be knocked down in a dose-dependent manner.

[0292] Example 9: Preparation of active drugs of some polypeptide oligonucleotide conjugates by injection into the cisterna magna in WT C57 mice

[0293] After freeze-drying to obtain the MC4R-targeting polypeptide oligonucleotide conjugate powder, artificial cerebrospinal fluid (CSF) was used as a solvent in a sterile environment to prepare a polypeptide oligonucleotide conjugate with a stock concentration of 1.8 nmol / ul (about 32-38 mg / ml).

[0294] Experimental grouping and drug administration

[0295] Four-week-old male WT C57 mice of similar weight were randomly divided into groups of 1 to 3 mice each. After anesthesia, the mice were injected with the peptide oligonucleotide conjugate via cisterna magna injection at a dose of 9 nmol / dose (approximately 0.16 to 0.19 mg / dose). On the day of drug injection, the mice were observed after recovery from anesthesia, and their weights were recorded daily.

[0296] Material collection and testing

[0297] On the 22nd day of administration, the experimental mice were sacrificed and samples were collected from the brainstem, hypothalamus, hippocampus, thalamus, and spinal cord. The samples were stored in RNALater to prevent RNA degradation. RNA from each brain tissue was then extracted using TRI REAGENT (MRC, Catalog No.: TR118) and reverse transcribed into cDNA using the PrimeScript RTregent Kit (Takara, Catalog No.: RR047A). The prepared QPCR system was added to a 96-well PCR plate, sealed with a plate film, and QPCR was performed on the StepOnePlus real-time PCR system (Applied Biosystems) to detect the expression level of mMC4R. At the same time, half the brain was taken to detect the content of the siRNA antisense chain in the brain.

[0298] The results are shown in the table below.

[0299] Table 14 Activity detection results and antisense chain content of polypeptide oligonucleotide conjugates in various brain tissues of mice on day 22

[0300]

[0301] The results showed that delivery vectors with ligands P3 / P9 / P10 could deliver siRNA into specific brain regions and exhibit varying degrees of activity in various brain tissues of mice.

[0302] Example 10: Preparation of active pharmaceutical ingredients of some polypeptide oligonucleotide conjugates by injection into the cisterna magna in SD rats

[0303] After freeze-drying to obtain the MC4R-targeting polypeptide oligonucleotide conjugate powder, artificial cerebrospinal fluid (CSF) was used as a solvent in a sterile environment to prepare a polypeptide oligonucleotide conjugate with a stock concentration of 3.5 nmol / ul (about 66-69 mg / ml).

[0304] Experimental grouping and drug administration

[0305] Male Sprague-Dawley rats weighing approximately 160-180 g were selected for the experiment and randomly divided into groups of 3-10 rats. After anesthesia, the rats were injected with the peptide-oligonucleotide conjugate via cisterna magna injection at a dose of 70 nmol / dose (approximately 1.32-1.37 mg / dose). On the day of drug injection, the rats were observed after recovery from anesthesia, and their body weights were recorded daily.

[0306] Material collection and testing

[0307] On the 17th day of administration, some experimental rats were sacrificed and samples were collected. The expression level of rMC4R was detected by QPCR, and the experimental method was referred to Example 9.

[0308] The results are shown in the table below.

[0309] Table 15 Activity detection results of polypeptide oligonucleotide conjugates in rat hypothalamus tissue on day 17

[0310] Peptide-oligonucleotide conjugates % of remaining mMC4R mRNA BiP3-8039 71.95 BiP3-8107 42.53 BiP3-8010 20.03

[0311] The results showed that the delivery vector with P3 as the ligand could deliver different siRNAs into the hypothalamus, and sequences 8039, 8107, and 8010 showed different degrees of activity.

[0312] Example 11: Preparation of active drugs of some polypeptide oligonucleotide conjugates by injection into the cisterna magna in WT C57 mice

[0313] After freeze-drying to obtain the MC4R-targeting polypeptide oligonucleotide conjugate powder, artificial cerebrospinal fluid (CSF) was used as a solvent in a sterile environment to prepare a polypeptide oligonucleotide conjugate with a stock concentration of 3.6 nmol / ul (about 70 mg / mL).

[0314] Experimental grouping and drug administration

[0315] Seven- to eight-week-old male WT C57 mice of similar weight were randomly divided into 10 groups. After anesthesia, the mice were injected with the peptide-oligonucleotide conjugate via cisterna magna injection at a dose of 18 nmol / dose (approximately 0.35 mg / dose). On the day of drug injection, the mice were observed after recovery from anesthesia, and their weights were recorded daily.

[0316] Weight Record

[0317] Based on the theoretical basis that knockdown of MC4R will cause weight gain in experimental animals, the weight data of experimental animals were recorded at different time points after administration. The experimental results are shown in Figure 1 .

[0318] The results showed that BiP3-8022 and BiP3-5-8022 could significantly increase the body weight of experimental mice.

[0319] Example 12: Preparation of active drugs of some polypeptide oligonucleotide conjugates by injection into the cisterna magna in WT C57 mice

[0320] After freeze-drying to obtain the MC4R-targeting polypeptide oligonucleotide conjugate powder, artificial cerebrospinal fluid (CSF) was used as a solvent in a sterile environment to prepare a polypeptide oligonucleotide conjugate with a stock concentration of 3.6 nmol / ul (about 70 mg / mL).

[0321] Experimental grouping and drug administration

[0322] Seven- to eight-week-old male WT C57 mice of similar weight were randomly divided into groups of six. After anesthesia, the mice were injected with the peptide-oligonucleotide conjugate via cisterna magna injection at a dose of 18 nmol / dose (approximately 0.35 mg / dose). On the day of drug injection, the mice were observed after recovery from anesthesia, and their weights were recorded daily.

[0323] Material collection and testing

[0324] On the eighth day of administration, the mice were sacrificed and samples were collected from the hypothalamus. QPCR was performed to detect the expression of mMC4R using the same method as described above. Simultaneously, half the brain was collected to detect the content of the siRNA antisense strand in the brain.

[0325] The results are shown in the table below.

[0326] Table 16 Activity detection results of polypeptide oligonucleotide conjugates in mouse hypothalamus on day 8

[0327]

[0328] The results showed that BiP3-8022-1 and BiP3-8022-2 had high activity in the hypothalamus, and the content of the antisense chain of BiP3-8022-1 drug in the brain was relatively high.

[0329] Example 13: Preparation of active drugs of some polypeptide oligonucleotide conjugates by injection into the cisterna magna in WT C57 mice

[0330] After freeze-drying to obtain the MC4R-targeting polypeptide oligonucleotide conjugate powder, the polypeptide oligonucleotide conjugate was prepared in a sterile environment using artificial cerebrospinal fluid (CSF) as a solvent to prepare stock solutions with concentrations of 1.2 nmol / ul (approximately 23 mg / mL) and 3.6 nmol / ul (approximately 70 mg / mL).

[0331] Experimental grouping and drug administration

[0332] Seven- to eight-week-old male WT C57 mice of similar weight were randomly divided into 10 groups. After anesthesia, the mice were injected with the peptide-oligonucleotide conjugate via cisterna magna injection at doses of 6 nmol / dose (approximately 0.12 mg / dose) and 18 nmol / dose (approximately 0.35 mg / dose). On the day of drug injection, the mice were observed after recovery from anesthesia, and their weights were recorded daily.

[0333] Material collection and testing

[0334] On day 30 of drug administration, the mice were sacrificed and samples were collected. The expression of mMC4R was detected by QPCR, using the same experimental method as described above.

[0335] The results are shown in the table below.

[0336] Table 17 Activity detection results of polypeptide oligonucleotide conjugates in mouse hypothalamus on day 30

[0337]

[0338]

[0339] The results showed that BiP3-8022-2 had high activity in the mouse hypothalamus and was dose-dependent.

[0340] Example 14: Exploring the Preparation of Active Drugs of Peptide Oligonucleotide Conjugates by Injection into the Cisterna magna in Humanized Mice

[0341] After freeze-drying to obtain the MC4R-targeting polypeptide oligonucleotide conjugate powder, artificial cerebrospinal fluid (CSF) was used as a solvent in a sterile environment to prepare a polypeptide oligonucleotide conjugate with a stock concentration of 3.6 nmol / ul (about 70 mg / mL).

[0342] Experimental grouping and drug administration

[0343] Male humanized MC4R mice of similar weight were selected as experimental subjects and randomly divided into groups of 8. After anesthesia, the mice were injected with the peptide-oligonucleotide conjugate via cisterna magna injection at a dose of 18 nmol / dose (approximately 0.35 mg / dose). On the day of drug injection, the mice were observed after recovery from anesthesia, and their weights were recorded daily.

[0344] Material collection and testing

[0345] On day 28 of drug administration, mice were sacrificed and samples were collected from the hypothalamus, liver, and kidney. hMC4R expression was detected by QPCR using the same protocol as described above.

[0346] The results are shown in the table below.

[0347] Table 18 Activity detection results of different polypeptide oligonucleotide conjugates in different tissues of humanized MC4R mice on day 28

[0348]

[0349] The results showed that when the dose was 18 nmol / dose, different polypeptide oligonucleotide conjugates showed higher activity in the hypothalamus, but had no activity in the liver and kidney.

[0350] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. siRNA or a pharmaceutically acceptable salt thereof for inhibiting the expression of melanocortin receptor 4 (MC4R) gene in human cells, wherein: The siRNA includes a sense strand and a corresponding complementary antisense strand, wherein the sense strand comprises at least 15, 16 or 17 consecutive nucleotides, which differs from any sense strand sequence shown in Table 1 by no more than 3 nucleotides; and the antisense strand comprises at least 15, 16 or 17 consecutive nucleotides, which differs from any antisense strand nucleotide sequence shown in Table 1 by no more than 3 nucleotides.

2. The siRNA or a pharmaceutically acceptable salt thereof according to claim 1, wherein The siRNA is selected from the following sequences, or sequences that differ from its sense strand or antisense strand by no more than 3 nucleotides: 1) 8001a, the sense strand sequence is shown in SEQ ID NO: 1, and the antisense strand sequence is shown in SEQ ID NO: 2, 2) 8006b, the sense strand sequence is shown in SEQ ID NO: 13, and the antisense strand sequence is shown in SEQ ID NO: 14, 3) 8010b, the sense strand sequence is shown in SEQ ID NO: 23, and the antisense strand sequence is shown in SEQ ID NO: 24, 4) 8022a, the sense strand sequence is shown in SEQ ID NO: 59, and the antisense strand sequence is shown in SEQ ID NO: 60, 5) 8023b, the sense strand sequence is shown in SEQ ID NO: 65, and the antisense strand sequence is shown in SEQ ID NO: 66, 6) 8039a, the sense strand sequence of which is shown in SEQ ID NO: 119, and the antisense strand sequence of which is shown in SEQ ID NO: 120, 7) 8064a, the sense strand sequence is shown in SEQ ID NO: 191, and the antisense strand sequence is shown in SEQ ID NO: 192, 8) 8103a, the sense strand sequence is shown in SEQ ID NO: 303, and the antisense strand sequence is shown in SEQ ID NO: 304, 9) 8107a, the sense strand sequence is shown in SEQ ID NO: 311, and the antisense strand sequence is shown in SEQ ID NO: 312, 10) 8110a, the sense strand sequence of which is shown in SEQ ID NO: 317, and the antisense strand sequence of which is shown in SEQ ID NO: 318, 11) 8113a, the sense strand sequence of which is shown in SEQ ID NO: 323, and the antisense strand sequence of which is shown in SEQ ID NO: 324, 12) 8012a, the sense strand sequence is shown in SEQ ID NO: 29, and the antisense strand sequence is shown in SEQ ID NO: 30, 13) 8020b, the sense strand sequence is shown in SEQ ID NO: 55, and the antisense strand sequence is shown in SEQ ID NO: 56, 14) 8026b, the sense strand sequence is shown in SEQ ID NO: 77, and the antisense strand sequence is shown in SEQ ID NO: 78, 15) 8061b, the sense strand sequence is shown in SEQ ID NO: 185, and the antisense strand sequence is shown in SEQ ID NO: 186, 16) 8065b, the sense strand sequence is shown in SEQ ID NO: 197, and the antisense strand sequence is shown in SEQ ID NO: 198, 17) 8066b, the sense strand sequence is shown in SEQ ID NO: 201, and the antisense strand sequence is shown in SEQ ID NO: 202, 18) 8100a, the sense strand sequence is shown in SEQ ID NO: 297, and the antisense strand sequence is shown in SEQ ID NO: 298, 19) 8106a, the sense strand sequence is shown in SEQ ID NO: 309, and the antisense strand sequence is shown in SEQ ID NO: 310, 20) 8070a, the sense strand sequence is shown in SEQ ID NO: 213, and the antisense strand sequence is shown in SEQ ID NO: 214, 21) 8096a, the sense strand sequence is shown in SEQ ID NO: 289, and the antisense strand sequence is shown in SEQ ID NO:

290.

3. The siRNA or a pharmaceutically acceptable salt thereof according to claim 1, wherein The nucleotides of the siRNA are modified, and the siRNA is selected from any pair of siRNAs in Table 2, or a sequence that differs from the sense strand or antisense strand thereof by no more than 3 nucleotides.

4. The siRNA or pharmaceutically acceptable salt thereof according to claim 3, wherein The siRNA is selected from the following sequences, or sequences that differ from its sense strand or antisense strand by no more than 3 nucleotides: 1) 8001.4-2, the 5'-3' sequence of the sense strand is mU*mU*mGmUmGmUmGmUfGfCfUfUmCfAmUmGmUmCmUmCmA, and the 5'-3' sequence of the antisense strand is VPU-S*fG*mAmGmAmCmAmUmGmAmAdGmCfAmCfAmCmAmCmAmA*mU*mA 2) 8006.4-2, the 5'-3' sequence of the sense strand is mG*mA*mGmGmAmAmAmAfCfCfUfUmCfAmAmAmGmAmGmAmA, and the 5'-3' sequence of the antisense strand is VPU-S*fU*mCmUmCmUmUmUmGmAmAdGmGfUmUfUmUmCmC*mU*mC 3) 8010.4-2, the 5'-3' sequence of the sense strand is mC*mU*mUmCmAmAmAmGfAfGfAfUmCfAmUmCmUmGmUmUmA, and the 5'-3' sequence of the antisense strand is VPU-S*fA*mAmCmAmGmAmUmGmAmUdCmUfCmUfUmUmGmA*mA*mG 4) 8022.4-2, the 5'-3' sequence of the sense strand is mU*mG*mGmGmAmUmCmAfUmCfAmUfAmAmGmUmUmGmUmAmA, and the 5'-3' sequence of the antisense strand is VPU-S*fU*mAmCmAmAmCmUmUmAmUdGmAfUmGfAmUmCmCmCmA*mA*mC 5) 8022.4-3, the 5'-3' sequence of the sense strand is mU*mG*mGmGmAmUmCmAfUfCfAfUmAfAmGmUmUmGmUmAmA, and the 5'-3' sequence of the antisense strand is VPU-S*fU*mAmCmAmAmCmUmUmAmUdGmAfUmGfAmUmCmCmCmA*mA*mC 6) 8023.5-3, the 5'-3' sequence of the sense strand is mA*mG*mCmGmUmUmUmCfAfAfAfUmGfGmAmUmCmAmGmAmA, and the 5'-3' sequence of the antisense strand is VPU-S*fU*mCmUmGmAmUmCmCmAmUdTmUfGmAfAmAmCmG*mC*mU 7) 8039.2-2, the 5'-3' sequence of the sense strand is mC*mA*mUmGmUmCmUmCfAfCfUfUmUfAmAmCmUmUmGmUmA, and the 5'-3' sequence of the antisense strand is VPU-S*fA*mCmAmAmGmUmUmAmAmAdGmUfGmAfGmAmCmAmUmG*mA*mA 8) 8064.3-2, the 5'-3' sequence of the sense strand is mA*mU*mGmUmCmUmCmAfCfUfUfUmAfAmCmUmUmGmUmAmA, and the 5'-3' sequence of the antisense strand is VPU-S*fU*mAmCmAmAmGmUmUmAmAdAmGfUmGfAmGmAmCmAmU*mG*mA 9)8103.2-1, the 5'-3' sequence of the sense strand is mC*mU*mCmCmAmGmUmAfCfCfAfUmAfAmCmAmUmUmAmUmA, and the 5'-3' sequence of the antisense strand is VPU-S*fA*mUmAmAmUmGmUmUmAmUdGmGfUmAfCmUmGmG*mA*mG 10) 8107.2-1, the 5'-3' sequence of the sense strand is mU*mG*mAmGmGmAmAmAfAfCfCfUmUfCmAmAmAmGmAmGmA, and the 5'-3' sequence of the antisense strand is VPU-S*fC*mUmCmUmUmUmGmAmAmGdGmUfUmUfUmCmCmU*mC*mA 11) 8110.2-1, the 5'-3' sequence of the sense strand is mU*mG*mUmCmAmUmCmAfCfCfCfUmAfUmUmAmAmAmCmAmA, and the 5'-3' sequence of the antisense strand is VPU-S*fU*mGmUmUmUmAmAmUmAmGdGmGfUmGfAmUmGmA*mC*mA 12) 8113.2-1, the 5'-3' sequence of the sense strand is mU*mU*mGmUmUmCmAmUfCfAfUfUmUfAmCmUmCmAmGmAmA, and the 5'-3' sequence of the antisense strand is VPU-S*fU*mCmUmGmAmGmUmAmAmAdTmGfAmUfGmAmAmC*mA*mA 13)8012.3-3, the 5'-3' sequence of the sense strand is mU*mA*mCmGmAmGmCmAfAfCfUfUmUfUmUmGmUmCmUmCmA, and the 5'-3' sequence of the antisense strand is VPU-S*fG*mAmGmAmCmAmAmAmAmAdGmUfUmGfCmUmCmGmUmA*mG*mC 14)8020.5-3, the 5'-3' sequence of the sense strand is mA*mG*mGmCmAmUmUmUfUfGfUfUmCfAmUmCmAmUmUmUmA, and the 5'-3' sequence of the antisense strand is VPU-S*fA*mAmAmUmGmAmUmGmAmAdCmAfAmAfAmUmGmC*mC*mU 15)8026.4-2, the 5'-3' sequence of the sense strand is mG*mG*mUmUmGmGmGmAfUfCfAfUmCfAmUmAmAmGmUmUmA, and the 5'-3' sequence of the antisense strand is VPU-S*fA*mAmCmUmUmAmUmGmAmUdGmAfUmCfCmCmAmA*mC*mC 16)8061.4-2, the 5'-3' sequence of the sense strand is mU*mC*mAmUmGmUmCmUfCfAfCfUmUfUmAmAmCmUmUmGmA, and the 5'-3' sequence of the antisense strand is VPU-S*fC*mAmAmGmUmUmAmAmAmGdTmGfAmGfAmCmAmU*mG*mA 17)8065.4-2, the 5'-3' sequence of the sense strand is mC*mU*mCmAmCmUmUmUfAfAfCfUmUfGmUmAmUmCmUmCmA, and the 5'-3' sequence of the antisense strand is VPU-S*fG*mAmGmAmUmAmCmAmAmGdTmUfAmAfAmGmUmG*mA*mG 18)8066.5-2, the 5'-3' sequence of the sense strand is mU*mC*mAmCmUmUmUmAfAfCfUfUmGfUmAmUmCmUmCmAmA, and the 5'-3' sequence of the antisense strand is VPU-S*fU*mGmAmGmAmUmAmCmAmAdGmUfUmAfAmAmGmU*mG*mA 19)8100.2-1, the 5'-3' sequence of the sense strand is mA*mC*mUmUmUmAmCmUfAfUfCfUmUfCmUmAmUmGmCmUmA, and the 5'-3' sequence of the antisense strand is VPU-S*fA*mGmCmAmUmAmGmAmAmGdAmUfAmGfUmAmAmA*mG*mU 20)8106.2-1, the 5'-3' sequence of the sense strand is mG*mG*mUmUmUmCmAmGfGfCfAfUmUfUmUmGmUmUmCmAmA, and the 5'-3' sequence of the antisense strand is VPU-S*fU*mGmAmAmCmAmAmAmAmUdGmCfCmUfGmAmAmA*mC*mC 21)8070.3-2, the 5'-3' sequence of the sense strand is mC*mG*mUmGmCmUfUfCfAfUmGfUmCmUmCmAmCmUmA, and the 5'-3' sequence of the antisense strand is VPU-S*fA*mGmUmGmAmGmAmCmAmUdGmAfAmGfCmAmCmG*mC*mA 22)8096.2-1, the 5'-3' sequence of the sense strand is mU*mU*mGmUmUmGmGmAfGfAfAfUmAfUmCmUmUmAmGmUmA, and the 5'-3' sequence of the antisense strand is VPU-S*fA*mCmUmAmAmGmAmUmAmUdTmCfUmCfCmAmAmC*mA*mA.

5. A biological agent or pharmaceutical preparation for inhibiting MC4R expression, wherein: The preparation comprises the siRNA or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, and a targeted delivery vector connected to or encapsulated with the siRNA, wherein the targeted delivery vector transports the siRNA or a pharmaceutically acceptable salt thereof to the central nervous system.

6. The biological preparation or pharmaceutical preparation according to claim 5, wherein The targeted delivery vector is composed of a targeting ligand and a linking group connecting the targeting ligand and siRNA, wherein the targeting ligand is a polypeptide targeting low-density lipoprotein receptor-related protein 1 (LRP1) or a polypeptide targeting transferrin receptor (TfR) or a polypeptide targeting melanocortin receptor (MCR).

7. The biological preparation or pharmaceutical preparation according to claim 6, wherein The ligand targeting LRP1 is ANG2, and its amino acid sequence from N-terminus to C-terminus is as follows: Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Gl u-Glu-Tyr; Preferably, the structure after the ANG2 polypeptide is linked to the siRNA is selected from any one of the following: wherein X and Y are O or CH2; R is O or S; p and q are integers of 0-4, k is 1, 2, 4 or 8, and Ac is acetyl; more preferably, The structure of the ANG2 polypeptide after being linked to the siRNA is one of the following:

8. The biological preparation or pharmaceutical preparation according to claim 6, wherein The amino acid sequence of the ligand targeting the melanocortin receptor is selected from any one of the following: Nle-c[Asp-Pro-D-Nal-Arg-Trp-Lys]-D-Val-D-Pro-Gly; Arg-c[Cys-D-Ala-His-D-Phe-Arg-Trp-Cys]; Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val; Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]; Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr; Tyr-Val-Met-Gly-His-Phe-Arg-D-Trp-Asp-Arg-Phe-Gly; Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-D-Trp-Gly-Lys-Pro-Val; Tyr-Val-Met-Gly-His-D-Phe-Arg-D-Trp-Asp-Arg-Phe-Gly; Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp(SCF3)-Gly-Lys-Pro-Val; Among them, Trp(SCF3) represents Nle represents L-norleucine, Dpr represents L-2,3-diaminopropionic acid, and D-Nal represents 3-(2-naphthyl)-D-alanine; See more of the sources of the photos and images of the [PAc-N-s-le] ro-D-Nal-Arg-Trp-Lys]-D-Val-D-Pro-Gly-Azk-NH2; Ac-Azk-Arg-c[Cys-D-Ala-His-D-Phe-Arg-Trp-Cys]-NH2\ Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-Azk-NH2;Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-Azk-NH2; Ac-Tyr-c[Asp-Arg-Phe-Asn-Ala-Phe-Dpr]-Tyr-Azk-NH2; Tyr-Val-Met-Gly-His-Phe-Arg-D-Trp-Asp-Arg-Phe-Gly-Azk-NH2; Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-D-Trp-Gly-Lys-Pro-Val-Azk-NH2;Tyr-Val-Met-Gly-His-D-Phe-Arg-D-Trp-Asp-Arg-2Phek-NH Ac-Azk-AEEA-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2; 6-Azido-hexanoic-AEEA-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Ly s-Pro-Val-NH2; Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys(AEEA-6-Azido-hexa noic)-Pro-Val-NH2; Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp(SCF3)-Gly-Lys-Pro-Val-AZK-NH2, wherein Ac is an acetyl group.

9. The biological preparation or pharmaceutical preparation according to claim 8, wherein The structure of the ligand and the siRNA after being connected via a linker is as follows: Wherein X and Y are O or CH2; R is O or S; p and q are integers of 0-4; k is 1, 2 or 8, and Peptide is the ligand targeting the melanocortin receptor as claimed in claim 8.

10. The biological preparation or pharmaceutical preparation according to claim 9, wherein: The structure of the ligand and siRNA after being connected through a linker group is as follows: Wherein R is O or S.

11. The biological preparation or pharmaceutical preparation according to claim 10, wherein: The structure of the ligand and siRNA after being connected through the linker group is as follows: Wherein, AEEA represents 2-(2-(2-aminoethoxy)ethoxy)acetic acid, Trp(SCF3) represents Nle represents L-norleucine, Dpr represents L-2,3-diaminopropionic acid, and D-Nal represents 3-(2-naphthyl)-D-alanine.

12. Use of the siRNA or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, or the biological preparation or pharmaceutical preparation according to any one of claims 5 to 11, in the preparation of a medicament for preventing or treating a disease mediated by the MC4R gene; or use of the siRNA or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, or the biological preparation or pharmaceutical preparation according to any one of claims 5 to 11, in the preparation of a medicament for inhibiting the expression of the melanocortin receptor 4 (MC4R) gene; preferably, the disease mediated by the MC4R gene is cachexia, sarcopenia, wasting syndrome, anorexia nervosa, and wasting diseases caused by chronic inflammation, cancer, and immune disorders.

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