A highly efficient oligonucleotide drug delivery system based on fusion peptide and its preparation method and application

Abstract

This invention discloses a highly efficient oligonucleotide drug delivery system based on a fusion peptide, its preparation method, and its applications. The system includes a fusion peptide and an oligonucleotide drug. The fusion peptide is formed by linking a targeting peptide with a sorting signal peptide of a lysosomal targeting receptor on the cell surface. The fusion peptide is linked to the oligonucleotide drug via a linker. The advantages of this invention include: targeted, efficient, and stable delivery of oligonucleotides into cells to achieve gene therapy effects; low immunogenicity; high safety; high delivery efficiency; and wide applicability.

Description

Technical Field

[0001] This invention relates to the field of gene therapy drug technology, and more particularly to oligonucleotide drugs delivered by fusion peptides. Background Technology

[0002] In eukaryotic cells, with the exception of mitochondria and chloroplasts in plant cells where a small amount of protein is synthesized, the vast majority of proteins are encoded by nuclear genes and synthesized either on free ribosomes or on ribosomes bound to the rough endoplasmic reticulum membrane. However, the sites where proteins perform their structural or functional functions are almost ubiquitous throughout various regions and components of the cell. Therefore, different mechanisms must exist to ensure protein sorting and transport to specific sites within the cell; this process is called protein targeting or protein sorting. Based on the different transport methods or mechanisms, protein sorting and transport pathways can be divided into three categories: gated transport, transmembrane transport, and vesicular transport. Material transfer between different parts of the intracellular membrane system often occurs through vesicular transport. Based on the different proteins coating the surface of transport vesicles, they are classified into three types: COPI (coat protein II) coated vesicles, COPI (coat protein I) coated vesicles, and clathrin / adaptor protein coated vesicles.

[0003] Clathin molecules consist of three heavy chains and three light chains, forming a triskelion shape with three curved arms. These arms intertwine to form a cage with mesh-like openings. The clathin coat also contains adaptin proteins (APs), which lie between the clathin and ligand-receptor complex. APs can bind to clathin and recognize the tail peptide signal on the cytoplasmic surface of the transmembrane receptor, thus mediating the selective transport of transmembrane receptors and their bound ligands through clathin-coated vesicles. Currently, adaptin proteins discovered in higher eukaryotes fall into two main categories: the AP complex family and the GGA monomeric protein family.

[0004] The AP family has five members (AP1-5), among which AP2 is located in the plasma membrane and participates in the transport of clathrin / aptamer-coated vesicles from the plasma membrane to the lysosome. For example... Figure 1 As shown, AP2 is a heterotetramer composed of two structurally related large subunits α and β2, a medium subunit μ2, and a small subunit σ2, as shown in the diagram. Figure 1As shown in the left figure, the β2 hinge region of AP2 binds to the terminal domain of the β-helix of clathrin. The α subunit binds to various regulatory and accessory proteins, participating in the formation of clathrin / aptimin-coated vesicles. The μ2 subunit of AP2 recognizes the tyrosine-based YXXØ (Ø represents a large hydrophobic residue, X represents any amino acid) peptide sequence in the tail peptide signal of the transmembrane receptor cytoplasm, and the σ2 subunit recognizes the dileucine-based (D / E)XXXL (L / I) peptide sequence in the tail peptide signal of the transmembrane receptor cytoplasm (D / E represents selecting D or E, L / I represents selecting L or I). In addition to these two peptide sequences, AP2 can also recognize another tyrosine peptide sequence, NPXY.

[0005] Extracellular ligands bind to lysosome-targeting receptors (LTRs) on the cell surface and then target the endocytosis process of lysosomes, such as... Figure 1 As shown in the right figure, AP2 is needed to recognize signals within the cytoplasmic domains of LTRs and recruit other related components to form the corresponding transport vesicles. The tyrosine-based YXXØ peptide sequence or the dileucine-based (D / E)XXXL(L / I) peptide sequence in the tail peptide signal of the LTR cytoplasmic surface can activate the μ2 or σ2 subunit of AP2. These peptide sequences are called the signal peptide for sorting of LTRs (SPSLTR).

[0006] like Figure 2 As shown, LTRs are transmembrane receptors located on the cell membrane, mediating the transport of proteins to lysosomes and subsequent degradation. When a ligand binds to an LTR, the tyrosine-based YXXØ peptide sequence or the dileucine-based (D / E)XXXL(L / I) peptide sequence in the tail peptide signal of the transmembrane receptor activates the μ2 or σ2 subunit of AP2. The β2 hinge region of AP2 recruits clathrin, which aggregates under the membrane, gradually forming a 50-100 nm diameter membrane indentation called a clathrin-coated pit. A small GTP-binding protein, dynamin, assembles into a loop at the neck of the indented clathrin pit. Dynamine hydrolyzes the GTP bound to it, causing neck constriction, and eventually detaches from the plasma membrane to form a clathrin-coated vesicle. A few seconds later, the clathrin detaches from the membrane-coated vesicle and returns to the vicinity of the plasma membrane for reuse. The uncoated vesicle fuses with the early endosome, thereby taking transport molecules and extracellular fluid into the cell.

[0007] Nucleic acid drugs need to be delivered to the cytoplasm (siRNA, mRNA) or the nucleus (ASO, DNA, and CRISPR) to achieve therapeutic effects. Naked nucleic acid drugs are susceptible to degradation by nucleases, which limits their half-life. Therefore, specialized delivery systems have been developed as a necessary measure to ensure the efficacy of nucleic acid drugs in vivo. However, current oligonucleotide delivery systems have high immunogenicity and significant toxic side effects. Most delivered oligonucleotides fail to reach the intracellular space, resulting in low delivery efficiency and necessitating the injection of high-dose drugs.

[0008] Lipid nanoparticles (LNPs) are one of the most widely researched and applied delivery systems for nucleic acid drugs. BioNTech, Moderna, and CureVac, the three giants in the field, all use LNP delivery technology for their mRNA COVID-19 vaccines. Statistics show that over 30 of the more than 40 mRNA vaccine projects currently in clinical trials worldwide utilize LNP technology. In 2018, Patisiran, the world's first siRNA drug and the first nucleic acid drug to use an LNP delivery system, was approved. In addition, N-acetyl-D-galactosamine (GalNAc) technology also plays an important role in the development pipelines of several pharmaceutical companies, most notably Alnylam, a leading nucleic acid drug company. Givosiran, developed by Alnylam and approved by the FDA in November 2019, achieves subcutaneous administration through enhanced chemical processes to stabilize ESC-GalNAc binding, resulting in higher efficacy, durability, and broader therapeutic applications. Currently, both LNP and GalNAc technologies are targeted delivery systems based on the liver's unique biological mechanisms, resulting in a narrow delivery range. Delivery technologies targeting other tissues or cells are not yet mature enough to reach the level of LNP or GalNAc technologies, and new breakthroughs are urgently needed. Most domestic nucleic acid drug development companies are also exploring along the existing lines of LNP and GalNAc. Several candidate drugs targeting the liver, kidneys, and eyes are still in clinical trials, and new drugs targeting the central nervous system and other non-hepatic tissues have not yet been developed.

[0009] Therefore, developing an oligonucleotide drug delivery system that is highly efficient, widely applicable, highly targeted, low in immunogenicity, and stable can provide a new foundation for gene therapy in treating diseases. Summary of the Invention

[0010] The purpose of this invention is to provide an efficient oligonucleotide drug delivery system based on fusion peptides, its preparation method and application, to solve the problems of low delivery efficiency, narrow applicability, low stability, high immunogenicity and weak targeting in existing oligonucleotide drug delivery systems.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] A highly efficient oligonucleotide drug delivery system based on a fusion peptide includes a fusion peptide and an oligonucleotide drug. The fusion peptide is formed by linking a targeting peptide to a sorting signal peptide of a lysosomal targeting receptor on the cell surface. The fusion peptide is linked to the oligonucleotide drug via a linker. A schematic diagram is shown below. Figure 3 As shown.

[0013] Furthermore, the targeting peptide is a small molecule peptide that can specifically bind to specific receptors on the cell surface.

[0014] Furthermore, the sorting signal peptide of the lysosomal targeting receptor on the cell surface is a tyrosine-based YXXØ peptide sequence, a dileucine-based (D / E)XXXL(L / I) peptide sequence, or a tyrosine-based NPXY peptide sequence in the tail peptide signal of the lysosomal targeting receptor on the cytoplasmic surface of the cell surface that can activate the μ2 or σ2 subunit of AP2, where Ø represents a large hydrophobic residue and X represents any amino acid.

[0015] like Figure 4 As shown, the working principle of the oligonucleotide drug delivery system based on fusion peptides is:

[0016] In drug delivery systems, the targeting peptide binds to specific receptors on the surface of cells in tissues or organs. The sorting signal peptide of the lysosomal targeting receptor on the cell surface activates the μ2 or σ2 subunit of AP2. The β2 hinge region of AP2 recruits clathrin; clathrin aggregates under the membrane, gradually forming a plasma membrane depression with a diameter of 50-100 nm, called a clathrin-coated pit.

[0017] A small GTP-binding protein, dynamin, assembles into a ring in the neck of a deep-seated coated pit. Dynammin hydrolyzes the GTP bound to it, causing neck constriction, and eventually detaches from the plasma membrane to form a clathrin-coated vesicle.

[0018] A few seconds later, the clathrin detaches from the membrane-coated vesicle and returns to the vicinity of the plasma membrane for reuse. The uncoated vesicle fuses with an early endosome, thereby taking up the lysosome with an oligonucleotide drug delivery system based on the fusion peptide.

[0019] Utilizing the low pH and specific enzymes of lysosomes, linkers and fusion peptides are degraded, releasing oligonucleotide drugs into the cytoplasm. After detaching from the fusion peptide-based oligonucleotide drug delivery system, the receptor returns to a region near the plasma membrane for reuse. The oligonucleotide drug binds to a specific target, achieving gene delivery.

[0020] Furthermore, the tyrosine-based YXXØ peptide sequence is selected from peptide sequences shown in SEQ ID No. 1~35.

[0021] Furthermore, the dileucine-based (D / E)XXXL (L / I) peptide sequence is selected from peptide sequences shown in SEQ ID No. 36~70.

[0022] Furthermore, the tyrosine-based NPXY peptide sequence is selected from peptide sequences shown in SEQ ID No. 71~89.

[0023] Table 1-1 Tyrosine-based YXXØ peptide sequence

[0024] Table 1-2 Tyrosine-based YXXØ peptide sequences

[0025] Table 1-3 Tyrosine-based YXXØ peptide sequences

[0026] Table 2-1 (D / E) XXXL (L / I) peptide sequences based on dileucine

[0027] Table 2-2 (D / E) XXXL (L / I) peptide sequences based on dileucine

[0028] Table 2-3 (D / E) XXXL (L / I) peptide sequences based on dileucine

[0029] Table 3-1 Tyrosine-based NPXY peptide sequence

[0030] Table 3-2 Tyrosine-based NPXY peptide sequences

[0031] Furthermore, the oligonucleotide drug is any one of antisense oligonucleotides (ASO), small interfering RNA (siRNA), small activating RNA (saRNA), microRNA (miRNA), or aptamer.

[0032] The present invention also provides a method for preparing an efficient oligonucleotide drug delivery system based on fusion peptides, the process comprising: linking a target peptide (TP) to a sorting signal peptide (SPSLTR) of a lysosomal target receptor on the cell surface to form a fusion peptide (FP), and then coupling an oligonucleotide to the fusion peptide to form an oligonucleotide drug delivery system based on fusion peptides (FP-Oligonucleotide).

[0033] Furthermore, oligonucleotides and fusion peptides are coupled via linkers to form an oligonucleotide drug delivery system based on fusion peptides.

[0034] The present invention also provides the application of an efficient oligonucleotide drug delivery system based on fusion peptides in drug preparation.

[0035] The advantages of this invention are:

[0036] 1. In the oligonucleotide drug delivery system based on fusion peptide (FP-Oligonucleotide delivery system), the targeting peptide first binds to a specific receptor on the cell surface to achieve targeting. Then, the sorting signal peptide of the lysosomal targeting receptor on the cell surface in the delivery system activates AP2, which enhances the clathrin-mediated lysosomal pathway to deliver oligonucleotides into the cell in a targeted, efficient and stable manner, thereby achieving the effect of gene therapy.

[0037] 2. Both the fusion peptide and the sorting signal peptide of the lysosomal target receptor on the cell surface are short peptides, and the sorting signal peptide of the lysosomal target receptor on the cell surface comes from the body itself, with low immunogenicity and high safety.

[0038] 3. The targeting peptide binds to specific receptors on the surface of cells in tissues or organs. The sorting signal peptide of the lysosomal targeting receptor on the cell surface activates AP2, which enhances the efficient delivery of oligonucleotides into the cell through the clathrin-mediated lysosomal pathway, resulting in high delivery efficiency.

[0039] 4. The drug system is suitable for targeting peptides that target different tissues and organs, and can achieve non-extrahepatic delivery, making it highly adaptable. Attached Figure Description

[0040] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0041] Figure 1This is a schematic diagram of the structure and recognition principle of AP2;

[0042] Figure 2 This is a schematic diagram of endocytosis targeting lysosomes;

[0043] Figure 3 This is a schematic diagram of an oligonucleotide drug delivery system based on fusion peptides;

[0044] Figure 4 This is a schematic diagram illustrating the working principle of an oligonucleotide drug delivery system based on fusion peptides.

[0045] Figure 5 This is a schematic diagram of the GLP1-CI-MPR-ASO delivery system;

[0046] Figure 6 It is a GLP1-CI-MPR-ASO delivery system architecture;

[0047] Figure 7 This is a schematic diagram of the GLP1-CI-MPR-ASO-Cy3 delivery system;

[0048] Figure 8 It is a GLP1-CI-MPR-ASO-Cy3 delivery system architecture;

[0049] Figure 9 This is an experimental diagram showing the internalization process of cells treated with the GLP1-CI-MPR-ASO-Cy3 delivery system. Detailed Implementation

[0050] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0051] 1. Construction of FP-Oligonucleotide delivery system

[0052] The targeting peptide GLP1 is linked to the sorting signal peptide CI-MPR of the lysosomal targeting receptor on the cell surface to form the fusion peptide FP. Then, the oligonucleotide ASO is coupled to the fusion peptide FP via the Ahx linker to form the fusion peptide-based oligonucleotide drug delivery system GLP1-CI-MPR-ASO, which is the FP-Oligonucleotide delivery system. Its structure is as follows: Figure 5-6 As shown.

[0053] The sequence of the targeting peptide TP (GLP1) is SEQ ID No. 90:

[0054] His-Aib-EGTFTSDVSSYLEGQAAK(Ahx)EFIAWLVRGRG;

[0055] The sorting signal peptide SPSLTR (CI-MPR) sequence is SEQ ID No. 91: SYKYSKVNKE;

[0056] The fusion peptide FP sequence is SEQ ID No. 92:

[0057] His-Aib-EGTFTSDVSSYLEGQAAK(Ahx)EFIAWLVRGRG-SYKYSKVNKE;

[0058] The ASO sequence is (5'-3') SEQ ID No. 93: CAAAGTCACATACGGCCAATCC.

[0059] 2. Validate the GLP1-CI-MPR-ASO delivery system at the cellular level.

[0060] To verify the delivery efficiency of the GLP1-CI-MPR-ASO delivery system, a fluorescent group Cy3 was attached to the 3' end of the ASO group to construct the GLP1-CI-MPR-ASO-Cy3 delivery system, the structure of which is shown below. Figure 7-8 As shown.

[0061] This invention uses HEK293 cells that stably express the human GLP1 receptor (GLP1 receptor expressing HEK293) to investigate the interaction between the GLP1-CI-MPR-ASO delivery system and the GLP1 receptor.

[0062] By treating cells with a fluorescently labeled GLP1-CI-MPR-ASO-Cy3 delivery system and monitoring the enhancement of cellular fluorescence signals, the cell internalization process can be visually visualized. Three experimental groups were set up as follows:

[0063] (1) Wild-type HEK293 was incubated with the GLP1-CI-MPR-ASO-Cy3 delivery system;

[0064] (2) GLP1 receptor expressing HEK293 was incubated with the GLP1-ASO-Cy3 delivery system; the main purpose was to verify that the sorting signal peptide in the fusion peptide is the one that promotes the cell internalization process.

[0065] (3) GLP1 receptor expressing HEK293 was incubated with the GLP1-CI-MPR-ASO-Cy3 delivery system.

[0066] The results are as follows Figure 9 As shown, fluorescently labeled GLP1-ASO peptide ( Figure 9B) with GLP1-CI-MPR-ASO ( Figure 9 C) Both GLP1R-HEK293 cells were rapidly internalized, and the delivery efficiency of GLP1-CI-MPR-ASO was higher than that of GLP1-ASO peptide, while no fluorescence was observed in wild-type HEK293 cells. Figure 9 A) indicates that the GLP1-CI-MPR-ASO-Cy3 delivery system was not internalized in this cell. Experimental results show that the fusion peptide, composed of a targeting peptide linked to a lysosomal targeting receptor sorting signal peptide on the cell surface, can deliver ASO into the cell, and the sorting signal peptide in the fusion peptide promotes the cell internalization process.

[0067] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A highly efficient oligonucleotide drug delivery system based on fusion peptides, characterized in that, The invention includes fusion peptides and oligonucleotide drugs, wherein the fusion peptide is formed by linking a targeting peptide to a sorting signal peptide of a lysosomal targeting receptor on the cell surface, and the fusion peptide is linked to the oligonucleotide drug via a linker.

2. The efficient oligonucleotide drug delivery system based on fusion peptide according to claim 1, characterized in that, The targeting peptide is a small molecule peptide that can specifically bind to specific receptors on the cell surface.

3. The efficient oligonucleotide drug delivery system based on fusion peptide according to claim 1, characterized in that, The sorting signal peptide of the lysosomal target receptor on the cell surface is a tyrosine-based YXXØ peptide sequence, a dileucine-based (D / E)XXXL(L / I) peptide sequence, or a tyrosine-based NPXY peptide sequence in the tail peptide signal of the lysosomal target receptor on the cytoplasmic surface of the cell surface that can activate the μ2 or σ2 subunit of AP2. Ø represents a large hydrophobic residue and X represents any amino acid.

4. The efficient oligonucleotide drug delivery system based on fusion peptide according to claim 3, characterized in that, The tyrosine-based YXXØ peptide sequence is selected from peptide sequences shown in SEQ ID No. 1~35.

5. The efficient oligonucleotide drug delivery system based on fusion peptide according to claim 1, characterized in that, The dileucine-based (D / E)XXXL(L / I) peptide sequence is selected from peptide sequences shown in SEQ ID No. 36~70.

6. The efficient oligonucleotide drug delivery system based on fusion peptide according to claim 1, characterized in that, The tyrosine-based NPXY peptide sequence is selected from peptide sequences shown in SEQ ID No. 71~89.

7. The efficient oligonucleotide drug delivery system based on fusion peptide according to claim 1, characterized in that, Oligonucleotide drugs are any of the following: antisense oligonucleotides, small interfering RNA, small activating RNA, microRNA, and aptamers.

8. A method for preparing a highly efficient oligonucleotide drug delivery system based on a fusion peptide as described in any one of claims 1-7, characterized in that, The process includes: linking the targeting peptide with the sorting signal peptide of the lysosomal targeting receptor on the cell surface to form a fusion peptide, and then coupling the oligonucleotide with the fusion peptide to form an oligonucleotide drug delivery system based on the fusion peptide.

9. The method for preparing a highly efficient oligonucleotide drug delivery system based on a fusion peptide according to claim 8, characterized in that, Oligonucleotides and fusion peptides are coupled via linkers to form an oligonucleotide drug delivery system based on fusion peptides.

10. The use of a highly efficient oligonucleotide drug delivery system based on fusion peptides as described in any one of claims 1-7 in the preparation of a drug.

Images