Preparation and application of a lysosome-targeted nucleic acid chimera

Abstract

The present invention provides a preparation and application of a lysosome-targeted nucleic acid chimera, and specifically relates to the application of constructing a lysosome-targeted chimera based on nucleic acid for specific degradation of intracellular and extracellular biomacromolecules and preparation of related drugs.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to the preparation and application of a lysosome-targeted nucleic acid chimera. Background Art

[0002] Most therapeutic approaches targeting single proteins rely on specific activity-modulating interactions with target proteins, such as enzyme inhibition or ligand blocking. However, most therapeutically relevant proteins are undruggable targets due to lack of enzymatic activity or lack of druggable sites on the surface. For difficult-to-target proteins, methods such as proteolysis-targeting chimaeras (PROTACs) have been developed as protein degradation platforms. However, these methods involve the degradation mechanism of intracellular proteins and are therefore limited to the types of proteins. For example, these proteins need to contain intracellular domains that can bind ligands and recruit essential cellular components. However, extracellular proteins and membrane-associated proteins that do not contain the above intracellular domains are key factors in some important diseases, such as cancer, aging-related diseases, and autoimmune diseases. Targeted degradation of these proteins is of great significance to human health and has important research potential.

[0003] Unlike the proteasome pathway, the lysosomal pathway for protein degradation is not limited to proteins with intracellular domains. The family of lysosome-targeting receptors (LTRs) on the cell surface can promote the transport of proteins to lysosomes. Proteins such as LTRs can be recognized by antibodies or target protein ligands to promote protein degradation. However, the method of constructing antibody-based targeted recognition of biological macromolecules such as lysosomes and proteins has certain technical difficulties and is time-consuming and labor-intensive. Therefore, the above method has the disadvantages of long production cycle, large workload, and high cost.

[0004] Therefore, there is an urgent need in the art to develop a method for degrading biological macromolecules such as proteins that is efficient, stable and suitable for production. Summary of the invention

[0005] The purpose of the present invention is to provide a method for degrading biological macromolecules such as proteins which is efficient, stable and suitable for production.

[0006] In the first aspect of the present invention, a nucleic acid chimera is provided, wherein the nucleic acid chimera has a structure shown in Formula I:

[0007] A1-L-A2 (I)

[0008] In the formula,

[0009] A1 is a nucleic acid aptamer element for a lysosome-targeting receptor (LTR), and the nucleic acid aptamer element specifically binds to the lysosome-targeting receptor;

[0010] L is none or linker sequence;

[0011] A2 is the aptamer element that targets the protein to be degraded;

[0012] Each "-" is independently a bond or a nucleotide linking sequence.

[0013] In another preferred example, the nucleic acid aptamer element comprises one or more LTR binding domains, and the LTR binding domains specifically bind to the lysosomal targeting receptor (LTR).

[0014] In another preferred embodiment, the LTR binding domain is composed of a nucleic acid sequence that specifically recognizes and binds to LTR.

[0015] In another preferred embodiment, the nucleic acid aptamer element further contains other domains besides the LTR binding domain.

[0016] In another preferred embodiment, the nucleic acid sequence of the LTR includes a single-stranded nucleic acid sequence.

[0017] In another preferred embodiment, the nucleic acid sequence includes DNA, RNA, LNA, PNA, HNA, CeNA, NAN, FANA, or a combination thereof.

[0018] In another preferred embodiment, the nucleic acid sequence includes natural and non-natural bases, such as bases selected from the following group: A, T, C, G, U, I, M-fC, I-fC, isoG, isoC, Ds, Pa, F, X, Y, Z, P.

[0019] In another preferred embodiment, the lysosomal targeting receptor (LTR) is located on the cell surface, preferably on the outer surface of the cell membrane.

[0020] In another preferred embodiment, the lysosomal targeting receptor (LTR) is located on the surface of the membrane structure within the cell, preferably on the surface of the endosome.

[0021] In another preferred embodiment, the lysosomal targeting receptor is selected from the following group: IGF2R, Rab, ESCRT, or a combination thereof.

[0022] In another preferred embodiment, the A1 is a nucleic acid aptamer element targeting IGF2R.

[0023] In another preferred embodiment, the cells are mammalian (such as human and non-human mammalian) cells.

[0024] In another preferred embodiment, the cells are selected from the following group: tumor cells, immune cells, neural cells, epithelial cells, and stem cells.

[0025] In another preferred embodiment, A1 and A2 are connected via a linker sequence, base complementary pairing or a phosphodiester bond.

[0026] In another preferred embodiment, the L is a nucleic acid linker formed by nucleic acid complementarity.

[0027] In another preferred embodiment, the length of the nucleic acid linker is 6-80 nt, preferably 10-50 nt, and more preferably 15-40 nt.

[0028] In another preferred embodiment, the nucleic acid linker includes a first linker single strand L1 and a second linker single strand L2, and a part or all of the first linker single strand L1 and the second linker single strand L2 form a nucleic acid complementary structure.

[0029] In another preferred embodiment, the nucleic acid adaptor element A1 is a first nucleic acid adaptor strand, and the first connecting strand L1 is connected to one end (such as 5' or 3') of the first nucleic acid adaptor strand.

[0030] In another preferred embodiment, the nucleic acid adaptor element A2 is a second nucleic acid adaptor strand, and the second connecting strand L2 is connected to one end (such as 5' or 3') of the second nucleic acid adaptor strand.

[0031] In another preferred embodiment, the first connecting single strand L1 is also connected to one end (such as 5' or 3') of the second nucleic acid adaptor single strand.

[0032] In another preferred embodiment, the second connecting single strand L2 is also connected to one end (such as 5' or 3') of the first nucleic acid adaptor single strand.

[0033] In another preferred embodiment, the proteins to be degraded include membrane proteins, secretory proteins, and intracellular proteins.

[0034] In another preferred embodiment, the protein to be degraded is selected from the following group: oncoprotein, neurodegenerative disease target protein, immune response-related protein, endocrine-related protein, reproduction-related protein or exogenous protein.

[0035] In another preferred embodiment, the protein to be degraded is selected from the following group: Met, PTK7, and EGFR.

[0036] In a second aspect of the present invention, a composition is provided, comprising:

[0037] (i) the nucleic acid chimera according to the first aspect of the present invention;

[0038] (ii) a pharmaceutically acceptable carrier.

[0039] In another preferred embodiment, the composition is a pharmaceutical composition.

[0040] In the third aspect of the present invention, there is provided a method for preparing the nucleic acid chimera as described in the first aspect of the present invention, comprising the steps of:

[0041] (S1) Provide A1 and A2;

[0042] (S2) connecting A1 and A2 to form a nucleic acid chimera with the structure of formula I.

[0043] In a fourth aspect of the present invention, there is provided a use of the nucleic acid chimera as described in the first aspect of the present invention for preparing a drug for degrading targeted proteins, nucleic acids or fats.

[0044] In another preferred embodiment, the drug is used to degrade extracellular proteins, cell membrane proteins or intracellular proteins.

[0045] In another preferred embodiment, the nucleic acid chimera is used in the preparation of anti-tumor therapeutic drugs.

[0046] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 A shows a schematic diagram of the nucleic acid chimera structure, wherein A1 and A2 represent two different nucleic acid aptamers (A1 is an IGF2R nucleic acid aptamer, and A2 is a c-Met nucleic acid aptamer), and Linker is a connector connecting A1 and A2; 1B shows three nucleic acid chimeras D1, D2 and D3 synthesized by different connection methods; 1C is a non-denaturing gel analysis of nucleic acids of D1, D2 and D3.

[0048] Figure 2 A shows a nucleic acid non-denaturing gel image of the stability analysis of D1, D2 and D3 nucleic acid chimeras; 2B shows a cell flow cytometry analysis of the cell affinity of D1, D2 and D3 nucleic acid chimeras.

[0049] Figure 3The western analysis and immunofluorescence analysis of the degradation ability of nucleic acid chimeras on cell membrane protein c-MET are shown; 3A shows the effect of D1, D2 and D3 nucleic acid chimeras on c-Met protein levels, CTR is the control group cells, GAPDH is the negative control; 3B is the concentration gradient effect of D3 on c-Met protein levels; 3C is the time gradient effect of D3 on c-Met protein levels; 3D shows the fixation and antibody staining of HeLa cells treated with D3 (scale bar is 10μm). In the figure, aptamer means aptamer.

[0050] Figure 4 A shows the cell flow cytometry analysis of the ability of D3 nucleic acid chimera to degrade cell membrane protein c-MET at different treatment times; 4B is the laser confocal analysis of the cells used in the cell flow cytometry analysis.

[0051] Figure 5 A shows the western analysis of the degradation ability of D3' nucleic acid chimera on cell membrane protein PTK7; 5B shows the cell flow cytometry analysis of D3' nucleic acid chimera on cell membrane protein PTK7.

[0052] Figure 6 The secondary structures of the nucleic acid aptamers and the corresponding nucleic acid chimeras in the present invention are shown, wherein aptamer represents the aptamer;

[0053] Among them, 6A is the secondary structure of the IGF2R aptamer;

[0054] 6B is the secondary structure of c-Met aptamer;

[0055] 6C is the secondary structure of the PTK7 aptamer;

[0056] 6D is the secondary structure of the D1 nucleic acid chimera, wherein the D1 nucleic acid chimera includes an IGF2R aptamer shown in 6A and a c-Met aptamer shown in 6B, wherein the linker is polyT (single-stranded T 10 );

[0057] 6E is the secondary structure of the D2 nucleic acid chimera, wherein the D2 nucleic acid chimera includes an IGF2R aptamer shown in 6A and a c-Met aptamer shown in 6B, wherein linker is a double-stranded nucleic acid of 17 bp;

[0058] 6F is the secondary structure of the D3 nucleic acid chimera, the D3 nucleic acid chimera includes an IGF2R aptamer shown in 6A and a c-Met aptamer shown in 6B, wherein the linker is a 23 bp double-stranded nucleic acid including a 10 bp double-stranded complementary structure from the c-Met aptamer itself;

[0059] 6G is the secondary structure of the D3' nucleic acid chimera, which includes an IGF2R aptamer shown in 6A and a PTK7 aptamer shown in 6C, wherein the linker is a 22 bp double-stranded nucleic acid including a 9 bp double-stranded complementary structure from the PTK7 aptamer itself. DETAILED DESCRIPTION

[0060] After extensive and in-depth research, the inventors have developed for the first time a unique nucleic acid chimera structure A1-L-A2 (Formula I), which simultaneously binds to a lysosomal targeting receptor and a protein to be degraded. Specifically, the nucleic acid aptamer of the lysosomal targeting receptor IGF2R and the nucleic acid aptamer of c-MET or PTK7 are connected by nucleic acid sequence, base complementary pairing or phosphodiester bond connection to form a nucleic acid chimera that simultaneously binds to IGF2R and the target protein. The present invention was completed on this basis.

[0061] The present invention constructs a nucleic acid chimera targeting lysosomes, which can be used for the specific degradation of biomacromolecules such as proteins (extracellular proteins, membrane proteins and intracellular proteins), nucleic acids, and the regulation of physiological processes and disease treatment related to degradation products. In particular, it relates to a nucleic acid structure targeting LTR (such as IGF2R) (including nucleic acid aptamers targeting IGF2R, etc.), and a chimera (A1-L-A2) is constructed based on the nucleic acid to simultaneously target LTR (A1) and specific biomacromolecules (A2), so that biomacromolecules such as proteins are transported to lysosomes through lysosomal targeting receptors such as IGF2R for targeted degradation, specifically regulating the physiological processes involved in biomacromolecules such as target proteins, and applying to the treatment of corresponding diseases.

[0062] the term

[0063] In the present invention, the "nucleic acid chimera of the present invention", "lysosome targeting chimera", and "lysosome targeting chimera of the present invention" can be used interchangeably and all refer to the nucleic acid chimera having the structure of Formula I in the present invention.

[0064] As used herein, the terms “containing”, “having” or “including” include “comprising”, “mainly consisting of…”, “substantially consisting of…”, and “consisting of…”; “mainly consisting of…”, “substantially consisting of…” and “consisting of…” are subordinate concepts of “containing”, “having” or “including”.

[0065] Lysosomal targeting receptors

[0066] Unlike the proteasomal pathway, the lysosomal pathway for protein degradation is not limited to proteins with intracellular domains. A family of lysosome-targeting receptors (LTRs) on the cell surface have been reported to promote the transport of proteins to lysosomes.

[0067] The IGF2R in the present invention is a lysosome targeting receptor, namely, the cation-independent mannose-6-phosphate receptor CI-M6PR.

[0068] Aptamer element

[0069] Aptamers are nucleic acid (NA) probes generated in an in vitro process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). By folding into different tertiary structures, aptamers can specifically recognize a group of targets, such as metal ions, organic molecules, and proteins, with dissociation constants up to picomolar values. Aptamers are based on low molecular weight, can quickly penetrate tissues and tumors, and can be quickly cleared from the blood. Since aptamers are composed of nucleotides and are non-immunogenic, they can be easily synthesized and modified for site-specific modification of the coupling of fluorescent dyes, radionuclides, drugs, and pharmacokinetic modifiers. Importantly, aptamers generated from SELEX can distinguish the molecular features of normal cells from cancer cells. Therefore, aptamers have received extensive attention as molecular probes in cancer diagnosis and treatment.

[0070] As used herein, the term "nucleic acid aptamer element" refers to an aptamer consisting of or substantially consisting of a nucleic acid sequence, and the binding of the nucleic acid aptamer element to a target (such as LTR or other target protein) is at least partially or entirely derived from the nucleic acid sequence. It should be understood that in the present invention, for nucleic acid aptamers, "substantially consisting of a nucleic acid sequence" means that at least 30%, preferably at least 50%, and more preferably at least 80% of the components of the aptamer element are nucleic acids.

[0071] It should be understood that in the present invention, at least one or both of A1 and A2 are nucleic acid aptamer elements.

[0072] Taking the nucleic acid aptamer element directed against or targeting LTR as an example, A1 is a nucleic acid aptamer element targeting IGF2R.

[0073] Linker

[0074] The linker sequence (L) can be a single-stranded nucleic acid, a double-stranded base pair, a peptide chain, or other chemical bonds, which connects the nucleic acid aptamer elements A1 and A2 together.

[0075] It should be understood that A1 or A2 in the present invention can be connected by other chemical or biological means, such as click chemistry, bioorthogonal reaction, etc.

[0076] In the present invention, there is no particular limitation on the linker sequence (L), as long as the linker sequence can link the nucleic acid aptamer elements A1 and A2 together and has no or substantially no influence on the respective functions of A1 and A2.

[0077] A preferred linker sequence is a nucleic acid linker sequence. A preferred linker sequence is a nucleic acid connecting A1 and A2, including a single-stranded nucleic acid (such as polyT), a double-stranded nucleic acid, or a combination thereof (such as a nucleic acid having a partial single-stranded region and a partial double-stranded region).

[0078] It should be understood that when the linker sequence is a nucleic acid linker sequence, its length is 1 nt-50 nt (or 1-50 bp), preferably 5-40 nt (or bp), and more preferably 8-30 nt (or bp).

[0079] Preferably, in the present invention, the nucleic acid linker sequence can be preliminarily connected to the nucleic acid aptamer element A1 and / or A2, and then annealed, extended and / or nucleic acid connected under appropriate conditions to form the nucleic acid chimera of the present invention.

[0080] In another preferred embodiment, any two of the nucleic acid aptamer elements A1 and A2 are connected in a head-to-head, head-to-tail, or tail-to-tail manner.

[0081] In another preferred embodiment, the “head” refers to the 5′ end of the nucleic acid aptamer element.

[0082] In another preferred embodiment, the “tail” refers to the 3′ end of the nucleic acid aptamer element.

[0083] Since nucleic acid aptamer elements pre-carrying a complete or partial nucleic acid linker sequence can be conveniently prepared by artificial synthesis methods, and the conditions for annealing, extension and / or nucleic acid ligation reactions are relatively mild and efficient, the nucleic acid chimera of the present invention can be prepared more efficiently and conveniently, while retaining the respective functions (such as respective binding properties) of the nucleic acid aptamer elements A1 and A2.

[0084] It should be understood that in the present invention, although the nucleic acid linker sequence can be completely composed of nucleotide sequences other than the structure of the nucleic acid aptamer element A1 and / or A2 itself (such as Figure 6The polyT used in the nucleic acid chimera D1 shown in D), but the nucleic acid linker sequence can also be a partial sequence derived from the structure of the nucleic acid aptamer element A1 and / or A2 itself, especially a partial sequence that has little effect on the respective functions of the nucleic acid aptamer elements A1 and A2, for example, from a certain end of the secondary structure of the nucleic acid aptamer element, for example Figure 6 The 8 bp complementary sequence at the end of the c-Met nucleic acid aptamer element in B and the two unpaired nucleotides at the 3' end (or a part thereof); or Figure 6 The 8 bp complementary sequence at the end of the PTK7 nucleic acid aptamer element in C and an unpaired nucleotide (or a part thereof) at the 5' end.

[0085] Therefore, in the present invention, in addition to the additionally added sequence, the linker sequence also includes a partial sequence of the nucleic acid aptamer A1 and / or a partial sequence of the nucleic acid aptamer A2.

[0086] Preferably, in the present invention, the aptamer element A1 has a sticky end ST1 (stick tail 1), and the aptamer element A2 has a sticky end ST2 (stick tail 2), wherein ST1 and ST2 can complement each other to form a pairing structure, thereby forming a linker structure connecting the aptamer elements A1 and A2 together.

[0087] In a specific embodiment, the linker can be a single-stranded DNA of 10 T bases (SEQ ID NO: 4), 17 base pairs (SEQ ID NO: 7), 23 base pairs (SEQ ID NO: 10, linker in D3) or 22 base pairs (linker in D3') nucleic acid linker.

[0088] Nucleic acid chimera

[0089] The present invention provides a nucleic acid chimera, wherein the nucleic acid chimera has a structure shown in Formula I:

[0090] A1-L-A2 (I)

[0091] In the formula,

[0092] A1 is a nucleic acid aptamer element for a lysosome-targeting receptor (LTR), and the nucleic acid aptamer element specifically binds to the lysosome-targeting receptor;

[0093] L is none or linker sequence;

[0094] A2 is the aptamer element that targets the protein to be degraded;

[0095] Each "-" is independently a bond or a nucleotide linking sequence.

[0096] The design and development of the lysosomal targeting chimera of the present invention can be as follows:

[0097] (1) connecting a nucleic acid aptamer element (A1) targeting a lysosomal targeting receptor and a nucleic acid aptamer element (A2) of a target biomacromolecule through a single-stranded nucleic acid linker sequence, base complementary pairing, a phosphodiester bond, etc., where L is a linker sequence connecting A1 and A2, thereby synthesizing a dual-targeting nucleic acid chimera (A1-L-A2);

[0098] (2) The nucleic acid aptamer (A1) targeting the lysosome targeting receptor is connected to the small molecule, peptide or other ligand (A2) of the target biological macromolecule by chemical, biological or other methods (L) to synthesize a dual-targeting nucleic acid chimera (A1-L-A2).

[0099] (3) The other nucleic acid structure (A1) targeting the lysosomal targeting receptor is connected to the ligand (A2) binding to the target biological macromolecule by chemical, biological or other methods (L) to synthesize a dual-targeting nucleic acid chimera (A1-L-A2).

[0100] (4) The peptide or other ligands (A1) that bind to the lysosomal targeting receptor are connected to the nucleic acid structure (A2) that binds to the target biological macromolecule by chemical, biological or other methods (L) to synthesize a dual-targeting nucleic acid chimera (A1-L-A2).

[0101] It should be understood that although the nucleic acid chimera of the present invention can be formed by assembling (including annealing, extension and / or connection) A1, L and A2, or by a connection reaction, a precursor comprising A1, L and A2 can also be directly synthesized. For example, when A1, L1, L2 and A2 are all nucleic acid sequences, a nucleic acid precursor sequence (single strand) comprising A1, L1, L2 and A2 can be directly synthesized, and then annealed and connected under appropriate conditions to form a nucleic acid chimera of Formula I.

[0102] The present invention also provides a composition, which contains an effective amount (such as 0.000001-90wt%; preferably 0.1-50wt%; more preferably, 5-40wt%) of the nucleic acid chimera of the present invention, and a pharmaceutically acceptable carrier.

[0103] Generally, the nucleic acid chimera of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably, about pH 6-8.

[0104] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0105] As used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

[0106] The pharmaceutical composition of the present invention contains a safe and effective amount of the nucleic acid chimera of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be prepared as a sustained-release preparation.

[0107] Preparation and application of nucleic acid chimeras

[0108] Methods such as enzyme inhibition or ligand blocking that rely on specific activity-regulated interactions with target proteins are not druggable for proteins that lack enzyme activity or lack druggable sites on their surfaces. The targeted degradation method (PROTACs) that relies on the proteasome pathway to ubiquitinate the target protein relies on the protein degradation mechanism, has restrictions on the types of proteins, and requires the search for a ligand that can bind to the target protein, which is somewhat difficult. The lysosomal degradation pathway is not limited to proteins with intracellular domains, and is applicable to a variety of biological macromolecules such as proteins and nucleic acids. However, the method of constructing chimeras based on antibodies that target and recognize lysosomes and biological macromolecules such as proteins has certain technical difficulties and is time-consuming and labor-intensive.

[0109] The purpose of the present invention is to provide a method for targeting lysosomes that is simple to synthesize and easy to artificially transform, which can transport biomacromolecules such as proteins and nucleic acids into lysosomes for specific degradation, break through the limitations of the above methods, and further enrich the types of targeted degradation products. In addition, the method of the present invention is a chimera developed based on the nucleic acid structure, which can be mass-produced and stored for a long time, can achieve precise site modification and labeling, has good stability, and has loose requirements for transportation conditions. At the same time, it has low immunogenicity and good tissue permeability. The invention further accelerates the drug-making process of targeting target biomacromolecules.

[0110] The invention provides a technology for simultaneously targeting lysosome targeting receptors and biological macromolecules such as proteins by nucleic acid chimeras, and transporting the biological macromolecules into lysosomes for specific degradation, as well as the application in physiological and pathological processes involving target biological macromolecules and the development of related drugs.

[0111] In addition, the present invention also provides an application of a lysosome-targeted nucleic acid chimera in physiological processes and diseases in which target biological macromolecules participate.

[0112] The present invention also provides an application of a lysosome-targeted nucleic acid chimera in a technology or medicine for regulating a corresponding physiological process.

[0113] The present invention also provides an application of a lysosome-targeted nucleic acid chimera in preparing drugs for treating corresponding diseases.

[0114] The present invention uses non-denaturing gel to identify whether two nucleic acid aptamers are connected and their stability in serum; uses binding test and cell flow cytometry to analyze the affinity of nucleic acid chimeras and cells with target proteins; and uses cell flow cytometry, laser confocal, immunofluorescence, western and other methods to detect the degradation ability of nucleic acid chimeras on target proteins.

[0115] In a preferred embodiment of the present invention, the preparation and characterization of nucleic acid chimeras are as follows:

[0116] 1) Several methods for synthesizing nucleic acid chimeras and non-denaturing gel analysis

[0117] Two nucleic acid aptamers (A1 and A2) are connected together through a linker to form a nucleic acid chimera (A1-L-A2) that simultaneously targets two proteins ( Figure 1 A). The IGF2R aptamer and c-Met aptamer were synthesized into nucleic acid chimeras D1, D2 and D3 ( Figure 1 B), and the synthesis results of nucleic acid chimeras were determined by non-denaturing gel ( Figure 1 C).

[0118] 2) Analysis of the stability of synthetic nucleic acid chimeras and their ability to bind to target cells

[0119] Simulating cell culture conditions, the synthesized nucleic acid chimeras were incubated with 10% FBS at 37°C for different time periods. It was found that D1 was completely degraded in about 3 hours, while D2 and D3 were relatively stable ( Figure 2 A). Through cell binding experiments and cell flow cytometry analysis, it was found that D3 had the best affinity for cells containing the target, while D2 had the lowest affinity for cells ( Figure 2 B).

[0120] 3) Analysis of the ability of nucleic acid chimeras to degrade cell membrane protein c-MET

[0121] Western experimental results showed that the aptamers, D1 and D2 alone had no protein degradation ability, while D3 could significantly degrade the c-Met protein on the cells ( Figure 3A), and has a good concentration gradient effect ( Figure 3 B) and time gradient effects ( Figure 3 C). Cell membrane immunofluorescence ( Figure 3 D) and the binding of c-Met aptamer to cell membrane ( Figure 4 A and B) further confirmed the degradation ability of D3.

[0122] 4) Analysis of the ability of nucleic acid chimeras to degrade cell membrane protein PTK7

[0123] The IGF2R aptamer and PTK7 aptamer were synthesized into a nucleic acid chimera D3' by T4 ligase. Western and flow cytometry results showed that D3' could effectively degrade PTK7 protein on cells ( Figure 5 A), and has a significant time gradient effect ( Figure 5 B).

[0124] The main advantages of the present invention include:

[0125] 1) The nucleic acid chimera used in the present invention has simple preparation conditions and steps, is easy to operate and artificially modified, and has the prospect of industrial synthesis;

[0126] 2) The nucleic acid chimera prepared in the present invention has low immunogenicity and can specifically target lysosomes;

[0127] 3) The nucleic acid chimera prepared in the present invention can specifically degrade the target protein very quickly (within 1 hour);

[0128] 4) The nucleic acid chimera prepared in the present invention accelerates the development of drugs targeting specific biomacromolecules and is used for physiological regulation and disease treatment.

[0129] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0130] sequence

[0131] The sequences of the nucleic acid chimeras and their constituent elements used in the examples are shown in Table 1.

[0132] Table 1. Nucleic acid chimera sequences of the present invention

[0133]

[0134] Note:

[0135] (a) "P-" represents the phosphorylated 5' structure, for example, PC represents the phosphorylation of the 5' C;

[0136] (b) The linker sequence in the stem-loop structure is marked by “underline”.

[0137] Example 1 Synthesis of Nucleic Acid Chimeras and Non-denaturing Gel Analysis

[0138] The nucleic acid chimera can simultaneously target the lysosomal targeting receptor IGF2R and the target cell membrane protein, and transport the target cell membrane protein into the lysosome for specific degradation through the lysosomal targeting receptor IGF2R. The structure of the nucleic acid chimera is A1-L-A2 ( Figure 1 A), where A1 and A2 are nucleic acid aptamers (one is an IGF2R nucleic acid aptamer and the other is a c-Met nucleic acid aptamer), and L is a linker connecting A1 and A2. We synthesized three nucleic acid chimeras ( Figure 1 B), where D1 connects A1 and A2 into one nucleic acid chain through 10 T bases; D2 connects A1 and A2 together through base complementary pairing; D3 phosphorylates the 5' ends of the nucleic acid chains of A1 and A2, and connects the 5' and 3' ends of A1 and A2 together through T4 ligase. Through non-denaturing PAGE gel analysis, the target molecular weight bands were observed in the lanes marked with D1, D2 and D3, indicating that through the different connection modes of D1, D2 and D3, A1 and A2 are connected together to form a nucleic acid chimera ( Figure 1 C).

[0139] Example 2 Analysis of the stability of synthetic nucleic acid chimeras and their ability to bind to target cells

[0140] In order to further detect the stability of the synthesized D1, D2 and D3, the synthesized D1, D2 and D3 were incubated with 10% FBS at 37°C for different time periods. It was found that D1 was completely degraded in about 3 h, while D2 and D3 were relatively stable ( Figure 2 A). HeLa cells express IGF2R and c-Met membrane proteins simultaneously. Cy5-labeled D1, D2, and D3 as well as individual IGF2R and c-Met aptamers were incubated with HeLa cells for binding experiments.

[0141] Flow cytometry analysis revealed that D3 had a high affinity for HeLa cells (K D=20.12 nM) was significantly higher than that of the single aptamer and D1 and D2. The affinity of D1 for HeLa cells was not significantly different from that of the single aptamer for HeLa cells, while D2 had the lowest affinity for HeLa cells ( Figure 2 B) These results indicate that D3 has both high cell affinity and better stability compared to D1 and D2.

[0142] Example 3 Analysis of the ability of nucleic acid chimeras to degrade cell membrane protein c-MET

[0143] Lysosomal targeting receptors (such as IGF2R receptors) on the cell surface can promote the transport of proteins to lysosomes and their degradation in lysosomes. After incubating HeLa cells with the synthesized D1, D2, D3, and the IGF2R aptamers and c-Met aptamers for 24 hours, the expression of proteins was analyzed by western blot. Figure 1 A, B, and C) and immunofluorescence ( Figure 3 D) Detect the changes in the level of c-Met protein. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the internal reference protein.

[0144] like Figure 3 As shown in A, compared with the untreated control group cells (CTR), D3 can significantly reduce the c-Met protein level in the cells, while D1, D2 and the single aptamer have no significant effect on the c-Met protein level. The effect of D3 on the c-Met protein level has a significant concentration gradient effect ( Figure 3 B) and time gradient effects ( Figure 3 C), and D3 treatment of cells can significantly reduce c-Met levels within 1 hour. At the same time, after fixing and antibody staining of HeLa cells treated with nucleic acid chimeras, it was found that after D3 treatment of cells, the level of c-Met protein on the cell membrane was significantly reduced ( Figure 3 D).

[0145] To further demonstrate the effect of D3, HeLa cells were treated with non-fluorescently labeled D3 for different time periods, and then the cy5-labeled c-Met nucleic acid aptamer was used to perform binding experiments with the treated HeLa cells. Figure 4 A), it was found that the fluorescence intensity of c-Met nucleic acid aptamers bound to the cell surface decreased significantly after D3 treatment for 1 hour, indicating that D3 significantly reduced the level of c-Met protein on the cell membrane. The cells used for cell flow cytometry analysis were subjected to laser confocal microscopy analysis ( Figure 4B). The figure shows that the fluorescence intensity on the cell membrane labeled with red fluorescence was significantly reduced after treatment with D3, further confirming the effect of D3 on the level of c-Met protein on the cell surface.

[0146] The above results indicate that D3 has the ability to specifically reduce the intracellular c-Met protein level and can exert its effect in a very short period of time (eg, 1 h), while D1 and D2 have no effect.

[0147] Example 4 Analysis of the ability of nucleic acid chimeras to degrade cell membrane protein PTK7

[0148] In order to further analyze the effect of A1-L-A2 chimera on the target protein level and degradation, we synthesized the nucleic acid chimera D3' by T4 ligase method with IGF2R nucleic acid aptamer and PTK7 nucleic acid aptamer. CEM cells were treated with IGF2R nucleic acid aptamer, PTK7 nucleic acid aptamer and D3' for 24 hours respectively, and western blot detection showed that ( Figure 5 A), D3' can significantly reduce the PTK7 protein level in CEM cells compared to untreated cells, while the aptamer alone has no effect on the PTK7 protein level. At the same time, after incubating D3' with CEM cells for different time periods, the cy5-labeled PTK7 aptamer was used to perform binding experiments with CEM cells.

[0149] Flow cytometry analysis revealed that Figure 5 B), D3' treatment of CEM cells for 1 h can significantly reduce the number of PTK7 nucleic acid aptamers bound to the surface of CEM cells, indicating that D3' significantly reduced the level of PTK7 protein on the surface of CEM and had a time gradient effect.

[0150] discuss

[0151] In the present invention, the inventors take membrane proteins as an example to illustrate that nucleic acid chimeras (A1-L-A2) developed based on nucleic acids that simultaneously target lysosomal targeting receptor IGF2R and membrane protein c-Met or PTK7 can specifically transport c-Met or PTK7 into lysosomes for degradation. The nucleic acid chimeras can simultaneously bind to cell surface IGF2R and cell membrane protein c-Met or PTK7 to induce lysosomal degradation of the target, thereby providing a means of accelerating protein degradation by a binding agent that acts in the extracellular space.

[0152] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Han Da <120> Preparation and application of a lysosome-targeted nucleic acid chimera <130> P2020-2092 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <400> 1 gggcgcgtag atgacgagca gtcctaacat cgtttaggac 40 <210> 2 <211> 51 <212> DNA <213> Artificial Sequence <400> 2 atcaggctgg atggtagctc ggtcggggtg ggtgggttgg caagtctgat a 51 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <400> 3 atctaactgc tgcgccgccg ggaaaatact gtacggttag a 41 <210> 4 <211> 10 <212> DNA <213> Artificial Sequence <400> 4 tttttttttt 10 <210> 5 <211> 102 <212> DNA <213> Artificial Sequence <400> 5 atcaggctgg atggtagctc ggtcggggtg ggtgggttgg caagtctgat aatttttttt 60 ttgggcgcgt agatgacgag cagtcctaac atcgtttagg ac 102 <210> 6 <211> 57 <212> DNA <213> Artificial Sequence <400> 6 gggcgcgtag atgacgagca gtcctaacat cgtttaggac cgtaaatcag tcatact 57 <210> 7 <211> 17 <212> DNA <213> Artificial Sequence <400> 7 cgtaaatcag tcatact 17 <210> 8 <211> 69 <212> DNA <213> Artificial Sequence <400> 8 atcaggctgg atggtagctc ggtcggggtg ggtgggttgg caagtctgat aaagtatgac 60 tgatttacg 69 <210> 9 <211> 53 <212> DNA <213> Artificial Sequence <400> 9 tgactgattt acggggcgcg tagatgacga gcagtcctaa catcgtttag gac 53 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <400> 10 agtctgataa tgactgattt acg 23 <210> 11 <211> 67 <212> DNA <213> Artificial Sequence <400> 11 cgtaaatcag tcattatcag gctggatggt agctcggtcg gggtgggtgg gttggcaagt 60 ctgataa 67 <210> 12 <211> twenty two <212> DNA <213> Artificial Sequence <400> 12 cggttagatt gactgattta cg 22 <210> 13 <211> 55 <212> DNA <213> Artificial Sequence <400> 13 cgtaaatcag tcaatctaac tgctgcgccg ccgggaaaat actgtacggt tagat 55

Claims

1. A nucleic acid chimera, It is characterized in that The nucleic acid chimera has the structure shown in Formula I: A1-L-A2(I) In the formula, A1 is a nucleic acid aptamer element for a lysosome-targeting receptor (LTR), and the nucleic acid aptamer element specifically binds to the lysosome-targeting receptor; L is the linker sequence; A2 is the aptamer element that targets the protein to be degraded; Each "-" is independently a bond; The nucleic acid aptamer element comprises one or more LTR binding domains, wherein the LTR binding domains specifically bind to the lysosomal targeting receptor (LTR); The A1 is a nucleic acid aptamer element targeting IGF2R; The L is a nucleic acid linker formed by nucleic acid complementation, the nucleic acid linker is a double-stranded base-paired double strand, and the length of the nucleic acid linker is 10-50 nt; The nucleic acid linker comprises a first linking strand L1 and a second linking strand L2, and the entire regions of the first linking strand L1 and the second linking strand L2 form a nucleic acid complementary structure; the nucleic acid aptamer element A1 is a first nucleic acid aptamer strand, and the first linking strand L1 is connected to one end of the first nucleic acid aptamer strand; the nucleic acid aptamer element A2 is a second nucleic acid aptamer strand, and the second linking strand L2 is connected to one end of the second nucleic acid aptamer strand; and L comprises a partial sequence of the nucleic acid aptamer A1 structure and a partial sequence of the nucleic acid aptamer A2 structure. The first connecting single strand L1 is also connected to one end of the second nucleic acid adaptor single strand, and / or the second connecting single strand L2 is also connected to one end of the first nucleic acid adaptor single strand; The protein to be degraded is selected from the following group: proto-oncoprotein, neurodegenerative disease target protein, immune response-related protein, endocrine-related protein, reproduction-related protein or exogenous protein.

2. The nucleic acid chimera according to claim 1, It is characterized in that The lysosomal targeting receptor (LTR) is located on the surface of the membrane structure within the cell.

3. The nucleic acid chimera according to claim 1, It is characterized in that The LTR binding domain is composed of a nucleic acid sequence that specifically recognizes and binds to LTR.

4. The nucleic acid chimera according to claim 1, It is characterized in that The nucleic acid aptamer element further contains other domains besides the LTR binding domain.

5. The nucleic acid chimera according to claim 1, It is characterized in that The nucleic acid sequence of the LTR includes a single-stranded nucleic acid sequence.

6. The nucleic acid chimera of claim 5, wherein the nucleic acid sequence comprises DNA, RNA, LNA, PNA, HNA, CeNA, NAN, FANA, or a combination thereof.

7. The nucleic acid chimera according to claim 5, It is characterized in that The nucleic acid sequence includes natural and non-natural bases, including bases selected from the following group: A, T, C, G, U, I, M-fC, I-fC, isoG, isoC, Ds, Pa, F, X, Y, Z, P.

8. The nucleic acid chimera according to claim 1, It is characterized in that The lysosomal targeting receptor (LTR) is located on the cell surface.

9. The nucleic acid chimera according to claim 8, It is characterized in that The cells are selected from the group consisting of tumor cells, immune cells, neural cells, epithelial cells, and stem cells.

10. The nucleic acid chimera according to claim 1, It is characterized in that The A1 is a nucleic acid aptamer element targeting IGF2R.

11. The nucleic acid chimera according to claim 1, It is characterized in that The L includes a partial sequence of its own structure that has no influence on the respective functions of the nucleic acid aptamer elements A1 and / or A2.

12. The nucleic acid chimera according to claim 11, It is characterized in that The length of the nucleic acid linker is 15-40 nt.

13. The nucleic acid chimera according to claim 1, It is characterized in that The linker sequence is 23 base pairs, and its sequence is shown in SEQ ID NO:10; or 22 base pairs, and its sequence is shown in SEQ ID NO:

12.

14. A composition, It is characterized in that The composition comprises: (i) the nucleic acid chimera according to claim 1; (ii) a pharmaceutically acceptable carrier.

15. A method for preparing the nucleic acid chimera according to claim 1, It is characterized in that Includes steps: (S1) Provide A1 and A2; (S2) connecting A1 and A2 to form a nucleic acid chimera with the structure of formula I.

16. A use of the nucleic acid chimera according to claim 1, It is characterized in that Used to degrade targeted proteins in vitro, and the use is for non-therapeutic purposes.

17. The use according to claim 16, It is characterized in that The targeting protein is selected from the following group: proto-oncoprotein, neurodegenerative disease target protein, immune response-related protein, endocrine-related protein, reproduction-related protein or exogenous protein.

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