WO2022095853A1

WO2022095853A1 - Preparation for and application of lysosome-targeting nucleic acid chimera

Info

Publication number: WO2022095853A1
Application number: PCT/CN2021/128209
Authority: WO
Inventors: 韩达; 苗彦彦; 张朝; 高倩倩
Original assignee: 臻智达生物技术（上海）有限公司
Priority date: 2020-11-03
Filing date: 2021-11-02
Publication date: 2022-05-12
Also published as: CN114438088A

Abstract

Provided is preparation for a lysosome-targeting nucleic acid chimera, and an application of constructing a lysosome-targeting chimera on the basis of the nucleic acid in specific degradation of intracellular and extracellular biological macromolecules and preparation of related drugs.

Description

Preparation and application of a lysosome-targeted nucleic acid chimera

technical field

The present invention relates to the technical field of molecular biology, and in particular, the present invention relates to the preparation and application of a lysosome-targeted nucleic acid chimera.

Background technique

Therapies targeting individual proteins mostly rely on specific activity-modulating interactions with the target protein, such as enzyme inhibition or ligand blockade. However, most therapeutically relevant proteins become undruggable targets due to lack of enzymatic activity or lack of druggable sites on the surface. For proteins that are difficult to target, methods for proteolysis platforms such as Proteolysis-targeting Chimaeras (PROTACs) have been developed. However, these methods involve the degradation mechanism of intracellular proteins and thus have limitations on protein species. 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-mentioned 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.

Unlike the proteasome pathway, the lysosomal pathway for degrading proteins is not limited to proteins with intracellular domains. The lysosome-targeting receptors (LTRs) family on the cell surface can facilitate protein transport to lysosomes. Proteins such as LTR can be recognized by antibodies or target protein ligands to promote protein degradation. However, methods for targeting and recognizing biological macromolecules such as lysosomes and proteins based on antibodies are technically difficult and time-consuming. Therefore, the above method has the disadvantages of long production cycle, large workload and high cost.

Therefore, there is an urgent need in the art to develop a method for degrading biological macromolecules such as proteins, which is efficient, stable and suitable for production.

SUMMARY OF THE INVENTION

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.

In a first aspect of the present invention, a nucleic acid chimera is provided, and 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 no or linker sequence (Linker);

A2 is an aptamer element targeting the protein to be degraded;

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

In another preferred embodiment, the nucleic acid aptamer element includes one or more LTR binding domains, and the LTR binding domains specifically bind to the lysosomal targeting receptor (LTR).

In another preferred embodiment, the LTR-binding domain consists of a nucleic acid sequence that specifically recognizes and binds to LTR.

In another preferred embodiment, the nucleic acid aptamer element also contains other domains in addition to the LTR binding domain.

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

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

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.

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

In another preferred embodiment, the lysosomal targeting receptor (LTR) is located on the surface of the intracellular membrane structure, preferably on the surface of the endosome.

In another preferred embodiment, the lysosomal targeting receptor is selected from the group consisting of IGF2R, Rab, ESCRT, or a combination thereof.

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

In another preferred embodiment, the cells are mammalian (eg, human and non-human mammalian) cells.

In another preferred embodiment, the cells are selected from the group consisting of tumor cells, immune cells, nerve cells, epithelial cells, and stem cells.

In another preferred embodiment, the A1 and A2 are connected through a linker sequence, complementary base pairing or phosphodiester bond.

In another preferred embodiment, the L is a nucleic acid linker formed by nucleic acid complementation.

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

In another preferred example, the nucleic acid linker comprises a first linked single strand L1 and a second linked single strand L2, and part or all of the regions of the first linked single strand L1 and the second linked single strand L2 form Nucleic acid complementary structures.

In another preferred example, the nucleic acid adapter element A1 is a first nucleic acid adapter single strand, and the first connecting single strand L1 is connected to one end of the first nucleic acid adapter single strand (eg, 5 ' or 3') connected.

In another preferred example, the nucleic acid adapter element A2 is a second nucleic acid adapter single strand, and the second connecting single strand L2 is connected to one end of the second nucleic acid adapter single strand (eg, 5 ' or 3') connected.

In another preferred example, the first linking single strand L1 is also linked to one end (eg, 5' or 3') of the second nucleic acid-adapting single strand.

In another preferred example, the second linking single strand L2 is also linked to one end (eg, 5' or 3') of the first nucleic acid-adapting single strand.

In another preferred embodiment, the proteins to be degraded include membrane proteins, secreted proteins, and intracellular proteins.

In another preferred embodiment, the protein to be degraded is selected from the group consisting of proto-oncoprotein, neurodegenerative disease target protein, immune response-related protein, endocrine-related protein, reproductive-related protein or exogenous protein.

In another preferred embodiment, the protein to be degraded is selected from the group consisting of Met, PTK7, and EGFR.

In a second aspect of the present invention, there is provided a composition comprising:

(i) the nucleic acid chimera of the first aspect of the invention;

(ii) A pharmaceutically acceptable carrier.

In another preferred embodiment, the composition is a pharmaceutical composition.

In another preferred example, the composition further includes other drugs for degrading targeted proteins, nucleic acids or fats.

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:

(S1) Provide A1, A2;

(S2) A1 and A2 are connected to form a nucleic acid chimera of the structure of formula I.

In the fourth aspect of the present invention, there is provided a use of the nucleic acid chimera according to the first aspect of the present invention for preparing a drug for degrading targeted proteins, nucleic acids or fats.

In another preferred example, the drug is used to degrade extracellular proteins, cell membrane proteins or intracellular proteins.

In another preferred embodiment, the nucleic acid chimera is used for the preparation of anti-tumor therapeutic drugs.

In another preferred embodiment, the tumors include: cervical cancer, lymphoma, lung cancer, breast cancer, liver cancer, intestinal cancer, and pancreatic cancer.

In a fifth aspect of the present invention, there is provided a method for treating tumors, comprising administering the nucleic acid chimera as described in the first aspect of the present invention or the composition as described in the second aspect of the present invention to a subject in need thereof .

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 in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

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

Figure 2A shows a nucleic acid native gel plot for stability analysis of D1, D2 and D3 nucleic acid chimeras; 2B shows a cytometric flow cytometry plot for cell affinity of D1, D2 and D3 nucleic acid chimeras.

Figure 3 shows the western analysis and immunofluorescence analysis of the nucleic acid chimera's ability to degrade the cell membrane protein c-MET; among them, 3A shows the effect of D1, D2 and D3 nucleic acid chimeras on the level of c-Met protein, and CTR is the control cell , GAPDH is the negative control; 3B is the concentration gradient effect of D3 on c-Met protein level; 3C is the time gradient effect of D3 on c-Met protein level; 3D shows the fixation and antibody staining of D3-treated HeLa cells (scale bar 10 μm). In the figure, aptamer represents an aptamer.

Figure 4A shows the cytometry analysis of the degradation ability of the D3 nucleic acid chimera on the cell membrane protein c-MET under different treatment times; 4B is the laser confocal analysis of the cells used for the cytometry analysis.

Figure 5A shows the western analysis of the degradation ability of the D3' nucleic acid chimera to the cell membrane protein PTK7; 5B is the cell flow analysis of the D3' nucleic acid chimera to the cell membrane protein PTK7.

Figure 6 shows the secondary structures of nucleic acid aptamers and corresponding nucleic acid chimeras in the present invention, in which aptamer represents aptamers;

Wherein, 6A is the secondary structure of the IGF2R aptamer;

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

6C is the secondary structure of PTK7 aptamer;

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 ). );

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 the linker is a 17bp double-stranded nucleic acid;

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 23bp double-stranded nucleic acid, including from c -Met aptamer's own 10bp double-stranded complementary structure;

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

Detailed ways

After extensive and in-depth research, the inventors developed a unique nucleic acid chimera structure A1-L-A2 (Formula I) for the first time, which combines lysosomal targeting receptors and proteins to be degraded at the same time. Specifically, the nucleic acid aptamer of the lysosome 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, and the two nucleic acid aptamers are connected. Ligands to form nucleic acid chimeras that simultaneously bind IGF2R and the target protein. The present invention has been completed on this basis.

The invention constructs a nucleic acid chimera targeting lysosomes, which can be used for the specific degradation of biological macromolecules such as proteins (extracellular proteins, membrane proteins and intracellular proteins) and nucleic acids, as well as the regulation of physiological processes related to the degradation products and the treatment of diseases. In particular, it relates to nucleic acid structures targeting LTR (eg, IGF2R) (including nucleic acid aptamers targeting IGF2R, etc.), based on which chimeras (A1-L-A2) are constructed to target both LTR (A1) and Specific biological macromolecules (A2), so that biological macromolecules such as proteins are transported to lysosomes for targeted degradation through lysosomal targeting receptors such as IGF2R, and specifically regulate the physiological processes involved in biological macromolecules such as target proteins. applied to the treatment of corresponding diseases.

the term

In the present invention, "nucleic acid chimera of the present invention", "lysosome-targeted chimera" and "lysosome-targeted chimera of the present invention" can be used interchangeably, and all refer to the structure of formula I in the present invention. Nucleic acid chimeras.

As used herein, the words "comprising", "having" or "including" include "comprising", "consisting essentially of", "consisting essentially of", and "consisting of"; " Consists essentially of", "consisting essentially of" and "consisting of" are subordinate concepts of "contains", "has" or "includes".

Lysosomal targeting receptor

Unlike the proteasome pathway, the lysosomal pathway for protein degradation is not limited to proteins with intracellular domains. It has been reported that a family of lysosome-targeting receptors (LTRs) on the cell surface can promote the transport of proteins to lysosomes.

The IGF2R in the present invention is a lysosomal targeting receptor, namely the cation-independent manno-6-phosphate receptor CI-M6PR.

aptamer element

An aptamer is a nucleic acid (NA) probe produced 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 set of targets, such as metal ions, organic molecules, and proteins, with dissociation constants up to picomolar values. Aptamers are based on low molecular weights, can rapidly penetrate tissues and tumors, and can quickly clear the blood. Since aptamers are composed of nucleotides and are non-immunogenic, they can be easily synthesized and modified to facilitate the conjugation of fluorescent dyes, radionuclides, drugs, and pharmacokinetic modifiers point-specific modification. Importantly, the aptamers generated from SELEX can distinguish molecular signatures from normal and cancer cells. Therefore, aptamers have received extensive attention as molecular probes in cancer diagnosis and therapy.

As used herein, the term "nucleic acid aptamer element" refers to an aptamer that consists or consists essentially of a nucleic acid sequence that is associated with a target (eg, LTR or other target protein) Binding is derived at least in part or in whole from the nucleic acid sequence. It should be understood that, in the present invention, for nucleic acid adaptor elements, "substantially consisting of nucleic acid sequences" means that at least 30%, preferably at least 50%, more preferably at least 80% of the adaptor elements are nucleic acid .

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

Taking the nucleic acid aptamer element for or targeting LTR as an example, A1 is the nucleic acid aptamer element for targeting IGF2R.

Linker sequence (Linker)

The linker sequence Linker (L) can be nucleic acid single-stranded, base-complementary paired double-stranded, peptide chain and other chemical bonds, etc., connecting the nucleic acid aptamer elements A1 and A2 together.

It should be understood that A1 or A2 in the present invention can be linked by other chemical or biological means, such as click chemistry, bioorthogonal reaction and the like.

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

A preferred linker sequence is a nucleic acid-based linker sequence. A preferred linker sequence is a nucleic acid linking A1 and A2, including single-stranded nucleic acid (eg polyT), double-stranded nucleic acid, or a combination thereof (eg, nucleic acid with a partial single-stranded region and a partial double-stranded region).

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

Preferably, in the present invention, the nucleic acid-based linker sequence can be connected to the nucleic acid aptamer element A1 and/or A2 in advance, and then annealing, extension and/or nucleic acid ligation are performed under suitable conditions, thereby forming the nucleic acid of the present invention. Chimera.

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.

In another preferred embodiment, the "head" refers to the 5' end of the nucleic acid aptamer element.

In another preferred embodiment, the "tail" refers to the 3' end of the end of the nucleic acid aptamer element.

Since nucleic acid aptamer elements with complete or partial nucleic acid-based linker sequences in advance can be easily prepared by synthetic methods, and the conditions for annealing, extension and/or nucleic acid ligation reactions are milder and more efficient, it is more efficient and convenient. The nucleic acid chimeras of the present invention are prepared and retain the respective functions (eg, respective binding properties) of the nucleic acid aptamer elements A1 and A2.

It should be understood that in the present invention, although the nucleic acid-based linker sequence can completely adopt the nucleotide sequence outside the structure of the nucleic acid aptamer element A1 and/or A2 (as shown in the nucleic acid chimera D1 shown in FIG. 6D ) polyT), but the nucleic acid-based linker sequence can also adopt a partial sequence derived from the structure of the nucleic acid aptamer element A1 and/or A2 itself, especially the partial sequence that basically has no 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, such as the 8 bp complementary sequence at the end of the c-Met nucleic acid aptamer element in Figure 6B and the two unpaired nucleotides at the 3' end (or wherein part of ); or the 8 bp complementary sequence at the end of the PTK7 aptamer element in Figure 6C and an unpaired nucleotide (or a part thereof) at the 5' end.

Therefore, in the present invention, in addition to the additional sequences, the linker sequence also includes the partial sequence of the nucleic acid aptamer A1 and/or the partial sequence of the nucleic acid aptamer A2.

Preferably, in the present invention, the nucleic acid aptamer element A1 has a sticky end ST1 (stick tail 1), and the nucleic acid aptamer element A2 has a sticky end ST2 (stick tail 2), wherein ST1 and ST2 can complement each other to form a paired structure , and then form a linker structure connecting the nucleic acid aptamer elements A1 and A2 together.

In a specific embodiment, the linker may 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: 7) 10, linker in D3) or a nucleic acid linker of 22 base pairs (linker in D3').

nucleic acid chimera

The present invention provides a nucleic acid chimera, the nucleic acid chimera has the structure shown in formula I:

A1-L-A2 (I)

In the formula,

L is no or linker sequence (Linker);

A2 is an aptamer element targeting the protein to be degraded;

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

The design and development of the lysosome targeting chimera described in the present invention can be as follows:

(1) The nucleic acid aptamer element (A1) targeting the lysosome-targeting receptor and the nucleic acid aptamer element (A2) of the target biological macromolecule are connected by the linker sequence of single-stranded nucleic acid, base complementary pairing, phosphoric acid Connect by methods such as diester bonds, L is the linker sequence connecting A1 and A2, and then synthesize a double-targeted nucleic acid chimera (A1-L-A2);

(2) The nucleic acid aptamer (A1) targeting the lysosome-targeting receptor is linked with the small molecule, peptide and other ligands (A2) of the target biological macromolecule by chemical, biological methods, etc. (L) to synthesize Dual-targeted nucleic acid chimeras (A1-L-A2).

(3) Connect other nucleic acid structures (A1) targeting lysosome-targeting receptors with ligands (A2) that bind to target biological macromolecules by chemical, biological and other methods (L) to synthesize dual-targeting nucleic acid chimeric Combination (A1-L-A2).

(4) Connect ligands such as peptides (A1) that bind to lysosomal targeting receptors and nucleic acid structures (A2) that bind to target biomacromolecules by chemical, biological and other methods (L) to synthesize dual-targeted nucleic acids Chimera (A1-L-A2).

It should be understood that although the nucleic acid chimeras of the present invention can be formed by assembly (including annealing, extension and/or ligation) of A1, L and A2, or by a ligation reaction, it is also possible to directly synthesize A1, L, A2 For example, when A1, L1, L2 and A2 are all nucleic acid sequences, the nucleic acid precursor sequences (single-stranded) comprising A1, L1, L2 and A2 can be directly synthesized, and then annealed and connected under suitable conditions , thereby forming a nucleic acid chimera of the structure of formula I.

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

Generally, the nucleic acid chimeras of the present invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably, about a pH of about 6-8.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, a substance 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.

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, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. Usually the pharmaceutical preparation should be matched with the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by using normal saline or an aqueous solution containing glucose and other adjuvants by conventional methods. The pharmaceutical compositions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be made into a sustained-release preparation.

Preparation method and application of nucleic acid chimera

Methods that rely on specific activity-modulating interactions with target proteins, such as enzyme inhibition or ligand blockade, are not druggable for proteins that lack enzymatic activity or lack druggable sites on the surface. Targeted degradation of target proteins after ubiquitination (PROTACs) relying on the proteasome pathway relies on the protein degradation mechanism, which limits the types of proteins and needs to find the binding ligands of the target protein, which is difficult. The lysosomal degradation pathway is not limited to proteins with intracellular domains, and is applicable to various biological macromolecules such as proteins and nucleic acids. However, the method of constructing chimeras targeting and recognizing biological macromolecules such as lysosomes and proteins based on antibodies is technically difficult and time-consuming.

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 biological macromolecules such as proteins and nucleic acids for specific degradation in lysosomes, and break through the limitations of the above methods. Further enrich the types of targeted degradants. And the method of the present invention is a chimera developed based on nucleic acid structure, which can be produced on a large scale and stored for a long time, can realize precise site modification and labeling, has good stability, has loose requirements on transportation conditions, and is immunogenic. Small, good tissue permeability, etc. The invention further accelerates the drug-forming process of targeting target biological macromolecules.

The nucleic acid chimera provided by the present invention simultaneously targets biological macromolecules such as receptors and proteins in lysosomes, and transports biological macromolecules into lysosomes for specific degradation, as well as the biological macromolecules involved in the biological macromolecules involved in the target biological macromolecules. Applications in pathological processes and related drug development.

In addition, the present invention also provides the application of a nucleic acid chimera targeting lysosomes in physiological processes and diseases involving target biological macromolecules.

The present invention also provides the application of the nucleic acid chimera targeting lysosome in the technology or medicine for regulating the corresponding physiological process.

The present invention also provides an application of the nucleic acid chimera targeting lysosomes in the preparation of medicines for the treatment of corresponding diseases.

The present invention identifies whether the two nucleic acid aptamers are connected and the stability in serum by non-denaturing gel; analyzes the affinity of the nucleic acid chimera and the cells with the target protein through the binding test and cell flow; Confocal laser, immunofluorescence, western and other methods were used to detect the degradation ability of nucleic acid chimeras to target proteins.

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

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

Two nucleic acid aptamers (A1 and A2) were linked together by a linker to form a nucleic acid chimera (A1-L-A2) targeting both proteins simultaneously (Fig. 1A). IGF2R aptamer and c-Met aptamer were synthesized into nucleic acid chimeras D1, D2 and D3 by the method of nucleic acid sequence, complementary base pairing and T4 ligase to form phosphodiester bond respectively (Fig. 1B), and The results of the synthesis of nucleic acid chimeras were determined by non-denaturing gels (Fig. 1C).

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

Simulating cell culture conditions, after incubating the synthesized nucleic acid chimeras with 10% FBS at 37°C for different times, it was found that D1 was completely degraded in about 3 h, while D2 and D3 were relatively stable (Fig. 2A). Through cell binding experiments and cell flow analysis, it was found that D3 had the best affinity for cells containing the target, while D2 had the lowest affinity (Fig. 2B).

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

The results of Western experiments showed that the nucleic acid aptamers alone, D1 and D2 had no protein degradation ability, and D3 could significantly degrade c-Met protein on cells (Fig. 3A), and had a good concentration gradient effect (Fig. 3B) and time Gradient effect (Figure 3C). Cell membrane immunofluorescence (Figure 3D) and the binding of c-Met aptamer to cell membrane (Figure 4A and B) further confirmed the degradation ability of D3.

4) Analysis of the degradation ability of nucleic acid chimeras to cell membrane protein PTK7

The nucleic acid chimera D3' was synthesized from the IGF2R aptamer and the PTK7 aptamer by the method of T4 ligase. The results of Western and flow cytometry experiments showed that D3' could effectively degrade PTK7 protein on cells (Fig. 5A), and had a significant time gradient effect (Fig. 5B).

The main advantages of the present invention include:

1) The preparation conditions and steps of the nucleic acid chimera used in the present invention are simple, easy to operate and artificially transform, and have the prospect of industrial synthesis;

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

3) The nucleic acid chimera prepared in the present invention can specifically degrade the target protein, and it is very rapid (within 1 hour);

4) The nucleic acid chimera prepared in the present invention accelerates the drug preparation targeting specific biological macromolecules, and is used for physiological regulation and disease treatment.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to manufacture conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise specified.

sequence

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

Table 1. Nucleic acid chimera sequences of the invention

Note:

(a) "P-" represents phosphorylated 5' structure, eg P-C represents phosphorylated C of 5';

(b) The linker sequence in the stem-loop structure is "underlined".

Example 1 Synthesis method of nucleic acid chimera and analysis of non-denaturing gel

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 (Fig. 1A), wherein A1 and A2 are nucleic acid aptamers (one is IGF2R nucleic acid aptamer, the other is c-Met nucleic acid aptamer), and L is connection Linker for A1 and A2. We synthesized three nucleic acid chimeras (Fig. 1B), in which D1 connects A1 and A2 into one nucleic acid strand through 10 T bases; D2 connects A1 and A2 through complementary base pairing; D3 connects A1 and A2 together by complementary base pairing; The 5' ends of A1 and A2 nucleic acid chains are phosphorylated, and the 5' and 3' ends of A1 and A2 are linked together by T4 ligase. Through non-denaturing PAGE gel analysis, the target molecular weight bands were observed in the lanes labeled D1, D2 and D3, indicating that A1 and A2 were linked together to form nucleic acid chimeras through the different connection methods of D1, D2 and D3 (Fig. 1C). .

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

In order to further test 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 respectively, and it was found that D1 was completely degraded in about 3h, while D2 and D3 were relatively stable ( Figure 2A). HeLa cells express both IGF2R and c-Met membrane protein. Binding experiments were performed by incubating cy5-labeled D1, D2 and D3 as well as IGF2R and c-Met aptamers alone with HeLa cells.

By flow cytometry analysis, it was found that the affinity of D3 for HeLa cells (K _D = 20.12 nM) was significantly higher than that of aptamer alone and D1 and D2. The affinity of D1 to HeLa cells was significantly higher than that of aptamer alone to HeLa cells There was no significant change in the affinity of D2, while D2 had the lowest affinity for HeLa cells (Fig. 2B). These results indicate that D3 has both high cell affinity and better stability relative to D1 and D2.

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

A family of lysosomal-targeting receptors on the cell surface, such as the IGF2R receptor, facilitates protein transport to and degradation in lysosomes. After incubating the synthetic D1, D2, D3 and individual IGF2R aptamers and c-Met aptamers with HeLa cells for 24 h, respectively, the results were analyzed by western blot (Fig. 1A, B, and C) and immunofluorescence (Fig. 3D). ) to detect changes in the level of c-Met protein. GAPDH is glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase) as an internal reference protein.

As shown in Figure 3A, D3 significantly reduced c-Met protein levels in cells compared to untreated control cells (CTR), whereas D1, D2 and aptamers alone did not. Influence. The effect of D3 on c-Met protein level had significant concentration gradient effect (Fig. 3B) and time gradient effect (Fig. 3C), and D3 treatment of cells could significantly reduce c-Met level within 1 h. At the same time, after the nucleic acid chimera-treated HeLa cells were fixed and stained with antibodies, it was found that the level of c-Met protein on the cell membrane was significantly reduced after D3-treated cells (Fig. 3D).

In order to further prove the effect of D3, after treating HeLa cells with D3 without fluorescent label for different time, the binding experiment was carried out using cy5-labeled c-Met nucleic acid aptamer and the treated HeLa cells. By flow cytometry analysis (Figure 4A), it was found that the fluorescence intensity of c-Met nucleic acid aptamer bound on the cell surface decreased significantly after D3 treatment of cells for 1 h, indicating that D3 significantly reduced the level of c-Met protein on the cell membrane. Cells for flow cytometry were subjected to confocal laser analysis (FIG. 4B). The figure shows that the fluorescence intensity on the cell membrane labeled with red fluorescence was significantly reduced after D3 treatment, which further confirmed the effect of D3 on the level of c-Met protein on the cell surface.

The above results show that D3 has the ability to specifically reduce the level of intracellular c-Met protein, and it can play a role in a very short time (eg 1h), while D1 and D2 have no effect.

Example 4 Analysis of the degradation ability of nucleic acid chimeras to cell membrane protein PTK7

In order to further analyze the effect of A1-L-A2 chimera on the level of target protein and its degradation, IGF2R nucleic acid aptamer and PTK7 nucleic acid aptamer were synthesized into nucleic acid chimera D3' by T4 ligase method. CEM cells were treated with IGF2R aptamer, PTK7 aptamer and D3' respectively for 24h, and it was found by western blot (Fig. 5A) that D3' could significantly reduce the level of PTK7 protein in CEM cells compared with untreated cells , while the nucleic acid aptamer alone had no effect on PTK7 protein levels. At the same time, after incubating D3' with CEM cells for different time, the binding experiment was carried out using cy5-labeled PTK7 nucleic acid aptamer and CEM cells.

It was found by flow cytometry analysis (Fig. 5B) that D3' treatment of CEM cells for 1 h could significantly reduce the number of PTK7 aptamers bound on the CEM cell surface, indicating that D3' significantly reduced the level of PTK7 protein on the surface of CEM, and had a time gradient effect .

discuss

In the present invention, the inventors take a membrane protein as an example to illustrate that the nucleic acid chimera (A1-L-A2) that simultaneously targets the lysosome targeting receptor IGF2R and the membrane protein c-Met or PTK7 based on nucleic acid development can be specific c-Met or PTK7 can be transported into lysosomes for degradation. The nucleic acid chimera 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 through a binding agent acting in the extracellular space.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content 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 appended claims of the present application.

Claims

A nucleic acid chimera, 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 no or linker sequence (Linker);

A2 is an aptamer element targeting the protein to be degraded;

Each "-" is independently a bond or a nucleotide linking sequence.
The nucleic acid chimera of claim 1, wherein the nucleic acid aptamer element comprises one or more LTR binding domains that specifically bind to the lysosome targeting receptor (LTR) binding.
The nucleic acid chimera of claim 2, wherein the LTR-binding domain is composed of a nucleic acid sequence that specifically recognizes and binds to LTR.
The nucleic acid chimera of claim 1, wherein the nucleic acid aptamer element further contains other domains in addition to the LTR binding domain.
The nucleic acid chimera of claim 3, wherein the nucleic acid sequence of the LTR comprises a single-stranded nucleic acid sequence.
The nucleic acid chimera of claim 3, wherein the nucleic acid sequence comprises DNA, RNA, LNA, PNA, HNA, CeNA, NAN, FANA, or a combination thereof.
The nucleic acid chimera of claim 3, wherein the nucleic acid sequence comprises natural and non-natural bases, such as bases selected from the group consisting of A, T, C, G, U, I, M-fC, I-fC, isoG, isoC, Ds, Pa, F, X, Y, Z, P.
The nucleic acid chimera of claim 1, wherein the lysosomal targeting receptor (LTR) is located on the cell surface, preferably on the outer surface of the cell membrane.
The nucleic acid chimera of claim 1, wherein the lysosomal targeting receptor (LTR) is located on the surface of the intracellular membrane structure, preferably on the surface of the endosome.
The nucleic acid chimera of claim 1, wherein the lysosomal targeting receptor is selected from the group consisting of IGF2R, Rab, ESCRT, or a combination thereof.
The nucleic acid chimera of claim 8 or 9, wherein the cells are mammalian (eg, human and non-human mammalian) cells.
The nucleic acid chimera of claim 8 or 9, wherein the cells are selected from the group consisting of tumor cells, immune cells, nerve cells, epithelial cells, and stem cells.
The nucleic acid chimera of claim 1, wherein the A1 and A2 are connected through a linker sequence, complementary base pairing or a phosphodiester bond.
The nucleic acid chimera of claim 1, wherein the L is a nucleic acid linker formed by nucleic acid complementation.
The nucleic acid chimera of claim 14, wherein the length of the nucleic acid linker is 6-80nt, preferably 10-50nt, more preferably 15-40nt.
The nucleic acid chimera of claim 1, wherein the proteins to be degraded include membrane proteins, secreted proteins, and intracellular proteins.
The nucleic acid chimera of claim 1, wherein the protein to be degraded is selected from the group consisting of proto-oncoprotein, neurodegenerative disease target protein, immune response-related protein, endocrine-related protein, reproductive-related protein or foreign protein.
The nucleic acid chimera of claim 1, wherein the protein to be degraded is selected from the group consisting of Met, PTK7, and EGFR. 19. A composition, characterized in that the composition comprises:

(i) the nucleic acid chimera of claim 1;

(ii) A pharmaceutically acceptable carrier.
A method of preparing nucleic acid chimera as claimed in claim 1, characterized in that, comprising the steps of:

(S1) Provide A1, A2;

(S2) A1 and A2 are connected to form a nucleic acid chimera of the structure of formula I.
A use of the nucleic acid chimera according to claim 1, characterized in that it is used to prepare a drug for degrading targeted proteins, nucleic acids or fats.
A use of the nucleic acid chimera of claim 1, wherein the drug is used to degrade extracellular proteins, cell membrane proteins or intracellular proteins.
The use according to claim 22, wherein the nucleic acid chimera can be used to prepare an anti-tumor therapeutic drug, and the tumors include cervical cancer, lymphoma, lung cancer, breast cancer, liver cancer, intestinal cancer, and pancreatic cancer.