Application of HDAC (histone deacetylase) inhibitor in improving in-vivo delivery of lipid nanoparticles

Abstract

The invention discloses application of an HDAC (histone deacetylase) inhibitor to improvement of in-vivo delivery of lipid nanoparticles, and belongs to the technical field of gene therapy. HDACi is a small molecule compound for inhibiting histone deacetylase (HDAC), and the gene expression is influenced by inhibiting the HDAC so as to improve the acetylation level of histone or other non-histone substrates. After an organism is treated by the HDAC inhibitor, the in-vivo delivery efficiency of the lipid nanoparticles can be improved by 62%, and the effectiveness and safety of the nucleic acid medicine based on the lipid nanoparticles are improved. Therefore, the method disclosed by the invention lays a solid foundation for gene therapy based on lipid nanoparticles.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene therapy, and particularly relates to application of an HDAC inhibitor in improving in vivo delivery of lipid nanoparticles. BACKGROUND

[0002] In recent years, nucleic acid drugs can achieve the effects of protein replacement, antigen expression, gene expression interference and gene editing, and have shown broad application prospects in the prevention and treatment of tumors, genetic diseases, metabolic diseases or pathogen infection diseases. Non-viral delivery carriers based on lipid nanoparticles are the most commonly used in vivo delivery carriers of nucleic acid drugs. Compared with viral vectors, lipid nanoparticles not only can achieve transient gene expression, but also have many advantages: large loading capacity, extremely low immunogenicity and no risk of genomic integration. Therefore, lipid nanoparticles have developed into a highly efficient nucleic acid delivery platform with good biocompatibility and biodegradability.

[0003] However, low delivery efficiency is the main obstacle limiting the in vivo application of lipid nanoparticles, especially in the application of protein replacement therapy and gene editing therapy, efficient delivery is a core element to achieve effective treatment. The in vivo delivery efficiency of lipid nanoparticles is closely related to the endogenous regulation system of the body. After entering the body, lipid nanoparticles need to interact with tissues and organs in the body to be taken up by target cells and expressed, which requires overcoming a series of extracellular and intracellular biological obstacles, including interaction with body fluids, immune system clearance, extravasation, cell uptake and endosome escape. Therefore, it is necessary to develop a method that can improve the in vivo delivery efficiency of lipid nanoparticles. SUMMARY

[0004] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide application of an HDAC inhibitor in improving in vivo delivery of lipid nanoparticles, so as to improve the in vivo delivery efficiency of lipid nanoparticles.

[0005] The technical solution of the present application to solve the above technical problems is as follows: providing application of an HDAC inhibitor in improving in vivo delivery efficiency of lipid nanoparticles.

[0006] The present application provides a method for improving in vivo delivery efficiency of lipid nanoparticles, comprising the following steps: injecting or orally administering an HDAC inhibitor before injecting a nanoparticle-nucleic acid complex.

[0007] Further, the amount of HDAC inhibitor is 30-40 μL.

[0008] Further, the HDAC inhibitor includes at least one of a tablet, a capsule, a drop pill, an aerosol, a pill, a powder, a solution, a suspension, an emulsion, a granule, a liposome, a transdermal agent, a buccal tablet, a suppository, and a freeze-dried powder injection.

[0009] Further, the HDAC inhibitor includes hydroxamic acid, cyclic peptide, fatty acid, and benzamide HDAC inhibitors.

[0010] Further, the HDAC inhibitor includes suberoylanilide hydroxamic acid, pracinostat, romidepsin, and a depsipeptide.

[0011] Further, the HDAC inhibitor is injected or orally administered before the injection of the lipid nanoparticle-mRNA complex, in particular, the HDAC inhibitor is injected or orally administered 2-48 hours before the injection of the lipid nanoparticle-mRNA complex.

[0012] Further, the nucleic acid is a nucleic acid sequence having a gene therapy, gene editing, gene vaccination, antisense oligonucleotide, or interfering RNA effect.

[0013] Further, the nucleic acid includes EPO mRNA and Cas9 mRNA.

[0014] Further, the injection amount of the lipid nanoparticle-nucleic acid is 0.1-0.5 μg / g.

[0015] The present application has the following beneficial effects: HDACi is a small molecule compound that inhibits histone deacetylase (HDAC), which improves the acetylation level of histone or other non-histone substrates by inhibiting HDAC, thereby affecting gene expression. The present application can improve the in vivo delivery efficiency of the lipid nanoparticle-nucleic acid complex by 62% after treating the organism with the HDAC inhibitor, thereby improving the effectiveness and safety of the nucleic acid drug based on the lipid nanoparticle. Therefore, the method of the present application improves the safety and effectiveness of the lipid nanoparticle in vivo gene therapy, and lays a solid foundation for the lipid nanoparticle-based gene therapy. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Flowchart of the method for improving the delivery efficiency of the lipid nanoparticle of the present application; Figure 2 Mouse in vivo imaging result graph of the effect of the HDAC inhibitor on LNP delivery of luciferase mRNA; Figure 3 Mouse ex vivo imaging result graph of the effect of the HDAC inhibitor on LNP delivery of luciferase mRNA; Figure 4 Protein expression detection result graph of the effect of the HDAC inhibitor on LNP delivery of EPO mRNA. DETAILED DESCRIPTION

[0017] The following examples are intended to illustrate but not limit the present application. Unless otherwise indicated, the particular conditions used in the examples were determined to be the most appropriate under the circumstances. Reagents or instruments not specified were obtained from commercial suppliers and used according to the manufacturer's instructions.

[0018] Example 1: A method for improving the in vivo delivery efficiency of lipid nanoparticles (see flow chart Figure 1 ), comprising the following steps: (1) 100 mg of suberoylanilide hydroxamic acid (SAHA) was dissolved in 500 uL of dimethyl sulfoxide (DMSO), and then diluted to 100 mg / mL; (2) 40 uL of SAHA was injected intraperitoneally 24 hours before the injection of lipid nanoparticle-EPO mRNA via the tail vein; (3) In vivo imaging and ex vivo imaging were performed 6 hours after the injection of lipid nanoparticle-EPO mRNA (injection amount: 0.1 ug / g) via the tail vein.

[0019] Example 2: A method for improving the in vivo delivery efficiency of lipid nanoparticles, comprising the following steps: (1) 100 mg of paribranib was dissolved in 500 uL of dimethyl sulfoxide (DMSO), and then diluted to 20 mg / mL; (2) 20 uL of paribranib was injected intraperitoneally 2 hours before the injection of lipid nanoparticle-EPO mRNA via the tail vein; (3) In vivo imaging and ex vivo imaging were performed 6 hours after the injection of lipid nanoparticle-EPO mRNA (injection amount: 0.2 ug / g) via the tail vein.

[0020] Example 3: A method for improving the in vivo delivery efficiency of lipid nanoparticles, comprising the following steps: (1) 100 mg of romidepsin was dissolved in 500 uL of dimethyl sulfoxide (DMSO), and then diluted to 5 mg / mL; (2) 20 uL of romidepsin was injected intraperitoneally 48 hours before the injection of lipid nanoparticle-EPO mRNA via the tail vein; (3) In vivo imaging and ex vivo imaging were performed 6 hours after the injection of lipid nanoparticle-EPO mRNA (injection amount: 0.3 ug / g) via the tail vein.

[0021] Example 4: A method for improving the in vivo delivery efficiency of lipid nanoparticles, comprising the following steps: (1) 100 mg SAHA was dissolved in 500 uL DMSO, and then diluted to 2 mg / mL; (2) 20 uL SAHA was injected intraperitoneally 20 h before the injection of LNPs-EPO mRNA via tail vein; (3) In vivo and ex vivo imaging was performed 6 h after the injection of LNPs-EPO mRNA (0.4 ug / g) via tail vein.

[0022] Example 5: A method for improving the in vivo delivery efficiency of lipid nanoparticles, comprising the following steps: (1) 100 mg SAHA was dissolved in 500 uL DMSO, and then diluted to 2 mg / mL; (2) 20 uL SAHA was injected intraperitoneally 20 h before the injection of LNPs-EPO mRNA via tail vein; (3) In vivo and ex vivo imaging was performed 6 h after the injection of LNPs-EPO mRNA (0.4 ug / g) via tail vein.

[0023] Test Example: Luciferase was used as a reporter system. Parallel experiments were performed according to the above administration strategy, and control group (CtrL) and different SAHA treatment groups (SAHA-1, SAHA-2, SAHA+1 and SAHA+2) were set. The fluorescence signal of the Region of Interest (ROI) was quantified. It can be seen from Figure 2 that SAHA enhances the fluorescence value of the LNP-luciferase complex, and the fluorescence value is increased by 62%. This indicates that SAHA can improve the protein expression efficiency of the LNP-luciferase complex in vivo.

[0024] To further verify whether the effect of SAHA on improving the efficiency of the LNP-luciferase complex has organ selectivity, ex vivo imaging was performed on the five internal organs of the experimental animals. It can be seen from Figure 3 that the fluorescence signal intensity of the SAHA treatment group is significantly improved compared with the control group. This indicates that SAHA can affect the fluorescence reporter signal in the body by regulating the epigenetic expression of related genes. The ROI signal of the SAHA treatment group (SAHA-1, SAHA-2, SAHA+1, and SAHA+2) is significantly enhanced, indicating that SAHA can promote the expression of mRNA loaded by lipid nanoparticles by inhibiting the activity of histone deacetylase, and thus present a stronger fluorescence signal in imaging; Figure 4is the ELISA detection result of EPO mRNA (erythropoietin), which reflects the expression level of EPO in different treatment groups through the absorbance (OD value), including the control group (CTR, black), the lipid nanoparticle group (LNP, orange) and the lipid nanoparticle combined with SAHA group (LNP+SAHA, green). It can be seen from Figure 4 that the OD value of the control group (CTR) is about 0.3, and the expression level of EPO mRNA is the lowest; the OD value of the lipid nanoparticle-EPO group is about 0.65, and the expression of EPO mRNA is significantly higher than that of the control group; the OD value of the lipid nanoparticle-EPO+SAHA group is close to 0.9, and the expression of EPO in the SAHA treatment group is greatly improved compared with the control group, which indicates that the lipid nanoparticle can promote the expression of EPO.

[0025] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of an HDAC inhibitor in improving the in vivo delivery efficiency of a lipid nanoparticle.

2. A method of improving the in vivo delivery efficiency of a lipid nanoparticle, comprising, comprising the following steps: injecting or orally administering the HDAC inhibitor before injecting the nanoparticle-nucleic acid complex.

3. The method of claim 2, wherein, The HDAC inhibitor is used in an amount of 30-40 μL.

4. The method of claim 2, wherein, The HDAC inhibitor comprises at least one of a tablet, a capsule, a dripping pill, an aerosol, a pill, a powder, a solution, a suspension, an emulsion, a granule, a liposome, a transdermal preparation, a buccal tablet, a suppository, and a freeze-dried powder injection.

5. The method according to any one of claims 2-4, characterized in that, The HDAC inhibitor comprises a hydroxamic acid, a cyclic peptide, a fatty acid, and a benzamide HDAC inhibitor.

6. The method of claim 5, wherein, The HDAC inhibitor comprises suberoylanilide hydroxamic acid, pracinostat, romidepsin, and a depsipeptide.

7. The method of claim 2, wherein, The injecting or orally administering the HDAC inhibitor before injecting the lipid nanoparticle-mRNA complex is specifically injecting or orally administering the HDAC inhibitor 2-48 h before injecting the lipid nanoparticle-mRNA complex.

8. The method of claim 2, wherein, The nucleic acid is a nucleic acid sequence having the effect of gene therapy, gene editing, gene vaccination, antisense oligonucleotide, or interfering RNA.

9. The method of claim 8, wherein, The nucleic acid comprises EPO mRNA and Cas9 mRNA.

10. The method of claim 2, wherein, The injection amount of the lipid nanoparticle-nucleic acid is 0.1-0.5 μg / g.

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