CN110859806A - System for delivering nucleic acid drugs with specific HBV gene cleavage function and application thereof - Google Patents

Abstract

The invention discloses a system for delivering nucleic acid drugs with specific HBV gene shearing function and application thereof. The invention wraps or loads CRISPR/Cas9 gene medicine by pH sensitive cationic liposome, has higher medicine delivery efficiency aiming at HBV positive cells, can obviously reduce the expression of HBV gene, inhibit the replication of hepatitis B virus in vivo and achieve the effect of preventing and treating HBV positive hepatocellular carcinoma.

Description

System for delivering nucleic acid drugs with specific HBV gene cleavage function and application thereof

Technical Field

The invention belongs to the field of genetic engineering and pharmaceutical preparations, and relates to a lipid non-viral vector-nucleic acid drug complex for targeted delivery of a sgRNA expression vector capable of clipping (specifically modifying) human Hepatitis B Virus (HBV) gene (S/X) and a CRISPR/Cas9 nuclease expression vector.

Background

Hepatitis B is caused by Hepatitis B Virus (HBV), become the main disease with hepatitis virus, and can cause the damage of multiple organs, hepatitis B is widely spread in all countries of the world, mainly invades children and young and old, a few patients can be converted into cirrhosis or liver cancer, has become a worldwide disease seriously threatening human health, more than two hundred million of chronic hepatitis B virus infectors all over the world, about 60 million of people die because of complications such as cirrhosis, end-stage liver disease and liver cancer caused by the hepatitis B virus infectors each year, at present, the clinical use of interferon- α, and nucleoside analogs such as lamivudine, entecavir, adefovir dipivoxil and other drugs for treating hepatitis B has long administration time and can not obtain good curative effect of inhibiting replication of HBV, therefore, the treatment method for Hepatitis B Virus (HBV) infection still needs to be innovated technology and breakthrough brought by new drugs.

Nucleic acid drugs are various Ribonucleotides (RNA) or Deoxyribonucleotides (DNA) having different functions, and mainly act at the gene level. Nucleic acid drugs are generally considered to include aptamers, antigenes (antibiotics), ribozymes (ribozymes), antisense nucleic acids (antisense acids), RNA interference agents. The nucleic acid medicine is specific to pathogenic genes, namely has specific targets and action mechanisms, so the nucleic acid medicine has wide application prospect.

Nucleic acid drugs represented by the CRISPR/Cas9 system show an increasing clinical application potential, and some CRISPR/Cas9 drugs are already in preclinical and clinical trials. The working principle of the CRISPR/Cas9 system is that crRNA (CRISPR-derived RNA) binds to tracrRNA (trans-activating RNA) by base pairing to form a tracrRNA/crRNA complex, which directs the nuclease Cas9 protein to cleave double-stranded DNA at sequence target sites paired with the crRNA. By artificially designing these two RNAs, sgrna (single guide RNA) with guiding function can be formed to guide site-directed cleavage of DNA by Cas 9.

In order to achieve therapeutic effect, nucleic acid drugs must enter target cells to exert their effects, and nucleic acid molecules are degraded by the body soon after entering the body. Therefore, ensuring efficient and complete entry of nucleic acid molecules into target cells is a major challenge facing nucleic acid drugs. The lipid non-viral vector mainly comprises a cationic liposome, a lipid micelle, a nanoparticle and the like, and has the advantages of low immunogenicity, large drug loading, easy industrial production and the like compared with the viral vector, and has great potential in clinical application. For example, in vivo and in vitro experiments show that the expression of HBV-S, HBV-X can be obviously reduced, the infection of HBV virus can be eliminated, hepatitis caused by H BV can be prevented, and the method has the outstanding characteristics of high efficiency and capability of knocking out and modifying a plurality of target genes simultaneously. However, the existing lipid non-viral vectors (e.g., cationic liposomes) still have to be improved in the delivery efficiency of nucleic acid drugs. The main factors causing the low delivery efficiency include poor stability after the carrier and the drug are compounded, low drug encapsulation efficiency and low drug targeting release efficiency, which also directly influences the treatment effect of the anti-HBV nucleic acid drug preparation based on the nucleic acid drug delivery system.

Currently, the gene editing vector can only achieve the effect of eliminating hepatitis B virus (for example, Chinese patent 201610133993.9), but the clinical problems are as follows: for most hepatitis B patients (e.g., chronic HBV carriers), there is a lack of effective therapies to prevent the progression to HBV-positive hepatocellular carcinoma stage (the main reason includes the inability to suppress replication of hepatitis B virus in vivo after gene knockout, thereby substantially or completely eliminating hepatitis B virus in vivo). Meanwhile, although experiments show that the replication of hepatitis B virus can be influenced by knocking down the expression of HBV genes, the means for clinically treating liver cancer mainly comprises radiotherapy and chemotherapy. At present, reports of preventing and treating liver cancer (for example, HBV positive hepatocellular carcinoma) by eliminating hepatitis B virus in vivo are not seen.

Disclosure of Invention

The invention aims to provide a system for delivering nucleic acid drugs with specific HBV gene splicing functions and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a nucleic acid drug delivery system targeting HBV positive cells comprises a lipid carrier (with the particle size of 100-150 nm) and one or more nucleic acid molecules for inhibiting HBV replication, wherein the carrier material of the lipid carrier comprises cationic lipid and auxiliary lipid, and the auxiliary lipid comprises a pH sensitive lipid component and an immune escape promoting lipid component.

Preferably, the lipid-based carrier is selected from pH-sensitive cationic liposomes.

Preferably, the cationic lipid is selected from one or more of DOTAP ((2, 3-dioleoyl-propyl) trimethylammonium chloride, N- [1- (2,3-dioleyloxy) propyl ] -N, N, N-trimethyllammonium chloride), DOSPA (dimethyl-2, 3-dioleyloxypropyl-2- (2-spermimido) ethylammonium trifluoroacetate), DOTMA (N- [1- (2,3-dioleyloxy) propyl ] -N, N, N-trimethyllammonium chloride), DOGS or DC-Chol (3 β - (N- (N ', N' -dimethylaminoethyl) carbamoyl) -cholesterol), the pH sensitive lipid component is selected from DOPE (dioleoylphosphatidylethanolamine), and the immunodevading lipid component is DSPE (distearoylphosphatidylethanolamine) and/or DSPE-PEG 2000.

Preferably, the lipid carrier is prepared by adopting the cationic lipid to pH sensitive lipid component molar ratio of 1 (0.1-6), and the dosage of the immunity escape promoting lipid component is not more than 10% of the total lipid mass of the carrier material.

Preferably, the auxiliary lipid further comprises a membrane fluidity-regulating lipid component, the membrane fluidity-regulating lipid component is DSPC and/or cholesterol, and the molar ratio of the cationic lipid to the membrane fluidity-regulating lipid component is 1 (0.1-5).

Preferably, the lipid carrier is selected from cationic liposome, the carrier material of the cationic liposome comprises cationic lipid and auxiliary lipid, wherein the cationic lipid is DOTAP, the auxiliary lipid is DOPE, DSPE-PEG2000 and cholesterol, the cationic liposome is prepared by adopting the molar ratio of DOTAP to DOPE to cholesterol of 1 (0.1-6) to (0.1-5) (for the pH sensitive cationic liposome of which the carrier material is DOTAP, cholesterol, DSPE-PEG2000 and DOPE, the molar ratio is 1 (1-3) to (0.5-1), the following is better, and the dosage of the DSPE-2000 PEG is 1-5% of the total lipid mass of the carrier material.

Preferably, the drug delivery system further comprises an auxiliary molecule for improving the stability of the system, the auxiliary molecule being selected from one or more of sodium citrate, disodium hydrogen phosphate, sodium dihydrogen phosphate or sodium chloride.

Preferably, the nucleic acid molecule is selected from nucleic acid drugs such as DNA, siRNA or MicroRNA.

Preferably, the nucleic acid molecule is selected from a gene editing vector, for example, DNA.

Preferably, the targeting localization sequence of the gene editing is selected from sgRNA which can specifically target human HBV cccDNA in CRISPR-Cas 9-specific modified human HBV gene.

Preferably, the gene editing vector comprises an expression vector of sgRNA and an expression vector of nuclease Cas9, and the expression vector of nuclease Cas9 can adopt pST1374-NLS-flag-Cas9-ZF plasmid in a Chinese patent 'specific sgRNA for inhibiting HBV replication by combining immune genes, an expression vector and application thereof' (application number is 201610133993.9).

Preferably, the sgRNA is the sgRNA of seq.id.no.3, seq.id.no.4 and the like capable of specifically targeting human HBV cccDNA.

Preferably, the expression vector of the sgRNA is pGL3-U6-HBV-S sg plasmid and/or pGL3-U6-HBV-X sg plasmid. pGL3-U6-HBV-S sg was obtained by linking the double strand of sgRNA of the sgRNA whose sequence is shown in SEQ.ID. NO.4 with a linearized pGL3-U6-sgRNA plasmid, pGL3-U6-HBV-X sg was obtained by linking the double strand of sgRNA whose sequence is shown in SEQ.ID. NO.3 with a linearized pGL3-U6-sgRNA plasmid, and was inserted into the multiple cloning site of pGL3-U6-sgRNA plasmid (the entire plasmid is named pGL3-U6-sgRNA-PGK-puromycin) by linking the sequence shown in SEQ.ID. NO.4 or SEQ ID. NO.3 (refer to the Chinese patent "specific sgRNA inhibiting HBV replication by combining immune genes, expression vector and application No. 201610133993.9").

Preferably, the mass ratio of the pGL3-U6-HBV-S sg plasmid to the pGL3-U6-HBV-X sg plasmid is (1-2): (1-2).

Preferably, the mass ratio of the expression vector of the nuclease Cas9 to the expression vector of the sgRNA is (1-4) to (1-3).

The preparation method of the nucleic acid drug delivery system comprises the following steps:

1) dissolving cationic lipid and auxiliary lipid in the same solvent to obtain carrier material solution;

2) evaporating the solvent of the carrier material solution to obtain a lipid film;

3) drying the lipid film, hydrating the lipid film with water or a non-ionic buffer solution (40-60 ℃), and then sequentially carrying out ultrasonic dispersion, stirring and microfiltration to obtain a lipid carrier solution;

4) and mixing the nucleic acid molecules with the lipid carrier solution, and then incubating at 10-30 ℃ (20-60 min) to enable the lipid carrier to wrap or load the nucleic acid molecules.

Preferably, the solvent is selected from organic solvents such as chloroform and chloroform.

Preferably, in the step 3), the non-ionic buffer is selected from HEPES solution with a concentration of 10-40 mmol/L (preferably 20-25 mmol/L); the ultrasonic dispersion condition is ultrasonic treatment for 1-20 min (preferably 5-10 min) in water bath at 40-60 ℃, the stirring condition is magnetic stirring in water bath at 40-60 ℃, and the aperture of the microporous filter membrane is less than or equal to 0.22 mu m.

Preferably, the molar ratio of nitrogen in the nucleic acid molecule to phosphorus in the lipid carrier is 1 to 10:1 (more preferably 3 to 6: 1).

Preferably, the auxiliary molecule is added to the incubated system at a final concentration of 1 to 150mmol/L (preferably 50 to 100 mmol/L), and then the incubation is continued at 10 to 30 ℃.

The nucleic acid drug delivery system targeting HBV positive cells is applied to the preparation of anti-hepatitis B virus drugs or drugs for preventing and treating human HBV positive hepatocellular carcinoma.

The invention has the beneficial effects that:

the invention utilizes lipid non-viral nano-carriers to wrap or load nucleic acid drug molecules such as gene editing carriers for inhibiting HBV replication and the like, constructs a nucleic acid drug delivery system with high specificity aiming at HBV virus-infected cells, wherein the lipid carriers are cationic liposomes formed by cationic lipids and auxiliary lipid components, can efficiently deliver the nucleic acid drugs to target cells (HBV-infected cells) through immune escape and pH response mechanisms, and release the nucleic acid drugs into the target cells, thereby ensuring that the drug activity of the nucleic acid drugs is not damaged in the delivery, the delivered nucleic acid drugs are subjected to targeted editing (such as shearing) HBV, not only inhibiting the gene expression of the HBV, but also realizing the effective elimination of the in-vivo hepatitis B virus by inhibiting the replication of the in-vivo hepatitis B virus, and the invention not only can remarkably improve the hepatitis B treatment (anti-virus) effect, moreover, the hepatitis B virus replication in vivo is inhibited, so that liver cancer such as HBV positive hepatocellular carcinoma can be effectively prevented and treated.

Further, the invention constructs a sgRNA expression vector for inhibiting HBV gene according to the human HBV gene (HBV-S, HBV-X) suitable for CRISPR-Cas9 target clipping. The nucleic acid and the expression vector of the nuclease Cas9 are combined into a CRI SPR-Cas9 system, and by means of the drug delivery system, the corresponding nucleic acid drugs are efficiently delivered to HBV positive cells, so that conditions are created for clinical application of gene drugs in liver cancer treatment.

Furthermore, the cationic lipid, DOPE, DSPE-PEG2000 and cholesterol are mixed according to a certain proportion, a pH sensitive cationic liposome can be constructed by a film dispersion method, and can be mixed and incubated with a nucleic acid drug to obtain a stable cationic liposome-nucleic acid compound.

Furthermore, the cationic liposome prepared by the invention has uniform particle size and stable system, can wrap or load CRISPR/Cas9 gene medicine, and has high medicine encapsulation efficiency.

Furthermore, the invention improves the stability of the nucleic acid drug delivery system and prevents the occurrence of coagulation by introducing auxiliary molecules.

Furthermore, the invention has higher drug delivery efficiency aiming at HBV positive cells, can obviously reduce the expression of HB V genes, and more importantly, can inhibit the replication of hepatitis B virus in vivo by combining specific gene knockout target genes (such as S + X), thereby obviously improving the treatment effect of hepatitis B and HBV positive hepatocellular carcinoma.

Drawings

FIG. 1 is a schematic diagram of the effect of cationic liposome delivery of nucleic acid drugs.

Fig. 2 is a graph showing the particle size of the composite prepared according to the present invention.

FIG. 3 shows the Zeta potential of the composites prepared according to the present invention.

Fig. 4 is an appearance of cationic liposomes prepared according to the present invention, wherein: (a) three months before, (b) three months after.

Fig. 5 is a schematic diagram of Cas9 achieving site-directed cleavage resulting in DNA and double strand break processes.

FIG. 6 is an agarose gel electrophoresis image of gene drug encapsulation efficiency analysis.

Figure 7 is the result of the change in HBsAg expression caused by liposome-mediated HBV-specific cleavage of the sgRNA/Cas 9-encapsulated plasmid composition.

FIG. 8 shows immunohistochemistry of liver after injection of compound in transgenic mice.

FIG. 9 shows the tumor inhibition results of tumor-bearing mice.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.

The action principle of the nucleic acid drug delivery system prepared in the invention is shown in figure 1. The nucleic acid drug delivery system is a pH sensitive cationic liposome-nucleic acid drug (e.g., DNA) complex, and the pH sensitive cationic liposome is used for effectively delivering the nucleic acid drug into a target cell and releasing the nucleic acid drug under the specific pH environment of the target cell so as to enable the nucleic acid drug to play a role.

The invention screens the carrier material and proportion of the cationic liposome according to the characteristics of stability, pH sensitivity and the like, and aims to find a feasible way for improving the delivery efficiency of the antiviral gene drug based on the cationic liposome.

Through experiments, DSPE-PEG2000 and DOPE are selected as auxiliary phospholipids, and a long-circulating pH-sensitive cationic liposome is designed. Wherein, the DSPE-PEG2000 can increase the stability of the liposome-D NA compound by utilizing the space configuration thereof, and can realize the in vivo immune escape of the compound. DOPE is used as a pH sensitive element of the liposome, is stable in a physiological environment (pH7.4) and is structurally changed under an acidic condition (pH5.0-6.8), and by utilizing the characteristic, the DOPE can help the liposome to escape from lysosomes under the acidic condition of the lysosomes and prevent DNA from being degraded. The cationic liposome can be used for constructing a medicinal-grade drug delivery system, and realizes multiple targeting and long circulation effects of gene drug delivery.

Preparation of blank cationic liposome

Dissolving medicinal synthetic phospholipid DOTAP, DOPE, DSPE-PEG2000 and medicinal synthetic cholesterol (4 components in total) in 5mL of chloroform, and transferring into a 25mL round-bottom flask, wherein the molar ratio of DOTAP to DOPE to cholesterol is 1:1:0.6, and the DSPE-PEG200 accounts for 1% of the total lipid mass (namely the sum of the 4 components); slowly rotary-evaporating at 30 deg.C under 0.08MPa (gauge pressure) to remove organic solvent (chloroform) to form a lipid film with uniform thickness at the bottom of the round-bottomed flask, and vacuum drying at room temperature under 0.1MPa (gauge pressure) for 2 hr (to completely remove organic solvent and steam); adding 10mL of HEPES solution (20mmol/L) preheated to 50 ℃ into a round-bottom flask, washing off the lipid film from the bottom of the round-bottom flask (washing the film), then placing the round-bottom flask on an ultrasonic cleaner for ultrasonic treatment in a 50 ℃ water bath for 5min, allowing the lipid film to disappear (dissolving the lipid film in the HEPES solution), transferring the round-bottom flask to a magnetic stirrer, and continuing to stir and hydrate in the 50 ℃ water bath at 900 rpm; stirring for 30min to obtain light blue opalescence without particle impurities (blue opalescence represents formation of cationic liposome, and liposome has complete shape and high quality), sequentially passing through 0.22 μm and 0.1 μm microporous filter membrane (to make cationic liposome uniform in particle size and also capable of sterilizing), introducing nitrogen gas, and storing at 4 deg.C for 5 times.

1) Particle size determination

And (3) measuring solution samples filtered by microporous filter membranes of different batches by using a laser particle size measuring instrument, finding that the average particle size of the dissolved cationic liposome is 100-150 nm, and measuring the Zeta potential average value by using a Zeta potentiometer to be 55-60 mV. FIGS. 2 and 3 show the results of the measurement of one batch of samples.

2) Stability of blank cationic liposomes

The blank liposome is aseptically filtered and subpackaged into a sterilizing tube, stored in a refrigerator at 4 ℃ for 28 days, and then kept still stable after standing for 3 months at room temperature, the liposome is white in color, good in uniformity and free of precipitation (figure 4).

3) pH sensitive Properties of blank cationic liposomes

The liposomes and PB solutions of different pH were incubated at 37 ℃ for 30min and the change in potential of the mixture was measured using a Zeta potentiometer. By measuring the surface potential of the liposome under different pH conditions, the results show that the surface charge of the liposome increases sharply with the decrease of pH (the cell inclusion is in an acidic condition, and the pH value is about 5.0).

(II) CRISPR/Cas9 system for targeted editing of HBV gene

Referring to fig. 5, the directional recognition and cleavage of genes by the CRISPR/Cas9 system is achieved by sgRNA, which determines targeting of Cas9 and also determines cleavage activity of Cas9, and nuclease Cas 9.

2.1 design and selection of HBV-targeting sgRNA oligonucleotides

1. The HBV gene may be selected from the 5 '-GGN (19) GG sequence, and if the HBV gene does not have the 5' -GGN (19) GG sequence, the 5 '-GN (20) GG sequence or the 5' -N (21) GG sequence may be used.

sgRNA is located in the S and X gene regions of HBV at targeted sites of HBV, respectively.

3. In UCSC databases, BLAT databases and NCBI databases, the uniqueness of the sgRNA target sequence is determined, and potential off-target sites are reduced.

4. If the targeted combined editing of HBV genes is realized by using sgRNA aiming at different regions of HBV, the HBV genes can be knocked out more effectively.

5. If two sgRNAs targeting different regions of HBV are used for knocking out target genes, the replication of HBV virus can be more effectively inhibited.

2.2 construction of oligonucleotide double strands of sgRNA

According to the sgRNA chosen, adding CCGG at its 5 'end gives a forward oligonucleotide (forward oligo), if the sequence itself already has 1 or 2 Gs at the 5' end, the corresponding omission of 1 or 2 Gs; obtaining a complementary strand of its corresponding DNA according to the selected sgRNA, and adding AAAC 5' thereof to obtain a reverse oligonucleotide (reverse oligo); the forward and reverse oligonucleotides were synthesized separately, and forward and reverse oligos of the synthesized sgRNA oligonucleotide were annealed in pairs.

The annealing reaction system is as follows:

run in a PCR instrument according to the following touch down program: 95 ℃ for 5 min; 95-85 ℃ at-2 ℃/s; at-0.1 ℃/s at 85-25 ℃; storing at 4 deg.C for use.

Annealing is followed by formation of sgRNA oligonucleotide duplexes that can be ligated into the U6 eukaryotic expression vector.

2.3 construction of sgRNA oligonucleotide plasmids (expression vectors)

1. Linearized pGL3-U6-sgRNA plasmid (trade name: pGL3-U6-sgRNA-PGK-puromycin, Addgene), enzyme digestion system and conditions were as follows: 2 μ g pGL3-U6-sgRNA (400 ng/. mu.L), 1 μ L CutSmart Buffer, and 1 μ L BsaI (NEB), ddH₂Supplementing O to 50 mu L, and incubating for 3-4 hours at 37 ℃; after the enzyme digestion is finished, the linearized pGL3-U6-sgRNA plasmid is purified and recovered into 20-40 mu L of sterilized water by AxyPrep PCR Clean up Kit (AP-PCR-250).

2. The annealed sgRNA oligonucleotide double strand was ligated with the linearized pGL3-U6-sgRNA plasmid to obtain pGL3-U6-HBV sg plasmid (specifically, pGL3-U6-HBV-S sg, pGL3-U6-HBV-X sg, etc., depending on the different gene regions of HBV targeted by the sgRNA).

3. Coli competent cells were transformed and plated on Amp + plates (50. mu.g/mL).

4. Positive clones were identified by sequencing with the U6 universal primer (see seq. id. No. 1).

The cells were shaken in a shaker at 5.37 ℃ and the positive clones were cultured overnight and pGL3-U6-HBV sg Plasmid was extracted with AxyPrep Plasmid Miniprep Kit (AP-MN-P-250).

(III) preparation of pH-sensitive cationic liposome-DNA Complex

Preparing a CRISPR/Cas9 system (DNA) for transfecting cells and the blank cationic liposome solution prepared in the step one into a mixed solution (a solution obtained by adding plasmids into a filter membrane for filtration) according to a nitrogen-phosphorus molar ratio of 6:1, incubating at room temperature for 20min, adding a certain amount of sodium citrate into the mixed solution to enable the concentration of the sodium citrate in the system to be 60mmol/L, and incubating at room temperature for 20min to form a uniform solution without granular impurities, wherein the solution is a pH sensitive cationic liposome-DNA complex solution.

The encapsulation efficiency of the liposome-DNA complex is detected by a gel electrophoresis experiment, which comprises the following steps:

1) preparing gel according to a gel electrophoresis experiment, wherein the gel concentration is 1-2%, and the gel contains EB;

2) after preparing the cationic liposome-DNA complex solution, adding a sample buffer solution into a quantitative sample, loading the sample into a sample loading hole, and performing electrophoresis;

3) the amount of free DNA was judged by comparing the fluorescence intensity of the free DNA in the sample with that of the Marker band under UV light, and the DNA encapsulated in the liposome was blocked in the well.

According to the gray scale analysis, the entrapment rate of DNA of the sample (213.5 ng/. mu.L, 20. mu.L) was estimated to be 95% or more (free plasmid DNA at the box of FIG. 6) preliminarily by agarose gel electrophoresis as compared with Maker (20 ng/. mu.L, 5. mu.L).

The cationic liposome-DNA complex was resuspended in a medium containing 10% fetal bovine serum and the change in particle size of the complex was determined by laser particle size/Zeta potentiometer. The existing cationic liposome complex and proteins in blood are easy to generate non-specific adsorption, and the application of the cationic liposome complex as a CRISPR/Cas9 drug delivery system is seriously influenced. In the invention, the particle size change of the cationic liposome-DNA complex in a culture medium containing 10% fetal calf serum is measured, and the complex is found to be stable in serum.

(IV) efficacy test of nucleic acid drug delivery System with high specificity for HBV-infected cells

The nucleic acid drug delivery system is a cationic liposome-nucleic acid compound constructed by wrapping a CRISPR/Cas9 system with liposome, takes human hepatitis B cells infected by HBV, hepatitis B transgenic mice and tumor-bearing mouse models as research objects, adopts a method of specific targeting single gene or cooperative combat, simultaneously delivers and intervenes aiming at main molecular targets of diseases, realizes effective knockout of HBV virus target pathogenic genes, can obviously inhibit HBV gene expression and replication, achieves the aim of completely eliminating viruses, and thus provides an effective way for in-vivo HBV gene elimination and prevention and treatment of hepatitis B and liver cancer.

1. Transfection of human hepatitis B HepG2.2.15 cells

1.1 cell culture

First, HepG2.2.15 cells in logarithmic growth phase were thoroughly digested with pancreatin, then the cells in the flask were gently pipetted with a non-calibrated elbow pipette and transferred into a centrifuge tube and centrifuged at 114g for 5min with cell culture medium containing 10% newborn calf serum to terminate the digestion. After centrifugation, the supernatant was decanted, the cells were resuspended in cell culture medium to make a single cell suspension, stained with trypan blue at room temperature for 2min, and the cells were counted on a hemocytometer under an inverted microscope. Then, the culture plate is rinsed 1 time with cell culture solution, the cell density is adjusted to 70% -90% of the confluence degree of the next day, the mixture is inoculated into a 12-hole plate, the volume of the culture solution is supplemented to 1mL, and the mixture is placed at 37 ℃ and 5% CO₂Culturing in a cell culture box.

1.2 Liposome-free Co-transfection of HepG2.2.15 cells

After the sgRNA oligonucleotides targeting HBV cccDNA were designed, selected and synthesized, sgRNA oligonucleotides targeting HBV cccDNA (i.e., sgrnas targeting S gene and X gene of HBV) were ligated to linearized pGL3-U6-sgRNA plasmids, respectively, to obtain vectors pGL3-U6-HBV sg (pGL3-U6-HBV-S sg and pGL3-U6-HBV-X sg) containing sgRNA oligonucleotides targeting HBV S gene and X gene, respectively, hepg2.2.15 cells were transfected as follows: according to the operating manual of Lipofectamine 2000Transfection Reagent (Invitrogen,11668-019), a vector pGL3-U6-HBV-S sg containing targeted HBV S gene sgRNA oligonucleotides and a vector pGL3-U6-HBV-X sg containing targeted HBV X gene sgRNA oligonucleotides are mixed with pST1374-NLS-flag-Cas9-ZF plasmid singly or in combination to transfect cells.

1.3 Co-transfection of HepG2.2.15 cells with liposomes

And (3) mixing and incubating pGL3-U6-HBV sg plasmid (targeted HBV-S and/or HBV-X) with sgRNA oligonucleotide of the corresponding HBV gene, pST1374-NLS-flag-Cas9-ZF plasmid (Addgene) and cationic liposome solution according to the third step, and standing for 15-20 min at 25 ℃. Then, the mixed solution is slowly and uniformly added into a 12-hole plate, and then the mixed solution is placed in a cell culture box for culture for 4-6 hours.

2. Grouping

The gene drug processing group is transferred into an sgRNA vector with shearing activity aiming at HBV-S, HBV-X (the group of Lipo + gRNA-HBV-S: pGL3-U6-HBV-S sg, namely pGL3-U6-HBV sg contains sgRNA oligonucleotide targeting HBV-S, the sgRNA is shown in SEQ ID.NO.4, the group of Lipo + gRNA-HBV-X is pGL3-U6-HBV-X sg, namely pGL 3-U6-sHBV g contains sgRNA oligonucleotide targeting HBV-X, the sgRNA is shown in SEQ ID.NO.3, and the group of Lipo + gRNA-HBV- (S + X) combines pGL3-U6-HBV sg aiming at HBV-S and HBV-X and pGL 1374-NLS-flag-9-ZF plasmid.

The control group (gRNA empty vector group) was transformed with sgRNA vector (pGL3-U6-HBVsg with sgRNA replaced with SEQ. ID. NO.2) and pST1374-NLS-flag-Cas9-ZF plasmid, which had no splicing activity.

Blank group (Blank group): hepg2.2.15 cells without transfection of any plasmid.

In the control group and the treatment group, the mixing ratio of the total pGL3-U6-HBV sg plasmid and the pST1374-NLS-flag-Cas9-ZF plasmid in each group is 1:1 (mass ratio), and HepG2.2.15 cells are co-transfected by cationic liposome.

Plasmid verification group: compared with the treatment group, the difference is that plasmids containing corresponding sgRNAs and pST1374-NLS-flag-Cas9-ZF plasmids are directly transfected without liposome coating (respectively marked as gRNA-HBV-S group, gRNA-HBV-X group and gRNA-HBV- (S + X) group).

Enzyme-linked immunosorbent assay for detecting change of hepatitis B virus surface antigen

1. According to the method and grouping of the step (IV), pGL3-U6-HBV sg plasmid (sgRNA of S gene and/or X gene of targeted HBV) with sgRNA oligonucleotide corresponding to HBV shearing activity and pST1374-NLS-flag-Cas9-ZF plasmid are respectively mixed uniformly, and HepG2.2.15 cells are co-transfected in two ways (IV; 1.2\ 1.3).

2. On the next day after transfection, the supernatant was collected and the Hepatitis B virus Surface Antigen was measured according to the Diagnostic Kit for Hepatitis BVirus Surface Antigen (ELISA) using instructions.

The control group (gRNA empty vector) was transformed with sgRNA vector pGL3-U6-HBVsg (corresponding to the sgRNA of SEQ. ID. NO.2) having no cleavage activity, and the treatment group (S + X) was sgRNA vectors pGL3-U6-HBV-S sg and pGL3-U6-HBV-X sg (corresponding to the sgRNAs of SEQ. ID. NO.4 and SEQ. ID. NO.3) to which the S gene and the X gene of HBV cccDNA were simultaneously added. HBsAg expression is detected by ELISA, and compared with a control group, the surface antigen of the CRISPR/Cas9 treatment group is obviously reduced; the liposome (Lipo) + CRISPR/Cas9 has more obvious effect compared with the CRISPR/Cas9 single treatment group (plasmid verification group). The expression of the hepatitis B virus surface antigen of the liposome + CRISPR/Cas9 treatment group is obviously reduced.

(VI) in vivo inhibition of HBV viral replication

HBV transgenic mice are selected as animal models and are divided into 3 groups, namely a control group, a CRISPR/Cas9 knockout HBV group (corresponding to a plasmid verification group) and a liposome-encapsulated CRISPR/Cas9 knockout HBV group (corresponding to a treatment group). The tail vein injection method is used, and the dose administered to each group is exemplified as follows:

control group: 40 μ g of pST1374-NLS-flag-Cas9-ZF +40 μ g of pGL3-U6-HBV sg (corresponding sgRNA is SEQ. ID. NO. 2);

CRISPR/Cas9 knockdown HBV panel (gRNA-HBV (X + S) panel): 40 μ g pST1374-NLS-flag-Cas9-ZF +20 μ g pGL3-U6-HBV-S sg +20 μ g pGL3-U6-HBV-X sg;

liposome-encapsulated CRISPR/Cas9 knockdown HBV panel (Lipo + gRNA-HBV (X + S) panel): 40 μ g of pST1374-NLS-flag-Cas9-ZF +20 μ g of pGL3-U6-HBV-S sg +20 μ g of pGL3-U6-HBV-X sg, added to 200 μ L of blank cationic liposome solution (in line with the nitrogen-phosphorus molar ratio of 6: 1).

After plasmid injection, mouse caudal venous blood is extracted on the first day and the third day, serum is separated, and the change of HBsAg is detected by an ELISA method, and the result is shown in figure 7, compared with a control group and a CRI SPR/Cas9 gene drug which is not delivered by liposome, the expression level of hepatitis B surface antigen can be more effectively reduced by delivering the CRISPR/Cas9 gene drug by liposome.

After 3 weeks, the mice were sacrificed and HBsAg expression in mouse liver cells was detected by immunohistochemistry. Referring to fig. 8, it can be seen that the expression level of hepatitis b surface antigen can be more effectively reduced by liposome delivery of CRISPR/Cas9 gene drug compared to the control group and CRISPR/Cas9 gene drug not delivered by liposome.

(VII) HBV-Positive hepatocellular carcinoma-bearing mouse experiment

Mouse HepG2.2.15 cells were cultured at 2X 10 per injection point⁶Number of individual cells Balb/c mice were inoculated subcutaneously on the back.

The tumor to be transplanted grows to 2mm³Size, grouped by (four): namely a control group, a gRNA-HBV-S group, a gRNA-HBV-X group, a gRNA-HBV- (S + X) group, a Lipo + gRNA-HBV-S group, a Lipo + gRNA-HBV-X group, and a Lipo + gRNA-HBV- (S + X) group. The tail vein injection method of the target gene plasmid is adopted, and the administration dosage of each group is respectively as follows:

1. control group: 40 μ g pST1374-NLS-flag-Cas9-ZF +20 μ g empty gRNA;

gRNA-HBV-S group: 40 μ g pST1374-NLS-flag-Cas9-ZF +40 μ g pGL 3-U6-HBV-Ssg;

gRNA-HBV-X group: 40 μ g pST1374-NLS-flag-Cas9-ZF +40 μ g pGL 3-U6-HBV-Xsg;

gRNA-HBV- (S + X) group: 40 μ g pST1374-NLS-flag-Cas9-ZF +20 μ g pGL3-U6-HBV-Ssg +20 μ g pGL3-U6-HBV-X sg;

lipo + gRNA-HBV-S group: adding 40 mu g of pST1374-NLS-flag-Cas9-ZF +40 mu g of pGL3-U6-HBV-Ssg into 200 mu L of blank cationic liposome solution (according to the molar ratio of nitrogen to phosphorus of 6: 1);

lipo + gRNA-HBV-X group: adding 40 mu g of pST1374-NLS-flag-Cas9-ZF +40 mu g of pGL3-U6-HBV-Xsg into 200 mu L of blank cationic liposome solution (according to the molar ratio of nitrogen to phosphorus of 6: 1);

lipo + gRNA-HBV- (S + X) group: adding 40 mu g of pST1374-NLS-flag-Cas9-ZF +20 mu g of pGL3-U6-HBV-S sg +20 mu g of pGL3-U6-HBV-X sg into 200 mu L of blank cationic liposome solution (according to the molar ratio of nitrogen to phosphorus of 6: 1);

mice were sacrificed at 3, 6, 9, 12, 15, 18, 21d dislocation after administration, and a tumor photograph was taken, see fig. 9, and the tumor-inhibiting effect of the gRNA-HBV-S group or the gRNA-HBV-X group delivered by liposome was superior to that of the gRNA-HBV-S group or the gRNA-HBV-X group not delivered by liposome, compared to the control group; the Lipo + gRNA-HBV- (S + X) group delivered by liposome has better effect of inhibiting tumor growth.

Since previous studies have focused on the elimination of hepatitis b virus (viral gene knockdown), no effective inhibition of viral replication in vivo has yet been found to provide a prophylactic and therapeutic approach for inhibiting HBV-positive hepatocellular carcinoma based on viral clearance.

In a word, the cationic liposome prepared by the invention has uniform particle size, stable system and high drug encapsulation rate, has high-efficiency drug delivery efficiency as a nucleic acid drug delivery system, and has the following advantages:

1. the drug delivery system has wide application range and can be used for delivering nucleic acid drugs such as DNA, siRNA, MicroRNA and the like to cells.

2. By screening liposome components and the ratio of the liposome components, the dilemma that the efficiency of a plurality of cationic liposomes is unstable when the cationic liposomes are used for delivering nucleic acid medicaments to cells is solved, and the nucleic acid medicaments can be efficiently delivered to target cells and play a role.

3. The gene expression vector disclosed by the invention is simple in preparation method, good in sgRNA targeting and high in knockout efficiency of the CRISPR/Cas9 system.

4. The drug delivery system is a cationic liposome with the function of splicing HBV S and/or X genes specifically, can precisely target and cut HBV genes, efficiently reduce the gene expression of HBV, obviously reduce the expression level of hepatitis B surface antigen, show the high efficacy of resisting hepatitis B virus infection, and provide an effective drug treatment way for targeted treatment of hepatitis B virus infection.

5. The drug delivery system can exert the advantages of CRISPR/Cas9 in cutting HBV gene rapidly, conveniently, efficiently and specifically, and can effectively inhibit virus replication by combining with the cationic liposome for delivery, thereby exerting the effect of eliminating HBV virus continuously existing in normal liver of human, and solving the problem that the existing human hepatitis B virus infection treatment (especially HBV positive hepatocellular carcinoma) lacks effective prevention and treatment drugs.

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Claims (10)

1. A nucleic acid drug delivery system targeting HBV positive cells, characterized in that: the drug delivery system comprises a lipid carrier and one or more nucleic acid molecules for inhibiting HBV replication, wherein the one or more nucleic acid molecules are wrapped or loaded by the lipid carrier, the carrier material of the lipid carrier comprises cationic lipid and auxiliary lipid, and the auxiliary lipid comprises a pH sensitive component and an immune escape promoting component.

2. The nucleic acid drug delivery system targeting HBV positive cells according to claim 1, wherein: the lipid carrier is selected from pH sensitive cationic liposome.

3. The nucleic acid drug delivery system targeting HBV positive cells according to claim 1, wherein: the cationic lipid is selected from one or more of DOTAP, DOSPA, DOTMA, DOGS or DC-Chol; the pH sensitive component is selected from DOPE, and the immunity escape promoting component is DSPE and/or DSPE-PEG 2000.

4. The nucleic acid drug delivery system targeting HBV positive cells according to claim 1, wherein: in the carrier material, the molar ratio of the cationic lipid to the pH sensitive component is 1 (0.1-6), and the dosage of the immune escape promoting component is not more than 10% of the total lipid mass of the carrier material.

5. The nucleic acid drug delivery system targeting HBV positive cells according to claim 1, wherein: the auxiliary lipid also comprises a membrane fluidity adjusting component, the membrane fluidity adjusting component is DSPC and/or cholesterol, and the molar ratio of the cationic lipid to the membrane fluidity adjusting component is 1 (0.1-5).

6. The nucleic acid drug delivery system targeting HBV positive cells according to claim 1, wherein: the lipid carrier is selected from cationic liposome, and the carrier material of the cationic liposome comprises cationic lipid and auxiliary lipid, wherein the cationic lipid is DOTAP, and the auxiliary lipid is DOPE, DSPE-PEG2000 and cholesterol; in the carrier material, the molar ratio of DOTAP to DOPE to cholesterol is 1 (0.1-6) to 0.1-5, and the dosage of DSPE-PEG2000 is 1-5% of the total lipid mass of the carrier material.

7. The nucleic acid drug delivery system targeting HBV positive cells according to claim 1, wherein: the nucleic acid molecule is selected from a gene editing vector; the gene editing vector is an expression vector of sgRNA and/or an expression vector of nuclease Cas9, and the expression vector of sgRNA comprises sgRNA which can specifically target human HBV gene cccDNA in CRISPR-Cas9 specific modified human HBV gene; the expression vector of the sgRNA is pGL3-U6-HBV-S sg plasmid and/or pGL3-U6-HBV-X sg plasmid, and the corresponding expressed sgRNA is SEQ ID No.4 and/or SEQ ID No. 3.

8. The nucleic acid drug delivery system targeting HBV positive cells according to claim 7, wherein: the mass ratio of the pGL3-U6-HBV-S sg plasmid to the pGL3-U6-HBV-X sg plasmid is (1-2) to (1-2); the mass ratio of the expression vector of the nuclease Cas9 to the expression vector of the sgRNA is (1-4) to (1-3).

9. Use of the nucleic acid drug delivery system of any one of claims 1 to 8 in the preparation of an anti-hepatitis B virus drug or in the preparation of a drug for the prevention and treatment of liver cancer.

10. The use according to claim, wherein: the liver cancer is selected from human HBV positive hepatocellular carcinoma.

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