CN106729753A - The delivery system and biological agent of anti-hepatitis B virus - Google Patents
The delivery system and biological agent of anti-hepatitis B virus Download PDFInfo
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- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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Abstract
Disclosed herein is the lipidic nanoparticles being made up of fat peptide molecule, DOPE, cholesterol and polyethylene glycol, the sgRNA molecules of its parcel Cas9mRNA and targeting HBV gene conservative region.There is disclosed herein the preparation method and purposes of the lipidic nanoparticles, and pharmaceutical composition and preparation comprising the lipidic nanoparticles.The lipidic nanoparticles and biological agent of this paper can be used as the Novel Delivery Systems of anti-hepatitis B virus.
Description
Technical field
The present invention relates to field of medicaments, and in particular to anti-hepatitis B virus field, a kind of new resisting is more particularly to
The Novel Delivery Systems and biological agent of hepatitis type B virus.
Background technology
Global Patients with Hepatitis B Virus Infection there are about 3.5 hundred million people, wherein about 100,000,000 people are in China.These people are latent due to hepatitis B
Volt cannot be effected a radical cure.The latent main cause of hepatitis B is its genome with a kind of covalently closed circular DNA (covalently
Closed circular DNA or cccDNA) form exist steadily in the long term in the liver cell nuclear of patient.Existing anti-hepatitis B
Medicine is mainly interferon and nucleosides (acid) analog, but this two classes medicine cannot remove cccDNA, thus cannot effect a radical cure second
Liver.
Emerging gene editing technology CRISPR-Cas9 systems are due to its efficiently special DNA shearing and edit capability
The extensive use in scientific research field is obtained.CRISPR-Cas9 is a kind of DNA cutting systems found in bacterium, by DNA
Restriction endonuclease Cas9 recognizes specific DNA sequence dna and is cut in specific site with the help of one section of tiny RNA (guide RNA)
Cut.Due to needing to express an a big PROTEIN C as9 and small guide RNA simultaneously, how to be carried out safely and effectively in human body
The conveying CRISPR-Cas9 systems that have special curative effect be a problem demanding prompt solution, and have become CRISPR-Cas9 and exist
The bottleneck of clinical practice.
Conveying is by expressing Cas9 and guide with baculovirus expression system in existing CRISPR-Cas9 bodies
RNA, but the security of adenovirus is still problematic.And, adenovirus this DNA of expression Cas9 steady in a long-term in the cell
Enzyme, it is also possible to bring long-term toxic and side effect.Nucleic acid induction system based on new lipidic nanoparticles (LNP) exists
In vivo good effect is shown on conveying tiny RNA such as siRNA and long-chain RNA such as mRNA.
Therefore, needs are still suffered from for safely and effectively conveying the CRISPR-Cas9 systems of specific curative effect.
The content of the invention
It is an object of the invention to provide a kind of efficient LNP delivery systems, by by LNP and Cas9mRNA and one section of list
Chain guide RNA (sgRNA) is obtained by mixing by microfluidic devices, and it can effectively be transported to mouse liver and right
Hepatitis B cccDNA is sheared, and reaches the purpose for suppressing hepatitis B.The reagent provides a kind of in human-body safety radical cure
The possibility of hepatitis B.
The first aspect of the invention is related to target the sgRNA molecules of HBV gene conservative region, and it has SEQ ID NO:
1 to SEQ ID NO:One of nucleotide sequence shown in 10：GAGGTGAAGCGAAGTGCACA(SEQ ID NO:1)；
CCACCCAAGGCACAGCTTGG(SEQ ID NO:2)；CGGGGAGTCCGCGTAAAGAG(SEQ ID NO:3)；
AAGCCACCCAAGGCACAGCT(SEQ ID NO:4)；GAAGCGAAGTGCACACGGTC(SEQ ID NO:5)；
AGAAGATGAGGCATAGCAGC(SEQ ID NO:6)；CAAGCCTCCAAGCTGTGCCT(SEQ ID NO:7)；
GGGGCGCACCTCTCTTTACG(SEQ ID NO:8)；GGACTTCTCTCAATTTTCTA(SEQ ID NO:9)；Or
TCCTCTGCCGATCCATACTG(SEQ ID NO:10)。
The second aspect of the invention is related to for the RNA molecule transport based on CRISPR-Cas9 systems to be arrived into liver
Lipidic nanoparticles.The lipidic nanoparticles are by fat peptide molecule, DOPE, cholesterol and polyethylene glycol
Constitute.The lipidic nanoparticles wrap up Cas9mRNA and as described above the sgRNA molecules described in one side.
In one embodiment of the invention, the fat peptide molecule is the polymer of fat polypeptide.In one kind of the invention
In specific embodiment, fat polypeptide is 1,3,5- tri- (N1,N3,N5- (the double dodecane aminopropyls of 3-) benzamide (TT3), gathers
Ethylene glycol is PEG2000.
In one embodiment of the invention, fat peptide molecule, DOPE, cholesterol and poly- second
The mol ratio of glycol is 15:25:45:0.75 or 15:30:40:0.75.The matter of Cas9mRNA and/or sgRNA and fat peptide molecule
Amount is (1 than scope:10)-(1:5).In a preferred embodiment, Cas9mRNA and/or sgRNA and fat peptide molecule
Quality than scope be 1:10.
The third aspect of the invention is related to a kind of biological agent, and it includes second aspect lipid nanometer of the invention
Particle, Cas9mRNA and/or the present invention the on one side described in sgRNA molecules, and pharmaceutically acceptable excipient and
Auxiliary material.
The fourth aspect of the invention is related to the preparation method of lipidic nanoparticles, the method to include：
I be dissolved in alcohol for fat peptide molecule, cholesterol and polyethylene glycol by (), obtain the first solution；And by Cas9mRNA
And/or sgRNA molecules are dissolved in water, the second solution is obtained；
(ii) mix first solution and second solution in microfluidic devices, form the lipidic nanoparticles.
Fat polypeptide in the preparation method is the (N of 1,3,5- tri-1,N3,N5- (the double dodecane aminopropyls of 3-) benzamide
(TT3)；The polyethylene glycol is PEG2000；And the sgRNA molecules are the sgRNA molecules described in present invention one side.
In a kind of specific embodiment, the radius of the lipidic nanoparticles is 80-160nm, preferably 80-120nm.
The lipidic nanoparticles for being enclosed with CRISPR-Cas9 systems of the invention and the biology comprising the lipidic nanoparticles
Preparation can safely and effectively convey the CRISPR-Cas9 systems of specific curative effect, while avoiding adenovirus delivery system band
The safety issue and toxic and side effect come.
Brief description of the drawings
Fig. 1：The method according to the invention synthesizes the lipidic nanoparticles comprising Cas9mRNA and sgRNA by microfluidic devices
Method schematic diagram.
Fig. 2：A kind of micro-fluid chip for synthesizing lipidic nanoparticles of implementation method of the invention it is micro-
Mirror photo, wherein being schematically illustrated in the passage that basis of microscopic observation is arrived.
Fig. 3：A kind of implementation method of the invention synthesizes the fat comprising Cas9mRNA or sgRNA by micro-fluid chip
The schematic diagram of matter nanoparticle.
Fig. 4：Target the sgRNA designs of HBV ayw subtype gene groups.A.HBV ayw genomic constitutions and sgRNA B1-B9's
Target area；B.sgRNA B1-B9 target the conservative Analysis of sequence.
Fig. 5：Transiently transfect anti-hepatitis virus of the CRISPR/Cas9 systems based on RNA components in HepAD38 cell lines
Effect.A. experiment flow schematic diagram；B. cells and supernatant sAg levels；C. intracellular HBsAg water
It is flat, as a result represented relative to the percentage of control group level with each group, control group only transfects Cas9mRNA, and t inspections calculate P values,
Conspicuousness is represented with *；D.Hirt methods extract HepAD38 cell lines cccDNA, T7E1 mispairing enzyme experiment detection Cas9/sgRNA targets
To shearing DNA double chain efficiency, the black triangles arrow of the purpose band after digestionMark.
Fig. 6：TT3 lipid nanometers particulate synthesizes and expression checking in vivo.A.TT3 lipid nanometer particulates synthetic method is illustrated
Figure；B. the particle diameter and distribution of dynamic light scattering measurement TT3-O3 and TT3-O14 lipid nanometer particulates；C. in extraction mouse liver cell
RNA, reverse transcription detects Cas9mRNA levels into after cDNA with real-time fluorescence quantitative PCR；D. in extraction mouse liver cell
RNA, reverse transcription detects sgRNA levels into after cDNA with real-time fluorescence quantitative PCR；6h after E.Western detection tail vein injections
Cas9 protein expression levels in mouse liver；Cas9 albumen after F.Western detection tail vein injections in 6h mouse spleens
Expression.
Fig. 7：Anti- second of the Cas9mRNA and sgRNA-B6 of TT3-O3 lipid nanometer microparticulate deliveries in HepAD38 cell lines
Hepatopathy toxic action.A. experiment flow schematic diagram；B. cells and supernatant sAg levels；C. intracellular HBeAg
Level, t inspections calculate P values, and conspicuousness is represented with *；D. real-time fluorescence quantitative PCR detection 3.5kb pregenome RNAs express water
It is flat；E.Hirt methods extract HepAD38 cell lines cccDNA, T7E1 mispairing enzyme experiment detection Cas9/sgRNA targeting shearing DNA doubles
Chain efficiency, the black triangles arrow of the purpose band after digestionMark.
Fig. 8：Anti- second of the Cas9mRNA and sgRNA-B6 of TT3-O3 lipid nanometer microparticulate deliveries in mouse hepatitis B model
Hepatopathy toxic action.A. mouse experiment schematic flow sheet；B.ELISA detects serum hepatitis B virus sAg levels；C.ELISA is examined
Survey serum hepatitis B virus e antigen levels；HBsAg level in D.ELISA detection livers；E.ELISA detects liver
Interior HBeAg level；F. hepatitis B 3.5kb pregenome RNA relative expression levels in liver；G. in liver
RcccDNA relative amounts；H.B10sgRNA causes special gene mutation in HBV DNA, and special without finding in control group
The mutation of the opposite sex.
Fig. 9：The Cas9mRNA and sgRNA-B5 or sgRNA-B10 of TT3-O3 lipid nanometer microparticulate deliveries are in mouse hepatitis B mould
Anti-HBV effect in type.Fig. 9 A show mouse experiment schematic flow sheet；Fig. 9 B show that ELISA detects serum hepatitis B
Viral surface antigen level；Fig. 9 C show that ELISA detects serum hepatitis B virus e antigen levels；Fig. 9 D show that ELISA is detected
HBsAg level in liver；Fig. 9 E show HBeAg level in ELISA detection livers；Fig. 9 F show
Hepatitis B 3.5kb pregenome RNA relative expression levels in liver are gone out；Fig. 9 G show that rcccDNA contains relatively in liver
Amount；Fig. 9 H show that B10sgRNA causes special gene mutation in HBV DNA, and specific without finding in control group
Mutation.
Specific embodiment
Unless otherwise stated, term used herein up and down of the invention has implication given below.Do not have herein
Having other terms for specifically giving implication has its common implication in the art.
1. define：
TT3：(the N of 1,3,5- tri-1,N3,N5- (the double dodecane aminopropyls of 3-) benzamide
PDMS：Dimethyl silicone polymer
DOPE：DOPE (1,2-Dioleoyl-sn-glycero-3-
phosphoethanolamine)
PEG：Polyethylene glycol
2. material and method
One embodiment of the present invention is related to new LNP delivery systems, comprising：Lipidic nanoparticles, Cas9mRNA and/
Or sgRNA, wherein lipidic nanoparticles are made up of fat peptide molecule with cholesterol and polyethylene glycol.
Show that the method according to the invention synthesizes the lipid comprising Cas9mRNA and sgRNA by microfluidic devices in Fig. 1
The schematic diagram of the method for nano particle.TT3, DOPE, PEG 2000 and cholesterol are dissolved in alcohol, and by Cas9mRNA and/
Or sgRNA is soluble in water, then two kinds of solution are rapidly mixed in microfluidic devices together, form radius for 80-160nm
Particle.
2.1. the sgRNA sequences Designs of HBVayw hypotypes are targetted
By CRISPR design tools (http://www.genome-engineering.org/crispr/) and sgRNA
Design principle, assessment HBVayw sequences on score it is higher target site design sgRNA.HBV different genes are obtained from HBVdb
Type and subtype sequences, with CLC Sequence Viewer software analysis and sequence alignment analysis target site sequence in different bases
Because of the conservative in the HBV gene sequence of type and hypotype, 10 sgRNA of targeting HBV gene conservative region are chosen and synthesize, point
B1-B10 is not named as it.
Shown in table 1 targeting HBV gene conservative region sgRNA sequence and its region in HBV gene group and
The HBV ORF of targeting.
Table 1：Target the sgRNA sequences of HBV gene conservative region
2.2.sgRNA-B1~B10 expression vector establishments
According to the sgRNA sequences of design, positive oligonucleotide sequence is obtained plus CACCG in its 5 ' end, it is complementary at it
5 ' ends of chain add AAAC, and 3 ' ends obtain reverse oligonucleotide sequence, are respectively synthesized forward and reverse oligonucleotides plus C
Sequence.The sequence of synthesis is made into its phosphorylation for 30 minutes with T4 polynueleotide kinases in 37 DEG C for the treatment of, 95 DEG C be denatured 5 minutes,
After being cooled to 25 DEG C of annealing with 1.5 DEG C/min, the double chain DNA fragment with BsmBI (or BbsI) cohesive end is obtained, it is as follows：
It is positive：5’-CACCGNNNNNNNNNNNNNNNNNNNN
Reversely：CNNNNNNNNNNNNNNNNNNNNCAAA-5’
Phosphorylation, denaturation and annealing system are：
1 100 μM of μ l forward direction oligonucleotide chains
1 100 μM of μ l reverse oligonucleotide chains
1μl 10×T4Ligation Buffer(NEB)
6.5μl ddH2O
0.5 μ l T4 polynueleotide kinases
Connected by double chain DNA fragment and with LentiCRISPRv2 (Addgene#52961) carrier of BsmbI digestions
Connect, connection product is transformed into bacillus coli DH 5 alpha competent cell, coat the LB flat boards with amicillin resistance
On, the positive bacterium colony of screening extracts positive bacterium colony plasmid and is analyzed and is sequenced, and determines the success of sgRNA expression vector establishments, name
It is LentiCRISPRv2-sgRNA (B1-B9).
By double chain DNA fragment and the PX330-U6-Chimeric_BB-CBh-hSpCas9 with BbsI digestions
(Addgene#42230) carrier is attached, and connection product is transformed into bacillus coli DH 5 alpha competent cell, coats band
Have on the LB flat boards of amicillin resistance, the positive bacterium colony of screening extracts positive bacterium colony plasmid and is analyzed and is sequenced, it is determined that
The success of gRNA expression vector establishments, is named as PX330-HBVgRNA (B1-B9).
2.3. the structure and sgRNA of the Huh7 cell lines of the sgRNA of expression Cas9 and targeting HBVayw are stablized for HBV
The analysis detection of antivirus action
The inoculation 8 × 10 per hole in six orifice plates5Individual 293T cells, treat that cell density reaches 70-80% and transfects 2 μ g
CRISPR expression plasmids, 0.4 μ g vsvG, 0.2 μ g Tat, 0.2 μ g Gag/Pol and 0.2 μ g Rev plasmids.6-8h is more after transfection
Change fresh culture, 48h collects 293T culture supernatants, 3000rpm is centrifuged 10 minutes removal precipitations, viral supernatants use immediately or
In -80 DEG C of storages.
The slow-virus infection Huh7 cell lines packed out with 293T cells, are screened with puromycin after infection 48h and obtain steady
Surely the Huh7 cell lines of Cas9 and corresponding sgRNA are expressed, Huh7-B1~B9 cell lines are named as.Condition of culture is cultivated for DMEM
Base, wherein containing 10%FBS, 2 μ g/ml Puromycin, penicillin (100U/ml) and streptomycin (100 μ g/
Ml), 37 DEG C, 5%CO2。
Huh7-B1~B9 cell lines are inoculated in 6 orifice plates respectively in the day before transfection, per hole 6 × 105It is individual, treat cell
Density is transfected when reaching 70%-80%.2.5 μ g prcccDNA, 2.5 μ g pCMV- are transfected with Lipofectamine3000
Cre, 8-12h change 37 DEG C of fresh cultures of preheating.Continue to cultivate the HBV collected in cell extraction cell after 72h
CccDNA, is detected by the Huh7 cell line transfers in stabilization expression Cas9 and the sgRNA of targeting HBVayw with T7E1 mispairing enzyme
Whether the HBV gene group sequence of dye generates mutation.
2.4. stablize expression Cas9 and targeting HBVayw sgRNA HepAD38 cell lines structure and sgRNA for
The analysis detection of HBV antivirus actions
Slow virus postoperative infection HepAD38 cell lines are packed out in 293T cells using above-mentioned same procedure, after infection 48h
The HepAD38 cell lines of the sgRNA for obtaining stabilization expression Cas9 and targeting HBV DNA are screened with puromycin, is named as
HepAD38-B1~B9 cell lines.Condition of culture is DMEM culture mediums, wherein containing 10%FBS, 2 μ g/ml Puromycin, 400 μ
G/ml G418,0.3 μ g/ml Tet, penicillin (100U/ml) and treptomycin (100 μ g/ml), 37 DEG C, 5%
CO2。
HepAD38-B1~B9 cell lines are inoculated in 6 orifice plates respectively, per hole 6 × 105It is individual, cell is collected after 72h.Carry
The HBV cccDNA in cell are taken, is detected by the sgRNA's in stabilization expression Cas9 and targeting HBVayw with T7E1 mispairing enzyme
Whether HBV gene group sequence generates mutation in HepAD38 cell lines.
2.5. detection transfection antivirus actions of the Cas9mRNA and sgRNA for HBV is analyzed in HepAD38 cell lines
HepAD38 cell lines are inoculated in 6 orifice plates, per hole 6 × 105It is individual, enter when cell density reaches 70%-80%
Row transfection.9.6ug Cas9mRNA and 9ul 20uM sgRNA, 8h 37 DEG C of preheatings of replacing are transfected with Lipofectamine3000
Fresh culture.Continue to collect cells and supernatant and cell after cultivating 72h, cells and supernatant and cell are detected with ELISA
Interior HBsAg level, intracellular HBeAg level.The HBV cccDNA in cell are extracted, is used
After T7E1 mispairing enzyme detection transfection Cas9mRNA and sgRNA in HepAD38 cell lines HBV gene group sequence whether generate it is prominent
Become.
2.6.Hirt method extracts intracellular HBVcccDNA
3000rpm is centrifuged 5 minutes after cell is collected in pancreatin digestion, is washed with PBS one time, and 3000rpm is centrifuged 5 minutes, receives
Take cell precipitation.Add 500 μ l lysates I (1%NP-40,1mM EDTA, 50mM NaCl, 10mM Tris-HCl, pH
8.0), crack 30 minutes on ice, 10000g, 4 DEG C of centrifugations collect precipitation in 20 minutes, retaining cracking supernatant is used for ELISA method detection
The change of HBsAg and e antigen presentations.1ml lysates II (1%SDS, 10mM EDTA, 150mM are added to precipitation
NaCl, 50mM Tris-HCl, pH 8.0), 4 DEG C of rotations, 60 minutes cell lysis core.Add 200 μ l 3M potassium chloride, 4 DEG C of rotations
Overnight.14000rpm, 4 DEG C are centrifuged 20 minutes, take supernatant, add isometric phenol：Chloroform：Isoamyl alcohol, acutely mixes, and room temperature is quiet
Put 7 minutes, 14000rpm, 4 DEG C are centrifuged 10 minutes, take supernatant.2 times of volume ethanols and 1/10 body are added by the supernatant volume collected
Long-pending 3M sodium acetates, precipitate 2 hours in -80 DEG C.14000rpm, 4 DEG C are centrifuged 20 minutes, abandon supernatant, add the ethanol of 1ml 75%
Washing precipitation, 14000rpm, 4 DEG C are centrifuged 20 minutes, abandon supernatant.Dry to remove residual ethanol, 20 μ l in 37 DEG C of baking ovens
DdH2O dissolution precipitations, Nanodrop determines DNA concentration.
2.7.T7E1 mispairing enzyme detection
Target site design primer PCR according to sgRNA B1-B9 on HBVayw genomes is amplified comprising sgRNA target position
The fragment of point, primer sequence is shown in table 2.
Table 2：The PCR primer of the target site design according to sgRNA B1-B9 on HBVayw genomes
PCR system is as follows：
PCR reaction cycles
PCR is reclaimed using the ultra-thin PCR primer purification kit of Axygen companies glue reclaim kit or Tiangeng company to obtain
Purpose fragment, specific method is carried out according to the specification that company provides, and Nanodrop determines DNA concentration.
To NEB Buffer 2 are added in the purpose fragment that recovery is obtained, anneal as follows：
To adding 1 μ l T7E1 restriction endonucleases, 37 DEG C, 30min in annealed product.6X Purple Loading Dye are added,
2% agarose gel electrophoresis is detected.
Cas9 cuts off target DNA double-strand in the position of the PAM upstreams 3bp of identification.The digestion purpose of prediction is shown in table 3
Clip size.
Table 3：The digestion purpose fragment size of the prediction of sgRNA
2.8.ELISA method determines the change of HBsAg and e antigen presentations
Supernatant after cells and supernatant or cell cracking is collected, according to hepatitis B virus surface after being diluted with proper ratio
Diagnostic antigen kit and hepatitis B virus e antigen diagnostic kit (China of Shanghai section) specification measurement HBsAg and e
Antigen, comprises the following steps that：
Sample to be tested (μ l of 75 μ l, e antigen of surface antigen 50) is added per hole, if positive and negative compare each holes, is added per hole
The negative control (or positive control) isometric with testing sample, and set the hole of blank 1.
Surface antigen reaction plate is incubated 1 hour (e antigen-reactives plate is without being incubated) in 37 DEG C, and enzyme conjugates 50 is added per hole
μ l (except blank), gently shake 10 seconds by hand, after covering reaction plate with mounting paper, reaction plate is put into 37 DEG C and is incubated 30
Minute.
Reaction plate is taken out, mounting paper, washing reaction plate 5 times is torn off：Liquid in hole is discarded, is filled with working concentration cleaning solution
Each hole, stands 30-60 seconds, dries, and after being repeated 5 times, is patted dry on clean blotting paper.
Washing adds developer A, each 50 μ l of developer B in each hole immediately after terminating, gently shake 10 seconds by hand.With
After mounting paper covering reaction plate, reaction plate is put 37 DEG C and is incubated 10-15 minutes.
50 μ l terminate liquids are added in each hole, concussion reaction plate 5 seconds is allowed to fully mixing.Use ELIASA reading, wavelength
450nm。
2.9. inhibitory action of detection sgRNA-B5, the B6 for hbv replication is analyzed in mouse model
6ug prcccDNA, 4ug pCMV-Cre and 20ug PX330/PX330-B5/PX330-B6 plasmids are dissolved in
In 1.7mlPBS, with 2ml injector for medical purpose add 1ml syringe needles will dissolved with the PBS of plasmid in 5-7 seconds by tail it is quiet
Arteries and veins is expelled in wild type C57BL/6 Mice Bodies.
Mouse after injection supports pneumoretroperitoneum injection 200-250 μ l Avertin anesthesia in 3 days, is taken a blood sample from eye socket with capillary
50-100ul.Pneumoretroperitoneum injection 200-250 μ l Avertin anesthesia in 5 days is supported, eyeball is won and is taken blood, place after death cuts open and takes liver,
Liquid nitrogen flash freezer is after -80 DEG C of preservations.The mouse blood of collection after 4 DEG C of refrigerators stand overnight, 3500rpm, 4 DEG C centrifugation
Serum is separated after 15min.After taking a small amount of mice serum dilution hepatitis b virus s antigen therein and e are detected with ELISA
Antigen levels.Protein concentration is determined after taking a small amount of liver lysate homogenate, cracking on ice, each mouse liver sample protein is dense
After degree standardization hepatitis b virus s antigen therein and e antigen levels are detected with ELISA.Hirt methods extract mouse liver
In cccDNA, with T7E1 mispairing enzyme test detection by sgRNA process mouse liver in HBV gene group sequence whether produce
Mutation is given birth to.
2.10. the synthesis of the lipid nanometer particulate of parcel Cas9mRNA or sgRNA
It is prepared by the PDMS micro-fluid chips for being 2.10.1 used to synthesize lipid nanoparticle
Dimethyl silicone polymer (PDMS) prepolymer and curing agent are sufficiently mixed in beaker, are put into vacuum tank and are removed
Bubble in mixture.It is cast on the SU-8 moulds using lithography process, baking oven internal heating curing is put into, from SU-8 moulds
Take the PDMS blocks after solidification off.The entrance and exit position punching of structure, PDMS blocks are cut along chip boundary on PDMS blocks.It is right
PDMS blocks do oxygen gas plasma cleaning, by PDMS block alignments, pressing bonding.Every time oxygen is used before lipid nanoparticle is synthesized
Plasma does hydrophilic treated.
2.10.2 lipid nanometer particulate synthesis
Two kinds of formulas (O3 is formulated and O14 formulas) of each lipid nanometer particulate are shown in table 4 below：
Table 4：The composition of lipid nanometer particulate
Synthetic method：Liquid storage is obtained with each lipid components of anhydrous alcohol solution, by the matter of the Cas9mRNA or sgRNA of parcel
The ratio of amount and each lipid components calculates each lipid components institute expense, lipid components and the water phase of the ethanol phase that will be prepared
RNA components push microfluidic chip devices together and make its quick mixing, being assembled into internal package by electrostatic interaction has
The lipid nanometer particulate of RNA.Lipid nanometer particulate for mouse experiment in vivo is needed with 3.5K MWCO's before tail vein injection
Dialysis is dialysed 1 hour in being stuck in PBS, and dialysis is completed after 4 DEG C of preservations.Of the invention one is showed in Fig. 3
Plant implementation method and pass through the schematic diagram that micro-fluid chip synthesizes the lipid nanometer particulate comprising Cas9mRNA or sgRNA.
The sign of lipid nanometer particulate：Lipid nanometer diameter of particle and the distribution of synthesis are measured with dynamic light scattering (DLS),
The parcel efficiency of lipid nanometer particulate is detected with Quant-iT RiboGreen RNA Kit.
2.10.3 Quant-iT RiboGreen kits detect the parcel efficiency of lipid nanometer particulate
Dilute 20 × TE solution with DEPC water and be configured to 1 × TE working solutions, the lipid nanometer particulate that will synthesize it is a with 1 ×
TE working solutions press 1：100 dilution proportion, portion is with containing Triton-X100, (1 × TE working solutions of final concentration of 1%) press 1：
100 dilution proportion, room temperature places 15 minutes to destroy lipid nanometer particulate so as to discharge the RNA of parcel.
It is each in transparent flat 96 orifice plate to add 100 μ l sample diluting liquids, while it is 0,10,50,90 to set concentration,
The RNA standard items dilution holes of 100ng/ml.100 μ l Reagent A working solutions are added in each hole, room temperature lucifuge is reacted 5 minutes
Fluorescence microplate reader reading, exciting light 480nm, launching light 520nm are used afterwards.Reading according to standard items draws standard curve, calculates
The RNA concentration of each sample, the parcel efficiency calculation formula of lipid nanometer particulate is：(1- is added without Triton-X100 reacting holes
RNA concentration/addition Triton-X100 reacting hole RNA concentration) × 100%
2.11. inhibitory action of the detection lipid nanometer particulate for hbv replication is analyzed in mouse model
PrcccDNA and pCMV-Cre is expelled in Mice Body by the method for Hydrodynamic injection, B-mode liver is built
The mouse model of scorching virus.With capillary from eye socket blood sampling 50-100 μ l after 2 days, B-type hepatitis are detected with ELISA after separating serum
Malicious sAg levels.It is grouped according to serum hepatitis B virus antigen levels, it is ensured that average level is without significant difference between group.Point
The lipid nanometer particulate of the sgRNA or control sgRNA of Cas9mRNA and targeting HBV He Cheng not be wrapped up, in Hydrodynamic injection
Carry out tail vein injection within the 3rd day and the 4th day afterwards.5th day intraperitoneal injection 200ul Avertin anesthetized mice, extracts eyeball and takes blood,
Place after death cuts abdominal cavity open, takes out liver, and liquid nitrogen flash freezer is preserved.Serum is separated, serum hepatitis B virus table is detected with ELISA after dilution
Face antigen and e antigen levels.To take after liver homogenate and determine protein concentration with BCA methods and standardize, detected with ELISA kit
HBsAg and e antigen levels in liver.Trizol methods extract liver rna, and reverse transcription is into using fluorescence reality after cDNA
When quantitative PCR detection HBV 3.5kb rna contents therein.Hirt methods extract HBV cccDNA, and use fluorescence real-time quantitative PCR
Detect the changes of contents of HBV cccDNA therein.Fluorescence real-time quantitative primer is listed in table 5.
Table 5：Fluorescence real-time quantitative primer
The ratio of the quality of Cas9mRNA or sgRNA and each lipid components can be with the property of lipid components and for exempting from
The animal of epidemic disease and change.Number of ways can be used for using the lipid nanometer for being enclosed with CRISPR-Cas9 systems of the invention
Grain and the biological agent comprising the lipidic nanoparticles：Including in subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal and spleen
Etc..
3. result
3.1. the sgRNA designs and checking of targeting HBV ayw subtype gene groups
The sgRNA that website obtains difference ORF in targeting HBV ayw subtype gene groups is designed according to CRISPR, from HBVdb
The genome sequence for obtaining HBV different subtypes is compared, and is selected from the target site for finding positioned at conservative upper zone
SgRNA sequences, are named as B1-B9.Target areas and conservative Analysis result such as figure of the B1-B9 in HBV ayw subtype gene groups
Shown in 4A and Fig. 4 B.Fig. 4 shows the sgRNA designs of targeting HBV ayw subtype gene groups, wherein Fig. 4 A.HBV ayw genomes
Into and sgRNA B1-B9 target area；Fig. 4 B are the conservative Analysis that sgRNA B1-B9 target sequence.
3.2. anti-hepatitis virus of the CRISPR/Cas9 systems based on RNA components in HepAD38 cell lines are transiently transfected
Effect and the external identification of sgRNA effects
Because the gene editing of CRISPR System-mediateds can be completed by snap, therefore demonstrated
The CRISPR systems of the targeting HBV ayw of LentiCRISPRv2 slow virus carriers delivering are in cell line to the antiviral of hepatitis B
After effect, we have evaluated transient transfection Cas9mRNA and external conjunction in the HepAD38 cell lines of stabilization expression HBV gene group
Into sgRNA B1-B9 antivirus action.Fig. 5 shows that transiently transfecting the CRISPR/Cas9 systems based on RNA components exists
Anti-HBV effect in HepAD38 cell lines, wherein Fig. 5 A are experiment flow schematic diagram；Fig. 5 B-5C are respectively cell training
The intracellular HBsAg level of supernatant is supported, is as a result represented relative to the percentage of control group level with each group, it is right
According to group, only transfection Cas9mRNA, t inspections calculate P values, and conspicuousness is represented with *, and Fig. 5 D are that Hirt methods extract HepAD38 cell lines
CccDNA, T7E1 mispairing enzyme experiment detection Cas9/sgRNA targeting shearing DNA double chain efficiency, the purpose band black after digestion
Triangular arrowheads are identified.
Method shown in Fig. 5 A comprises the following steps：With TransIT-mRNA points in the HepAD38 cell lines of inoculation
Cell and culture supernatant Zhuan Ran not be collected after Cas9mRNA and B1-B9sgRNA, 72h.ELISA detection cells and supernatants are neutralized
Intracellular hepatitis B surface antigen level, as a result shows compared with the only control group of transfection Cas9mRNA, sgRNA B3, B5, B6
Intracellular and secretion the hepatitis B surface antigen level (Fig. 5 B-5C) of HepAD38 systems can be significantly reduced with B7.Hirt methods are extracted
CccDNA in HepAD38 cells, T7E1 mispairing enzyme are test result indicate that B1, B3, B4, B5, B6, B7, B8 can target shearing
HBV cccDNA, but it is relatively low (Fig. 5 D) to be compared to the cell line shear efficiency of stabilization expression Cas9/sgRNA.
3.3. the synthesis of TT3 lipid nanometers particulate and expression checking in vivo of parcel CRISPR system RNA components
Far apart especially on molecular weight because mRNA and siRNA have different characteristics, Cas9mRNA is about
4200nt, makes it increasingly difficult when being wrapped up using lipid nanometer particulate.We are obtained for passing by in-vitro screening and optimization
The new TT3 lipid nanometer particulates of mRNA are sent, synthetic method schematic diagram is as shown in Figure 5A.The two kinds of formulas obtained by optimization
TT3 lipid nanometer particulates：TT3-O3 and TT3-O14.It is corresponding as the synthesis of the initial concentration of Cas9mRNA using 60 μ g/ml
TT3-O3 and TT3-14 lipid nanometer particulates, its particle diameter of dynamic light scattering measurement and distribution, as a result display are compared to TT3-14,
The lipid nanometer particle radius of TT3-O3 formula synthesis are smaller and distribution more concentrates (Fig. 6 B).TT3 fat is shown in Fig. 6 A-F
Matter nanoparticle synthesizes and expression checking in vivo, and wherein Fig. 6 A are TT3 lipid nanometer particulate synthetic method schematic diagrames；Fig. 6 B show
The particle diameter and distribution of dynamic light scattering measurement TT3-O3 formulas and TT3-O14 formula lipid nanometer particulates；Fig. 6 C are small to extract
RNA in hepatocytes, reverse transcription detects Cas9mRNA levels into after cDNA with real-time fluorescence quantitative PCR；Fig. 6 D show and carry
Take the RNA in mouse liver cell, reverse transcription with real-time fluorescence quantitative PCR into detecting sgRNA levels after cDNA；Fig. 6 E show
Cas9 protein expression levels after Western detection tail vein injections in 6h mouse livers；Fig. 6 F show that Western detects tail
Cas9 protein expression levels after intravenous injection in 6h mouse spleens.
TT3-O3 formula lipids using 60 μ g/ml as the initial concentration of RNA synthesis parcel Cas9mRNA and B6sgRNA are received
Rice particulate, wraps up the TT3-O3 lipid nanometer particulates of Cas9mRNA and B6sgRNA in C57BL/6 mouse by tail vein injection
(5 μ g/20g body weight), mouse liver detection TT3-O3 formula lipid nanometer microparticulate deliveries are collected in different time points
The expression of Cas9mRNA and sgRNA.After RT-qPCR results in Fig. 6 C and Fig. 6 D are displayed in tail vein injection 6h, in liver cell
Have been able to detect Cas9mRNA, sgRNA, Cas9mRNA, sgRNA have descended to relatively low level after 12h.Tail vein is noted
After penetrating the TT3-O3 formula lipid nanometer particulates 6h of parcel Cas9mRNA, the protein expression of Cas9 is able to detect that in liver
(Fig. 6 E) and (Fig. 6 F) is can't detect in spleen, it was demonstrated that TT3-O3 formula lipid nanometer microparticulate delivery RNA components mainly exist
Expression high in liver.
Embodiment
Embodiment 1:The Cas9mRNA and sgRNA-B6 of TT3-O3 formula lipid nanometer microparticulate deliveries are in HepAD38 cell lines
In anti-HBV effect
The RNA groups of parcel are delivered in the cell line that TT3-O3 formulas are cultivated in vitro by adding other materials without external source
Point.Therefore we have evaluated TT3-O3 lipid nanometer particulates and pass in the HepAD38 cell lines of stabilization expression HBV gene group first
The antivirus action of the Cas9mRNA and sgRNA B6 for sending.
Fig. 7 shows the Cas9mRNA and sgRNA-B6 of TT3-O3 formula lipid nanometer microparticulate deliveries in HepAD38 cells
Anti-HBV effect in system.Fig. 7 A show the schematic diagram of experiment flow；Fig. 7 B-7C respectively illustrate cells and supernatant
With intracellular HBeAg level, t inspection calculate P values, conspicuousness is represented with *；Fig. 7 D show real time fluorescent quantitative
PCR detects 3.5kb pregenome RNA expressions；Fig. 7 E show that Hirt methods extract HepAD38 cell lines cccDNA, T7E1
Mispairing enzyme experiment detection Cas9/sgRNA targeting shearing DNA double chain efficiency, the purpose band black triangles arrow mark after digestion
Know.
Show that TT3-O3 is formulated the preparation method of lipid nanometer particulate in Fig. 7 A：In the HepAD38 cell lines of inoculation
Addition is enclosed with the TT3-O3 lipid nanometer particulates of Cas9mRNA and sgRNA-B6, sgRNA-sgGFP and sgRNA-B6S2 respectively
(sgRNA-B6 sequences are upset at random) collects cell and culture supernatant as a control group, after 48h.Cas9mRNA and sgRNA-B6 roots
Form lipid nanometer particulate respectively according to the method shown in Fig. 7, its can with it is sequential delivering or deliver simultaneously.ELISA detections are thin
With intracellular hepatitis B surface antigen and e antigen levels in born of the same parents' culture supernatant, as a result show compared with control group, sgRNA-B6 realities
Testing group can significantly reduce intracellular and secretion the hepatitis B virus e antigen level (Fig. 7 B-7C) of HepAD38 systems, base before intracellular 3.5kb
Because a group RNA relative expression levels also have significant decline (Fig. 7 D).Hirt methods extract the cccDNA, T7E1 in HepAD38 cells
Mispairing enzyme is test result indicate that the Cas9mRNA and sgRNA-B6 of TT3-O3 lipid nanometer microparticulate deliveries can target shearing HBV
CccDNA, but shear efficiency is relatively low (Fig. 7 E), this be because most of cccDNA is degraded after shearing, thus cannot
Detected in final T7E1 mispairing enzyme experiment.
Confirming the Cas9mRNA and sgRNA-B6 of TT3-O3 formula lipid nanometer microparticulate deliveries in HepAD38 cell lines
In anti-HBV effect after, we further have evaluated its anti-HBV effect in mouse hepatitis B model.
Fig. 8 shows that the Cas9mRNA and sgRNA-B5 or sgRNA-B10 of TT3-O3 formula lipid nanometer microparticulate deliveries exist
Anti-HBV effect in mouse hepatitis B model.Fig. 8 A show mouse experiment schematic flow sheet；Fig. 8 B show that ELISA is examined
Survey serum hepatitis B virus e antigen levels；Fig. 8 C show that ELISA detects serum hepatitis B virus rna level；Fig. 8 D show
Hepatitis B virus DNA level in ELISA detection livers.
Show the Cas9mRNA and sgRNA-B6 of TT3-O3 formula lipid nanometer microparticulate deliveries in mouse hepatitis B in Fig. 8 A
Experiment flow in model：Built in C57BL/6 mouse by Hydrodynamic injection prcccDNA and pCMV-Cre plasmid
Hepatitis B infected model, is grouped at random according to Mouse Weight, according to Cas9 albumen and the dynamic variation of sgRNA expressions, in
The TT3-O3 of 24h tail vein injections parcel Cas9mRNA is formulated lipid nanometer particulate (200 μ l/10g body weight), tail vein note after 6h
The TT3-O3 for penetrating parcel sgRNA-B5 or control group sgRNA is formulated lipid nanometer particulate (100 μ l/10g body weight).Gathered after 48h
Mouse peripheral blood separates serum, collects mouse liver, the HBeAg level in ELISA detection livers.Result shows,
The anti-hepatitis virus that the Cas9mRNA and sgRNA-B6 of TT3-O3 formula lipid nanometer microparticulate deliveries have in mouse hepatitis B model
Effect, and have significantly for HBeAg (Fig. 8 B) in mouse hepatitis B model, RNA (Fig. 8 C) and cccDNA (Fig. 8 D)
Targeting destruction.
Embodiment 2：The Cas9mRNA and sgRNA-B5 or sgRNA-B10 of TT3-O3 formula lipid nanometer microparticulate deliveries are small
Anti-HBV effect in mouse hepatitis B model
Its anti-HBV effect in mouse hepatitis B model is have evaluated in the method similar to embodiment 1.
Fig. 9 shows that the Cas9mRNA and sgRNA-B5 or sgRNA-B10 of TT3-O3 formula lipid nanometer microparticulate deliveries exist
Anti-HBV effect in mouse hepatitis B model.Fig. 9 A show mouse experiment schematic flow sheet；Fig. 9 B show that ELISA is examined
Survey serum hepatitis B virus sAg levels；Fig. 9 C show that ELISA detects serum hepatitis B virus e antigen levels；Fig. 9 D show
HBsAg level in ELISA detection livers；Fig. 9 E show HBeAg in ELISA detection livers
Level；Fig. 9 F show hepatitis B 3.5kb pregenome RNA relative expression levels in liver；Fig. 9 G are shown in liver
RcccDNA relative amounts；Fig. 9 H show that B10sgRNA causes special gene mutation in HBV DNA, and do not have in control group
It is found specific mutation.
Show that TT3-O3 is formulated the Cas9mRNA and sgRNA-B5 or sgRNA-B10 of lipid nanometer microparticulate delivery in Fig. 9 A
In the experiment flow in mouse hepatitis B model：By Hydrodynamic injection prcccDNA and pCMV-Cre plasmid in C57BL/
Hepatitis B infected model is built in 6 mouse, is grouped at random according to Mouse Weight, according to the dynamic of Cas9 albumen and sgRNA expressions
Mechanical change, lipid nanometer particulate (200 μ l/10g body weight) is formulated in the TT3-O3 of 24h tail vein injections parcel Cas9mRNA,
The TT3-O3 formula lipid nanometer particulates of tail vein injection parcel sgRNA-B5 or sgRNA-B10 or control group sgRNA after 6h
(100 μ l/10g body weight).Mouse peripheral blood is gathered after 48h and separates serum, collect mouse liver, ELISA detection serum and liver
Interior HBsAg and e antigen levels.ELISA results are shown compared to sgGFP control groups, sgRNA:B5 and B10
The serum hepatitis B virus surface antigen and e antigens (Fig. 9 B and Fig. 9 C) of experimental mice, liver inner surface antigen and e antigens have aobvious
Write and decline (Fig. 9 D-9E), 3.5kb pregenome RNAs relative expression levels are also decreased significantly (Fig. 8 F) in liver.Extract liver
STb gene, its changes of contents is detected after Plasmid-Safe DNaseI digestion process with cccDNA special primers, is as a result shown
Control group is compared to, cccDNA contents are decreased significantly (Fig. 9 G) in sgRNA B10 experimental mice livers.Surveyed by depth
Sequence, we have further confirmed that there is specific mutation (Fig. 9 H) really in the hepatitis B virus DNA of the mouse of B10 treatment.With
Upper result shows that the Cas9mRNA and sgRNA-B5 or sgRNA-B10 of TT3-O3 formula lipid nanometer microparticulate deliveries are in mouse second
There is certain anti-HBV effect in liver model, and have significant targeting destruction for HBV cccDNA in mouse hepatitis B model
Effect.
******
All publications and patents mentioned hereinabove are incorporated herein by reference.Methods described of the invention and
The various change of system is to those skilled in the art obviously without departing from scope and spirit of the present invention.Although
With reference to specifically preferred embodiment present invention is described, it should be appreciated that the invention is not restricted to these specific embodiments.
In fact, those skilled in the art realizes that the various changes of the mode of the invention are included in this hair at can be used for of knowing
In bright scope, the scope of the present invention is defined by the claims.
Claims (11)
1. the sgRNA molecules of HBV gene conservative region are targetted, and it has one of following nucleotide sequence：
GAGGTGAAGCGAAGTGCACA(SEQ ID NO:1)；
CCACCCAAGGCACAGCTTGG(SEQ ID NO:2)
CGGGGAGTCCGCGTAAAGAG(SEQ ID NO:3)
AAGCCACCCAAGGCACAGCT(SEQ ID NO:4)
GAAGCGAAGTGCACACGGTC(SEQ ID NO:5)
AGAAGATGAGGCATAGCAGC(SEQ ID NO:6)
CAAGCCTCCAAGCTGTGCCT(SEQ ID NO:7)
GGGGCGCACCTCTCTTTACG(SEQ ID NO:8)
GGACTTCTCTCAATTTTCTA(SEQ ID NO:9)；Or
TCCTCTGCCGATCCATACTG(SEQ ID NO:10)。
2. lipidic nanoparticles, by fat peptide molecule, DOPE, cholesterol and polyethylene glycol with 15:25:
45:0.75 or 15:30:40:0.75 mol ratio is constituted, wherein the lipidic nanoparticles wrap up Cas9mRNA and as right will
The sgRNA molecules described in 1 are sought, and wherein Cas9mRNA and/or sgRNA and the quality of fat peptide molecule are (1 than scope:
10)–(1:5)。
3. lipidic nanoparticles according to claim 2, wherein, the fat peptide molecule is 1,3,5- tri- (N1,N3,N5-
(the double dodecane aminopropyls of 3-) benzamide (TT3), the polyethylene glycol is PEG2000, and the sgRNA molecules are such as right
It is required that the sgRNA molecules described in 1.
4. lipidic nanoparticles according to Claims 2 or 3, wherein, the fat peptide molecule is the polymer of fat polypeptide.
5. lipidic nanoparticles according to Claims 2 or 3, wherein, the quality of Cas9mRNA and sgRNA and fat peptide molecule
It is 1 than scope:10.
6. lipidic nanoparticles according to Claims 2 or 3, wherein, the radius of the lipidic nanoparticles is 80-
160nm。
7. lipidic nanoparticles according to claim 6, wherein, the radius of the lipidic nanoparticles is 80-120nm.
8. lipidic nanoparticles, Cas9mRNA and as described in the appended claim 1 as any one of claim 2 to 7 are included
SgRNA molecules, and pharmaceutically acceptable excipient and auxiliary material pharmaceutical composition.
9. the preparation method of lipidic nanoparticles, methods described includes：
I be dissolved in alcohol for fat peptide molecule, DOPE, cholesterol and polyethylene glycol by (), obtain first
Solution；And Cas9mRNA and/or sgRNA molecules are dissolved in water, obtain the second solution；
(ii) mix first solution and second solution in microfluidic devices, form the lipidic nanoparticles.
10. method according to claim 9, wherein, the fat peptide molecule is 1,3,5- tri- (N1,N3,N5- (3- double ten
Dialkylamino propyl group) benzamide (TT3), the polyethylene glycol is PEG2000, and the sgRNA molecules are as in claim 1
Described sgRNA molecules.
A kind of 11. methods for treating hepatitis B, methods described include to subject give effective dose such as claim 1-7 institutes
The step of stating lipidic nanoparticles or claim 8 described pharmaceutical composition.
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