CN111363761A - Method for promoting AAV-mediated gene expression by using cationic polymer DNA complex - Google Patents

Method for promoting AAV-mediated gene expression by using cationic polymer DNA complex Download PDF

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CN111363761A
CN111363761A CN202010184761.2A CN202010184761A CN111363761A CN 111363761 A CN111363761 A CN 111363761A CN 202010184761 A CN202010184761 A CN 202010184761A CN 111363761 A CN111363761 A CN 111363761A
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邵嘉红
张宗德
谈鹏程
赵晓明
吴相�
盛必成
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Suzhou Jiheng Gene Technology Co ltd
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Abstract

The invention discloses a method for promoting AAV-mediated gene expression by using a cationic polymer DNA complex. The gene expression method comprises the following steps: co-transfecting the host cell, preferably after co-incubating the AAV vector comprising the gene of interest, the helper DNA with a positively charged cell transfection reagent; alternatively, a host cell is transfected with either one of a cationic multimeric DNA complex formed by incubating helper DNA with a positively charged cell transfection reagent and an AAV vector comprising a gene of interest, and then transfected with the other within a set time. The invention combines the cationic polymer with AAV-mediated gene therapy by utilizing the characteristics of the cationic polymer, thereby improving AAV-mediated gene expression effect, improving gene expression efficiency, enhancing AAV-mediated gene therapy effect, and having important significance for promoting the development of the fields of gene therapy, cell engineering and the like.

Description

Method for promoting AAV-mediated gene expression by using cationic polymer DNA complex
Technical Field
The invention relates to a method for promoting AAV-mediated gene expression, in particular to a method for promoting AAV-mediated gene expression by using a cationic polymer DNA complex, belonging to the technical field of genetic engineering.
Background
Gene therapy is an important technical means for treating tumors, infectious diseases and genetic diseases of human beings at present, and means an effective scheme for introducing specific exogenous genes into target cells in various ways to treat or even completely cure the diseases. The most critical technology of gene therapy is to stably and safely introduce exogenous genes for therapy into target cells by selecting a proper mode or a proper vector, and to perform efficient therapy. The delivery efficiency of different modes and vectors greatly influences subsequent research and clinical treatment effects. Therefore, it is necessary to develop a method for efficiently promoting expression of a vector.
At present, vectors for gene delivery are largely classified into viral vectors and non-viral vectors. AAV has been widely used as an important viral vector in scientific research and clinical applications. AAV is a parvovirus with natural defects, no coating and no pathogenicity. The AAV genome is a linear, single-stranded dna (ssdna) molecule consisting of 4680 nucleotides, containing a terminal repeat (ITR) of 145 bases at each end. With the development of research of AAV, it has become a major genetic vector in scientific research and clinical application. It is therefore highly desirable to effectively improve AAV-mediated gene therapy.
Disclosure of Invention
The main objective of the present invention is to provide a method for promoting AAV-mediated gene expression using cationic polymer DNA complex, so as to overcome the disadvantages of the prior art.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a method for promoting AAV-mediated gene expression by using a cationic polymer DNA complex, which comprises the following steps:
co-transfecting host cells after co-incubation of an AAV vector containing a target gene, auxiliary DNA and a positively charged cell transfection reagent;
alternatively, a host cell is transfected with either one of a cationic multimeric DNA complex formed by incubating helper DNA with a positively charged cell transfection reagent and an AAV vector comprising a gene of interest, and then transfected with the other within a set time.
In some preferred embodiments, the gene expression method comprises:
co-transfecting host cells after co-incubation of an AAV vector containing a target gene, auxiliary DNA and a positively charged cell transfection reagent;
or, transfecting host cells after co-incubation of helper DNA with a positively charged cell transfection reagent, and then transfecting the host cells with an AAV vector comprising a gene of interest for a selected time period, the set time period being less than 24 hours, preferably less than 12 hours, more preferably within 6 hours;
or, transfecting a host cell with an AAV vector comprising a target gene, followed by transfecting said host cell after co-incubation of helper DNA with a positively charged cell transfection reagent for a selected time, said set time being less than 24 hours, preferably less than 6 hours; wherein simultaneous transfection is optimal.
Further, the gene expression method comprises:
mixing the auxiliary DNA and the cell transfection reagent with positive charge in a selected buffer solution for reaction to form a cationic polymer DNA complex; and
transfecting a host cell with the cationic multimeric DNA complex.
Compared with the prior art, the invention has the beneficial effects that:
the method for promoting the AAV-mediated expression capacity of the exogenous gene and enhancing the expression time of the exogenous gene by using the cationic polymer DNA compound combines the cationic polymer with AAV-mediated gene therapy by using the characteristics of the cationic polymer, so that the AAV-mediated gene expression effect is improved, the gene expression efficiency is improved, the AAV-mediated gene therapy effect is enhanced, and the method has a vital significance in promoting the development of the fields of gene therapy, cell engineering and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of SDS-PAGE electrophoresis to detect the purity of rAAV vector in example 1 of the present invention.
FIG. 2A is a fluorescent microscope image of AAV expression mediated by cationic polymer PEI-DNA at different cell densities in example 1 of the present invention.
FIG. 2B is a graph showing the flow analysis results of AAV expression mediated by cationic polymer PEI-DNA at different cell densities in example 1 of the present invention.
FIG. 3A is a fluorescent microscope image of gene expression of AAV vectors of different MOI mediated by cationic polymer PEI-DNA in example 1 of the present invention.
FIG. 3B is a graph showing the results of flow analysis of gene expression of AAV vectors of different MOI mediated by cationic polymer PEI-DNA in example 1 of the present invention.
FIG. 4A is a fluorescent microscope image of gene expression of AAV vector mediated by cationic polymer PEI-DNA at various times in example 1 of the present invention.
FIG. 4B is a graph showing the results of flow analysis of gene expression of AAV vectors mediated by cationic polymer PEI-DNA at various times in example 1 of the present invention.
FIG. 5A is a fluorescent microscope image of gene expression of AAV vector mediated by cationic polymer PEI-DNA at various time points in example 1 of the present invention.
FIG. 5B is a graph showing the results of flow analysis of gene expression of AAV vectors mediated by cationic polymer PEI-DNA at various time points in example 1 of the present invention.
FIG. 6A is a fluorescent microscope graph of the gene expression of AAV vector mediated by cationic polymer PEI-DNA at different ratios in example 1 of the present invention.
FIG. 6B is a graph showing the results of flow analysis of the gene expression of AAV vectors mediated by cationic polymer PEI-DNA at different ratios in example 1 of the present invention.
Detailed Description
The rAAV vector becomes the most promising virus vector with the advantages of non-pathogenicity, low immunogenicity, stable expression of target genes and the like, and is widely applied to gene therapy of retinopathy. The cationic polymer as non-viral vector has the advantages of no infectivity, wide source, capacity of being prepared in great amount, etc. and is used widely in gene therapy, cell therapy and other basic research. Cationic polymer polyvinyl amide (PEI) is a synthetic polymer with molecular weight of about 200Da to 1500kDa, mainly in two structural forms of branching and linear, because the PEI has good proton sponge effect and high gene delivery efficiency, and the PEI has more excellent delivery effect.
In view of the deficiencies of the prior art, the inventors of the present invention have made extensive studies and practice to propose a technical solution of the present invention, which combines the cationic multimer with AAV-mediated gene therapy by mainly utilizing the properties of the cationic multimer, thereby improving AAV-mediated gene expression effects and enhancing AAV-mediated gene therapy effects. Through the joint use of the cationic polymer and the AAV vector, a method for effectively improving AAV-mediated gene expression is provided, namely, a method for efficiently improving AAV-mediated foreign gene expression capacity and enhancing AAV-mediated foreign gene expression time by using a cationic polymer DNA complex has a vital significance in promoting the development of the fields of gene therapy, cell engineering and the like.
The technical solution, its implementation and principles, etc. will be further explained as follows.
One aspect of the embodiments of the present invention provides a method for promoting AAV-mediated gene expression using a cationic multimeric DNA complex, comprising:
co-transfecting host cells after co-incubation of an AAV vector containing a target gene, auxiliary DNA and a positively charged cell transfection reagent;
alternatively, a host cell is transfected with either one of a cationic multimeric DNA complex formed by incubating helper DNA with a positively charged cell transfection reagent and an AAV vector comprising a gene of interest, and then transfected with the other within a set time.
In some preferred embodiments, the gene expression method comprises:
co-transfecting host cells after co-incubation of an AAV vector containing a target gene, auxiliary DNA and a positively charged cell transfection reagent;
or, transfecting host cells after co-incubation of helper DNA with a positively charged cell transfection reagent, and then transfecting the host cells with an AAV vector comprising a gene of interest for a selected time period, the set time period being less than 24 hours, preferably less than 12 hours, more preferably within 6 hours;
alternatively, the host cell is transfected with an AAV vector comprising the gene of interest and then transfected after co-incubation of the helper DNA with a positively charged cell transfection reagent for a selected time, said set time being less than 24h, preferably less than 6 h.
Further, simultaneous transfection is the best protocol in the above methods.
In some preferred embodiments, the gene expression method comprises:
co-transfecting host cells after co-incubation of an AAV vector containing a target gene, auxiliary DNA and a positively charged cell transfection reagent, and performing gene expression for more than 24h, preferably more than 48h, and particularly preferably more than 72 h;
alternatively, a host cell is transfected with either one of a cationic multimeric DNA complex formed by incubating a helper DNA with a positively charged cell transfection reagent and an AAV vector containing a target gene, and then the other one is transfected within a set time period, and gene expression is performed for 24 hours or more, preferably 48 hours or more, and particularly preferably 72 hours or more.
In some preferred embodiments, the gene expression method comprises:
when the host cell is transfected with either one of the AAV vector containing the target gene, the helper DNA, and the positively charged cell transfection reagent after co-incubation, or the cationic multimeric DNA complex formed by incubation of the helper DNA with the positively charged cell transfection reagent, and the AAV vector containing the target gene, the cell concentration is 10% to 100%, preferably 80% to 90%.
In some preferred embodiments, the AAV vector comprising the gene of interest has a MOI <100000, preferably < 10000.
In some preferred embodiments, the gene expression method comprises:
mixing the auxiliary DNA and the cell transfection reagent with positive charge in a selected buffer solution for reaction to form a cationic polymer DNA complex; and
transfecting a host cell with the cationic multimeric DNA complex.
In some more specific embodiments, the gene expression method comprises:
mixing the auxiliary DNA and the cell transfection reagent with positive charge in a selected buffer solution for reaction to form a cationic polymer DNA complex; and
transfecting a host cell with the cationic multimeric DNA complex.
Further, the gene expression method specifically comprises: preparing auxiliary DNA and cell transfection reagent into a first transfection solution and a second transfection solution by using a selected buffer solution respectively;
the second transfection solution is added in portions to the first transfection solution, thereby reacting to form cationic polymer DNA complexes.
Further, the gene expression method specifically comprises: the second transfection solution is added dropwise to the first transfection solution and incubated to react to form a cationic multimeric DNA complex.
Further, the selected buffer includes HBS buffer, but is not limited thereto.
In some preferred embodiments, the mass to volume ratio of the helper DNA to the cell transfection reagent is 1 μ g: 1 to 5. mu.l.
In some preferred embodiments, the cell transfection reagent comprises a polycationic polymer, preferably selected from linear polyethylenimine, especially preferably linear Polyethylenimine (PEI) with a molecular weight of 25kDa, but is not limited thereto.
In some preferred embodiments, the helper DNA in the cationic multimeric DNA complexes to which the present invention relates may be various forms of DNA molecules including plasmids.
Further, the auxiliary DNA includes any one of circular plasmid, linear fragment, double-stranded DNA, single-stranded DNA, cell genome enzyme-cut fragment, and the like, preferably circular plasmid such as pUC57 plasmid (pUC57-SaCas 9-shRNA-bakbone), but is not limited thereto.
In some preferred embodiments, the AAV vector containing the gene of interest includes AAV6-EGFP, but is not limited thereto.
To further optimize the protocol, the present invention involves multiple validation, including different forms of helper DNA, amount of helper DNA used, and timing of transfection.
For example, the present invention can employ a three plasmid system to achieve packaging of AAV viruses. Wherein the three plasmids needed respectively comprise AAV genomes, AAV mutant capsid proteins and replication proteins. The skeletons of the three plasmids can be derived from pFastbacadual plasmid (purchased from Invitrogen), namely, the target gene expression cassette is amplified by PCR and cut into a Multiple Cloning Site (MCS) of the pFastbacadual plasmid by enzyme.
The first plasmid related in the invention is AAV genome plasmid pFastbacual-ITR-EGFP, comprises two inverted terminal repeat sequences (ITR) of AAV serotype 2(AAV2), and also comprises an exogenous gene expression cassette (comprising a promoter, an enhancer, an intron and a polyA sequence, wherein the exogenous gene expression cassette comprises a green fluorescent protein gene EGFP and the like) expressed in eukaryotic cells.
The second plasmid related in the invention is a plasmid pFastbinary-inrep for coding AAV replication protein (Rep), which comprises a Rep gene expression frame of AAV2, an intron sequence for enhancing expression and the like (Litamine and the like, an AAV-ITR gene expression microcarrier prepared by insect cells, a report of bioengineering, 2015,31(8), page 1232, and a method of 'constructing pFastbinary-ITR-EGFP plasmid' in 1.2.1).
The third plasmid involved in the present invention is plasmid pFastbacadual-inCap 6 encoding AAV6 capsid protein. The plasmid contains AAV6 cap gene, intron sequence, and its expression is regulated by p10 promoter and HSV tk polyA element on pFastbinary plasmid vector (Litamine et al, AAV-ITR gene expression microcarrier prepared by insect cell, Biotechnology report, 2015,31(8), page 1232, 1.2.2 method "construct pFastbinary-incap plasmid").
The AAV vectors involved in the invention are all produced in insect cells:
firstly, respectively transforming escherichia coli DH10Bac competent cells of the three recombinant plasmids pFastbacal-inCap 6/pFastbacal-ITR-EGFP/pFastbacal-inrep by a conventional method, selecting white colonies containing recombinant Bacmid and blue colonies which are not recombined by two-round blue-white screening, selecting the white colonies for amplification, and extracting the recombinant Bacmid-inCap 6/Bacmid-ITR-EGFP/Bacmid-inrep.
Then, using an insect cell transfection reagent to transfect the three recombinant Bacmid-inCap6/Bacmid-ITR-EGFP/Bacmid-inrep into an insect cell Sf9, after 4-5 days, collecting cell supernatant, filtering the cell supernatant by using a 0.22 mu m filter to obtain P1 generation recombinant Baculovirus-inCap6/Baculovirus-ITR-EGFP/Baculovirus-inrep, infecting the P1 generation recombinant Baculovirus twice with Sf9 cells, amplifying to obtain P3 generation recombinant Baculovirus, and measuring the titer of the P3 generation Baculovirus by using a plaque method, wherein the titer (pfu/mL) is equal to 1/dilution multiple × plaque number × 1/per hole inoculation volume.
Finally, three recombinant baculoviruses (Baculovir-inCap 6/Baculovir-ITR-EGFP/Baculovir-inrep) of P3 generation are co-infected with Sf9 cells, and packaged to obtain rAAV 6.
And a method for purifying and concentrating high-concentration recombinant AAV by using a CsCl density gradient centrifugation method, a method for detecting rAAV6 titer by using fluorescence quantitative PCR, and a method for detecting rAAV6 purity by using SDS-PAGE.
The DNA in the cationic polymer DNA complex of the present invention may be a DNA molecule in various forms including a plasmid.
The present invention relates to cationic polymeric cell transfection reagents comprising Polyethyleneimine (PEI). Taking linear PEI with the molecular weight of 25kDa as an example, plating on the day before transfection, and performing transfection when the cell density reaches 70% -80%, wherein the ratio of DNA to PEI is 1: 2(μ g: μ l). Respectively mixing plasmids to be transferred and PEI reagents with HBS to prepare 15 mul A, B mixed solution, standing at room temperature for 10-15 minutes, gradually swirling the test tube filled with the solution A, gradually adding the solution B into the solution A (the sequence cannot be reversed), fully mixing (without swirling, one drop of the solution can be uniformly mixed), standing at room temperature for 10-15 minutes to form DNA-PEI complex, gradually adding the complex into a cell culture hole which is converted into an antibiotic-free cell culture medium in advance, and gently shaking for uniform mixing. The cell culture medium is replaced in time in the subsequent culture process to ensure the activity of the cells.
The invention proves that the cationic polymer DNA complex can promote the expression of AAV-mediated genes by various technical schemes:
firstly, the method comprises the following steps: co-transfecting the pUC57 plasmid with PEI and AAV6-EGFP into HEK293 cells; AAV6-EGFP is singly transfected, and after 24h, the cationic polymer-DNA complex is compared by using a fluorescence microscope and a flow analysis technology to promote AAV-mediated EGFP protein expression.
Secondly, the method comprises the following steps: different AAV vectors are set to transfect MOI, HEK293 cells are cotransfected with PEI transfection reagent-pUC 57 complex, after 24 hours, results are observed by using a fluorescence microscope and a flow cytometer, compared with the result of singly transfecting the AAV vectors, the cationic polymer has good promotion effect on the MOI of different AAV vectors, and the promotion effect is most obvious when the MOI is low (10000 <).
Thirdly, the method comprises the following steps: firstly, the PEI-DNA complex is transfected for 6h, 12h and 24h, and then AAV6-EGFP is transfected; or, the AAV6-EGFP is transfected firstly, and the PEI-DNA complex is transfected after 6 hours; simultaneously transfecting the PEI-DNA complex and the AAV 6-EGFP; AAV6-EGFP was singly transfected. And analyzing the results by using a fluorescence microscope and a flow cytometer after 24h to determine the transfection promoting effect under different transfection sequences and different time intervals, and finding that the effect is the best when the transfection is carried out simultaneously.
In conclusion, the results show that the cationic polymer DNA compound obviously improves the AAV-mediated gene expression.
By the technical scheme, the method for promoting the AAV-mediated foreign gene expression capacity and enhancing the AAV-mediated foreign gene expression time by using the cationic polymer DNA compound provided by the invention combines the cationic polymer with AAV-mediated gene therapy by using the characteristics of the cationic polymer, so that the AAV-mediated gene expression effect is improved, the gene expression efficiency is improved, the AAV-mediated gene therapy effect is enhanced, and the method has a vital significance for promoting the development of the fields of gene therapy, cell engineering and the like.
The invention is further illustrated by the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise specified, various reagents used in the following examples are well known to those skilled in the art and available from commercial sources and the like. However, the experimental methods in the following examples, in which specific conditions are not specified, are generally performed under conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or under the conditions recommended by the manufacturers.
Example 1
The embodiment relates to a cationic polymer DNA compound for efficiently promoting AAV-mediated gene expression, and the specific experimental method comprises the following steps:
(1) preparation of recombinant plasmid:
preparation of pFastbacadual-ITR-inCap 6
The plasmid contains AAV2 cap gene, intron sequence, the expression of which is regulated by p10 promoter and HSV tk polyA element on pFastbacdual plasmid vector, and is constructed and stored in laboratory (reference: Litamine et al, AAV-ITR gene expression microcarrier prepared by insect cell, Biotechnology report 2015,31(8), page 1232, method "construct pFastbinary-incap plasmid" in 1.2.2).
Preparation of pFastbacadual-ITR-EGFP
The pFastbacdual-ITR-EGFP plasmid contains two ITRs of AAV2 and a CMV promoter, a beta-globin intron, a coding EGFP gene and an hGH polyA sequence, and is constructed and stored in a laboratory (reference: Litamine et al, AAV-ITR gene expression microcarrier prepared by insect cells, bioengineering, 2015,31(8), page 1232, method "pFastbacdual-ITR-EGFP plasmid" in 1.2.1).
Preparation of pFastbacadual-inrep plasmid
The pFastbacdual-inrep plasmid contains AAV2 rep gene, intron sequence, the expression of which is regulated by p10 promoter and HSV tk polyA element on pFastbacdual plasmid vector, and is constructed and stored in laboratory (reference: Litamine et al, AAV-ITR gene expression microcarrier prepared by insect cell, report of bioengineering, 2015,31(8), p 1232, 1.2.2 method "pFastbinary-inrep plasmid construction").
(2) Preparation of recombinant Bacmid
The three recombinant plasmids in the last step are respectively used for preparing recombinant Bacmid, and the specific method is as follows:
1) 100 μ L of DH10Bac was slowly thawed on ice.
2) Add 50ng plasmid DNA and mix gently.
3) Standing on ice for 30min, heat shocking at 42 deg.C for 90s, immediately transferring to ice and standing for 2 min.
4) Add 900. mu.L of SOC medium and shake at 37 ℃ and 225rpm for 4 h.
5) 40 μ L of 2% (20mg/mL) indoxyl substrate Blue-gal and 7 μ L of 20% (200mg/mL) IPT were added dropwise to the center of a pre-prepared 90mm agar plate containing 50 μ g/mL kanamycin Kan, 7 μ g/mL gentamicin Gen, 10 μ g/mL tetracycline Tet. The plates were spread over the entire surface using a sterile spreader and incubated at room temperature until all liquid disappeared.
6) Cells (10-1, 10-2, 10-3) were diluted in SOC medium in 10-fold gradients, and 100. mu.L of each gradient was plated on LB plates.
7) After 48h at 37 ℃ 10 white colonies were picked and dipped on fresh LB agar plates (resistant as above) overnight at 37 ℃. The confirmed white spots were picked and inoculated into LB liquid medium (containing 50. mu.g/mL Kan, 7. mu.g/mL Gen, 10. mu.g/mL Tet).
8) The mixture was left overnight at 4 ℃ to allow the blue color to develop sufficiently during this period.
9) Sf9 can be transfected after the correct PCR identification can be carried out for white spots.
10) Extracting and separating recombinant bacmid DNA by using an OMEGA kit, and measuring the concentration of bacmid by the experimental method according to the kit specification, subpackaging and freezing at-20 ℃ to avoid repeated freezing and thawing.
11) And (3) PCR identification of Bacmid, wherein the used primers are respectively as follows: an upstream primer 5'-CCC AGT CAC GAC GTT GTAAAA CG-3' and a downstream primer 5'-GCT CTA GAT TAC TTG TAC AGC TCG TCC AT-3'.
12) Taking out the Bacmid strain with correct identification, and inoculating the Bacmid strain to 3mL LB (Kan +, Gen +, Tet +) for 12h according to the proportion of 1: 300. Then inoculating 150mL LB (Kan +, Gen +, Tet +) shake bacteria for 16h according to the proportion of 1:100, and extracting Bacmid in large quantity according to the instruction of a large-scale/large-scale plasmid extraction kit so as to prepare baculovirus by transfecting cells.
(3) Preparation of baculovirus
① Sf9 cell culture
Sf9 cells were plated one day in advance in six well plates at 50% well density using complete medium, 95% viability. The recombinant Bacmid DNA is subjected to warm bath in a 70 ℃ water bath for 20min, and then 12000g of the recombinant Bacmid DNA is centrifuged for 10min to obtain supernatant.
② cell plating
Operation was performed while ensuring cell density at 1.5-2.5 × 106cells/mL (medium without antibiotics.) 2mL Grace insect cell culture medium without additives Grace medium (without antibiotics and serum) was added to 6-well plates, 8 × 105 cells/mL Sf9 in step 1 (medium not changed and cells washed) was seeded and cells allowed to adhere for 15min at room temperature.
③ preparation of transfection reagents
a) The transfection reagent cellfectin II was mixed well, added to 8. mu.L to 92. mu.L of Grace medium without additives (without antibiotics and serum), vortexed and mixed well.
b) And taking 5 mu L of bacmid DNA (500 ng/mu L, ensuring that the quantity of bacmid is 2-3 mu g) to 95 mu L of Grace culture medium without additives (containing no antibiotics and serum), and gently mixing the bacmid DNA and the Grace culture medium.
c) Mixing the above two solutions, and incubating at room temperature for 30 min.
④ the DNA-Lipid mixture was added dropwise to the wells plated with cells, and the cells were incubated at 27 ℃ for 5 hours.
⑤ remove the medium from the plate and replace it with 2mL of complete medium.
⑥ were incubated at 27 ℃ for 72h and observed for signs of viral infection.
Isolation P1:
after confirming that the cells are in the late stage of infection (usually 4-5 days after transfection), 2mL of virus-containing medium per well was collected into a sterile 15mL centrifuge tube and centrifuged at 1000g for 5min to remove cell debris.
The supernatant was filtered through a 0.22 μm filter into a sterile 15mL centrifuge tube and stored at 4 ℃ in the dark. If long-term storage is desired, subpackaging and freezing at-80 ℃.
And (3) virus amplification:
taking 10mL suspension culture cells with the MOI of 0.05-0.1, wherein the density is 2 × 106cells/mL, or taking cells in a 6-well plate with the density of 2 × 106 cells/well, and calculating the required volume of P1.
① Sf9 cells were plated on six-well plates, 2 × 106 cells/well, and left to adhere at room temperature for 1 hour and observed under a microscope.
② adding appropriate amount of P1 into each well, and culturing at 27 deg.C for 48-72 h.
③ collect 2mL of virus-containing medium per well in a sterile 15mL centrifuge tube and centrifuge at 1000g for 5min.
④ the supernatant was transferred to a sterile 15mL centrifuge tube and the virus supernatant was stored at P2.4 ℃ in the dark and frozen at-80 ℃ if long term storage was desired.
⑤ was amplified as described above to obtain P3 (commonly obtained P1 virus titers were 1 × 10)6～1×107P2 titer was 1 × 107～1×108In between).
Viral titers were determined by plaque assay. The detailed experimental procedure is as follows:
① 2 mL/well (5 × 105 cells/mL) were seeded in 6-well plates, incubated at room temperature for 1h to adhere, and after incubation, their degree of adherence was examined under a microscope.
② A water bath at 70 ℃ was used to melt 4% agarose gel, and 2 × Grace was preheated in a 100mL sterile bottle at 40 ℃.
③ gradient dilution of baculovirus with serum-free supplemented Grace Medium 10-1～10-8。
④ discard the supernatant in the 6-well plate, add diluted virus quickly, and incubate 1 mL/well (duplicate) for 1h at room temperature.
⑤ preparing upper agar, adding 20mL high temperature inactivated FBS to 2 × Grace 100mL, 25mL 2 × Grace (containing FBS) +12.5mL sterile water +12.5mL 4% agarose geL L, placing into a preheated 100mL sterile bottle, gently mixing, and placing into a 37 ℃ water bath for standby.
⑥ discarding supernatant in 6-well plate, adding 2mL upper agar rapidly to prevent bacterial layer from drying, standing for 10-20 min to solidify, placing 6-well plate in 27 deg.C incubator, and culturing for 5 days.
⑦ A1 mg/mL neutral red solution was prepared and sterile filtered through Grace complete medium.
⑧ 1.5.5 mL of the above solution, 16.5mL of Grace complete medium, 6mL of 4% agar prepared as neutral red top agar.
⑨ after 4 days of virus infection, 1mL of neutral red top agar was added.
⑩, continuously placing the mixture in an incubator, observing plaques after 4-5 days, counting the number of plaques, and obtaining the virus titer.
Note that the virus titer (pfu/mL) is 1/dilution × plaque number × 1/inoculation volume per well
(4) Preparation and purification of rAAV vector
Purifying and concentrating the high-concentration recombinant AAV by a CsCl density gradient centrifugation method, detecting the titer of rAAV6 by fluorescence quantitative PCR, and detecting the purity of rAAV6 by SDS-PAGE. FIG. 1 is a schematic diagram of the SDS-PAGE electrophoresis for detecting the purity of the rAAV vector in this example, in FIG. 1, A represents rAAV6-CMV-EGFP, B represents rAAV6-S663L-CMV-EGFP, and C represents rAAV 6-S663L-GFAP-EGFP.
(5) Preparation method of HBS buffer solution and PEI transfection reagent
When preparing HBS buffer solution, 0.954g of hydroxyethylpiperazidine ethanethiosulfonic acid Hepes and 1.754g of NaCl are weighed and dissolved in 180ml ddH2In O, the pH value is adjusted to 7.4 by 1mol/L NaOH or HCl, and ddH is added2O is added to 200mL, and the mixture is filtered and sterilized through a 0.22-micron filter membrane and stored at the temperature of minus 20 ℃.
When 50mmol/L PEI solution was prepared, 0.0215g PEI was dissolved in 9mL ddH preheated to 75 deg.C2Adding HCl into O, stirring for 3h, adding NaOH to adjust the pH value to 7.4 after PEI is completely dissolved, and then adding ddH2O is added to 10mL, and the mixture is filtered and sterilized through a 0.22-micron filter membrane and stored at the temperature of minus 20 ℃. The PEI storage solution was dissolved well at room temperature before each use to ensure the same concentration for each use.
(6) Cell culture method
HEK293 cells (purchased from American type culture Collection) grown adherent to cells at 37 ℃ and 5% CO in complete high-glucose DMEM medium (supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin double antibody) were cultured2The constant temperature incubator. One day before transfection, pouring out the culture medium in a T25 cell culture bottle, adding 1ml of 0.05% pancreatin after rinsing with 1ml of PBS once, placing the cells in an incubator at 37 ℃ for digestion for 2-3 minutes, adding 1.5ml of serum-containing high-sugar DMEM complete culture medium after observing cell slice drifting under a microscope, sucking cell sap into a 15ml centrifuge tube after gently blowing adherent cells, centrifuging 250g for 5 minutes, counting by using a blood counting plate after gently resuspending the cells adherent to 1ml of culture medium, inoculating about 30 ten thousand cells into a 24-well plate for culture, and enabling the cell density to reach 70% -80% the next day.
(7) Method for transfecting plasmids using PEI transfection reagent
The ratio of DNA to PEI was controlled at 1:1(μ g: μ l). And when the cell density reaches 70-80%, the culture medium in the hole is replaced by a high-glucose DMEM culture medium without antibiotics, and then transfection is carried out. Preparing DNA to be transferred and PEI into two 15-microliter transfection systems by using HBS buffer solution, recording as A, B solution, immediately mixing by vortex oscillation, standing for 10-15min at room temperature after instantaneous centrifugation, adding B solution dropwise while slightly swirling A solution (the sequence cannot be reversed, if no condition exists, one B solution can be dropped and then is quickly mixed), standing for 10-15min at room temperature, finally slightly adding A, B mixed solution into a 24-well plate, slightly shaking and mixing uniformly, and then placing into an incubator at 37 ℃. The complete culture medium is replaced after 4h, and the culture medium is replaced in time for subsequent culture to ensure that the cell state is intact.
(8) Cationic multimers promote AAV-mediated gene expression
By using PEI as a transfection reagent, 400ng of pUC57-SaCas9-shRNA-Backbone was co-transfected into HEK293 cells according to the transfection method, AAV6-EGFP was simultaneously transfected, the expression condition of EGFP protein was observed by using a fluorescence microscope after 24h, and the fluorescence expression percentage and intensity of EGFP were analyzed by using a flow cytometer.
The cell processing method in flow cytometry analysis is as follows:
1) the medium was aspirated from the 24 wells and approximately 250. mu.l of PBS was added to rinse the cells per well.
2) About 250. mu.l of 0.05% pancreatin was added to each well and digested in an incubator at 37 ℃ for 2-3 minutes until cells were observed under a microscope to be exfoliated.
3) Digestion was stopped by adding about 500. mu.l of 10% serum-containing high-glucose DMEM medium to each well.
4) After gently pipetting the cells, the cell fluid was aspirated into a 1.5ml EP tube and centrifuged at 250g for 5 minutes.
5) 700. mu.l of PBS was added to the EP tube, and after gently adherent pipetting, the cells were rinsed with heavy suspension and centrifuged at 250g for 5 minutes.
6) And adding 500 mu l of PBS into the EP tube, gently blowing and rinsing the cells adherent to the wall, transferring the cell sap into a flow tube, and preparing for on-machine analysis.
(9) Through fluorescence microscope observation and flow analysis, the AAV gene expression mediated by the cationic polymer is obviously improved. In this experiment, the inventors selected multiple cell concentrations (10% -100%) for transfection.
FIG. 2A is a fluorescent microscope image showing AAV expression mediated by cationic polymer PEI-DNA at different cell densities in this example, wherein four groups of ABCD are co-transfected and ABCd is a single-transfected AAV virus. FIG. 2B is a graph showing the flow analysis results of cationic polymer PEI-DNA mediated AAV expression at different cell densities in this example.
(10) Cationic polymer-mediated gene expression of AAV vectors of different MOIs
Different AAV vectors are set to transfect the MOI (1000, 10000, 100000 and 500000), the AAV vectors with different MOI and cationic polymer PEI-DNA (400ng,1:1) are co-transfected into the HEK293 cell according to the transfection method, the expression condition of the EGFP protein is observed by using a fluorescence microscope after 24h, the fluorescence expression percentage and intensity of the EGFP are analyzed by using a flow cytometer, and the regulation result is compared with the result of singly transfecting the AAV vectors.
Fluorescence microscopic observation and flow analysis show that when the MOI is lower than 100000, cotransfection has a remarkable expression promoting effect, and both fluorescence intensity and overall transfection efficiency have remarkable improving effects, so that the cationic polymer PEI-DNA complex has a remarkable gene transfection regulation effect on AAV vectors, and the effect is most remarkable under the condition of low MOI.
FIG. 3A shows fluorescent microscopy observations of gene expression of AAV vectors of different MOIs mediated by cationic polymer PEI-DNA in this example. FIG. 3B shows the results of flow analysis of gene expression of AAV vectors for different MOIs mediated by cationic polymer PEI-DNA in this example.
(11) Cationic Polymer PEI-DNA mediated changes in AAV Gene expression over time
According to the transfection method, HEK293 cells are co-transfected by cationic polymer PEI-DNA (400ng,1:1) and AAV (MOI:30000), expression conditions of EGFP protein are observed by using a fluorescence microscope after 24h, 48h and 72h, fluorescence expression percentage and intensity of EGFP are analyzed by using a flow cytometer, and the regulation and control result is compared with the result of singly transfecting AAV vectors.
Through fluorescence microscope observation and flow analysis, the gene expression of the AAV vector is obviously enhanced under the mediation of the cationic polymer PEI-DNA along with the prolongation of the gene expression time.
FIG. 4A shows fluorescent microscopy observations of AAV gene expression mediated by cationic polymer PEI-DNA at different times in this example, where three sets of ABCs are single transfected AAV vectors and ABC is co-transfected. FIG. 4B is a graph showing the results of flow analysis of gene expression of AAV vectors mediated by cationic polymer PEI-DNA at various times in this example.
(12) The cationic polymer PEI-DNA mediates the gene expression of AAV at different time points: 1) firstly, cationic polymer PEI-DNA is transfected, AAV (MOI:30000) is transfected after 6h, 24h and 48 h; according to the transfection method, HEK293 cells are respectively transfected by cationic polymer PEI-DNA (400ng,1:1) according to a time sequence, AAV vectors (MOI:30000) are then transfected respectively when the time reaches 6h, 12h and 24h, the expression condition of EGFP protein is observed by using a fluorescence microscope after 24h, the fluorescence expression percentage and intensity of EGFP are analyzed by using a flow cytometer, and the regulation and control result is compared with the result of simultaneous co-transfection. 2) AAV vector (MOI:30000) is transfected, HEK293 cells are transfected by cationic polymer PEI-DNA (400ng,1:1) according to the transfection method when the transfection time is 6h, the expression condition of EGFP protein is observed by using a fluorescence microscope after 24h, the fluorescence expression percentage and intensity of EGFP are analyzed by using a flow cytometer, and the regulation and control result is compared with the result of simultaneous cotransfection. Through fluorescence microscope observation and flow analysis, 1) when the time interval is within 24h, the mediated enhancement effect of the cationic polymer PEI-DNA is most obvious, no matter the cationic polymer PEI-DNA or AAV is transfected first.
FIG. 5A shows fluorescent microscopy observations of gene expression of AAV vectors mediated by cationic multimeric PEI-DNA at different time points in this example. FIG. 5B is a graph showing the results of flow analysis of gene expression of AAV vectors mediated by cationic polymer PEI-DNA at various time points in this example.
(13) Effect of different ratios of cationic Polymer PEI-DNA on AAV vector transfection
Selecting different PEI-DNA ratios, namely PEI-DNA (1:1,1:2,1:3,2:1 and 3:1), co-transfecting HEK293 cells with cationic polymer PEI-DNA and AAV vector (MOI:20000) according to the transfection method, observing the expression condition of EGFP protein by using a fluorescence microscope after 24h, and analyzing the fluorescence expression percentage and intensity of EGFP by using a flow cytometer. The effect was most pronounced at 1:1 as observed by fluorescence microscopy and flow analysis.
FIG. 6A shows fluorescent microscopy observations of the gene expression of AAV vectors mediated by cationic multimeric PEI-DNA under different ratio conditions in this example. FIG. 6B is a graph showing the results of flow analysis of the gene expression of AAV vectors mediated by cationic polymer PEI-DNA at various ratios in this example.
The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.
Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.
It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Claims (10)

1. A method for promoting AAV-mediated gene expression using a cationic multimeric DNA complex, comprising:
co-transfecting host cells after co-incubation of an AAV vector containing a target gene, auxiliary DNA and a positively charged cell transfection reagent;
alternatively, a host cell is transfected with either one of a cationic multimeric DNA complex formed by incubating helper DNA with a positively charged cell transfection reagent and an AAV vector comprising a gene of interest, and then transfected with the other within a set time.
2. The method of gene expression according to claim 1, comprising:
co-transfecting host cells after co-incubation of an AAV vector containing a target gene, auxiliary DNA and a positively charged cell transfection reagent;
or, transfecting host cells after co-incubation of helper DNA with a positively charged cell transfection reagent, and then transfecting the host cells with an AAV vector comprising a gene of interest for a selected time period, the set time period being less than 24 hours, preferably less than 12 hours, more preferably within 6 hours;
or, transfecting a host cell with an AAV vector comprising a target gene, followed by transfecting said host cell after co-incubation of helper DNA with a positively charged cell transfection reagent for a selected time, said set time being less than 24 hours, preferably less than 6 hours; wherein simultaneous transfection is optimal.
3. The method of gene expression according to claim 1, comprising:
co-transfecting host cells after co-incubation of an AAV vector containing a target gene, auxiliary DNA and a positively charged cell transfection reagent, and performing gene expression for more than 24h, preferably more than 48h, and particularly preferably more than 72 h;
alternatively, a host cell is transfected with either one of a cationic multimeric DNA complex formed by incubating a helper DNA with a positively charged cell transfection reagent and an AAV vector containing a target gene, and then the other one is transfected within a set time period, and gene expression is performed for 24 hours or more, preferably 48 hours or more, and particularly preferably 72 hours or more.
4. The method of gene expression according to claim 1, comprising: when the host cell is transfected after incubating the AAV vector containing the target gene, the helper DNA and the positively charged cell transfection reagent, or when the host cell is transfected with any one of the cationic multimeric DNA complex formed by incubating the helper DN a and the positively charged cell transfection reagent and the AAV vector containing the target gene, the cell concentration is 10% to 100%, preferably 80% to 90%.
5. The method of gene expression according to claim 1, wherein: the AAV vector comprising the gene of interest has a MOI <100000, preferably < 10000.
6. The method of gene expression according to claim 1, comprising: mixing the auxiliary DNA and the cell transfection reagent with positive charge in a selected buffer solution for reaction to form a cationic polymer DNA complex; and
transfecting a host cell with the cationic multimeric DNA complex.
7. The method of claim 6, comprising: preparing auxiliary DNA and cell transfection reagent into a first transfection solution and a second transfection solution by using a selected buffer solution respectively;
adding the second transfection solution to the first transfection solution in portions, thereby reacting to form cationic polymer DNA complexes;
preferably, the gene expression method specifically comprises: adding a second transfection solution drop to the first transfection solution, and incubating so that a cationic polymer DNA complex is formed by reaction; preferably, the selected buffer comprises HBS buffer.
8. The method of gene expression according to claim 1, wherein: the mass-to-volume ratio of the auxiliary DNA to the cell transfection reagent is 1 mug: 1 to 5. mu.l.
9. The method of gene expression according to claim 1, wherein: the cell transfection reagent comprises a polycationic high molecular compound, preferably selected from linear polyethyleneimine;
and/or, the auxiliary DNA comprises any one of a circular plasmid, a linear fragment, a double-stranded DNA, a single-stranded DNA and a cell genome, and is preferably a circular plasmid.
10. The method of gene expression according to claim 1, wherein: the AAV vector containing the target gene comprises AAV 6-EGFP.
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