A transfection auxiliary reagent for improving gene vector transfection efficiency and / or transfection accuracy and its application

Abstract

The present invention provides a transfection auxiliary reagent for improving the transfection efficiency and / or transfection accuracy of a gene vector and its application, wherein the transfection auxiliary reagent comprises polyethylene glycol and / or its derivatives. The present invention creatively uses polyethylene glycol as an auxiliary reagent for gene transfection, and finds that: (1) polyethylene glycol or its derivatives can significantly improve the transfection efficiency of gene vectors for in vitro cells, including some cells that cannot be transfected by gene vectors; (2) polyethylene glycol or its derivatives can significantly improve the transfection efficiency of gene vectors for living tissues, and promote the local expression of gene vectors in living tissues, thereby achieving efficient and accurate transgenic expression in target tissues; (3) polyethylene glycol or its derivatives can promote the efficient and accurate transfection of gene vectors for living tissues for a long time, and will not cause significant side effects in living tissues, such as cell apoptosis, severe immune response, and significant changes in animal behavior functions.

Description

Technical Field

[0001] The invention belongs to the fields of bioengineering technology and gene technology, and relates to a transfection auxiliary reagent for improving gene vector transfection efficiency and / or transfection accuracy and application thereof. Background Art

[0002] Wild-type adeno-associated virus (AAV) is a non-pathogenic, replication-deficient single-stranded DNA parvovirus with a genome size of approximately 4.7 kb, which mainly consists of three parts: ITR, Rep, and Cap. ITR is an inverted terminal repeat sequence that plays a decisive role in viral replication and packaging; Rep encodes proteins related to AAV replication, packaging, and genome integration (Rep78, Rep68, Rep52, and Rep40); and Cap encodes three structural proteins that make up the viral capsid (VP1, VP2, and VP3, in a ratio of 1:1:10). These three structural proteins can interact to form a symmetrical icosahedral structure. Wild-type AAV was first discovered in the laboratory in 1965 as a contaminant in the adenovirus preparation process. It requires the participation of auxiliary factors (such as adenovirus, vaccinia virus, and herpes simplex virus) to complete replication; when there are no auxiliary factors, it can only latently infect cells.

[0003] Recombinant adeno-associated virus (rAAV) is a gene vector modified from wild-type AAV. It has good biosafety, is non-pathogenic to host cells, has low immunogenicity, and generally does not integrate into the host cell genome. When transfecting cells, rAAV first binds to specific receptor proteins on the surface of the cell membrane, activates related signaling pathways in the cell, and then stimulates the cell's endocytosis to engulf it into the cell; then, with the assistance of organelles such as the Golgi apparatus, rAAV enters the cell nucleus and cleaves, and after the single-stranded DNA is replicated into double-stranded DNA, the reporter protein can be expressed. rAAV can achieve long-term and stable transgenic expression in host cells, and its host cell range is wide (including a variety of cell lines, muscles, lungs, liver, retina, and central nervous system, etc.). Transgenic expression mediated by rAAV has been widely used in basic research and clinical treatment, such as: cell-based gene therapy, cytogenetics, gene editing, neuronal morphology and neuronal population imaging, neural circuit regulation, neurological disease model development and treatment, etc.

[0004] Despite this, there are still many problems to be solved in the use of rAAV vectors to deliver genes for transgenic expression. For example: (1) rAAV has low infection efficiency for many in vitro cultured cells, and some cells cannot even be transfected. (2) When using rAAV to transfect the living nervous system, in order to produce sufficient transgenic expression, it is usually necessary to inject a large dose of high-titer AAV into the target brain area. On the one hand, due to the limited transport efficiency of viral vectors in the body, a considerable portion of viral vectors cannot be delivered to the cell nucleus. They will be degraded by host cell proteases, thereby triggering the body's immune response. On the other hand, high-titer rAAV transfection of the nervous system will damage neurons and cause them to regress to an immature state. Many genes in neurons are upregulated, including some glial genes. This will cause gene sequences with glial cell promoters to leak into neurons for transgenic expression, thereby indirectly changing the cell type tropism of rAAV and misleading experimental results. (3) Stereotactic brain injection of viral vectors is currently the most common method for transfecting the nervous system. However, viral vectors will spread rapidly in neural tissue filled with cerebrospinal fluid, causing a large area of ​​non-target tissue to be transfected. This is very unfavorable for studying the function of small-volume nuclei in the brain and studying the projection relationship between closely spaced brain regions.

[0005] Therefore, how to provide a gene transfection method that can achieve more efficient transfection of in vitro cells, more accurate and efficient transfection of living tissues, and does not cause side effects has become an urgent problem to be solved in this field. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a transfection auxiliary reagent and its application for improving the transfection efficiency and / or transfection accuracy of gene vectors.

[0007] The transfection efficiency of the present invention refers to the percentage of successfully transfected cells in in vitro transfection and / or in vivo transfection; the transfection accuracy refers to the range of tissues or organs transfected by the virus in in vivo transfection.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a transfection auxiliary reagent for improving the transfection efficiency and / or transfection accuracy of a gene vector, wherein the transfection auxiliary reagent comprises polyethylene glycol (PEG) and / or its derivatives.

[0010] The derivatives include PEG derivatives with an active group at one end (such as Glucose PEG NHS Ester, Methoxy PEG Carboxyl, Methoxy PEG Succinimidyl Carbonate, Methoxy PEG Thiol, etc.), disubstituted derivatives with the same functional group (such as Carboxyl PEG Carboxyl, Amine PEG Amine, Acrylate PEG Acrylate, etc.), Y-type PEG derivatives (such as Y-Shape PEG NHS Ester, Y-Shape PEG Carboxyl, Y-Shape PEG Maleimide, Y-Shape PEG Acetaldehyde, Y-Shape PEG Propionaldehyde, Y-Shape PEG Amine, etc.), etc., preferably polyethylene glycol (PEG).

[0011] Preferably, the average molecular weight of the polyethylene glycol is 1000-10000, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, etc., preferably 2000-6000.

[0012] When the average molecular weight of polyethylene glycol is in the range of 2000-6000, the effect of improving the transfection efficiency and transfection accuracy of gene vectors is better. When the average molecular weight of polyethylene glycol is 4000, the effect of improving the transfection efficiency and transfection accuracy of gene vectors is best.

[0013] Preferably, the gene vector comprises any one of plasmid, retrovirus, herpes simplex virus, adenovirus, adeno-associated virus or lentivirus, or a combination of at least two thereof, preferably adeno-associated virus.

[0014] Preferably, the adeno-associated virus includes human serotype adeno-associated virus and / or non-human primate serotype adeno-associated virus, preferably human serotype adeno-associated virus (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAV12).

[0015] Preferably, the adeno-associated virus promoter includes any one of CMV, hSyn, GFAP, CaMKIIα, TH, c-fos, NSE, MBP, TBG, aMHC, Rpe65, PDX1, SM22a, CD68, ICAM2, MCK or NPHS1, preferably any one of CMV, hSyn, GFAP, CaMKIIα, TH, c-fos, NSE or MBP, and further preferably CMV or hSyn.

[0016] In a second aspect, the present invention provides use of the transfection auxiliary reagent for improving gene vector transfection efficiency and / or transfection accuracy as described in the first aspect in gene transfection, wherein the gene transfection includes in vitro transfection and / or in vivo transfection.

[0017] In a third aspect, the present invention provides an in vitro transfection method, which comprises mixing the transfection auxiliary reagent for improving the transfection efficiency and / or transfection accuracy of the gene vector as described in the first aspect, the gene vector and cells for transfection.

[0018] The mixing may be direct mixing or indirect mixing.

[0019] Preferably, the cells include cells susceptible to viral transfection and / or cells difficult to be transfected by viruses.

[0020] The cells that are easily transfected by viruses include, but are not limited to, cervical cancer cells (Hela), human kidney cell lines (HEK293T), human bronchial epithelial cells (HBEC), human hepatoma cells (HepG2), human osteosarcoma cells (Saos-2), human osteosarcoma cells (U2OS), human umbilical vein endothelial cells (HUVEC), African green monkey SV40-transformed kidney cells (COS-7), mouse fibroblast cell lines (3T3), astrocytes, etc. Those skilled in the art should know that, in addition to the above-mentioned cells, other cells that are easily transfected by viruses in the art are feasible and are within the protection scope of the present invention.

[0021] The cells that are difficult to be transfected by viruses include, but are not limited to, human chronic myeloid leukemia cells (K562), adrenal pheochromocytoma cells (PC12), epithelial colorectal cells (Caco-2), human breast cancer cells (MCF-7), directed hematopoietic stem cells (Hematopoietic Progenitor), embryonic stem cells (ES cell), Chinese hamster ovary cells (CHO), human colon cancer cells (HT29), human colorectal adenocarcinoma cells (CaCo-2), etc. Those skilled in the art should know that, in addition to the above cells, other cells that are difficult to be transfected by viruses in the art are feasible and are within the scope of protection of the present invention.

[0022] Preferably, in the transfection system obtained after the mixing, the mass fraction of the transfection auxiliary reagent is 0.1%-20%, for example, 0.1%, 0.2%, 0.5%, 0.7%, 1%, 1.2%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.7%, 4%, 4.2%, 4.5%, 4.8%, 5%, 5.2%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 5.1%, 5.2 ... .7%, 6%, 6.2%, 6.5%, 6.7%, 7%, 7.2%, 7.5%, 7.7%, 8%, 8.2%, 8.5%, 8.7%, 9%, 9.2%, 9.5%, 9.7%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc., preferably 0.2%-4.8%, more preferably 2.8%-4.2%.

[0023] Preferably, the mixing time is 1s-72h, for example, 1s, 30s, 1min, 10min, 40min, 1h, 3h, 5h, 7h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 45h, 50h, 55h, 60h, 65h, 70h, 72h, etc., preferably 12h-36h.

[0024] In a fourth aspect, the present invention provides an in vivo transfection method, which comprises delivering the transfection auxiliary reagent for improving the transfection efficiency and / or transfection accuracy of the gene vector as described in the first aspect and the gene vector into a living tissue or living organ for transfection.

[0025] The transfection auxiliary reagent and the gene vector can be delivered separately or mixed and delivered together, and the delivery method can be injection, for example.

[0026] Preferably, if the transfection auxiliary reagent is mixed with the gene carrier and then introduced, the mass fraction of the transfection auxiliary reagent in the mixed system is 1%-99%, for example, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 99%, etc., preferably 20%-60%.

[0027] Preferably, the living tissue or living organ includes any one of the brain, spine, liver, kidney, heart, lung, eye, muscle, pancreas or blood vessel or a combination of at least two thereof, preferably the brain.

[0028] Preferably, the transfection time is 1 week to 5 years, for example 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 year, 2 years, 3 years, 4 years, 5 years, etc., preferably 2-10 weeks.

[0029] In a fifth aspect, the present invention provides the use of polyethylene glycol and / or its derivatives in the preparation of transfection auxiliary reagents, characterized in that the transfection auxiliary reagents are used to improve the transfection efficiency and / or transfection accuracy of gene vectors.

[0030] Preferably, the average molecular weight of the polyethylene glycol is 1000-10000, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, etc., preferably 2000-6000.

[0031] Preferably, the gene vector comprises any one of plasmid, retrovirus, herpes simplex virus, adenovirus, adeno-associated virus or lentivirus, or a combination of at least two thereof, preferably adeno-associated virus.

[0032] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention creatively utilizes PEG or its derivatives as auxiliary reagents for gene transfection and finds that:

[0035] (1) PEG or its derivatives can significantly improve the transfection efficiency of gene vectors on cells in vitro, including some cells that cannot be transfected by gene vectors alone without auxiliary transfection reagents (K562 cells, PC12 cells, etc.).

[0036] (2) PEG or its derivatives can significantly improve the transfection efficiency of gene vectors into living tissues or living organs, and promote the local expression of gene vectors in living tissues or living organs, thereby achieving efficient and precise transgenic expression in target tissues / organs.

[0037] (3) PEG or its derivatives can promote the efficient and precise transfection of gene vectors into living tissues in the long term without causing significant side effects in living tissues, such as cell apoptosis, severe immune response, and significant changes in animal behavioral functions.

[0038] The present invention creatively provides a method for gene transfection using PEG or its derivatives as auxiliary agents. The present invention significantly improves the transfection efficiency of AAV on in vitro cultured cells (including some cells that cannot be transfected) by dissolving a small amount of PEG or its derivatives in a viral solution or a cell culture medium. The present invention significantly improves the transfection efficiency of AAV on living tissues (or organs) by dissolving a small amount of PEG or its derivatives in a viral solution. Taking brain tissue as an example, the present invention uses PEG or its derivatives as auxiliary agents, which not only significantly improves the transfection efficiency of AAV on brain tissue, but also promotes AAV long-term and accurate transfection of small-volume nuclei in the brain without any side effects. The gene transfection method proposed in the present invention has very important application value in the fields of precision gene therapy and fine neural circuit research. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The expression of GFP fluorescent protein in Hela cells, HEK293T cells, K562 cells and PC12 cells of AAV-CMV::GFP and AAV-CMV::GFP / PEG virus solutions in Example 1, CMV: broad-spectrum promoter, GFP: green fluorescent protein.

[0040] Figure 2 The statistical results of the transfection efficiency of AAV-CMV::GFP and AAV-CMV::GFP / PEG virus solutions in Hela cells, HEK293T cells, K562 cells and PC12 cells in Example 1 are shown.

[0041] Figure 3 The expression of GFP fluorescent protein in Hela cells by AAV-CMV::GFP / PEG virus solution when the mass fraction of PEG in Example 2 is 0.2%, 0.9%, 1.6% and 3.6%, respectively.

[0042] Figure 4 These are the statistical results of the transfection efficiency of AAV-CMV::GFP / PEG virus solution in Hela cells when the mass fractions of PEG in Example 2 are 0.2%, 0.9%, 1.6% and 3.6%, respectively.

[0043] Figure 5 For Example 3, the titer is 4.2×10 11 The expression of EGFP fluorescent protein in the mouse brain after 3 weeks of transfection with v·g / mL AAV-hSyn::EGFP virus solution. From left to right, the PEG mass fractions are 0%, 20% and 40%, respectively. The scale bar in the figure is 200μm.

[0044] Figure 6 For Example 3, the titer is 4.2×1011 The expression of EGFP fluorescent protein in the mouse brain after 3 weeks of transfection with AAV-hSyn::EGFP virus solution (injection volume 300nL) at 40% v·g / mL. The virus solution in the left brain did not contain PEG, while the mass fraction of PEG in the virus solution in the right brain was 40%. hSyn: neuron-specific promoter, EGFP: enhanced green fluorescent protein.

[0045] Figure 7 For Example 3, the titer is 4.2×10 11 The expression of EGFP fluorescent protein in the mouse brain after 3 weeks of transfection with AAV-hSyn::EGFP virus solution (injection volume of 1000nL) at v·g / mL. The virus solution in the left brain does not contain PEG, and the mass fraction of PEG in the virus solution in the right brain is 40%. hSyn: neuron-specific promoter, EGFP: enhanced green fluorescent protein.

[0046] Figure 8 For Example 3, the titer is 4.2×10 11 Statistical results of the EGFP expression range when the mouse brain was transfected with AAV-hSyn::EGFP virus solution at v·g / mL for 3 weeks.

[0047] Fig. 9 For Example 4, the titer is 4.2×10 11 Three weeks after the mouse brain was transfected with v·g / mL AAV-hSyn::EGFP virus solution, the fluorescence results of neurons in brain slices were labeled by immunofluorescence method, NeuN: neuron-specific nuclear protein.

[0048] Fig.10 For Example 4, the titer is 4.2×10 11 Statistical results of transfection efficiency of mouse brains 3 weeks after transfection with v·g / mL AAV-hSyn::EGFP virus solution.

[0049] Fig.11 For Example 5, the titer is 4.2×10 11 The expression of EGFP fluorescent protein in the mouse brain after 3 and 8 weeks of transfection with AAV-hSyn::EGFP virus solution at v·g / mL. The left picture shows that the virus solution does not contain PEG, and the right picture shows that the mass fraction of PEG in the virus solution is 40%.

[0050] Fig.12 For Example 6, the titer is 4.2×10 11 The fluorescence results of astrocytes in brain slices were marked by immunofluorescence method 3 and 8 weeks after the mouse brain was transfected with AAV-hSyn::EGFP virus solution at v·g / mL.

[0051] Fig.13 For Example 7, the titer is 4.2×10 11 Statistical results of the movement speed and rest time of mice in the behavioral experiment 3 and 8 weeks after the mouse brain was transfected with AAV-hSyn::EGFP virus solution without PEG or with 40% PEG by mass (v·g / mL). DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0053] In the following examples, unless otherwise specified, all reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

[0054] Example 1. PEG improves the transfection efficiency of AAV in various cell lines.

[0055] (1) Cell culture

[0056] HeLa and HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody. PC12 cells were cultured in 1640 medium containing 5% fetal bovine serum, 10% inactivated horse serum and 1% double antibody. K562 cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% double antibody. The CO2 concentration of the incubator was 5% and the temperature was 37°C.

[0057] (2) Transfection of cells with AAV transfection solution without auxiliary transfection reagents

[0058] 1 mL of Hela cells, HEK293T cells, PC12 cells or K562 cells in good condition were seeded into a 24-well plate at a cell number of 10 4 indivual.

[0059] Dilute the rAAV9-CMV::GFP transfection solution to 5×10 with 1×PBS. 10 v·g / mL.

[0060] v·g refers to viral genomes.

[0061] Then the diluted rAAV9-CMV::GFP transfection solution was added to the 24-well plate seeded with cells, and the final transfection multiplicity was 5×10 5 v·g / cell.

[0062] The 24-well plate was then placed in a cell culture incubator with a CO2 concentration of 5% and a temperature of 37°C.

[0063] After culturing for 24 h, fresh medium was replaced and culture was continued for 48 h.

[0064] Then, a laser confocal microscope (Zeiss LSM710) was used to observe the GFP fluorescence expression. The excitation light of the microscope was 488 nm laser, and the objective lens magnification was 10× or 20×. The observation results are shown in Figure 1 .

[0065] Then, all cells and GFP-positive cells in the fluorescent photos were counted using ImageJ software. Each set of experimental parameters was repeated 3 times. For PC12 cells, 3 areas with more than 500 cells were randomly counted in each experiment. For HeLa cells, HEK293T cells, and K562 cells, 3 areas with more than 1000 cells were randomly counted in each experiment.

[0066] (3) Transfect cells with AAV / PEG transfection solution.

[0067] The difference between the method and (2) transfection of cells with AAV transfection solution without auxiliary transfection reagent is that PEG is added as an auxiliary reagent for transfection. The rest is referred to (2), which is as follows:

[0068] 1 mL of Hela cells, HEK293T cells, PC12 cells or K562 cells in good condition were seeded into a 24-well plate at a cell number of 10 4 indivual.

[0069] A certain amount of rAAV9-CMV::GFP virus and a certain amount of polyethylene glycol 4000 (PEG4000) were added to the PBS solution to prepare an rAAV titer of 5×10 10 v·g / mL, PEG4000 concentration is 40% original solution.

[0070] The above rAAV9-CMV::GFP / PEG4000 solution was added to a 24-well plate with 1×PBS, and the final transfection multiplicity was 5×10 5 v·g / cell, and the mass fraction of PEG was 3.6%.

[0071] The 24-well plate was then placed in a cell culture incubator with a CO2 concentration of 5% and a temperature of 37°C.

[0072] After culturing for 24 h, fresh medium was replaced and culture was continued for 48 h.

[0073] Then, a laser confocal microscope (Zeiss LSM710) was used to observe the GFP fluorescence expression. The excitation light of the microscope was 488 nm laser, and the objective lens magnification was 10× or 20×. The observation results are shown in Figure 1 .

[0074] Then, all cells and GFP-positive cells in the fluorescent photos were counted using ImageJ software. Each set of experimental parameters was repeated 3 times. For PC12 cells, 3 areas with more than 500 cells were randomly counted in each experiment. For HeLa cells, HEK293T cells, and K562 cells, 3 areas with more than 1000 cells were randomly counted in each experiment.

[0075] (4) Analyze and compare data.

[0076] The ratio of the number of GFP-positive cells to the number of all cells was defined as the transfection efficiency of AAV cells, and the standard error was calculated. Then, the corresponding results were plotted as a bar graph for comparison in the Origin software (see Figure 2 ).

[0077] The results showed that adding PEG to transfection significantly improved the transfection efficiency of AAV in various cell lines.

[0078] Example 2. Effects of PEG molecular weight and addition amount on AAV transfection efficiency in cells.

[0079] (1) HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody. The CO2 concentration of the incubator was 5% and the temperature was 37°C.

[0080] (2) Inoculate 1 mL of Hela cells in good condition into a 24-well plate. The number of cells is 10 4 indivual.

[0081] (3) Dilute the rAAV9-CMV::GFP transfection solution to 10 with 1×PBS. 11 v·g / mL.

[0082] (4) Then add the diluted rAAV9-CMV::GFP transfection solution into the 24-well plate.

[0083] (5) Then, different amounts of PEG4000 were added to the 24-well plate, so that the final mass fractions of PEG were 0.2%, 0.9%, 1.6%, and 3.6%, respectively. The final transfection multiplicity was 5×10 5 v·g / cell.

[0084] (6) The 24-well plate was then placed in a cell culture incubator with a CO2 concentration of 5% and a temperature of 37°C.

[0085] (7) After 24 h of culture, replace the culture medium with fresh one and continue culturing for 48 h.

[0086] (8) Then, a laser confocal microscope (Zeiss LSM710) was used to observe the GFP fluorescence expression. The excitation light of the microscope was 488 nm laser, and the objective lens magnification was 10× or 20×. The observation results are shown in Figure 3 .

[0087] (9) ImageJ software was used to count all cells and GFP-positive cells in the fluorescent photos. Each set of experimental parameters was repeated 3 times, and 3 areas with a cell count greater than 1000 were randomly counted in each experiment.

[0088] (10) The ratio of the number of GFP-positive cells to the number of all cells was defined as the transfection efficiency of AAV on Hela cells, and the standard error was calculated. Figure 4 .

[0089] The results showed that the amount of PEG added had a significant effect on the transfection efficiency. When the added mass fraction was 3.6%, the transfection efficiency of AAV on the cell line was the highest. Continuing to increase the PEG concentration would lead to toxicity, so the optimal concentration of PEG4000 in in vitro transfection was 3.6%.

[0090] Example 3. PEG promotes localized transgene expression of AAV in the living brain.

[0091] (1) Prepare AAV / PEG mixed transfection solution.

[0092] Accurately weigh 4 g of PEG4000 with a balance, pour it into a 15 mL centrifuge tube, and then add an appropriate amount of 1×PBS to dissolve PEG4000. Use a vortexer or metal bath heating to promote the dissolution of PEG4000. After it is completely dissolved, use 1×PBS to accurately make it up to 10 mL and shake it with a vortexer to obtain a PEG4000 solution with a mass fraction of 40%.

[0093] Use a pipette to accurately take 3 mL of a 40% PEG4000 solution, dilute it to 6 mL with 1×PBS, and shake it with a vortex to obtain a 20% PEG4000 solution.

[0094] The titer of rAAV9-hSyn::EGFP was increased from 1.4×10 14 v·g / mL diluted to 4.2×10 11 After being shaken well with a vortexer, the virus solutions of each group were obtained and stored in a 4°C refrigerator for use on the same day.

[0095] (2) Craniotomy in mice.

[0096] Male C57 mice aged 7-8 weeks were purchased from the company. After being raised in a laboratory environment for 3 days, they were injected with Avertin anesthetic by intraperitoneal injection at a dose of 0.04 mL / g, divided into two injections. After the mice were deeply anesthetized, the hair above the scalp of the mice was shaved. The mice were fixed to the brain stereotaxic instrument with ear bars, and the scalp was disinfected with cotton swabs dipped in iodine, and the scalp of the mice was cut to expose the skull. The mouse skull was then leveled using three-dimensional coordinates and marked above the target brain area. Then, under a stereomicroscope, the skull at the marked position was removed with a cranial drill to expose the cranial window. The diameter of the cranial drill used was about 0.4 mm, and the cranial window was a square with a side length of about 1 mm. After carefully peeling off the dura mater under the skull, cover the exposed brain tissue with a wet cotton swab.

[0097] (3) Brain localization injection.

[0098] Assemble a Hamilton syringe with a range of 10 μL and a No. 33 injection needle with an outer diameter of 210 μm. Fix the movable end of the microinjection pump to the movable arm of the brain stereotaxic instrument. Connect the microinjection syringe to the microinjection pump. Then set the parameters on the microinjection syringe display screen to absorb the virus solution prepared in step (1) and set the absorption speed to 0.1 μL / s. Then implant the injection needle into the target brain area through the movable arm of the brain stereotaxic instrument. Set the injection parameters on the display screen, the injection speed is 0.1 μL / min, and the injection volume is 300nL or 1000nL. After the injection of the virus solution is completed, in order to prevent the virus solution from flowing back, it is necessary to stay in place for 10 minutes before slowly pulling the injection needle out of the brain tissue. Finally, fill the cranial window with a single-component isolation glue and suture the scalp.

[0099] (4) Remove the brain.

[0100] Three weeks after the virus transfected the brain, the mouse was anesthetized by intraperitoneal injection of anesthetics again. Then 20 mL of normal saline was perfused transcardially to drain the blood from the mouse body. Then 20 mL of paraformaldehyde solution was slowly perfused transcardially to perform pre-fixation on the mouse. After half an hour of placement, the mouse brain was carefully removed and soaked in paraformaldehyde solution overnight for post-fixation.

[0101] (5) Brain slice sample preparation.

[0102] Prepare a 30% sucrose solution. Then dehydrate the mouse brain fixed overnight with 30% sucrose. The dehydration time is about 24 hours. After the brain tissue sinks to the bottom in the 30% sucrose solution, take it out, embed it with OCT, and freeze it quickly at -25°C for 1 hour. Then use a freezing microtome to cut it into 30μm thick brain slice samples and attach them to a slide. Finally, use a pipette to add anti-fluorescence quenching agent, cover with a coverslip, and fix the four corners of the coverslip with nail polish.

[0103] (6) Acquisition, analysis and processing of fluorescence photographs.

[0104] The brain slice samples prepared in step (5) were observed and photographed using a laser confocal microscope. The excitation light of the microscope was 488 nm laser, and the objective lens magnification was 5× or 20×. The fluorescence expression results are shown in Figure 5 , Figure 6 and Figure 7 . Take 6 brain slices and use ImageJ software to count the EGFP fluorescence expression range. Finally, use Origin software to draw the results into a bar graph for comparison. Figure 8 .

[0105] The results showed that after adding PEG, the AAV transfection efficiency showed local high efficiency, while the transfection efficiency of AAV without auxiliary transfection reagents showed diffuse low density characteristics, indicating that PEG significantly promoted the local transgene expression of AAV in the living brain.

[0106] Example 4. PEG promotes high-density transgene expression of AAV in the living brain.

[0107] (1) Brain slice sample preparation:

[0108] The titer was 4.2×10 11 v·g / mL of virus transfection solution, wherein the mass fraction of PEG4000 is 0% and 40%, respectively. 300nL or 1000nL of the above transfection solution is injected into the mouse brain, respectively. After 3 weeks of virus expression, the mouse brain is frozen and sliced ​​into 30μm thick brain slices according to the steps described in Example 3, attached to a glass slide, and then a hydrophobic pen is used to draw a circle around the brain slice, and it is stored in a 4°C humidity box away from light.

[0109] (2) Immunofluorescence staining:

[0110] Next, the neuron-specific nuclear protein NeuN was labeled by immunofluorescence staining to reflect the distribution of neurons in the brain slice. The specific staining experimental process is as follows: First, TritonX-100 was diluted to 0.3% volume fraction with 3% bovine serum albumin (BSA). Then 100 μL of 0.3% TritonX-100 was added to the brain slice with a pipette to treat the nerve cells through the membrane for 15 minutes. Then 3% BSA was replaced to block the sample for 1 hour to occupy non-specific binding sites on the brain slice. Then a mouse monoclonal primary antibody (Millipore, MAB377) that can specifically recognize NeuN was selected to mark neurons, and the primary antibody was diluted with 3% bovine serum albumin at a volume ratio of 1:200. After incubation in a refrigerator at 4°C overnight, wash 3 times with 1×PBS, each time for 15 minutes. Then, goat anti-mouse secondary antibody with Alexa Fluor 633 fluorescent group (Thermal Fisher, A-21050) was selected to incubate the brain slices at room temperature in the dark for 2 hours, where the secondary antibody was diluted with 3% bovine serum albumin at a volume ratio of 1:1000. After the secondary antibody incubation, the slices were washed three times with 1×PBS, each time for 15 minutes. Then, anti-fluorescence quencher was added to the brain slice samples to protect the fluorescence, and the coverslip was slowly covered, and the four corners of the coverslip were fixed with nail polish.

[0111] (3) Fluorescence photo acquisition and analysis and processing:

[0112] The brain slice samples after immunofluorescence staining were observed and photographed by laser confocal microscopy. The objective lens magnification was selected to be 20×, and EGFP fluorescence and Alexa Fluor633 fluorescence were excited by 488nm and 633nm lasers respectively. Six brain slices were taken (typical fluorescence expression results are shown in Fig. 9 ), the number of EGFP / NeuN co-positive cells and the number of NeuN-positive cells within the EGFP fluorescence expression range were counted using ImageJ software, and the ratio of the two was defined as the transfection efficiency. Finally, the results were plotted into a bar graph for comparison using Origin software. Fig.10 .

[0113] The results showed that after adding PEG, the AAV transfection efficiency was significantly improved, indicating that PEG can significantly promote high-density transgene expression of AAV in the living brain.

[0114] Example 5. PEG promotes local and efficient transfection of AAV into the living brain over a long period of time.

[0115] (1) Prepare the titer of 4.2×10 11v·g / mL virus transfection solution, wherein the mass fraction of PEG4000 is 0% and 40%, respectively. 300 nL of the virus transfection solution is injected into the mouse brain. After 3 weeks and 8 weeks of virus expression, the mouse brain is removed, cut into brain slices, and brain slice samples are prepared according to the method described in Example 3.

[0116] (2) The expression of fluorescent proteins in brain slice samples was observed by laser confocal microscopy. The excitation wavelength of the confocal microscope was selected as 488 nm, the objective magnification was selected as 20×, and the 3×3 large image stitching function was selected to characterize a large range of neurons. The results are shown in Fig.11 .

[0117] From the results of EGFP fluorescence expression, at weeks 3 and 8, the transfection efficiency of AAV / PEG showed local high efficiency, while the transfection efficiency of AAV without auxiliary transfection reagent showed diffuse low density. This result fully confirms that PEG can promote the local high efficiency transfection of AAV in the living brain for a long time.

[0118] Example 6. Verification of the good biocompatibility of PEG in the living brain: immune response.

[0119] (1) Prepare AAV / PEG mixed transfection solution.

[0120] The same step (1) as in Example 3 was used to prepare a titer of 4.2×10 11 v·g / mL of virus transfection solution, wherein the mass fraction of PEG4000 was 0% and 40% respectively.

[0121] (2) Craniotomy in mice.

[0122] Male C57 mice aged 7-8 weeks were purchased from the company. After being raised in a laboratory environment for 3 days, they were injected with Avertin anesthetic by intraperitoneal injection at a dose of 0.04 mL / g, divided into two injections. The surgical instruments were sterilized with high temperature and high pressure in advance. After the mice were deeply anesthetized, the hair above the scalp of the mice was shaved. The mice were fixed to the brain stereotaxic instrument through ear bars, and the scalp was disinfected with cotton swabs dipped in iodine, and then the mouse scalp was cut open with straight scissors to expose the skull. The mouse skull was then leveled using three-dimensional coordinates and marked above the target brain area. Then, under a stereomicroscope, the skull at the marked position was removed with a cranial drill to expose the cranial window. The cranial drill used had a diameter of about 0.4 mm, and the cranial window was a square with a side length of about 1 mm. After carefully peeling off the dura mater under the skull, the exposed brain tissue was covered with a wet cotton swab.

[0123] (3) Brain localization injection.

[0124] Assemble a Hamilton syringe with a range of 10 μL and a No. 33 injection needle with an outer diameter of 210 μm. Fix the movable end of the microinjection pump to the movable arm of the brain stereotaxic instrument. Connect the microinjection syringe to the microinjection pump. Then set the parameters on the microinjection syringe display screen, and absorb the virus solution prepared in step (1), and the absorption speed is set to 0.1 μL / s. Then implant the injection needle into the target brain area through the movable arm of the brain stereotaxic instrument. Set the injection parameters on the display screen, the injection speed is 0.1 μL / min, and the injection volume is 300 nL. After the injection of the virus solution is completed, in order to prevent the virus solution from flowing back, it is necessary to stay in place for 10 minutes before slowly pulling the injection needle out of the brain tissue. Finally, fill the cranial window with a single-component isolation glue and suture the scalp.

[0125] (4) Remove the brain.

[0126] After 3 and 8 weeks of virus transfection, the mice were anesthetized by intraperitoneal injection of anesthetics again. Then 20 mL of saline was perfused transcardially to drain the blood from the mice. Then 20 mL of paraformaldehyde solution was slowly perfused transcardially to perform pre-fixation on the mice. After half an hour of placement, the mouse brain was carefully removed and soaked in paraformaldehyde solution overnight for post-fixation.

[0127] (5) Brain slice sample preparation.

[0128] Prepare a 30% sucrose solution. Then dehydrate the mouse brain fixed overnight with 30% sucrose. The dehydration time is about 24 hours. After the brain tissue sinks to the bottom in the 30% sucrose solution, take it out, embed it with OCT, and freeze it at -25℃ for 1 hour. Then use a freezing microtome to cut it into 30μm thick brain slice samples, attach it to a slide, and then use a hydrophobic pen to draw circles around the brain slices, and store them in a 4℃ humidity box away from light.

[0129] (6) Immunofluorescence staining:

[0130] Next, the glial fibrillary acid protein GFAP was labeled by immunofluorescence staining to reflect the distribution of astrocytes in the brain slices. The specific staining experimental process is as follows: First, Triton X-100 was diluted to 0.3% volume fraction with 3% bovine serum albumin (BSA). Then 100 μL of 0.3% volume fraction Triton X-100 was added to the brain slice with a pipette, and the cells in the brain slice were treated for 15 minutes. Then 3% BSA was used to block the sample for 1 hour to occupy nonspecific binding sites on the brain slice. Then a rabbit primary antibody (Sigma-Aldrich, HPA056030) that can specifically recognize GFAP was selected to mark astrocytes, and the primary antibody was diluted with 3% bovine serum albumin at a volume ratio of 1:1000. After incubation in a refrigerator at 4°C overnight, wash 3 times with 1×PBS, each time for 15 minutes. Then, goat anti-rabbit secondary antibody (Thermal Fisher, A-11012) with Alexa Fluor 594 fluorescent group was selected to incubate the brain slices at room temperature in the dark for 2 hours. The secondary antibody was diluted with 3% bovine serum albumin at a volume ratio of 1:1000. After the secondary antibody incubation, the slices were washed three times with ×PBS, each time for 15 minutes. Then, anti-fluorescence quencher was added to the brain slice samples to protect the fluorescence, and the coverslip was slowly covered, and the four corners of the coverslip were fixed with nail polish.

[0131] (7) Fluorescence photo acquisition and analysis and processing:

[0132] Astrocytes in brain slice samples were observed by laser confocal microscopy to reflect the immune response in the brain. The excitation wavelength of the confocal microscope was selected to be 633nm, the objective magnification was selected to be 20×, and the 3×3 large image stitching function was selected. The results are shown in Fig.12 .

[0133] From the results of GFAP fluorescence staining, there was no significant difference in the immune response induced by AAV / PEG injection and AAV injection at weeks 3 and 8. Therefore, the introduction of a small amount of PEG will not significantly aggravate the immune response in the mouse brain.

[0134] Example 7. Verification of the good biocompatibility of PEG in the living brain: animal behavior.

[0135] (1) Prepare AAV / PEG mixed transfection solution.

[0136] The same step (1) as in Example 3 was used to prepare a titer of 4.2×10 11 v·g / mL of virus transfection solution, wherein the mass fraction of PEG4000 was 0% and 40% respectively.

[0137] (2) Craniotomy in mice.

[0138] Male C57 mice aged 7-8 weeks were purchased from the company. After being raised in a laboratory environment for 3 days, they were injected with Avertin anesthetic by intraperitoneal injection at a dose of 0.04 mL / g, divided into two injections. The surgical instruments were sterilized with high temperature and high pressure in advance. After the mice were deeply anesthetized, the hair above the scalp of the mice was shaved. The mice were fixed to the brain stereotaxic instrument through ear bars, and the scalp was disinfected with cotton swabs dipped in iodine, and then the mouse scalp was cut open with straight scissors to expose the skull. The mouse skull was then leveled using three-dimensional coordinates and marked above the target brain area. Then, under a stereomicroscope, the skull at the marked position was removed with a cranial drill to expose the cranial window. The cranial drill used had a diameter of about 0.4 mm, and the cranial window was a square with a side length of about 1 mm. After carefully peeling off the dura mater under the skull, the exposed brain tissue was covered with a wet cotton swab.

[0139] (3) Brain localization injection.

[0140] Assemble a Hamilton syringe with a range of 10 μL and a No. 33 injection needle with an outer diameter of 210 μm. Fix the active end of the microinjection pump to the active arm of the stereotaxic instrument, and connect the microinjection pump to the microinjection pump. Then set the suction speed to 0.1 μL / s on the microinjection display. For the control group mice, a virus solution without PEG was aspirated, and then the injection needle was implanted into the target brain areas of the left and right brains of the mice in sequence through the active arm of the stereotaxic instrument for injection. For the experimental group mice, a virus solution with a PEG mass fraction of 40% was aspirated, and then the injection needle was implanted into the target brain areas of the left and right brains of the mice in sequence through the active arm of the stereotaxic instrument for injection. Set the injection parameters on the display screen, the injection speed is 0.1 μL / min, and the injection volume is 300 nL. After the injection, in order to prevent the injection solution from flowing back, it is necessary to stay in place for 10 minutes before slowly pulling the injection needle out of the brain tissue. Finally, fill the cranial window with a single-component isolation glue and suture the scalp.

[0141] (4) Open-field behavior.

[0142] Three weeks after surgery, the mice were placed in a 50×50 cm 2 The mice were allowed to move freely in the open-field behavior box for 15 minutes to adapt to the environment. Then the SuperMaze video tracking software was turned on to record the coordinates of the center of gravity and the corresponding time of the mice for 15 minutes. There were 4 mice in each experimental group and control group, and the test was performed once during the day and once at night to avoid experimental errors caused by individual rhythm differences. Every time the mice were changed, 75% medical alcohol was sprayed on the open-field behavior box to remove odors.

[0143] (5) Motion data processing and analysis.

[0144] The average movement speed and rest time of mice within 15 minutes were analyzed by SuperMaze software, and the standard deviation was calculated. The analysis results were plotted into a bar graph using Origin software to compare the exercise capacity of the two groups of mice. Fig.13 .

[0145] The results showed that there was no significant difference in the movement speed and rest time of the two groups of mice. Therefore, the introduction of a small amount of PEG will not significantly affect the brain function of mice.

[0146] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate a transfection auxiliary reagent and its application for improving the transfection efficiency and / or transfection accuracy of a gene vector of the present invention, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

[0147] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0148] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A gene transfection method for non-disease diagnosis and treatment purposes, characterized in that: The gene transfection is in vitro transfection; The gene transfection method comprises mixing a transfection auxiliary reagent that improves the transfection efficiency and / or transfection accuracy of the gene vector, the gene vector and cells for transfection; The transfection auxiliary reagent is polyethylene glycol 4000; the mass fraction of the transfection auxiliary reagent in the transfection system is 2.8%-4.2%; The gene vector is an adeno-associated virus; The cell is any one of cervical cancer cells, human kidney cells, mouse astrocytes, human chronic myeloid leukemia cells or rat adrenal pheochromocytoma cells.

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