In-vitro transduction enhancer, and method for detecting infection efficacy

Abstract

The present invention relates to an in-vitro transduction enhancer and a method for detecting infection efficacy, and particularly, provided is an in-vitro transduction enhancer which comprises one or more of neuraminidase (NA), sodium butyrate (NaBu) and Poly-Lysine. It is found for the first time that the enhancer can greatly reduce a usage amount of viruses or vectors (such as viral vectors) and has a better efficacy detection effect, so that a technical bottleneck is broken through, and a technical obstacle in the art is solved.

Description

An in vitro transduction enhancer and infection efficacy detection method Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an in vitro transduction enhancer and an infection efficacy detection method. Background Art

[0002] Over the past decade, adeno-associated virus (AAV)-mediated gene therapy has shown great potential in treating inherited monogenic diseases, and an increasing number of AAV-based gene therapy products have entered clinical development. Currently, seven AAV-based drugs have been approved for marketing worldwide. One of the challenging aspects of gene therapy development is the characterization and quantification of vector potency, which is a key method for product quality control and is required for marketing applications for AAV-based drugs.

[0003] Potency assays for rAAV products should reflect viral physical titer, infectious activity, and biological activity. Quantitative polymerase chain reaction (qPCR) is generally used in early clinical trials to measure viral physical titer, while infectious activity and target product expression in sensitive or targeted cells can be used to characterize biological activity. After pivotal clinical trials are underway, further in vitro enzyme activity assays or in vivo functional assays reflecting the product's mechanism of action should be developed.

[0004] In vivo functional assays generally require corresponding animal models, which are relatively scarce given the wide range of indications for AAV gene therapy. Furthermore, animal experiments require long timelines, and individual differences between batches of animals can exist, making in vivo efficacy testing difficult to validate and standardize. Furthermore, in vivo efficacy assessment is a time-consuming and labor-intensive process, resulting in high costs and an increased risk of introducing variability.

[0005] The in vitro potency assessment method for AAV vectors reduces testing time and the amount of vector required, thereby lowering testing costs. Due to the homogeneity of in vitro cultured cells, the method's reproducibility is expected to be improved. Furthermore, the in vitro potency assessment method for AAV vectors complies with the EU's principles on the replacement, reduction, and improvement of animal experiments used for scientific purposes.

[0006] AAV9 transduction of 293T cells in vitro is challenging, typically requiring the use of large amounts of virus at very high MOIs to achieve optimal gene expression levels. A higher MOI results in a larger viral volume, and the volume of virus added in cell-based experiments cannot be increased indefinitely due to limitations in the culture medium, thus limiting the scope of detection methods. High MOIs in cell-based experiments require high titers of AAV vector, which also presents challenges in virus purification.

[0007] Therefore, there is an urgent need in the art to develop a new method for improving the preparation and efficacy detection of vector transduction in vitro.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to provide an in vitro transduction enhancer and an infection efficacy detection method.

[0010] A first aspect of the present invention provides an in vitro transduction enhancer comprising one or more of neuraminidase (NA), sodium butyrate (NaBu), and poly-lysine.

[0011] In another preferred embodiment, the in vitro transduction enhancer includes a combination of two or more of neuraminidase (NA), sodium butyrate (NaBu), and poly-lysine.

[0012] In another preferred embodiment, the in vitro transduction enhancer comprises a combination of sodium butyrate and polylysine.

[0013] In another preferred embodiment, the in vitro transduction enhancer comprises a combination of sodium butyrate and neuraminidase.

[0014] In another preferred embodiment, the in vitro transduction enhancer comprises a combination of polylysine and neuraminidase.

[0015] In another preferred embodiment, the in vitro transduction enhancer comprises a combination of polylysine, neuraminidase and sodium butyrate.

[0016] In another preferred embodiment, the poly-lysine includes poly-D-lysine (PDL).

[0017] In another preferred embodiment, the enzyme activity concentration of the neuraminidase (NA) is 3-120 mU / mL, preferably 5-110 mU / mL, and more preferably 6.25-100 mU / mL.

[0018] In another preferred embodiment, the molar concentration of sodium butyrate (NaBu) is 1-20 mM, preferably 2.5-20 mM, more preferably 5-20 mM.

[0019] In another preferred embodiment, the mass concentration of the poly-lysine is 0.1-200 μg / mL, preferably 0.5-150 μg / mL, and more preferably 1-100 μg / mL.

[0020] In another preferred embodiment, the in vitro transduction enhancer is used to enhance the transduction efficiency of the vector to cells.

[0021] In another preferred embodiment, the vector comprises a viral vector.

[0022] In another preferred embodiment, the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector (AAV), a DNA viral vector, a retroviral vector, or a combination thereof.

[0023] In another preferred embodiment, the vector is an adeno-associated virus AAV vector.

[0024] The second aspect of the present invention provides a kit comprising the in vitro transduction enhancer according to the first aspect of the present invention.

[0025] In another preferred embodiment, the kit further comprises a label or instructions.

[0026] The third aspect of the present invention provides a use of the in vitro transduction enhancer according to the first aspect of the present invention for preparing a composition or a kit for enhancing the transduction efficiency of a vector to cells.

[0027] In another preferred embodiment, the vector comprises a viral vector.

[0028] In another preferred embodiment, the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector (AAV), a DNA viral vector, a retroviral vector, or a combination thereof.

[0029] In another preferred embodiment, the vector is an adeno-associated virus AAV vector.

[0030] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0031] In another preferred embodiment, the composition further comprises other substances that enhance the transduction efficiency of the vector to cells.

[0032] In another preferred embodiment, the dosage form of the composition is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0033] In another preferred embodiment, the composition is in the form of a liquid preparation.

[0034] In another preferred embodiment, the composition is in the form of an injection.

[0035] The fourth aspect of the present invention provides a use of a substance for preparing a drug for gene therapy, wherein the substance comprises the in vitro transduction enhancer according to the first aspect of the present invention and a vector.

[0036] In another preferred embodiment, the vector comprises a viral vector.

[0037] In another preferred embodiment, the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector (AAV), a DNA viral vector, a retroviral vector, or a combination thereof.

[0038] In another preferred embodiment, the vector is an adeno-associated virus AAV vector.

[0039] A fifth aspect of the present invention provides a method for enhancing the in vitro transduction efficiency of a vector to cells, comprising:

[0040] The cells are contacted with the vector in the presence of the in vitro transduction enhancer according to the first aspect of the present invention.

[0041] In another preferred embodiment, the cells include eukaryotic cells.

[0042] In another preferred embodiment, the cells include human or non-human mammalian cells.

[0043] In another preferred embodiment, the non-human mammals include rodents (such as mice, rats, rabbits), cows, pigs, sheep, horses, dogs, cats, and non-human primates (such as monkeys).

[0044] In another preferred embodiment, the cells include somatic cells, stem cells, germ cells, non-dividing cells, or a combination thereof. In another preferred embodiment, the cells include renal cells, epithelial cells, endothelial cells, or a combination thereof.

[0045] In another preferred embodiment, the cells include: 293T cells, 293 cells, HEK 293.

[0046] In another preferred embodiment, the cells are cells cultured in vitro.

[0047] In another preferred embodiment, the method is an in vitro method.

[0048] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG1 shows a comparison of the effects of different formulations of in vitro transduction enhancers on AAV9-EGFP vector transduction of 293T cells.

[0050] FIG2 shows the effect of sodium butyrate on the transduction of 293T cells by AAV9-CBH-EGFP vector.

[0051] FIG3 shows a comparison of the effects of different combinations of poly-lysine and neuraminidase on AAV9-CBH-EGFP vector transduction in 293T cells.

[0052] FIG4 shows a comparison of the effects of different concentrations of sodium butyrate on AAV9-CBH-TGPD vector transduction of 293T cells.

[0053] FIG5 shows a comparison of the effects of different concentrations of neuraminidase on AAV9-CBH-TGPD vector transduction of 293T cells.

[0054] FIG6 shows a comparison of the effects of different combinations on AAV9-CBH-TGPD vector transduction of 293T cells.

[0055] FIG7 shows a comparison of the effects of different poly-lysine hand shapes and concentrations on AAV9-CBH-TGPD vector transduction of 293T cells.

[0056] FIG8 shows a schematic diagram of the structure of AAV9-CBH-TGPD.

[0057] FIG9 shows a schematic diagram of the specific structure of AAV9-CBH-EGFP. DETAILED DESCRIPTION

[0058] After extensive and in-depth research, the inventors discovered for the first time that adding one or more of neuraminidase (NA), sodium butyrate (NaBu), and poly-lysine to the in vitro potency assessment of AAV vectors can significantly reduce the amount of virus or vector (e.g., viral vector) used while achieving better potency testing results. This breakthrough overcomes technical bottlenecks and technical obstacles in this field. Based on this, the inventors completed the present invention.

[0059] Neuraminidase (NA)

[0060] Neuraminidase, also known as sialidases, belongs to a family of exoglycosaccharidases (EC 3.2.1.18) that hydrolyze terminal sialic acid residues in glycolipids, oligosaccharides, and glycoproteins as the first step in their degradation. Neuraminidase has been identified in a range of organisms, including viruses, protozoa, bacteria, fungi, and vertebrates. The best-known neuraminidase is viral neuraminidase (viNEU), a drug target for preventing the spread of influenza infection.

[0061] Sodium butyrate (NaBu)

[0062] As described herein, sodium butyrate (NaBu) is a deacetylase inhibitor that can increase histone acetylation levels, facilitate transcription, and thus enhance gene expression.

[0063] Poly-Lysine

[0064] As described herein, poly-lysine is a polycationic polymer with a high cationic charge density, enabling the polymer network to act as an effective "proton sponge" at any pH. This polycation can transfer various reporter genes into cells of various species.

[0065] In the present invention, poly-lysine includes poly-D-lysine and poly-L-lysine.

[0066] Enhancer

[0067] As used herein, the terms "in vitro transduction enhancer" and "enhancer" are used interchangeably and refer to a composition containing one or more of the following components: neuraminidase (NA), sodium butyrate (NaBu), and poly-lysine.

[0068] There is no limitation on the ratio of the components of the enhancer of the present invention. Generally, each component should satisfy its minimum effective concentration. In a preferred embodiment, the minimum effective concentration of each component in the enhancer is as follows:

[0069] The neuraminidase (NA) activity concentration is 3-120 mU / mL, preferably 5-110 mU / mL, more preferably 6.25-100 mU / mL; the molar concentration of sodium butyrate (NaBu) is 1-20 mM, preferably 2.5-20 mM, more preferably 5-20 mM;

[0070] The mass concentration of poly-lysine is 0.1-200 μg / mL, preferably 0.5-150 μg / mL, and more preferably 1-100 μg / mL.

[0071] In the present invention, it was demonstrated that one or more of neuraminidase (NA), sodium butyrate (NaBu), and poly-lysine have excellent activity in improving the efficiency of vector-to-cell transduction. Of course, based on the teachings of the present invention, those skilled in the art can also combine any of the above neuraminidase (NA), sodium butyrate (NaBu), and poly-lysine to develop new enhancers that have the activity of improving the efficiency of vector-to-cell transduction.

[0072] Specifically, the present invention adds one or more of neuraminidase (NA), sodium butyrate (NaBu), and poly-lysine (Poly-Lysine) during the in vitro efficacy evaluation of AAV vectors, which can greatly reduce the amount of virus used while achieving better efficacy detection results, breaking through technical bottlenecks, and solving technical obstacles in this field.

[0073] The reasons are speculated to be as follows: neuraminidase (NA) can hydrolyze sialic acid, exposing terminal galactose residues in cultured cells, enhancing AAV9's ability to bind to cells and thus improving transduction efficiency; sodium butyrate (NaBu), as a deacetylase inhibitor, can increase histone acetylation levels, facilitating transcription and thus enhancing gene expression; and poly-lysine is a polycationic polymer with a high cationic charge density, enabling the polymer network to act as an effective "proton sponge" at any pH. This polycation can transfer various reporter genes into cells of various species.

[0074] The rate of dopamine biosynthesis is limited by three enzymes expressed in substantia nigra neurons. Tyrosine hydroxylase (TH) and cyclohydrolase 1 (GCH1) promote the conversion of tyrosine to levodopa, while amino acid decarboxylase (AADC) converts levodopa to dopamine. The AAV9-CBH-TGPD vector contains three genes: TH, GCH1, and AADC. After transduction into 293T cells, CBH initiates the expression of these three genes. Adding tyrosine as a substrate allows the three enzymes to convert tyrosine to dopamine. We evaluated the cell transduction ability of AAV9-CBH-TGPD by measuring the amount of dopamine produced by 293T cells.

[0075] During the AAV9-CBH-TGPD vector transduction of 293T cells, sodium butyrate (NaBu), poly-D-lysine (PDL), neuraminidase (NA) and their combination were used to treat the cells at different stages to detect the ability of the AAV9-CBH-TGPD vector to convert tyrosine to dopamine in 293T cells, thereby evaluating its ability to enhance the transduction of 293T cells by the AAV9-CBH-TGPD vector.

[0076] Pharmaceutical composition

[0077] The present invention provides a pharmaceutical composition comprising the enhancer of the present invention and a pharmaceutical composition comprising the in vitro transduction enhancer of the present invention and a vector (such as a viral vector).

[0078] The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient: the enhancer of the present invention.

[0079] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0080] As used herein, a "pharmaceutically acceptable carrier" is a substance that is suitable for administration to humans and / or mammals without excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., has a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.

[0081] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be compatible with the mode of administration. The pharmaceutical compositions of the present invention are available in the form of injections, oral preparations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, they can be prepared using conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions are preferably manufactured under sterile conditions.

[0082] The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's body weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg / kg animal body weight (preferably 0.0001 mg-10 mg / kg animal body weight) per day, satisfactory results can be obtained. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0083] The pharmaceutically acceptable carriers of the present invention include, but are not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the mode of administration, as is well known to those skilled in the art.

[0084] The present invention also provides uses of the pharmaceutical composition for enhancing transduction of cells by vectors (such as viral vectors); and / or for gene therapy. The pharmaceutical composition of the present invention can be administered in vivo via a local or systemic route, for example, by injection via an intramuscular, intravenous, intraarterial, intraperitoneal, or intracranial route. Therefore, the present invention also relates to a method for gene therapy, comprising administering a pharmaceutical composition as described above to a patient in need thereof.

[0085] The method further comprises administering a viral vector for gene therapy before, after, or together with the enhancer of the present invention. As is well understood in the art, the virus or viral vector for gene therapy of the present invention comprises a therapeutic gene.

[0086] Methods for enhancing vector transduction of cells in vitro

[0087] The present invention provides a method for enhancing in vitro cell transduction by a vector, comprising:

[0088] The cells are contacted with the vector in the presence of the in vitro transduction enhancer according to the first aspect of the present invention.

[0089] In the present invention, the method of the present invention further comprises verifying the effective transduction or transduction level of the virus or vector (such as a viral vector) to the cell. In addition, according to this aspect, the enhancer of the present invention may be used to improve the infection or transduction of the target virus or vector (such as a viral vector), which has many applications as described below.

[0090] A preferred embodiment of the present invention is the use of the enhancers of the present invention as general enhancers of viral infection or transduction efficiency in routine laboratory practice or gene therapy approaches based on viral vector systems. The compositions enhance the entry of vectors, such as vectors designed for gene therapy, diagnosis, or any other purpose (e.g., vectors for functional studies), into cells in vitro and ex vivo. The enhancers can be administered in combination with viral vectors, such as vectors for gene therapy or diagnosis, and mediate the entry of the viral vector into target cells. The compositions are also useful in vitro because they promote viral uptake by cells. Therefore, they can be used as tools for studying viruses and their mechanisms of action.

[0091] The enhancers of the present invention can generally be used to enhance the entry of viral particles into target cells. The enhancers can also be used as general enhancers of the infection / transduction rate of viral particles, such as non-enveloped viral particles such as AAV vectors.

[0092] The enhancers of the present invention can also be used to enhance gene delivery rates in ex vivo gene therapy methods based on vector systems, particularly those based on non-enveloped or enveloped vector systems. Therefore, the present invention also relates to the use of enhancers as described above in gene therapy to promote infection of eukaryotic cells by a virus or viral vector in a subject in need thereof. The enhancers of the present invention can be used in combination with a virus or viral vector for gene therapy. In particular, the present invention relates to methods for using the enhancers of the present invention in treating a disease by gene therapy, wherein the enhancers of the present invention are used in combination with a virus or viral vector that contains in its genome an appropriate therapeutic transgene for treating the disease. In one embodiment, the treatment is performed by administering a virus or viral vector containing one of the above-mentioned transgenes for treating the corresponding disease (e.g., thalassemia, sickle cell anemia, Parkinson's disease, AMD, hemophilia, AIDS, hyperlipidemia, diabetes, etc.). The enhancer can be administered simultaneously, separately, or sequentially with the gene therapy vector. Transduction efficiencies are typically low when viral vectors are tested for efficacy in vitro. However, in the presence of the enhancers of the present invention, cells can be effectively transduced by the viral vector, resulting in higher efficiency of gene delivery to target cells compared to samples without the enhancers of the present invention. Another advantage of the enhancer of the present invention is that it allows for the use of less viral vector than would be the case without the enhancer of the present invention.

[0093] The main advantages of the present invention include:

[0094] 1. The present invention has for the first time discovered that the efficiency of vector transduction of cells in vitro is improved, and the level of transduction of related vectors in target cells is improved.

[0095] 2. Adding one or more of neuraminidase (NA), sodium butyrate (NaBu), and poly-lysine during the in vitro potency evaluation of AAV vectors can greatly reduce the amount of virus used while achieving better potency detection results, breaking through technical bottlenecks, and solving technical obstacles in this field.

[0096] Example

[0097] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0099] Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

[0100] Vector construction information:

[0101] The specific construction information of AAV9-CBH-TGPD is described in the patent application with patent application number CN202311094965.7.

[0102] AAV9-CBH-TGPD vector:

[0103] TH-GS15-GCH1-P2A-AADC was digested with AgeI and SalI / ligated and cloned into the adeno-associated virus backbone vector pAAV-MCS-CBH-SV40 between CBH and SV40, and the vector was named AAV9-CBH-TGPD vector.

[0104] The schematic diagram of the structure of the AAV9-CBH-TGPD vector is shown in Figure 8;

[0105] Among them, TH is tyrosine hydroxylase, CBH is promoter, GCH1 is GTP-cyclohydrolase 1, and AADC is aromatic amino acid dopa decarboxylase.

[0106] AAV9-CBH-EGFP vector:

[0107] The EGFP sequence was cloned and used to replace the TH-GS15-GCH1-P2A-AADC sequence in the AAV9-CBH-TGPD vector. The vector was named CBH-EGFP vector.

[0108] The specific structural diagram of AAV9-CBH-EGFP is shown in FIG9 .

[0109] Reagent Information:

[0110] Unless otherwise specified, the reagents used in this example are commercially available. The following reagents are schematically listed:

[0111] Example 1: The enhancing effect of poly-D-lysine, neuraminidase and sodium butyrate on AAV9-EGFP vector transduction of 293T cells.

[0112] 48-well plates were coated with 200 μl / well of 10 μg / mL poly-D-lysine (purchased from Beyotime, Catalog No. C0312) and refrigerated at 4°C overnight. The next morning, the poly-D-lysine solution was aspirated, washed three times with D-PBS, and air-dried. 293T cells (purchased from ATCC) were seeded into 48-well plates at 1.0E+05 cells / well. After 3 hours of culture, neuraminidase (purchased from Sigma, Catalog No. N7885-1UN) was added to a final concentration of 50 mU / mL. After 2 hours of culture, AAV9-CBH-EGFP (obtained from Kanglin Biotechnology (Hangzhou) Co., Ltd., structure shown in Figure 9) was transduced at an MOI of 5000. After overnight, an equal volume of DMEM medium (purchased from Cytiva, Catalog No. SH30022.01) containing 5 mM sodium butyrate (purchased from Aladdin, Catalog No. S102956) was added. 24 hours after transduction, the medium was replaced with 250 μL of DMEM medium (5% FBS) (purchased from ExcellBio, product number FSP500). Two days later, cells were collected and the GFP positive rate was detected by flow cytometry.

[0113] The results are shown in Figure 1. Poly-D-lysine can increase the transduction efficiency by 1.5 times, sodium butyrate can increase the transduction efficiency by 1.25 times, neuraminidase can increase the transduction efficiency by 2 times, the combination of poly-D-lysine and sodium butyrate can increase the transduction efficiency by 1.9 times, the combination of poly-D-lysine and neuraminidase can increase the transduction efficiency by 2.6 times, and the combination of poly-D-lysine, sodium butyrate and neuraminidase can increase the transduction efficiency by 3 times.

[0114] Example 2: Sodium butyrate enhances the transduction effect of AAV9-CBH-EGFP vector in 293T cells.

[0115] 293T cells were seeded into 48-well plates at 1.0E+05 cells / well and transduced with AAV9-CBH-EGFP at an MOI of 10,000 after 6 hours of culture. An equal volume (250 μL) of DMEM medium containing 0 / 5 mM sodium butyrate was added overnight. After 6-8 hours, 250 μL of medium was aspirated and an equal volume of DMEM was added. After overnight, the GFP-positive rate of 293T cells was determined by flow cytometry.

[0116] The results are shown in Figure 2. The results show that after adding sodium butyrate, although the GFP positive rate of 293T cells only increased by 6%, the average fluorescence intensity of GFP increased by about 1 times, indicating that sodium butyrate can significantly increase the expression of GFP in 293T cells.

[0117] Example 3: Different combinations of polylysine and neuraminidase enhance the transduction effect of 293T cells after AAV9-CBH-EGFP vector transduction.

[0118] A 96-well plate was coated with 100 μl / well of 10 μg / mL poly-D-lysine and refrigerated at 4°C overnight. The next morning, the poly-D-lysine solution was aspirated, the cells were washed three times with D-PBS, and air-dried. 293T cells were seeded into the 96-well plate at 2.0E+04 cells / well. After 3 hours of culture, neuraminidase was added to a final concentration of 50 mU / mL. After 2 hours of culture, AAV9-CBH-EGFP was transduced at an MOI of 30,000. Two days after transduction, the GFP-positive rate of the 293T cells was determined by flow cytometry.

[0119] The results are shown in Figure 3. Neuraminidase alone increased the GFP-positive rate of 293T cells by approximately 6%, while poly-D-lysine alone increased the GFP-positive rate by 2-fold. Simultaneous use of neuraminidase and poly-D-lysine increased the GFP-positive rate of 293T cells by more than 4-fold and the average GFP fluorescence intensity by approximately 2-fold.

[0120] Example 4: Sodium butyrate enhances the transduction effect of AAV9-CBH-TGPD vector in 293T cells.

[0121] 48-well plates were coated with 200 μl / well of 10 μg / mL poly-D-lysine and refrigerated at 4°C overnight. The next morning, the poly-D-lysine solution was aspirated, washed three times with D-PBS, and air-dried. 293T cells were seeded into 48-well plates at 1.0E+05 cells / well and cultured for 6 hours before transduction with AAV9-CBH-TGPD at an MOI of 200,000. Overnight, equal volumes (250 μl) of DMEM medium containing varying sodium butyrate concentrations (Cytiva, Cat. No. SH30022.01) were added, resulting in final sodium butyrate concentrations of 20, 10, 5, 2.5, 1.25, and 0 mM. Twenty-four hours after transduction, the medium was replaced with 250 μl of DMEM medium containing 0.2 mM tyrosine (5% FBS) (ExcellBio, Cat. No. FSP500). After 2 days, the cell culture supernatant was collected and 1 / 4 volume of perchloric acid (0.8 M) and cysteine ​​(3 mM) solution was added. The mixture was then centrifuged at 10,000 rpm for 5 minutes. The supernatant was collected and stored at -70°C or below. The test sample was removed, dissolved, and then the dopamine content was determined by HPLC.

[0122] The results are shown in FIG4 , which show that the dopamine content produced by 293T cells is proportional to the concentration of sodium butyrate within 10 mM, and the dopamine content is increased by 1.7-3.8 times compared with the control.

[0123] Example 5: Neuraminidase enhances the transduction effect of AAV9-CBH-TGPD vector in 293T cells.

[0124] A 48-well plate was coated with 200 μl / well of 10 μg / mL poly-D-lysine and refrigerated at 4°C overnight. The next morning, the poly-D-lysine solution was aspirated, washed three times with D-PBS, and air-dried. 293T cells were seeded into a 48-well plate at 1.0E+05 cells / well. After 3 hours of culture, neuraminidase was added to final concentrations of 100, 50, 25, 12.5, 6.25, and 0 mU / mL, respectively. After 2 hours of culture, AAV9-CBH-TGPD was transduced at an MOI of 200,000. An equal volume of DMEM medium containing 5 mM sodium butyrate was added overnight. 24 hours after transduction, the medium was replaced with 250 μL of DMEM medium (5% FBS) containing 0.2 mM tyrosine. After 2 days, the cell culture supernatant was collected and 1 / 4 volume of perchloric acid (0.8 M) and cysteine ​​(3 mM) solution was added. The mixture was then centrifuged at 10,000 rpm for 5 minutes. The supernatant was collected and stored at -70°C or below. The test sample was removed, dissolved, and then the dopamine content was determined by HPLC.

[0125] The results are shown in FIG5 , which show that the dopamine content produced by 293T cells is proportional to the neuraminidase concentration within 25 mU / mL, and the dopamine content is increased by 2.5-2.8 times compared to the control.

[0126] Example 6: The transduction-enhancing effect of polylysine and its different combinations with sodium butyrate and neuraminidase on 293T cells transduced with AAV9-CBH-TGPD vector.

[0127] A 48-well plate was coated with 200 μL / well of 10 μg / mL poly-D-lysine and refrigerated at 4°C overnight. The next morning, the poly-D-lysine solution was aspirated, the cells were washed three times with D-PBS, and air-dried. 293T cells were seeded at 1.0E+05 cells / well in a 48-well plate. After a 3-hour culture, neuraminidase was added to a final concentration of 50 mU / mL. After a 2-hour culture, AAV9-CBH-TGPD was transduced at an MOI of 200,000. An equal volume of DMEM medium containing 5 mM sodium butyrate was added overnight. 24 hours after transduction, the medium was replaced with 250 μL of DMEM medium containing 0.2 mM tyrosine (5% FBS). Two days later, the cell culture supernatant was collected and 1 / 4 volume of a 0.8 M perchloric acid solution containing 3 mM cysteine ​​was added. The cells were mixed, centrifuged at 10,000 rpm for 5 minutes, and the supernatant was collected and stored at -70°C or below. The test sample was taken out, dissolved and then the dopamine content was detected by HPLC.

[0128] The results are shown in Figure 6, which show that the use of poly D lysine alone can increase the dopamine content of 293T cells by 2.5-3.2 times, the simultaneous use of sodium butyrate and poly D lysine can increase the dopamine content of 293T cells by 6 times, the simultaneous use of sodium butyrate and neuraminidase can increase the dopamine content of 293T cells by 5 times, the simultaneous use of poly D lysine and neuraminidase can increase the dopamine content of 293T cells by 7 times, and the simultaneous use of poly D lysine, neuraminidase and sodium butyrate can increase the dopamine content of 293T cells by 15 times.

[0129] Example 7: Different poly-lysine hand shapes and concentrations enhance the transduction effect of AAV9-CBH-TGPD vector in 293T cells.

[0130] 48-well plates were coated with 200 μl / well of poly-D-lysine (PDL) or poly-L-lysine (PLL) at varying concentrations (1, 10, and 100 μg / mL) and refrigerated at 4°C overnight. The next morning, the poly-L-lysine solution was aspirated, washed three times with D-PBS, and air-dried. 293T cells were seeded into 48-well plates at a density of 1.0E+05 cells / well and cultured for 6 hours before transduction with AAV9-CBH-TGPD at an MOI of 200,000. An equal volume of DMEM medium supplemented with 5 mM sodium butyrate was added overnight. Twenty-four hours after transduction, the medium was replaced with 250 μL of DMEM medium supplemented with 0.2 mM tyrosine (5% FBS). Two days later, the cell culture supernatant was collected, and 1 / 4 volume of a 0.8 M perchloric acid solution containing 3 mM cysteine ​​was added. The cells were mixed, centrifuged at 10,000 rpm for 5 minutes, and the supernatant was collected and stored at -70°C or below. The test sample was taken out, dissolved and then the dopamine content was detected by HPLC.

[0131] The results are shown in Figure 7, which show that poly-D-lysine has a stronger ability to transduce 293T cells with AAV9-CBH-TGPD than poly-L-lysine, and there is no significant difference in the ability of poly-D-lysine to transduce 293T cells with AAV9-CBH-TGPD at concentrations of 1-100 μg / mL.

[0132] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An in vitro transduction enhancer, characterized in that include: One or more of neuraminidase (NA), sodium butyrate (NaBu), and poly-lysine (Poly-Lysine).

2. The in vitro transduction enhancer according to claim 1, characterized in that The enhancer includes two or more of neuraminidase (NA), sodium butyrate (NaBu) and poly-lysine (Poly-Lysine).

3. The in vitro transduction enhancer according to claim 1, characterized in that The enhancer includes a combination of sodium butyrate and poly-lysine.

4. The in vitro transduction enhancer according to claim 1, characterized in that The enhancer includes a combination of sodium butyrate and neuraminidase.

5. The in vitro transduction enhancer according to claim 1, characterized in that The enhancer comprises a combination of poly-lysine and neuraminidase.

6. The in vitro transduction enhancer according to claim 1, characterized in that The enhancer comprises a combination of poly-lysine, neuraminidase and sodium butyrate.

7. The in vitro transduction enhancer according to claim 1, characterized in that The poly-lysine includes poly-D-lysine (PDL).

8. The in vitro transduction enhancer according to claim 1, characterized in that The enzyme activity concentration of the neuraminidase (NA) is 3-120 mU / mL, preferably 5-110 mU / mL, and more preferably 6.25-100 mU / mL.

9. The in vitro transduction enhancer according to claim 1, characterized in that The molar concentration of the sodium butyrate (NaBu) is 1-20 mM, preferably 2.5-20 mM, more preferably 5-20 mM.

10. The in vitro transduction enhancer according to claim 1, characterized in that The mass concentration of the poly-lysine is 0.1-200 μg / mL, preferably 0.5-150 μg / mL, and more preferably 1-100 μg / mL.

11. The in vitro transduction enhancer according to claim 1, characterized in that The transduction enhancer is used to enhance the transduction efficiency of the vector to cells.

12. The in vitro transduction enhancer according to claim 11, characterized in that The vector includes a viral vector.

13. A kit, characterized in that: Comprising the in vitro transduction enhancer according to claim 1.

14. A use of the in vitro transduction enhancer according to claim 1, characterized in that: A composition or kit for preparing a composition or kit for enhancing the transduction efficiency of a vector to a cell.

15. The use according to claim 14, characterized in that The vector includes a viral vector.

16. A use of a substance, characterized in that: Used for preparing a drug for gene therapy, the substance comprises the in vitro transduction enhancer according to claim 1 and the vector according to claim 11.

17. The use according to claim 16, characterized in that The vector includes a viral vector.

18. A method for enhancing the in vitro transduction efficiency of a vector to a cell, characterized in that: include: The cells are contacted with the vector in the presence of the in vitro transduction enhancer of claim 1.

19. The method according to claim 18, characterized in that The cells include human or non-human mammalian cells.

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