Amino acid-mediated gene delivery and its uses

Abstract

The present invention provides an application of amino acids as carriers for delivering nucleic acids into the nervous system. Single-stranded oligonucleotides are effectively delivered to brain cells by local injection, preferably in combination with stereotactic injection, immunofluorescence staining, confocal microscopy and the like, and plasmid vectors are also delivered to astrocytes in the cerebellum.

Description

Technical Field

[0001] The present invention relates to amino acid-mediated gene delivery and uses thereof. Background Art

[0002] Many central nervous system diseases are associated with abnormal gene expression. In the past few decades, gene therapy has become an important means for treating various central nervous system diseases. However, safe and effective strategies for delivering genes into the central nervous system are very limited. At present, the main method of gene delivery in gene therapy usually uses viral vectors, but viruses, as naturally occurring pathogens, have a series of potential risks in gene therapy. In addition, several antisense oligonucleotide drugs have been used to treat neurodegenerative diseases, but the delivery efficiency of antisense oligonucleotide drugs in the central nervous system is still limited and lacks cell specificity, so long-term repeated high-dose administration is often required for effective treatment. Although other non-viral vectors (such as cationic polymers, liposomes, inorganic nanocarriers, etc.) have attracted widespread attention, due to the heterogeneity of the blood-brain barrier and brain tissue, it is still challenging to achieve high-efficiency and low-cytotoxicity treatments through these non-viral vectors. Therefore, the development of new delivery strategies, such as new vectors of nucleic acid materials, is of great significance for the treatment of neurological disorders, especially for gene therapy of neurodegenerative diseases.

[0003] Many neurological diseases are related to abnormal gene expression, and gene therapy has brought hope for the treatment of these neurological diseases. In recent years, more and more studies have found that astrocyte dysfunction can cause a series of central nervous system diseases. Therefore, the relationship between astrocyte function and related diseases has attracted extensive attention from researchers.

[0004] At present, the main method of gene delivery in the central nervous system is to use viral vectors. However, several defects of viral vectors limit their application in the treatment of nervous system diseases, especially in clinical practice, such as limited exogenous gene loading capacity, strong immunogenicity and potential carcinogenic risks. Therefore, the development of non-viral gene delivery methods has become an important research direction for gene delivery in the central nervous system. The non-viral methods currently used usually have disadvantages such as low delivery efficiency, high cytotoxicity and lack of cell type specificity. There is an urgent need for gene therapy to find non-viral-based vectors that can meet high delivery efficiency, low cytotoxicity and cell type specificity. Summary of the invention

[0005] The present invention focuses on improving the delivery efficiency of nucleic acid materials (such as oligonucleotides or plasmid vectors) into brain tissue. By local injection, preferably, combined with means such as stereotaxic injection, immunofluorescence staining, confocal microscopy, single-stranded oligonucleotides are effectively delivered to brain cells, and plasmid vectors are also delivered to astrocytes in the mouse brain. And using these strategies, we have achieved knockout and knock-in of genes in astrocytes in vivo. The present invention first found that amino acids can be directly used as nucleic acid delivery to tissues or organs of living organisms, particularly in the central nervous system.

[0006] First, the present invention provides a delivery system for delivery into a living organism, wherein the delivery system comprises an amino acid and a nucleic acid.

[0007] The nucleic acid is selected from oligonucleotides and plasmids. The oligonucleotides may be short lengths of single stranded DNA, RNA, such as antisense RNA and siRNA.

[0008] Preferably, the delivery system comprises amino acids and one or more plasmids.

[0009] Preferably, the amino acid is glycine, gamma-aminobutyric acid (GABA), proline, alanine, serine, asparagine, glutamine, sarcosine, citrulline, hydroxyproline, lily of the valley, betaine, hypotaurine, histidine or threonine. The amino acid can be L-type or D-type. Preferably, the amino acid is L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, glycine, gamma-aminobutyric acid (GABA), L-histidine or L-threonine. The amino acid can be a peptide composed of amino acids as basic units. Preferably, the peptide is diglycine or NL-leucylglycine.

[0010] Prior to delivery, the nucleic acid is dissolved in a solution containing the amino acid. Alternatively, the nucleic acid and amino acid solutions are delivered separately.

[0011] The delivery system of the present invention can be delivered directly into the tissue or organ of a living organism. Living organisms include, but are not limited to, plants and animals. Animals are invertebrates or vertebrates. Preferably, the animal is a bird, chicken, duck, goose. Preferably, the animal is a mammal, such as a mouse, rat, cat, rabbit, dog, horse, cow, sheep, goat, pig, tree shrew, monkey, chimpanzee, human, etc.

[0012] Preferably, the tissue or organ is the central nervous system, including the brain and spinal cord.

[0013] The brain involves, but is not limited to, the midbrain, thalamus, hypothalamus, brainstem, cerebellum, globus pallidus, cerebral cortex, and hippocampus.

[0014] Preferably, the delivery system of the present invention can be directly delivered to astrocytes in the midbrain, thalamus, hypothalamus, brainstem, cerebellum, globus pallidus lateralis, cerebral cortex and hippocampus via the delivery system of the present invention.

[0015] Preferably, the delivery system of the present invention can directly enter Bergmann glia, velate astrocytes and basket / stellate interneurons in the cerebellum via the delivery system of the present invention.

[0016] Preferably, the uptake of plasmids by astrocytes through the delivery system of the present invention is concentration-dependent. The concentration of amino acids in the solution of the delivery system can be as high as 300 mM. Preferably, the concentration of amino acids in the solution of the delivery system is in the range of 50 mM-300 mM.

[0017] Preferably, glycine specifically promotes astrocyte uptake of plasmid vectors. Preferably, GABA specifically promotes astrocyte uptake of plasmid vectors.

[0018] The osmotic pressure of a 300 mM glycine solution is approximately equal to that of physiological saline, and the osmotic pressure of a solution is proportional to its concentration. In the present invention, it is determined that the pH values ​​of glycine solutions of different concentrations remain stable and are maintained at about pH=6.

[0019] Similarly, solutions of L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, L-histidine, and L-threonine all promoted astrocyte uptake of plasmid vectors.

[0020] Preferably, in addition to amino acids, the delivery system of the present invention also comprises a selective inhibitor of amino acid transporters. For example, astrocytes are 3-4 times more efficient at taking up plasmids by a delivery system comprising glycine and bitopertin (a selective inhibitor of glycine transporter 1) than a delivery system comprising glycine alone.

[0021] The delivery system of the present invention effectively delivers an ultra-large plasmid vector carrying the CRISPR / Cas9 system to astrocytes and successfully knocks out genes in the genome of transgenic mice.

[0022] The delivery system of the present invention also successfully knocks exogenous genes into the genome of astrocytes to achieve permanent expression of the exogenous genes.

[0023] At the same time, the piggyBac transposon system was used to successfully integrate the exogenous gene eGFP into the astrocyte genome to achieve permanent expression of the exogenous gene.

[0024] The delivery system of the present invention can facilitate the efficient co-transfection of multiple plasmid vectors without the use of specific promoters, viral vectors and transgenic mice. Preferably, the delivery system of the present invention can achieve efficient co-transfection of two or three plasmid vectors into astrocytes.

[0025] The delivery system of the present invention can be used for gene therapy for the treatment of nervous system diseases, particularly central nervous system diseases. For example, the disease can be a neurodegenerative disease, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia. The disease can be a neurogenetic disease, such as congenital spinal muscular atrophy. The disease can be a tumor in the brain and / or spinal cord, such as a glioma.

[0026] The efficient co-transfection of multiple plasmid vectors provides a powerful tool for simultaneously manipulating the expression of multiple genes in astrocytes.

[0027] Second, the present invention provides the use of amino acids as vehicles for delivering nucleic acids into living organisms.

[0028] The nucleic acid is selected from oligonucleotides and plasmids. The oligonucleotides may be short lengths of single stranded DNA, RNA, such as antisense RNA and siRNA.

[0029] Preferably, the delivery vehicle is a solution comprising an amino acid.

[0030] Preferably, the delivery vehicle of the present invention can deliver one or more plasmids simultaneously.

[0031] Preferably, the amino acid is glycine, gamma-aminobutyric acid (GABA), proline, alanine, serine, asparagine, glutamine, sarcosine, citrulline, hydroxyproline, lily of the valley, betaine, hypotaurine, asparagine, glutamine, sarcosine, citrulline, hydroxyproline, lily of the valley, betaine, hypotaurine, histidine or threonine. The amino acid may be L-type or D-type. Preferably, the amino acid is L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, L-glutamine, D-glutamine, glycine, gamma-aminobutyric acid (GABA), L-histidine or L-threonine. The amino acid may be a peptide composed of amino acids as basic units. Preferably, the peptide is diglycine or NL-leucylglycine. The amino acid may be a peptide composed of amino acids as basic units. Preferably, the peptide is diglycine or NL-leucylglycine.

[0032] Prior to delivery, the nucleic acid is dissolved in a solution containing the amino acid. Alternatively, the nucleic acid and amino acid solutions are delivered separately.

[0033] The nucleic acid can be delivered directly into a tissue or organ of a living organism. Living organisms include, but are not limited to, plants and animals. The animal is an invertebrate or a vertebrate. Preferably, the animal is a bird, chicken, duck, goose. Preferably, the animal is a mammal, such as a mouse, rat, cat, rabbit, dog, horse, cow, sheep, goat, pig, tree shrew, monkey, chimpanzee, human, etc.

[0034] Preferably, the tissue or organ is the central nervous system, including the brain and spinal cord.

[0035] The brain involves, but is not limited to, the midbrain, thalamus, hypothalamus, brainstem, cerebellum, globus pallidus, cerebral cortex, and hippocampus.

[0036] Preferably, the nucleic acid can be delivered directly to astrocytes in the midbrain, thalamus, hypothalamus, brainstem, cerebellum, globus pallidus, cerebral cortex and hippocampus by the vector of the present invention.

[0037] Preferably, the nucleic acid can be delivered directly to Bergmann glia, velate astrocytes and basket / stellate interneurons in the cerebellum by the vector of the present invention.

[0038] Preferably, the uptake of plasmids by astrocytes via the vectors of the invention is concentration dependent. The concentration of the amino acid may be as high as 300 mM. Preferably, the concentration of the amino acid is in the range of 50 mM-300 mM.

[0039] Preferably, glycine specifically promotes astrocyte uptake of plasmid vectors. Preferably, GABA specifically promotes astrocyte uptake of plasmid vectors.

[0040] The osmotic pressure of a 300 mM glycine solution is approximately equal to that of physiological saline, and the osmotic pressure of a solution is proportional to its concentration. In the present invention, it is determined that the pH values ​​of glycine solutions of different concentrations remain stable and are maintained at about pH=6.

[0041] Similarly, solutions of L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, L-histidine, and L-threonine all promoted astrocyte uptake of plasmid vectors.

[0042] Preferably, the vector of the present invention also contains a selective inhibitor of amino acid transporters. For example, the efficiency of astrocyte uptake of plasmids by a delivery vehicle composed of glycine and bitopertin (a selective inhibitor of glycine transporter 1) is 3-4 times higher than that of a delivery vehicle containing only glycine.

[0043] The vector of the present invention effectively delivers an ultra-large plasmid carrying the CRISPR / Cas9 system to astrocytes and successfully knocks out genes in the genome of transgenic mice.

[0044] The vector of the present invention also successfully knocks exogenous genes into the genome of astrocytes to achieve permanent expression of the exogenous genes.

[0045] The vector of the present invention can promote the efficient co-transfection of multiple plasmid vectors without the need for specific promoters, viral vectors and transgenic mice. Preferably, the delivery system of the present invention can achieve the efficient co-transfection of two or three plasmid vectors into astrocytes.

[0046] The efficient co-transfection of multiple plasmid vectors provides a powerful tool for simultaneously manipulating the expression of multiple genes in astrocytes.

[0047] The vector of the present invention can be used for treating nervous system diseases, especially gene therapy of central nervous system diseases. For example, the disease can be a neurodegenerative disease, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia. The disease can be a neurogenetic disease, such as congenital spinal muscular atrophy. The disease can be a tumor in the brain and / or spinal cord, particularly for example a glioma.

[0048] The efficient co-transfection of multiple plasmid vectors provides a powerful tool for simultaneously manipulating the expression of multiple genes in astrocytes.

[0049] Third, the present invention provides a method for delivering nucleic acids into living organisms by using amino acids as mediators.

[0050] The nucleic acid is selected from oligonucleotides and plasmids. The oligonucleotides may be short lengths of single stranded DNA, RNA, such as antisense RNA and siRNA.

[0051] Preferably, the delivery vehicle is a solution comprising an amino acid.

[0052] Preferably, the delivery system of the present invention can deliver one or more plasmids simultaneously.

[0053] Prior to delivery, the nucleic acid is dissolved in a solution containing the amino acid. Alternatively, the nucleic acid and amino acid solutions are delivered separately.

[0054] Preferably, the amino acid is glycine, gamma-aminobutyric acid (GABA), proline, alanine, serine, asparagine, glutamine, sarcosine, citrulline, hydroxyproline, lily of the valley, betaine, hypotaurine, histidine or threonine. The amino acid can be L-type or D-type. Preferably, the amino acid is L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, glycine, gamma-aminobutyric acid (GABA), L-histidine or L-threonine. The amino acid can be a peptide composed of amino acids as basic units. Preferably, the peptide is diglycine or NL-leucylglycine.

[0055] The nucleic acid can be delivered directly into a tissue or organ of a living organism. Living organisms include, but are not limited to, plants and animals. The animal is an invertebrate or a vertebrate. Preferably, the animal is a bird, chicken, duck, goose. Preferably, the animal is a mammal, such as a mouse, rat, cat, rabbit, dog, horse, cow, sheep, goat, pig, tree shrew, monkey, chimpanzee, human, etc.

[0056] Preferably, the tissue or organ is the central nervous system, including the brain and spinal cord.

[0057] The brain involves, but is not limited to, the midbrain, thalamus, hypothalamus, brainstem, cerebellum, globus pallidus, cerebral cortex, and hippocampus.

[0058] Preferably, the nucleic acid can be delivered directly to astrocytes in the midbrain, thalamus, hypothalamus, brainstem, cerebellum, globus pallidus, cerebral cortex and hippocampus by the vector of the present invention.

[0059] Preferably, the nucleic acid can be delivered directly to Bergmann glia, velate astrocytes and basket / stellate interneurons in the cerebellum by the vectors of the present invention.

[0060] Preferably, the uptake of plasmids by astrocytes via the vectors of the invention is concentration dependent. The concentration of the amino acid may be as high as 300 mM. Preferably, the concentration of the amino acid is in the range of 50 mM-300 mM.

[0061] Preferably, glycine specifically promotes astrocyte uptake of plasmid vectors. Preferably, GABA specifically promotes astrocyte uptake of plasmid vectors.

[0062] The osmotic pressure of a 300 mM glycine solution is approximately equal to that of physiological saline, and the osmotic pressure of a solution is proportional to its concentration. In the present invention, it is determined that the pH values ​​of glycine solutions of different concentrations remain stable and are maintained at about pH=6.

[0063] Similarly, solutions of L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, L-histidine, and L-threonine all promoted astrocyte uptake of plasmid vectors.

[0064] Preferably, the delivery vehicle of the present invention also comprises a selective inhibitor of an amino acid transporter. For example, astrocytes take up plasmids 3-4 times more efficiently through a delivery vehicle comprising glycine and bitopertin (a selective inhibitor of glycine transporter 1) than a delivery vehicle containing only glycine.

[0065] The vector of the present invention effectively delivers an ultra-large plasmid vector carrying the CRISPR / Cas9 system to astrocytes and successfully knocks out genes in the genome of transgenic mice.

[0066] The vector of the present invention also successfully knocks exogenous genes into the genome of astrocytes to achieve permanent expression of the exogenous genes.

[0067] The vector of the present invention can promote the efficient co-transfection of multiple plasmid vectors without using specific promoters, viral vectors and transgenic mice. Preferably, the delivery system of the present invention can achieve the efficient co-transfection of two or three plasmid vectors into astrocytes.

[0068] The efficient co-transfection of multiple plasmid vectors provides a powerful tool for simultaneously manipulating the expression of multiple genes in astrocytes.

[0069] Fourthly, the present invention first provides the use of a delivery system for delivering a nucleic acid into a living organism.

[0070] The nucleic acid is selected from oligonucleotides and plasmids. The oligonucleotides may be short lengths of single stranded DNA, RNA, such as antisense RNA and siRNA.

[0071] Preferably, the delivery system comprises amino acids and one or more plasmids.

[0072] Prior to delivery, the nucleic acid is dissolved in a solution containing the amino acid. Alternatively, the nucleic acid and amino acid solutions are delivered separately.

[0073] Preferably, the amino acid is glycine, gamma-aminobutyric acid (GABA), proline, alanine, serine, asparagine, glutamine, sarcosine, citrulline, hydroxyproline, lily of the valley, betaine, hypotaurine, histidine or threonine. The amino acid can be L-type or D-type. Preferably, the amino acid is L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, glycine, gamma-aminobutyric acid (GABA), L-histidine or L-threonine. The amino acid can be a peptide composed of amino acids as basic units. Preferably, the peptide is diglycine or NL-leucylglycine.

[0074] The delivery system of the present invention can be delivered directly into the tissue or organ of a living organism. Living organisms include, but are not limited to, plants and animals. Animals are invertebrates or vertebrates. Preferably, the animal is a bird, chicken, duck, goose. Preferably, the animal is a mammal, such as a mouse, rat, cat, rabbit, dog, horse, cow, sheep, goat, pig, tree shrew, monkey, chimpanzee, human, etc.

[0075] Preferably, the tissue or organ is the central nervous system, including the brain and spinal cord.

[0076] The brain involves, but is not limited to, the midbrain, thalamus, hypothalamus, brainstem, cerebellum, globus pallidus, cerebral cortex, and hippocampus.

[0077] Preferably, the delivery system of the present invention can be directly delivered to astrocytes in the midbrain, thalamus, hypothalamus, brainstem, cerebellum, globus pallidus lateralis, cerebral cortex and hippocampus via the delivery system of the present invention.

[0078] Preferably, the delivery system of the present invention can directly enter Bergmann glia, velate astrocytes and basket / stellate interneurons in the cerebellum via the delivery system of the present invention.

[0079] Preferably, the uptake of plasmids by astrocytes through the delivery system of the present invention is concentration-dependent. The concentration of amino acids in the delivery system solution can be as high as 300 mM. Preferably, the concentration of amino acids in the delivery system solution is in the range of 50 mM-300 mM.

[0080] Preferably, glycine specifically promotes astrocyte uptake of plasmid vectors. Preferably, GABA specifically promotes astrocyte uptake of plasmid vectors.

[0081] Similarly, solutions of L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, L-histidine, and L-threonine all promoted astrocyte uptake of plasmid vectors.

[0082] Preferably, in addition to amino acids, the delivery system of the present invention also comprises a selective inhibitor of amino acid transporters. For example, astrocytes take up plasmids 3-4 times more efficiently with a delivery system comprising glycine and bitopertin (a selective inhibitor of glycine transporter 1) than with a delivery system comprising only glycine.

[0083] The delivery system of the present invention effectively delivers super-large plasmids carrying the CRISPR / Cas9 system to astrocytes and successfully knocks out genes in the genome of transgenic mice.

[0084] The delivery system of the present invention also successfully knocks exogenous genes into the genome of astrocytes to achieve permanent expression of the exogenous genes.

[0085] The delivery system of the present invention can facilitate the efficient co-transfection of multiple plasmid vectors without using specific promoters, viral vectors and transgenic mice. Preferably, the delivery system of the present invention can achieve the efficient co-transfection of two or three plasmid vectors into astrocytes.

[0086] The efficient co-transfection of multiple plasmid vectors provides a powerful tool for simultaneously manipulating the expression of multiple genes in astrocytes.

[0087] The delivery system of the present invention can be used for gene therapy for treating diseases of the nervous system, particularly central nervous system diseases. For example, the disease can be a neurodegenerative disease, such as epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia. The disease can be a neurogenetic disease, such as congenital spinal muscular atrophy. The disease can be a tumor in the brain and / or spinal cord.

[0088] Fifth, the present invention first provides the use of a delivery system in gene therapy for treating a nervous system disease, particularly a central nervous system disease. For example, the disease can be a neurodegenerative disease, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia. The disease can be a neurogenetic disease, such as congenital spinal muscular atrophy. The disease can be a tumor in the brain and / or spinal cord, such as a glioma.

[0089] The delivery system of the present invention can be delivered directly to the nervous system of an animal, particularly the central nervous system. The animal is an invertebrate or a vertebrate. Preferably, the animal is a bird, chicken, duck, goose. Preferably, the animal is a mammal, such as a mouse, rat, cat, rabbit, dog, horse, cow, sheep, goat, pig, tree shrew, monkey, chimpanzee, human, etc.

[0090] Preferably, the nervous system is the central nervous system, including the brain and spinal cord.

[0091] The brain involves, but is not limited to, the midbrain, thalamus, hypothalamus, brainstem, cerebellum, globus pallidus, cerebral cortex, and hippocampus.

[0092] Preferably, the delivery system of the present invention can be directly delivered to astrocytes in the midbrain, thalamus, hypothalamus, brainstem, cerebellum, globus pallidus lateralis, cerebral cortex and hippocampus via the delivery system of the present invention.

[0093] Preferably, the delivery system of the present invention can directly enter Bergmann glia, velate astrocytes and basket / stellate interneurons in the cerebellum via the delivery system of the present invention.

[0094] Preferably, the uptake of plasmids by astrocytes through the delivery system of the present invention is concentration-dependent. The concentration of amino acids in the delivery system solution can be as high as 300 mM. Preferably, the concentration of amino acids in the delivery system solution is in the range of 50 mM-300 mM.

[0095] Preferably, glycine specifically promotes astrocyte uptake of plasmid vectors. Preferably, GABA specifically promotes astrocyte uptake of plasmid vectors.

[0096] The osmotic pressure of a 300 mM glycine solution is approximately equal to that of physiological saline, and the osmotic pressure of a solution is proportional to its concentration. In the present invention, it is determined that the pH values ​​of glycine solutions of different concentrations remain stable and are maintained at about pH=6.

[0097] Similarly, solutions of L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, L-histidine, and L-threonine all promoted astrocyte uptake of plasmid vectors.

[0098] Preferably, in addition to amino acids, the delivery system of the present invention also comprises a selective inhibitor of amino acid transporters. For example, astrocytes take up plasmids 3-4 times more efficiently with a delivery system comprising glycine and bitopertin (a selective inhibitor of glycine transporter 1) than with a delivery system comprising glycine alone.

[0099] The delivery system of the present invention effectively delivers the super-large plasmid carrying the CRISPR / Cas9 system to astrocytes and successfully knocks out genes in the genome of transgenic mice. Preferably, the delivery system is a delivery system comprising glycine.

[0100] The delivery system of the present invention also successfully integrates exogenous genes into the astrocyte genome to achieve permanent expression of the exogenous genes.

[0101] Specifically, the transposon system was delivered into astrocytes, thereby achieving permanent expression of foreign genes in adult mouse astrocytes.

[0102] The delivery system of the present invention can facilitate the efficient co-transfection of multiple plasmid vectors without the use of specific promoters, viral vectors and transgenic mice. Preferably, the delivery system of the present invention can achieve efficient co-transfection of two or three plasmid vectors into astrocytes.

[0103] The efficient co-transfection of multiple plasmid vectors provides a powerful tool for simultaneously manipulating the expression of multiple genes in astrocytes.

[0104] The gene delivered to the animal nervous system (particularly the central nervous system) by the delivery system of the present invention can be selected according to the disease to be treated.

[0105] Sixth, the present invention provides a delivery system for delivering the CRISPR / Cas9 system into the central nervous system to knock out one or more genes in an animal. In particular, the delivery system for delivering the CRISPR / Cas9 system can be delivered to astrocytes.

[0106] The delivery system contains amino acids and the CRISPR / Cas9 system.

[0107] The CRISPR / Cas9 system is dissolved in a solution containing amino acids before delivery. Alternatively, the CRISPR / Cas9 system and the amino acid solution are delivered separately.

[0108] Preferably, the amino acid is glycine, gamma-aminobutyric acid (GABA), proline, alanine, serine, asparagine, glutamine, sarcosine, citrulline, hydroxyproline, lily of the valley, betaine, hypotaurine, histidine or threonine. The amino acid may be L-type or D-type. Preferably, the amino acid is L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, glycine, gamma-aminobutyric acid (GABA), L-histidine or L-threonine. The amino acid may be a peptide composed of amino acids as basic units. Preferably, the peptide is diglycine or NL-leucylglycine. Preferably, the amino acid is glycine.

[0109] The animal is an invertebrate or a vertebrate. Preferably, the animal is a bird, chicken, duck, goose. Preferably, the animal is a mammal, such as a mouse, rat, cat, rabbit, dog, horse, cow, sheep, goat, pig, tree shrew, monkey, chimpanzee, human, etc.

[0110] Therefore, the delivery system for delivering the CRISPR / Cas9 system in the present invention can be used for gene therapy of tumors, neurodegenerative diseases (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia) and neurogenetic diseases (e.g., congenital spinal muscular atrophy). The tumor can be in the brain and / or spinal cord, such as glioma.

[0111] Seventh, the present invention provides a delivery system for delivering a transposon system into astrocytes, wherein the delivery system comprises amino acids.

[0112] The transposon system was delivered into astrocytes, thereby achieving permanent expression of foreign genes in adult mouse astrocytes.

[0113] Preferably, the transposon system is a piggyBac transposon system. A multicolor labeling technique for long-term tracking of adult mouse astrocytes can be constructed using the piggyBac transposon system.

[0114] Preferably, the amino acid is glycine, gamma-aminobutyric acid (GABA), proline, alanine, serine, asparagine, glutamine, sarcosine, citrulline, hydroxyproline, lily of the valley, betaine, hypotaurine, histidine or threonine. The amino acid can be L-type or D-type. Preferably, the amino acid is L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, glycine, gamma-aminobutyric acid (GABA), L-histidine or L-threonine. The amino acid can be a peptide composed of amino acids as basic units. Preferably, the peptide is diglycine or NL-leucylglycine.

[0115] The transposon plasmid is dissolved in a solution containing amino acids prior to delivery. Alternatively, the transposon plasmid and the amino acid solution are delivered separately.

[0116] The concentration of the amino acid in the solution of the delivery system may be up to 300 mM. Preferably, the concentration of the amino acid in the solution of the delivery system is in the range of 50 mM-300 mM.

[0117] Eighth, the present invention provides a delivery system for delivering one or more vectors expressing one or more fluorescent proteins into astrocytes.

[0118] The delivery system is a solution comprising amino acids and one or more vectors expressing one or more fluorescent proteins. Preferably, the vector is a plasmid.

[0119] Preferably, the amino acid is glycine, gamma-aminobutyric acid (GABA), proline, alanine, serine, asparagine, glutamine, sarcosine, citrulline, hydroxyproline, lily of the valley, betaine, hypotaurine, histidine or threonine. The amino acid may be L-type or D-type. Preferably, the amino acid is L-proline, D-alanine, L-serine, L-glutamine, D-glutamine, glycine, gamma-aminobutyric acid (GABA), L-histidine or L-threonine. The amino acid may be a peptide composed of amino acids as basic units. Preferably, the peptide is diglycine or NL-leucylglycine. Preferably, the amino acid is glycine.

[0120] Preferably, the delivery system comprises two or three plasmids.The fluorescent protein may be RFP (red), YFP or GFP (green) or CFP or BFP (blue).

[0121] The present invention successfully constructed a new "brain rainbow" multi-color labeling technology for astrocytes by simultaneously expressing two or three fluorescent proteins.

[0122] The carriers and delivery systems of the present invention are both effective and safe.

[0123] L-proline can induce a large number of astrocytes to take up plasmid vectors and express green fluorescent protein, while high concentration of L-proline solution will not cause apoptosis of brain tissue cells.

[0124] Astrocytes not only provide structural and energy support for neurons, but also play an important role in the blood-brain barrier. They are also involved in the development of neurons and inflammatory responses in the brain. Recently, the role of astrocytes in neural circuits has also attracted more and more attention. Corresponding to their various functions, astrocytes may vary greatly in morphology and structure in different brain regions or even in the same brain region. In the present invention, the glycine transfection system is used to express two or three plasmid vectors simultaneously. The effective co-transfection of multiple plasmid vectors provides a powerful tool for manipulating the expression of multiple genes in astrocytes at the same time.

[0125] We also found that the uptake of different plasmid vectors by different astrocytes resulted in very different expressions of various fluorescent proteins. Using this result, we successfully constructed a new "Brain Rainbow" multicolor labeling technology for astrocytes by simultaneously expressing two or three fluorescent proteins. Compared with the previous "Brain Rainbow" strategy, there is no need to construct transgenic mice or viral vectors with specific promoters, which significantly reduces the economic and time costs required for multicolor labeling of astrocytes. It provides a convenient and direct visualization tool for studying the interactions between astrocytes and between astrocytes and other brain cells.

[0126] Amino acids are a group of organic molecules that consist of a basic amino group (-NH2), an acidic carboxyl group (-COOH), and an organic R group (or side chain) that is unique to each amino acid. Some amino acids form proteins when linked together with other amino acids. Essential amino acids cannot be made by the body, so they must come from food. The 9 essential amino acids are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Non-essential amino acids are amino acids that the body is able to produce. Non-essential amino acids include: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine.

[0127] The brain is an organ with a very complex structure and function. Different parts of the brain tissue have their own unique functions. The complex and precise coordination of interactions between different parts of the brain controls many different behaviors, such as cognition, sensation, movement, and emotion. Even in parts with similar macroscopic structures, there are considerable differences in the composition of cell types, interactions between cells, and molecular expressions at the microscopic level. For a long time, astrocytes have generally been considered a class of cell types with similar morphology and function. However, in recent years, the study of the diversity of astrocytes has attracted much attention.

[0128] Tree shrews are mainly distributed in South Asia, Southeast Asia and southwestern China. Due to their small adult size, high brain-to-body mass ratio, short breeding cycle, simple breeding and low cost, they have become emerging experimental animals in vision, hepatitis virus infection and neurological diseases, and have been widely studied.

[0129] Microglia are innate immune cells present in the brain. Increasing evidence shows that activated microglia are a chronic source of multiple neurotoxic factors, including TNF, nitric oxide, interleukin-1, and reactive oxygen species (ROS). Ibal (ionized calcium binding adaptor molecule-1) protein is a microglia-specific marker protein. When brain tissue is damaged, microglia proliferate and undergo a series of morphological changes. Therefore, observing microglial changes by immunofluorescence staining of Ibal protein is often used to evaluate inflammation or damage to brain tissue.

[0130] sgRNA can guide the nuclease (Cas9 protein) to accurately cut the target gene sequence, so it has the potential to treat central nervous system diseases. Despite its great application potential, it is still challenging to effectively and low-toxicly deliver genes related to the CRISPR / Cas9 system to the central nervous system. At present, gene editing in the adult mouse brain is mainly achieved by delivering sgRNA to the target gene through a viral vector, but this method requires the construction of transgenic mice that constitutively express the Cas9 protein. Due to the size limitation of viral particles, it is difficult for viral vectors to load genes with larger open reading frame sequences (ORFs) such as Cas9 protein, which significantly limits the application of the CRISPR / Cas9 system in the central nervous system. At the same time, due to the inherent immunogenicity of viral vectors and the long-term expression of CRISPR-Cas9 and sgRNA, off-target effects are easily caused, thereby inducing potential carcinogenic risks. Therefore, in the central nervous system, gene knockdown using the CRISPR-Cas9 system by constructing viral vectors or transgenic mice remains challenging.

[0131] Transposon. Non-viral vector nucleic acid delivery has great potential in genetic research and treatment due to its convenient and direct preparation method and high biosafety. Standard plasmid DNA delivered by non-viral vectors usually cannot integrate into the host genome, so these gene vectors are only transiently expressed in cells. However, the treatment of some hereditary or chronic diseases requires long-term gene expression. One of the methods to achieve long-term or even permanent stable expression of exogenous genes is to use a transposon system, which is a genetic element that can be transferred between the vector and the host genome or within the genome. Typically, transposases recognize specific reverse terminal repeats (IR) at both ends of the transposon and cut transposon elements from their original positions to reintegrate them into other locations. Using these characteristics of transposons, inserting a target gene between the terminal repeat sequences IRS at both ends of the transposon can integrate the exogenous gene into the host genome and achieve extended or even permanent expression of the exogenous gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 A schematic diagram of the administration method of amino acid-mediated delivery of plasmid vectors is shown.

[0133] Figure 2 Glycine solution was shown to promote cellular uptake of plasmid vectors in the midbrain.

[0134] Figure 3 The glycine transfection strategy was shown to specifically promote astrocyte uptake of plasmid vectors.

[0135] Figure 4 Validation of the cell specificity of the glycine transfection strategy-mediated delivery by transgenic mice (Aldh111-EGFP X Ai9) is shown.

[0136] Figure 5 Delivery efficiency and tissue damage of the transfection strategy with different concentrations of glycine are shown.

[0137] Figure 6 Changes in microglia by immunofluorescence staining are shown.

[0138] Figure 7 It was shown that the cellular uptake efficiency of plasmid vectors using the glycine transfection strategy depends on the glycine concentration.

[0139] Figure 8 The glycine transfection strategy was shown to promote astrocyte uptake of plasmid vectors in different brain regions.

[0140] Fig. 9 showed that the glycine transfection strategy promoted cellular uptake of plasmid vectors in the cerebellar cortex.

[0141] Fig.10 The application of the glycine transfection strategy in tree shrews is shown.

[0142] Fig.11 Amino acids were shown to promote astrocyte uptake of plasmid vectors.

[0143] Fig.12 showed that the L-proline transfection system promotes plasmid uptake by astrocytes in different brain regions.

[0144] Fig.13 An evaluation of the safety of L-proline delivery systems to the central nervous system is shown.

[0145] Fig.14 The GlyT1-selective inhibitor Bitopertin was shown to increase the uptake efficiency of plasmid vectors by 3-4 fold via the glycine transfection system.

[0146] Fig.15 The glycine transfection strategy was shown to facilitate the simultaneous delivery of two plasmid vectors.

[0147] Fig.16 Simultaneous delivery of three plasmid vectors using the glycine transfection strategy is shown.

[0148] Fig.17 Different quantifications of astrocyte cell body color are shown.

[0149] Fig.18 Distinct “regions” of astrocytes visualized by the multicolor strategy are shown.

[0150] Fig.19 The L-proline transfection system was shown to facilitate efficient co-transfection using dual-plasmid vectors and triple-plasmid vectors.

[0151] Fig. 20 The construction of the gene targeting vector for knocking out the eYFP gene is shown.

[0152] Fig.21 Expression of large plasmid vectors in astrocytes using the glycine transfection strategy is shown.

[0153] Fig. 22 A schematic diagram showing the knockout of the eYFP gene in Ai3 transgenic mice by the CRISPR-Cas9 system.

[0154] Fig.23 Efficient eYFP knockdown in astrocytes using the glycine transfection strategy is shown.

[0155] Fig.24 Validation of knockout of the eYFP gene by sequencing analysis is shown.

[0156] Fig.25A schematic diagram of the integration of foreign genes into the genome via the piggyBac transposon system is shown.

[0157] Fig.26 The integration of the eGFP gene into the genome of astrocytes by the piggyBac transposon system is shown.

[0158] Fig. 27 Shown is piggyBac transposon-mediated long-term eGFP expression in astrocytes of adult mouse brain. DETAILED DESCRIPTION

[0159] The descriptions of specific embodiments and examples are provided by way of illustration and not limitation. Those skilled in the art will readily recognize that a variety of noncritical parameters can be changed or modified to produce substantially similar results.

[0160] Example

[0161] Example 1: Plasmid delivery in mouse midbrain using the glycine transfection system

[0162] like Figure 1 As shown, a plasmid (pBT140) solution (1.5 μg / uL) expressing Cre recombinase and a glycine solution (300 mM) were injected into the midbrain of Ai9 mice containing the Cre-LoxP system. Cre recombinase will delete the translation termination sequence between the LoxP sequences, thereby eliminating the inhibition of the expression of the fluorescent protein tdTomato by the termination sequence ( Figure 2 A).

[0163] After 5 days, a large number of cells around the injection site were observed to express tdTomato in the slices. This result indicates that the transfection system composed of glycine solution can effectively promote the uptake of plasmids by midbrain cells, and there is no visible tissue necrosis, indicating that the transfection system has low toxicity ( Figure 2 B). In addition, comparative analysis of the structure and morphology of cells expressing fluorescent proteins under a high-power microscope showed that all cells successfully transfected under this transfection system were astrocytes. Aldh111-eGFP transgenic mice are a reporter line that specifically expresses fluorescent protein (GFP) in astrocytes. By comparing astrocytes in Aldh111-eGFP mice with astrocytes expressed in the glycine transfection system in Ai9 mice, the glycine transfection system was in a densely expressed area. The density of expressing cells reached 67.7% ( Figure 2 C).

[0164] The fluorescent protein tdTomato is expressed in astrocyte-specific marker Aldh111-eGFP transgenic mice ( Figure 3). By laser confocal microscopy imaging analysis, it was found that all cells expressing tdTomato were astrocytes labeled in Aldh111-eGFP mice, indicating that glycine solution specifically promoted the uptake of plasmids by astrocytes.

[0165] like Figure 4 As shown in A, hybrid mice of Aldh111-eGFP mice and Ai9 mice (Aldh111-eGFP XAi9) were constructed, both of which specifically expressed eGFP in astrocytes and expressed the fluorescent protein tdTomato through Cre recombinase. Cre recombinase can cut LoxP sites even in the case of extremely small or short-term expression, so it can continuously express the fluorescent protein tdToamto, thereby eliminating the problem that some cells express weakly and cannot be accurately observed. The glycine transfection system was injected into Aldh111-eGFP X Ai9 hybrid mice, and the cells expressing the tdTomato fluorescent protein were still all astrocytes, which once again proved that the transfection method has cell specificity ( Figure 4 B).

[0166] Example 2. Evaluation of the Glycine Delivery System in Central Nervous System Injury

[0167] To evaluate whether the glycine transfection system causes local or large-scale tissue damage to brain tissue, we used the transfection system with different glycine concentrations and observed the sections 5 days after injection. Nuclear damage is an important mechanism for evaluating cytotoxicity. When a large number of cells die and tissue necrosis occurs, the nucleus will appear condensed, the chromatin will be condensed and deformed, and the nucleus will be fragmented and dissolved.

[0168] like Figure 5 As shown, the 300mM glycine transfection system induced the expression of a large number of astrocytes. The cell morphology was normal and no visible tissue necrosis was observed. When a higher concentration of the transfection system (500mM and 1000mM) was used, the nuclei of a large number of cells around the injection site condensed and the tissue was severely damaged. At the same time, very few astrocytes were expressed, and only a few were scattered around the damaged tissue. The cause of these injuries may be the ultra-high concentration of glycine in the local tissue, or it may be caused by the osmotic pressure of the glycine transfection system exceeding 300mM under normal physiological conditions. In contrast, under the 300mM glycine transfection system, the cell transfection efficiency was high and the damage to the brain tissue was not obvious, indicating that the glycine transfection system is both effective and safe.

[0169] To evaluate whether the glycine delivery system would lead to overt inflammatory responses and tissue damage, we observed changes in microglia. Microglia are innate immune cells in the brain. Accumulating evidence suggests that activated microglia are a chronic source of multiple neurotoxic factors, including TNFα, nitric oxide, interleukin-1, and reactive oxygen species (ROS).

[0170] To evaluate whether the glycine transfection system activates microglia, we performed fluorescent immunostaining using the microglia-specific marker protein Ibal. Figure 6 As shown in A, 6B, and 6C, microglia activation and cell proliferation were present in a limited area (about 100 μm) around the injection path, whereas in areas away from the injection path, although a large number of astrocytes expressing tdTomato were present, microglia were not significantly activated ( Figure 6 D), indicating that mechanical damage caused by the injection electrode in brain tissue induces a certain degree of inflammatory response, which in turn activates microglia. In contrast, the glycine transfection system does not induce significant inflammatory response and tissue damage.

[0171] Example 3. Glycine concentration-dependent plasmid uptake by astrocytes

[0172] Equal volumes of 300 mM, 200 mM, 100 mM, and 50 mM glycine transfection system were injected into the midbrain tissue of mice. Five days later, the expression of fluorescent proteins was analyzed by laser confocal microscopy and ImageJ software. Figure 7 As shown in A and 7B, when the concentration of glycine is reduced, the efficiency of astrocyte uptake of the plasmid vector is significantly reduced. When 50 mM glycine is used, only a few astrocytes take up and express green fluorescent protein (eGFP).

[0173] In addition, the plasmid vector pBT140 (expressing Cre recombinase) was injected into the midbrain of Ai9 transgenic mice, and astrocytes expressing the fluorescent protein tdTomato were counted using ImageJ software. Figure 7 As shown in C and 7D, when a 300 mM glycine transfection system was used, the average number of expressing astrocytes was as high as 8000, and when a 200 mM system was used, the number of cells was significantly reduced. These results indicate that changes in glycine concentration have a significant effect on the efficiency of astrocyte uptake of plasmids, and that the uptake efficiency is significantly reduced as the glycine concentration decreases.

[0174] Example 4. Glycine transfection system promotes plasmid uptake by astrocytes in different brain regions

[0175] Due to the great differences in brain tissue function and structure, it is necessary to explore the role of the glycine transfection system in promoting the efficiency of astrocyte uptake of plasmids in different brain regions.

[0176] We attempted to use glycine to promote the uptake of a plasmid (pBT140, which expresses Cre recombinase) into different brain regions of adult Ai9 mice. These brain regions included the hypothalamus ( Figure 8 A) Thalamus Figure 8 B) preoptic area of ​​the hypothalamus ( Figure 8 C), globus pallidus lateralis ( Figure 8 D) Hippocampus Figure 8 E) Brain stem ( Figure 8 F) Cerebral cortex ( Figure 8 G) and cerebellar cortex ( Fig. 9 ). The results showed that tdTomato fluorescent protein was expressed in a large number of astrocytes in the hypothalamus, thalamus, lateral globus pallidus, and brainstem, while only a few cells in the hippocampus and cortex expressed the fluorescent protein tdTomato. At the same time, neurons were present in cells expressing in the hippocampus. Interestingly, in the cerebellum, the glycine transfection system could effectively promote plasmid uptake ( Fig. 9 A), and there are multiple cell types ( Fig. 9 B), including Bergmann glia surrounding Purkinje cells, velate astrocytes surrounding granule cells, and basket / stellate interneurons located in the molecular layer of the cerebellar cortex.

[0177] Example 5. Use of the glycine transfection system in other species

[0178] The Chinese tree shrew was used as the experimental subject. 300mM glycine transfection system was injected into the midbrain of the tree shrew to deliver the plasmid vector expressing tdTomato fluorescent protein. Fig.10 A and Fig.10 As shown in B, many astrocytes in the tree shrew midbrain took up and expressed the plasmid vector. Interestingly, when the glycine transfection system (two fluorescent protein vectors: eGFP and tdToamto) was injected into the tree shrew's striatum, many neurons were found to express the two fluorescent proteins ( Fig.10 C) These results suggest that the use of the glycine transfection system can enhance the uptake of plasmid vectors by astrocytes from other species.

[0179] Example 6. Other amino acids promote astrocyte uptake of plasmid vectors

[0180] Other amino acids were tested, including serine, alanine, histidine, γ-aminobutyric acid, proline, asparagine, glutamine, ornithine, citrulline, sarcosine, hydroxyproline, β-alanine, taurine, hypotaurine, betaine, lilysine, lysine, threonine, and peptides composed of amino acids as basic units, including diglycyl peptide, diglycylglycine, L-alanyl-L-aminopropionic acid, glycyl-L-histidine, histidylglycine, NL-leucylglycine. It was found that these amino acids can effectively promote the uptake of plasmid vectors by astrocytes. From the analysis of the transfection efficiency of various amino acids, it can be seen that, except for alanine, the efficiency of L-type amino acids is much higher than that of D-type amino acids (see Fig.11 ).

[0181] Histidine, lysine, and arginine, which are also positively charged, have significant differences in delivery efficiency, and there are also huge differences in gene delivery efficiency for threonine, serine, lysine, etc. These results suggest that the delivery efficiency of amino acid solutions to gene carriers may not be related to their physical and chemical properties, but may be related to the biological functions of amino acids in the brain (scale bar = 200 μm).

[0182] Example 7. L-proline transfection system promotes plasmid uptake by astrocytes in different brain regions

[0183] The L-proline transfection system was used to promote the uptake of plasmid (pBT140, which expresses Cre recombinase) in different brain regions of adult Ai9 mice. These brain regions include the hypothalamus, thalamus, hippocampus, brainstem, cerebral cortex, and cerebellum. The results showed that tdTomato fluorescent protein was expressed in a large number of astrocytes in the hypothalamus, thalamus, and brainstem, while only a few cells in the hippocampus and cortex expressed the fluorescent protein tdTomato. Interestingly, in the cerebellum, the L-proline transfection system can effectively promote plasmid uptake ( Fig.12 ), and there are multiple cell types, including Bergman glia surrounding Purkinje cells, velate astrocytes surrounding granule cells, and basket / stellate interneurons in the molecular layer of the cerebellar cortex ( Fig.12 ).

[0184] Example 8. Evaluation of L-proline delivery system on central nervous system injury

[0185] To evaluate whether high concentration of L-proline solution induced astrocyte uptake of plasmid vectors which may cause visible central nervous system damage, fluorescent immunostaining was performed using microglia-specific marker protein Iba1. Fig.13The results showed that compared with the 1× PBS control group, there was no significant increase in the fluorescence intensity of Iba1 protein immunofluorescence staining and Iba1-labeled microglia in the test group, indicating that 300 mM L-proline solution did not cause significant tissue damage.

[0186] During apoptosis, genomic chromosomes are randomly cut by nucleases and produce a large number of sticky 3′-OH ends. Under the action of terminal deoxyribonucleotidyl transferase (TdT), fluorescent molecules can be labeled to the 3′ end of DNA fragments. Normal cells have almost no 3′-OH ends due to DNA breaks, so the TUNEL method is a common method for detecting apoptosis. In order to explore the effect of high concentration L-proline solution (300mM) on apoptosis of brain tissue cells, the TUNEL method was used to detect apoptosis in brain slices injected with proline. It was found that L-proline can induce a large number of astrocytes to take up plasmid vectors and express green fluorescent protein, but almost no TUNEL fluorescently labeled cells were found, indicating that high concentration L-proline solution does not cause apoptosis of brain tissue cells ( Fig.13 ).

[0187] Example 9. Bitopertin, a GlyT1 selective inhibitor, increases the uptake efficiency of plasmid vectors by 3-4 times via the glycine transfection system

[0188] Astrocyte GlyT1 plays an important role in the clearance of glycine in the extracellular space of brain tissue. Therefore, we used Bitopertin, a selective inhibitor of GlyT1, to inhibit the clearance of glycine by astrocytes. We found that the efficiency of cellular uptake of plasmid vectors increased by 3-4 times, which may be due to the inhibition of astrocytes ( Fig.14 ). Removal of extracellular glycine via glycine transporters prolonged the duration of action of high-concentration glycine solutions, thereby improving astrocyte uptake of plasmid vectors.

[0189] Example 10. Glycine transfection system facilitates efficient co-transfection of dual plasmid vectors

[0190] Select plasmid vectors of green fluorescent protein and tdTomato fluorescent protein as markers. Fig.15 As shown in A, two plasmid vectors were injected into the midbrain at the same time. Five days later, brain tissue was obtained by perfusion. Observation of brain slices by fluorescence microscopy showed that both fluorescent proteins were highly expressed. Each astrocyte can be given a unique color marker. Therefore, the glycine transfection system not only achieves the efficient transfection of two plasmids, but also develops a very convenient and simple astrocyte "brain rainbow" labeling technology. Fig.15As can be seen in the three-dimensional display of B, because each astrocyte has a different color from its neighboring cells, we can clearly distinguish the "domain" range of each astrocyte. To check the co-transfection efficiency of the two plasmids, we counted a total of 918 astrocytes expressing fluorescent proteins ( Fig.15 C). The statistical results showed that the co-transfection efficiency of the two plasmids was as high as 87.6%, indicating that the glycine transfection system can simultaneously induce the effective co-transfection of two plasmid vectors.

[0191] Example 11. Glycine transfection system facilitates efficient co-transfection of three plasmid vectors

[0192] On the basis of eGFP and tdTomato plasmid vectors, TagBFP vector was added to test the effective co-transfection of the three plasmid vectors by glycine transfection system. The results showed that all three plasmid vectors were highly expressed ( Fig.16 A). At the same time, the "Brain Rainbow" multicolor labeling strategy constructed using a dual plasmid vector has a maximum color combination of 255×255 in RGB display mode. After the introduction of the BFP blue channel, the color mode is expanded to three-dimensional information, reaching 255×255×255 in RGB display mode. The number of distinguishable colors is significantly expanded.

[0193] Confocal microscopy was used to capture astrocytes in different colors and statistical analysis was performed on 593 astrocytes labeled on multiple brain sections ( Fig.16 B). Almost half of the labeled cells expressed three plasmid vectors (47%), and astrocytes expressed only two plasmid vectors (32.3%). Fig.16 C). These results indicate that the glycine transfection system can effectively promote the simultaneous uptake and expression of multiple plasmid vectors by astrocytes and provide an effective gene expression tool for the simultaneous manipulation and study of multiple genes in adult mouse astrocytes. It also suggests that the brain “rainbow” strategy constructed using the glycine transfection system has excellent potential for multicolor labeling.

[0194] To further analyze the color distribution generated by the three-plasmid “brainbow” labeling strategy constructed using the glycine transfection system, confocal microscopy was used to capture and the color of each astrocyte cell body was obtained using ImagJ software, which was presented in RGB mode. Fig.17As shown in A, the specific value of each channel is directly plotted on the three-dimensional coordinate graph. It can be seen that in the entire three-dimensional coordinate, there are a certain number of cells distributed in different areas, but most of the cells are concentrated in the darker areas. The results are similar to the expression of the dual-plasmid vector. The three-dimensional coordinate drawing method using the RGB mode can directly reflect the specific color of each astrocyte, but the color brightness of each cell depends on many factors, such as imaging depth, cell morphology, and cell expression activity. Therefore, the RGB mode can be transformed into a two-dimensional HSB mode (hue-saturation-brightness mode) for display (such as Fig.17 B). The colors of different astrocytes are distributed in each area of ​​the color ring. Another way of presentation is through a ternary graph with three axes, each axis representing a different color percentage, which can reflect the relative proportion of each fluorescent protein expression in different cells. Fig.17 As shown in C, the amount of TagBFP (blue channel) expressed by the majority of cells (85.5%) is distributed below 50% of the highest fluorescence intensity (cells expressing the most TagBFP). Fig.17 As shown in D, the distribution of fluorescence intensity of the three fluorescent proteins is also slightly different.

[0195] Example 12. The "Brainbow" multicolor labeling strategy constructed by the glycine transfection system can effectively distinguish the "territories" of adjacent astrocytes.

[0196] Given the unique and complex three-dimensional structure of astrocytes, clear distinctions between different astrocytes can facilitate studies of interactions with neurons within an astrocyte “territory” and between neighboring astrocytes.

[0197] A simple and convenient "Brain Rainbow" multicolor technique was constructed using the glycine transfection system. Fig.18 As shown in A and 18B, in local areas, adjacent astrocytes are labeled with different colors, and the "territory" of each astrocyte is clearly distinguishable. Even in the extremely small space where adjacent astrocytes are in contact with each other, single-color labeling is usually difficult to accurately determine whether the tiny structure belongs to one cell or multiple cells. However, multi-color labeling can clearly distinguish ( Fig.18 C and 18D).

[0198] Example 13. L-proline transfection system promotes efficient co-transfection of dual plasmid vectors, L-proline transfection system promotes efficient co-transfection of three plasmid vectors

[0199] like Fig.19As shown in A, two plasmid vectors (p-eGFP-C1 and p-tdTomato-N1) were simultaneously expressed in the midbrain of mice using 300 mM L-proline solution. The two fluorescent proteins were expressed in a large number of astrocytes. At the same time, astrocytes showed different colors due to the difference in fluorescence expression (scale bar = 200 μm). Fig.19 As shown in B, among all astrocytes expressing fluorescent proteins, 91.8% of the cells expressed tdTomato, 97.9% of the cells expressed eGFP, and the co-expression ratio reached 89.7%. Fig.19 C-19D shows the cell color distribution of different astrocytes. The fluorescence intensity of the two types of cell bodies is distributed throughout the brightness area, but mainly in the darker area. The number of cells with cell body fluorescence intensity with RGB values ​​less than 50 accounts for 60.9% (green) and 58.3% (red), respectively. Fig.19 E shows the simultaneous expression of three plasmid vectors (p-EGFP-C1, p-tdTomato-N1, and p-TagBFP-N at a concentration of 1 μg / uL, respectively) in the mouse midbrain with 300 mM L-proline solution (scale bar = 50 μm).

[0200] Example 14. Expression of very large plasmid vectors in astrocytes

[0201] 1. Construction of pX330-U6-sgRNA-CBh-hSpCas9-mCherry plasmid

[0202] The pX330-U6-sgRNA-CBh-hSpCas9-mCherry plasmid knocks out the EYFP gene of Ai3 mice and expresses the red fluorescent mCherry fluorescent protein.

[0203] Plasmids p-eGFP-c1, pBT140, pCAG-PBase, and PBCAG-eGFP were purchased from Addgene. pX330-U6-sgRNA-CBh-hSpCas9-mCherry was provided by Jiankui Zhou from Xingxu Huang's laboratory.

[0204] The pX330-U6-Chimeric_BB-CBh-hSpCas9-mCherry plasmid was used as a vector, and the sgRNA target binding sequence was inserted into the BbsI restriction site behind the U6 promoter. The target sequence of the sgRNA is EYFP sgRNA1-S: CAC CGGGCG AGG AGC TGT TCA CCG, EYFP sgRNA1-a: AAA CCG GTG AAC AGC TCC TCG CC. First, the vector plasmid was digested using a digestion system (2 μL of BbsI endonuclease, 1-2 μg of vector plasmid, digestion buffer was added, and finally ddH2O was added to 50 μL), and the reaction was carried out at 37 ° C for 30 minutes to 1 hour. The digestion product was purified using the Takara MiniBEST DNA fragment purification kit Ver4.0 kit, and the digestion was detected by 1% agarose gel electrophoresis. The recovered digested fragments were connected to the target sequence using T4 ligase. The enzyme ligation reaction system is: 100ng fragment, 1μL 5mM target sequence hybrid double strand, 0.2-1μL T4 ligase, ligation buffer, add ddH2O to 10μL), and react at constant temperature for 15-30 minutes. After bacterial transformation, pick a single colony and sequence it. At the same time, mix 500μL of bacterial solution with an equal volume of 50% sterile glycerol, and then select the corresponding bacterial solution for plasmid extraction according to the sequencing results. The constructed pX330-U6-sgRNA-CBH-HspCas9-mCherry plasmid is as follows Fig. 20 shown.

[0205] 2. Expression of super-large plasmid vectors in astrocytes

[0206] The pX330-U6-sgRNA-CBh-hSpCas9-CMV-mCherry vector with a size of approximately 10,000 base pairs was selected to express sgRNA through the U6 promoter, SpCas9 protein through the SpB9 promoter, and mCherry fluorescence through the CMV promoter. The glycine transfection system was used to deliver the pX330-U6-sgRNA-CBh-hSpCas9-CMV-mCherry vector. Fig.21 As shown in A, under a fluorescence confocal microscope, a large number of astrocytes expressed the fluorescent protein mCherry, indicating that the vector can be effectively taken up by astrocytes under the glycine transfection system. In addition, unlike other fluorescent proteins (eGFP, eYFP, tdTomato, TagBFP), the expressed mCherry fluorescent protein aggregated into small particles in astrocytes ( Fig.21 B) Analysis using ImageJ software showed that these small particles have a diameter of approximately 1-3 μm.

[0207] Example 15. Knockout of the eYFP gene in AI3 transgenic mice using the CRISPR-Cas9 system

[0208] Ai3 mice (RCL-EYFP) were selected as reporter mice, and the CRISPR / Cas9 system was used to knock out the eYFP gene in the genome of its astrocytes. Fig. 22 As shown, two plasmid vectors (pBT140 vector expressing Cre recombinase and pX330-U6-sgRNA-CBh-hSpCas9-mCherry vector expressing CRISPR / Cas9 system) were delivered using glycine transfection system, and when the Cre recombinase vector was expressed in astrocytes, the translation termination sequence "STOP" in front of the eYFP gene sequence could be excised, thereby successfully achieving the expression of the eYFP gene, and when the CRISPR / Cas9 vector targeting the EYFP gene was simultaneously expressed in cells, the eYFP gene was knocked out. Therefore, even in the presence of Cre recombinase, active eYFP fluorescent protein cannot be effectively expressed.

[0209] First, construct a suitable sgRNA targeting the eYFP gene. Fig. 20 As shown in A, by 1% agarose gel electrophoresis, it can be seen that the digested plasmid vector produces a band of about 5 kb (lane 1). Because in the vector pX330-U6-sgRNA-CBh-hSpCas9-mCherry, in addition to the two BbsI restriction endonuclease sites in the sgRNA position for inserting new sgRNA in the mCherry fluorescent protein sequence, there is also a BbsI digestion site, which produces two fragments of similar size after digestion. In addition, we used bacterial liquid sequencing ( Fig. 20 B) Confirmed that the pX330-U6-sgRNA(eYFP)-CBh-hSpCas9-mCherry vector targeting the eYFP gene was successfully constructed.

[0210] Next, to confirm whether the designed sgRNA-eYFP can effectively target and excise the eYFP gene in astrocytes from Ai3 transgenic mice. Fig. 22 ), firstly, two plasmid vectors pBT140 and pX330-U6-sgRNA(ctrl)-CBh-hSpCas9-mCherry were injected into 8-week-old Ai3 transgenic mice. Fig.23 As shown in A, both eYFP and mCherry fluorescent proteins are highly expressed, and most (average 88.7%) astrocytes expressing mCherry fluorescent protein also express eYFP fluorescent protein ( Fig.23D), indicating that the expression of the CRISPR / Cas9 vector as a control group does not affect the expression of the eYFP fluorescent protein. Two plasmid vectors pBT140 and pX330-U6-sgRNA(eYFP)-CBh-hSpCas9-mCherry were also injected at the same time. Fig.23 As shown in B and 23C, most (average 89.8%) astrocytes expressing mCherry did not express eYFP fluorescent protein, and further statistical analysis showed that sgRNA(eYFP) expression could effectively knock out the eYFP gene in astrocytes of Ai3 transgenic mice.

[0211] Therefore, to further verify the removal efficiency of CRISPR / Cas9, we sequenced the genome at the target location. Fig.24 As shown in A, we designed a pair of primers pCAG-F and eYFP-R to amplify the sequence containing the sgRNA target site by PCR. After Cre recombinase cleavage, the amplified fragment will be shortened. By optimizing the PCR conditions, a cutoff value of 300 bp was obtained for sequencing analysis ( Fig.24 B) Sequencing results are shown in Fig.24 C. In astrocytes expressing Cre recombinase, insertion mutations, deletion mutations, and point mutations were detected. Point mutations have limited effects on the structure of fluorescent proteins, which may be the reason why a small number (10.2%) of astrocytes expressing mCherry fluorescent protein can also see eYFP fluorescence.

[0212] Example 16. Knock-in of the eGFP gene in C57BL / 6 mice using the piggyBac transposition system

[0213] The piggyBac transposon system requires two plasmid vectors to express the transposase (pCAG-Pbase plasmid vector) and the transposon carrying the foreign gene with terminal repeat sequences (PBCAG-eGFP plasmid vector) ( Fig.25 ). First, inject a common plasmid vector (p-eGFP-C1). Since common vectors cannot be integrated into the cell genome, the exogenous plasmid DNA can only be expressed transiently and eventually degraded by nucleases in the cell. Fig.26 As shown in A, after 25 days of expression, the intensity of green fluorescent protein (eGFP) decreased significantly, only about one-tenth of the fluorescence expression at 5 days ( Fig.26 B). In contrast, when the piggyBac transposon system was used, fluorescent protein expression was higher after 25 days of expression compared to 5 days ( Fig.26 C and Fig.26D) The increase in fluorescence intensity is due to the integration of the EGFP gene into the genome via the transposon system, and the continuous and stable expression of eGFP increases the total amount of fluorescent protein in the cell.

[0214] To further investigate the ability of the piggyBac transposon system to achieve long-term or even permanent expression of foreign genes, the expression of PB plasmid vectors was observed for two months after injection ( Fig. 27 A) and six months ( Fig. 27 B). It was found that eGFP expression remained stable and sustained even for half a year. These results fully demonstrated that the piggyBac transposon system can be induced to permanently express foreign genes in astrocytes of adult mice using the glycine transfection system.

Claims

1. A delivery system for preparing a drug for delivering a nucleic acid into a living organism, the delivery system comprising an amino acid and a nucleic acid; The amino acid is L-proline, D-alanine or L-histidine; the concentration of the amino acid is in the range of 50mM-300mM; The delivery of the nucleic acid to a living organism is delivery of the nucleic acid to astrocytes in the midbrain, thalamus, hypothalamus, brainstem, cerebellum, globus pallidus, cerebral cortex or hippocampus, Bergmann glial cells, velate astrocytes or stellate interneurons in the cerebellum in the living organism.

2. The method according to claim 1, wherein the nucleic acid is selected from an oligonucleotide or a plasmid.

3. The use according to claim 2, wherein the oligonucleotide is a short length of single-stranded DNA or RNA. The use according to claim 3 , wherein the short-length single-stranded RNA is antisense RNA or siRNA.

5. The use according to claim 1, wherein the delivery system comprises amino acids and one or more plasmids.

6. The use according to claim 1, wherein the delivery system comprises amino acids and two or three plasmids.

7. The use according to claim 1, wherein the nucleic acid is dissolved in the amino acid solution before delivery, or the nucleic acid and the amino acid solution are delivered separately.

8. The use according to claim 1, wherein the nucleic acid is a CRISPR / Cas9 system.

9. The use according to claim 1, wherein the nucleic acid is a transposon system.

10. The use according to claim 9, wherein the transposon system is the piggyBac transposon system.

11. The use according to claim 1, wherein the concentration of the amino acid in the delivery system is 300 mM.

12. The use according to claim 1, wherein The nucleic acid is one or more plasmids expressing one or more fluorescent proteins selected from RFP, YFP, GFP, CFP and BFP.

13. The use according to claim 1, wherein the nucleic acid is two or three plasmids expressing two or three fluorescent proteins selected from RFP, YFP, GFP, CFP and BFP.

14. The use according to claim 1, wherein the delivery system is in the form of a solution.

15. The use according to claim 1, wherein the living organism comprises a bird, chicken, duck, goose, mouse, rat, cat, rabbit, dog, horse, cow, sheep, goat, pig, tree shrew, monkey, chimpanzee or human.

16. Use of a delivery system for preparing a drug for gene therapy for treating a disease, the delivery system comprising amino acids and nucleic acids; The amino acid is L-proline, D-alanine or L-histidine, and the concentration of the amino acid is in the range of 50mM-300mM; The disease is a neurodegenerative disease of Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia or a tumor in the brain.

17. Use according to claim 16, wherein the nucleic acid is selected from an oligonucleotide or a plasmid.

18. The use according to claim 17, wherein the oligonucleotide is a short length of single-stranded DNA or RNA.

19. The use according to claim 18, wherein the short-length single-stranded RNA is antisense RNA or siRNA.

20. The use according to claim 16, wherein the delivery system comprises amino acids and one or more plasmids.

21. The use according to claim 16, wherein the delivery system comprises amino acids and two or three plasmids.

22. The use according to claim 16, wherein the nucleic acid is dissolved in the solution of the amino acid before delivery, or the nucleic acid and the solution of the amino acid are delivered separately.

23. The use according to claim 16, wherein the nucleic acid is a CRISPR / Cas9 system.

24. The use according to claim 16, wherein the nucleic acid is a transposon system.

25. The use according to claim 24, wherein the transposon system is the piggyBac transposon system.

26. The use according to claim 16, wherein the concentration of the amino acid in the delivery system is 300 mM.

27. The use according to claim 16, wherein The nucleic acid is one or more plasmids expressing one or more fluorescent proteins selected from RFP, YFP, GFP, CFP and BFP.

28. The use according to claim 16, wherein the nucleic acid is two or three plasmids expressing two or three fluorescent proteins selected from RFP, YFP, GFP, CFP and BFP.

29. The use according to claim 16, wherein the delivery system is in the form of a solution.

30. Use of a delivery system expressing one or more fluorescent proteins in the preparation of drugs in "brain rainbow" multicolor labeling technology, wherein the delivery system comprises amino acids and nucleic acids; The amino acid is L-proline, D-alanine or L-histidine, and the concentration of the amino acid is in the range of 50mM-300mM; The nucleic acid is one or more plasmids expressing one or more fluorescent proteins selected from RFP, YFP, GFP, CFP and BFP.

31. The use according to claim 30, wherein the nucleic acid is two or three plasmids expressing two or three fluorescent proteins selected from RFP, YFP, GFP, CFP and BFP.

32. The use according to claim 30, wherein the nucleic acid is selected from an oligonucleotide or a plasmid.

33. The use according to claim 32, wherein the oligonucleotide is a short length of single-stranded DNA or RNA.

34. The use according to claim 33, wherein the short-length single-stranded RNA is antisense RNA or siRNA.

35. The use according to claim 30, wherein the nucleic acid is dissolved in the solution of the amino acid before delivery, or the nucleic acid and the solution of the amino acid are delivered separately.

36. The use according to claim 30, wherein the nucleic acid is a CRISPR / Cas9 system.

37. The use according to claim 30, wherein the nucleic acid is a transposon system.

38. The use according to claim 37, wherein the transposon system is the piggyBac transposon system.

39. The use according to claim 30, wherein the concentration of the amino acid in the delivery system is 300 mM.

40. The use according to claim 30, wherein the delivery system is in the form of a solution.

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