CN114931652B - Application of Hmga2 gene in repairing damaged retina - Google Patents
Application of Hmga2 gene in repairing damaged retina Download PDFInfo
- Publication number
- CN114931652B CN114931652B CN202210716606.XA CN202210716606A CN114931652B CN 114931652 B CN114931652 B CN 114931652B CN 202210716606 A CN202210716606 A CN 202210716606A CN 114931652 B CN114931652 B CN 114931652B
- Authority
- CN
- China
- Prior art keywords
- hmga2
- adeno
- associated virus
- gene
- hmga2 gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 101150073387 Hmga2 gene Proteins 0.000 title claims abstract description 44
- 101100395338 Mus musculus Hmga2 gene Proteins 0.000 title claims abstract description 44
- 210000001525 retina Anatomy 0.000 title claims abstract description 44
- 241000702421 Dependoparvovirus Species 0.000 claims abstract description 56
- 239000013598 vector Substances 0.000 claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 239000003814 drug Substances 0.000 claims abstract description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 3
- 230000014509 gene expression Effects 0.000 claims description 20
- 238000001890 transfection Methods 0.000 claims description 20
- 241000700605 Viruses Species 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 12
- 239000013612 plasmid Substances 0.000 claims description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 238000010367 cloning Methods 0.000 claims description 9
- 238000010276 construction Methods 0.000 claims description 9
- 238000001976 enzyme digestion Methods 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 239000002299 complementary DNA Substances 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 238000003753 real-time PCR Methods 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- 238000001962 electrophoresis Methods 0.000 claims description 3
- 238000011013 endotoxin removal Methods 0.000 claims description 3
- 239000013613 expression plasmid Substances 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 230000004927 fusion Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000012096 transfection reagent Substances 0.000 claims description 3
- 239000013603 viral vector Substances 0.000 claims 3
- 230000006378 damage Effects 0.000 abstract description 14
- 206010057430 Retinal injury Diseases 0.000 abstract description 10
- 230000002207 retinal effect Effects 0.000 abstract description 7
- 210000000608 photoreceptor cell Anatomy 0.000 abstract description 5
- 230000004243 retinal function Effects 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 201000007737 Retinal degeneration Diseases 0.000 abstract description 2
- 208000015114 central nervous system disease Diseases 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 208000001738 Nervous System Trauma Diseases 0.000 abstract 1
- 208000015122 neurodegenerative disease Diseases 0.000 abstract 1
- 230000004258 retinal degeneration Effects 0.000 abstract 1
- 230000001225 therapeutic effect Effects 0.000 abstract 1
- 230000004382 visual function Effects 0.000 abstract 1
- 210000005156 Müller Glia Anatomy 0.000 description 41
- 210000004027 cell Anatomy 0.000 description 28
- 241000699670 Mus sp. Species 0.000 description 16
- 230000000694 effects Effects 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 230000008672 reprogramming Effects 0.000 description 11
- 208000027418 Wounds and injury Diseases 0.000 description 10
- 208000014674 injury Diseases 0.000 description 10
- 230000002018 overexpression Effects 0.000 description 10
- NALMPLUMOWIVJC-UHFFFAOYSA-N n,n,4-trimethylbenzeneamine oxide Chemical compound CC1=CC=C([N+](C)(C)[O-])C=C1 NALMPLUMOWIVJC-UHFFFAOYSA-N 0.000 description 8
- 229940032753 sodium iodate Drugs 0.000 description 8
- 235000015281 sodium iodate Nutrition 0.000 description 8
- 239000011697 sodium iodate Substances 0.000 description 8
- 239000007924 injection Substances 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 108091008695 photoreceptors Proteins 0.000 description 7
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 6
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 6
- 210000003986 cell retinal photoreceptor Anatomy 0.000 description 6
- 206010018341 Gliosis Diseases 0.000 description 5
- 102000049982 HMGA2 Human genes 0.000 description 5
- 108700039143 HMGA2 Proteins 0.000 description 5
- 230000007387 gliosis Effects 0.000 description 5
- 230000035755 proliferation Effects 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 101150025841 CCND1 gene Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 210000000683 abdominal cavity Anatomy 0.000 description 4
- 210000003169 central nervous system Anatomy 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000003364 immunohistochemistry Methods 0.000 description 4
- 210000002569 neuron Anatomy 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 description 3
- 238000000116 DAPI staining Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000004300 dark adaptation Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000004438 eyesight Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000001879 gelation Methods 0.000 description 2
- 238000003208 gene overexpression Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000001178 neural stem cell Anatomy 0.000 description 2
- 230000004766 neurogenesis Effects 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 210000001116 retinal neuron Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 239000013607 AAV vector Substances 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 101150010353 Ascl1 gene Proteins 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 241000252212 Danio rerio Species 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101150092239 OTX2 gene Proteins 0.000 description 1
- 208000030768 Optic nerve injury Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 206010038910 Retinitis Diseases 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 101150109085 TSA gene Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 206010064930 age-related macular degeneration Diseases 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 108091006090 chromatin-associated proteins Proteins 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000007831 electrophysiology Effects 0.000 description 1
- 238000002001 electrophysiology Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000001328 optic nerve Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of genetic engineering; in retinal degenerative diseases, when the retina is damaged, there is a secondary loss of photoreceptor cells, and the lost photoreceptor cells are difficult to regenerate, which is a main cause of difficulty in recovering visual functions. How to effectively treat the retinal degeneration diseases and recover the damaged retinal functions is always a great difficulty facing clinical ophthalmology. The invention provides an adeno-associated virus vector carrying Hmga2 genes, which is a DNA sequence carrying Hmga2 genes shown in SEQ ID NO. 1. The invention also provides application of the adeno-associated virus vector carrying Hmga2 gene in preparing medicaments for repairing damaged retina. The invention provides a new therapeutic target and theoretical basis for clinical treatment of retinal injury, and has important reference significance for treatment of central nervous system diseases and injuries.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to application of Hmga2 gene in preparation of a medicament for repairing damaged retina.
Technical Field
With the advent of aging society, the prevalence of irreversible and refractory blindness-causing diseases including age-related macular degeneration and retinitis has increased. A typical pathological change in the retina of these blind patients is the loss of photoreceptors and retinal pigment epithelial cells (RPE). As with other parts of the central nervous system, the retina of adult mammals has no ability to regenerate itself upon injury or degeneration. There is currently no effective treatment for the loss of RPE, photoreceptors and other neurons. In experimental studies, there has been some progress in repairing damaged neurons by transplanting exogenous stem cells, however, the source, safety and effectiveness of donor cells have been unavoidable bottleneck problems, and in order to break through these bottlenecks, it is possible to reprogram them in situ into photoreceptors or other neurons by activating endogenous stem cells, which is a new research direction for repairing or regenerating damaged retina.
In lower vertebrates, such as zebra fish, after the retina is damaged, muller Glia (MG) cells can act as endogenous stem cells in the retina, activated, dedifferentiated, proliferated, and the resulting offspring can differentiate into functional neurons, restoring impaired vision. In contrast, mammalian MG loses the ability to reprogram pluripotent stem cells, and damage or degeneration often results in retinal stress, leading to MG hypertrophy and increased stiffness, eventually overactivating, forming gliosis, and difficult to progress to neurogenesis.
Studies on mouse MG reprogramming have shown that by activating a series of specific signaling pathways, e.g., in combination with the deacetylase inhibitors TSA and Ascl1 gene overexpression, MG reprogramming of adult mice can be activated and new retinal neurons generated; new photoreceptor cells can be generated by combining three transcripts of Wnt/beta-catenin and Otx2, crx and Nrl. Although 20% -40% of MGs change the fate of cells from gliosis to reprogramming, a few MG-derived precursor cells can also differentiate into structurally and functionally mature retinal neurons, but most MGs fail to reprogram. Therefore, the target molecules and genes affecting the MG reprogramming of the mice are searched and regulated, the MG reprogramming capacity is enhanced, the retina regeneration is promoted, and the target molecules and genes are the subject to be solved urgently, and the target molecules and genes have bright clinical application prospect.
Hmga2, the most abundant non-histone chromatin-associated protein, plays a key role in gene transcription regulation, determining fate and neurogenesis of stem cells during development. Hmga2 is highly expressed in embryonic stem cells, while its expression is extremely limited in the later stages of development and adulthood. Overexpression of Hmga2 allows reprogramming of human somatic cells into neural stem cells while improving the self-renewal capacity of mouse neural stem cells.
In combination with the above-described related actions of Hmga2, hmga2 is presumed to be a key molecule affecting MG reprogramming, so it is assumed that Hmga2 can promote MG reprogramming ability after retinal damage, thereby saving retinal function.
At present, research of Hmga2 is focused on development and tumor, related research on Hmga2 genes in preparation of drugs for repairing damaged retina is not seen, and preparation of drugs for repairing damaged retina by constructing adeno-associated virus vectors carrying Hmga2 genes is not reported in literature.
Disclosure of Invention
In view of the above, the present invention aims to provide an adeno-associated virus vector carrying Hmga2 gene, a construction method thereof, and an application thereof in preparing a medicament for repairing damaged retina.
The invention injects sodium iodate solution into the abdominal cavity of adult mice to make retinal injury models.
In RNA sequencing of mouse retina and detection of Real-time PCR, hmga2mRNA expression level was found to be significantly higher than that of normal adult mouse by about 3.07 times in retina 1 day after injury; 3 days after injury, hmga2mRNA expression in the retina was 2.04-fold that in normal adult mice, and decreased to normal levels by day 5. Immunohistochemical results showed that the expression of Hmga2 in the retinal MG nuclei increased significantly 3 to 5 days after retinal injury, followed by a gradual decrease. Western Blot results show that Hmga2 protein expression levels in the retinas of mice 3 days after injury were significantly higher than that in normal adult mice, approximately 1.20-fold higher than in normal adult rats, and decreased to normal levels by day 14 after injury.
The binding of Hmga 2-related effects, in particular its close relationship to development, cancer and the altered nature of the gene expression following retinal damage, suggests that treatment of Hmga2 adeno-associated virus overexpression prior to retinal damage may promote MG reprogramming and survival of damaged retinal cells.
According to the technical scheme, an adeno-associated virus vector of Hmga2 is mainly constructed, in an adult mouse retina injury model, the mouse retina MG cells are specifically transfected through intravitreal injection of the adeno-associated virus vector of Hmga2, and in vivo observation and discussion are carried out to activate MG reprogramming through Hmga2 gene overexpression so as to repair damaged retina.
In order to achieve the above object, according to a first aspect of the present invention, there is provided an adeno-associated virus vector carrying Hmga2 gene, wherein the adeno-associated virus vector is a DNA sequence carrying Hmga2 gene as shown below.
ATGAGCGCACGCGGTGAGGGCGCCGGGCAGCCGTCCACATCAGCCCAGGGACAACCT
GCCGCCCCGGTGCCACAGAAGCGAGGACGCGGCCGACCCAGGAAGCAGCAGCAAGAG
CCAACCTGTGAGCCCTCTCCTAAGAGACCCAGAGGAAGACCCAAAGGCAGCAAAAAC
AAGAGCCCCTCTAAAGCAGCCCAGAAGAAAGCAGAGACCATTGGAGAAAAACGGCCA
AGAGGCAGACCTAGGAAATGGCCACAACAAGTCGTTCAGAAGAAGCCTGCTCAGGGG
GGTGAGTTTGGAGAACGCACCAGGTGCCATGAATCACAAACCTCAAGTCTGCCCCACA
ATCCTTATGGGCAGGAGCTCATCGCTTCCACTCTCATGGGAGAC
(SEQ ID NO:1)；
In a second aspect of the present invention, there is provided a method for constructing the aforementioned adeno-associated virus vector carrying Hmga2 gene, comprising the steps of:
cloning construction of adeno-associated Virus vector of Hmga2 Gene
The following PCR primers were designed and synthesized based on the Hmga2 gene (GenBank ID: NM-001347170.1):
hmga2 upstream primer:
5’-CCGCTGCTCGCGGGGTCTAGAGCCACCATGAGCGCACGCGGTGAGGGCG-3’
(SEQ ID NO:2)；
hmga2 downstream primer:
5’-AGTAGCTCCGCTTCCGGATCCGTCTCCCATGAGAGTGGAAGCG-3’
(SEQ ID NO:3)；
amplifying Hmga2 target gene from a cDNA template containing Hmga2 gene as shown in SEQ ID NO. 1 by using a PCR method; enzyme cutting is carried out on the adeno-associated virus vector, enzyme cutting product purification is carried out after enzyme cutting is completed, connection reaction is carried out on the enzyme-cut adeno-associated virus vector and Hmga2 gene PCR product, hmga2 expression vector is obtained, after the connection product is obtained, the product is transformed into competent escherichia coli, cloning identification is carried out on Hmga2 gene adeno-associated virus plasmid on the grown transformant, positive electrophoresis result is positive cloning, and the fact that the target gene is directionally connected into the target vector is proved; performing biological sequencing and analysis comparison on clones positive to the PCR identification, and if the sequences are consistent with the GenBank ID: NM_001347170.1 sequences in the database, the comparison is correct, so that the plasmid construction is successful; the plasmid contains a Muller cell specific promoter, and can effectively target and infect Muller cells by adopting a Muller cell specific adeno-associated virus serotype.
Adeno-associated virus concentration of Hmga2 Gene
Extracting the constructed expression plasmid by using an ultrapure endotoxin removal kit, and transfecting AAV-293 cells by using a transfection reagent to determine that the transfection and the expression of the adeno-associated virus vector of the Hmga2 gene are successful; before transfection, AAV-293 cells have a fusion degree of more than 80%, after 72 hours of transfection, cells are collected by centrifugation, then the cells are lysed, after centrifugation at 4 ℃ and 48000 rpm for 130min, cell supernatants rich in adeno-associated virus particles are collected, concentrated and the virus titer is determined by quantitative PCR. Adding glycerol to high titer adeno-associated virus concentrate to final concentration of 5%, packaging, and storing at-80deg.C.
In-vitro and in-vivo experiments have different requirements on the titer of the adeno-associated virus, and adeno-associated virus particles with different titers can be obtained according to the requirements in the production process and used for corresponding experiments.
In a third aspect, the invention provides an application of the adeno-associated virus vector carrying Hmga2 gene in preparing a medicament for repairing damaged retina.
The adenovirus-associated virus vector carrying Hmga2 gene constructed by the invention is used for preparing the medicine for repairing damaged retina. Firstly, a mouse retina MG cell is transfected by an adeno-associated virus vector of Hmga2 gene, then, a damage model is established by intraperitoneal injection of sodium iodate, and the effect of the Hmga2 gene over-expressed in MG on damaged retina repair is observed.
The application of the adeno-associated virus vector carrying Hmga2 gene in preparing medicaments for repairing damaged retina is disclosed, wherein the adeno-associated virus of Hmga2 gene can be used for transfecting MG, and in clinic, the virus can be used for transfecting MG by intravitreal injection for carrying out gene therapy after retina damage.
The AAV is a single-chain linear defective DNA virus without envelope, and DNA of AAV can be integrated into chromosome of host in low frequency in form of dsDNA, so that AAV vector has the characteristics of stable latency state, wide host range, high transfection efficiency, long expression time, weak immune response and the like, so that it is regarded as the safest virus vector for in vivo experiment at present. There are now a variety of AAV serotypes for specific transfection of different tissues, and selection of serotype adeno-associated viruses and MG-specific promoters with greater transfection and specificity in MG can achieve efficient targeted transfection of MG.
The invention is based on the unique effect of Hmga2 in tumor, cerebral cortex, subventricular zone, retina and other tissues, and uses the adeno-associated virus vector carrying Hmga2 gene to transfect MG, observe the effect of reprogramming MG, photoreceptor survival, vision function recovery and the like.
The retina and optic nerve are part of the Central Nervous System (CNS) and are ideal sites for studying CNS injury and regeneration. Research on repair and regeneration after retinal injury provides a new powerful means and theoretical basis for clinical treatment of optic nerve injury, and has important reference significance for treatment of CNS diseases and injury.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in the following preferred detail with reference to the accompanying drawings, in which:
FIG. 1 is a graph showing the effect of adenovirus-associated (green) transfection of mouse MG (left side) and a partial graph showing overexpression of Hmga2 protein in retina after transfection (right side). 3 weeks after the mice are subjected to intravitreal injection of the virus, retina sections are taken, cells transfected by the virus in the retina can be seen, and the cells are in a typical MG arbor form, and the transfection efficiency is about 70%; and Hmga2 protein (red) can be expressed correctly.
FIG. 2 shows the effect of Hmga2 adeno-associated virus on MG-gelatinization. The expression of the gliosis-related protein GFAP in the non-injured group (SI-PBS and Hmga 2-SI-PBS), the purely injured group (SI-14 d, SI-21d, SI-28 d) and the treated group (Hmga-SI-14 d, hmga-SI-21d, hmga-SI-28 d) was detected by immunohistochemistry at 3 weeks after the respective injections of PBS and AAV-Hmga2, and Sodium Iodate (SI) solution was injected into the abdominal cavity, and 4 weeks after the injury, and it was seen that the expression level of the MG gliosis GFAP (red) was significantly reduced 14-21 days after the AAV-Hmga2 treatment (left graph of FIG. 2). The right panel of fig. 2 shows statistics of GFAP protein relative expression amounts (n=3, P <0.05, P < 0.01). This result suggests that MG gelation degree is reduced.
FIG. 3 shows the effect of Hmga2 adeno-associated virus on MG proliferation. The expression of proliferation-related protein Ccnd1 was examined by immunohistochemistry in the non-injured group (SI-PBS and Hmga 2-SI-PBS), the purely injured group (SI-14 d, SI-21d, SI-28 d) and the treated group (Hmga-SI-14 d, hmga-SI-21d, hmga-SI-28 d), and it was found that the expression level (white) of Ccnd1 was significantly increased 14-28 days after AAV-Hmga2 treatment (left panel of FIG. 3). The right graph in fig. 3 shows the statistics of the proportion of the proliferated MG to the total MG (n=3, ×p <0.01, ×p < 0.001). This result suggests that MG proliferation capacity is enhanced.
FIG. 4 is a graph showing the effect of Hmga2 adeno-associated virus on survival of retinal photoreceptor layer (ONL) cells, wherein the left panel of FIG. 4 is stained with nuclei (DAPI) to observe survival of retinal photoreceptor layer cells in the non-injured (SI-PBS and Hmga 2-SI-PBS), the purely injured (SI-14 d, SI-21d, SI-28 d) and the treated (Hmga-SI-14 d, hmga-SI-21d, hmga-SI-28 d) groups; the right panel of fig. 4 shows quantitative statistics of the number of photoreceptor layers (n=5, ×p < 0.001). It can be seen that the thickness of the retinal photoreceptor layer was greater than that of the purely damaged group 14-28 days after AAV-Hmga2 treatment, and that viable photoreceptor cells were significantly increased.
FIG. 5 is a rescue effect of Hmga2 adeno-associated virus on retinal function. Mice from the non-injured group (SI-PBS and Hmga 2-SI-PBS), the singly injured group (SI-14 d, SI-21d, SI-28 d) and the treated group (Hmga-SI-14 d, hmga-SI-21d, hmga-SI-28 d) were examined by electroretinogram technique for light intensity of 3.0cd.s.m under dark adaptation environment -2 At this time, the condition of the retinal waveform (upper graph of fig. 5) and the magnitudes of the a-wave and b-wave (lower graph of fig. 5) are counted (n=4-7, P<0.05,**P<0.01,***P<0.001). It can be seen that the amplitude of the a wave and b wave is significantly increased, i.e. retinal function is greatly improved, 14-28 days after AAV-Hmga2 treatment.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be noted that the illustrations provided in the following embodiments merely illustrate the basic idea of the present invention by way of illustration, and the following embodiments and features in the embodiments may be combined with each other without conflict.
Examples:
1. obtaining the Gene fragment of interest
The sequence of Hmga2 gene was queried from GenBank, primers were designed using Primer5 software, and Hmga2 gene fragment was amplified using PCR with mouse cDNA as template.
PCR amplification of the target gene: the high-fidelity PrimeSTAR enzyme is used for amplifying the target gene, and the reaction system and the conditions are as follows:
table 1: hmga2 gene amplification system using mouse cDNA as template
2. Cloning construction of adeno-associated Virus vector of Hmga2 Gene
Amplifying Hmga2 target genes, carrying out enzyme digestion on adeno-associated virus vectors, purifying enzyme digestion products after enzyme digestion is completed, carrying out seamless connection reaction on the enzyme digestion adeno-associated virus vectors and Hmga2 gene PCR products to obtain Hmga2 expression vectors, after obtaining connection products, converting the products into competent escherichia coli, carrying out cloning identification on Hmga2 gene adeno-associated virus plasmids on the grown transformants, and carrying out positive cloning on electrophoresis results to prove that the target genes are directionally connected to the target vectors; performing biological sequencing and analysis comparison on clones positive to the PCR identification, and if the sequences are consistent with the GenBank ID: NM_001347170.1 sequences in the database, the comparison is correct, so that the plasmid construction is successful; the plasmid contains a Muller cell specific promoter, and can effectively target and infect Muller cells by adopting a Muller cell specific adeno-associated virus serotype.
3. Adeno-associated virus concentration of Hmga2 Gene
Extracting the constructed expression plasmid by using an ultrapure endotoxin removal kit, and transfecting AAV-293 cells with auxiliary plasmids by using a transfection reagent to determine that the transfection and the expression of the adeno-associated virus vector of the Hmga2 gene are successful; before transfection, AAV-293 cells have a fusion degree of more than 80%, after 72 hours of transfection, cells are collected by centrifugation, then the cells are lysed, after centrifugation at 4 ℃ and 48000 rpm for 130min, cell supernatants rich in adeno-associated virus particles are collected, concentrated and the virus titer is determined by quantitative PCR. Adding glycerol to high titer adeno-associated virus concentrate to final concentration of 5%, packaging, and storing at-80deg.C.
4. Grouping of laboratory animals
(1) Non-injured group:
intraperitoneal injection of PBS, intravitreal injection of PBS or AAV-Hmga2 virus (SI-PBS and Hmga 2-SI-PBS);
(2) Pure injury group:
injecting sodium iodate into the abdominal cavity, injecting PBS (SI-14 d, SI-21d, SI-28 d) into the vitreous cavity;
(3) Treatment group:
injecting sodium iodate into abdominal cavity, injecting AAV-Hmga2 virus (Hmga-SI-14 d, hmga-SI-21d, hmga-SI-28 d) into vitreous cavity;
5. hmga2 adeno-associated virus transfected mouse retina and overexpression of Hmga2 after transfection
After 3 weeks of injection of AAV-Hmga2 virus into the vitreous cavity of mice, the mice were fixed in 4% paraformaldehyde for 2 hours after taking out the eyeballs, and after sucrose was dehydrated overnight, frozen sections were performed, and then the fluorescent (green) expression of EGFP virus was observed, and the virus was found to be transfected with most of MG with an efficiency of about 70%. The expression of Hmga2 protein was observed by immunohistochemistry after virus transfection on the retinas of mice (as shown in FIG. 1). It can be seen that in the retina, the virus transfected cells are in the form of typical MG arbor, with a transfection efficiency of about 70%; and Hmga2 protein (red) can be expressed correctly.
6. Construction of mouse model for retinal injury
After the Hmga2 adeno-associated virus transfects the retinas of mice, retinal injury model preparation is carried out, after the conventional disinfection of each group of experimental animals, intraperitoneal injection is carried out according to 30mg/kg Sodium Iodate (SI) of each mouse, and the clinical condition of retinal pigment degeneration disease is simulated.
7. Effect of Hmga2 overexpression on MG
(1) Effect of Hmga2 overexpression on the gliosis marker GFAP of MG
After the mice retina was transfected with the Hmga2 adeno-associated virus by immunohistochemical method, a significant decrease in the GFAP expression level (red) of the MG-gelatinized protein was observed 14-21 days after AAV-Hmga2 treatment (left panel of FIG. 2), and the statistical result of the relative GFAP protein expression level (n=3, P <0.05, P < 0.01) was shown in the right panel of FIG. 2. This result suggests that MG gelation degree is reduced.
(2) Effect of Hmga2 overexpression on the proliferation marker Ccnd1 of MG
After the Hmga2 adeno-associated virus transfection of the retinas of mice was observed by immunohistochemistry, a significant increase in Ccnd1 expression (white) was seen 14-28 days after AAV-Hmga2 treatment (left panel of FIG. 3). The right graph in fig. 3 shows the statistics of the proportion of the proliferated MG to the total MG (n=3, ×p <0.01, ×p < 0.001). This result suggests that MG proliferation capacity is enhanced.
8. Protective effect of Hmga2 overexpression on damaged retina
(1) Effect of Hmga2 overexpression on cell number of photoreceptor layer
DAPI staining: DAPI staining was performed on frozen sections, and survival of cells on the retinal photoreceptor layers was observed in the non-damaged group, the purely damaged group, and the treated group in terms of the number of layers (left graph of fig. 4); the right panel of fig. 4 shows the quantitative result of the number of photoreceptor layers (n=5, ×p < 0.001). It can be seen that the thickness of the retinal photoreceptor layer was greater than that of the purely damaged group 14-28 days after AAV-Hmga2 treatment, and that viable photoreceptor cells were significantly increased.
(2) Rescue effect of Hmga2 overexpression on retinal electrophysiology
1) Electroretinogram (ERG) detection: ERG detection was performed on both eyes after anesthesia by 1% sodium pentobarbital injection in each group of mice
2) 4 weeks of retinal injury, mice from the non-injured, purely injured and treated groups were tested for light intensity of 3.0cd s.m in dark adaptation environment -2 At this time, the condition of the retinal waveform, as shown in the upper graph of fig. 5, is counted and the amplitudes of the a wave and the b wave (lower graph of fig. 5) are counted (n=4-7, P<0.05,**P<0.01,***P<0.001). It can be seen that the amplitude of the a wave and b wave is significantly increased, i.e. retinal function is greatly improved, 14-28 days after AAV-Hmga2 treatment.
The preferred embodiments of the invention have been described above in detail, but the invention is not limited to these
Various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the invention, and are intended to be included within the scope of the present invention as defined in the appended claims.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the claims of the present invention.
Claims (4)
1. The application of Hmga2 gene in preparing medicament for repairing damaged retina is characterized in that Hmga2 gene is applied in preparing medicament for repairing damaged retina.
2. The use of the Hmga2 gene of claim 1 in the manufacture of a medicament for repairing damaged retina, wherein the Hmga2 gene is carried in the form of an adeno-associated virus vector.
3. The use of the Hmga2 gene according to claim 2 in the manufacture of a medicament for repairing damaged retina, wherein the adeno-associated viral vector is a DNA sequence carrying the Hmga2 gene as shown in SEQ ID No. 1.
4. The use of the Hmga2 gene according to claim 3 for the preparation of a medicament for repairing damaged retina, wherein the construction method of the adeno-associated viral vector carrying the Hmga2 gene comprises the steps of:
a) Cloning construction of adeno-associated viral vector of Hmga2 Gene PCR primers were designed and synthesized as follows:
hmga 2-forward primer is shown as SEQ ID NO. 2; the Hmga 2-reverse primer is shown as SEQ ID NO. 3;
amplifying Hmga2 target gene from a cDNA clone template containing Hmga2 gene as shown in SEQ ID NO. 1 by using a PCR method; carrying out enzyme digestion on the adeno-associated virus vector, purifying enzyme digestion products after enzyme digestion is completed, carrying out seamless connection reaction on the enzyme digestion adeno-associated virus vector and Hmga2 gene PCR products to obtain Hmga2 expression vectors, after obtaining connection products, converting the products into competent escherichia coli, carrying out cloning identification on Hmga2 gene adeno-associated virus plasmids on the grown transformants, and carrying out positive cloning on electrophoresis results to prove that target genes are directionally connected into the target vectors; performing biological sequencing and analysis comparison on clones positive to the PCR identification, and if the sequences are consistent with the GenBank ID: NM_001347170.1 sequences in the database, the comparison is correct, so that the plasmid construction is successful; the plasmid contains a Muller cell specific promoter, and adopts a Muller cell specific adeno-associated virus serotype to effectively target and infect the Muller cell;
b) Adeno-associated virus concentration of Hmga2 Gene
Extracting the constructed expression plasmid by using an ultrapure endotoxin removal kit, and transfecting AAV-293 cells with auxiliary plasmids by using a transfection reagent to determine that the transfection and the expression of the adeno-associated virus vector of the Hmga2 gene are successful; before transfection, AAV-293 cells have a fusion degree of more than 80%, after 72 hours of transfection, the cells are collected by centrifugation, then the cells are lysed, after centrifugation at a high speed of 48000 ℃ for 130min, the cell supernatant rich in adeno-associated virus particles is collected, concentrated, and then the virus titer is determined by quantitative PCR; adding glycerol to high titer adeno-associated virus concentrate to final concentration of 5%, packaging, and storing at-80deg.C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210716606.XA CN114931652B (en) | 2022-06-23 | 2022-06-23 | Application of Hmga2 gene in repairing damaged retina |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210716606.XA CN114931652B (en) | 2022-06-23 | 2022-06-23 | Application of Hmga2 gene in repairing damaged retina |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114931652A CN114931652A (en) | 2022-08-23 |
| CN114931652B true CN114931652B (en) | 2024-01-26 |
Family
ID=82868849
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210716606.XA Active CN114931652B (en) | 2022-06-23 | 2022-06-23 | Application of Hmga2 gene in repairing damaged retina |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114931652B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174514A (en) * | 2010-11-24 | 2011-09-07 | 华侨大学 | Double-stranded siRNA (Small Interfering Ribonucleic Acid) for suppressing Hmga2 gene expression and application thereof |
| CN102375064A (en) * | 2010-08-26 | 2012-03-14 | 杭州华得森生物技术有限公司 | In-vitro diagnostic kit for detecting HMGA2 (High Mobility Group A) content with enzyme-linked immuno sorbent assay |
| CN103224956A (en) * | 2013-05-17 | 2013-07-31 | 中国人民解放军第二军医大学 | Type-II adeno-associated virus carrying neuritin genes and application thereof in restoring optic nerve injuries |
| CN106755104A (en) * | 2016-12-07 | 2017-05-31 | 中国人民解放军第二军医大学 | A kind of adeno-associated virus 2 of carrying genes of Pim 1 and its application in crushed optic nerve is repaired |
| CN109868313A (en) * | 2018-06-25 | 2019-06-11 | 山东大学 | Application of the HMGA2 gene in Stein-Leventhal syndrome disease |
| CN114350664A (en) * | 2022-01-11 | 2022-04-15 | 四川省医学科学院·四川省人民医院 | Promoter specifically expressed in retina Muller cell and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB201318347D0 (en) * | 2013-10-16 | 2013-11-27 | Ucl Business Plc | Retroviral vectors |
| WO2017062659A1 (en) * | 2015-10-06 | 2017-04-13 | University Of Virginia Patent Foundation | Compositions and methods for treating diabetic retinopathy |
-
2022
- 2022-06-23 CN CN202210716606.XA patent/CN114931652B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375064A (en) * | 2010-08-26 | 2012-03-14 | 杭州华得森生物技术有限公司 | In-vitro diagnostic kit for detecting HMGA2 (High Mobility Group A) content with enzyme-linked immuno sorbent assay |
| CN102174514A (en) * | 2010-11-24 | 2011-09-07 | 华侨大学 | Double-stranded siRNA (Small Interfering Ribonucleic Acid) for suppressing Hmga2 gene expression and application thereof |
| CN103224956A (en) * | 2013-05-17 | 2013-07-31 | 中国人民解放军第二军医大学 | Type-II adeno-associated virus carrying neuritin genes and application thereof in restoring optic nerve injuries |
| CN106755104A (en) * | 2016-12-07 | 2017-05-31 | 中国人民解放军第二军医大学 | A kind of adeno-associated virus 2 of carrying genes of Pim 1 and its application in crushed optic nerve is repaired |
| CN109868313A (en) * | 2018-06-25 | 2019-06-11 | 山东大学 | Application of the HMGA2 gene in Stein-Leventhal syndrome disease |
| CN114350664A (en) * | 2022-01-11 | 2022-04-15 | 四川省医学科学院·四川省人民医院 | Promoter specifically expressed in retina Muller cell and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Genetic instability and diminished differentiation capacity in long-term cultured mouse neurosphere cells;Vladimir Vukicevic等;《Mech Ageing Dev . 》;第131卷(第2期);124-32 * |
| let-7 microRNA regulates neurogliogenesis in the mammalian retina through Hmga2;Xiaohuan Xia等;《Dev Biol . 》;第410卷(第1期);70-85 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114931652A (en) | 2022-08-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20210260217A1 (en) | GENERATING GABAergic NEURONS IN BRAINS | |
| Chan et al. | Osteopontin expression in acute immune response mediates hippocampal synaptogenesis and adaptive outcome following cortical brain injury | |
| US20230250407A1 (en) | Silencing of dux4 by recombinant gene editing complexes | |
| WO2021031810A1 (en) | Application of ptbp1 inhibitor in preventing and/or treating nervous system disease related to functional neuronal death | |
| Zhou et al. | Targeting RPTPσ with lentiviral shRNA promotes neurites outgrowth of cortical neurons and improves functional recovery in a rat spinal cord contusion model | |
| WO2021032068A1 (en) | Application of ptbp1 inhibitor in preventing and/or treating nervous system disease related to functional neuronal death | |
| Povysheva et al. | Post–spinal cord injury astrocyte-mediated functional recovery in rats after intraspinal injection of the recombinant adenoviral vectors Ad5-VEGF and Ad5-ANG | |
| WO2020176862A1 (en) | Neuroprotection of neuronal soma and axon by modulating er stress/upr molecules | |
| CN114931652B (en) | Application of Hmga2 gene in repairing damaged retina | |
| CN112386699A (en) | Use of Ptbp1 inhibitors for the prevention and/or treatment of neurological disorders associated with functional neuronal death | |
| CA3188895A1 (en) | Method for the treatment of wwox associated diseases | |
| US20220193265A1 (en) | Methods and compositions for reprogramming müller glia | |
| WO2021228050A1 (en) | Method for inducing glial cells transdifferentiation into functional neurons, and application thereof | |
| CN117279668A (en) | Glial cell transdifferentiation into neurons for preventing or treating diseases related to neuronal loss of function or death | |
| WO2019210320A2 (en) | Methods and compositions to stimulate retinal regeneration | |
| Liu et al. | Dual-targeting AAV9P1-mediated neuronal reprogramming in a mouse model of traumatic brain injury | |
| US20230119699A1 (en) | Diagnostic methods using sirt1 expression | |
| US20230121720A1 (en) | Diagnostic methods using pcg-1a expression | |
| CN115838725B (en) | Promoter sequence of specific promoter gene in mammal heart and application thereof | |
| US20230126157A1 (en) | Mirna-485 inhibitor for gene upregulation | |
| WO2023051802A1 (en) | Direct transdifferentiation for treatment of neurological disease | |
| JP2023543361A (en) | NEUROD1 and DLX2 vectors | |
| JP2024506869A (en) | Use of MIRNA-485 inhibitors to treat diseases or disorders associated with abnormalities in NLRP3 expression | |
| KR20230123926A (en) | DLX2 vectors | |
| CN116761812A (en) | NEUROD1 and DLX2 vectors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |