CN112795595A - Gene therapy system for hereditary transthyretin amyloidosis - Google Patents
Gene therapy system for hereditary transthyretin amyloidosis Download PDFInfo
- Publication number
- CN112795595A CN112795595A CN202011556490.5A CN202011556490A CN112795595A CN 112795595 A CN112795595 A CN 112795595A CN 202011556490 A CN202011556490 A CN 202011556490A CN 112795595 A CN112795595 A CN 112795595A
- Authority
- CN
- China
- Prior art keywords
- gene therapy
- grna
- cas9
- aav
- therapy system
- 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.)
- Pending
Links
Images
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
- 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
- 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
- A61K48/005—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 'active' part of the composition delivered, i.e. the nucleic acid delivered
- A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- 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
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- 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
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The invention provides a gene therapy system for hereditary transthyretin amyloidosis, which utilizes a CRISPR/Cas9 system to treat hATTR, selects Nme2Cas9 applicable to The Transthyretin (TTR) gene therapy of the disease to be applied to the system, optimizes and designs high-efficiency gRNA simultaneously, realizes the knockout of functional genes, shows strong capacity of inhibiting the expression of mutant TTR, and shows the application prospect of the system in the aspect of treating the hereditary transthyretin amyloidosis.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a gene therapy system for hereditary transthyretin starch-like degeneration diseases.
Background
Hereditary transthyretin amyloidosis (hATTR) is a rare type of advanced autosomal dominant hereditary disease caused by mutations in The Transthyretin (TTR) gene. Normally, most of the TTR proteins in vivo are synthesized by liver cells and transport thyroxine and vitamin a in a stable tetrameric form. To date, about 120 more mutations occurring in exons 2, 3, and 4 have been reported to be associated with hATTR, with c.148g > a, the protein level p.v30m point mutation, being the most common type of causative mutation in the clinic. The TTR gene generates mutant TTR protein monomer after mutation, the TTR protein tetramer containing the mutant monomer has unstable structure and is easily depolymerized into TTR monomer, and the depolymerized monomer can be further misfolded. The misfolded monomers aggregate to form amyloid fibrin, which is deposited in the intercellular space to cause dysfunction of tissues and organs, thereby causing disease.
The liver is the most major organ for generating TTR, and clinical practice proves that liver transplantation can obviously reduce the generation of mutant TTR, further reduce the concentration of TTR in peripheral blood and obviously improve the prognosis of patients. Liver transplantation remains the only treatment that can radically cure hATTR to date. However, liver transplantation has obvious limitations such as scarcity of healthy donors, large trauma of transplantation operation, and the need of long-term administration of immunosuppressive agents after operation.
Patent US10060921B2 discloses that the gene therapy drug Patisiran, which is a siRNA-like drug, can effectively alleviate the condition of hATTR, and acts to reduce TTR protein expression by indiscriminate targeted degradation of wild-type and mutant TTR mRNA in hepatocytes. In addition, Inotersen was also shown to be effective in alleviating the hATTR condition in clinical trials, and two drugs were approved by the U.S. FDA for the treatment of hATTR in months 8 and 10 of 2018, respectively. Inotersen is a complex of antisense oligonucleotide molecules and proteins, and similarly reduces TTR protein levels by targeted binding, degradation of wild-type and mutant TTR mrnas. However, because both Patisiran and Inotersen act by degrading TTR mRNA, neither can fundamentally cure the disease, and patients need to receive medication for life, but both are expensive and face a serious economic burden and life threat to hATTR patients.
At the core of the CRISPR/Cas9 system is an RNA-protein complex consisting of a gRNA that binds complementarily to a genomic target DNA sequence and a Cas9 nuclease with DNA cleavage activity. In the system, the gRNA guides the Cas9 to specifically recognize a target DNA sequence, a Cas9 endonuclease is combined and cuts double-strand DNA, the cut double-strand DNA is broken, a cell repairs the double-strand DNA through two mechanisms of homologous recombination and non-homologous end connection, and the insertion or deletion of a DNA fragment at a target site of the DNA can cause frame shift mutation, so that the target gene is lost, and the functional gene is knocked out. The repairing mechanism is utilized to knock out the pathogenic gene with high efficiency, and the aim of treating the acquired functional hereditary diseases is expected to be achieved.
However, the application of the CRISPR/Cas9 system has more complex influences depending on different application objects and target genes, and no report on progressive autosomal dominant hereditary diseases caused by TTR gene mutation by using the CRISPR/Cas9 system is available at present.
Disclosure of Invention
The invention aims to solve the technical problem that no medicine or method for radically treating hereditary transthyretin amyloidosis exists in the prior art, and aims to provide a gene therapy system for hereditary transthyretin amyloidosis, wherein the system treats hATTR by using a CRISPR/Cas9 system for the first time, creatively selects Nme2Cas9 suitable for human transthyretin (TTR) gene therapy of the disease to be applied to the system, and simultaneously optimally designs high-efficiency gRNA. In the gene therapy system, a gRNA sequence can guide the Nme2Cas9 to specifically recognize a target DNA sequence, an endonuclease Nme2Cas9 is combined and cuts double-strand DNA, the cut double-strand DNA is broken, cells are repaired by homologous recombination and non-homologous end connection, and the latter can generate insertion or deletion of a DNA fragment at a DNA target site to cause frame shift mutation, so that the function of the target gene is lost, the functional gene is knocked out, the strong capacity of inhibiting the expression of mutant TTR is displayed, and the gene therapy system has an application prospect of fundamentally curing hereditary transthyretin amyloidosis.
Accordingly, it is an object of the present invention to provide a gene therapy system for hereditary transthyretin amyloidosis.
Another object of the present invention is to provide a method for constructing a gene therapy system for hereditary transthyretin amyloidosis.
The above purpose of the invention is realized by the following technical scheme:
the invention provides a gene therapy system for hereditary transthyretin amyloidosis, in particular to a CRISPR/Cas9 system, wherein a delivery vector of the CRISPR/Cas9 system can be AAV, liposome nanoparticles or cationic polymer vectors and the like.
Preferably, the Cas9 is Nme2Cas 9.
Preferably, the gene therapy system further comprises a gRNA capable of targeted cleavage of the TTR gene.
More preferably, the gene therapy system further comprises a gRNA capable of targeted cleavage of the first exon of the TTR gene.
Further preferably, the gRNA sequence is 5'-gcagaggaggagcagacgatgagaagcc-3' or 5'-ggccagcctcagacacaaataccagtcc-3'.
The gene therapy system adopts an AAV-mediated CRISPR/Cas9 system, and innovatively selects Nme2Cas9 suitable for transthyretin (TTR) gene therapy of the disease to be applied to the system. In the gene therapy system, a gRNA sequence can guide the Nme2Cas9 to specifically recognize a target DNA sequence, an endonuclease Nme2Cas9 is combined and cuts double-stranded DNA, the cut double-stranded DNA is broken, a cell is repaired by homologous recombination and non-homologous end connection, and the insertion or deletion of a DNA fragment can be generated at a DNA target site to cause frame shift mutation, so that the function of the target gene is lost, the functional gene is knocked out, and the strong capacity of inhibiting the expression of mutant TTR is displayed.
Preferably, AAV is packaged with packaging plasmid pAAV2/8 to obtain AAV 8.
AAV has no self-replication ability, can infect non-dividing cells, has high safety and effectiveness, while AAV of different serotypes has relatively specific tropism to different tissues and organs in vivo, and AAV8 is homophilic to human and mouse liver and shows certain liver targeting specificity, and can be used as an ideal vector of the gene therapy system.
Meanwhile, the invention also provides a construction method of the gene therapy system for the hereditary transthyretin amyloidosis disease, which comprises the following steps:
s1, connecting a gRNA fragment to a Cas9_ AAV vector to construct a plasmid of Cas9_ AAV-gRNA:
s2, co-transfecting a target plasmid Cas9_ AAV-gRNA, a helper plasmid and a packaging plasmid into HEK293T cells to produce a gene therapy system for simultaneously expressing Cas9 and gRNA.
Among them, preferably, the Cas9_ AAV plasmid is cleaved using SapI endonuclease in step S1, and the gRNA fragment is ligated to Cas9_ AAV vector using T4 ligase to construct a plasmid of Cas9_ AAV-gRNA.
Preferably, the helper plasmid used in step S2 is pHelper and the packaging plasmid is pAAV 2/8.
Preferably, in step S2, AAV which simultaneously expresses Cas9 and gRNA is obtained by separation and purification 48 to 120 hours after co-transfection into HEK293T cells.
Specifically, when the Cas9 is Nme2Cas9, the construction method of the gene therapy system for hereditary transthyretin amyloidosis is as follows:
s1, connecting a gRNA fragment to an Nme2Cas9_ AAV vector to construct a plasmid of an Nme2Cas9_ AAV-gRNA:
s2, co-transfecting a target plasmid Nme2Cas9_ AAV-gRNA, a helper plasmid pHelper and a packaging plasmid pAAV2/8 into HEK293T cells to produce the gene therapy system for simultaneously expressing the Nme2Cas9 and the gRNA.
More specifically, as an alternative, the method of step S2 is: co-transfecting Nme2Cas9_ AAV-gRNA, helper plasmid pHelper and packaging plasmid pAAV2/8 into HEK293T cells by using polyethyleneimine to produce AAV8, collecting cells after 96 hours, and separating the cells by using ultrasonic disruption to obtain an AAV8 crude product; the AAV8 crude product is subjected to iodixanol density gradient centrifugation and ultrafiltration concentration to obtain purified and concentrated AAV8, namely the gene therapy system: AAV 8-mediated CRISPR/Nme2Cas9-gRNA system.
Preferably, the mass ratio of the polyethyleneimine, the Nme2Cas9_ AAV-gRNA, the helper plasmid and the packaging plasmid is 120 μ g: 15 μ g: 15 μ g: 10 μ g.
The invention has the following beneficial effects:
the invention treats hereditary transthyretin amyloidosis by using CRISPR/Cas9 system for the first time, and creatively selects Nme2Cas9 suitable for TTR gene therapy of the disease to be applied to the system.
In addition, the invention also designs two gRNAs capable of efficiently targeting and cutting the first exon of the TTR gene, and the gRNA sequence cannot be directly obtained by the means of the prior art, but is the sequence which can efficiently cut the target site and is determined by the applicant after a plurality of attempts.
The AAV 8-mediated CRISPR/Nme2Cas9-gRNA system provided by the invention has a good application prospect in the aspect of treating hATTR.
Drawings
FIG. 1A is a map of a pCCL-HCR-hAAT-hTTR (c.148G > A) -P2A-GFP lentiviral vector; FIG. 1B shows the results of reporter gene GFP expression; FIG. 1C shows the results of the immunoblot experiment.
FIG. 2A shows the results of PCR verification of F0 mice; FIG. 2B shows the results of reporter gene GFP expression in F0 mouse; FIG. 2C shows the results of reporter GFP expression in F1 mouse; FIG. 2D is the BSP methylation sequencing result; fig. 2E is a liver slice.
Fig. 3A is a Nme2Cas9_ AAV plasmid map; fig. 3B is gRNA targeting position and sequence; FIG. 3C shows the results of the T7E1 experiment.
FIG. 4A shows the results of the T7E1 experiment; FIG. 4B shows the real-time fluorescent quantitative PCR results; FIG. 4C shows the results of immunoblotting experiments; FIG. 4D is a statistical chart of immunoblot results; fig. 4E is a liver slice.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 Generation of humanized mutant TTR transgenic mice Using lentiviruses
(1) Construction of lentiviral plasmids: taking a pCCL lentivirus framework as a base, cloning a coding sequence of a specific promoter ApoE-HCR-hAAT of the hepatocyte and a human TTR protein containing point mutation c.148G > A into the pCCL framework; meanwhile, the P2A short peptide is linked with a GFP coding sequence behind the hTTR (c.148G > A) sequence to achieve the purpose of characterizing the expression of hTTR (V30M) by using GFP as a reporter gene, and a pCCL-HCR-hAAT-hTTR (c.148G > A) -P2A-GFP lentiviral vector is constructed (see FIG. 1A). After 3 days of infecting the mouse hepatoma cell line Hepa1-6 cells cultured in vitro with the plasmid-packaged lentivirus, the reporter gene GFP expression can be observed under a fluorescence microscope (figure 1B), and meanwhile, the hTTR (V30M) mutant protein can be detected by an immunoblotting experiment (figure 1C), thereby confirming that the lentivirus packaged by the plasmid can normally express the target protein in the hepatocytes.
(2) Constructing a transgenic mouse: a lentivirus expressing hTTR (c.148G > A) -P2A-GFP is delivered into a fertilized egg of a CD1 mouse by microinjection to infect the fertilized egg, and the fertilized egg after injection is transplanted into the uterus of a surrogate mouse to prepare a transgenic mouse. After the generation of pregnant mice, the transfer gene F0 generation of initial mice was obtained by PCR verification, and the liver GFP reporter gene was found to be in a chimeric expression mode (FIG. 2A, B). Further propagation resulted in F1 generation mice, and most of the F1 generation transgenic mice were found to be unable to express the gene of interest due to methylation silencing of its transgenic promoter and hTTR (c.148G > A) DNA fragments (FIG. 2C, D). Further propagation yielded F2 generation mice with relatively consistent genetic background, which successfully activated lentiviral-mediated transgene expression by subcutaneous injections of 25. mu.g and 50. mu.g of 5-azacytidine administered at postnatal days 2 and 9, respectively (FIG. 2E).
Example 2 screening to obtain gRNA capable of efficiently targeting and cutting first exon of hTTR gene
Plasmid Nme2Cas9_ AAV (#119924, fig. 3A) for expression of Nme2Cas9 and gRNA was purchased from adddge corporation. In order to effectively knock out hTTR (c.148G > A), 4 gRNAs targeting the Nme2Cas9 of the hTTR gene exon 1 are selected through a large amount of optimization design and screening (FIG. 3B), and further four gRNAs are subjected to experimental verification.
The Nme2Cas9_ AAV plasmid was cleaved using SapI endonuclease, the gRNA sequence was cloned into the Nme2Cas9_ AAV plasmid vector with T4 ligase, and the plasmid was introduced into HEK293T cells cultured in vitro by transient transfection. 2 days after transfection, genomic DNA was extracted and the target site sequence was amplified by PCR, and the cleavage efficiency of 4 gRNAs was verified by performing T7E1 experiment using the PCR product. As a result, it was found that two grnas, G3 and G4, could efficiently cleave the target site (fig. 3C).
The two gRNA sequences are respectively:
G3:5’-gcagaggaggagcagacgatgagaagcc-3’；
G4:5’-ggccagcctcagacacaaataccagtcc-3’。
example 3AAV 8-mediated application study of CRISPR/Nme2Cas9-gRNA System
Presented below is AAV 8-mediated CRISPR/Nme2Cas9-G3 highly effective in inhibiting hTTR mutein expression in mice:
the plasmid expressing the Nme2Cas9_ AAV-G3 obtained in example 2 was used to obtain AAV8 expressing both the Nme2Cas9 and the G3 gRNA. The preparation method comprises the following steps:
mu.g of Nme2Cas9_ AAV-G3, 15. mu.g of helper plasmid pHelper, and 10. mu.g of packaging plasmid pAAV2/8 were co-transfected with 120. mu.g of polyethyleneimine into HEK293T cells cultured in 15cm dishes to produce AAV8, which were harvested 96 hours later and isolated by sonication of the cells to give crude AAV 8. The AAV8 crude product is subjected to iodixanol density gradient centrifugation and ultrafiltration concentration to obtain purified and concentrated AAV 8. AAV8 was further titered using real-time fluorescent quantitative PCR.
The AAV 8-mediated Nme2Cas9-G3 is verified to efficiently inhibit the expression of the hTTR mutant protein in a mouse, and the specific implementation steps are as follows:
2 x 10 to12AAV8 from GC was diluted to 200 μ l with physiological saline, and AAV8 was delivered by tail vein injection into 4-week-old hTTR (V30M) -expressing F2-generation transgenic mice obtained in example 1.
Mice were sacrificed 12 weeks after injection and liver tissue was taken for analysis. Genomic DNA of liver tissue was extracted and the target site sequence was amplified by PCR, and T7E1 experiment was performed with the PCR product to verify that the target site was efficiently cut in the genomic of liver tissue (fig. 4A).
Liver tissue RNA was extracted for mRNA expression analysis, and real-time fluorescent quantitative PCR experiments showed that hTTR mRNA expression was reduced by 65% in mice injected with AAV8 (i.e., Nme2Cas 9-AAV 8-G3) compared with controls injected with Nme2Cas9 and non-targeted gRNA (FIG. 4B).
Liver tissue proteins were extracted and it was found by immunoblotting that the reporter GFP protein expression used to characterize hTTR (V30M) protein expression was reduced by 71% in the group of mice injected with Nme2Cas9_ AAV8-G3 compared to the control group (fig. 4C, D).
Upon observation of the tissue sections, a significant reduction in the proportion of GFP positive cells in the mouse liver was observed (fig. 4E).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A gene therapy system for hereditary transthyretin amyloidosis, characterized in that the gene therapy system is a CRISPR/Cas9 system, and the delivery vector of the CRISPR/Cas9 system is AAV, liposome nanoparticles or cationic polymer vector.
2. A gene therapy system according to claim 1, wherein the Cas9 is Nme2Cas 9.
3. A gene therapy system according to claim 1 or 2, characterized in that the gene therapy system further comprises a gRNA capable of targeted cleavage of the TTR gene.
4. A gene therapy system according to claim 3, characterized in that the gene therapy system further comprises a gRNA capable of targeted cleavage of the first exon of the TTR gene.
5. A gene therapy system according to claim 4, wherein the gRNA sequence is 5'-gcagaggaggagcagacgatgagaagcc-3' or 5'-ggccagcctcagacacaaataccagtcc-3'.
6. The gene therapy system according to claim 1, wherein AAV is packaged with packaging plasmid pAAV 2/8.
7. A method for constructing a gene therapy system according to any one of claims 1 to 6, which comprises the steps of:
s1, connecting a gRNA fragment to a Cas9_ AAV vector to construct a plasmid of Cas9_ AAV-gRNA:
s2, co-transfecting a target plasmid Cas9_ AAV-gRNA, a helper plasmid and a packaging plasmid into HEK293T cells to produce a gene therapy system for simultaneously expressing Cas9 and gRNA.
8. The method of construction of claim 7 wherein the Cas9_ AAV plasmid is cleaved using SapI endonuclease in step S1, and the gRNA fragment is ligated to Cas9_ AAV vector using T4 ligase to construct a plasmid of Cas9_ AAV-gRNA.
9. The method according to claim 7 or 8, wherein the helper plasmid used in step S2 is pHelper and the packaging plasmid is pAAV 2/8.
10. The construction method according to claim 7, wherein in step S2, AAV simultaneously expressing Cas9 and gRNA is obtained by separation and purification 48-120 hours after co-transfection into HEK293T cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011556490.5A CN112795595A (en) | 2020-12-24 | 2020-12-24 | Gene therapy system for hereditary transthyretin amyloidosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011556490.5A CN112795595A (en) | 2020-12-24 | 2020-12-24 | Gene therapy system for hereditary transthyretin amyloidosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112795595A true CN112795595A (en) | 2021-05-14 |
Family
ID=75804700
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011556490.5A Pending CN112795595A (en) | 2020-12-24 | 2020-12-24 | Gene therapy system for hereditary transthyretin amyloidosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112795595A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115125316A (en) * | 2022-08-19 | 2022-09-30 | 中国医学科学院北京协和医院 | Application of intestinal flora in diagnosing transthyretin amyloidosis |
| WO2024003805A1 (en) * | 2022-06-30 | 2024-01-04 | Geneditbio Limited | Methods and compositions for ttr gene editing and therapy using crispr system |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018007871A1 (en) * | 2016-07-08 | 2018-01-11 | Crispr Therapeutics Ag | Materials and methods for treatment of transthyretin amyloidosis |
| US20180110877A1 (en) * | 2015-04-27 | 2018-04-26 | The Trustees Of The University Of Pennsylvania | DUAL AAV VECTOR SYSTEM FOR CRISPR/Cas9 MEDIATED CORRECTION OF HUMAN DISEASE |
| US20190098879A1 (en) * | 2017-09-29 | 2019-04-04 | Regeneron Pharmaceuticals, Inc. | Non-Human Animals Comprising A Humanized TTR Locus And Methods Of Use |
| CN111417728A (en) * | 2017-09-29 | 2020-07-14 | 因特利亚治疗公司 | Compositions and methods for TTR gene editing and treatment of ATTR amyloidosis |
| WO2020240876A1 (en) * | 2019-05-27 | 2020-12-03 | 株式会社トランスジェニック | Exon-humanized mouse |
-
2020
- 2020-12-24 CN CN202011556490.5A patent/CN112795595A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180110877A1 (en) * | 2015-04-27 | 2018-04-26 | The Trustees Of The University Of Pennsylvania | DUAL AAV VECTOR SYSTEM FOR CRISPR/Cas9 MEDIATED CORRECTION OF HUMAN DISEASE |
| WO2018007871A1 (en) * | 2016-07-08 | 2018-01-11 | Crispr Therapeutics Ag | Materials and methods for treatment of transthyretin amyloidosis |
| US20190098879A1 (en) * | 2017-09-29 | 2019-04-04 | Regeneron Pharmaceuticals, Inc. | Non-Human Animals Comprising A Humanized TTR Locus And Methods Of Use |
| CN111163633A (en) * | 2017-09-29 | 2020-05-15 | 瑞泽恩制药公司 | Non-human animals comprising humanized TTR loci and methods of use thereof |
| CN111417728A (en) * | 2017-09-29 | 2020-07-14 | 因特利亚治疗公司 | Compositions and methods for TTR gene editing and treatment of ATTR amyloidosis |
| WO2020240876A1 (en) * | 2019-05-27 | 2020-12-03 | 株式会社トランスジェニック | Exon-humanized mouse |
Non-Patent Citations (4)
| Title |
|---|
| LI B 等: "Strategies for the CRISPR-based therapeutics", 《TRENDS IN PHARMACOLOGICAL SCIENCES》 * |
| TITZE-DE-ALMEIDA SS 等: "Leading RNA Interference Therapeutics Part 1: Silencing Hereditary Transthyretin Amyloidosis, with a Focus on Patisiran", 《MOL DIAGN THER》 * |
| 余怡文 等: "野生型转甲状腺素蛋白淀粉样变的研究进展", 《中华心血管病杂志》 * |
| 王凤君 等: "转甲状腺素蛋白与神经系统疾病的关系研究进展", 《中国实用神经疾病杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024003805A1 (en) * | 2022-06-30 | 2024-01-04 | Geneditbio Limited | Methods and compositions for ttr gene editing and therapy using crispr system |
| CN115125316A (en) * | 2022-08-19 | 2022-09-30 | 中国医学科学院北京协和医院 | Application of intestinal flora in diagnosing transthyretin amyloidosis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7004328B2 (en) | High transduction efficiency rAAV vector, composition, and method of use | |
| US11963982B2 (en) | CRISPR/RNA-guided nuclease systems and methods | |
| US20200157515A1 (en) | Systems and methods for the treatment of hemoglobinopathies | |
| JP2021536244A (en) | Editing of RNA and DNA bases via recruitment of genetically engineered ADAR | |
| JP2019519221A (en) | Gene therapy for age-related diseases and conditions | |
| US20220073951A1 (en) | Systems and methods for the treatment of hemoglobinopathies | |
| CN102282259A (en) | Use of SIRNA to achieve down regulation of an endogenous gene in combination with the use of a sense construct to achieve expression of a desired polynucleotide | |
| AU2023237106A1 (en) | Aav vectors for treatment of dominant retinitis pigmentosa | |
| EP1824519A2 (en) | Inhibition of li expression in mammalian cells | |
| CN112795595A (en) | Gene therapy system for hereditary transthyretin amyloidosis | |
| CN111225682A (en) | Use of syncytin for targeted delivery of drugs and genes to lung tissue | |
| WO2019010091A1 (en) | Methods and compositions for facilitating homologous recombination | |
| Chemello et al. | CRISPR-editing therapy for Duchenne Muscular Dystrophy | |
| US20210230638A1 (en) | Systems and methods for the treatment of hemoglobinopathies | |
| US20220047637A1 (en) | Systems and methods for the treatment of hemoglobinopathies | |
| US20200263206A1 (en) | Targeted integration systems and methods for the treatment of hemoglobinopathies | |
| US20230044220A1 (en) | Treatment of chronic pain | |
| CN112813063A (en) | Construction of animal model for lipid metabolism disorder and repair by AAV-CRISPR/CAS9 | |
| WO2023131345A1 (en) | Gene treatment drug and method for x-linked adrenoleukodystrophy | |
| US20090060889A1 (en) | Ii-RNAi involved Ii suppression in cancer immunotherapy | |
| EP3851532A1 (en) | Gene editing for the treatment of epidermolysis bullosa | |
| WO2024078345A1 (en) | Snrna nucleic acid molecule and application thereof | |
| CN115998867A (en) | Application of TBL1XR1 in treatment of liver diseases and liver regeneration | |
| EP4263818A1 (en) | Therapeutic lama2 payload for treatment of congenital muscular dystrophy | |
| AU2022379633A1 (en) | Compositions and methods for expressing factor ix for hemophilia b therapy |
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 |