WO2020258078A1 - Procédé d'édition dirigée par l'arn pour inhiber la néovascularisation choroïdienne, et réactif - Google Patents

Procédé d'édition dirigée par l'arn pour inhiber la néovascularisation choroïdienne, et réactif Download PDF

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WO2020258078A1
WO2020258078A1 PCT/CN2019/093030 CN2019093030W WO2020258078A1 WO 2020258078 A1 WO2020258078 A1 WO 2020258078A1 CN 2019093030 W CN2019093030 W CN 2019093030W WO 2020258078 A1 WO2020258078 A1 WO 2020258078A1
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sequence
casrx
grna
growth factor
vascular endothelial
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PCT/CN2019/093030
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Chinese (zh)
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杨辉
周昌阳
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中国科学院脑科学与智能技术卓越创新中心
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    • C—CHEMISTRY; METALLURGY
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    • 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

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  • the present invention belongs to the technical field of genetic modification. More specifically, the present invention relates to methods and reagents for inhibiting choroidal neovascularization based on RNA-directed editing technology.
  • Age-related macular degeneration characterized by the development of choroidal neovascularization (CNV) is the main cause of vision deterioration in adults over 50 years of age. In its clinical manifestations, visual acuity can be normal or severely decreased. The main complaint is often dyslexia and the need to increase light intensity to help reading; the fundus manifests as drusen, focal hyperpigmentation, patchy depigmentation foci, geographic atrophy, etc. .
  • vascular endothelial growth factor A vascular endothelial growth factor A (vascular endothelial growth factor A, VEGFA) plays a key role in it.
  • Anti-VEGFA therapy using humanized antibodies has been used clinically to treat AMD, and the therapeutic effect is maintained by regular injection of antibodies.
  • the defect of this type of antibody has also been found clinically, that is, the antibody is prone to degradation in the body, resulting in a short effective time and requires relatively frequent administration, for example, it takes 1 to several months depending on the condition. Frequent administration on the one hand increases the cost of medical treatment, on the other hand it also reduces the compliance of patients, which is called the bottleneck restricting the widespread use of such drugs.
  • VEGFA VEGFA
  • choroidal neovascularization occurs in the organ of the eye, it is extremely prone to side effects or uncertain risks, and drug research is more difficult.
  • the purpose of the present invention is to provide an RNA targeting tool based on the CRISPR system that can efficiently and specifically target the VEGFA gene, achieve the inhibition of choroidal neovascularization, and achieve the purpose of preventing and treating macular degeneration.
  • a method for targeted inhibition of vascular endothelial growth factor A in cells includes: using adeno-associated virus to deliver CasRx and gRNA directed against vascular endothelial growth factor A into cells, thereby targeted Inhibit intracellular vascular endothelial growth factor A.
  • both ends of the CasRx encoding gene also include a nuclear localization signal sequence.
  • the CasRx expression cassette uses EFS, CMV, CAG, CBH or EF1a as a promoter to drive the expression of CasRx; preferably, EFS is used as a promoter.
  • the CasRx expression cassette includes the following operatively linked sequence elements: promoter sequence, nuclear localization signal 1 sequence, CasRx encoding nucleic acid sequence, nuclear localization signal 2 sequence; preferably, in the promoter
  • the 5'end of the sequence also includes a 5'end inverted repeat sequence; preferably, the 3'end of the nuclear localization signal 2 sequence also includes a PolyA sequence.
  • the gRNA expression cassette for vascular endothelial growth factor A uses U6 as a promoter to drive the expression of gRNA.
  • the gRNA expression cassette for vascular endothelial growth factor A includes the following operatively linked sequence elements: U6 promoter, gRNA for vascular endothelial growth factor A; preferably, in the The 3'end of the gRNA also includes a 3'end inverted repeat sequence.
  • the CasRx has a polypeptide sequence encoded by the nucleotide sequence 1699-4596 in SEQ ID NO:1 or its degenerate sequence (also includes variants or fragments of the same function) .
  • the gRNA for vascular endothelial growth factor A is targeted to the region of the sequence shown in SEQ ID NO: 9 (AGACCCTGGTGGACATCTTCCAGGAGTACC) or SEQ ID NO: 10 (CACATAGGAGAGATGAGCTTCCTACAGCAC) in the following vascular endothelial growth factor A segment.
  • the coding sequence of CasRx and gRNA for vascular endothelial growth factor A are assembled in an adeno-associated virus vector.
  • the adeno-associated virus vector includes the nucleotide sequence shown in SEQ ID NO:4.
  • the CasRx expression cassette includes the nucleotide sequence shown in SEQ ID NO:1.
  • the gRNA expression cassette includes the nucleotide sequence shown in SEQ ID NO: 2 and/or the nucleotide sequence shown in SEQ ID NO: 2.
  • the method for targeted inhibition of vascular endothelial growth factor A expression in cells is a non-therapeutic method.
  • a recombinant vector expressing CasRx and gRNA directed against vascular endothelial growth factor A is provided.
  • the recombinant vector is an adeno-associated virus vector, including an expression cassette of CasRx and an expression cassette of gRNA of vascular endothelial growth factor A .
  • both ends of the CasRx encoding gene also include a nuclear localization signal sequence; and/or use EFS, CMV, CAG, CBH or EF1a as a promoter to drive the expression of CasRx
  • the expression cassette includes the following operatively linked sequence elements: promoter sequence, nuclear localization signal 1 sequence, CasRx encoding nucleic acid sequence, nuclear localization signal 2 sequence; preferably, 5'of the promoter sequence
  • the end also includes a 5'end inverted repeat sequence; preferably, the 3'end of the nuclear localization signal 2 sequence also includes a PolyA sequence.
  • the expression cassette for vascular endothelial growth factor A U6 is used as a promoter to drive the expression of gRNA; more preferably, the expression cassette includes the following operably linked sequence Element: U6 promoter, gRNA directed against vascular endothelial growth factor A; preferably, at the 3'end of the gRNA, it also includes a 3'end inverted repeat sequence.
  • the CasRx has a polypeptide sequence encoded by the nucleotide sequence 1699-4596 in SEQ ID NO:1 or its degenerate sequence (also includes variants or fragments of the same function) .
  • the gRNA for vascular endothelial growth factor A is targeted to a segment of the sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10 in vascular endothelial growth factor A.
  • the coding sequence of CasRx and the gRNA for vascular endothelial growth factor A are assembled in an adeno-associated virus vector; more preferably, the adeno-associated virus vector includes the nucleoside shown in SEQ ID NO: 4 Acid order.
  • any of the aforementioned recombinant vectors is provided for packaging recombinant viruses, and the recombinant viruses are recombinant adeno-associated viruses.
  • a recombinant virus is provided.
  • the virus is an adeno-associated virus, which is packaged by the recombinant vector.
  • the recombinant virus is used to prepare a reagent for targeted inhibition of intracellular vascular endothelial growth factor A.
  • the recombinant virus is used to prepare a medicine or composition for inhibiting choroidal neovascularization.
  • the recombinant virus is used to prepare a medicine or composition for alleviating or treating macular degeneration (such as age-related macular degeneration).
  • kits or a medicine kit comprising: the recombinant virus or the recombinant vector.
  • AAV-mediated CasRx delivery reduces the area of CNV in a mouse model of AMD.
  • FIG. 1 (a) Schematic diagram of integrated AAV vector and experimental procedure. 21 days before laser burn, AAV-CasRx-Vegfa was injected intravitreally into one eye, and PBS was injected into the other eye as a control. Three weeks after AAV infection, the transcription level of Vegfa mRNA was analyzed without laser burn. The VEGFA protein level was quantified by ELISA 3 days after laser burn. Measure CasRx and Vegfa mRNA levels jm and CNV area 7 days after laser burn.
  • the inventors constructed an adeno-associated virus for the delivery of CasRx and gRNA targeting vascular endothelial growth factor A (Vegfa), and realized the use of CasRx and gRNA targeted inhibition to efficiently and accurately knock down Vegfa mRNA and inhibit the cause
  • the virus obtained by the invention can continuously and effectively realize the down-regulation of Vegfa mRNA, has a long action time and good stability.
  • the "element” refers to a series of functional nucleic acid sequences useful for protein expression.
  • the “element” is systematically constructed to form an expression construct.
  • the sequence of the "element” may be those provided in the present invention, and also include their variants, as long as these variants basically retain the function of the "element” by inserting or deleting some bases (such as 1-50bp; preferably 1-30bp, more preferably 1-20bp, more preferably 1-10bp), or by random or site-directed mutagenesis.
  • operably linked refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences.
  • the promoter region is placed at a specific position relative to the nucleic acid sequence of the target gene, so that the transcription of the nucleic acid sequence is guided by the promoter region, so that the promoter region is "operably linked” to the nucleic acid sequence.
  • the "expression cassette” refers to a gene expression system that contains all the necessary elements required to express a target gene, and usually includes the following elements: promoter, target gene sequence, terminator; in addition, it can also optionally Including signal peptide coding sequence and so on. These elements are operatively connected.
  • the "construct (or construct)” refers to a single-stranded or double-stranded DNA molecule that has been made through human intervention to contain DNA fragments assembled and arranged according to sequences that do not exist in nature .
  • the "construct” includes an expression vector; or, the “construct” is included in an expression vector as a part of the expression vector.
  • the "gRNA target” refers to a target region suitable for gene editing operations in the Vegfa mRNA of interest in the present invention.
  • the inventor is committed to the study of inhibiting choroidal neovascularization.
  • the permanent Vegfa gene destruction was induced through editing such as spCas9; however, because this editing is aimed at the DNA level
  • the risks associated with permanent DNA modification cannot be avoided, including unwanted off-target and targeting effects; and the inventors also found that Cas9 is difficult to package into AAV viruses, and the editing efficiency is not ideal.
  • the inventors improved the previous plan, using AAV to deliver CasRx and Vegfa gRNA, which can efficiently and accurately knock down Vegfa mRNA and inhibit the formation and development of pathogenic choroidal neovascularization (CNV).
  • One of the differences between the technical solution of the present invention and other gene editing technical solutions is that the present invention targets Vegfa mRNA to suppress, rather than target DNA or protein, which avoids the risk of permanent DNA modification.
  • Adeno-associated virus is a virus that cannot replicate itself and has low immunogenicity.
  • AAV viral vectors is limited, which limits its use. In this field, there are relatively few examples of successful transfection with AAV virus.
  • AAV vector is a vector that can be artificially transgenic, which is produced after genetic engineering using certain characteristics of naturally occurring adeno-associated virus. In the preferred mode of the present invention, the AAV vector is optimized.
  • a gRNA target suitable for targeted operations is provided, which can achieve high-efficiency and precise targeted inhibition of Vegfa without off-target effects and other adverse side effects.
  • an expression cassette for expressing CasRx which includes the following operatively linked sequence elements: a promoter, a nuclear localization signal 1 sequence, a CasRx encoding nucleic acid sequence, a nuclear localization signal 2 sequence;
  • the 5'end of the subsequence also includes a 5'end inverted repeat sequence; preferably, the 3'end of the nuclear localization signal 2 sequence also includes a PolyA sequence.
  • the promoter can be CMV, CAG, CBH, EF1a, EFS, etc. In a more preferred manner, the promoter is an EFS promoter.
  • the inventors found that applying it to the present invention not only has a short sequence but also has ideal gene expression activity, and can also selectively drive CasRx expression.
  • an expression cassette for expressing gRNA targeting Vegfa including the following operatively linked sequence elements: a promoter, a gRNA targeting vascular endothelial growth factor A; preferably, in the 3 of the gRNA 'End, also includes 3'end inverted repeat sequence.
  • the promoter is U6 promoter.
  • the simplified SV40PolyA is connected after the expression frame, which can ensure that the target packaging system is reduced to a minimum, effectively reduce the difficulty of virus packaging, and improve the packaging efficiency of AAV.
  • guide-1 and guide-2 in series can effectively improve editing efficiency.
  • the expression cassette for expressing CasRx and the expression cassette for expressing gRNA for Vegfa are placed in the same AAV expression vector for virus packaging.
  • the inventor's optimized design overcomes the problem of low AAV packaging capacity, and successfully integrates the two groups of expression cassettes into one expression vector, which helps simplify the subsequent operation process, and the drug delivery scheme is simple and easy to operate.
  • variants of the aforementioned elements that have been appropriately changed and still retain their original functions are also included in the present invention.
  • a sequence variant that hybridizes with the sequence defined in the present invention under stringent conditions and has the same function.
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the homology between the two sequences is at least 70%, More preferably 75% or more, 80% or more, 85% or more or 90% or more, and more preferably 95% or more before hybridization occurs.
  • the sequence may also be the complement of these defined sequences.
  • the full-length nucleotide sequence of the gene pointed to by each element of the present invention or its fragments can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences.
  • the upstream and downstream positions of the aforementioned elements in the vector may also include restriction enzyme cleavage sites, which facilitates the organic connection of the elements.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells.
  • the vector containing the above-mentioned appropriate polynucleotide sequence and appropriate promoter or control sequence can be used for virus packaging.
  • the packaged AAV virus expressing CasRx and gRNA is injected into the vitreous of an animal model eye, successfully reducing the area of CNV in the animal eye, and the effect is particularly significant.
  • the present invention also provides a composition (such as a pharmaceutical composition), which contains an effective amount (such as 0.000001-50wt%; preferably 0.00001-20wt%; more preferably, 0.0001-10wt%)
  • a composition such as a pharmaceutical composition
  • an effective amount such as 0.000001-50wt%; preferably 0.00001-20wt%; more preferably, 0.0001-10wt%
  • the adeno-associated virus obtained by packaging of the present invention and a pharmaceutically acceptable carrier such as 0.000001-50wt%; preferably 0.00001-20wt%; more preferably, 0.0001-10wt
  • the term “effective amount” or “effective dose” refers to those that can produce function or activity on humans and/or animals and can be accepted by humans and/or animals as used herein.
  • pharmaceutically acceptable ingredients are substances that are suitable for humans and/or mammals without excessive adverse reactions (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • the adenovirus can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, and preferably, the pH is about 6-8.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the dosage of the active ingredient (adenovirus) is a therapeutically effective amount, which is within the skill range of a skilled physician.
  • the adeno-associated virus can be administered systemically or locally.
  • topical administration is preferred, especially intravitreal injection.
  • the present invention also provides a kit/kit containing the expression cassette for expressing CasRx and the expression cassette for expressing gRNA targeting Vegfa, or a virus packaged by the vector.
  • kit/kit for the convenience of those skilled in the art.
  • kit may also include instructions for instructing those skilled in the art to operate.
  • the technical scheme of the present invention demonstrates the feasibility of the RNA-targeted CRISPR system for in vivo gene therapy, and provides effective clinically for choroidal neovascularization and macular degeneration (such as age-related macular degeneration) caused by high expression of VEGFA. , Precise new treatment tools.
  • Choroidal neovascularization is currently treated with monoclonal antibodies or inhibitors, which has a short duration.
  • AAV is used for the first time to carry an efficient and accurate RNA editing tool to target VEGFA, and the therapeutic effect can be achieved for several years.
  • gRNA-1 5’-gtgctgtaggaagctctctctcctatgtg-3’;
  • gRNA-2 5'-ggtactcctggaagatgtccaccagggtct-3'.
  • pcDNA3.1 as the backbone plasmid, adding multiple cloning sites and CAG promoters in it, to obtain the pCAG-EGFP plasmid, and then clone into it in sequence SV40NLS1, CasRx, SV40NLS2, P2A, GFP, WPRE, BGH PolyA elements, thereby The pCAG-SV40NLS1-CasRx-SV40NLS2-P2A-GFP-WPRE-PolyA plasmid was obtained.
  • the sequence was optimized by codons, and gene synthesis was performed by Shanghai Huajin Biological Co., Ltd.
  • SEQ ID NO: 1 to 1648 are the pCAG promoter sequence
  • SV40NLS1 Sequence ID NO: 1669-1689;
  • CasRx the sequence of positions 1699-4596 in SEQ ID NO:1;
  • SV40NLS2 The sequence of positions 4612 to 4632 in SEQ ID NO:1;
  • P2A SEQ ID NO: 4639-4695 sequence
  • GFP SEQ ID NO: 4702 ⁇ 5421 sequence
  • WPRE SEQ ID NO: 5433 ⁇ 6021 sequence
  • PolyA SEQ ID NO:1, the sequence of positions 6042 to 6249.
  • nucleotide sequence of pCAG-SV40NLS1-CasRx-SV40NLS2-P2A-GFP-WPRE-PolyA is shown in SEQ ID NO:1.
  • U6-gRNA plasmid is obtained through gene synthesis, which in turn includes U6, DR1, guide 1, DR2, CMV promoter (pCMV), mCherry, WPRE, and PolyA elements to obtain U6-DR1-guide 1-DR2-pCMV- mCherry-WPRE-PolyA plasmid.
  • the coding sequence of each element is:
  • DR1 SEQ ID NO: 2 257 to 282 sequence
  • DR2 the sequence of the 323th to 359th positions in SEQ ID NO: 2;
  • pCMV the sequence of positions 365 to 872 in SEQ ID NO: 2;
  • mCherry sequence of 906th to 1616th in SEQ ID NO: 2;
  • WPRE the 1617 to 2204th sequence of SEQ ID NO: 2;
  • PolyA SEQ ID NO: 2 the 2246 to 2471 sequence.
  • the nucleotide sequence of U6-DR1-guide1-DR2-pCMV-mCherry-WPRE-PolyA is shown in SEQ ID NO: 2.
  • DR1 and DR2 are used to guide CasRx to target RNA.
  • U6-gRNA plasmid is obtained through gene synthesis, which in turn includes U6, DR1, guide 2, DR2, CMV promoter (pCMV), mCherry, WPRE, and PolyA elements to obtain U6-DR1-guide 2-DR2-pCMV- mCherry-WPRE-PolyA plasmid.
  • the coding sequence of each element is:
  • DR1 SEQ ID NO: 3, 257 to 282;
  • DR2 SEQ ID NO: the 323-359th sequence
  • pCMV SEQ ID NO: the 365 to 872 sequence
  • mCherry the 906th to 1616th sequence in SEQ ID NO: 3;
  • WPRE the sequence of the 1617 to 2204 in SEQ ID NO: 3;
  • PolyA SEQ ID NO: 2246-2471 sequence.
  • the nucleotide sequence of the U6-DR1-guide2-DR2-pCMV-mCherry-WPRE-PolyA plasmid is shown in SEQ ID NO: 3. Among them, DR1 and DR2 are used to guide CasRx to target RNA.
  • AAV plasmid (#60231) purchased from Addgene, ITR, EFS, SV40NLS1, CasRx, HA, SV40NLS2, SV40PolyA, U6, DR1, guide1, DR2, guide2, DR3, ITR elements were sequentially cloned in it to obtain ITR -EFS-SV40NLS1-CasRx-HA-SV40NLS2-SV40PolyA-U6-DR1-guide1-DR2-guide2-DR3-ITR plasmid.
  • the coding sequence of each element is:
  • ITR SEQ ID NO: the sequence from 1 to 130 in 4;
  • EFS SEQ ID NO: No. 143 to 398 sequence
  • SV40NLS1 SEQ ID NO: the 414th to 434th sequence
  • CasRx the sequence of positions 444 to 3341 in SEQ ID NO: 4;
  • HA SEQ ID NO: 3351 ⁇ 3377 sequence
  • SV40NLS2 Sequence ID NO: 3378-3398 in SEQ ID NO: 4;
  • SV40 PolyA SEQ ID NO: 3414 ⁇ 3548 sequence
  • DR1 the sequence of positions 3805 to 3834 in SEQ ID NO: 4;
  • DR2 the sequence of positions 3865 to 3900 in SEQ ID NO: 4;
  • ITR SEQ ID NO: 3998-4138 sequence.
  • nucleotide sequence of ITR-EFS-SV40NLS1-CasRx-HA-SV40NLS2-SV40PolyA-U6-DR1-guide1-DR2-guide2-DR3-ITR plasmid is shown in SEQ ID NO: 4.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • penicillin/streptomycin penicillin/streptomycin
  • the control group was only transfected with 2 ⁇ g/well vector containing CasRx. Three days after transfection, GFP+mCherry+ cells (GFP+ cells in the control group) were isolated using flow cytometry. First, use Trizol (Ambion) to purify total RNA, and then transcribe it into complementary DNA (HiScript QRT SuperMix for qPCR, Vazyme, Biotech). The qPCR reaction is tracked through SYBR green probes (AceQ qPCR SYBR Green Master Mix, Vazyme, Biotech).
  • AAV-CasRx-Vegfa (AAV-PHP.eb capsid) was packaged by transfecting HEK293T cells with polyethyleneimine (PEI) (50 ⁇ g/ml) (Chan,KYet al.Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.Nature neuroscience 20,1172-1179, doi:10.1038/nn.4593(2017)].
  • PEI polyethyleneimine
  • the virus was harvested 3-7 days after three transfections, purified and concentrated. Mice (C57BL/6) aged 6-8 weeks were anesthetized for intravitreal injection.
  • AAV-CasRx-Vegfa (7.5 ⁇ 10 9 viral genomes in 1 ⁇ l) or PBS was injected intravitreally under an Olympus microscope (Olympus, Tokyo, Japan) using a Hamilton syringe with a 34G needle. Exclude mice with retinal hemorrhage.
  • mice Two to three weeks after AAV injection, the mice were used for laser burns to induce CNV models [Gong, Y.et al. Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice.Plos One 10, doi:ARTN e0132643 10.1371/journal.pone.0132643 (2015)]. In short, the mice were anesthetized and the pupils were dilated with dilating eye drops to dilate the pupil size.
  • Use NOVUS Spectra (LUMENIS) for laser photocoagulation.
  • the laser parameters used in the present invention are: 532nm wavelength, 70ms exposure time, 180mW or 240mW power and 50 ⁇ m spot size.
  • IB4 isolectin-B4
  • RPE complexes for ELISA.
  • 30 laser burns were induced in each eye 3 weeks after AAV injection.
  • the eyes were removed 3 days after induction, and the RPE complex was dissociated from the retina and lysed with RPA.
  • Quantikine ELISA kit (MMV00, R&D SYSTEMS) was used to determine the level of VEGFA protein.
  • CasRx targeting sites suitable for the targeted operation of the present invention have been identified.
  • the targeting site is a CasRx targeting site that is conserved in human and mouse Vegfa genes.
  • the inventors separately designed two guide RNAs (gRNAs) targeting these two sites ( Figure 1a) to achieve effective Vegfa mRNA knockdown.
  • Human 293T cells and mouse N2a cells were used to study the effectiveness of the targeted operation of the present invention. Firstly, the mRNA level was investigated, and the pCAG-SV40NLS1-CasRx-SV40NLS2-P2A-GFP-WPRE-PolyA plasmid and U6-gRNA-pCMV-mCherry-PolyA plasmid constructed above were co-transfected into human 293T cells or Mouse N2a cells.
  • Example 2 Adeno-associated virus (AAV)-mediated CasRx delivery reduces the area of CNV
  • AAV-CasRx-Vegfa double gRNA array targeting Vegfa
  • FIG. 2a The schematic diagram of the construction of the AAV-CasRx-Vegfa recombinant plasmid is shown in the left figure in Figure 2a, and the operation flow of injection and detection is shown in the right figure in Figure 2a.
  • PBS injection in the other eye was used as a control Figure 2a.
  • the therapeutic effect of the CasRx method was evaluated by quantifying the CNV area 7 days after laser treatment.
  • the inventors' results showed that compared with the control eyes injected with PBS, Vegfa targeting AAV significantly reduced the CNV area of two different laser irradiation levels.
  • the laser parameter is 180mW power
  • the laser parameter is 240mW power
  • the inventors conducted phased observations on the transfected animal model, and no side effects caused by off-target effects and other visible side effects were observed.
  • CasRx is suitable for containing multiple gRNAs in a single AAV vector for in vivo delivery.
  • CasRx provided by AAV has the potential to continuously correct protein expression for up to 2 years in a single injection. This makes the risk associated with mRNA editing significantly lower than the risk of DNA editing because there are a large number of transcripts, many of which may maintain normal functions.
  • the CasRx knockdown method can complement existing therapeutic strategies such as monoclonal antibodies, antisense oligonucleotides and DNA nuclease editing.

Abstract

L'invention concerne un procédé à base d'édition dirigée par l'ARN pour inhiber la néovascularisation choroïdienne, et un réactif. Par construction d'un virus adéno-associé pour l'administration de CasRx et de gARN ciblant le facteur de croissance endothéliale vasculaire A (Vegfa), CasRx et gARN sont utilisés pour inhiber de manière ciblée l'ARNm de Vegfa et inhiber la formation et le développement de néovascularisations choroïdiennes pathogènes (CNV).
PCT/CN2019/093030 2019-06-26 2019-06-26 Procédé d'édition dirigée par l'arn pour inhiber la néovascularisation choroïdienne, et réactif WO2020258078A1 (fr)

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WO2019051278A1 (fr) * 2017-09-07 2019-03-14 The Board Of Trustees Of The Leland Stanford Junior University Systèmes de nucléases pour génie génétique

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KONERMANN S. ET AL.: "Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors", CELL, vol. 173, no. 3, 15 March 2018 (2018-03-15), XP055529705, ISSN: 0092-8674, DOI: 20200325102043Y *

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