WO2023078099A1 - Agent thérapeutique génétique pour le traitement des lésions nerveuses - Google Patents
Agent thérapeutique génétique pour le traitement des lésions nerveuses Download PDFInfo
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- C12N2310/141—MicroRNAs, miRNAs
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- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/32—Special delivery means, e.g. tissue-specific
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- C—CHEMISTRY; METALLURGY
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- 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
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- C—CHEMISTRY; METALLURGY
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- 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
Definitions
- the invention relates to a gene medicine for treating nerve injury diseases, and belongs to the technical field of gene therapy.
- the optic nerve is formed by the axons sent by the retinal ganglion cells (RGC) converging at the optic disc and then passing through the sclera. , essentially belonging to the central nervous system.
- RRC retinal ganglion cells
- Optic nerve injury also known as traumatic optic neuropathy, is one of the common and serious complications of traumatic brain injury. Car accident injuries, falling injuries and blow injuries are prone to optic nerve damage, especially car accident injuries.
- the direct injury caused by the stabbing of the optic nerve by a sharp instrument and the direct injury of other parts of the optic nerve are relatively rare in clinical practice, and more than 90% of the optic nerve injuries are indirect injuries of the optic canal.
- indirect optic nerve injury refers to the outer side of the orbit, generally refers to the impact on the upper temporal part of the brow arch, and the external force is transmitted to the optic canal through the skull, causing deformation or fracture of the optic canal, resulting in vision and visual field impairment caused by optic nerve damage.
- the current common treatment methods mainly include conservative treatment, hormone therapy, surgical treatment (eg, optic canal decompression, autologous transplantation, etc.), protection and regeneration of the optic nerve.
- Traditional conservative treatment, hormone therapy or surgical treatment cannot achieve good therapeutic effect.
- Due to the low regenerative ability of RGCs it is difficult for RGCs to regenerate after optic nerve injury, which leads to optic atrophy and blindness; therefore, how to repair and regenerate the damaged optic nerve and restore the function of the optic nerve is a difficult problem in clinical research in ophthalmology at home and abroad.
- Adeno-associated virus (adeno associated virus, AAV) belongs to Parvoviridae, and is the most simple type of single-stranded DNA-deficient virus with no envelope and icosahedral structure found so far. It needs helper virus (usually Adenovirus) involved in replication.
- helper virus usually Adenovirus
- the inverted repeat sequence (ITR) at both ends of the adeno-associated virus contains cap and rep genes, wherein the cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration.
- the probability of wild-type AAV infecting human chromosome 19 is about 19%, but the modified recombinant adeno-associated virus (rAAV) lacks the ability of site-specific integration because it cannot synthesize Rep protein, mainly in the form of episomes Existence, the probability of insertional mutation and activation of oncogenes is extremely low; in view of the characteristics of good safety of recombinant adeno-associated virus, wide range of host cells, low immunogenicity, and long time of expressing foreign genes in vivo, it is considered the most promising One of the promising gene transfer vectors is widely used in gene therapy research worldwide.
- rAAV modified recombinant adeno-associated virus
- AAV vectors can be divided into 12 serotypes (AAV-1-AAV-12) and more than 100 variants (such as some derived chimeric AAV vectors).
- AAV-1-AAV-12 serotypes
- AAV2 and AAV9 have been proved to have relatively high transfection efficiency for the nervous system.
- the present invention discloses that the effect of the Porf-2 gene on the treatment/improvement of optic nerve damage diseases is studied by means of RNA interference (RNAi), and thus a method for treating/improving optic nerve damage diseases is provided, the method comprising A molecule capable of specifically inhibiting the transcription or translation of the Porf-2 gene, or the expression or activity of the Porf-2 protein, is administered to the patient's optic nerve tissue, thereby treating/improving the optic nerve injury disease.
- RNAi RNA interference
- the Porf-2 gene exists in other nerve tissues besides the optic nerve tissue; the inventor of the present invention discloses "the role of the Porf-2 gene revealed by RNAi in the treatment/improvement of optic nerve damage diseases" On the basis of ", those skilled in the art can reasonably extend to: the Porf-2 gene can also be applied to the treatment/improvement of other nerve injury diseases.
- the first aspect of the present invention provides the use of Porf-2 gene in treating/improving neurological injury diseases.
- the dosage of the molecule administered to the nervous tissue of the patient is a dosage sufficient to reduce the transcription or translation of Porf-2 gene, or the dosage sufficient to reduce the expression or activity of Porf-2 protein.
- the expression of the Porf-2 gene is at least reduced by 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%.
- the molecule administered to the patient's nervous tissue may be selected from but not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering viruses.
- the nucleic acid includes, but is not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA) or short hairpin RNA (shRNA).
- dsRNA double-stranded RNA
- shRNA short hairpin RNA
- the double-stranded RNA (dsRNA) or short hairpin RNA (shRNA) contains the promoter sequence or coding region sequence information of the Porf-2 gene.
- the double-stranded RNA is small interfering RNA (siRNA).
- the small interfering RNA comprises a first strand and a second strand complementary to each other to form an RNA dimer; the sequence of the first strand is consistent with 15-27 continuous nucleotide sequences in the Porf-2 gene (target sequence) are basically the same.
- the siRNA can specifically bind to the mRNA fragment encoded by the target sequence, and specifically silence the expression of the Porf-2 gene.
- the target sequence in the Porf-2 gene is: when the siRNA specifically silences the expression of the Porf-2 gene, the fragment in the Porf-2 gene corresponding to the mRNA fragment complementary to the siRNA.
- the Porf-2 gene is a human-derived Porf-2 gene.
- the nerve damage disease is an optic nerve damage disease.
- the second aspect of the present invention provides the use of Porf-2 gene or Porf-2 protein in preparing or screening drugs for treating/improving nerve injury diseases.
- the dosage of the molecule administered to the nervous tissue of the patient is a dosage sufficient to reduce the transcription or translation of Porf-2 gene, or the dosage sufficient to reduce the expression or activity of Porf-2 protein.
- the expression of the Porf-2 gene is at least reduced by 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%.
- the molecule administered to the patient's nervous tissue may be selected from but not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering viruses.
- the nucleic acid includes, but is not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA) or short hairpin RNA (shRNA).
- dsRNA double-stranded RNA
- shRNA short hairpin RNA
- the double-stranded RNA (dsRNA) or short hairpin RNA (shRNA) contains the promoter sequence or coding region sequence information of the Porf-2 gene.
- the double-stranded RNA is small interfering RNA (siRNA).
- the small interfering RNA comprises a first strand and a second strand complementary to each other to form an RNA dimer; the sequence of the first strand is consistent with 15-27 continuous nucleotide sequences in the Porf-2 gene (target sequence) are basically the same.
- the siRNA can specifically bind to the mRNA fragment encoded by the target sequence, and specifically silence the expression of the Porf-2 gene.
- the target sequence in the Porf-2 gene is: when the siRNA specifically silences the expression of the Porf-2 gene, the fragment in the Porf-2 gene corresponding to the mRNA fragment complementary to the siRNA.
- the Porf-2 gene is a human-derived Porf-2 gene.
- the nerve damage disease is an optic nerve damage disease.
- the third aspect of the present invention provides the use of the isolated Porf-2 gene or Porf-2 protein in screening drugs for treating/improving nerve injury diseases.
- the "use of the isolated Porf-2 gene or Porf-2 protein in screening for the treatment/improvement of nerve injury diseases” includes: using the isolated Porf-2 gene or Porf-2 protein as a target for screening nerve injury Disease treatment/improvement drugs.
- the isolated Porf-2 gene or Porf-2 protein is used as the object/target of action, and the drug is screened to find a drug that can inhibit the expression of the Porf-2 gene as a candidate drug for treating/improving nerve damage diseases.
- the siRNA and shRNA of the Porf-2 gene as described in the present invention are obtained by screening the Porf-2 gene as an object, and can be used as candidate drugs for treating/improving nerve injury diseases; in addition, small molecule chemical drugs, antibody drugs, polypeptides Or protein, etc. can also use Porf-2 gene or its protein as the target.
- the drug for treating/improving nerve injury diseases is a molecule that can specifically inhibit the transcription or translation of the Porf-2 gene, or can specifically inhibit the expression or activity of the Porf-2 protein, thereby reducing the expression or activity of the Porf-2 gene in nerve tissue. expression level, to achieve the purpose of treating/improving nerve injury diseases.
- the administration dose of the medicine for treating/improving nerve injury disease is enough to reduce the transcription or translation of Porf-2 gene, or the dosage enough to reduce the expression or activity of Porf-2 protein.
- the expression of the Porf-2 gene is at least reduced by 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%.
- Drugs for the treatment/improvement of nerve injury diseases obtained by screening the isolated Porf-2 gene or Porf-2 protein can be selected from but not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering viruses.
- the nucleic acid includes, but is not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA) or short hairpin RNA (shRNA).
- dsRNA double-stranded RNA
- shRNA short hairpin RNA
- the double-stranded RNA (dsRNA) or short hairpin RNA (shRNA) contains the promoter sequence or coding region sequence information of the Porf-2 gene.
- the double-stranded RNA is small interfering RNA (siRNA).
- the small interfering RNA comprises a first strand and a second strand complementary to each other to form an RNA dimer; the sequence of the first strand is consistent with 15-27 continuous nucleotide sequences in the Porf-2 gene (target sequence) are basically the same.
- the siRNA can specifically bind to the mRNA fragment encoded by the target sequence, and specifically silence the expression of the Porf-2 gene.
- the target sequence in the Porf-2 gene is: when the siRNA specifically silences the expression of the Porf-2 gene, the fragment in the Porf-2 gene corresponding to the mRNA fragment complementary to the siRNA.
- the Porf-2 gene is a human-derived Porf-2 gene.
- the nerve damage disease is an optic nerve damage disease.
- the fourth aspect of the present invention provides an isolated nucleic acid molecule for the treatment/improvement of nerve injury diseases, wherein the isolated nucleic acid molecule includes targeting the Porf-2 gene in the nerve tissue, and knocking down the Porf-2 gene in the nerve tissue.
- Double-stranded RNA and/or shRNA for gene expression are provided.
- the double-stranded RNA contains a nucleotide sequence capable of hybridizing with the Porf-2 gene under stringent conditions.
- the shRNA contains a nucleotide sequence capable of hybridizing with the Porf-2 gene under stringent conditions.
- the double-stranded RNA comprises a first strand and a second strand complementary to each other to form an RNA dimer; the sequence of the first strand is identical or substantially identical to the target sequence of the Porf-2 gene. More preferably, the double-stranded RNA is siRNA (small interfering RNA). More preferably, the siRNA is obtained by performing RNA interference sequence design with the Porf-2 gene sequence as the target sequence.
- the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop segment connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, and
- the sequence of the sense strand fragment is identical or substantially identical to the target sequence of the Porf-2 gene. More preferably, the sense strand fragment and the antisense strand fragment of the shRNA are obtained by using the Porf-2 gene sequence as the target sequence and performing RNA interference sequence design.
- the shRNA is transformed into siRNA through enzymatic cleavage in the cell, and then plays the role of specifically knocking down the expression of the Porf-2 gene.
- the target sequence in the Porf-2 gene is: when the siRNA specifically silences the expression of the Porf-2 gene, the fragment in the Porf-2 gene corresponding to the mRNA fragment complementary to the siRNA.
- the target sequence refers to 15-27 continuous nucleotide sequences in the Porf-2 gene; preferably, the target sequence refers to 19-23 continuous nucleotide sequences in the Porf-2 gene more preferably, the target sequence refers to 19, 20 or 21 consecutive nucleotide sequences in the Porf-2 gene.
- the Porf-2 gene is a human-derived Porf-2 gene. More preferably, the target sequence of the Porf-2 gene is as shown in any one of SEQ ID NO: 1-6.
- the mRNA sequence of the coding region (CDS region) of the human Porf-2 gene was obtained from NCBI query, and the siRNA interference sequence for specifically knocking down the human Porf-2 gene was designed using software , and filter by rating. After screening by the inventors, the target sequence of the human Porf-2 gene as shown in any sequence of SEQ ID NO: 1-6 was selected.
- sequence of SEQ ID NO: 1 is: gagaaggactatgagatttac;
- sequence of SEQ ID NO: 2 is: gcctccaagcacttcaacaag;
- sequence of SEQ ID NO: 3 is: gctgatccagatgtacatggg;
- sequence of SEQ ID NO: 4 is: gcgataagcacgtatgccaag;
- sequence of SEQ ID NO: 5 is: gccaagtactgttaccacaag;
- sequence of SEQ ID NO: 6 is: gggacattgacgaggtgaatg.
- the fifth aspect of the present invention provides an expression vector of interfering nucleic acid of Porf-2 gene, wherein the expression vector comprises a gene fragment encoding the above shRNA, and the expression vector can express the shRNA.
- the expression vector of the interfering nucleic acid of the Porf-2 gene is obtained by cloning the gene fragment encoding the above shRNA into a known vector.
- the known vector may be a lentiviral vector, an adeno-associated viral vector or a retroviral vector, etc.
- the expression vector of the interfering nucleic acid of the Porf-2 gene is a recombinant viral vector, which is formed by cloning the gene fragment encoding the above-mentioned shRNA into the coding region of the viral vector;
- the viral vector is a lentiviral vector , adeno-associated viral vector, or retroviral vector.
- the expression vector of the interfering nucleic acid of the Porf-2 gene after being packaged by the virus, becomes an infectious virus particle, infects the nerve tissue, and then transcribes the above-mentioned shRNA, and then undergoes steps such as enzyme cutting and processing in the cell to become siRNA , and finally achieve specific knockdown of the expression of the Porf-2 gene.
- the expression vector of the interfering nucleic acid of the Porf-2 gene also contains a promoter sequence and/or a nucleotide sequence encoding a marker that can be detected in nerve tissue; the detectable marker , such as green fluorescent protein (GFP).
- GFP green fluorescent protein
- the expression vector of the interfering nucleic acid of the Porf-2 gene is a recombinant adeno-associated virus vector obtained by inserting the gene fragment encoding the above-mentioned shRNA into the coding region between the two ITR sequences of the adeno-associated virus vector.
- the "adeno-associated virus vector” can be serotype AAV1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or chimeric AAV derived from these serotypes, such as AAV2-AAV3, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAVHSC15, AAV-HSC17, AAVhu.37, AAVrh.8, etc.
- the adeno-associated viral vector is a serotype AAV2 or 5 vector. More preferably, the adeno-associated virus vector is a serotype AAV2 vector.
- the sixth aspect of the present invention provides a virus, wherein the virus is obtained by transfecting a eukaryotic cell with a virus packaging system; the virus packaging system comprises the above-mentioned recombinant adeno-associated virus vector.
- the virus packaging system is an adeno-associated virus packaging system, which comprises the above-mentioned recombinant adeno-associated virus vector containing the Porf-2 gene interference nucleic acid, the packaging plasmid of the adeno-associated virus, and the adeno-associated virus helper plasmid.
- the virus packaging system adopts the adeno-associated virus packaging system of the three-plasmid system, which includes the packaging plasmid pAAV-RC (containing the AAV2 coat protein gene), the helper plasmid pHelper (containing the gene) and the above-mentioned recombinant adeno-associated virus vector containing Porf-2 gene interference nucleic acid.
- the adeno-associated virus packaging system transfects eukaryotic cells, and the adeno-associated virus is obtained through virus packaging.
- the adeno-associated virus infects nerve tissue, and transcribes the above-mentioned shRNA, which is processed into siRNA by enzyme digestion, and finally achieves specific knockdown of the expression of the Porf-2 gene.
- the seventh aspect of the present invention provides a pharmaceutical composition, which comprises the above-mentioned virus, and a pharmaceutically acceptable carrier or excipient.
- the pharmaceutical composition comprises 1-99% wt of the virus, and a pharmaceutically acceptable carrier or excipient.
- the active ingredient is usually mixed with an excipient, or diluted with an excipient, or encapsulated in a pharmaceutical carrier.
- the pharmaceutical composition can be tablets, pills, powders, solutions, syrups, sterile injection solutions and the like.
- an injection form is used. More preferably, a dosage form suitable for subretinal injection or vitreous injection is used.
- the eighth aspect of the present invention provides the expression vector of the above-mentioned Porf-2 gene interfering nucleic acid, or the above-mentioned virus, or the application of the above-mentioned pharmaceutical composition in the preparation of drugs for the treatment/improvement of nerve injury diseases.
- the above virus or pharmaceutical composition can be used to treat/improve nerve damage diseases.
- the method for treating/improving nerve injury diseases comprises administering an effective dose of the virus or the pharmaceutical composition to the nerve tissue of the subject.
- the expression of the Porf-2 gene in the nerve tissue of the subject is knocked down. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the Porf-2 gene expression is knocked down.
- the subject can be a human.
- the nerve damage disease is an optic nerve damage disease.
- the expression vector of the above-mentioned Porf-2 gene interfering nucleic acid, or the above-mentioned virus, or the above-mentioned pharmaceutical composition is used to promote the regeneration of optic nerve axons after optic nerve injury.
- the expression vector of the interfering nucleic acid of the above-mentioned Porf-2 gene, or the above-mentioned virus, or, as the above-mentioned pharmaceutical composition is used to improve the survival rate of RGC cells after optic nerve injury.
- the expression vector of the above-mentioned Porf-2 gene interfering nucleic acid, or the above-mentioned virus, or the above-mentioned pharmaceutical composition is used to prevent the compound layer of ganglion cells from becoming thinner after optic nerve injury.
- the above-mentioned Porf-2 gene interfering nucleic acid expression vector, or the above-mentioned virus, or the above-mentioned pharmaceutical composition is used to promote the recovery of visual function after optic nerve injury.
- Fig. 1 is the map of the first empty plasmid described in the embodiment 1 of the present invention.
- Fig. 2 is the result figure of the Porf-2 expression level analysis obtained at different time points (after injury) obtained by immunofluorescence detection;
- Fig. 3 is the result figure of the Porf-2 expression level analysis obtained at different time points (after injury) detected by qPCR;
- Fig. 4 is two weeks after virus expression, the Porf-2 expression level analysis result figure of qPCR detection experimental group and control group;
- Fig. 5 is a graph showing the statistical results of the regenerated axons at different distances from the injury in the experimental group and the control group;
- Fig. 6 is a graph showing the survival results of retinal cells after immunofluorescent staining of the retinal slices.
- Porf-2 protein inhibits the proliferation of neural stem cells. Specifically, Porf2 protein inactivates Rac1 through its GAP domain, which leads to the continuous activation of Wnt/ ⁇ -Catenin pathway, which leads to the blockage of neural stem cell division.
- the inventors of the present application found in the research on the Porf-2 gene that after a period of optic nerve injury, the expression level of the Porf-2 gene in RGC cells increased;
- the expression of Porf-2 gene unexpectedly found that the knockdown of Porf-2 gene in mouse optic nerve tissue can promote the regeneration of optic nerve axons after optic nerve injury, especially it can also improve the survival rate of RGC cells after optic nerve injury, and prevent the The ganglion cell complex layer becomes thinner and promotes the recovery of visual function; this finding suggests that the Porf-2 gene can be used as a therapeutic target after optic nerve injury by knocking down the expression of the Porf-2 gene by RNAi, especially in combination with The gene therapy technology of adeno-associated virus can realize direct drug delivery to the eye, and it can become an effective means of treating/improving optic nerve damage in the future, and has considerable clinical application prospects.
- siRNA-1-6 an appropriate stem-loop fragment sequence was designed (in this example, the DNA sequence corresponding to the stem-loop fragment is: TTCAAGAGA), and thus the corresponding 6 siRNAs were obtained.
- shRNA shRNA1 ⁇ 6
- the sense strands of shRNA1-6 are the corresponding sense strands of siRNA-1-6, and the antisense strands of shRNA1-6 are the corresponding antisense strands of siRNA-1-6, which will not be repeated here.
- primer sequences introducing a SmaI restriction site
- Table 2 the primer sequences are shown in Table 2 below.
- the steps of the build process are as follows:
- the interfering fragments to be cloned and inserted in the above step 1) and the digested first empty plasmid in step 2) were all recovered using the AxyPrep PCR cleaning kit.
- AxyPrep PCR cleaning kit For specific operations, please refer to the instructions and will not repeat them.
- Plasmid ligation The above-mentioned cleaning and recovery of the interfering fragment and the first empty plasmid were ligated by T4 ligase (20 ⁇ l system).
- Transformation, recombinant plasmid identification and amplification transform the ligation product of step 3) into the competent cell Top10, spread the Amp resistance plate, and obtain monoclonal colonies for small shaking and small extraction (using Axygen small extraction kit);
- the plasmids obtained by the small extraction were digested and identified, and the plasmids with the same size as the target fragment were selected and sent to Shanghai Sangong Co., Ltd. for sequencing and identification, and the nucleic acid sequence comparison was performed.
- the recombinant plasmid with the correct sequencing ratio was selected and shaken, and the Axygen large extraction kit was used for extraction and purification to obtain a recombinant plasmid knocking down the Porf-2 gene.
- the recombinant plasmids obtained above to knock down the Porf-2 gene are recombinant adeno-associated virus vectors expressing shRNA1-6 respectively (named RNAi-1, RNAi-2, ... RNAi- 6).
- the shRNA1-6 sequence that interferes with the expression of the Porf-2 gene is inserted into the coding region between the ITR sequences at both ends of the adeno-associated virus vector (the above-mentioned first empty plasmid), Specifically, insert between the two Esp3I restriction sites in Figure 1.
- the inventors constructed an overexpression plasmid of the Porf-2 gene and an interference control plasmid.
- the overexpression plasmid (hereinafter also referred to as OV, i.e. overexpression construct) is obtained by inserting the cDNA sequence of the Porf-2 gene into the second empty plasmid (pAAV-CMV bGlobin-Arhgap39-mCherry-3xFlag-WPRE-hGHpA).
- the expression plasmid can make the fusion expression of Porf-2 protein and red fluorescent protein (mCherry);
- the overexpressed control plasmid (hereinafter also referred to as OC, i.e. overexpression empty control) refers to the above-mentioned second empty plasmid;
- the interference control plasmid (hereinafter also referred to as RNAi-NC) is obtained by inserting an shRNA with no interference activity (no corresponding target) into the first empty plasmid pAAV-H1-MCS-gRNA-CAG-EGFP-WPRE-SV40pA .
- RNAi-1-6 adeno-associated virus vectors
- shRNA1-6 shRNA1-6
- OV overexpression plasmid
- Knockdown groups 1-6 respectively set the following group names: OV+RNAi-1, OV+RNAi-2, ... OV+RNAi-6;
- Control groups 1-6 are respectively set the following group names: OC+RNAi-1, OC+RNAi-2, ... OC+RNAi-6;
- control group OV+RNAi-NC and a control group OV (only OV was added) were also set up.
- the red fluorescence effects of the knockdown group 1-6 were significantly weaker than that of the control group OV; however, 1) compared with the control group OV, the red fluorescence of the two groups There is no significant difference in the expression level, which indicates that the interference control plasmid (RNAi-NC) does not have the knockdown ability; 2) Normal red fluorescent expression was observed in the control group 1-6, and its expression level was compared with that of the control group OV , there is no significant difference, which shows that the knockdown plasmid RNAi-1 ⁇ 6 only specifically knocks down the Porf-2 gene; 3) Among the 6 knockdown groups, the effects of knockdown groups 4, 6, 2 and 3 are better; , the red fluorescence effect is the weakest in knockdown group 4, followed by knockdown group 6.
- the inventors selected the knockdown plasmid RNAi-4 (ie, the recombinant adeno-associated virus vector expressing shRNA4) to prepare an adeno-associated virus.
- the adeno-associated virus packaging system in this example uses a three-plasmid system, which includes the packaging plasmid pAAV-RC (containing the AAV2 coat protein gene), the helper plasmid pHelper (containing the gene that can help AAV replicate) and the above-mentioned knockdown plasmid RNAi-4 (recombinant adeno-associated virus vector expressing shRNA4).
- Step 1) Cultivation: On the first day, 293T cells with a degree of polymerization above 90% were subcultured at a ratio of 1:3, and cultured with high-glucose DMEM containing 10% FBS. About 1-2 hours before transfection the next day, replace the culture medium with serum-free culture medium;
- Step 2) Co-transfect 293T cells with Lipofectamine 2000, the above-mentioned knockdown plasmid RNAi-4, packaging plasmid pAAV-RC and helper plasmid pHelper; about 24 hours after transfection, change the medium; after about 72 hours after transfection, Use PEG8000 to precipitate the virus in the medium supernatant, and collect the virus after precipitation overnight;
- Step 3) purification and concentration the virus mixture collected in step 2) was purified by iodixanol density gradient centrifugation, and then concentrated by an ultrafiltration tube to obtain the adeno-associated virus solution of this embodiment, namely Solution of adeno-associated virus expressing shRNA4.
- virus titer Take a small amount of the adeno-associated virus solution obtained above, add proteinase K, incubate at 37°C for half an hour to break the virus capsid; then heat at 95°C for 5 minutes to inactivate the enzyme; centrifuge at 12000rp for 2min, collect the supernatant ; Then dilute the collected supernatant into different gradient concentrations, and perform qPCR amplification. After calculation, the virus titer is about 1.82E+13, which meets the requirements of virus products;
- the AAV2 vector (the first empty vector pAAV-H1-MCS-gRNA-CAG-EGFP-WPRE-SV40pA) has several characteristic sequences, such as the promoter CAG and the marker gene EGFP; thus, The supernatant collected in the identification of the above part 2) was added with primers of different characteristic sequences for qPCR detection; after detection, the amplified products of the promoter CAG and the marker gene EGFP of the adeno-associated virus prepared in this example The signal value is very close to that of the standard sample (the first empty vector), which shows that the knockdown plasmid RNAi-4 (i.e., the recombinant adeno-associated virus vector expressing shRNA4) contained in the adeno-associated virus obtained in this example is correct , no recombination between the knockdown plasmid RNAi-4 and the AAV genome occurred.
- the knockdown plasmid RNAi-4 i.e., the recombinant aden
- the build steps are as follows:
- the control group exposed the optic nerve without clamping.
- mice Select 5-week-old C57BL/6 male mice and randomly divide them into a control group (normal) and a 7-day-injury group; the 7-day-injury group was established according to the above-mentioned part 5, and the mice were killed 7 days after modeling obtained after.
- the retinal tissues of the normal control group and each injury group (non-injury side) were collected, total RNA was extracted, and after reverse transcription PCR, the expression of Porf-2 in the retina was detected by qPCR. As shown in FIG. 3 , the analysis results of Porf-2 expression at different time points (after injury) obtained by qPCR detection.
- the adeno-associated virus of this example that is, the adeno-associated virus obtained in the above section 4, that is, the adeno-associated virus containing the recombinant adeno-associated virus vector expressing shRNA4
- the adeno-associated virus of this example that is, the adeno-associated virus obtained in the above section 4, that is, the adeno-associated virus containing the recombinant adeno-associated virus vector expressing shRNA4
- mice in the control group were intravitreally injected with the interference control virus—the interference control plasmid (RNAi-NC) constructed in the above-mentioned part 3, and the adeno-associated virus obtained by the virus packaging method in the above-mentioned part 4.
- the interference control virus the interference control plasmid (RNAi-NC) constructed in the above-mentioned part 3
- the adeno-associated virus obtained by the virus packaging method in the above-mentioned part 4.
- the temperature of the case and the freezing head is adjusted to -20°C. Transfer the tissue from the -80°C freezer to the microtome half an hour in advance to thaw. Retina and optic nerve were sliced longitudinally, retina 20 ⁇ m/layer, optic nerve 14 ⁇ m/layer, the slices were pasted on anti-slip slides, and stored at -20°C for later use.
- the retina was placed in a 24-well plate containing blocking solution, and blocked at room temperature for 2 hours. After the blocking, the blocking solution was sucked off, and the primary antibody (TUJ11:300) was added to incubate at 4°C for 48 hours.
- the retinal tissues of each group were obtained for qPCR detection to verify whether the expression of the Porf-2 gene was effectively knocked down in the retinal tissues of the mice in the experimental group.
- the operation of qPCR is the same as the qPCR in Section 6 above.
- the expression level of the Porf-2 gene in the experimental group was significantly lower than that in the control group—a drop of about 50%.
- the expression level of the Porf-2 gene in the retinal tissue of mice injected with the adeno-associated virus of this example was significantly reduced, which verified the adeno-associated virus obtained in this example.
- a related virus can efficiently knock down the Porf-2 gene in retinal cells.
- the optic nerve clamp injury was performed on the mice of the experimental group and the control group according to the method of the above-mentioned part 5 (construction of the mouse optic nerve clamp injury model);
- Step 1) After 2 weeks of optic nerve damage, perform intraperitoneal injection anesthesia with 1% pentobarbital sodium injection (10ul/g) again, treat that after the mouse is anesthetized, add compound tropicamide eye drops at the left eyeball The liquid dilates the pupil.
- Step 2 At about 1mm behind the corneoscleral limbus, use a micro-syringe to insert the needle obliquely at 45° downwards. Be careful not to damage the lens and retina. After extracting 1.5ul of vitreous humor, pull out the syringe, which will help the subsequent vitreous cavity Injection can also prevent the increase of intraocular pressure caused by injection.
- Step 3 Use the micro-syringe again to insert the needle from the original needle port, and slowly inject 1.5ul of the prepared CTB-555 solution into the vitreous cavity of the mouse. After the injection, stay for 5 minutes until the reagent fully infiltrates the vitreous cavity, and then pull out the needle to prevent reflux. Tobramycin and dexamethasone eye ointment was applied after the operation to prevent infection, and the animals were transferred to the incubator until they recovered from anesthesia and returned to the cage for feeding. After close observation, those with lens opacity, massive hemorrhage in vitreous cavity and large-scale retinal detachment were excluded.
- Step 4 After 3 days, after the tracer is fully traced, the mice are anesthetized and sacrificed.
- Step 5 Count the number of axons at 0.1, 0.2, 0.5, 1.0, and 1.5 mm from the injury.
- the axons on the CTB marker after the injury were regarded as regenerated axons, and the regenerated axons at different positions away from the injury point were counted, and the same slice layers of each optic nerve were used for statistics and the average number was taken.
- the optic nerve clamp injury was performed on the mice of the experimental group and the control group according to the method of the above-mentioned part 5 (construction of the mouse optic nerve clamp injury model);
- OCT detection and pupillary light reflex detection were performed on the mice to observe the changes in the visual function of the mice.
- A. OCT detection method is as follows:
- mice were anesthetized by intraperitoneal injection of pentobarbital sodium (100 mg/kg), and their pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine hydrochloride eye drops;
- each volume consists of 100 b-scans and 1000 a-scans;
- GCC ganglion cell complex
- NNL nerve fiber layer
- GCL ganglion cell layer
- IPL inner plexiform layer
- the device for measuring and analyzing the dynamic change of pupil diameter is provided by the research group of Academician Yang Xiongli, School of Medicine, Fudan University. Through this device, the change of pupil diameter of the mouse can be digitally photographed and automatically analyzed when the mouse is awake.
- mice were dark-adapted for 24 hours before the test. During the test, the head of the mouse was braked, and the position between the camera and the pupil was adjusted so that the pupil was in the center of the screen and a clear and stable pupil image was obtained;
- the GCC thickness of the mice in the control group was about 30.14 ⁇ 1.82 ⁇ m, and the mice in the experimental group
- the GCC thickness (injected with the adeno-associated virus of the present embodiment) is about 40.78 ⁇ 2.57 ⁇ m; the data of the experimental group are significantly higher than the data of the control group (p ⁇ 0.05, there is a significant difference), which shows that using the method of the present embodiment Knockdown of the Porf-2 gene in retinal tissue by adeno-associated virus (containing a recombinant adeno-associated virus vector expressing shRNA4) prevented the thinning of the ganglion cell complex layer after optic nerve injury.
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Abstract
Agent thérapeutique génétique pour le traitement des lésions nerveuses. La présente invention concerne une molécule d'acide nucléique isolée destinée à traiter ou à atténuer des lésions nerveuses. La molécule d'acide nucléique isolée contient un ARN double brin ou un ARNsh ciblant un gène Porf-2 dans un tissu nerveux ciblé et annulant l'expression du gène Porf-2 dans le tissu nerveux, un vecteur d'expression contenant l'ARN double brin ou l'ARNsh ; un virus utilisant le vecteur d'expression et une composition pharmaceutique de celui-ci ; et l'utilisation de celui-ci dans un agent thérapeutique destiné à traiter ou à atténuer des lésions nerveuses.
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Non-Patent Citations (7)
Title |
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HU, SHUANG ET AL.: "Expression Pattern of Different Serotypes of Adeno-Associated Viral Vectors in Mouse Retina", JOURNAL OF PEKING UNIVERSITY(HEALTH SCIENCES), vol. 52, no. 5, 31 October 2020 (2020-10-31), XP009546055 * |
HUANG GUO-HUI, YANG XI-TAO, CHEN KUI, XING JIN, GUO LIN, ZHU LIANG, LI HONG-JIANG, LI XIN-CAI, ZHANG SHENG-YI, FENG DONG-FU: "Porf-2 Inhibits Neural Stem Cell Proliferation Through Wnt/β-Catenin Pathway by Its GAP Domain", FRONTIERS IN CELLULAR NEUROSCIENCE, vol. 10, XP093064368, DOI: 10.3389/fncel.2016.00085 * |
HUANG, GUOHUI ET AL.: "Neuronal GAP-Porf-2 Transduces EphB1 Signaling to Brake Axon Growth.", CELLULAR AND MOLECULAR LIFE SCIENCES, vol. 75, no. 22, 25 June 2018 (2018-06-25), XP036608643, DOI: 10.1007/s00018-018-2858-0 * |
HUANG, GUOHUI ET AL.: "Research Progress of Preoptic Regulatory Factor-2", THE JOURNAL OF PRACTICAL MEDICINE, vol. 29, no. 17, 10 September 2013 (2013-09-10), XP009546056 * |
NOWAK FELICIA V.: "Porf-2 = Arhgap39 = Vilse: A Pivotal Role in Neurodevelopment, Learning and Memory", ENEURO, vol. 5, no. 5, 1 September 2018 (2018-09-01), pages 1 - 12, XP093064375, DOI: 10.1523/ENEURO.0082-18.2018 * |
SHUANG MA; FELICIA V. NOWAK;: "The RhoGAP domain-containing protein, Porf-2, inhibits proliferation and enhances apoptosis in neural stem cells", MOLECULAR AND CELLULAR NEUROSCIENCES., SAN DIEGO, US, vol. 46, no. 3, 10 December 2010 (2010-12-10), US , pages 573 - 582, XP028153395, ISSN: 1044-7431, DOI: 10.1016/j.mcn.2010.12.008 * |
YANG XI-TAO, HUANG GUO-HUI, LI HONG-JIANG, SUN ZHAO-LIANG, XU NAN-JIE, FENG DONG-FU: "Rac1 Guides Porf-2 to Wnt Pathway to Mediate Neural Stem Cell Proliferation", FRONTIERS IN MOLECULAR NEUROSCIENCE, vol. 10, XP093064362, DOI: 10.3389/fnmol.2017.00172 * |
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