CN118256500A - SiRNA targeting FASN gene, carrier complex and application thereof - Google Patents
SiRNA targeting FASN gene, carrier complex and application thereof Download PDFInfo
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Abstract
The invention provides siRNA targeting FASN gene, a compound and application thereof, belonging to the technical field of gene therapy medicines, and the sense strand and the antisense strand of the siRNA targeting FASN gene provided by the invention have sequences shown as SEQ ID No.1 and SEQ ID No. 2; or has the sequences shown as SEQ ID No.3 and SEQ ID No. 4; or has the sequence shown as EQ ID No.5 and SEQ ID No. 6; the siRNA can inhibit the expression of FASN gene and reduce the protein abundance of FASN in liver, thereby reducing the deposition of liver fat, reducing lipotoxicity and inflammatory reaction and realizing accurate treatment of MAFLD; the siRNA provided by the invention is subjected to chemical modification, so that the stability of the siRNA is improved.
Description
Technical Field
The invention belongs to the technical field of gene therapy medicines, and particularly relates to siRNA targeting FASN genes, a compound and application thereof.
Background
Metabolic-related fatty liver disease (MAFLD) is the most common form of chronic liver disease worldwide, and is a collective term for a range of liver diseases including steatosis, nonalcoholic steatohepatitis, cirrhosis, and eventually hepatocellular carcinoma. At present, MAFLD is known to be a complex systemic metabolic disease, which is influenced by genetic factors, behavioral habits and other factors, and the multi-channel participation, the ectopic deposition of lipid and the insulin resistance are two accepted causes, but the specific pathogenesis is still unknown.
The main treatment methods at present are lifestyle intervention treatment, weight reduction operation treatment, drug treatment and the like; targeting abnormal fatty acid metabolism to prevent liver fat accumulation and create a pro-fibrotic environment may be a promising therapeutic strategy. However, MAFLD related metabolic pathways are complex, and at present, on the basis of undefined pathogenesis of MAFLD, accurate treatment is difficult to realize.
Disclosure of Invention
In view of the above, the present invention aims to provide a siRNA and a complex targeting a FASN gene, and applications thereof, which combine RNA interference technology with the FASN gene, and provide a therapeutic approach for targeted inhibition of expression of the FASN gene against MAFDL diseases.
Furthermore, the siRNA targeting the FASN gene provided by the invention is subjected to chemical modification, so that the stability of the siRNA in vivo is improved on the basis of keeping the biological activity of the siRNA.
The invention provides an siRNA targeting FASN gene, which can inhibit the expression of FASN gene.
Preferably, the sense strand and the antisense strand of the siRNA have the sequences shown as SEQ ID No.1 and SEQ ID No. 2;
Or has the sequences shown as SEQ ID No.3 and SEQ ID No. 4;
or has the sequence shown as EQ ID No.5 and SEQ ID No. 6.
Preferably, the sense and antisense strands of the siRNA also have tails of 2 to 3 nucleotides.
Preferably, the sense strand of the siRNA is modified as follows;
All of the nucleotides were PS modified and,
2'Ome modification is carried out on 1 st to 4 th nucleotides of the 5' end, and 2'ome modification is carried out on 3 rd to 6 th nucleotides of the 3' end; 2 'deoxythymines are connected at the 3' end;
the antisense strand of the siRNA was modified as follows:
All of the nucleotides were PS modified and,
The 5' -end 6 th and 7 th nucleotides are subjected to GNA modification, and the 3' -end is connected with 2' -deoxythymine.
The invention provides a carrier complex of a target FASN gene, which is characterized in that GalNAc is connected to the 3' end of a sense strand of siRNA.
The invention provides application of the siRNA and the carrier complex in preparing a reagent for inhibiting FASN gene expression.
The invention also provides application of the siRNA and the carrier complex in preparing medicaments for preventing and/or treating metabolic-related fatty liver diseases.
Compared with the prior art, the invention has the following beneficial effects: the invention provides an siRNA targeting FASN gene, which can inhibit the expression of the FASN gene, and can reduce the protein abundance of FASN in liver, thereby reducing the deposition of liver fat, reducing lipotoxicity and inflammatory reaction, and realizing the accurate treatment of metabolic related fatty liver disease (MAFLD).
Furthermore, the siRNA provided by the invention is subjected to chemical modification, so that the stability of the siRNA in the delivery process is improved.
The carrier compound of the targeting FASN gene provided by the invention is different from the traditional small molecule drug, has more accurate targeting property, plays a role continuously, can reduce lipid for a long time, has single metabolic pathway, is removed along with kidneys, and reduces the load of livers; the traditional small molecule drugs are metabolized from the liver to cause serious liver injury, and the MAFLD patients have metabolic dysfunction to cause a certain burden on the diseases. In addition, the carrier compound provided by the invention is subjected to subcutaneous administration, is inhibited for a long time, reduces the medicine taking frequency of patients, and brings more convenience and more effective treatment experience for the patients.
Drawings
FIG. 1 is a graph showing the effect of siRNA silencing FASN gene in example 1, wherein the left graph is human hepatic cell C3A, and the right graph is human hepatic stellate cell LX2; the upper part of the left and right graphs is the WB result, and the lower part is the QPCR result;
FIG. 2 shows the effect of siRNA silencing of the FASN gene and determination of IC50 values, the points of the two broken lines intersecting the red dotted line being IC50 values;
siRNA-1 is the result of transfection of naked strand FASN SIRNA-289 on C3A cells, siRNA-2 is the result of transfection of naked strand FASN SIRNA-289 on LX2 cells;
FIG. 3 is a schematic diagram of siRNA modification scheme;
FIG. 4 shows comparison of IC50 values of FASN SIRNA-289 modified and naked strand silencing FASN gene, respectively transfected naked strand siRNA and modified siRNA in C3A and LX2 cells, grouped into C3A+ naked strand (siRNA-1) and C3A+ modified strand (siRNA-1P); LX2+ bare chain (siRNA-2) and LX2+ modified chain (siRNA-2P), the silencing efficiency of modified siRNA is obviously improved;
FIG. 5 shows the effect of CCK8 method on cell activity of FASN SIRNA-289 modified at different concentrations, wherein cell is untreated cell, INTERFERIN is transfection reagent added only to cell, negative control is siRNA added to cell without effect on GADPH gene, positive control is siRNA added to cell against GADPH gene; 0nm, 1nm, 10nm, 100nm, 1 μm means that different concentrations of modified FASN SIRNA-289 are added to the cells;
FIG. 6 shows that the effect of FASN SIRNA-289 on apoptosis is detected, wherein the apoptosis rates of two cells, namely C3A and LX2, are not obviously increased (the early apoptosis rate of C3A is 7.12 percent, and the early apoptosis rate of LX2 is 5.24 percent), so that FASN SIRNA-289 has no obvious toxicity on cells and is safer;
FIG. 7 shows the results of FASN gene silencing by introducing FASN SIRNA-289 naked sequences into human liver cells (C3A), different treatment groups C3A oil red staining;
FIG. 8 is a schematic structural diagram of a carrier complex;
FIG. 9 shows the silencing efficiency of FASN gene at different times after injection of different concentrations of vector complex.
Detailed Description
The invention provides an siRNA targeting FASN gene, which can inhibit the expression of FASN gene. In the present invention, the sense strand and the antisense strand of the siRNA have sequences as shown in SEQ ID No.1 and SEQ ID No. 2; or has the sequences shown as SEQ ID No.3 and SEQ ID No. 4; or has the sequence shown as EQ ID No.5 and SEQ ID No. 6. Further, the sense strand and the antisense strand of the siRNA also have a tail of 2 to 3 nucleotides, which tail is preferably "TT"; in the present invention, the sequences of the sirnas are specifically shown in table 1.
TABLE 1 sequence of siRNA
In the present invention, the sense strand of the siRNA is preferably modified as follows;
All of the nucleotides were PS modified and,
2'Ome modification is carried out on 1 st to 4 th nucleotides of the 5' end, and 2'ome modification is carried out on 3 rd to 6 th nucleotides of the 3' end; 2 'deoxythymines are connected at the 3' end;
the antisense strand of the siRNA was modified as follows:
All of the nucleotides were PS modified and,
The 5' -end 6 th and 7 th nucleotides are subjected to GNA modification, and the 3' -end is connected with 2' -deoxythymine.
In the present invention, the preferred modified sequences of FASN SIRNA-289 are as follows:
Sense strand:
FASN siRNA-289-ss：
(ome-C)*(ome-C)*(ome-A)*(ome-G)*A*U*U*C*A*C*U*C*C*G*A*G*G*(ome-A)*(ome-A)*(ome-C)*(ome-A)*/idT/*/idT/*
FASN siRNA-289-as：
U*G*U*U*C*/GNA-C*/GNA-U*C*G*G*A*G*U*G*A*A*U*C*U*G*G*/idT/*/idT/*；
Wherein ome represents modification of 2' -O-methylation; * Represents replacement of sulfate linkages with Phosphorothioate (PS) linkages; GNA stands for Glycerol nucleic acid, glycerol nucleic acid and idT is 2' deoxythymine.
The invention also provides a carrier complex of the target FASN gene, which is conjugated and coupled with GalNAc at the 3' end of the sense strand of the siRNA; the GalNAc is preferably GalNAc-L96.
The invention provides application of the siRNA and the carrier complex in preparing a reagent for inhibiting FASN gene expression. The siRNA or the carrier complex can obviously inhibit the expression of FASN genes when entering cells or organisms, and has specificity.
The invention also provides application of the siRNA and the carrier complex in preparing medicaments for preventing and/or treating metabolic-related fatty liver diseases. In the invention, the siRNA can inhibit the expression of FASN gene, thereby reducing the protein abundance of FASN in liver, reducing the deposition of liver fat, reducing lipotoxicity and inflammatory reaction, and realizing the prevention and treatment of metabolic related fatty liver disease (MAFLD).
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Sequence design of siRNA
Multiple siRNAs are designed according to the sequence of the FASN gene, 3 siRNAs are screened out, and the specific sequence is as follows:
FASN siRNA-289(FASN1)：
UGUUCC UCGGAGU GAAUC U GG (antisense strand)
CCAGAU UCAC UCC GAGGAA CA (sense strand)
The sequence design key points are as follows:
1. binding target in CDS region of FASN gene
The first base at the 5' end of the SS strand is G or C
The first base at the 5' end of the AS strand is A or U
SS chain base preference near silencing principle (A at position 19, C at position 10, C at position 13)
5. The sequence not forming a hairpin or palindromic structure
6. Does not contain any form of repetition of the three base combinations
AS-strand base preference meeting the requirements (C at position 6)
8. Binding target position 289..309;
FASN siRNA-5309(FASN2)：
UCUGAGAAAG GUC GAAUUU GC (sense strand)
GCAAAU UCGA CCU UUCUCA GA (antisense strand)
The sequence design key points are as follows:
1. binding target in CDS region of FASN gene
The first base at the 5' end of the SS strand is G or C
The first base at the 5' end of the AS strand is A or U
SS strand base preference near silencing principle (A at position 19, U at position 10, U at position 13)
5. The sequence not forming a hairpin or palindromic structure
6. Repeat containing three base combinations
AS-strand base preference meeting the requirements (G at position 6)
8. Binding to target position 5309..5329 (closer to the 3' end).
Sequence modification of FASN-siRNA-289
FASN SIRNA-289 modified sequence is as follows:
Sense strand:
FASN siRNA-289-ss：
(ome-C)*(ome-C)*(ome-A)*(ome-G)*A*U*U*C*A*C*U*C*C*G*A*G*G*(ome-A)*(ome-A)*(ome-C)*(ome-A)*/idT/*/idT/*
FASN siRNA-289-as：
U*G*U*U*C*/GNA-C*/GNA-U*C*G*G*A*G*U*G*A*A*U*C*U*G*G*/idT/*/idT/*；
Wherein ome represents modification of 2' -O-methylation; * Represents replacement of sulfate linkages with Phosphorothioate (PS) linkages; GNA stands for Glycerol nucleic acid, glycerol nucleic acid and idT is 2' deoxythymine.
FASN siRNA-749(FASN3)：
GCACCAAUACAGAUGGCUU TT (sense strand)
AAGCCAUCUG UAU UGGUGC TT (antisense strand)
The sequence design key points are as follows:
1. binding target in CDS region of FASN gene
The first base at the 5' end of the SS strand is G or C
The first base at the 5' end of the AS strand is A or U
SS base preference deficiency (U at 19, C at 10, A at 13)
5. The sequence structure forms a hairpin
6. Does not contain any form of repetition of the three base combinations
AS-strand base preference deficiency (A at position 6)
8. Binding to target site 749.
Comprehensive analysis: the optimized sequence FASN SIRNA-289 has the advantages that the sequence design meets various requirements, but the target position of the optimized sequence FASN SIRNA-5309 is far away from the 3' end of the target gene. The optimized sequence FASN SIRNA-5309 has the advantage of being closer to the 3' end of the target gene (silencing efficiency may be better), but with consecutive 3 base repeats (AAA/UU) in the sequence, it is possible to terminate RNA Polymerase III-mediated transcription, thus two pairs of siRNAs were screened in parallel experiments.
In addition, a hairpin structure is formed on the FASN SIRNA-749 sequence, and the existence of the hairpin structure can reduce the effective concentration and silencing efficiency of siRNA.
Sequence modification of FASN-siRNA-749
FASN SIRNA-749 modified sequence is as follows:
(ome-G)*(ome-C)*(ome-A)*(ome-C)*C*A*A*U*A*C*A*G*A*U*G*(om e-G)*(ome-C)*(ome-U)*(ome-U)*idT*idT*
A*A*G*C*C*/GNA-A/*/GNA-U/*C*U*G*U*A*U*U*G*G*U*G*C*idT*idT*
3 siRNA bare sequences obtained through design are respectively introduced into human liver cells C3A and human hepatic stellate cells LX2 to silence FASN genes, and the specific steps are as follows:
Cells were cultured at 37℃under 5% CO 2. siRNA transfection was performed using INTERFERIN transfection reagents according to the manufacturer's instructions. Setting 6 groups: specific siRNA groups (siRNAs against C3A cells (1; 2; 3) and LX2 cells (1; 2; 3)), non-specific siRNA groups (negative control). Preparation of transfection reagent: 400. Mu.L of nuclease water was added to the tube, and the tube was shaken for 10s to dissolve the lipid. After shaking, the reagent is kept at-20 ℃ and is also required to be shaken before use. Cells were transfected with appropriate mixing ratios (1:1-1:2/liposome volume: siRNA mass). A suitable volume of serum-free medium was added to one transfection tube. 10nM/ml siRNA was added and after shaking 16. Mu.l of transfection reagent was added and shaking was again performed. The mixture was left at room temperature for 10-15min. The medium in the plates was aspirated and washed once with PBS or serum-free medium. Adding the mixed solution, putting the C3A and LX2 cells growing to 70-80% back into the incubator for culturing for one hour, and then adding the complete medium for culturing for 24-48 hours. RNA extraction and quantitative real-time PCR (qPCR): 48 hours after transfection, total RNA was extracted using an RNA extraction kit and reverse transcribed into cDNA using a reverse transcription reagent. mRNA expression levels of the target genes were quantified by qPCR technique using specific primers. To verify the protein-level changes in the silencing effect of siRNA. Furthermore, for confirming the siRNA silencing efficacy, gradients of siRNA concentrations (1 e-8,1e-7,1e-6,1e-5,1 e-4) were set to calculate the optimal concentrations for IC50 selection intervention C3A and LX 2. As shown in FIG. 2, siRNA1 in FIG. 2 is FASN SIRNA-289 and siRNA2 in FASN SIRNA-749; the points of the two broken lines intersecting the red broken line are the IC50 values, and the concentration of 1e-4 was selected for intervention according to the experimental requirements.
And comparing the concentration gradients of FASN SIRNA-289 and FASN SIRNA-749, respectively setting the concentration of 1X 10 -8,1×10-7,1×10-6,1×10-5,1×10-4 mol/hole, and observing the silencing efficiency of the two siRNAs under different concentration gradients. As shown in FIG. 4, siRNA-1 was the result of transfection of naked strand FASN SIRNA-289 on C3A cells, and siRNA-1p was the result of modification of FASN SIRNA-289 on C3A cells; siRNA-2 is the result of FASN SIRNA-289 bare strand on LX2 cells, and siRNA-2P is the result of FASN SIRNA-289 modified on LX2 cells. As the concentration gradually increases, the difference in silencing efficiencies of FASN SIRNA-289 and FASN SIRNA-749 is significant, and the silencing efficiency of FASN SIRNA-289 is higher; the trend is consistent before and after modification, and the silencing efficiency after modification is higher. Safety was assessed by using CCK-8 to detect the effect of modified FASN SIRNA-289 on cell activity (fig. 5), where in fig. 5 cell is the cell without any treatment, INTERFERIN is the addition of transfection reagent only to the cell, negative control is the addition of universal Negative siRNA to the cell (N.C-S UUCUCCGAACGUGUCACGUTT; N.C-AACGUGACACGUUCGGAGAATT), positive control is the addition of siRNA(human-GADPH-S：GUAUGACAACAGCCUCAAGTT;human-GADPH-A：CUUGAGGCUGUUGUCAUACTT);0nm、1nm、10nm、100nm、1μm targeting endogenous gene GADPH to the cell refers to the addition of different concentrations of modified FASN SIRNA-289 to the cell; the results showed that the modified FASN SIRNA-289 was safe in both C3A and LX2 cells.
The effect of FASN SIRNA-289 on apoptosis was detected by flow cytometry, and the results show that the apoptosis rates of both C3A and LX2 are not obviously increased (7.12% of C3A early apoptosis rate and 5.24% of LX2 early apoptosis rate) as shown in FIG. 6, which indicates that FASN SIRNA-289 has no obvious toxicity to cells and is safer.
Silencing of FASN Gene by introducing FASN SIRNA-289 naked sequences into human liver cells (C3A)
The comparison of the high sugar group and the high sugar + FASN-siRNA-289 (labeled high sugar + FASN1 in fig. 7) was set, the C3A cells were treated and then stained with oil red O, and then observed under a microscope, and fat deposition was observed, as shown in fig. 7, and the result was that the transfected FASN SIRNA-289 into the high sugar-cultured C3A cells, and a reduction in lipid deposition was observed.
Example 2
The carrier complex targeting FASN gene is conjugated and coupled with GalNAc at the 3' end of FASN SIRNA-289 sense strand modified in example 1.
A total of 240C 57BL6 male 6 week old mice, divided into 24 groups of 10 mice each; the vector complex was subcutaneously injected into C57BL6 mice at concentrations of 1mg/kg (low dose), 5mg/kg (medium dose), 10mg/kg (high dose), respectively, and the FASN silencing efficiency was examined every four weeks up to 24 weeks using QPCR. Mice without injected vector complex were used as controls and were designated NC.
The results are shown in FIG. 9, where Week represents the number of weeks, e.g., W4 represents the fourth Week; the vector complex was found to silence FASN for 24 weeks.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. An siRNA targeting a FASN gene, wherein the siRNA is capable of inhibiting expression of the FASN gene.
2. The siRNA of claim 1, wherein the sense strand and the antisense strand of the siRNA have the sequences shown in SEQ ID No.1 and SEQ ID No. 2;
Or has the sequences shown as SEQ ID No.3 and SEQ ID No. 4;
or has the sequence shown as EQ ID No.5 and SEQ ID No. 6.
3. The siRNA of claim 1, wherein the sense strand and the antisense strand of the siRNA further have 2 to 3 nucleotide tails.
4. The siRNA of claim 2, wherein the sense strand of the siRNA is modified as follows;
All of the nucleotides were PS modified and,
2'Ome modification is carried out on 1 st to 4 th nucleotides of the 5' end, and 2'ome modification is carried out on 3 rd to 6 th nucleotides of the 3' end; 2 'deoxythymines are connected at the 3' end;
the antisense strand of the siRNA was modified as follows:
All of the nucleotides were PS modified and,
The 5' -end 6 th and 7 th nucleotides are subjected to GNA modification, and the 3' -end is connected with 2' -deoxythymine.
5. A vector complex targeting a FASN gene, wherein GalNAc is linked to the 3' -end of the sense strand of the siRNA of any one of claims 1 to 3.
6. Use of the siRNA of any one of claims 1 to 4, the vector complex of claim 5, for the preparation of a reagent for inhibiting expression of FASN gene.
7. Use of the siRNA of any one of claims 1 to 4, the vector complex of claim 5, for the preparation of a medicament for the prevention and/or treatment of metabolic-related fatty liver disease.
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