CN113403313B - sgRNA, plasmid and nano-composite for specifically recognizing human PLK1 locus and application - Google Patents

Abstract

The invention belongs to the technical field of medicines, and particularly relates to sgRNA (small guide ribonucleic acid), plasmid, nano compound and application for specifically identifying a human PLK1 locus, wherein the sequence of the sgRNA is SEQ ID NO: 1. SEQ ID NO:2 or SEQ ID NO:3, by adopting the sgRNA sequence, the gene editing efficiency is higher, the duration is long, and plasmids containing the sgRNAs can smoothly enter cell nuclei to play a role; the liposome iLP181 has a proper pKa value, a better endosome escape effect and better targeting property; the obtained nano-composite has better safety in vivo and in vitro, effectively realizes escape and release of load nucleic acid, still has higher gene editing efficiency for a long time, and can effectively inhibit proliferation of tumor cells.

Description

sgRNA, plasmid and nano-composite for specifically recognizing human PLK1 locus and application

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to sgRNA, plasmid, nano composite and application for specifically identifying a human PLK1 locus.

Background

Polo-like kinase (Polo-like kinase1, PLK1), a serine/threonine protein kinase widely present in eukaryotes, has been widely spotlighted because of its important role in the cell cycle. PLK1 was found to be highly expressed in most tumors in humans and to be closely related to tumor cell proliferation and patient prognosis. The preclinical results show that the interference of the expression of PLK1 can obviously inhibit the growth of various tumors including non-small cell lung cancer, lymphoma and colorectal cancer, and has no obvious influence on normal cells. Therefore, PLK1 is considered as a new malignant tumor target with good application prospect. Therefore, a PLK1 gene is disclosed to regulate a tumor proliferation mechanism by utilizing a gene editing technology aiming at a PLK1 target spot in a tumor, a new thought is provided for the research and development of human PLK1 site-related cancer treatment and medicines, and the method has important significance.

The current mature gene editing techniques include: ZFN, TALEN and CRISPR/Cas gene editing technology. Although the ZFN and TALEN gene targeting technology is two mature site-directed mutation technologies at present, the two technologies are complex in construction procedures, and each site needs to construct a pair of responsive nucleases. And the CRISPR/Cas gene editing technology is used for guiding a specifically recognized site by virtue of a small crRNA. Each specifically recognized crRNA has only dozens of bases, and the whole vector is small. It is easier to construct than ZFN and TALEN vectors.

CRISPR/Cas is an evolving adaptive immune defense mechanism for archaea. At present, the development of CRISPR/Cas gene editing technology enters a hopeful era, and a new treatment method is provided for diseases which cannot be cured at present. Can be used for treating cancer, vesicular skin disease, Huntington's disease, Sickle Cell Disease (SCD), HIV-1 infection, and establishing specific animal model with improved genome. To some extent, the emergence of CRISPR/Cas reflects a significant advance in human gene editing technology, providing an effective and powerful platform for further understanding gene function, the underlying biological or pathological mechanisms of various diseases, and ultimately developing new therapeutic regimens.

Common CRISPR/Cas delivery vectors are viral vectors and non-viral vectors. Although adenovirus AAV and lentivirus are widely used in clinical studies, 70% of the population already contain antibodies that neutralize AAV and have potential gene integration and concern for eliciting an immune response. Among non-viral delivery vehicles, Lipid Nanoparticles (LNPs) are the most widely used mRNA vectors, and the currently marketed mRNA vaccines mRNA-1273, BNT162b2, and the new coronavirus mRNA vaccines CVnCoV, ARCT-021 and ARCOV, which are still in clinical trials, are all LNPs delivery vehicles. LNPs consist primarily of ionizable lipids, phospholipids, cholesterol, and PEG lipids, where ionizable lipids are key lipid molecules for LNPs to escape from the endosome in response to the cellular microenvironment. Research shows that ionizable lipid with dissociation constant pKa of 6.2-6.5 has no charge under normal physiological environment (pH7.4), but has positive charge under micro-acid environment such as lysosome, and effectively promotes escape and release of loaded nucleic acid through proton sponge effect and colloid osmotic pressure effect. Compared to cationic lipids, ionizable lipids are clearly more advantageous, since the latter are easily associated with biological macromolecules in physiological environments, which poses safety problems. The key component of LNPs used by the siRNA drug Patisiran marketed by Alylam is Dlin-MC3-DMA with a pKa of 6.44, and the acid response ability, effectiveness and safety of Dlin-MC3-DMA have been verified.

Disclosure of Invention

The invention aims to provide sgRNA, plasmid, nano-composite for specifically identifying human PLK1 locus and application thereof.

The invention relates to a sgRNA specifically recognizing human PLK1 locus, wherein the sequence of the sgRNA is SEQ ID NO: 1. the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3, preferably SEQ ID NO: 2. SEQ ID NO:2 is as set forth in SEQ ID NO:1 is represented by SEQ ID NO:1, adding a restriction enzyme recognition site sequence in front of the sequence, wherein the restriction enzyme recognition site sequence is SEQ ID NO:3 is the peptide set forth in SEQ ID NO:2 is as set forth in SEQ ID NO:2, adding a base G for stabilizing the sgRNA sequence in front of the sequence. The sequence of a target fragment which is identified by the sgRNA is SEQ ID NO. 12.

A plasmid comprising the sgRNA.

A nanocomposite comprising liposome iLP181, and the above plasmid; the liposome iLP181 is composed of key lipid iLP181, phospholipid, cholesterol and PEG-lipid; the key lipid iLP181 has the structural formula:

the phospholipid comprises at least one selected from the following components: distearoylphosphatidylcholine (DSPC), Dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (POPC), Dioleoylphosphatidylethanolamine (DOPE), Dilauroylphosphatidylcholine (DLPC), Dimyristoylphosphatidylcholine (DMPC) and egg yolk lecithin (EPC).

The PEG-lipid comprises at least one selected from the group consisting of: distearoyl phosphatidyl ethanolamine-PEG (DSPE-PEG), dimyristyl glycerol (DMG-PEG), and dimethacrylate (DMA-PEG).

The molar ratio of the key lipid, the phospholipid, the cholesterol and the PEG-lipid is (25-57): 1-37): 25-67): 0.1-19.

The liposome iLP181 is prepared by dissolving key lipid, phospholipid, cholesterol and PEG-lipid in ethanol solvent, stirring, and adding into sodium citrate solution to obtain liposome iLP 181.

An application of the sgRNA in gene editing.

An application of the sgRNA in preparation of a medicine for treating cancer.

The sgRNA sequence has the advantages that the gene editing efficiency is high, the gene editing duration is long, and plasmids containing the sgRNAs can smoothly enter cell nuclei to play a role; the liposome iLP181 has a proper pKa value, a better endosome escape effect and better targeting property; the obtained nano-composite has better safety in vivo and in vitro, effectively realizes escape and release of load nucleic acid, still has higher gene editing efficiency for a long time, and can effectively inhibit proliferation of tumor cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the structure of key lipids in liposomes iLP181 used in the examples of the present invention.

FIG. 2 is a nuclear magnetic hydrogen spectrum of key lipid iLY1809 used in the examples of the present invention.

FIG. 3A is a schematic representation of the structure of nanocomposite iLP181/psgPLK1 in an embodiment of the invention;

FIG. 3B shows the particle size and potential of nanocomposite iLP181/psgPLK 1.

FIG. 4A is a cell viability assay of the nanocomplex iLP181/psgPLK 1;

fig. 4B is the pKa value of nanocomposite iLP 181.

FIG. 5A is a particle size measurement of iLP181/psgPLK1 in buffer and Human serum (Human serum, HS) for up to two weeks.

FIG. 5B is a graph of iLP181/psgPLK1 for two weeks of zeta potential measurements in buffer and Human serum (Human serum, HS).

FIG. 6A is a functional test of the ability of Lipo2000/psgPLK1 to enter the nucleus to function as a plasmid;

FIG. 6B is a quantitative analysis of GFP signals in FIG. 6A.

FIG. 7A is a screen of sgRNAs sequences in an example of the present invention;

FIG. 7B is a long-term assessment of iLP181/psgPLK1 mediated gene editing in HepG2-Luc cells.

FIG. 8A is a study of the cellular entry mechanism of iLP181 in example of the present invention;

FIG. 8B is a diagram illustrating the quantitative analysis of the fluorescence signal in FIG. 8A.

FIG. 9A is a study of inclusion escape of nanocomplexes iLP181/Cy5-NA in an example of the invention;

FIG. 9B is a quantitative analysis of the Cy5 fluorescence signal in FIG. 9A;

FIG. 9C is a co-localization analysis of Cy 5-labeled nucleic acid with lysotracker-stained endosomes/lysosomes in FIG. 9A.

Fig. 10A shows fluorescence imaging analysis of whole body and isolated major organs using a live imaging system at various times after tail vein administration of a liposome iLP181 delivering Cy5 labeled nucleic acid molecules according to an embodiment of the present invention.

FIG. 10B is a quantitative analysis of Mean Fluorescence Intensities (MFIs) at 6h, 12h and 24h for the isolated major organs of FIG. 10A.

FIG. 11A is the distribution of the plasmid expressing red fluorescent protein delivered by the liposome iLP181 in the mouse in the main organs of the mouse according to the present invention;

FIG. 11B is a quantitative analysis of RFP of the isolated major organ of FIG. 11A;

FIG. 12A is a schematic representation of the in vivo anti-tumor effect of iLP181/psgPLK 1;

FIG. 12B is luciferase expression in mouse tumor tissue at the end of the experiment;

FIG. 12C shows tumor volume growth throughout the experiment;

FIG. 12D is the expression of PLK1 mRNA in tumor tissue at the end of the experiment.

FIG. 13 shows the pathological analysis results of the major organs of iLP181/psgRNA complex after treatment of liver cancer in mice in the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1 sgRNA design specifically recognizing the human PLK1 site

4 sgRNAs targeting PLK1 genes were designed (sgRNA1, sgRNA2, sgRNA3, and sgRNA 4). The sequence of sgRNA1 is SEQ ID NO. 2, the sequence of sgRNA2 is SEQ ID NO. 3, the sequence of sgRNA3 is SEQ ID NO. 4, and the sequence of sgRNA4 is SEQ ID NO. 5.

First, polymeric sgRNAs were obtained by gradient annealing PCR and cloned onto PX458 plasmid via Bbs1 enzyme cleavage site, named Cas9-sgPLK 1. The constructed Cas9-sgPLKs plasmids (psgPLK1-1, psgPLK1-2, psgPLK1-3 and psgPLK1-4) were then transfected into competent E.coli DH5 a. The specific method comprises the following steps: adding the Cas9-sgPLKs plasmid into competent Escherichia coli DH5 alpha, gently mixing, placing on ice for 30min, then thermally shocking at 42 ℃ for 90s, placing on ice for 2-3min, adding a proper amount of LB culture medium for culture (without antibiotics), mixing, placing on a shaking table at 37 ℃, and shaking and culturing at 150rpm for 45min to recover the thallus. And then, after the recovered bacterial liquid is subjected to brief centrifugal concentration, adding a certain volume of the bacterial liquid into an LB agar culture medium (containing the aminobenzyl antibiotic), placing the mixture into an incubator at 37 ℃ for culture for 12-16h, picking the colony of the monoclonal antibody the next day into an LB liquid culture medium (containing the aminobenzyl antibiotic), placing the colony into a shaking table at 37 ℃, and performing shake culture at 180rpm for 16h to expand the thallus. After culturing, E.coli was harvested and Cas9-sgPLK1s plasmids (psgPLK1-1, psgPLK1-2, psgPLK1-3 and psgPLK1-4) were extracted using an Endo Free plasmid kit (Kangwei century).

CRISPR-PX458(PX458-E) is an empty vector plasmid, Cas9-sgPLK1 (namely psgPLK1) is a plasmid for specifically recognizing human PLK1 locus after PX458-E is modified, wherein the functional structural units comprise: a U6 promoter, an SV40 nuclear localization signal, an sgRNA modification functional box, a T2A, a Cas9 Protein sequence and an enhanced Green Fluorescence signal (GFP); as a result of sequencing information of Cas9-sgPLKs (psgPLK1-1, psgPLK1-2, psgPLK1-3 and psgPLK1-4) plasmids specifically recognizing human PLK1 sites, no mismatched base pairs are shown, and the success of constructing recombinant Cas9-sgPLKs (psgPLK1-1, psgPLK1-2, psgPLK1-3 and psgPLK1-4) is verified.

Example 2 Synthesis of liposomes iLP181

First is the synthesis of iLY1809 (key lipid): 1) adding N-tert-butyloxycarbonyl-1, 3-propanediamine, 1, 2-epoxydodecane and ethanol into a three-neck flask, and refluxing and reacting at 50 ℃ under the protection of nitrogen overnight. After the reaction was stopped by TLC, the mixture was concentrated under reduced pressure. Crude product was purified with developing solvent dichloromethane: separating and purifying methanol (v/v is 30:1) by a silica gel column to obtain transparent liquid (3- (bis (2-hydroxy dodecyl) amino) propyl) carbamic acid tert-butyl ester; 2) adding the compound (3- (bis (2-hydroxydodecyl) amino) propyl) carbamic acid tert-butyl ester and dichloromethane into a three-neck flask, stirring, adding hydrochloric acid alcohol solution, reacting at normal temperature overnight, and stopping reaction after TLC detection. Extracting the product, adjusting the pH value to 7-8, collecting an organic phase, concentrating under reduced pressure, and treating a crude product with a developing solvent of dichloromethane: separating and purifying methanol (v/v-15: 1) by a silica gel column to obtain transparent liquid 1,1' - ((3-aminopropyl) azanediyl) bis (dodecane-2-ol); 3) adding a compound 1,1' - ((3-aminopropyl) azanediyl) bis (dodecane-2-ol), succinic acid and catalysts 1-hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a three-neck flask containing dichloromethane, reacting at normal temperature overnight under the protection of nitrogen, and stopping the reaction after TLC detection. And washing the crude product with water and salt, drying the organic phase, filtering, and concentrating under reduced pressure. Crude product was purified with developing solvent dichloromethane: the methanol (v/v ═ 30:1) was separated and purified by silica gel column. The clear liquid, compound N1, N4-bis (3- (bis (2-hydroxydodecyl) amino) propyl) succinamide, was obtained and was named iLY 1809.

Nuclear magnetic hydrogen spectrum: ¹ H NMR(700MHz，

CDCl3)δ(ppm)0.88(t,12H,J＝13.24Hz,4×-CH3)1.26-1.59(m,76H,38×-CH2-)2.49(m，8H,2×-CH2-O,2×-CH2-)2.67(t,4H,J＝11.78Hz,4×N-CH2-)3.32(m，4H，2×N-CH2-)3.67(s,2H,2×-CH-OH)5.30(s,4H,4×-OH)。

HR-EIMS m/z calcd for C58H118N4O6(M+)967.5843,found 966.9051。

then, liposome iLP181 was prepared from iLY1809 (key lipid), PEG-lipid, phospholipid, cholesterol by a self-assembly process into liposome iLP 181. Firstly, cholesterol, phospholipid, iLY1809 and PEG-lipid are respectively dissolved in ethanol solution to prepare 20mg/mL solution (the concentration of 20mg/mL is the concentration of each of the 4 components), then the solution is mixed and inhaled into a 1mL insulin syringe (shown in Table 1), and the mixed solution is added into 3 volumes of pH 4 sodium citrate solution under the rotation of a magnetic rotor to obtain the liposome iLP 181.

Table 1 the ratio of the components in the liposome iLP181

Example 3 encapsulation of psgPLK1 by liposomes iLP181 to form iLP181/psgPLK1 nanocomplex

The final total required cell level of psgPLK1 was first calculated (total: 1): for example, in a 6-well plate transfection, one well would need to be loaded with 1.6. mu.g of psgPLK1(600 ng/. mu.L); 2) animal level: the typical dose was 0.5/1mg/kg, i.e., one mouse weighing 20g required 10/20 μ g of psgPLK1(600 ng/. mu.L)) prepared as a 1.2-fold excess of the total amount required. The psgPLK1 concentration was then diluted to 1000 ng/. mu.L. psgpplk 1 was mixed with buffer (50% aqueous ethanol) at a volume ratio of 1:1, mixing; an aqueous solution of psgPLK1 (1000 ng/. mu.L) was then mixed with liposomes iLP181 in a 1:1 volume ratio. Subsequently, the mixture was incubated at 50 ℃ for 20min, and then the mixture was dialyzed in 1 XPBS for 2 hours at room temperature in a 100kDa dialysis tube (1 XPBS is used in an amount according to the amount of the particular liposome solution, and generally 1L of 1 XPBS is required per 5mL of the liposome solution), and the beaker containing 1 XPBS was placed on a magnetic stirrer, and the magnetic stirring was maintained at a moderate speed during the dialysis. The uncoated iLP181 and ethanol were replaced by 1 × PBS during dialysis, and the dialyzed liquid was the final liposome nanocomposite.

Examples of the experiments

The advantages of sgrnas, plasmids, nanocomposites are illustrated by the following test experiments.

FIG. 3 is a physical characterization analysis of nanoparticles formed by encapsulating the psgPLK1 plasmid with liposomes iLP 181. FIG. 3A is a schematic representation of the formation of Liposomal Nanoparticles (LNPs). FIG. 3B shows the physicochemical parameters of iLP181/psgPLK1 nanocomposite.

Fig. 4A shows safety of the nanocomplexes at the cellular level, as measured by MTT method, and the cell survival rate of iLP181 treated in fig. 4A is more than 85%, indicating that liposome iLP181 has ideal biocompatibility in vitro.

FIG. 4B shows the pKa values of iLP181 determined by TNS (2- (p-tolyl) -6-naphthalenesulfonic acid) fluorescence probe method. First, iLP181(10mM) solutions were prepared in PBS, followed by a series of iLP181 solutions ranging in pH from 3.00 to 10.00 containing 1. mu.M TNS, 10mM ammonium acetate, 10mM 4-morpholinoethanesulfonic acid (MES), 10mM HEPES, and 130mM NaCl. The fluorescence intensity of each solution was measured with a spectrophotometer at an excitation wavelength of 321nm and an emission wavelength of 445 nm. Fluorescence data were analyzed using sigmoidal best fit analysis. The pKa value is calculated and defined as the pH value causing the half-maximum fluorescence intensity, and the pKa value of 6.43 is obtained, and researches show that ionizable lipid with the dissociation constant pKa of 6.2-6.5 is uncharged under the condition of normal physiological environment (pH7.4) and positively charged under the micro-acid environment such as lysosome and the like, and the escape and the release of loaded nucleic acid are effectively promoted through the proton sponge effect and the colloid osmotic pressure effect.

FIG. 5 is an iLP181/psgPLK1 stability assessment in buffer and Human serum (Human serum, HS). The particle size (A) and the potential (B) of the nano-composite in a buffer solution and Human serum (Human serum, HS) can be obtained from the figure, and the size and the zeta potential of iLP181/psgPLK1 are stable and uniform after being incubated for 2 weeks in a PBS buffer solution or 10% Human serum, which shows that iLP181/psgPLK1 has good stability and relative safety.

FIG. 6 shows that GFP is expressed at 24h and 48h in PX458-E or psgPLK1 (FIG. 6A) after Lipo2000 is transfected into SiPLK1 (the sense strand sequence of SiPLK1 is SEQ ID NO:10 and the antisense strand sequence of SiPLK1 is SEQ ID NO:11 in HEK293A cells to evaluate whether Lipo2000/psgPLK1 can enter the nucleus to function as a plasmid, and during practical use, a terminal overhang, namely dT, is added to the 3' ends of the sense strand sequence and the antisense strand sequence to enhance the stability of the antisense strand sequence), and PX458-E or psgPLK1 is treated. FIG. 6B is the quantification of FIG. 6A, showing that Lipo2000/psgPLK1 can bring psgPLK1 into the HEK293A nucleus.

FIG. 7 is a study progression of gene editing and cellular entry mechanisms mediated by iLP181/psgPLK 1. FIG. 7A shows the results of evaluation of the gene editing performance of Cas9-sgPLKs (psgPLK1-1, psgPLK1-2, psgPLK1-3 and psgPLK1-4) plasmids specifically recognizing human PLK1 site with respect to human PLK1 gene, and the gene editing efficiency of Cas9-sgPLKs (psgPLK1-1, psgPLK1-2, psgPLK1-3 and psgPLK1-4) transfected with Lipo2000 in HEK293A cells, and the internal reference primer sequences used for psgPLK1-1, psgPLK1-2, psgPLK1-3 and psgPLK1-4 were hGAPDH: (5 'end sequence is SEQ ID NO:6, 3' end sequence is SEQ ID NO:7), and PLK1 gene primer sequence is as follows: hPLK1(5 'end sequence is SEQ ID NO:8, 3' end sequence is SEQ ID NO:9), the used sense strand sequence of sipLK1 is SEQ ID NO:10, and the antisense strand sequence of sipLK1 is SEQ ID NO: 11. The results showed that the sgPLK1-1 sequence was most active and that the gene editing efficiency reached 33% (FIG. 7A). The psgPLK1 plasmid is wrapped by iLP181 in HepG2-Luc cells, and the DNA knockout of the PLK1 gene can be obviously mediated. FIG. 7B is a long-term evaluation of iLP181/psgPLK1 mediated gene editing in HepG2-Luc cells, which revealed that this system could achieve gene editing up to 7 days at the cellular level and 32% gene editing efficiency in the iLP181/psgPLK1 group at the seventh day after transfection.

FIG. 8A shows iLP181 determination of whether the uptake by HepG2-Luc cells is ApoE dependent, FIG. 8B shows a quantitative analysis of FIG. 8A by flowjo7.6.1 software, and the transfection procedures of Opti-MEM and DMEM representing iLP181/Cy5-NA, respectively, were performed in serum-free medium (Opti-MEM) and complete medium (DMEM), respectively; DMEM + ApoE represents iLP181/Cy5-NA/ApoE mixture transfected cells, the transfection procedure being carried out in DMEM; iLP181/Cy5-NA represents the binding of iLP181 to Cy5 labeled nucleic acids, as described above. From the results, it is found that the Cy5 fluorescence signal is stronger when the gene is transfected by the iLP181/Cy5-NA/ApoE group, which indicates that iLP181/Cy5-NA enters cells more, and iLP181/Cy5-NA is absorbed by HepG2-Luc cells and is ApoE-dependent.

Figure 9 is an endosome/lysosome escape effect and co-localization analysis of iLP 181. FIG. 9A is a graph of subcellular localization and endosome escape analysis of iLP181/Cy5-NA in HepG2-Luc cells at different transfection time points by confocal laser microscopy, scale: 20 μm. FIG. 9B shows the Mean Fluorescence Intensities (MFIs) of iLP181/Cy5-NA at different time points after entering HepG2-Luc cells, and the results demonstrate that the MFI of iLP181/Cy5-NA gradually increases with the time of transfection, indicating that more and more nucleic acid is accumulated in HepG2-Luc cells. FIG. 9C is a co-localization analysis of Cy 5-labeled nucleic acid with lysotracker-stained endosomes/lysosomes. As can be seen, iLP181/Cy5-NA entered the cell 1-3 hours after transfection and was internalized by lysosomes/endosomes; about 3-5h after transfection, iLP181/Cy5-NA escaped from the endosome/lysosome, indicating that iLP181 had a better effect of endosome escape.

FIG. 10 shows the distribution of iLP181/Cy5-NA in mice. The experiments were divided into 3 groups: PBS group, Naked Cy5-NA and iLP181/Cy 5-NA. At various times after dosing, fluorescence imaging was performed on major organs both systemically and ex vivo using an in vivo imaging system (fig. 10A). Isolated organs were quantitatively analyzed for Mean Fluorescence Intensities (MFIs) at 6h, 12h, and 24h (fig. 10B). Wherein G1 is PBS group (negative control), G2 is Naked Cy5-NA, and G3 is iLP181/Cy 5-NA. Cy5-NA represents Cy 5-labeled nucleic acid, which is bound to iLP181 in the same manner as mentioned above. First, 3 formulations were injected into the body of mice via tail vein. After 1, 6, 12 and 24h of administration, the fluorescence distribution of Cy5 in mice or isolated major organs was examined using a living body imaging system, and the results showed that the Cy5 fluorescence signal was mainly enriched in mouse liver and tumor sites, the Cy5 fluorescence signal was only enriched in mouse liver and tumor sites at 24h, and the fluorescence signals of other sites were metabolized (FIGS. 10A and 10B).

FIG. 11 shows iLP181 distribution of 181/Plasmid in mice. The experiments were divided into 3 groups: PBS group, Naked Plasmid and iLP 181/Plasmid. At various times after dosing, (a) fluorescence imaging of isolated major organs and (B) quantification of Mean Fluorescence Intensities (MFIs) at tumor sites using a live imaging system. (A) RFP imaging was performed on isolated organs at 2d, 3d and 5d post-dose, respectively. iLP181/Plasmid represents that iLP181 binds to a reporter Plasmid (RFP) expressing Red Fluorescent Protein in the same manner as iLP 181. First, 3 formulations were injected into the body of mice via tail vein. After 2d, 3d and 5d administration, RFP expression in isolated major organs of mice was observed using a living imaging system, respectively, and the results showed that RFP expression began at the tumor site of mice 24h after tail vein administration, while the signal was expressed only at the tumor site and continued until the 5d RFP fluorescence signal remained (fig. 11B).

FIG. 12 shows the in vivo anti-tumor profile of iLP181/psgPLK1 formulations, divided into 4 groups: the PBS group, iLP181/PX458-E, iLP181/siPLK1 and iLP181/psgPLK1, 4 formulations were injected intratumorally into mice and the tumor volume of the mice was recorded. Wherein FIG. 12A is a tumor suppression protocol; FIG. 12B is bioluminescent imaging of animal tumors at the end of the experiment; FIG. 12C is a growth curve of tumor volume throughout the experiment; FIG. 12D is the expression of PLK1 mRNA in tumor tissue at the end of the experiment; g1 is PBS, G2 is iLP181/PX458-E, G3 is iLP181/sipLK1, G4 is iLP181/psgPLK 1. The results show that iLP181/psgPLK1 formulation significantly reduced the number of tumor cells (decreased fluorescence signal) by intratumoral local administration of this treatment modality (fig. 12B), significantly controlled the proliferation of HepG2-Luc tumors (fig. 12C), and also knocked down the expression of the PLK1 gene (fig. 12D).

FIG. 13 is an in vivo antitumor safety factor evaluation of iLP181/psgPLK1 formulations, with experiments divided into 4 groups: PBS, iLP181/siPLK1, iLP181/PX458-E, and iLP181/psgPLK1, 4 formulations were injected intratumorally into mice, and the main organs were paraffin-embedded, sectioned, and analyzed for H & E staining at the experimental end-point. The results showed that iLP181/psgPLK1 did not cause any damage to major organs and was a safe administration method.

Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; features from the above embodiments, or from different embodiments, may also be combined, steps may be implemented in any order, and there are many other variations of the different aspects of one or more embodiments in this application, as described above, which are not provided in detail for the sake of brevity.

It is intended that the one or more embodiments of the present application embrace all such alternatives, modifications and variations as fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements, and the like that may be made without departing from the spirit and principles of one or more embodiments of the present disclosure are intended to be included within the scope of the present disclosure.

<110> Beijing university of science and technology

<120> sgRNA, plasmid, nano-composite for specifically recognizing human PLK1 locus and application

<160>12

<210>1

<211>20

<212>DNA

<213> Artificial sequence

<400>1

tacctacggc aaattgtgct 20

<210>2

<211>24

<212>DNA

<213> Artificial sequence

<400>2

cacctaccta cggcaaattg tgct 24

<210>3

<211>25

<212>DNA

<213> Artificial sequence

<400>3

caccgtacct acggcaaatt gtgct 25

<210>4

<211>24

<212>DNA

<213> Artificial sequence

<400>4

caccctcccc gtcatattcg actt 24

<210>5

<211>25

<212>DNA

<213> Artificial sequence

<400>5

caccgctccc cgtcatattc gactt 25

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<400>6

agaaggctgg ggctcatttg 20

<210>7

<211>20

<212>DNA

<213> Artificial sequence

<400>7

aggggccatc cacagtcttc 20

<210>8

<211>20

<212>DNA

<213> Artificial sequence

<400>8

gcccctcaca gtcctcaata 20

<210>9

<211>20

<212>DNA

<213> Artificial sequence

<400>9

tacccaaggc cgtacttgtc 20

<210>10

<211>21

<212>RNA

<213> Artificial sequence

<400>10

ugaagaagau cacccuccuu a 21

<210>11

<211>21

<212>RNA

<213> Artificial sequence

<400>11

uaaggagggu gaucuucuuc a 21

<210>12

<211>2160

<212>DNA

<213> Artificial sequence

<400>12

ggaggctctg ctcggatcga ggtctgcagc gcagcttcgg gagcatgagt gctgcagtga 60

ctgcagggaa gctggcacgg gcaccggccg accctgggaa agccggggtc cccggagttg 120

cagctcccgg agctccggcg gcggctccac cggcgaaaga gatcccggag gtcctagtgg 180

acccacgcag ccggcggcgc tatgtgcggg gccgcttttt gggcaagggc ggctttgcca 240

agtgcttcga gatctcggac gcggacacca aggaggtgtt cgcgggcaag attgtgccta 300

agtctctgct gctcaagccg caccagaggg agaagatgtc catggaaata tccattcacc 360

gcagcctcgc ccaccagcac gtcgtaggat tccacggctt tttcgaggac aacgacttcg 420

tgttcgtggt gttggagctc tgccgccgga ggtctctcct ggagctgcac aagaggagga 480

aagccctgac tgagcctgag gcccgatact acctacggca aattgtgctt ggctgccagt 540

acctgcaccg aaaccgagtt attcatcgag acctcaagct gggcaacctt ttcctgaatg 600

aagatctgga ggtgaaaata ggggattttg gactggcaac caaagtcgaa tatgacgggg 660

agaggaagaa gaccctgtgt gggactccta attacatagc tcccgaggtg ctgagcaaga 720

aagggcacag tttcgaggtg gatgtgtggt ccattgggtg tatcatgtat accttgttag 780

tgggcaaacc accttttgag acttcttgcc taaaagagac ctacctccgg atcaagaaga 840

atgaatacag tattcccaag cacatcaacc ccgtggccgc ctccctcatc cagaagatgc 900

ttcagacaga tcccactgcc cgcccaacca ttaacgagct gcttaatgac gagttcttta 960

cttctggcta tatccctgcc cgtctcccca tcacctgcct gaccattcca ccaaggtttt 1020

cgattgctcc cagcagcctg gaccccagca accggaagcc cctcacagtc ctcaataaag 1080

gcttggagaa ccccctgcct gagcgtcccc gggaaaaaga agaaccagtg gttcgagaga 1140

caggtgaggt ggtcgactgc cacctcagtg acatgctgca gcagctgcac agtgtcaatg 1200

cctccaagcc ctcggagcgt gggctggtca ggcaagagga ggctgaggat cctgcctgca 1260

tccccatctt ctgggtcagc aagtgggtgg actattcgga caagtacggc cttgggtatc 1320

agctctgtga taacagcgtg ggggtgctct tcaatgactc aacacgcctc atcctctaca 1380

atgatggtga cagcctgcag tacatagagc gtgacggcac tgagtcctac ctcaccgtga 1440

gttcccatcc caactccttg atgaagaaga tcaccctcct taaatatttc cgcaattaca 1500

tgagcgagca cttgctgaag gcaggtgcca acatcacgcc gcgcgaaggt gatgagctcg 1560

cccggctgcc ctacctacgg acctggttcc gcacccgcag cgccatcatc ctgcacctca 1620

gcaacggcag cgtgcagatc aacttcttcc aggatcacac caagctcatc ttgtgcccac 1680

tgatggcagc cgtgacctac atcgacgaga agcgggactt ccgcacatac cgcctgagtc 1740

tcctggagga gtacggctgc tgcaaggagc tggccagccg gctccgctac gcccgcacta 1800

tggtggacaa gctgctgagc tcacgctcgg ccagcaaccg tctcaaggcc tcctaatagc 1860

tgccctcccc tccggactgg tgccctcctc actcccacct gcatctgggg cccatactgg 1920

ttggctcccg cggtgccatg tctgcagtgt gccccccagc cccggtggct gggcagagct 1980

gcatcatcct tgcaggtggg ggttgctgta taagttattt ttgtacatgt tcgggtgtgg 2040

gttctacagc cttgtccccc tccccctcaa ccccaccata tgaattgtac agaatatttc 2100

tattgaattc ggaactgtcc tttccttggc tttatgcaca ttaaacagat gtgaatattc 2160

Claims (4)

1. A nanocomposite, comprising a liposome iLP181, and a plasmid, the plasmid comprising a sgRNA that specifically recognizes the human PLK1 site, the sequence of the sgRNA being SEQ ID NO:2 or SEQ ID NO: 3;

the liposome iLP181 consists of key lipid iLY1809, phospholipid, cholesterol and PEG-lipid; the key lipid iLY1809 has the structural formula:

；

the molar ratio of the key lipid iLY1809 to the phospholipid to the cholesterol to the PEG-lipid is (25-57) to (1-37) to (25-67) to (0.1-19);

liposome iLP181 is prepared by dissolving key lipid iLY1809, phospholipid, cholesterol and PEG-lipid in ethanol solvent, stirring, and adding into sodium citrate solution to obtain liposome iLP 181.

2. The nanocomposite of claim 1, wherein the phospholipid comprises one or more of distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylcholine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, egg yolk lecithin.

3. The nanocomposite of claim 1, wherein the PEG-lipid comprises one or more of distearoylphosphatidylethanolamine-PEG, dimyristyl glycerol, and dimethacrylate.

4. Use of the nanocomposite of claim 1 in the preparation of a medicament for the treatment of cancer.

Images