A peptide imprinting and transfection complex and its preparation method, and a method for inducing cell differentiation into dental stem cells using the peptide imprinting and transfection complex.

Abstract

This invention relates to a peptide imprinting and transfection complex, its uses and preparation method, and a method for inducing cell differentiation into dental stem cells (DSCs) using the peptide imprinting and transfection complex. Specifically, this invention relates to a peptide imprinting and transfection complex comprising dCas9 peptide-imprinted chitosan composite nanoparticles and a dCas9 protein-guide RNA complex, wherein the dCas9 peptide-imprinted chitosan composite nanoparticles bind to the dCas9 protein-guide RNA complex. This peptide imprinting and transfection complex can be mixed with target cells, allowing it to enter the target cells and thereby regulate at least one gene related to inducing cell differentiation into dental stem cells, thus promoting the successful differentiation of target cells into dental stem cells. These dental stem cells can be applied to tooth tissue regeneration, thereby solving the problem of damaged teeth that cannot regenerate.

Description

Technical Field

[0001] This invention relates to the technical field of peptide imprinting combined with the CRISPR / dCas9 system, and more particularly to a peptide imprinting transfection complex comprising dCas9 peptide-imprinted chitosan composite nanoparticles and a dCas9 protein-guide RNA complex, wherein the dCas9 peptide-imprinted chitosan composite nanoparticles bind to the dCas9 protein-guide RNA complex. The peptide imprinting transfection complex of this invention can be mixed with target cells, allowing the complex to enter the target cells and thereby regulate at least one gene related to inducing cell differentiation into dental stem cells (DSCs), thus successfully differentiating the target cells into dental stem cells. Prior Technology

[0002] After human baby teeth fall out, permanent teeth grow in. Once permanent teeth have formed, teeth cannot be replaced or grown again. Therefore, when teeth are damaged and broken or decayed and need repair, dentists often replace them with dentures or fill them with light-cured resin as substitutes. With the increasing prevalence of an aging society, the oral health of the elderly has become an important public health issue. Therefore, how to regenerate damaged teeth is also a focus of current medical technology.

[0003] Another emerging and popular technology is molecularly imprinted polymer (MIP), which has the characteristics of relative stability, simple operation process and low price. It has been rapidly developed to create new identification materials, and thus applied to the separation of optically active drugs or cells, biosensors, drug imprinting polymer particles as drug carriers, etc. These imprinted molecules are similar to natural antibodies, with high specificity and selectivity.

[0004] Currently, transplanting stem cells and differentiating them into dental stem cells is considered one of the most promising methods for treating damaged or non-regenerating teeth. However, a technique combining molecular imprinting with nanoparticle carriers to induce target cells to differentiate into dental stem cells has not yet been developed. Summary of the Invention

[0005] [The technical problem that the invention aims to solve]

[0006] One common method for regulating cellular gene expression is the use of vectors. Specifically, this involves transfecting cells with plasso DNA containing a specific gene or small interfering RNA that inhibits the expression of a specific gene, thereby amplifying or suppressing the expression of the target gene. Commonly used vectors include viral and non-viral vectors.

[0007] Although viral vectors (such as adenoviruses, lentiviruses, or retroviruses) have high transfection efficiency, they pose safety risks to cells, including immunogenicity, toxicity, carcinogenicity, and the risk of insertion and integration into the host DNA.

[0008] On the other hand, non-viral vectors (such as cationic liposomes or degradable cationic polymers) can overcome the problems of viral vectors in terms of immunogenicity and safety. They also have advantages such as controllable physicochemical properties, good reproducibility, ease of manufacture, low manufacturing cost, strong DNA loading capacity, and large-scale production capability, making them a popular target in gene vector research. However, non-viral vectors still have disadvantages, the biggest of which is that their transfection efficiency is much lower than that of viral vectors.

[0009] Therefore, in order to induce target cells into dental stem cells by regulating the amount of gene expression in cells, it is urgent to develop a drug carrier that has the advantages of low cytotoxicity, high gene transfection efficiency, good biocompatibility, controllable degradation performance, safety and practicality and low manufacturing cost. It is also necessary to develop a gene expression regulation method that uses a carrier with excellent cell transfection efficiency and can effectively regulate gene expression. [Technical means]

[0010] Therefore, the objective of this invention is to utilize peptide-imprinted nanoparticles combined with CRISPR / dCas9 technology to induce target cells into pluripotent stem cells (iPSCs), which can then differentiate into dental stem cells. To achieve the above objective, this invention provides the following technical means.

[0011] In a single-state sample, a peptide imprinting and transfection complex comprises: Chitosan composite nanoparticles imprinted with dCas9 peptide; and dCas9 protein-guide RNA complex; The chitin-based nanoparticles imprinted with dCas9 peptides bind to the dCas9 protein-guide RNA complex.

[0012] In some embodiments, the dCas9 peptide is peptide Q, and its sequence is SEQ ID NO:1.

[0013] In some embodiments, the dCas9 peptide-imprinted chitin composite nanoparticles include chitin imprinted with dCas9 peptide and a plurality of magnetic nanoparticles (MNPs).

[0014] In some implementations, the dCas9 protein is a dCas9 fusion protein.

[0015] In some embodiments, the dCas9 fusion protein system comprises the dCas9 protein and a protein with transcriptional activation function.

[0016] In some embodiments, the guide RNA system comprises transactivating CRISPR RNA (tracrRNA) and crRNA, wherein the crRNA is selected from the group consisting of SEQ ID NO:2~10.

[0017] In some embodiments, the crRNA sequence is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

[0018] In some embodiments, the crRNA sequence is SEQ ID NO:6.

[0019] In some implementations, the crRNA sequence is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

[0020] In some implementations, the gRNA system, composed of tracrRNA and crRNA, is used to guide the dCas9 fusion protein to bind to the target gene site. Specifically, the gRNA contains a complementary fragment to the target gene sequence and a protospacer adjacent motif (PAM). The PAM sequence consists of three specific nucleotides (NGG, PAM sequence) following the complementary fragment of the gRNA. The dCas9 fusion protein can activate the target gene by recognizing the PAM sequence of the target gene.

[0021] In another embodiment, the present invention provides a method for inducing cell differentiation into dental stem cells using the aforementioned peptide imprinting transfection complex, which includes the following steps: Pluripotent stem cells were obtained by transfecting target cells with the peptide imprinting transfection complex containing crRNA sequences selected from SEQ ID NO:2~SEQ ID NO:4; Dental stem cells were obtained by transfecting the pluripotent stem cells with the peptide imprinting transfection complex containing the crRNA sequence SEQ ID NO:6. The target cells are living cells in vitro.

[0022] In another embodiment, the present invention provides a method for inducing target cells to differentiate into dental tissue using the aforementioned peptide imprinting and transfection complex, comprising: Pluripotent stem cells were obtained by transfecting target cells with the peptide imprinting transfection complex containing crRNA sequences selected from SEQ ID NO:2~SEQ ID NO:4; Dental stem cells were obtained by transfecting the pluripotent stem cells with the peptide imprinting transfection complex containing the crRNA sequence SEQ ID NO:6. Tooth tissue cells were obtained by transfecting the tooth stem cells with the peptide imprinting transfection complex containing the crRNA sequence SEQ ID NO:7~SEQ ID NO:10; The target cells are living cells obtained in vitro.

[0023] In another embodiment, the present invention provides a use of the aforementioned peptide imprinting and transfer complex for manufacturing tooth tissue. The aforementioned tooth tissue may be, for example, odontogenic mesenchyme, dental placode, condensed dental mesenchyme, or enamel knot. In another embodiment, the peptide imprinting and transfer complex can be used to promote the calcification of tooth tissue.

[0024] In some embodiments, the target cells used to induce differentiation into dental stem cells or mature dental tissue may be human embryonic kidney cells (HEK293).

[0025] In another embodiment, the present invention provides a method for preparing a peptide imprinting and transfection complex, characterized by comprising: Step 1: Mix the dCas9 peptide aqueous solution, chitin solution, and magnetic nanoparticle solution to obtain a mixture for molecular imprinting; Step 2: Stir the molecular imprinting mixture thoroughly for 2-60 minutes to coat the magnetic nanoparticles with chitin; Step 3: Using a strong magnet, the molecular imprinting mixture is separated into a supernatant layer and a chitin composite nanoparticle layer, and the supernatant layer is removed; Step 4: Add deionized water, 0.5-95% ethanol, and 0.001-0.05% sodium dodecyl sulfate, and mix thoroughly. Step 5: Using a strong magnet, the molecular imprinting mixture is separated again into a supernatant layer and a chitin composite nanoparticle layer. The supernatant layer is then removed to remove the dCas9 peptide. This step is repeated at least twice. Step 6: Mix the dCas9 peptide-imprinted chitin composite nanoparticles obtained in Step 5 with guide RNA to form a peptide imprinting transfection complex; The aqueous solution of this dCas9 peptide contains peptide Q, the sequence of which is SEQ ID NO:1; This guide RNA system comprises tracrRNA and crRNA, and the sequence of the crRNA is selected from the group consisting of SEQ ID NO:2~10; This chitin solution contains chitin with a molecular weight of 370~1000 K. [Effects of the Invention]

[0026] This invention provides a peptide imprinting transfection complex using the CRISPR / dCas9 system, and provides a technique for inducing cell differentiation into dental stem cells using the peptide imprinting transfection complex. This technique can regulate Sox2 (SEQ ID NO:2), KLF-4 (SEQ ID NO:3), c-Myc (SEQ ID NO:4), OCT4 (SEQ ID NO:5), Pitx2 (SEQ ID NO:6), Msx2 (SEQ ID NO:7), Lef1 (SEQ ID NO:8), Edar (SEQ ID NO:9), and Runx2 (SEQ ID NO:10). [Table 1] Target gene name SEQ ID NO Genome location coordinates PAM sequence Sox2 2 +chr3:181711907 TGG KLF-4 3 +chr9:107489875 TGG c-Myc 4 +chr8:127735326 GGG OCT4 5 -chr6:31165750 GGG Pitx2 6 +chr4:110618111 GGG Msx2 7 +chr5:174729467 TGG Lef1 8 +chr4:108083348 GGG Edar 9 +chr2:108906309 TGG Runx2 10 -chr6:45512300 AGG

[0027] The induction of human embryonic kidney cells to differentiate into dental stem cells is divided into the following two stages: the first stage involves activating the Sox2, KLF-4, c-Myc, and OCT4 genes to induce human embryonic kidney cells (HEK293) to dedifferentiate into induced pluripotent stem cells (iPSCs), which takes approximately 3 to 10 days; the second stage involves activating the Pitx2 gene to induce iPSCs to differentiate into dental stem cells, which also takes approximately 3 to 10 days.

[0028] Inducing dental stem cells to differentiate into mature tooth tissue can be divided into two stages: Stage 1: Activating Msx2, Lef1, and Edar genes to induce dental stem cells to differentiate into odontogenic mesenchyme and dental placode, which takes about 3 days; Stage 2: Activating Runx2, Msx2, Lef1, and Edar genes to induce odontogenic mesenchyme and dental placode to differentiate into condensed dental mesenchyme and enamel knot, which also takes about 3 days.

[0029] Molecular imprinting technology can significantly increase the recognition specificity of nanoparticle carriers for dCas9 fusion proteins. Furthermore, the highly efficient peptide imprinting transfection complex of this invention can effectively promote the differentiation of target cells into dental stem cells. In addition, because the transfection complex of this invention has extremely low cytotoxicity, it will not harm normal cells. This invention can reduce the cost and time spent producing various dental stem cells. Since dental stem cells can be used to produce mature tooth tissue, this invention can be further used to produce autologous tooth tissue from patients, thereby treating damaged teeth. Simple Explanation of the Diagram

[0030] [Figure 1] Figure 1A is an immunofluorescence staining image of the proteins of the inducible dedifferentiation genes c-Myc, Sox2, OCT4 and KLF-4 of iPSC obtained in Example 3 of the present invention; Figure 1B is an enzyme AP staining image of iPSC obtained in Example 3. [Figure 2] Figure 2A is an immunofluorescence staining image of the target protein Pitx2 in the iPSCs obtained in Example 3 of the present invention after being induced with 10 nM and 20 nM Pitx2 crRNA for 2 days; Figure 2B is the area optical density (AOD) analysis result of the staining image in Figure 2A; Figure 2C is the expression of each gene in the obtained tooth stem cells by qPCR after being induced with different concentrations of Pitx2 crRNA in the iPSCs obtained in Example 3 of the present invention for 2 days. [Figure 3] Analysis results of cell survival rate and number of surviving cells of iPSCs obtained in Example 3 of the present invention after being induced with different concentrations of Pitx2 crRNA for 2 days. [Figure 4] Figure 4A shows the results of qPCR detection of Msx2, Lef1, and Edar genes after the dental stem cells obtained in Example 4 of this invention were induced with different concentrations of Msx2, Lef1, and Edar crRNA for 2 days. Figure 4B shows the results of qPCR detection of genes related to dental interstitium after the dental stem cells obtained in Example 4 of this invention were induced with different proportions of Msx2, Lef1, and Edar crRNA for 2 days. Figure 4C shows the results of qPCR detection of genes related to dental lamina after the dental stem cells obtained in Example 4 of this invention were induced with different proportions of Msx2, Lef1, and Edar crRNA for 2 days. Figure 4D shows the results of qPCR detection of these four genes and genes related to concentrated dental interstitium and enamel after the dental interstitium and dental lamina obtained in Example 5 of this invention were transfected with different proportions of Runx2, Msx2, Lef1, and Edar crRNA for 2 days. [Figure 5] Figure 5A shows the immunofluorescence staining results of CD49f, CD90, CD71, and CD140a at various time points after induction treatment with different ratios of crRNA in Example 6 of the present invention; Figure 5B shows the qPCR detection results of CD49f, CD90, CD71, and CD140a at various time points after induction treatment with different ratios of crRNA in Example 6 of the present invention; Figure 5C shows the AOD value analysis results of CD49f, CD90, CD71, and CD140a at various time points after induction treatment with different ratios of crRNA in Example 6 of the present invention; Figure 5D shows the immunofluorescence staining results of CD49f, CD90, CD71, and CD140a at various time points after induction treatment with different ratios of crRNA in Example 6 of the present invention. Figure 5E shows the AOD values ​​of Vimentin and CK14 at each time point after RNA induction treatment in Example 6 of the present invention, after induction treatment with different ratios of crRNA; Figure 5F shows the immunofluorescence staining results of DMP1, Amelogenin and STOR1 at each time point after crRNA induction treatment with different ratios of crRNA; Figure 5G shows the AOD values ​​of DMP1, Amelogenin and STOR1 at each time point after crRNA induction treatment with different ratios of crRNA in Example 6 of the present invention. [Figure 6] Results of Msx2, Lef1 and Edar immunofluorescence staining of dental stem cells in Example 4 of the present invention. [Figure 7] Immunofluorescence staining results of Runx2, Msx2, Lef1, and Edar of dental stem cells in Example 4 of the present invention. Implementation

[0031] The following describes embodiments of the present invention, but it is not intended to limit the present invention to being implemented in the following manner, but rather to illustrate the details of the present invention and the effects of its implementation.

[0032] The definitions of terms used in this invention are based on the generally accepted definitions in the fields of chemical engineering and biotechnology, and apply to the entire specification, unless otherwise specified in the specification based on specific circumstances.

[0033] [Example 1]: Preparation of dCas9 peptide-imprinted chitin composite nanoparticles

[0034] [Preparation of magnetic nanoparticle solutions]

[0035] The experimental steps for preparing magnetic nanoparticle solutions are as follows: 1. Mix FeCl₃·6H₂O and FeSO₄·7H₂O in a molar ratio of 1:1 to 5 to prepare a mixed solution of 200 to 1000 mL; 2. Take 1-100 mL of the mixed solution into a 500 mL beaker, place a magnet inside, and cover with aluminum foil; 3. Inject 1~100 mL of 1~14 N sodium hydroxide (NaOH) to obtain an iron ion / sodium hydroxide solution; 4. Place the beaker on a strong magnet to separate the solution into two layers: a clear supernatant and a precipitate. Remove the supernatant. The precipitate contains magnetic nanoparticles obtained by magnetizing iron ions. 5. Add deionized water, stir on a magnetic stirrer for 1-30 minutes, then place the beaker on a strong magnet to separate the solution into layers, producing a clear supernatant and a precipitate. Remove the supernatant. Repeat this step 1-5 times to obtain a high-purity magnetic nanoparticle solution. 7. Remove the magnet and add 1-100 mL of deionized water. Store the solution in a sample bottle.

[0036] [Preparation of dCas9 peptide-imprinted chitosan composite nanoparticles]

[0037] The experimental steps for preparing dCas9 peptide-imprinted chitosan composite nanoparticles are as follows: 1. Pour 2-10 mL of deionized water into an empty sample bottle and pre-cool it at 4°C for 5-60 minutes, then place it in a prepared ice bath. 2. Prepare an aqueous solution of dCas9 peptide with a concentration of 0.01~20 μg / mL (the dCas9 peptide to be imprinted in this example is peptide Q, whose peptide sequence is SEQ ID NO:1), a 0.001~5 wt% chitin microacid solution (using chitin with a molecular weight of 370~1000 K, dissolved in 0.001~20 wt% acetic acid solution), and a magnetic nanoparticle solution; choosing chitin as a carrier has many advantages, such as chitin being positively charged, thus it can easily enter negatively charged cells through endocytosis, low cytotoxicity, and high transfection efficiency; 3. Remove water from 10–1000 μL of magnetic nanoparticle solution, and mix thoroughly with 10–1000 μL of chitin microacid solution and 10–1000 μL of dCas9 peptide aqueous solution to obtain a mixture for molecular imprinting; 4. Gradually add the molecular imprinting mixture to the sample vials and stir thoroughly for 2–60 minutes to coat the magnetic nanoparticles with chitin; 5. After the reaction is complete, place the mixed solution on a strong magnetic plate and let it stand. The solution will separate into layers, forming a clear supernatant layer and a chitin composite nanoparticle layer. Remove the supernatant, add 0.5–10 mL of deionized water, 0.5–95% ethanol, and 0.001–0.05% sodium dodecyl sulfate (SDS), and shake for 1–30 minutes. Place the mixed solution on the strong magnetic plate again and remove the supernatant to complete one washing step, which is used to remove the peptide. Repeat the above washing step once more, remove the supernatant, and obtain dCas9 peptide-imprinted chitin composite nanoparticles (MPIPs). In the following examples, the dCas9 peptide-imprinted chitin composite nanoparticles obtained using peptide Q imprinting are referred to as MQIPs.

[0038] [Example 2]: dCas9-VPR transfection and dCas9-VPR fusion protein extraction

[0039] The dCas9 fusion protein used in this embodiment is obtained by fusing a mutant Cas9 gene (dCas9) without endonuclease activity with a protein gene with transcription activation activity using molecular biotechnology to obtain a plastid DNA, which is then transfected into cells to make the cells express the dCas9 fusion protein.

[0040] The dCas9 fusion protein used in this embodiment is the dCas9-VPR fusion protein (dCas9-VPR) which is a fusion of dCas9 and VP64-p65-Rta (VPR).

[0041] The cell transfection and dCas9-VPR fusion protein extraction steps are as follows: On day 0 (before transfection), HEK293 cells were isolated from the cell culture dish using trypsin. The cells were agitated to suspend them in the trypsin solution. Then, 1 mL of the cell suspension was added to 9 mL of culture medium and mixed thoroughly. 5 mL of this mixture was then added to two separate cell culture dishes and incubated for 6–96 hours. On day 1 of culture, 1–1000 μL of dCas9-VPR plasso DNA (1 μg / mL) was mixed with 0.05–10 mL of jetPRIME buffer, followed by the addition of another 1–1000 μL of jetPRIME buffer. After standing for 5–60 minutes, half of this mixture was added to each of the two cell culture dishes and incubated for 24–96 hours for cell transfection. On day 2, cell proteins were extracted, all at low temperature. First, the culture medium was removed from the culture dish, and 1–1000 μL of protease inhibitor and 0.01–2 mL of cell lysis buffer were prepared. The buffer was evenly added to two culture dishes. After scraping the dishes back and forth once with a spatula, the mixture was incubated for 30 minutes. After the incubation period, the cell culture from both dishes was collected into 15-100 mL centrifuge tubes. The cells were then broken up using an ultrasonic cell disruptor to release the dCas9 fusion protein. All cell culture was then aliquoted into microcentrifuge tubes, and the supernatant was collected. The magnetic composite nanoparticle solution was added to this supernatant, and the dCas9 fusion protein on the magnetic particles was washed away with deionized water to obtain the dCas9 protein solution.

[0042] [Example 3]: Evaluation of the efficacy of dCas9 peptide-imprinted chitosan composite nanoparticles in inducing HEK293 cells to dedifferentiate into iPSCs

[0043] dCas9 peptide-imprinted chitin composite nanoparticles were delivered to human HEK293 cells, inducing dedifferentiation of HEK293 cells into iPSCs. In this embodiment, the amount of crRNA added to the inducing genes Sox2, KLF-4, c-Myc, and OCT4 was equal and in equal proportions.

[0044] When the dCas9 fusion protein of CRISPR / dCas9 technology functions, it needs to form a dCas9-RNPs complex (dCas9-ribonucleoprotein) with guide RNA (gRNA), thereby using the gRNA sequence to guide dCas9 to a nucleic acid sequence in the cell nucleus that can pair with the gRNA.

[0045] gRNA consists of transactivating CRISPR RNA (tracrRNA) and CRISPR RNA with a specific sequence (crRNA). The selected crRNA sequence determines the nucleic acid sequence in which dCas9 functions in the cell. This example designs crRNAs for genes such as Sox2, KLF-4, c-Myc, and OCT4. The crRNA sequence corresponding to the Sox2 gene is SEQ ID NO:2; the crRNA sequence corresponding to the KLF-4 gene is SEQ ID NO:3; the crRNA sequence corresponding to the c-Myc gene is SEQ ID NO:4; and the crRNA sequence corresponding to the OCT4 gene is SEQ ID NO:5.

[0046] The tracrRNA used in this embodiment is a product (Edit-R tracrRNA) purchased from Horizon Discovery.

[0047] The steps to induce dedifferentiation are as follows: In this embodiment, human HEK293 cells were used as the target cells for transfection. On day 0, HEK293 cells (500–10 x 10³ cells / mL) were seeded. On day 1, 0.5–10 μL each of Sox2, KLF-4, c-Myc, and OCT4 (SEQ ID NO: 2–SEQ ID NO: 5) in equal proportions of crRNA solution and tracrRNA solution (1–100 μL) were mixed to form gRNA. Next, this mixture containing gRNA was mixed with a solution containing purified dCas9 protein (1–100 μL) to form the dCas9-RNPs complex. After removing the water from the dCas9 peptide-imprinted chitin composite nanoparticle solution using a high-power disk, the culture medium, dCas9-RNPs solution, and dCas9 peptide-imprinted chitin composite nanoparticles were mixed, allowing the dCas9 peptide-imprinted chitin composite nanoparticles to adsorb for 5–120 minutes to obtain MQIP-dCas9-RNPs. Next, a magnet was used to separate the residual solution and unreacted dCas9-RNPs from the MQIP-dCas9-RNPs to collect them. The adsorbed MQIP-dCas9-RNPs were then added to fresh culture medium, which was then added to HEK293 cell culture dishes from which the original culture medium had been removed. The cells were then incubated for 12–96 hours for transfection.

[0048] As shown in Figure 1, after culturing HEK293 cells for 24-96 hours, immunofluorescence staining was performed to detect the expression of target proteins Sox2, KLF-4, c-Myc, and OCT4 within the HEK293 cells, verifying whether HEK293 cells had been successfully induced to dedifferentiate into iPSCs. The staining pattern of the nucleic acid staining agent DAPI was used as an indicator of cell viability. After culturing the cells for another 7 days, enzyme AP staining was performed to confirm whether the differentiation into iPSCs had been successful. In Figure 1A, the images of each group, from left to right, are: cell appearance observed by optical microscope (OM), DAPI staining results, staining results of target proteins c-Myc, Sox2, OCT4, and KLF-4, and an overlay image of DAPI and target protein staining results (the upper right image shows the overlay of DAPI with c-Myc and Sox2 staining results, and the lower right image shows the overlay of DAPI with OCT4 and KLF-4 staining results). Figure 1B shows the enzyme AP staining results. The staining results in Figures 1A-1B confirm that the c-Myc, Sox2, OCT4, and KLF-4 genes were indeed delivered to HEK293 cells and activated, thus successfully inducing HEK293 cells to dedifferentiate into iPSCs.

[0049] [Example 4]: Evaluation of the efficacy of dCas9 peptide-imprinted chitosan composite nanoparticles in inducing iPSC differentiation into dental stem cells.

[0050] The experimental steps are as follows: In this embodiment, iPSCs were used as the target cells for transfection. On day 0, iPSCs (500–10 x 10³ cells / mL) were seeded. On day 1, 0.1–100 μL of 1–50 μM Pitx2 (SEQ ID NO: 6) crRNA solution and 0.1–100 μL of 1–50 μM trace RNA solution were mixed to form a gRNA solution. This gRNA-containing solution was then mixed with a solution containing purified dCas9 protein to form the dCas9-RNPs complex. After removing the water from the dCas9 peptide-imprinted chitosan complex nanoparticle solution using a high-performance disk, the culture medium, dCas9-RNPs solution, and dCas9 peptide-imprinted chitosan complex nanoparticles (10 μg–1 mg) were mixed with 50–5000 μL of culture medium to obtain MQIP-dCas9-RNPs. Next, MQIP-dCas9-RNPs were collected using a magnet. After the MQIP-dCas9-RNPs that had completed the adsorption process were added to fresh culture medium, they were added to iPSC cell culture plates from which the original culture medium had been removed, so that the individual transfection concentrations were 1-5000 nM. The cells were then placed back into the incubator and cultured for 12-96 hours for transfection.

[0051] After culturing iPSCs for 12–96 hours, immunofluorescence staining and quantitative real-time PCR (qPCR) were performed to detect the expression of the target protein Pitx2 within iPSC cells, verifying whether iPSCs had been successfully induced to differentiate into dental stem cells. The staining pattern of the nucleic acid dye DAPI was used as an indicator of cell viability. Figure 2A shows, from left to right, the cell appearance observed under an optical microscope (OM), DAPI staining results, target protein Pitx2 staining results, and an overlay of DAPI and target protein staining results. Figure 2A shows that the Pitx2 gene was indeed delivered to and activated in each iPSC cell, inducing iPSC cells to differentiate into dental stem cells. Figure 2B shows the AOD analysis values ​​of Pitx2 protein fluorescence staining results for dental stem cells induced by 10 nM and 20 nM Pitx2. These values ​​indicate that Pitx2 has a better activation effect at higher concentrations of crRNA. Figure 2C shows the qPCR results of WNT1, WNT3A, BMP4, FGF3, FGF10, SHH, TNF, and EDA genes in the dental stem cells obtained after treatment with 5 nM, 10 nM, 20 nM, and 50 nM Pitx2 crRNA in Example 3. These genes are related to the development of the oral ectoderm and dental lamina and can be used to confirm whether the cells differentiate into deciduous tooth cells. Based on these results, since 20 nM and 50 nM Pitx2 crRNA showed similar gene expression, 20 nM was chosen as the concentration used for Pitx2 crRNA induction in subsequent examples.

[0052] Three days after culturing iPSCs, an MTT assay was performed, and the number of surviving cells was calculated based on DAPI staining results to analyze the cell viability of iPSCs treated with Pitx2 crRNA. Figure 3 shows that compared to unactivated iPSCs, iPSCs treated with 5 nM, 10 nM, and 20 nM Pitx2 crRNA did not show a decrease in cell viability or the number of surviving cells; in fact, they increased by approximately 10%. Therefore, it can be concluded that Pitx2 crRNA treatment does not inhibit the cell activity and growth status of iPSCs.

[0053] [Example 5]: Evaluation of the efficacy of dCas9 peptide-imprinted chitosan composite nanoparticles in inducing dental stem cell differentiation into mature dental tissue.

[0054] The experimental steps are as follows:

[0055] In this embodiment, the dental stem cells obtained in Example 4 were used as the target cells for transfection. 0.1-20 μL of each of three crRNAs (Msx2, Lef1, and Edar, corresponding to SEQ ID NO:7-SEQ ID NO:9) at concentrations of 1-50 μM were mixed with 0.3-30 μL of tracrRNA solution at concentrations of 1-50 μM and allowed to stand for 20-120 minutes to form gRNA. Next, this mixture containing gRNA was mixed with a solution containing purified dCas9 protein (0.6-2000 μL) and allowed to stand for another 20-120 minutes to form dCas9-RNPs complexes. The culture medium, dCas9-RNPs solution, and dCas9 peptide-imprinted chitosan composite nanoparticles were mixed, allowing the dCas9 peptide-imprinted chitosan composite nanoparticles to adsorb for 20-120 minutes to obtain MQIP-dCas9-RNPs. Next, a magnet was used to separate the residual solution and unreacted dCas9-RNPs from the MQIP-dCas9-RNPs to collect the MQIP-dCas9-RNPs. The MQIP-dCas9-RNPs that had completed adsorption were added to a culture dish, and transfection was performed at individual transfection concentrations of 5–500 nM.

[0056] Two days after culturing dental stem cells, immunofluorescence staining and qPCR were performed to detect the expression of target genes Msx2, Lef1, Edar, and tooth development-related genes BMP4, FGF3, FGF10, ACTIVIN, SHH, WNT1, and WNT3A, to verify whether Msx2, Lef1, and Edar had been successfully activated and whether the dental stem cells had been induced to differentiate into dental interstitium and dental lamina. In Figures 4A to 4C, different proportions of three crRNAs (Msx2, Lef1, and Edar) were added to each group along the X-axis. The different colors in the figures correspond to the Msx2:Lef1:Edar addition ratios from left to right as follows: 0.5:1:1, 1:1:1, 2:1:1, 1:0.5:1, 1:1:1, 1:2:1, 1:1:0.5, 1:1:1, and 1:1:2. The 1:1:1 ratio is repeated three times in the figure for comparison with other groups. Figure 4B lists genes related to dental interstitial development, and Figure 4C lists genes related to dental lamina development. Figure 4A shows that Msx2, Lef1, and Edar were successfully activated through transfection. Figures 4B and 4C show changes in the expression of BMP4, FGF3, FGF10, WNT1, and WNT3A after transfection. The expression levels of the above genes were higher when the addition ratio of the three crRNAs was 1:2:1, while the expression of the SHH gene was similar under other addition ratios. Therefore, Msx2:Lef1:Edar=1:2:1 was selected as the better induction concentration.

[0057] In the top row of Figure 6, the images for each group, from left to right, show the cell appearance observed under an optical microscope, the DAPI staining results, the staining results of the target proteins Edar and Msx2, and an overlay image of the staining results of DAPI and these two target proteins. In the bottom row of Figure 6, the images for each group, from left to right, show the cell appearance observed under an optical microscope, the DAPI staining results, the staining results of the target protein Lef1, and an overlay image of the staining results of DAPI and Lef1. The staining results in Figure 6 show that Msx2, Lef1, and Edar were successfully activated, and the dental stem cells differentiated into mature cells.

[0058] After culturing dental stem cells for three days, 0.1-20 μL, 0.1-20 μL, 0.1-10 μL, and 0.1-10 μL of four different crRNAs (Msx2, Lef1, Edar, and Runx2, corresponding to SEQ ID NO:7-SEQ ID NO:10) at concentrations of 1-50 μM were mixed with 0.4-80 μL of tracrRNA solution at concentrations of 1-50 μM and allowed to stand for 20-120 minutes to form gRNA. Next, this mixture containing gRNA was mixed with a solution containing purified dCas9 protein (0.8-2000 μL) and allowed to stand for another 20-120 minutes to form the dCas9-RNPs complex. The culture medium, dCas9-RNPs solution, and dCas9 peptide-imprinted chitin composite nanoparticles were mixed, and the dCas9 peptide-imprinted chitin composite nanoparticles were subjected to adsorption for 20-120 minutes to obtain MQIP-dCas9-RNPs. Next, the residual solution and unreacted dCas9-RNPs were separated from the MQIP-dCas9-RNPs using a magnet to collect the MQIP-dCas9-RNPs. The adsorbed MQIP-dCas9-RNPs were then added to culture dishes to form individual concentrations of 5-500 nM for transfection.

[0059] Five days after culturing dental stem cells, qPCR was performed to detect the expression of target genes Msx2, Lef1, Edar, Runx2, and tooth development-related genes BMP4, FGF3, FGF10, SHH, WNT1, and WNT3A. This verified whether Msx2, Lef1, Edar, and Runx2 had been successfully activated, and whether the dental stem cells were further induced to differentiate into concentrated interdental matrix and enamel. Figure 4D shows different ratios of four crRNAs (Msx2, Lef1, Edar, and Runx2) added, including 0.5:1:2:1, 1:1:2:1, 2:1:2:1, and 1:1:1:1. Figure 4D shows that as the concentration of Runx2 increases, the expression levels of each gene also increase. Therefore, Runx2:Msx2:Lef1:Edar = 2:1:2:1 was selected as the optimal induction concentration.

[0060] In the top row of Figure 7, the images for each group, from left to right, show the cell appearance observed under an optical microscope, the DAPI staining results, the staining results of the target proteins Edar and Msx2, and an overlay image of the staining results of DAPI and these two target proteins. In the bottom row of Figure 7, the images for each group, from left to right, show the cell appearance observed under an optical microscope, the DAPI staining results, the staining results of the target proteins Runx2 and Lef1, and an overlay image of the staining results of DAPI and these two target proteins. The staining results in Figure 7 show that Runx2, Msx2, Lef1, and Edar were successfully activated, and the dental stem cells differentiated into more mature cells.

[0061] [Example 6]: Biomarker Analysis of Tooth Stem Cells and Mature Tooth Tissue

[0062] The iPSCs obtained from Example 3 of this invention were induced with Pitx2 crRNA for 2 days, followed by immunofluorescence staining, AOD analysis, and qPCR detection. On the fourth day, three crRNAs (Msx2, Lef1, and Edar) were induced for 2 days in a 1:2:1 ratio, followed by qPCR detection. On the seventh day, four crRNAs (Runx2, Msx2, Lef1, and Edar) were induced for 2 days in a 2:1:2:1 ratio, followed by qPCR detection. Figure 5A shows the immunofluorescence staining of dental stem cell biomarkers on days 0, 3, 6, and 9. Figure 5B shows the results of qPCR detection of dental stem cell biomarkers on days 0, 3, 6, and 9. Figure 5C shows the AOD analysis results of dental stem cell biomarkers on days 0, 3, 6, and 9. Figure 5D shows the immunofluorescence staining of Vimentin and CK14 on days 3, 6, and 9. Figure 5E shows the AOD analysis results of Vimentin and CK14 on days 3, 6, and 9. Figure 5F shows the immunofluorescence staining patterns of DMP1, Amelogenin, and STOR1 on days 3, 6, and 9. Figure 5G shows the AOD analysis results of DMP1, Amelogenin, and STOR1 on days 3, 6, and 9.

[0063] As shown in Figures 5A to 5C, after induction with different ratios of crRNA, the cell lines expressed CD49f, CD90, CD71, and CD140α, indicating that this induction successfully differentiated into four types of dental stem cells: periapical papillary stem cells (SCAP), dental pulp stem cells (DPSC), deciduous tooth pulp stem cells (SHED), and periodontal stem cells (PDLSC). These results also indicate that as dental stem cells differentiate into later stages, the expression levels of CD49f and CD90 decrease, suggesting that the number of DPSC dental pulp stem cells and SCAP periapical papillary stem cells gradually decreases.

[0064] Furthermore, as shown in Figures 5D and 5E, after induction with the aforementioned different ratios of crRNA, the cell lines exhibited Vimentin and CK14, which are biomarkers for the early differentiation of mesenchymal cells and ameloblasts, respectively. Therefore, the induction system of the transfection complex of this invention successfully induced cells to differentiate into mesenchymal cells and ameloblasts.

[0065] The results in Figures 5F and 5G show that as dental stem cells continue to differentiate into later stages, the expression of the human unique osteodifferentiation protein (STOR-1) increases, indicating a tendency for cell calcification. Simultaneously, the expression of Amelogenin and DMP1 also gradually increases. Amelogenin is a dental ammonia protein secreted by ameloblasts. DMP1 is a matrix protein secreted by odontoblasts.

[0066] none

[0067] TW202629795A_114100502_SEQL.xml

Claims

1. A peptide-imprinted transfection complex, characterized in that it comprises: chitin composite nanoparticles imprinted with dCas9 peptide; and a dCas9 fusion protein-guide RNA complex; wherein the chitin composite nanoparticles imprinted with dCas9 peptide are bound to the dCas9 protein-guide RNA complex; wherein the dCas9 fusion protein comprises dCas9 protein and VP64-p65-Rta (VPR); and wherein the guide RNA comprises tracrRNA (transactivating crRNA) and crRNA, and the sequence of the crRNA is SEQ ID NO:6; or the sequence of the crRNA is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:

10.

2. The peptide imprinting and transfection complex as described in claim 1, wherein, The dCas9 peptide-imprinted chitin composite nanoparticles contain chitin imprinted using dCas9 peptides and multiple magnetic nanoparticles.

3. A method for inducing cell differentiation into dental stem cells using the peptide imprinting transfection complex as described in claim 1, comprising the following steps: transfecting target cells with a peptide imprinting transfection complex containing crRNA sequences SEQ ID NO:2~SEQ ID NO:5 to obtain pluripotent stem cells; transfecting the pluripotent stem cells with the peptide imprinting transfection complex containing crRNA sequence SEQ ID NO:6 as described in claim 1 to obtain dental stem cells; the peptide imprinting transfection complex containing crRNA sequences SEQ ID NO:2~SEQ ID NO:5 comprises chitin composite nanoparticles imprinted with dCas9 peptide and a dCas9 fusion protein-guide RNA complex; the chitin composite nanoparticles imprinted with dCas9 peptide bind to the dCas9 protein-guide RNA complex; the dCas9 fusion protein comprises dCas9 protein and VP64-p65-Rta; the guide RNA comprises tracrRNA and crRNAs containing SEQ ID NO:2~SEQ ID NO:5; The target cells are live human embryonic kidney cells, specifically HEK293 cells.

4. A method for inducing cell differentiation into dental tissue using the peptide imprinting transfection complex as described in claim 1, comprising the following steps: transfecting target cells with a peptide imprinting transfection complex containing crRNA sequences SEQ ID NO:2~SEQ ID NO:5 to obtain pluripotent stem cells; transfecting the pluripotent stem cells with the peptide imprinting transfection complex containing crRNA sequence SEQ ID NO:6 as described in claim 1 to obtain dental stem cells; transfecting the dental stem cells with the peptide imprinting transfection complex containing crRNA sequences SEQ ID NO:7~SEQ ID NO:10 as described in claim 1 to obtain dental tissue cells; wherein the peptide imprinting transfection complex containing crRNA sequences SEQ ID NO:2~SEQ ID NO:5 comprises chitin composite nanoparticles imprinted with dCas9 peptides and a dCas9 fusion protein-guide RNA complex; wherein the chitin composite nanoparticles imprinted with dCas9 peptides bind to the dCas9 protein-guide RNA complex; The dCas9 fusion protein line contains dCas9 protein and VP64-p65-Rta; the guide RNA line contains tracrRNA and crRNA of SEQ ID NO:2~SEQ ID NO:5; the target cell is a live in vitro human embryonic kidney cell, specifically HEK293 cells.

5. A method for preparing a peptide imprinting and transfection complex as described in claim 1 or 2, characterized by comprising: Step 1: mixing an aqueous solution of dCas9 peptide, a chitin solution, and a magnetic nanoparticle solution to obtain a molecular imprinting mixture; Step 2: thoroughly stirring the molecular imprinting mixture for 2-60 minutes to coat the magnetic nanoparticles with chitin; Step 3: using a strong magnet, separating the molecular imprinting mixture into a supernatant layer and a chitin composite nanoparticle layer, and removing the supernatant layer; Step 4: adding deionized water, 0.5-95% ethanol, and 0.001-0.05% sodium dodecyl sulfate, and mixing them uniformly; Step 5: using a strong magnet, separating the molecular imprinting mixture again into a supernatant layer and a chitin composite nanoparticle layer, and removing the supernatant layer to remove the dCas9 peptide; this step is repeated at least twice. Step 6: The chitin composite nanoparticles imprinted with the dCas9 peptide obtained after Step 5 are mixed with the dCas9 protein-guide RNA complex to form a peptide imprinting transfection complex; The dCas9 peptide aqueous solution contains peptide Q, the sequence of which is SEQ ID NO:1; The guide RNA contains tracrRNA and crRNA, and the sequence of the crRNA is SEQ ID NO:6; or the sequence of the crRNA is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10; The chitin solution contains chitin with a molecular weight of 370~1000 K.

Images