A method for t cell gene editing and applications

Abstract

The application discloses a kind of to the method for gene editing of isolated T cell, the Cas9 / sgRNA of target T cell purpose gene is transferred into T cell by the degradable nanoparticle formed by the protein as inner core and the macromolecule as shell as carrier to make T cell purpose gene inactivation by gene editing.The application also provides the application of CD7 and PD1 defect T cell prepared by the method in the preparation of chimeric antigen receptor T cell.The method provided by the application is simple, green, low in cost, good in biocompatibility, low in cytotoxicity, has high cell uptake rate and gene editing rate, can also realize quantitative control to gene editing system, reduces off-target effect, and has wide application prospect in the field of gene editing and treatment.

Description

Technical Field

[0001] This invention discloses a gene editing method for T cells, belonging to the field of cell engineering technology. Background Technology

[0002] For CRISPR gene editing applications, direct delivery of the Cas9 protein and sgRNA complex into cells can achieve higher specificity and safety. However, delivering the Cas9 / sgRNA complex is challenging. First, both Cas9 protein and sgRNA are susceptible to degradation and inactivation by intracellular enzymes. Second, while assembling Cas9 / sgRNA into a tight, stable structure that can be taken up by cells, the dissociation and release of Cas9 / sgRNA within the cell must be considered; overly tight binding of Cas9 / sgRNA to the vector can also reduce its activity. Currently available Cas9 / sgRNA delivery vectors typically suffer from complex synthetic routes, low gene editing efficiency, and cytotoxicity due to insufficient degradation of the vector material. Furthermore, for primary T cells—suspension cells with unique structures and small sizes—no vector has been reported to deliver Cas9 / sgRNA to generate gene-edited T cells, and electroporation causes significant cell damage and is dependent on specific equipment.

[0003] Chimeric antigen receptor T cells (CAR T cells) are currently one of the most effective treatments for malignant tumors. Similar to other immunotherapies, its basic principle is to utilize the patient's own immune cells to eliminate cancer cells. However, because some endogenous genes in T cells can cause immune rejection, limiting the effectiveness of CAR T cell therapy, gene editing technology combined with CAR T cells shows broad application prospects in tumor immunotherapy. Using gene-edited T cells as the source of CAR T cells has the potential to enhance the efficacy of CAR T cell therapy for cancer and infectious diseases.

[0004] CD7 and PD1 are two clinically significant targets associated with CAR T cells. CD7 is a transmembrane protein highly expressed in T-cell malignancies and can serve as a CAR molecular target antigen for treating these malignancies. Since normal T cells also express CD7, the expression of CD7-specific CAR molecules can lead to T-cell cannibalization, thus hindering the in vitro expansion of CAR T cells. Knocking out CD7 on normal T cells... CD7Genes can prevent this self-destructive process and improve treatment efficacy. PD1 is an immunosuppressive molecule expressed on T cells that regulates the level of immune activation. It participates in suppressing sustained immune responses in peripheral tissues and preventing autoimmune damage, playing a protective role in the human immune system. Since tumor cells typically highly express PD-L1 (programmed death-ligand 1), the binding of the immune checkpoint molecule PD1 on T cells to PD-L1 on tumor cells initiates programmed death of T cells, allowing tumor cells to escape immune responses. Knocking out PD-L1 on T cells... PD1 Genes can weaken immunosuppressive signals and enhance the killing effect of CAR T cells on tumor cells.

[0005] Existing Cas9 / sgRNA delivery technologies to T cells have only been reported via electroporation. Therefore, a delivery system that uses degradable nanoparticles to achieve gene editing of CD7 and PD1 in T cells to overcome the above limitations is a technological necessity in this field for promoting the clinical application and development of CRISPR gene editing systems, especially for gene editing of T cells. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for gene editing of isolated T cells, the method comprising:

[0007] (1) Constructing biodegradable nanoparticles consisting of a protein as the core and a polymer as the shell, wherein the polymer is formed by in-situ polymerization of a neutral monomer and a cationic monomer in the presence of a free radical initiator after the primary amino group of lysine in the protein is modified to introduce a double bond.

[0008] (2) Using the nanoparticles obtained in step (1) as a carrier, mix them with Cas9 / sgRNA that targets the T cell target gene to obtain a self-assembled complex formed by mixing the nanoparticles and the Cas9 / sgRNA complex.

[0009] (3) The self-assembled complex obtained in step (2) is transferred into isolated T cells.

[0010] In a preferred embodiment, the cationic monomer in step (1) is ethyl 2-(dimethylamino)methacrylate, the electrically neutral monomer is acrylamide, the free radical initiator is ammonium persulfate and tetramethylethylenediamine, and the modification of the primary amino group of lysine in the protein is acrylation modification.

[0011] In a more preferred embodiment, N-hydroxysuccinimide acrylate is used to modify the primary amino group of lysine in the protein.

[0012] More preferably, the protein is any polypeptide having two or more lysine residues.

[0013] Preferably, the protein is bovine serum albumin. As is well known to those skilled in the art, chemically modified (e.g., fluorescently labeled) peptides are also included in embodiments of the present invention, provided that the modification does not interfere with the synthesis of nanoparticles.

[0014] Particularly preferred is that the molar ratio of bovine serum albumin: acrylamide: ethyl 2-(dimethylamino)methacrylate: ammonium persulfate: tetramethylethylenediamine is 1:3000:3000:250:1000.

[0015] In a preferred embodiment, the sequence of sgRNA in the Cas9 / sgRNA in step (2) is shown in SEQ ID NO.1, and the T cell target gene is the gene encoding CD7.

[0016] In another preferred embodiment, the sequence of sgRNA in the Cas9 / sgRNA in step (2) is shown in SEQ ID NO.2, and the T cell target gene is the gene encoding PD1.

[0017] Secondly, the present invention provides a CD7-deficient T cell prepared according to the above method.

[0018] Third, the present invention provides a PD1-deficient T cell prepared according to the above method.

[0019] Fourth, the present invention provides the application of the above-mentioned CD7-deficient T cells and / or PD1-deficient T cells in the preparation of chimeric antigen receptor T cells.

[0020] Finally, this invention provides the application of the above-mentioned T cells in the preparation of drugs for the treatment of tumors or autoimmune diseases.

[0021] Compared with existing T-cell gene editing methods, the method of using biodegradable nanoparticles as Cas9 / sgRNA vectors described in this invention has the following outstanding advantages:

[0022] 1) The Cas9 / sgRNA vector provided by this invention utilizes in-situ protein polymerization of polymers, and the material preparation method is simple, green, and low in cost; the design concept with protein as the core makes the material biocompatibility good and cytotoxic.

[0023] 2) The nanoparticles described in this invention can be well loaded with Cas9 / sgRNA complexes through simple mixing. Without the need for any other transfection reagents, Cas9 / sgRNA can be efficiently delivered into cells, significantly improving cell uptake.

[0024] 3) The cationic monomer of the polymer shell of the nanoparticles of this invention is designed as ethyl 2-(dimethylamino)methacrylate with tertiary amine groups and ester bonds. The tertiary amine group structure generates a proton sponge effect, which facilitates endosome escape. The ester bond connecting the tertiary amine group undergoes hydrolysis at physiological temperatures, causing the cationic group to degrade and promoting the release of Cas9 / sgRNA from the self-assembled complex. Simultaneously, cytoplasmic components such as negatively charged proteins and amino acids compete with the binding sites of the nanoparticles and Cas9 / sgRNA, further accelerating the dissociation of Cas9 / sgRNA from the self-assembled complex in the cytoplasm. The nanoparticles of this invention can achieve high levels of Cas9 / sgRNA release, significantly improving gene editing efficacy.

[0025] 4) In T-cell gene editing applications, the nanoparticle-mediated Cas9 / sgRNA complex can achieve an intracellular gene editing efficiency of up to 14%. Furthermore, the direct presentation of the Cas9 / sgRNA complex facilitates quantitative regulation of its intracellular content and duration of action, reducing off-target effects.

[0026] 5) Apart from electroporation, existing Cas9 / sgRNA delivery systems cannot be used for difficult-to-transfect cell types such as primary T cells. Cas9 / sgRNA delivery systems based on degradable nanoparticles provide a novel gene knockout method for primary T cells, laying the foundation for next-generation immunotherapies such as the preparation of universal CAR T cells. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the preparation of nanoparticles and their Cas9 / sgRNA delivery system.

[0028] Figure 2 The images show the morphology and size of the nanoparticles. (a) Transmission electron microscope image; (b) Atomic force microscope image.

[0029] Figure 3 Comparison of the structural properties of nanoparticles and core proteins. (a) Dynamic light scattering diagram; (b) Zeta potential diagram; (c) Fourier transform infrared spectrum; (d) Ion chromatogram.

[0030] Figure 4 The uptake and gene editing efficiencies of HEK293T cells transfected with self-assembled complexes of nanoparticles and Cas9 / sgRNA at different molar ratios were evaluated. Uptake efficiency was expressed as the mean fluorescence intensity of Cas9-AF647 uptake by cells after 12 h, and gene editing efficiency was expressed as the percentage of EGFP-negative cells after 4 days. Cas9-AF647 is AF64-labeled Cas9.

[0031] Figure 5(a) Flow cytometry results, (b) fluorescence microscopy observation results, and (c) sequencing and monoclonal sequencing analysis results of DNA fragments near the target sites of the cell genome for transfecting HEK293T cells with nanoparticles / Cas9 / sgRNA.

[0032] Figure 6 (a) Flow cytometry results, (b) Fluorescence microscopy observation results, and (c) Sequencing and monoclonal sequencing analysis results of DNA fragments near the target sites of the cell genome for A549 cells transfected with nanoparticles / Cas9 / sgRNA.

[0033] Figure 7 Transmission electron microscopy images of Cas9 / sgRNA and its self-assembled complex with nanoparticles. (a) Cas9 / sgRNA; (b) Nanoparticles / Cas9 / sgRNA; (c) Nanoparticle-37 / Cas9 / sgRNA, where nanoparticle-37 refers to nanoparticles placed at 37°C for 10 days.

[0034] Figure 8 (a) shows the change in zeta potential of the nanoparticles before and after being placed at 37°C for 10 days; (b) shows the fluorescence spectra of NP-FITC / Cas9-RBITC / sgRNA in different media. The protein medium concentration was 0.4 mg / ml; NP-FITC represents FITC-labeled nanoparticles; RNP-RBITC represents the complex formed by RBITC-labeled Cas9 and sgRNA.

[0035] Figure 9 This describes the in vitro CRISPR digestion reactions of nanoparticles / Cas9 / sgRNA under different conditions.

[0036] Figure 10 The Cas9-AF647 internalization efficiency in unactivated T cells was measured by flow cytometry 6 hours after transfection with nanoparticles / Cas9-AF647 / sgRNA.

[0037] Figure 11 To evaluate the internalization and genome editing efficiency of T cells under different activation conditions. (a) Cas9-AF647 internalization efficiency of activated T cells 6 h after transfection with nanoparticles / Cas9-AF647 / sgRNA, measured by flow cytometry; (b) CD7 APC expression measured by flow cytometry after culturing for 7 days following transfection with nanoparticles / Cas9 / CD7-sgRNA.

[0038] Figure 12 The results are from a single-clone Sanger sequencing of T cells targeting the (a) CD7 and (b) PD1 genes. PAM is the protospacer motif.

[0039] Figure 13 For deep sequencing analysis of T cells transfected with nanoparticles / Cas9 / CD7-sgRNA. (a) Frequency of each inserted base at each site, expressed as the number of inserted reads at each site / total number of reads; (b) Percentage of inserted reads of a specific length to the total number of inserted reads; (c) Frequency of each deleted base at each site, expressed as the number of deleted reads at each site / total number of reads; (d) Percentage of deleted reads of a specific length to the total number of deleted reads.

[0040] Figure 14 The main deletion or insertion sequences and their frequencies after transfection of T cells with nanoparticles / Cas9 / CD7-sgRNA.

[0041] Figure 15 For deep sequencing analysis of T cells transfected with nanoparticles / Cas9 / PD1-sgRNA. (a) Frequency of each inserted base at each site, expressed as the number of inserted reads at each site / total number of reads; (b) Percentage of inserted reads of a specific length to the total number of inserted reads; (c) Frequency of each deleted base at each site, expressed as the number of deleted reads at each site / total number of reads; (d) Percentage of deleted reads of a specific length to the total number of deleted reads.

[0042] Figure 16 The main deletion or insertion sequences and their frequencies after transfection of T cells with nanoparticles / Cas9 / PD1-sgRNA.

[0043] Figure 17 The effect of nanoparticle delivery systems on T cell activity. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0045] Example 1: Preparation method of biodegradable nanoparticles

[0046] 1) Bovine serum albumin (Sigma-Aldrich #SRE0096) was dissolved in pH 8.5, 100mM borate buffer and centrifuged in this buffer four times through a 30kD ultrafiltration tube (Amicon #UFC5030BK). The concentration was then determined by the quinolinic acid (BCA) method and the protein was diluted to 10mg / ml with this buffer to obtain the stock protein solution.

[0047] 2) Dissolve N-hydroxysuccinimide acrylate (Sigma-Aldrich #A8060) in dimethyl sulfoxide (Sigma-Aldrich #276855) to prepare a 20 mg / ml solution. Take 5.1 μl and mix it with 100 μl of the reaction protein stock solution (where N-hydroxysuccinimide acrylate: bovine serum albumin = 40:1 molar ratio). React at room temperature (25°C) for 6 h to obtain protein solution A with surface double bond modification.

[0048] 3) Dissolve acrylamide (Sigma-Aldrich #A8887) in deionized water to prepare a 200 mg / ml solution B; dissolve ammonium persulfate (Sigma-Aldrich #A3678) in deionized water to prepare a 100 mg / ml solution C; ethyl 2-(dimethylamino)methacrylate (Sigma-Aldrich #234907) and tetramethylethylenediamine (Sigma-Aldrich #T9281) are both liquid reagents and can be used directly in the reaction.

[0049] 4) Add 300 μl of pH 7.4, 100 mM phosphate buffer to the solution A system, then add 16 μl of solution B, 7.6 μl of 2-(dimethylamino)methacrylate, 8.6 μl of solution C, and 2.1 μl of tetramethylethylenediamine and mix thoroughly (wherein, the molar ratio of bovine serum albumin:acrylamide:2-(dimethylamino)methacrylate:ammonium persulfate:tetramethylethylenediamine = 1:3000:3000:250:1000). React at room temperature (25°C) for 4 h.

[0050] 5) See the schematic diagram of the reaction. Figure 1 The first two steps. After the polymerization reaction, non-target reaction product components generally remain in the system, so purification is required. The product solution was centrifuged and the buffer was changed four times in a 30kD ultrafiltration tube with pH 7.4, 10mM phosphate buffer (PBS) to obtain 300μl of nanoparticle solution. The concentration was determined to be 3.28mg / ml by BCA method, and it was diluted to 1.5mg / ml with PBS.

[0051] Example 2: Materials Characterization of Biodegradable Nanoparticles

[0052] The nanoparticles were observed using a transmission electron microscope (HT7700, Hitachi) and an atomic force microscope (Dimension Icon, Bruker). Figure 2 The particles are spherical, with a size distribution ranging from a few nanometers to 20 nanometers. Dynamic light scattering (Zetasizer Nano, Malvern) measured the average particle size of the nanoparticles to be 10 nm, which is much larger than the 5.5 nm particle size of its synthetic raw material, bovine serum albumin. Figure 3 (a). The average Zeta potential of the nanoparticles is 9.7 mV ( Figure 3 (b) Compared with bovine serum albumin with a zeta potential of -20.8mV, it can be seen that the design of cationic polymer surface modification changes its electrical properties. Figure 3 Further evidence from point c indicates that the nanoparticles are composed of a copolymer of ethyl 2-(dimethylamino)methacrylate and acrylamide coupled with proteins, as evidenced by the observation of a 2950 cm⁻¹ in the infrared spectrum of the nanoparticles. -1 (C–H stretching vibration peaks of –CH3 and –CH2), 2820 cm⁻¹ -1 and 2780cm -1 (C–H stretching vibration peak of –N(CH3)2), 1730 cm⁻¹ -1 (C=O stretching vibration peak in ester bond), 1460 cm⁻¹ -1 (–CH2– bending vibration peak), 1395cm -1 (CH3 bending vibration peak), 1148cm -1 (C–N and C–O stretching vibration peaks), these are characteristic absorption peaks of the ethyl 2-(dimethylamino)methacrylate unit. Additionally, at 1680 cm⁻¹... -1 This is a characteristic absorption peak of the C=O stretching vibration in the amide bond, which belongs to the linkage between the acrylamide unit and bovine serum albumin and N-hydroxysuccinimide acrylate. 3317 cm⁻¹ -1 The broad peak at 1540cm -1 The small peaks at the locations belong to the N–H stretching vibration and bending vibration peaks of the primary amine group in the acrylamide unit, respectively. Figure 3 Figure d shows the elution chromatogram detected at 280 nm using a Dionex ICS-5000+ ion chromatography system and a TOSOH TSKgel CM-STAT column (equilibration buffer: pH 4.5, 10 mM sodium acetate solution; eluent: pH 4.5, 10 mM sodium acetate solution with added 0.5 M NaCl). The results further indicate that the in-situ growth of the cationic polymer on the surface of the core protein imparts positive charge to the material. Furthermore, the absence of a distinct separation peak in the elution curve of the nanoparticles confirms the relative homogeneity of the nanoparticle material.

[0053] Example 3: Experimental optimization and gene editing results of nanoparticle delivery of Cas9 / sgRNA

[0054] In this embodiment, the Cas9 protein is... Escherichia coliBL21(DE3) expression and purification were performed using the pET-NLS-Cas9-6xHis plasmid, catalog number Addgene #62934. EGFP-sgRNA was transcribed and purified in vitro from a DNA template, the sequence of which is: GTTTTTTTTAATACGACTCACTATAgggcgaggagctgttcaccgGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT. As a control, cgRNA not targeting any gene was also transcribed and purified in vitro from a DNA template, the sequence of which is: GTTTTTTTTAATACGACTCACTATAgggtaaccgtgcggtcgtacGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT. The cells used were laboratory-constructed single-copy expression cells. EGFP- PEST HEK293T and A549 cell lines containing the gene.

[0055] The nanoparticles prepared in Example 1 above can be used to prepare a Cas9 / sgRNA delivery system. The process is as follows:

[0056] 1) Mix Cas9 protein and sgRNA and let stand at room temperature for 20 min to obtain Cas9 / sgRNA complex.

[0057] 2) Mix the nanoparticles with the Cas9 / sgRNA complex solution and let stand at room temperature for 20 min to obtain the self-assembled complex of nanoparticles and Cas9 / sgRNA.

[0058] 3) The above self-assembled complex was co-incubated with cells to obtain a Cas9 / sgRNA cell delivery system based on nanoparticles.

[0059] 4) Evaluate the cell gene editing effect produced by the Cas9 / sgRNA delivery system based on the above nanoparticles.

[0060] The following are the more specific experimental steps:

[0061] 1.18 μl of Cas9 protein (1354 μg / ml) and 1.59 μl of targeted... EGFPThe EGFP-sgRNA of the gene (200 μg / ml) was mixed at 25°C (molar ratio 1:1) and allowed to stand for 20 min to obtain the Cas9 / sgRNA complex. 2.2 μl, 4.4 μl, 8.8 μl, and 13.2 μl of nanoparticles (150 μg / ml) were mixed with the Cas9 / sgRNA complex prepared under the above conditions at 25°C and allowed to stand for 20 min to obtain self-assembled complexes of nanoparticles and Cas9 / sgRNA at different molar ratios (molar ratios of 1:2, 1:1, 2:1, and 3:1, respectively). The nanoparticle / Cas9 / sgRNA complex was added to serum-free DMEM medium to a final volume of 100 μl, containing 100 nM of Cas9 / sgRNA. HEK293T cells in the exponential growth phase were then added to 96-well plates. After 12 hours of transfection, the culture medium was replaced with DMEM containing 10% serum, and the cells were cultured for another 4 days. EGFP fluorescence expression was analyzed using flow cytometry (Guava easyCyte HT, Merck Millipore). EGFP-negative cells were those that did not express EGFP due to gene editing. The experimental control was achieved by replacing sgRNA with cgRNA (200 μg / ml), which does not target any gene. The difference in the percentage of EGFP-negative cells produced by sgRNA and cgRNA represents the gene editing efficiency.

[0062] Simultaneously, after incubating Cas9 protein (100kD ultrafiltration tube, pH 8.5, 100mM borate buffer) with AF647 fluorescent dye (Molecular Probes #A20006) at a 1:10 molar ratio in the dark for 1 h, the mixture was centrifuged four times with PBS in a 100kD ultrafiltration tube to remove free AF647, yielding fluorescently labeled Cas9-AF647. The same process was used to obtain self-assembled complexes of nanoparticles and Cas9-AF647 / sgRNA at a molar ratio of 1:2–3:1, which were then incubated with HEK293T cells under the same conditions for 12 h. Flow cytometry was then used to analyze AF647 fluorescence in the cells to evaluate the cellular uptake efficiency of Cas9 / sgRNA.

[0063] The above experimental results are as follows Figure 4As shown, increasing the proportion of nanoparticles generally enhanced the internalization efficiency of Cas9 / sgRNA. However, the internalization efficiency did not perfectly correlate with the gene editing efficiency. This indicates that partial complementarity between nanoparticles and Cas9 / sgRNA is crucial for efficient presentation and CRISPR function, possibly because the loading and release of Cas9 / sgRNA need to be balanced. The gene editing effect was optimal when the molar ratio of nanoparticles to Cas9 / sgRNA was 1:1. HEK293T and A549 cells were transfected under optimal conditions, and the intracellular EGFP fluorescence expression of the cells was characterized by flow cytometry and fluorescence microscopy after 4 days. Flow cytometry showed that the gene editing efficiency reached 28% ( Figure 5 (a) The proportions of EGFP-negative cells generated by targeted and non-targeted nanoparticle delivery systems were 34.4% and 6.04%, respectively, and 40% (b) and 40%, respectively. Figure 6 (a) The proportions of EGFP-negative cells generated by targeted and non-targeted nanoparticle delivery systems were 44.5% and 4.45%, respectively. From Figure 5 b and Figure 6 As can be clearly seen in step b, the nanoparticle / Cas9 / sgRNA targeting system produced a certain number of cells without EGFP expression, while the nanoparticle / Cas9 / cgRNA non-targeting system had virtually no effect on the fluorescence expression of the cells after delivery. This difference further confirms that nanoparticles, as a delivery vector, can achieve the gene knockout function of Cas9 / sgRNA. Genomic DNA was extracted from the cells, DNA fragments near the editing site were amplified by PCR, the PCR product was purified and ligated into the pCloneEZ TOPO cloning vector (CloneSmarter #C5865), and then transformed into... Escherichia coli In DH5α, the results of direct Sanger sequencing of PCR products and Sanger sequencing of more than 50 clones are as follows: Figure 5 c and Figure 6 As shown in Figure c, the overlapping peaks of the PCR products indicate the presence of fragments with different sequences, meaning that gene editing has occurred in the target region; the single-clone sequencing results show the details of gene editing, such as insertions, deletions, or single nucleotide variations.

[0064] Example 4: Study on the intracellular delivery mechanism of Cas9 / sgRNA based on nanoparticles

[0065] 1. Intrinsic stability of nanoparticles

[0066] The experimental condition of 37℃ for 10 days was chosen as the experimental condition for evaluating the stability of the nanoparticle material itself. The nanoparticles placed at 37℃ for 10 days were named nanoparticle-37.

[0067] Transmission electron microscopy revealed that the size of the Cas9 / sgRNA complex was close to that of nanoparticles. Figure 7 (a) The nanoparticles / Cas9 / sgRNA form regular spherical macroaggregates. Figure 7 (b). The nanoparticle-37 / Cas9 / sgRNA exhibits a loosely structured, dissociated structure. Figure 7 (c) This indicates that an intracellular 37°C environment can accelerate the dissociation and release of Cas9 / sgRNA from aggregates.

[0068] Measurements taken with a potential meter (Zetasizer Nano, Malvern) showed that the Zeta potential of the nanoparticles decreased significantly at 37°C, from ~8.4 mV to ~1.9 mV. Figure 8 (a). This shows that the instability of the nanoparticle's own structure is an important factor in its ability to release Cas9 / sgRNA intracellularly.

[0069] 2. Competitive binding from other molecules

[0070] Nanoparticles and Cas9 protein were labeled using FITC fluorescent labeling reagent (Sigma-Aldrich #F3651) and RBITC (Aladdin #R105502), respectively. Nanoparticles (NP) and FITC were incubated in the dark for 1 h at a 1:4 molar ratio. The mixture was then centrifuged four times with PBS in a 30 kD ultrafiltration tube to remove free FITC, yielding FITC-labeled nanoparticles (NP-FITC). Cas9 protein was incubated in the dark for 1 h with RBITC at a 1:5 molar ratio. The mixture was then centrifuged four times with PBS in a 100 kD ultrafiltration tube to remove free RBITC, yielding RBITC-labeled Cas9 (Cas9-RBITC). This was then mixed with sgRNA and incubated for 20 min to obtain RBITC-labeled Cas9 / sgRNA (RNP-RBITC).

[0071] The self-assembly formation and dissociation release of nanoparticles with Cas9 / sgRNA were evaluated using fluorescence resonance energy transfer (FRET) based on the aforementioned fluorescent molecular pairs. Figure 8(b) Fluorescence spectra were recorded using a SpectraMax Paradigm, Molecular Devices system, with excitation and emission wavelengths of 470 nm and 505–750 nm, respectively. During self-assembly, RNP-RBITC partially quenched the fluorescence of NP-FITC, resulting in a bimodal fluorescence spectrum. Adding bovine serum albumin or DMEM containing fetal bovine serum to the self-assembled complex solution triggered the release of Cas9 / sgRNA, manifested as a decrease in the intensity of the 580 nm peak in the fluorescence spectrum. This suggests that abundant cytoplasmic proteins can trigger the dissociation of Cas9 / sgRNA, possibly due to competition between negatively charged proteins and Cas9 / sgRNA for binding to nanoparticles.

[0072] 3. In vitro enzyme digestion experiments simulate the intracellular Cas9 / sgRNA release mechanism.

[0073] In this embodiment, the pcDNA3.1-EGFP plasmid was constructed by cloning the EGFP sequence (GenBank: DQ389577.1) into the multiple cloning site of plasmid pcDNA3.1 (+) (Invitrogen #V79020). Since pcDNA3.1-EGFP contains the EGFP-sgRNA targeting sequence, it can be used in Cas9 / sgRNA-mediated in vitro DNA digestion experiments to simulate intracellular RNP release conditions. Before the in vitro DNA digestion experiment, pcDNA3.1-EGFP was linearized by digestion with PvuI-HF (NEB #R3150S), making the double bands generated by further Cas9 / sgRNA digestion easier to detect.

[0074] Nanoparticles / Cas9 / sgRNA (molar ratio 1:1:1, final concentration 50 nM) were added to 250 ng of linearized pcDNA3.1-EGFP, followed by the addition of enzyme-free water or culture medium to a final volume of 20 μL. The mixture was then incubated at 37 °C for 1 h. The reaction was terminated by incubation with proteinase K (Sigma-Aldrich #P6556) for 30 min. The CRISPR digestion status of the DNA was analyzed by 1% agarose gel electrophoresis. Figure 9In the diagram, lane M represents the DL15000 DNA molecular marker; lane 1 represents the in vitro digestion reaction of nanoparticles / Cas9 / sgRNA; lane 2 represents the in vitro digestion reaction of nanoparticle-37 / Cas9 / sgRNA; lane 3 represents the in vitro digestion reaction of nanoparticles / Cas9 / sgRNA in DMEM; lane 4 represents the in vitro digestion reaction of nanoparticle-37 / Cas9 / sgRNA in DMEM; and lane 5 represents the in vitro digestion reaction of nanoparticle-37 / Cas9 / sgRNA in complete culture medium. The results show that nanoparticles / Cas9 / sgRNA cannot directly exert CRISPR digestion activity due to the overly tight binding between the vector and the loading material. Nanoparticle-37 / Cas9 / sgRNA exhibits partial Cas9 / sgRNA activity, further confirming that the effect of 37℃ on the nanoparticle structure weakens the binding between the loading material and the vector. In the presence of DMEM (containing anionic molecules such as negatively charged amino acids), nanoparticles / Cas9 / sgRNA also exhibit partial gene editing activity. When fetal bovine serum (FBS) was added to DMEM (rich in anionic molecules such as amino acids and proteins, serving as a rough mimicry of the cytoplasm), the nanoparticle-37 / Cas9 / sgRNA retained the complete enzymatic cleavage function of Cas9 / sgRNA. This is because anionic molecules such as negatively charged proteins can also bind to the nanoparticles, competing with Cas9 / sgRNA. These results simulate the principle that a 37°C cytoplasmic environment triggers the dissociation and release of the load from the vector, thereby causing breakage of the target DNA.

[0075] Example 5: Nanoparticle delivery of Cas9 / sgRNA to primary T cells

[0076] Chimeric antigen receptor T cells (CAR T cells) are currently one of the most effective treatments for malignant tumors. Similar to other immunotherapies, its basic principle is to utilize the patient's own immune cells to eliminate cancer cells. Because some endogenous genes of T cells can affect the efficacy of CAR T cell therapy, gene editing technology combined with CAR T cells shows broad application prospects in tumor immunotherapy. Using gene-edited T cells as the source of CAR T cells has the potential to enhance the efficacy of CAR T cell therapy for cancer and infectious diseases.

[0077] Extending CAR T cells to treat T-cell malignancies is problematic because most target antigens are shared between normal and malignant cells, leading to CAR T cell self-destruction. CD7, a transmembrane protein highly expressed in acute T-lymphoblastic leukemia and peripheral T-cell lymphoma, is an attractive therapeutic target for T-cell malignancies. Since the T cells used to create CAR T cells also express CD7, the expression of CD7-specific CARs (CD7-CARs) can cause T cell self-destruction, thus hindering CAR T cell proliferation. Knockout of the CD7 gene can prevent this self-destruction, and these T cells can be used to create CAR T cells that target T-cell tumors. Therefore, gene editing systems targeting CD7 have clear clinical significance.

[0078] Furthermore, since tumor cells typically highly express PD-L1 (programmed death-ligand 1), the binding of the immune checkpoint molecule PD-1 (programmed death receptor 1) on T cells to the highly expressed PD-L1 on tumor cells initiates programmed death of T cells, allowing tumor cells to escape the immune system. PD1 Gene knockout can weaken immunosuppressive signals, thereby enhancing the killing effect of CAR T cells on tumor cells.

[0079] Existing delivery technologies for Cas9 / sgRNA on T cells only include electroporation. This embodiment attempts to use the biodegradable nanoparticles of the present invention to achieve gene editing of CD7 and PD1 on T cells.

[0080] In this embodiment, the sequence of CD7-sgRNA is shown in SEQ ID NO.1, and the sequence of PD1-sgRNA is shown in SEQ ID NO.2. Both sgRNAs were transcribed and purified in vitro from a DNA template. The DNA template sequence of CD7-sgRNA is as follows:

[0081] GTTTTTTTTAATACGACTCACTATAggagcaggtgatgttgacggGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.

[0082] The DNA template sequence of PD1-sgRNA is as follows:

[0083] GTTTTTTTTAATACGACTCACTATAggccaggatggttcttaggtGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.

[0084] The lowercase letters in the two sequences above represent the 20bp sequence of sgRNA targeting the T cell genome. T cells were cultured in X-VIVO 15 medium (LONZA #04-418Q), which was supplemented with 100 U / ml recombinant human interleukin-2 (PeproTech, #200-02-100) and 5% fetal bovine serum.

[0085] 5.9 μl of Cas9 protein (1354 μg / ml) and 15.9 μl of targeted... CD7 The CD7-sgRNA of the gene (200 μg / ml) was mixed at 25°C (molar ratio 1:2) and incubated for 20 min to obtain the Cas9 / sgRNA complex. 33 μl of nanoparticles (150 μg / ml) were mixed with the Cas9 / sgRNA complex prepared under the above conditions and incubated for 20 min to obtain a self-assembled nanoparticle / Cas9 / sgRNA complex (molar ratio 1.5:1:2, which is more optimal for T cell transfection). The nanoparticle / Cas9 / sgRNA complex was added to serum-free DMEM medium to a final volume of 500 μl, containing 100 nM Cas9. Six hours after transfection, the medium was replaced with X-VIVO 15 medium, and cells were cultured for another 7 days. Cells were then stained with APC anti-human CD7 antibody (BioLegend#343108), and CD7 expression was detected by flow cytometry. CD7-negative cells were considered the target cells. CD7 Gene knockout cells. Single-clone sequencing of genomic DNA (see Experimental Methods in Example 3). Sanger sequencing results from more than 100 clones can be used to estimate gene editing efficiency on T cells and to show details of insertions, deletions, or single nucleotide variants edited.

[0086] After labeling Cas9 with AF647 (see Preparation Method in Example 3), a self-assembled complex of nanoparticles / Cas9-AF647 / sgRNA was prepared and incubated with T cells under the same conditions for 6 h. Flow cytometry was then used to analyze AF647 fluorescence in the cells to evaluate the uptake efficiency of Cas9 / sgRNA by T cells. Dynabeads® CD3 / CD28 CTS™ (Gibco #40203D) are magnetic beads incorporating anti-CD3 and anti-CD28 antibodies, providing the stimulatory signals required for T cell activation and expansion. To investigate the effects of activation on T cells, we compared endocytosis and genome editing during activation under different conditions. Cells were named according to their activation status at the time of transfection (e.g., "AC-1D" indicates transfection of cells activated 24 hours prior; cells activated once were cryopreserved and thawed for reactivation, and "RE-3D" indicates transfection of cells reactivated 3 days prior).

[0087] T cells that had not undergone magnetic bead activation stimulation showed an intracellular Cas9 / sgRNA uptake efficiency of only 67.7% after 6 hours of transfection under the above conditions. Figure 10 In contrast, the AF647 positivity rate of all cells activated with magnetic beads (according to the instructions, the ratio of cell number to magnetic bead number was 1:3) was close to 100%, indicating that activation significantly increased Cas9 / sgRNA uptake. Figure 11 (a). Meanwhile, the internalization level of Cas9 / sgRNA in reactivated cells was higher than in cells activated only once, but activation for 1–3 days had little effect on internalization. Figure 11 (a) After cells transfected with nanoparticles / Cas9 / sgRNA were cultured for 7 days, the proportion of CD7-negative cells was calculated by flow cytometry. Effective gene editing was detected in both single-activation and reactivation cells, and the gene knockout efficiency was correlated with the uptake of Cas9 / sgRNA. Compared with untransfected cells, approximately 13.3% of transfected RE-3D cells showed reduced CD7 expression (a). Figure 11 (b) Sanger sequencing of 100 clones revealed that 15 clones had insertions or deletions. Figure 12 (a) The gene editing efficiency (15%) is similar to the editing results detected by flow cytometry.

[0088] When using PD1-sgRNA targeting PD1, it can also affect T cells. PD1 Gene editing ( Figure 12 (b). Of the 200 clones, 27 had insertions or deletions, resulting in a gene editing efficiency of 13.5%. Compared to single-clone sequencing data from the 293T or A549 cell lines, the primary T clones had longer insertion or deletion sequences (e.g., 16bp and 35bp insertions, 106bp, 127bp, and 222bp deletions).

[0089] Example 6: Deep sequencing results of T cells after nanoparticle-mediated gene editing

[0090] In this embodiment, the PCR primers used for amplifying fragments near the CD7 target region were 5′-AGCTGCCTCAGGTAGATCCCA-3′ and 5′-GATCTGCTCCATGCCCCGTA-3′; the PCR primers used for amplifying fragments near the PD1 target region were 5′-TCTGGGCGGTGCTACAACT-3′ and 5′-AAGCCACACAGCTCAGGGT-3′. Genomic DNA was amplified into 250-280 bp fragments using primers. After end repair and the addition of an A-base tail, adapters were ligated to both ends of the fragments to construct a DNA library. Paired-end sequencing was performed using a high-throughput sequencing platform (NovaSeq 6000, Illumina), with 150 bp sequenced from each end. The high-throughput sequencing reads were aligned with reference reads using the BWA tool (https: / / sourceforge.net / projects / bio-bwa / ), and analyzed using SAMtools version 1.9 (http: / / www.htslib.org / ).

[0091] Analysis of the deep sequencing results of T cells (RE-3D) transfected with nanoparticles / Cas9 / CD7-sgRNA in Example 5 showed that ( Figure 13 Insertion occurred most frequently at the 5th site upstream of the PAM, followed by the 4th site upstream of the PAM. The probability of insertion was highest when the insertion sequence length was 1, accounting for 87.3% of all inserted reads. Deletion occurred much more frequently than insertion during gene editing. The highest frequency of deletion occurred after the 6th site of the 20bp CD7-sgRNA targeting sequence, followed by the 5th and 15th sites of the same length. Reads with a deletion length of 12 accounted for the highest proportion of all deleted reads, reaching 62.6%. Figure 14 The top 6 most frequent specific insertion or deletion sequences and their percentages are listed: 12bp deletion, 1bp insertion, 15bp deletion, two types of 1bp deletion at different positions, and 4bp deletion.

[0092] Analysis of deep sequencing results of T cells (RE-3D) transfected with nanoparticles / Cas9 / PD1-sgRNA revealed that ( Figure 15The highest frequency of insertions occurred at the 5th site upstream of PAM, followed by the 4th site upstream of PAM, consistent with insertions when targeting CD7. Similarly, the highest probability of insertion occurred when the insertion sequence length was 1, accounting for 75.6% of all insertion reads. Deletions occurred much more frequently than insertions in gene editing. The highest frequency of deletions occurred after the 9th site of the 20bp target for PD1-sgRNA, followed by the 4th site of the 20bp target. The number of reads with deletion lengths was relatively evenly distributed, with reads of lengths of 8, 12, 16, and 1 accounting for 8%-20% of all deletion reads. From the top 6 most frequent specific insertion or deletion sequences and their proportions, we can conclude that... Figure 16 Compared to gene editing targeting CD7, the specific bias of editing targeting PD1 is reduced. These results contribute to a clearer understanding of the details of T cell editing mediated by nanoparticle-mediated Cas9 / sgRNA delivery.

[0093] Example 7: Effects of Nanoparticle Delivery Systems on T Cell Viability

[0094] Primary T cells were transfected with nanoparticles / Cas9 / sgRNA and cultured for 7 days. Cells were then stained with acridine orange / propidium iodide dye (AO / PI, Nexcelom Bioscience #CS2-0106), and cell viability was assessed using an automated cell counter (Cellometer Auto 2000, Nexcelom Bioscience). Figure 17 The viable cell percentage of single-activated cells after treatment with the delivery system was 85.3%, a difference of only 6.2% compared to untreated single-activated cells; while the viable cell percentage of reactivated cells after treatment with the delivery system was 80.5%, also a difference of only 6.3% compared to untreated reactivated cells. These data indicate that, regardless of whether single-activated or reactivated cells are used, the nanoparticle delivery system exhibits low toxicity to primary T cells, making it a promising method for T cell transfection.

[0095] The above embodiments demonstrate the use of the present invention to target endogenous genes in T cells. CD7 and PD1The gene editing performed using the delivery system of this invention can also be used for knocking out other endogenous genes in T cells, enabling CAR T cells to target various T-cell antigens, thereby expanding the range of tumors that can be targeted; or modifying signaling pathways in T cells to enhance activation signals or weaken inhibitory signals, further enhancing the function of CAR T cells; or reducing the immune rejection response of xenogeneic CAR T cells by knocking out genes involved in immune surveillance in T cells. The prepared universal CAR T cells can overcome the quality and quantity defects of the patient's own T cells and can also reduce the time and cost of manufacturing autologous T cell products. In summary, this invention has very broad application prospects in tumor immunotherapy.

Claims

1. A method for gene editing of isolated T cells, the method comprising: (1) constructing degradable nanoparticles formed by a protein as a core and a polymer as a shell, the polymer being in-situ polymerized with an electrically neutral monomer and a cationic monomer in the presence of a free radical initiator after a primary amine group of lysine in the protein is modified to introduce a double bond, wherein the cationic monomer is 2-(dimethylamino)ethyl methacrylate, the electrically neutral monomer is acrylamide, and the free radical initiator is ammonium persulfate and tetramethyl ethylenediamine, the primary amine group of lysine in the protein is acrylated by using N-hydroxysuccinimidyl acrylate, and the protein is bovine serum albumin; (2) mixing the nanoparticles obtained in step (1) as a carrier with Cas9 / sgRNA capable of targeting a gene of interest of T cells to obtain a self-assembled complex formed by mixing the nanoparticles and the Cas9 / sgRNA complex; (3) transferring the self-assembled complex obtained in step (2) into isolated T cells, the isolated T cells being T cells activated by magnetic beads combined with anti-CD3 and anti-CD28 antibodies.

2. The method of claim 1, wherein, The molar ratio of bovine serum albumin: acrylamide: 2-(dimethylamino)ethyl methacrylate: ammonium persulfate: tetramethyl ethylenediamine is 1:3000:3000:250:1000.

3. The method of claim 1, wherein, The sequence of sgRNA in the Cas9 / sgRNA in step (2) is shown in SEQ ID NO. 1, and the gene of interest of the T cells is a gene encoding CD7.

4. The method of claim 1, wherein, The sequence of sgRNA in the Cas9 / sgRNA in step (2) is shown in SEQ ID NO. 2, and the gene of interest of the T cells is a gene encoding PD1.

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