CD26-targeted double-AAV delivery system based on CRISPR base editing technology

Abstract

The invention provides a CD26-targeted double-AAV delivery system based on a CRISPR base editing technology, and belongs to the field of gene engineering. According to the invention, a targeted CD26 editing system is constructed for the first time, in-vivo delivery is carried out through double AAV vectors, and selective removal of CD26 + CAFs in PDAC is successfully realized. The CD26 gene splicing donor site is precisely edited, the CD26 expression in the CAFs is remarkably reduced, lymphatic vessel generation and immunosuppressive cell infiltration are further reduced, LNM generation is blocked, and the tumor immune microenvironment is improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and in particular relates to a dual AAV delivery system for CD26 based on CRISPR base editing technology. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and metastatic malignant tumors of the digestive system, with an extremely poor prognosis, a very low five-year survival rate, and early distant metastasis. Studies have shown that lymph node metastasis (LNM) is an important marker of early PDAC progression and a key factor affecting patient prognosis. PDAC tumor cells tend to spread early through the lymphatic system, forming regional metastases, significantly impacting surgical resection rates and postoperative recurrence rates. Therefore, early prevention of LNM development is of significant clinical importance for improving the survival rate of PDAC patients.

[0003] CD26, also known as DPP4 (Dipeptidyl Peptidase-4), is a membrane-bound serine protease widely expressed in T cells, fibroblasts, and tumor-associated stromal cells. Recent studies have found that in the PDAC tumor microenvironment, CD26 is highly expressed in a class of aging-associated cancer-associated fibroblasts (CAFs) and participates in regulating multiple pro-metastatic signaling pathways. Research has found that CD26... + CAFs can promote lymph node metastasis in pancreatic cancer, making them potential therapeutic targets. Currently, treatment strategies targeting CD26 mainly focus on antibody blockade. While monoclonal antibodies such as Bevacizumab can interfere with the CD26 signaling axis to some extent, their efficacy in PDAC is minimal. This is because PDAC has a significantly immunosuppressive tumor microenvironment, including abundant immunosuppressive cells (such as Tregs and M2 macrophages) and a highly dense matrix structure, making it difficult for antibodies to penetrate deep into the tumor, and limiting ADCC effects. Therefore, traditional monoclonal antibody therapy struggles to target CD26. + Effective clearance of CAFs urgently requires the exploration of more efficient and tissue-penetrating treatment strategies. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a dual AAV delivery system for targeting CD26 based on CRISPR base editing technology.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides an editing system targeting CD26, the editing system comprising:

[0007] CRISPR base editor and sgRNA;

[0008] The CRISPR base editor includes an adenine base editor or a cytosine base editor;

[0009] The sequence of the sgRNA matches the encoding of the CRISPR base editor;

[0010] The sequences of the sgRNAs that match the adenine base editor are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, or SEQ ID NO.6;

[0011] The sequences of the sgRNAs that match the cytosine base editors are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6.

[0012] This invention provides a dual AAV delivery system for CD26 targeting based on CRISPR base editing technology, comprising a first AAV vector and a second AAV vector;

[0013] The structure of the first AAV vector is that AAV is used as the initial vector, and the Rep and Cap genes in AAV are replaced with target sequence 1;

[0014] The target sequence 1 is obtained by sequentially linking the CMV promoter, the sgRNA, Cas9n, the inteptide, and the CRISPR base editor from the 5' end to the 3' end.

[0015] The structure of the second AAV vector is that AAV is used as the initial vector, and the Rep and Cap genes in AAV are replaced with target sequence 2;

[0016] The target sequence 2 is obtained by sequentially linking the CMV promoter, the sgRNA, Cas9n, the inteptide, and the N-terminal fragment of the CRISPR base editor from the 5' end to the 3' end.

[0017] Preferably, the AAV is AAV9.

[0018] This invention provides the application of the aforementioned dual AAV delivery system in the preparation of a medicament for treating pancreatic ductal adenocarcinoma.

[0019] Preferably, the dual AAV carrier is delivered via local injection into the pancreas.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention is the first to construct a CD26-targeting editing system and deliver it in vivo via dual AAV vectors, successfully achieving selective clearance of CD26+CAFs in PDAC. By precisely editing the CD26 gene splicing donor site, CD26 expression in CAFs is significantly reduced, thereby decreasing lymphangiogenesis and immunosuppressive cell infiltration, blocking LNM occurrence, and improving the tumor immune microenvironment. This invention provides a CD26-targeting AAV dual-vector delivery system based on CRISPR base editing technology, which can efficiently and specifically edit CD26+CAFs in the PDAC microenvironment in vivo, achieving functional silencing, thereby significantly inhibiting tumor lymph node metastasis (LNM), reshaping the immune microenvironment, and improving treatment prognosis. This invention also employs a dual AAV-mediated intein split system to deliver in vivo gene editing elements, solving the problem of insufficient packaging capacity of existing AAV vectors, and has advantages such as high tissue specificity, high editing efficiency, and stable delivery system. This invention provides a novel CD26+CAF clearance treatment strategy for PDAC, with good translational potential and broad clinical application prospects. Attached Figure Description

[0022] Figure 1 To demonstrate the editing efficiency of various sgRNA+ base editor combinations using Sanger sequencing;

[0023] Figure 2 To detect the inhibition of protein expression levels after transfection with sgRNA in 293T cells and CAF cells, respectively;

[0024] Figure 3 A schematic diagram of the construction of a dual AAV system;

[0025] Figure 4 AAV therapy targeting CD26 significantly reduced the lymph node metastasis rate in pancreatic cancer. Detailed Implementation

[0026] This invention provides an editing system targeting CD26, the editing system comprising:

[0027] CRISPR base editor and sgRNA;

[0028] The CRISPR base editor includes an adenine base editor or a cytosine base editor;

[0029] The sequence of the sgRNA matches the encoding of the CRISPR base editor;

[0030] The sequences of the sgRNAs that match the adenine base editor are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, or SEQ ID NO.6;

[0031] The sequences of the sgRNAs that match the cytosine base editors are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6;

[0032] In this invention, the editing system is preferably an adenine base editor and an sgRNA with the sequence SEQ ID NO.5.

[0033] The sequences of SEQ ID NO.1 to 6 are detailed in Table 1.

[0034] Table 1 Nucleotide sequences of SEQ ID NO.1–6

[0035]

[0036]

[0037] This invention provides a dual AAV delivery system for CD26 targeting based on CRISPR base editing technology, comprising a first AAV vector and a second AAV vector;

[0038] The structure of the first AAV vector is that AAV is used as the initial vector, and the Rep and Cap genes in AAV are replaced with target sequence 1;

[0039] The target sequence 1 is obtained by sequentially linking the CMV promoter, the sgRNA, Cas9n, the inteptide, and the CRISPR base editor from the 5' end to the 3' end.

[0040] In this invention, the N-terminal and C-terminal fragments of the CRISPR base editor are obtained using the Npu DnaE inteptide splitting system described by Levy et al. (NatBiomedEng 2020, DOI:10.1038 / s41551-019-0501-5), with the splitting point located between E573 and C574 of SpCas9, which respectively encode the N-terminal and C-terminal functional domains of the base editor.

[0041] The structure of the second AAV vector is that AAV is used as the initial vector, and the Rep and Cap genes in AAV are replaced with target sequence 2;

[0042] The target sequence 2 is obtained by sequentially linking the CMV promoter, the sgRNA, Cas9n, the inteptide, and the N-terminal fragment of the CRISPR base editor from the 5' end to the 3' end.

[0043] In this invention, the AAV is preferably AAV9.

[0044] This invention provides the application of the aforementioned dual AAV delivery system in the preparation of a medicament for treating pancreatic ductal adenocarcinoma.

[0045] In this invention, the dual AAV carrier is preferably delivered via local injection into the pancreas.

[0046] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] sgRNA design and editing site screening

[0049] (1) Target screening

[0050] Using the SpliceR platform (https: / / z.umn.edu / splicer), the Ensembl transcript ID (ENSG00000197635) of the human CD26 gene (DPP4) was input, and the SpliceR algorithm was used to screen for high-throughput base editing targets in the key splice donor (GT) and splice acceptor (AG) regions of CD26. The editing window was set to a standard 5nt window for CBE (cytidine base editor) and ABE (adenine base editor) to screen for sgRNA sequences that could potentially interfere with splicing structures.

[0051] (2) Construction of sgRNA sequence

[0052] Step ①: Oligonucleotide synthesis and annealing:

[0053] The upstream and downstream primers contain the BbsI recognition site. They are diluted to 100 μM, mixed in equal volumes, and annealed according to the following procedure: 95℃ for 5 min; then the temperature is reduced by 1℃ per minute to 25℃ to obtain the dsDNA insert fragment (~25 bp).

[0054] An equal volume reaction system of 10 μL consists of: 1 μL upstream oligonucleotide (100 μM) + 1 μL downstream oligonucleotide (100 μM) + 1 μL 10×T4 DNA ligase buffer (NEB, B0202) + 7 μL RNase-free water; place in a PCR instrument at 95℃ for 2–5 min, then slowly cool to room temperature to complete annealing (or follow the 95→25℃ slow cooling program for 45 min).

[0055] Step 2: Vector digestion and ligation:

[0056] The specific reaction system for BbsI-HF (50 μL, total volume) is as follows: 1 μg of sgRNA expression vector DNA to be digested + 5 μL of 10×rCutSmart buffer + 10 U of BbsI-HF (usually 1 μL; calculated based on enzyme activity) + RNase-free water to a final volume of 50 μL; 37℃ for 1 h. BbsI-HF exhibits 100% activity in rCutSmart.

[0057] Ligation reaction formulation (20 μL, sticky ends): 50 ng of BbsI-HF linearized vector and annealed double-stranded oligonucleotide insert (dsDNA insert) were mixed at a 1:3 molar ratio + 2 μL of 10×T4 DNA ligase buffer + 1 μL of T4 DNA ligase (NEB, M0202) + water to make up the volume; reaction was carried out at 16℃ for 16 h.

[0058] Antibiotic concentration: pX330 carrier is Amp antibody, final concentration of ampicillin in plate is 100 μg / mL

[0059] Transformation method: Heat-shocked chemocompetent cells (using NEB 5-alpha / DH5α as an example): Resuscitate DH5α competent bacteria on ice, add 1-5 μL of ligation product, incubate on ice for 30 min → heat shock at 42℃ for 30 s → incubate on ice for 5 min → add 950 μL of room temperature SOC, incubate at 37℃ with shaking for 60 min → plate onto LB plates containing 100 μg / mL ampicillin (Amp), and incubate overnight at 37℃ with the plates inverted.

[0060] Step 3: Screening and sequencing of positive clones:

[0061] Single clones were selected, plasmids were extracted using Miniprep, and Sanger sequencing was performed using U6 / scaffold primers. The sequences were then compared with the original sequences to confirm their accuracy before being used in subsequent experiments.

[0062] Miniprep kit: QIAGEN QIAprep Spin Miniprep Kit (50 doses, catalog number 27104).

[0063] The sequencing primers (used to verify the 20bp sgRNA inserted into the U6 driver region) were U6-F and Scaffold-R, the sequence of which is shown in SEQ ID NO.7 and the sequence of which is shown in SEQ ID NO.8.

[0064] SEQ ID NO.7 (U6 promoter preprime):

[0065] 5'-CGTAACTTGAAAGTATTTCGATTTCTTGGC-3';

[0066] SEQ ID NO.8 (sgRNA scaffold universal reverse primer):

[0067] 5'-AAAAGCACCGACTCGGTGCC-3'.

[0068] Step 4: Plasmid extraction and cryopreservation: High-purity plasmids were prepared using the Endotoxin-free Maxiprep kit; the concentration was adjusted to 1 μg / μL, and the OD260 / 280 was between 1.8 and 2.0; the plasmids were then cryopreserved at -20℃ for later use.

[0069] (3) Construction of ABE expression plasmid

[0070] Vector: pCMV-ABEmax (Addgene#112095), from David Liu's laboratory; expression elements include: CMV promoter, nCas9 (D10A), TadA-TadA* fusion enzyme, NLS and polyA tail.

[0071] (4) Verification of 293T cell editing efficiency

[0072] Cells: ATCC-HEK293T, cultured in 6-well plates until 70% confluence; Transfection reagent: Lipofectamine 3000 (Invitrogen);

[0073] System: pCMV-ABEmax (1 μg); sgRNA expression plasmid (1 μg). After transfection, the cells were cultured for 7 days, and gDNA was extracted and Sanger sequencing was performed to analyze mutations.

[0074] Results: The sgRNA5+ABE combination showed the highest mutation efficiency (GT→GC, approximately 62%) and was selected as the subsequent system.

[0075] (3) Verification of sgRNA editing efficiency

[0076] The above-mentioned sgRNA was co-transfected into 293T cells with ABE and CBE base editors, respectively. Seven days after editing, Sanger sequencing was used to detect changes in the target sequence. The results showed that sgRNA5 had the highest editing efficiency when used in combination with ABE, primarily achieving the GT→GC splicing donor disruptive mutation (see...). Figure 1 Therefore, subsequent in vitro and in vivo experiments will preferentially use the sgRNA5-ABE pairing editing system.

[0077] Transfection method:

[0078] Cell preparation: The day before transfection, the cells should be at approximately 70%–90% confluence. The culture medium is DMEM (high glucose) + 10% FBS, cultured at 37°C and 5% CO2.

[0079] Reagents and carriers: Lipofectamine was used. TM 3000 (L3000008) and Opti-MEM TM (31985-062).

[0080] 6-well plate formulation (single well): Total DNA 2.5 μg: ABE plasmid 1.5 μg + sgRNA plasmid 1.0 μg; P3000 TM 5 μL; Lipofectamine TM 30007.5μL; Opti-MEM TM 125 μL each (two tubes, A and B).

[0081] Operating steps: ① Tube B (DNA mixture): Add total DNA to Opti-MEM TM 125 μL, add P3000 TM 5 μL mixed; ② Tube A (liposomes): Lipofectamine TM 30007.5μL of Opti-MEM was added. TM 1. Mix 125 μL; 2. Slowly add tube B to tube A and let stand at room temperature for 10-15 min to form a complex; 3. Add 2 mL of complete culture medium to each well, add the total amount of the complex to the target well, and shake gently; 4. Replace with fresh complete culture medium after 6-8 h.

[0082] Example 2

[0083] Gene editing validation and functional testing

[0084] In vitro CD26 knockdown validation: ABE+sgRNA5 was transfected into 293T cells and primary CAF cells (transfection method as above). Western blot results showed a significant decrease in CD26 protein expression, validating that the base editor successfully blocked splicing and inhibited transcription (see...). Figure 2 ).

[0085] Example 3

[0086] (1) Construction of dual AAV vectors: Dual AAV vectors were constructed using an intein-split strategy (Reference: DOI: 10.1038 / s41551-019-0501-5):

[0087] AAV9-N: CMV-gRNA5-Cas9n-intein-N;

[0088] AAV9-C: CMV-gRNA5-Cas9n-intein-C.

[0089] Skeleton: pAAV-MCS; Connector type: GibsonAssembly;

[0090] (2) AAV virus packaging and purification

[0091] Cells: HEK293T, 10cm culture dish; Triple plasmid system: AAV9-N or AAV9-C; pHelper; pAAV-RC9 (RepCap9); Transfection reagent: PEI 1mg / mL; Cultured for 5 days, cells and supernatant were collected;

[0092] Purification method: PEG8000 precipitation; Iodixanol gradient ultracentrifugation; Tit: qPCR detection >5×10⁻⁶ 10 vg / μL.

[0093] For detailed structural diagrams, please refer to... Figure 3 .

[0094] Packaging and virus titer detection were performed using the VectorBuilder platform (5 × 10⁶ per strain). 10 This design breaks through the upper limit of AAV monomer packaging capacity (<4.7kb), ensuring complete expression by the base editor.

[0095] Example 4

[0096] In vivo validation experimental procedure

[0097] (1) Establishment of humanized PDAC model

[0098] ① Animal source: Purchased from Vitalstar (China), 6-week-old hCD34 + NSG humanized mice (NOD-scid IL2Rgnull background, with established human immune system).

[0099] ② Rearing conditions: SPF barrier environment, constant temperature 22℃, 12-hour light and dark cycle, using autoclaved feed and water.

[0100] Tumor model construction:

[0101] ③ Cell preparation: Preparation of human PDAC organoids and CD26 + CAFs;

[0102] Mixing ratio: Organoids: CD26 + CAFs = 1:2 (total cell count approximately 2 × 10⁻⁶) 6 );

[0103] Matrix suspension: After mixing the cells, add Matrigel (Corning, 356231) to a final concentration of 50% to prepare an injection suspension.

[0104] ④ Surgical procedure: Mice were anesthetized (isoflurane inhalation, general sedation); a small incision was made in the left abdomen to expose the tail of the pancreas;

[0105] 30 μL of suspension was slowly injected using the pancreatic parenchyma injection method to ensure the formation of a tumor mass. Postoperative management: The incision was sutured, and ibuprofen analgesia (0.2 mg / mL) was administered continuously for 3 days; the patient was fed for 7 days postoperatively to stabilize the tumor microenvironment.

[0106] (2) Local delivery of AAV dual-carrier system

[0107] ①AAV formulations: AAV9-N and AAV9-C (both containing gRNA5 and a separate ABE structure), packaging titer: 5 × 10⁻⁶ 10 vg / μL.

[0108] ②Injection time: 7 days after tumor transplantation;

[0109] ③ Injection method: Re-anesthetize the mice; re-expose the original tumor injection site; use Hamilton microsyringe to slowly inject 20 μL each of AAV9-N and AAV9-C into the tumor area; n=6 mice per group, and suture in situ after injection.

[0110] 3) In vivo editing verification and mechanism detection

[0111] ① Sample collection: Mice were sacrificed on day 14 after injection; pancreatic tumor tissue and mesenteric / abdominal lymph nodes were collected; part of the tissue was used for genomic extraction and part was used for IHC and flow cytometry detection.

[0112] ②Genomic extraction: Tumor DNA was extracted using the QIAamp DNAMini Kit (Qiagen); the target editing region was amplified by PCR, and after library construction, Illumina high-throughput sequencing was performed.

[0113] ③ Lymph node metastasis assessment (LNM): Immunohistochemical analysis was performed using CK19 (tumor marker) and Vimentin (CAF marker).

[0114] The results showed that on the 7th day post-transplantation, AAV9-N and AAV9-C (5×10⁻⁶ each) were administered via local injection into the pancreas. 10 (vg / mouse). Editing efficiency was verified by high-throughput sequencing, with an average mutation rate >50%.

[0115] Sanger sequencing of mouse tumor masses revealed that AAV treatment significantly reduced CD26 expression levels in vivo and inhibited lymph node metastasis (LNM). Figure 4 ).

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An editing system targeting CD26, characterized in that, The editing system includes: CRISPR base editor and sgRNA; The CRISPR base editor includes an adenine base editor or a cytosine base editor; The sequence of the sgRNA matches the encoding of the CRISPR base editor; The sequences of the sgRNAs that match the adenine base editor are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, or SEQ ID NO.6; The sequences of the sgRNAs that match the cytosine base editors are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.

6.

2. A dual AAV delivery system targeting CD26 based on CRISPR base editing technology, characterized in that, Including the first AAV carrier and the second AAV carrier; The structure of the first AAV vector is that AAV is used as the initial vector, and the Rep and Cap genes in AAV are replaced with target sequence 1; The target sequence 1 is obtained by sequentially linking the CMV promoter, the sgRNA of claim 1, Cas9n, the inteptide, and the C-terminal fragment of the CRISPR base editor of claim 1 from the 5' end to the 3' end. The structure of the second AAV vector is that AAV is used as the initial vector, and the Rep and Cap genes in AAV are replaced with target sequence 2; The target sequence 2 is obtained by sequentially linking the CMV promoter, the sgRNA of claim 1, Cas9n, the integrin, and the N-terminal fragment of the CRISPR base editor of claim 1 from the 5' end to the 3' end.

3. The dual AAV delivery system according to claim 2, characterized in that, The AAV is AAV9.

4. The use of the dual AAV delivery system according to claim 2 or 3 in the preparation of a medicament for treating pancreatic ductal adenocarcinoma.

5. The application according to claim 4, characterized in that, The dual AAV vector is delivered via local injection into the pancreas.

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