Bionic bispecific nano editing system and application thereof
An editing system and bispecific technology, applied in the field of bionic bispecific nano editing system and its application, to reduce genotoxicity and improve the safety of treatment
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Embodiment 1
[0035] Example 1: Preparation and morphological characterization of a biomimetic bispecific nanoediting system
[0036] Preparation:
[0037] Synthesis of polydisulfide cationic polymer PD: Lipoic acid (4.12 g, 20 mmol) and CDI (4.28 g, 26.4 mmol) were dissolved in dry DCM (100 ml) and added dropwise to anhydrous DCM (30 ml). in a solution of diethylenetriamine 2 (DET, 16 g, 155 mmol). The reaction mixture was stirred at 0°C for 1 hour and allowed to recover at room temperature for 1 hour. Brine (3 x 100ml) was used to wash the end product. The organic layer was dried and concentrated to obtain monomer 1 (1.9 g, 50%) containing diethylenetriamine. Subsequently, guanidine group-containing monomer 2 was synthesized by adding lipoic acid (4.12 g, 20 mmol) and CDI (3.24 g, 20 mmol) to anhydrous DMF (50 ml), and then stirring at room temperature for 2 h. The above mixture was added to a solution of methyl L-arginine methyl ester dihydrochloride (2.61 g, 10 mmol) and DIEA (1.74 ...
Embodiment 2
[0048] Example 2: Biomimetic nano-bispecific nano-editing elements down-regulate the expression of related disease genes at the cellular level
[0049] Evaluation method:
[0050] T7E1 detection and deep sequencing: Assessment of indel frequencies at target genomic loci by T7E1 assay. DNA was extracted from transfected cells and liver tissue using DNeasy Blood & Tissue Kit (Vazyme, China). The target region was amplified by PCR using QIAquick PCR Purification Kit (Vazyme, China), and 200 ng of PCR product was used for T7E1 assay according to the recommended protocol. The products were analyzed by agarose gel electrophoresis (2% agarose gel) and visualized by a gel exposure system (c150, Azure Biosystems, USA). Finally, quantification of DNA band grayscale was measured using Image J. The calculation formula for indel frequency analysis is: [1-(1-fraction cleaved)1 / 2] × 100%, where cleavage rate = sum of intensities of each digested band / (sum of intensities of each digested b...
Embodiment 3
[0056] Example 3: Biomimetic bispecific nanoediting system for preventing hepatic ischemia-reperfusion injury
[0057] Establishment of surgically induced liver ischemia-reperfusion injury model: C57BL / 6 mice (6-8 weeks) were administered PBS, PD, PD / Pnt@M, PD / Pcas9@M or PD / PCasRx@M by tail vein injection. Complexes containing 30 μg plasmid and 150 μL PD / Pnt@M complex (1.5 μg / μL) or PBS were administered on days 1, 3, 5 and 7, respectively. The injected mice were divided into six groups (control group, Con A injected with PBS, ConA and PD, ConA and PD / Pnt@M, ConA and PD / Pcas9@M, and ConA and PD / PCasRx@M), each group 5 mice. On day 7, mice were anesthetized with isoflurane and then fixed supine on the operating table. After abdominal depilation and sterilization, mice were subjected to a midline laparotomy to expose the liver. The left and median liver lobes were blocked with microvascular clips to block the blood supply for 60 min, followed by reperfusion by removing the cl...
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