Preclinical study of suicide gene as a safety switch to control CAR-T cell cytotoxicity / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate whether AP1903, a small-molecule chemical inducer, can terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vivo and in vitro. Methods: CD19CAR-T cells over-expressing iCasp9 (iCasp9-CD19CAR-T) were constructed and co-incubated with AP1903. Then, the cell phenotype and apoptosis were detected by Flow cytometry, and the iCasp9/CID suicide gene system was verified on K562 and T cells, respectively. The cytotoxicity of iCasp9-CD19CAR-T cells was detected in vivo (survival rate of NCG mice bearing Raji cell transplanted xenograft) and in vitro (cell killing function was detected by Flow cytometry) under the administration of AP1903. Results: Compared with CD19CAR-T cells, iCasp9-CD19CAR-T cells showed in significant difference in proliferation, phenotype and cytotoxicity both in vitro and in vivo (all P>0.05). At 2 h after AP1903 administration, the apoptosis rates of K562 and T cells co-expressing iCasp9 and CD19CAR were (33.8±0.9)% and (27.95±0.35)%, respectively; and at 24 h after AP1903 administration, the apoptosis rates reached 100% in both cell lines. The in vitro cytotoxicity of iCasp9-CD19CAR-T cells induced by AP1903 was significantly lower than that without AP1903 treatment (P<0.01); the 60-day survival rate of mice bearing Raji cell transplanted tumor treated with AP1903-induced iCasp9-CD19CAR-T cells was also significantly lower than those treated with iCasp9-CD19CAR-T cells alone (P<0.01). Conclusion: AP1903 can effectively terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vitro and in vivo.

Genome editing for the treatment of tumorigenic viral infections and virus-related carcinomas / 医学前沿

Viral infections cause at least 10%-15% of all human carcinomas. Over the last century, the elucidation of viral oncogenic roles in many cancer types has provided fundamental knowledge on carcinogenetic mechanisms and established a basis for the early intervention of virus-related cancers. Meanwhile, rapidly evolving genome-editing techniques targeting viral DNA/RNA have emerged as novel therapeutic strategies for treating virus-related carcinogenesis and have begun showing promising results. This review discusses the recent advances of genome-editing tools for treating tumorigenic viruses and their corresponding cancers, the challenges that must be overcome before clinically applying such genome-editing technologies, and more importantly, the potential solutions to these challenges.

Advances in CRISPR/Cas9 gene-editing system / 基础医学与临床

CRISPR/Cas9 gene-editing system has been broadly used in various fields of bioscience and medicine in recent years. The system can be guided by RNA to specific DNA site thus achieving targeted gene editing.Off-target effect and editing efficiency remain to be two crucial challeges to the system. Currently, a number of researches have been focused on the optimization of the system by reducing off-target effects and increasing editing efficiency, which may enhance its safety and expand its application.

Advance and application of CRISPR/ Cas9 mediated genome editing technique on pathogenic microorganism / 中国人兽共患病学报

Clustered regularly interspaced short palindromic repeats (CRISPR) is an acquired immune system existing in archaea and bacteria with the long-term process of evolutionary.CRISPR/Cas9 gene editing system is a new type of gene editing technology developed based on the system.CRISPR/Cas9 is a more efficient method for gene targeting than the previous methods.It has been successfully applied for gene-modified of eukaryotes since 2012,but the reports about pathogenic microorgaisms are rarely.Here,the research progress in the structure,mechanism of CRISPR/Cas9 system and its applications on pathogenic microorgaisms is reviewed.

Mouse MAD2L1 gene editing by CRISPR/Cas9 and analysis of its off-target effect / 中国实验动物学报

Objective To establish a method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9, and analyze its off-target effect. Methods The gene editing site for MAD2L1 gene was designed by CHOP?CHOP, and the Cas9?MAD2L1 vector was constructed based on the designed editing site. Cas9?MAD2L1 was then transfected into NIH/3T3 cells and screened with puromycin, followed by observing GFP expression using fluorescence microscopy. The genomic DNA from transfected cells was extracted and a partial fragment of MAD2L1 gene was amplified by PCR. T7E1 analy?sis and Sangger sequencing were used for gene editing and off?target analysis. Results After Cas9?MAD2L1 transfection and puromycin screening, a large number of GFP?expressing cells were observed under the fluorescence microscope. Combined the PCR result with TE71 analysis, the amplified 228 bp PCR products can be digested into 166 bp and 62 bp fragments. The se?quencing result showed that the second exon of MAD2L1 gene was successfully edited, and the off?target effect was undetected in our system. Conclusions The method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9 is successfully established, and off?target effect of MAD2L1 gene is not detected.

Off-target Effects of RNAi and Design of Highly Effective Modified siRNAs / 生物化学与生物物理进展

RNA interference mediated by short interfering RNA is widely used to study functional genes and also being developed for therapeutic applications. However, recent study demonstrated that siRNA might activate innate immune system and induce huge production of inflammatory cytokines in mammals, and also randomly inhibit expression of undesired genes. Designing highly effective siRNAs or modifying the siRNA to retain or enhance the silence efficiency and meanwhile abolish the off-target effects associated with immunostimulation then become the key techniques in application of siRNAs as safe and effective therapeutic agents.