Abstract 3617: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (CAS) system mediated endogenous CD19 gene knockout model in burkitt lymphoma

BACKGROUND: Pediatric Burkitt lymphoma (PBL) is an aggressive mature B-cell malignancy and is the most common non-Hodgkin lymphoma (NHL) in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo et al., BJH 2012). The survival of Pediatric Burkitt lymphoma (PBL) has significantly improved over the past 30 years through the introduction of short and intensive multi-agent chemotherapeutic regimens (Cairo et al., JCO, 2012). CD19 is an antigen expressed on the surface of cells of the B lineage, and is recognized as a potential target for immunotherapy. CD19 immunotherapy has been emerged as a promising approach for B cell malignancies (Porter/June et al., NEJM, 2011). However, the mechanism(s) and function of CD19 is still poorly understood in BL. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (CAS) genome engineering has been developed for precision targeted genome mutagenesis in vitro and in vivo for new experimental and therapeutic tools (Jao et al., PNAS, 2013; Morgan et al., Nat. Met., 2013; Fu et al., Nat. Biotech, 2013). OBJECTIVES: We hypothesize that 1) CRISPR/Cas system is suitable for silencing of CD19 expression in BL, and 2) CRISPR/Cas-mediated CD19 knockout model will be provide useful tool for understanding the mechanism and function or CD19 and the potential use of CD19 antibodies as therapeutic agents in BL. METHODS: Synthetic gRNAs targeting CD19 locus were constructed using modified CRISPR/Cas system from Hwang et al (Nat. Biotech, 2013) for CD19 knockout Raji cells. CRISPR-Cas/RNA-Guided Nuclease Plasmids were obtained from Addgene (ID: 43860 and 43861). Surveyor mutation detection assay was used for CD19 CRISPR validities and confirmed by sequencing analysis. Western blotting was performed for measurement of protein level and statistical significance was determined by one-tailed Student t test using GraphPad Prism software. RESULTS: Two pairs of functional CD19 CRISPR (C1 and C2) targeting start (exon 1) and stop codon sequences (exon 14) of endogenous CD19 gene were generated. Mutated sequences by non-homologous end joining (NHEJ) were confirmed in CRISPR mediated CD19 knockout pool Raji cells. We successfully obtained stably CD19 knockout out single clone (hemizygote) which is entire CD19 gene (about 7.0kb, exon1 to exon 14) was deleted by CRISPR/Cas (Clone CD19-SC8). CD19-SC8 showed that a significant decrease of CD19 protein expression (92±0.2% reduction, p CONCLUSIONS: This preliminary data suggest that CRISPR/Cas system is useful tool for the modification of endogenous CD19 gene expression and CRISPR/Cas mediated CD19 knockout model will be useful for studying CD19 function in BL and the efficacy of second generation CD19 antibodies and antibody conjugates against CD19 in BL. Citation Format: Sanghoon Lee, Changhong Yin, Janet Ayello, Carmella van de Ven, Mitchell S. Cairo. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (CAS) system mediated endogenous CD19 gene knockout model in burkitt lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3617. doi:10.1158/1538-7445.AM2014-3617