유전자 도입을 위한 non-viral TcBuster mRNA Transposase & Transposon | 고마바이오텍

1) TcBuster 시약은 mRNA 전위효소(transposase)와 DNA 전위인자(transposon)로 구성되며, 전기천공법(electroporation)을 통해 세포 내로 도입됩니다.

2) 세포 내로 들어가면 TcBuster mRNA는 전위효소 단백질 (TcBuster transposase)로 번역됩니다.

3) 전위효소는 DNA transposon 내 유전자 관심 부위(GOI)를 감싸는 특정 부위인 역말단 반복서열(ITR, inverted terminal repeats)에 결합합니다.

4) TcBuster는 transposon으로부터 DNA 운반체(cargo)를 절단해냅니다.

5) TcBuster transposase는 DNA 운반체를 숙주 세포의 게놈 내 AT 뉴클레오타이드 부위에 무작위로 삽입합니다.

6) 삽입된 DNA 운반체는 mRNA로 전사되고,

7) 이후 단백질로 번역되어 안정적인 유전자 발현을 일으킵니다.

Primary T cells from 3 donors were expanded for 7 days after genetic modification in GMP Human T Cell Media (Catalog # CCM038-GMP-1L) supplemented with 5% hAB serum and 10 ng/mL each of IL-7 (Catalog # BT-007-GMP) and IL-15 (Catalog # BT-015-GMP) in a 6 well G-Rex®. (A). Representative flow plots of genetically modified T cells with a 5.1 kb insert containing a GFP sequence, introduced on the ThermoFisher Neon™, Lonza 4D- Nucleofector®, or MaxCyte®. (B).Three DNA cargos introduced on the same electroporation platforms achieve high editing efficiency in T cells (>40%). Data points represent the average of 3 donors with technical duplicates ± SD.

T cells from 3 donors were expanded for 7 days after genetic modification in GMP Human T Cell Media (Catalog # CCM038-GMP-1L) supplemented with 5% hAB serum and 10 ng/mL each of IL-7 (Catalog # BT-007-GMP) and IL-15 (Catalog # BT-015-GMP) in a 6 well G-Rex®. (A) TcBuster efficiently integrates cargos of different sizes regardless of electroporation platform, resulting in a similar number of total modified T cells produced. (B) T cells from 3 donors modified with the 5.1 kb plasmid were cryopreserved in CS10 following 7 days of culture after electroporation. T cells were thawed and immediately added to either CD19+ or CD19- target cells at different E:T ratios and controlled target cell growth after 24 hours of co-culture.

(A) T cells from 3 donors were collected after 9 days of culture for digital PCR analysis of the population’s average adjusted vector copy number (VCN). (B) Integration site analysis (ISA) was performed on 3 different T cell donors that were modified with a different range of plasmid sizes with TcBuster or a lentiviral vector. DNA libraries were Illumina sequenced by GeneWerk (now ProtaGene).

T cells from 3 donors were expanded for 9 days in GMP Human T Cell Media, supplemented with 5% hAB serum and 10 ng/mL each of IL-7 and IL-15, in a 6 well G-Rex®. The GOI is refers to the promoter and genes integrated in the genome. Graph shows the efficiency of genetic modification for both TcBuster and lentivirus, with TcBuster showing comparable or better editing efficiency. Data points represent the average of 3 donors ± SD.