TcBuster™-M Transposase mRNA

Mechanistic Diagram of the TcBuster Non-Viral Gene Editing System. The TcBuster system components are introduced into cells via electroporation (1). The TcBuster-M transposase mRNA is then translated into the transposase enzyme (2), which binds to the inverted terminal repeats (ITRs) on the DNA transposon (3). The transposase enzyme excises the genes of interest (GOI) from the transposon (4) and inserts them into the host genome (5). The GOI mRNA is transcribed from the host genome (6), and the protein is stably expressed in the edited cells (7).

Efficient gene editing of T cells modified with different sized cargos on different electroporation platforms. 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.

TcBuster edited CD19-CAR-T cells specifically kill CD19+ target cells at low E:T ratios. 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.

Genetic characterization of TcBuster genetically modified primary T cells shows a favorable insertional profile and low copy number. (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).

TcBuster edits primary T cells as efficiently or better than lentivirus. 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.