TcBuster 非病毒基因工程服务

Theoretically transposons can be used for in vivo delivery and is an active area of research in the field. Current delivery mechanisms don’t allow for efficient enough transfer and editing efficiency.

Foreign DNA from the transposon can activate intracellular DNA sensing mechanisms and trigger apoptosis. The toxicity can be managed with specific cell handling techniques during transfection. One reason for delivering the transposase as mRNA is to avoid triggering these responses.

The transposon must be delivered as a DNA template as this will be directly inserted into the genome. The transposase is delivered as mRNA to prevent any possible random integration of the enzyme and decrease DNA mediated toxicity. Transposases are difficult to manufacture as purified protein in high enough concentrations for transfection due to their relatively insoluble nature.

Gene transfer is considered stable if the DNA is integrated into the cellular genome. Once incorporated, we have not observed any issues with silencing or loss of expression. In transformed cell lines we routinely use a selection cassette as part of the transposon to allow for re-selection of the cells if necessary. In T cells we have performed repeated stimulations of transposed cells and do not observe loss of transgene expression over time.

Multicistronic vectors can be used with viruses, although due to packaging limitations in viruses the yield and titer of the virus are often negatively impacted when delivering multiple genes. Transposons can move larger cargo and are therefore better suited to multicistronic vectors.

TcBuster has been engineered to have increased enzymatic activity over the wildtype version of the enzyme. This increases the efficiency of gene transfer to rates that are equal to or better than alternate gene engineering systems.

Resting cells have a relatively larger nucleus, making it easier to get DNA into the nucleus. Proliferating cells have enlarged cytoplasm, so the nucleus is a relatively smaller target.

No, TcBuster can work with both resting or pre-activated T cells.

The gene insertion profile of TcBuster has been studied and has been found to be more random than that of lentivirus. While viruses do “randomly” integrate they preferentially insert into more active regions, i.e gene-coding regions. TcBuster-M and transposons in general are more random in their insertional profile decreasing the likelihood of disrupting gene transcription as there is less coding genome than non-coding.

TcBuster can consistently integrate a single CAR into primary CD4+/CD8+ T-cells with an efficiency of 50-70% depending on the exact construct. As construct size and complexity increase, the transposition rates do tend to decline modestly. Transposition of multicistronic vectors encoding either 3 or 4 transgenes have achieved rates from 25-40%.

TcBuster can be used in cell therapy for gene modification of T cells, NK cells, and iPSCs. In addition, it can be used to generate stable producer cell lines for either antibody or protein production, and it is used to over express genes in cell lines for drug discovery or screening purposes.