Gene editing experiments in vitro and in vivo with Cre-lox recombination
In vitro experiments
Following transfer, LentiFlash-Cre particles induced the steady excision of the dsRed transgene (Figure 2, sample 4 for the polyclonal population and sample 7 for clone 14) with high efficiency in all target cells. Of note the stability of excision suggested 100% efficacy following LentiFlash particles mRNA delivery.
Equivalent excision was observed with an IDLV expressing Cre from an EF1 promoter (Figure 2, sample 3 for the polyclonal population and sample 6 for clone 14). The main difference is that unlike IDLV, LentiFlash particles allows the excision without residual integration.
Next, we determined whether LentiFlash enables systemic and localized in vivo expression in mice. The ability to deliver RNA in vivo would be valuable for several research and clinical applications.
In vivo experiments
The mouse model Figure 4 highlighted the efficiency of the non-integrating lentiviral particle LentiFlash to induce stable excision of the gene of interest (Here YFP).
The excision led to YFP expression in a wide zone of injected muscle (Figure 4, a, b and c, as compared to non-affected muscle zone d). LentiFlash particles spread in approximately 1 cm of the muscle, and this is comparable to the area reached with purified lentiviral vectors.
Altogether, in vivo data confirmed the potency of LentiFlash particles for delivering functional mRNA locally.
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