Engineering hepatocyte-targeted LNPs for RNA and DNA delivery to primary human T cells

As gene therapy moves towards bigger, more complex payloads. Our industry partners need platforms that can actually keep up and go beyond what current viral and non-viral can offer today.

At ElevateBio, we have developed a proprietary library of ionized lipids and PEG-lipids that enable us to develop LNPs for delivering to different organs and cell types. In one of my colleagues’ talk earlier, we shared that one of our lead formulations, LNP, demonstrated safe and potent nucleic acid delivery to the liver in NHPs. Today, I’d like to focus on our effort in developing LNPs for primary T cell engineering.

We first benchmark our LNPs against a commercially available T cell transfection reagent using eGFP mRNA. Our results showed that our LNPs achieve higher transfection efficiency while also maintain better cell variability. Next, we compared our platform with electroporation for CD19-CAR mRNA delivery. While electroporation results in relatively transient CAR expression our LNPs achieve comparable initial expression, but with improved durability. We still observe more than 60% CAR-positive cells on day four post-transfection. We then evaluate gene editing performance using our proprietary editors to achieve TRAC knock out at a dose of two microgram per million cells. Our LNPs achieve TRAC knock out efficiency comparable to electroporation.

So far we have demonstrated efficient delivery of reporter mRNA,  CD19 -CAR mRNA and gene editors using our RNA LNP platform. However, in some more advanced gene editing scenario, efficient DNA delivery becomes essential, especially for large gene insertion. So, to address this challenge, we optimize our formulation and develop a DNA LNP platform for DNA delivery. We were able to get a DNA LNP that capable of potently delivering plasma DNA to primary human T cells.  We were able to achieve over 90% GFP positive cells at relatively low dose. Finally, we apply both our RNA and DNA LNP platforms to enable large serine recombinase or LSR- mediated CAR insertion. In this workload, LSR mRNA was encapsulated in our RNA LNP formulation, while the CAR DNA payload was delivered using our DNA LNP formulation. Using electroporation, we achieve about 20% CAR insertion efficiency, but when we switch to our LNP-based delivery system, the CAR insertion efficiency increased to over 85%. Our LNPs dose-dependently increase the CAR insertion efficiency.

So, in summary, our LNP platform enables highly efficient DNA and RNA delivery to primary human T cells. Importantly, our system outperforms electroporation in delivering challenging payloads, such as large DNA payloads required for gene insertion, helping address an important unmet need in next generation cell engineering.

This gives our partners a true alternative, one that handles large and complex payloads and really opens things up for advanced cellular engineering.