Cytiva, 1001144, GenVoy-ILM™ T cell kits for mRNA

GenVoy-ILM™ T cell Kit for mRNA, Ignite™, 3 mL GenVoy-ILM™ T cell Kit for mRNA is a lipid nanoparticle (LNP) reagent mix optimized for the delivery of mRNA or CRISPR Cas9 mRNA with gRNA into human primary T cells using LNPs prepared on the NanoAssemblr™ Spark™ and Ignite™ systems. GenVoy-ILM™ T cell Kits for mRNA offers the following benefits: Highly efficient knockout and expression: The mRNA or mRNA/sgRNA can be delivered into activated human primary T cells consistently with high gene knockout efficiency. High cell viability: Maintain high T cell viability, even after sequential genetic manipulation. Scalable: Scale your cell therapy research on the NanoAssemblr™ platform from discovery to preclinical. Validated with T cell manufacturing workflow: Optimized and validated for downstream T cell culture workflow. The kits enable researchers to establish a clinically relevant and scalable method for ex vivo gene delivery and gene editing to advance the development of T cell therapies. These kits can be used at various stages of cell therapy research and development from discovery to preclinical across the NanoAssemblr™ platforms. Greater ratio of engineered T cells compared to electroporation (EP) Using the GenVoy-ILM™ T cell Kit for mRNA to deliver mRNA into activated human primary T cells results in an increased number of engineered T cells. This method demonstrates a more effective gene delivery method compared to EP. Transfection efficiency was measured by protein expression via flow cytometry at 24 and 48 hours post-treatment. EP was performed by following the manufacturer’s protocol, and LNP treatment was performed following the GenVoy-ILM™ T cell Kit for mRNA on Spark™ protocol. Untreated cells (UT) were used as control. **p<0.01, ****p<0.0001 by one-way ANOVA. Uniform cellular protein expression Engineering human primary T cells using the GenVoy-ILM™ T cell Kit for mRNA results in highly uniform protein expression across the treated cell population. Representative mean fluorescence intensity (MFI) plots of cell surface protein expression were generated using flow cytometry at 24 and 48 hours post-treatment. EP was performed by following the manufacturer’s protocol. Untreated cells (UT) were used as controls. Highly viable T cells Activated human primary T cells remain highly viable after ex vivo gene delivery using the GenVoy-ILM™ T cell Kit for mRNA. Cell viability was measured via flow cytometry at 24 and 48 hours post-treatment. EP was performed by following the manufacturer’s protocol, and LNP treatment was performed following the GenVoy-ILM™ T cell Kit for mRNA on Spark™ protocol. Untreated cells (UT) were used as controls. ****p<0.0001 by one-way ANOVA. Multi-step engineering of T cells for off-the-shelf cell therapy Achieve highly efficient genome editing knockouts in combination with high levels of CD19 CAR expression. This allows for the engineering of functional and viable “universal” CAR T cells that effectively kill CD19+ tumor cells. A) Schematic illustration of experiment. mRNA-LNPs containing sgRNA + Cas9 mRNA were added to primary T cells. Cells expanded prior to treatment with CD19 CAR mRNA-LNPs. At 24 hours post CAR mRNA-LNP treatment, CD19+ T cell killing assay was conducted for 16 hours. B) TCR knockout efficiency was assessed. Starting sample was TCR negative selected to further purify the TCR- population. C) Left: Percent CD19 CAR expression 24 hours after treatment with CAR mRNA-LNPs at 3.2 μg RNA/million cells, when population is TCR+, or TCR−. Right: Corresponding cell viabilities, normalized to the untreated population. D) Functional killing of CD19+ B cells (SUP-B15) by either UT, TCR+/CAR+, or gene-edited TCR−/CAR+ T cells at the indicated effector to target ratios (E:T). For all, a dose of 3.2 μg RNA/million cells was applied. Error bars represent standard deviation. Statistical significance was evaluated using t-tests or one-way ANOVA among the shown groups (2). Scale up from discovery to preclinical The data demonstrates that the GenVoy-ILM™ T cell Kits and the scalable NanoAssemblr™ platform enable robust and equivalent performance on both the Spark™ and Ignite™ instruments enabling scale up from discovery to preclinical. A) GFP and B) CD19 CAR transfection efficiency 24 hours post mRNA-LNP addition. C) Levels of single target (T cell receptor, TCR) knockout, and D) Double target (TCR and CD52) knockout through sgRNA and Cas9 mRNA delivery. E) Functional killing of CD19+ B cells (SUP-B15) in a 16-hour co-culture experiment. For all: a dose of 3.2 μg RNA/million cells was applied to human primary T cells. RNA-LNPs were prepared according to the GenVoy-ILM™ T cell Kit for mRNA on Spark™ or Ignite™ user guides. Average gene expression and gene knockout was detected by flow cytometry showing at minimum n=8 independent RNA-LNP preparations and n=2 donors. Functional killing performance was detected by flow cytometry showing n=2 independent RNA-LNP preparations and n=2 donors. Error bars represent standard deviation with statistical significance evaluated using t-tests among selected groups (2). Kit components

	GenVoy-ILM™ T cell Kit for mRNA, Spark™, (Part no. 1000700)	GenVoy-ILM™ T cell Kit for mRNA w/ 5 Spark™ cartridge, (Part no. 1000683)	GenVoy-ILM™ T cell Kit for mRNA, Ignite™, 3 mL, (Part no. 1001144)	GenVoy-ILM™ T cell Kit for mRNA, Ignite™, 6 mL, (Part no. 1001161)
Lipid mix	90 µL	90 µL	3 mL	6 mL
Formulation buffer (10X)	30 µL	30 µL	2 mL	4 mL
Dilution buffer (10X)	900 µL	900 µL	40 mL	80 mL
Cryopreservation buffer (2X)	-	-	3 mL	6 mL
Apolipoprotein-E (ApoE)	50 µg	50 µg	500 µg	500 µg x 2
Spark Cartridges	-	5	-	-