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Novel Lipid Nanoparticle Technology for Effective Non-Viral Engineering of Primary Human T Cells Towards T Cell Therapy ApplicationsAndrew Brown*, Rebecca De Souza, Nikita Jain, Tara Fernandez, Andrea Bernardo, Raymond Sabaten, Samuel Clarke and Anitha Thomas Contact us at:
Precision NanoSystems Inc, Vancouver, BC, [email protected]
Introduction▪ Currently approved cell therapies employ viral gene transfer
which has significant manufacturing challenges and safety concerns
▪ Non-viral gene delivery methods, such as electroporation, also suffer from issues of scalable manufacturing, and can result in significant cellular toxicity
▪ PNI’s novel lipid nanoparticle (LNP) technology enables nucleic acid LNPs to be rapidly manufactured at any scale and provides high performance in sensitive and difficult to transfect cells
Objectives▪ Demonstrate the manufacturing and performance advantages of
PNI’s LNP reagent for messenger RNA (mRNA) delivery to humanprimary T cells
▪ Showcase the therapeutic utility of PNI’s LNP reagent throughexpression of a chimeric antigen receptor (CAR) construct inhuman primary T cells
MethodsGene Delivery via Lipid Nanoparticles
www.precisionnanosystems.com [email protected] @PrecisionNano
Results
Conclusions▪ mRNA LNPs were reproducibly manufactured in less than 5
minutes using the NanoAssemblr Spark
▪ mRNA delivery to T cells using PNI’s LNP reagent resulted inprotein expression in up to 90% of cells with minimal impacton toxicity and normal proliferation
▪ Robust performance was observed across a wide range of cellprocessing and culture conditions
▪ Delivery and expression of a mRNA CAR constructdemonstrates the utility PNI’s novel LNP technology forenabling the development of next generationimmunotherapies
1. Robust Transfection While Preserving Viability in Primary Human T Cells
Further DetailsHuman T cells were isolated using pan T-cell markers and treated with LNPs containing CleanCap eGFP mRNA (Trilink). After 48 hours, T cells were stained with viability stain, FVS 570 (BD), and analyzed by flow cytometry. Cells were gated for only live cells and GFP fluorescence was quantified (A). Histograms are representative replicates from duplicate samples. Live cells were quantified and expressed as a percentage of the population (B).
A. Titratable transgene expression for 14 days following mRNA LNP delivery.
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B. Greater than 90% viability was observed for all treatment conditions.
▪ LNPs encapsulate nucleic acids and delivers them into the cytoplasm of cells using natural endocytic pathways
▪ The features of PNI’s novel LNP technology include:
▪ High transfection efficiencies
▪ Minimal impact on cell physiology and viability
▪ Nucleic acids are protected from degradation to maintain potency
▪ Simple 1-step administration to cells amenable to culture workflows
Scalable Manufacturing using NxGen Microfluidic Technology
• Fast, simple and scalable LNP manufacturing using the NanoAssemblr® platform
• LNPs are rapidly optimized for new cell types, payloads and culture conditions using the NanoAssemblr Spark in vitro screening workflow
5. Transfection is Amenable to Various T Cell Activation Methods High expression of GFP observed in T cells activated with either CD3/CD28 (Duo Activated) or CD3/CD28/CD2 (Triple Activated) reagents.
Further DetailsHuman T cells were isolated using pan T-cell markers and activated using human CD3/CD28 or CD3/CD28/CD2 activator reagents (Stemcell). T cells treated with mRNA LNPs were stained with a viability stain, FVS 570 (BD) and analyzed by flow cytometry. Cells were gated for only live T cells and GFP fluorescence was quantified. Histograms are representative replicates from duplicate samples.
6. Similar Performance in Both Fresh and Cryopreserved T Cells
High expression of GFP+ cells observed in both fresh and previously-frozen cells.
Further DetailsHuman pan T cells were isolated from whole blood and were either treated directly with mRNA LNPs or cryopreserved (Cryostor, Stemcell) and then subsequently treated. Flow cytometry analysis was performed by staining cells with a viability stain, FVS 570 (BD). Cells were gated for only live T cells and GFP fluorescence was quantified.
7. Therapeutic Utility Demonstrated by Delivery of a CD19 CAR Construct to Primary Human T Cells
A. T cells treated with mRNA LNP carrying CD19 CAR mRNA indicate successful delivery and expression of the CAR construct.
B. Viability was unaffected by CAR delivery with mRNA LNPs, an important consideration for a high yield of engineered T cells.
Further DetailsCD19 CAR plasmid was purchased from Creative Biolabs and transcribed into mRNA by Trilink. Human T cells were isolated using pan T-cell markers and treated with mRNA LNPs. Flow cytometry analysis was performed by staining T cells with FVS 570 (BD) for viability, and CD19 CAR was detected using CD19 CAR detection biotin reagent (Miltenyi). Cells were gated for only live cells and fluorescence of anti-biotin-PE was quantified (A). Live cells were quantified and expressed as a percentage of the population (B).
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4. Equivalent Transgene Expression in Both Positive and Negative T Cell Selection ProtocolsSimilar GFP expression in T cell populations isolated by either positive or negative immunomagnetic selection from the same single donor.
Further DetailsHuman T cells were isolated using either immunomagnetic positive (CD3+) or negative selection kits (Stemcell) and treated with LNPs containing CleanCap eGFP mRNA (Trilink). After 48 hours, T cells were stained with viability stain, FVS 570 (BD),and analyzed by flow cytometry. Cells were gated for only live cells and GFP fluorescence was quantified. Histograms are representative replicates from duplicate samples. Live cells were quantified and expressed as a percentage of the population.
3. T Cell Proliferation Is Not Affected Following Transfection
A. High GFP Expression Levels Maintained after 96h
B. Proliferation Analysis Shows Cell Division Is The Same Between Treated and Untreated Controls
Further DetailsHuman T cells were treated with proliferation dye followed by mRNA LNPs, and viability dye and analyzed by flow cytometry. Proliferation analysis was conducted using the proliferation module in FlowJo software. Data points represent the average of two replicates, and error bars represent standard deviation.
2. Versatile Transgene Expression in Multiple T Cell SubpopulationsmRNA LNPs Perform Consistently Across All T Cell Subtypes
Further DetailsHuman T cells were isolated using pan T-cell markers or CD4 or CD8, then treated with LNPs containing CleanCap eGFP mRNA (Trilink). After 48 hours, T cells were stained with a viability stain, FVS 570 (BD) and analyzed by flow cytometry. Populations were gated for live T cells only or CD4+ or CD8+ and GFP fluorescence was quantified. Histograms are representative replicates from duplicate samples.
eGFP mRNA LNP
