Description
*LNP composition in this research-use-only kit include ionizable lipid excipients that can be licensed for future clinical evaluation. Easily integrated into standard CD34+ HSC cell culture protocols The CD34+ HSC LNP kits can be easily integrated into a CD34+ HSC workflow for both mRNA-based gene expression and CRISPR/Cas gene editing applications. A general workflow is outlined below. Schematic diagram of the LNP treatment and HSC cell culture workflow. LNP production may be decoupled from the cell culture workflow, allowing greater flexibility and simplicity of workflow. Dose-dependent gene knockout of CD45 Achieve dose-dependent highly efficient genome editing of CD34+ HSCs sourced from human cord blood and human mobilized peripheral blood using the CD34+ HSC LNP kit, 100 µL on the NanoAssemblr™ Spark™ system, and CD34+ HSC LNP kit, 2 mL on the NanoAssemblr™ Ignite™ instrument. A) CD45 dose response of CRISPR-Cas9 mRNA and sgRNA LNPs produced on the NanoAssmblr™ Spark™ instrument. B) Corresponding cell viability. C) Representative images of untreated and CRISPR RNA-LNP treated CFU plates. D) Normalized erythroid and myeloid colony yields of untreated (UT), empty LNP treated, and CRISPR RNA-LNP treated samples. Cell proliferation and viability following LNP treatment LNP-treated HSCs show high cell proliferation and viability. A) Cell proliferation and B) viability monitored for over 1 week after LNP-mediated CD45 targeted CRISPR RNA-LNP treated gene editing of HSCs. C) LNP-mediated gene editing compared across n = 2 donors using flow cytometry via CD45 surface expression analysis and D) corresponding histogram. HSC cryopreservation and freeze-thawing post-LNP treatment Cryopreservation of LNP-treated HSCs showed high cell viability and yield. Furthermore, cells retained their engraftable HSC phenotype distribution while maintaining CD45 knockout efficiency. Cryopreservation of HSCs post-LNP-mediated CRISPR Cas9 gene editing. A) Schematic diagram of the freeze-thaw (F/T) cycles of HSCs. B) Post-thaw, or non-frozen long-term repopulating HSC phenotype levels, at either 24h or 48h post-LNP treatment freeze time points. C) Cell viability and live cell recovery post 24h-LNP treatment F/T cycle. D) CD45 knockout efficiency observed in freeze-thawed and fresh LNP-treated HSCs in total live cells and engraftable phenotype (CD34+ CD38- CD90+ CD133+). Scale-up of cell culture and RNA-LNP production The CD34+ HSC LNP kits are available on the NanoAssemblr™ Spark™ and NanoAssemblr™ Ignite™ instruments. Reagents are also available for scaling process development and GMP manufacturing through BioPharma Services. The CD34+ HSC LNP kits combined with the NanoAssemblr™ platform enables robust and equivalent performance on both the Spark™ and Ignite™ instruments and offers seamless scalability from discovery to preclinical studies. A) CD45 knockout and B) CD33 knockout of the total live cell population, as well as C) corresponding cell viability. A-C includes >20 unique experiments with >4 unique donors. D) Untreated, empty-LNP, and CRISPR-Cas9 RNA-loaded LNP treated HSCs were assessed for proliferation using an automated cell counter. E) Untreated, empty-LNP and CRISPR-Cas9 RNA-loaded LNP treated HSCs were assessed for clonogenicity using an automated CFU counter, 5 independent experiments with Spark™ and 2 independent experiments with the Ignite™ instrument. Kit components CD34+ HSC LNP kit, 100 µL, (Part No. 1003000) CD34+ HSC LNP kit, 100 µL with cartridges, (Part No. 1004000) CD34+ HSC LNP kit, 2 mL, (Part No. 105000) Lipid mix 100 µL 100 µL 2 mL Formulation buffer type 1 400 µL 400 µL 1.6 mL Dilution buffer type 1 1.5 mL 1.5 mL 100 mL (10X) Cryopreservation buffer type 2 - - 6 mL Apolipoprotein-E3 (ApoE3) 100 µg 100 µg 500 do Spark™ Cartridges - 5 -
