*LNP composition in this research-use-only kit includes ionizable lipid excipients that can be licensed for future clinical evaluation.

Figure 1. A) Schematic diagram of the LNP formulation process using the NanoAssemblr technology. Physicochemical data B) size, C) PDI, D) encapsulation efficiency, and E) Cryo-TEM images of CRISPR Cas9 mRNA and sgRNA-loaded LNPs produced on the Ignite (preclinical scale) instruments. 

Figure 2. A) CD45 knockout efficiency using various LNP compositions and B) cell viability as analyzed by flow cytometry. 

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 Spark and CD34+ HSC LNP kit, 2 mL on the Ignite instrument.  

Figure 4. 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. 

Cryopreservation of LNP-treated HSCs showed high cell viability and yield. Furthermore, cells retained their HSC phenotype distribution while maintaining CD45 knockout efficiency. 

Figure 5. 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+).  

*Treatment dose likely to fall between 2-6 µg RNA/million cells

Scale up from discovery to preclinical with peace of mind. The NxGen NanoAssemblr platform enables robust and equivalent performance of the CD34+ HSC LNP kit, 100 µL on the Spark instrument to the CD34+ HSC LNP kit, 2 mL on the Ignite instrument. These data demonstrate the seamless scalability from discovery to preclinical studies.

Figure 7. 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. 

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CD34+ HSC LNP kit, 100 µL and CD34+ HSC LNP kit, 2 mL are optimized for the delivery of RNA into CD34+ HSCs using RNA-LNPs formulated on the NanoAssemblr Spark and Ignite instruments and cartridges, respectively.