Abstract: Human induced pluripotent stem cells (iPSCs) have emerged as novel autologous cell resources for studying degenerative diseases. Their ability to differentiate into clinically relevant phenotypic cells makes them invaluable for in vitro disease models. iPSC-derived neurons have been used for in vitro neurotoxicity testing and the evaluation of neuronal transfection efficiency. In this study, we utilized iPSC-derived cortical neurons (iPSC CN) as an in vitro neuron model and demonstrated the efficiency of dual gene knock-out (KO) delivered through lipid nanoparticles (LNPs) containing CRISPR encoding for ALS target 1 and 2 (confidential targets). We hypothesise that target 1 and 2 can be KO in iPSC CN. SgRNA encoding for target 1/2 has been designed and validated by in vitro nuclease assay. LNPs were formulated and characterised using DLS. Encapsulation efficiency of nucleic acids was measured by RiboGreen™ assay. LNPs were incubated in serum to assess their ability to protect NA from degradation. Single and dual knockout efficiency (target 1 & 2) were assessed in iPSC CN using T7 endonuclease assay (T7EI), Sanger Sequencing and Inference of CRISPR Edits (ICE) analysis and western blotting. Off-target effect was assessed using T7EI. Serum-stable LNPs produced were 130-140 nm in diameter with -90% nucleic acid loading efficiency. Successful transfection with LNP-mediated single gene KO was achieved with a 23.3% and 23.7% gene KO in gene 1 and 2, respectively, leading to 40.5% and 54.3% reduction in target 1 and 2 protein expression. The dual gene KO LNPs encapsulating the combination of sgRNAs encoding target 1 and 2 achieved significant reductions in both gene and protein levels, with 29% and 42% KO of target 1 and 2 genes, respectively. This led to a reduction of 51.43% and 56.42% in target 1 and 2 protein levels. No off-target effects were generated by the LNPs, indicating that our LNPs are specific and efficient for delivering the dual gene KO CRISPR in iPSCs. LNPs achieved efficient single and dual gene knockout in the iPSC CNs, lays the foundation for predicting therapeutic effects in humans and facilitates the translation of in vitro results into potential in vivo clinical applications. The next stages of the project involve assessing functional studies of target 1 and 2 in iPSCs to evaluate their effects in ALS therapy.
Funding Source: Sanofi Innovation Awards; King's-China Scholarship Council
