Poster V10 - Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells

Introduction: CRISPR gene editing allows unprecedented control over cell fate through precise manipulation of target gene expression, but the clinical translation of many CRISPR strategies is challenged by limitations in safe yet effective cellular delivery. Non-viral CRISPR delivery overcomes the risk of genomic integration characteristic of viral vectors, but common nanoparticle-based approaches such as liposomes and cationic polymers induce cytotoxicity and unexpected perturbations of broad cellular processes(1). These effects are especially concerning when targeting the CRISPR modification of primary stem cells such as mesenchymal stromal cells (MSCs), where multipotency and self-renewal must be maintained for successful therapies. Furthermore, current nanoparticles for CRISPR delivery lack versatility due to their high specificity for just one type of CRISPR cargo (e.g., plasmid DNA, different RNAs, or proteins). This limitation hinders their applicability to a wide repertoire of CRISPR therapies and increases manufacturing burdens in cell therapy pipelines. Previously, we have demonstrated that the arginine-alanine-leucine-alanine (RALA) cell penetrating peptide (CPP) can deliver different types of cargos such as pDNA and inorganic particles to primary MSCs with superior efficiencies and lower toxicity than cationic polymers and lipid vectors(1,2,3). Therefore, we hypothesize that RALA will enable efficient CRISPR delivery in different molecular cargo formats for a wide range of CRISPR application while preserving key stem cell functions.

Materials and

Methods: Different CRISPR molecular cargos were delivered to primary rat bone marrow-derived MSCs through the RALA CPP as previously described1. To investigate uptake efficiency, RALA was complexed to mRNA or pDNA encoding green fluorescent protein (GFP) and Cas9-GFP respectively. After MSC transfection, cell viability, transfection efficiency, and overall transfected cell yield were assessed using flow cytometry. This RALA transfection system was then used for CRISPR knock-in by co-delivering 2 different pDNAs: 1) pDNA encoding Cas9 with a guide RNA (gRNA) targeting the ROSA26 safe harbor locus and 2) pDNA containing a red fluorescent protein (RFP) and nanoLUC donor sequences. Knock-in was monitored using fluorescent and luminescent imaging and a pure RFP+ cell population was purified using flow assisted cell sorting (FACS). Next, gene knock-out was performed through the RALA-mediated delivery of Cas9-gRNA ribonucleoprotein (RNP) complex targeting the RFP sequence in the edited MSCs. Knock-out efficiency was assessed using flow cytometry and both gene knock-in and knock-out were confirmed through whole genome sequencing. Gene activation was then performed using RALA to co-deliver mRNA expressing a nuclease null Cas9 (dCas9) fused to the tripartite activator VPR (dCas9-VPR) with gRNA targeting BMP2 and TNMD genes and confirming activation through qPCR.

Results, Conclusions, and Discussions: RALA-mediated delivery of mRNA and pDNA effectively transfected over 50% and 20% of cells respectively with reduced cytotoxicity in comparison commercially available vectors, lipofectamine and polyethylenimine (PEI). Additionally, RALA resulted in significant (p < 0.05) higher cell-viability (79.3 ± 1.02 % and 57.7 ± 5.15 % respectively) and shorter doubling time (1.09 ± 0.25 days) with both mRNA and pDNA transfection. Despite a lower percentage of GFP+ cells, the disparity in cell viability and proliferation gave RALA a higher overall GFP+ cell count at day 3 than lipofectamine or PEI suggesting a greater overall biomanufacturing potential. The co-delivery of pDNA encoding Cas9-ROSA26 gRNA and RFP donor sequence effectively knocked-in the RFP sequence as seen by constitutive fluorescence which enabled FACS sorted resulted in a 95% pure RFP reporter population. Subsequent delivery of Cas9 RNP targeting the RFP gene knocked-out RFP expression resulted in ~40% RFP- cells according to flow cytometry. The presence of knock-in and knock-out was further confirmed by whole genome sequencing. RALA also effectively activates therapeutic gene expression via mRNA delivery. The co-delivery of CRISPRa mRNA with gRNA targeting BMP2 and TNMD resulted in 2- and 20- fold activation respectively (p < 0.05). Activation of these endogenous chondrogenic/tenogenic genes represents a promising CRISPR strategy in regenerative medicine since it encourages safe yet reliable lineage commitment without mutation risks associated with endonuclease activity. Furthermore, the safety of RALA nanoparticles allows repeated transfections and therefore allows temporal activation of diverse genetic target genes for fine control over the complete differentiation process. In summary, we demonstrate the potential of the RALA peptide for the delivery of a wide range of CRISPR therapeutics and the engineering of primary stem and progenitor cells with minimal cytotoxicity.

Acknowledgements (Optional): 1. Gonzalez-Fernandez, T. et al. Mesenchymal stem cell fate following non-viral gene transfection strongly depends on the choice of delivery vector. Acta Biomater. 55, 226–238 (2017).
2. Sathy, B. N. et al. RALA complexed α-TCP nanoparticle delivery to mesenchymal stem cells induces bone formation in tissue engineered constructs in vitro and in vivo. J. Mater. Chem. B 5, 1753–1764 (2017).
3. McCarthy, H. O. et al. Development and characterization of self-assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery. J. Controlled Release 189, 141–149 (2014).