The delivery challenge : synthetic solutions for next-generation gene editing tools

Gene therapy has progressed rapidly in recent years, with several treatments- such as CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-based therapies for sickle cell disease and beta thalassemia- receiving FDA approval. Permanent gene editing generally follows two strategies: gene replacement, where a new copy of a gene is introduced, and gene repair, where the existing gene is precisely corrected. Gene repair is preferred when feasible, as it preserves the gene's natural regulatory environment.

The field has seen an explosion of new gene editing tools. Innovations like base editing and prime editing now allow for the correction of nearly any type of genetic mutation with high precision. The CRISPR/Cas9 system, which revolutionized gene editing and earned a Nobel Prize, stands out for its simplicity: retargeting it only requires changing the RNA guide, unlike earlier systems such as TALENs (Transcription Activator-Like Effector Nuclease) or zinc finger nucleases. TALENs and zinc finger nucleases were the first programmable gene editors; however, because they rely solely on protein-DNA interactions, they require custom protein engineering for each new DNA target, significantly limiting their versatility and scalability Moreover, CRISPR/Cas9 can be engineered into versatile fusion proteins for applications beyond cutting DNA (DeoxyriboNucleic Acid), including epigenetic modulation and targeted base conversions.

However, a major barrier remains: efficient delivery into target cells. Cells are inherently resistant to foreign macromolecules, and current delivery methods often face inefficiency, toxicity, cost limitations, etc.

This PhD project focused on developing a synthetic delivery system for CRISPR/Cas9 RNPs (RiboNucleoProteins)-the active protein-RNA complexes- rather than delivering the system as DNA or mRNA (messenger RiboNucleic Acid). The aim was to create a cost-effective, scalable method capable of transfecting even challenging cell types such as T cells, iPSCs (induced Pluripotent Stem Cells), and primary human MuSCs (Muscle Stem Cells). By simplifying and expanding access to efficient RNP delivery, this work seeks to advance both research and therapeutic gene editing applications.

Paper I - Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP.

This study demonstrates a proof-of-concept for repurposing nucleotide- delivering CPPs (Cell-Penetrating Peptides) for Cas9 RNP delivery. We show that Cas9 RNP can form nanoparticles with the PF14 CPP, enabling efficient uptake across multiple cell lines. Gene editing efficiency was evaluated using both the Stoplight reporter system and endogenous targets via fluorescence- and genomic analysis. Our results highlight CPP-mediated delivery as a simple, effective strategy that outperforms nearly all non-viral methods available at the time in terms of efficiency.

Paper II - Design and screening of novel endosomal escape compounds that enhance functional delivery of oligonucleotide in vitro

This study builds on previous work from our lab using endosomolytic small molecules. In collaboration with AstraZeneca, we designed new variations of a previously identified EEE (Endosomal Escape Enhancers), resulting in the development of EEE4. This molecule efficiently delivers ONs (OligoNucleotides) to a broad range of cell types with minimal cellular toxicity. The findings establish a foundation for future studies, with EEE4 representing a significant improvement over earlier generations both in terms of efficiency and safety.

Paper III - Nanoparticle-based delivery of RNP gene editors using modified cell-penetrating peptides

This study expands on our previous work by introducing a new peptide family, hPep, for delivering a broader range of gene editing proteins to an expanded set of cell types. These peptides maintain the high efficiency of PF14 while being gentler on cells, demonstrating the ability to deliver cargo with diverse charges - an ability not previously seen in nanoparticle-forming CPPs. A key finding was the role of silica nanoparticles (sub-40 nm) in enhancing editing efficiency. These nanoparticles likely serve as a protein and peptide adsorption core, forming more effective nanoparticles than those produced in HBG (HEPES Buffered Glucose) buffer alone. Additionally, we achieved efficient delivery to hard-to-transfect cells, including iPSCs and MuSCs, highlighting the potential of this method for advanced gene editing applications ex vivo.

Paper IV - A Small-Molecule-Based Universal Endosomal Escape Strategy for Protein Delivery

Building on Paper II, this study uses the recently developed EEE4 endosomolytic small molecule for protein delivery of gene editing tools. It introduces a novel concept: naked gene editors can remain stable in whole serum and undergo sufficient endocytosis to enable genome editing, as long as they are efficiently released from endosomes. The method is highly scalable, easy to implement, and effective in therapeutically relevant cell types, including T cells and iPSCs. Furthermore, the study reveals a strong correlation between protein endocytosis and editing efficiency, with increased uptake leading to a significant rise in editing. This insight opens a promising path for further optimization.

List of scientific papers

I. Oskar Gustafsson, Julia Rädler, Samantha Roudi, Tõnis Lehto, Mattias Hällbrink, Taavi Lehto, Dhanu Gupta, Samir El Andaloussi, Joel Z Nordin. Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP. https://doi.org/10.3390/pharmaceutics13060878

II. H. Yesid Estupiñán, Tom Baladi, Samantha Roudi, Michael J. Munson, Jeremy Bost, Oskar Gustafsson, Daniel Velásquez-Ramírez, Deepak Kumar Bhatt, Daniel Hagey, Dennis Hekman, Shalini Andersson, Samir EL Andaloussi and Anders Dahlen. Design and screening of novel endosomal escape compounds that enhance functional delivery of oligonucleotide in vitro. https://doi.org/10.1016/j.omtn.2025.102522

III. Oskar Gustafsson, Supriya Krishna, Sophia Borate, Marziyeh Ghaeidamini, Xiuming Liang, Osama Saher, Raul Cuellar, Björn K. Birdsong, Samantha Roudi, H. Yesid Estupiñán, Evren Alici, CI Edvard Smith, Elin K. Esbjörner, Olivier Gerrit de Jong, Simone Spuler, Helena Escobar, Joel Z. Nordin, Samir EL Andaloussi. Nanoparticle-based delivery of RNP gene editors using modified cell-penetrating peptides. [Manuscript]

IV. Oskar Gustafsson, H. Yesid Estupiñán, Juliette Suermondt, Samantha Roudi, Julia Radler, Sophia Borate, Xiuming Liang, Evren Alici, Joel Z. Nordin, Samir EL Andaloussi. A Small-Molecule-Based Universal Endosomal Escape Strategy for Protein Delivery. [Manuscript]