Extracellular vesicles (EVs) are naturally occurring nano-sized carriers that are secreted by cells and facilitate cell-to-cell communication by their unique ability to transfer biologically active cargo. Despite the pronounced increase in our understanding of EVs over the last decade, from disease pathophysiology to therapeutic drug delivery, improved molecular tools to track their therapeutic delivery are still needed. Unfortunately, the present catalogue of tools utilised for EV labelling lacks sensitivity or are not sufficiently specific. Here, we have explored the bioluminescent labelling of EVs using different luciferase enzymes tethered to CD63 to achieve a highly sensitive system for in vitro and in vivo tracking of EVs. Using tetraspanin fusions to either NanoLuc or ThermoLuc permits performing highly sensitive in vivo quantification of EVs or real-time imaging, respectively, at low cost and in a semi-high throughput manner. We find that the in vivo distribution pattern of EVs is determined by the route of injection, but that different EV subpopulations display differences in biodistribution patterns. By applying this technology for real-time non-invasive in vivo imaging of EVs, we show that their distribution to different internal organs occurs just minutes after administration. Graphical abstract

The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.

Non-viral transfection vectors are commonly used for oligonucleotide (ON) delivery but face many challenges before reaching the desired compartments inside cells. With the support of additional compounds, it might be more feasible for a vector to endure the barriers and achieve efficient delivery. In this report, we screened 18 different excipients and evaluated their effect on the performance of peptide dendrimer/lipid vector to deliver single-stranded, splice-switching ONs under serum conditions. Transfection efficiency was monitored in four different reporter cell lines by measuring splice-switching activity on RNA and protein levels. All reporter cell lines used had a mutated human β-globin intron 2 sequence interrupting the luciferase gene, which led to an aberrant splicing of luciferase pre-mRNA and subsidence of luciferase protein translation. In the HeLa Luc/705 reporter cell line (a cervical cancer cell line), the lead excipients (Polyvinyl derivatives) potentiated the splice-switching activity up to 95-fold, compared to untreated cells with no detected cytotoxicity. Physical characterization revealed that lead excipients decreased the particle size and the zeta potential of the formulations. In vivo biodistribution studies emphasized the influence of formulations as well as the type of excipients on biodistribution profiles of the ON. Subsequently, we suggest that the highlighted impact of tested excipients would potentially assist in formulation development to deliver ON therapeutics in pre-clinical and clinical settings.

Efficient delivery of the CRISPR/Cas9 system and its larger derivatives, base editors, and prime editors remain a major challenge, particularly in tissue-specific stem cells and induced pluripotent stem cells (iPSCs). This study optimized a novel family of cell-penetrating peptides, hPep, to deliver gene-editing ribonucleoproteins. The hPep-based nanoparticles enable highly efficient and biocompatible delivery of Cre recombinase, Cas9, base-, and prime editors. Using base editors, robust and nearly complete genome editing was achieved in the human cells: HEK293T (96%), iPSCs (74%), and muscle stem cells (80%). This strategy opens promising avenues for ex vivo and, potentially, in vivo applications. Incorporating silica particles enhanced the system's versatility, facilitating cargo-agnostic delivery. Notably, the nanoparticles can be synthesized quickly on a benchtop and stored as lyophilized powder without compromising functionality. This represents an important advancement in the feasibility and scalability of gene-editing delivery technologies.

The therapeutic potential of mRNA is vast, and yet translating this potential into effective treatments requires overcoming significant challenges of achieving safe and efficient delivery. This process is hampered by biological barriers that limit cellular uptake, degrade exposed mRNA, and thus necessitate effective endosomal escape to reach the cytoplasm. To address these challenges, we developed a hPep peptide-based nanoparticle (PNP) system that encapsulates mRNA, forming stable and biocompatible particles, which are rapidly taken up by the cells and enable efficient mRNA delivery across various cell culture models. Following systemic administration in mice, lead hPep3/mRNA PNPs achieve broad mRNA expression across multiple tissues, including the lungs, liver, and spleen, and enable effective mRNA delivery to the central nervous system upon local administration. Furthermore, we established a high-yield (≥70%) microfluidics-based protocol to scale up the production of well-defined, sterile hPep3/mRNA PNP formulations (approximately 70 nm, PDI around 0.170). Most importantly, in a proof-of-concept study in nonhuman primates (NHPs), we demonstrate that hPep PNPs loaded with human erythropoietin (hEPO) mRNA induce dose-dependent expression of hEPO protein in monkey serum, reaching up to 10 ng/ml at 1.0 mg/kg dose, following both single and repeated administration, while remaining systemically well-tolerated. These findings underscore the potential of hPep peptide-based nanoparticles as a versatile platform for mRNA delivery across multiple tissues, highlighting their promise in advancing the development of mRNA therapeutics.

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 DeliveryBuilding 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 papersI. 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/pharmaceutics13060878II. 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.2025.102522III....