Nano-Enabled CRISPR-Cas Gene Editing for Cancer Therapeutics

Opening the Floodgates: Clinically Applicable Hydrodynamic Delivery of Plasmid DNA to Skeletal Muscle

<p dir="ltr">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.</p><p dir="ltr">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.</p><p dir="ltr">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.</p><p dir="ltr">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.</p><p dir="ltr"><b>Paper I - Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP.</b></p><p dir="ltr">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.</p><p dir="ltr"><b>Paper II - Design and screening of novel endosomal escape compounds that enhance functional delivery of oligonucleotide in vitro </b></p><p dir="ltr">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.</p><p dir="ltr"><b>Paper III - Nanoparticle-based delivery of RNP gene editors using modified cell-penetrating peptides </b></p><p dir="ltr">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.</p><p dir="ltr"><b>Paper IV - A Small-Molecule-Based Universal Endosomal Escape Strategy for Protein Delivery</b></p><p dir="ltr">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.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Oskar Gustafsson</b>, 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. <a href="https://doi.org/10.3390/pharmaceutics13060878" target="_blank">https://doi.org/10.3390/pharmaceutics13060878</a></p><p dir="ltr">II. H. Yesid Estupiñán, Tom Baladi, Samantha Roudi, Michael J. Munson, Jeremy Bost, <b>Oskar Gustafsson</b>, 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. <a href="https://doi.org/10.1016/j.omtn.2025.102522" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.omtn.2025.102522</a></p><p dir="ltr">III. <b>Oskar Gustafsson</b>, 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]</p><p dir="ltr">IV. <b>Oskar Gustafsson</b>, 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]</p>

<p dir="ltr">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.</p><p dir="ltr">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.</p><p dir="ltr">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.</p><p dir="ltr">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.</p><p dir="ltr"><b>Paper I - Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP.</b></p><p dir="ltr">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.</p><p dir="ltr"><b>Paper II - Design and screening of novel endosomal escape compounds that enhance functional delivery of oligonucleotide in vitro </b></p><p dir="ltr">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.</p><p dir="ltr"><b>Paper III - Nanoparticle-based delivery of RNP gene editors using modified cell-penetrating peptides </b></p><p dir="ltr">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.</p><p dir="ltr"><b>Paper IV - A Small-Molecule-Based Universal Endosomal Escape Strategy for Protein Delivery</b></p><p dir="ltr">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.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Oskar Gustafsson</b>, 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. <a href="https://doi.org/10.3390/pharmaceutics13060878" target="_blank">https://doi.org/10.3390/pharmaceutics13060878</a></p><p dir="ltr">II. H. Yesid Estupiñán, Tom Baladi, Samantha Roudi, Michael J. Munson, Jeremy Bost, <b>Oskar Gustafsson</b>, 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. <a href="https://doi.org/10.1016/j.omtn.2025.102522" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.omtn.2025.102522</a></p><p dir="ltr">III. <b>Oskar Gustafsson</b>, 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]</p><p dir="ltr">IV. <b>Oskar Gustafsson</b>, 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]</p>

An ASGCT Perspective on the National Academies Genome Editing Summit.

Plasmids are widely used gene vectors in gene therapy, yet their efficient delivery remains a major challenge for achieving optimal therapeutic outcomes. Recently, poly(β-amino esters) (PBAEs) have emerged as promising carriers for non-viral gene delivery due to their tunable structures and high delivery efficiency. Nonetheless, the cationic nature of PBAEs raises toxicity concerns, and their lack of tissue-specific targeting capability limits their clinical application. Herein, a novel PBAE for efficient plasmid delivery is constructed, which contains disulfide bonds in the backbone and N-acetylgalactosamine (GalNAc) moieties at the terminals (GalNAc-PBAEs). To address the potential reduction in nucleic acid condensing capacity caused by end-capping PBAE with GalNAc, the commonly used end-capping amines in conventional PBAE are employed as monomers to create GalNAc-PBAEs with tertiary amine side chains. Through side-chain screening, the optimized GalNAc-PBAE exhibits enhanced transfection efficiency, surpassing that of the conventional top-performing PBAE, C28-E7, and rivaling the commercial transfection reagent Lipo2000. Importantly, the GalNAc moieties enable the plasmid-PBAE polyplex to efficiently target the liver. In a mouse model with acute liver fibrosis, the efficient delivery and expression of a representative plasmid encoding HGF significantly mitigate liver fibrosis and improve liver function, demonstrating the potential of the designed PBAE in gene therapy.

Many drugs have been designed to act on intracellular targets and to affect intracellular processes inside target cells. For the desired effects to be exerted, these drugs should permeate target cells and reach specific intracellular organelles. This subcellular drug targeting approach has been proposed for enhancement of accumulation of these drugs in target organelles and improved efficiency. This approach is based on drug encapsulation in drug delivery systems (DDSs) and/or their decoration with specific targeting moieties that are intended to enhance the drug/DDS accumulation in the intracellular organelle of interest. During recent years, there has been a constant increase in interest in DDSs targeted to specific intracellular organelles, and many different approaches have been proposed for attaining efficient drug delivery to specific organelles of interest. However, it appears that in many studies insufficient efforts have been devoted to quantitative analysis of the major formulation parameters of the DDSs disposition (efficiency of DDS endocytosis and endosomal escape, intracellular trafficking, and efficiency of DDS delivery to the target organelle) and of the resulting pharmacological effects. Thus, in many cases, claims regarding efficient delivery of drug/DDS to a specific organelle and efficient subcellular targeting appear to be exaggerated. On the basis of the available experimental data, it appears that drugs/DDS decoration with specific targeting residues can affect their intracellular fate and result in preferential drug accumulation within an organelle of interest. However, it is not clear whether these approaches will be efficient in in vivo settings and be translated into preclinical and clinical applications. Studies that quantitatively assess the mechanisms, barriers, and efficiencies of subcellular drug delivery and of the associated toxic effects are required to determine the therapeutic potential of subcellular DDS targeting.

Since the advent of the clustered regularly interspaced short palindromic repeats (CRISPR) system in the gene editing field, diverse CRISPR-based gene editing tools have been developed for treating genetic diseases. Of these, base editors (BEs) are promising because they can carry out precise gene editing at single-nucleotide resolution without inducing DNA double-strand breaks (DSBs), which pose significant risks of genomic instability. Despite their outstanding advantages, the clinical application of BEs remains challenging due to their large size, which limits their efficient delivery, particularly in adeno-associated virus (AAV)-based systems. To address this issue, various strategies have been explored to reduce the size of BEs. These approaches include truncating the nonessential domains and replacing the bulky components with smaller substitutes without compromising the editing efficiency. In this review, we highlight the importance of downsizing BEs for therapeutic applications and introduce recent advances in size-reduction strategies. Additionally, we introduce the ongoing efforts to overcome other limitations of BEs, providing insights into their potential for improving in vivo gene editing.

Tuning the bioactivity of bone morphogenetic protein-2 with surface immobilization strategies

Lipid nanoparticles containing zwitterionic lipids versatilely enhance the efficiency of mRNA delivery.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR-Cas9) is an RNA-guided gene editing tool which offers several advantageous characteristics in comparison with the conventional methods (e.g., zinc finger nucleases and transcription activator-like effector nucleases) such as cost-effectiveness, flexibility, and being easy-to-use. Despite some limitations such as efficient delivery and safety, CRISPR-Cas9 is still the most convenient tool for gene editing purposes. Due to the potential capability of the CRISPR-Cas9 system in genome editing and correction of casual mutations, it can be considered as a possible therapeutic system in the treatment of disorders associated with the genome mutations and in particular cancer treatment. In this review, we will discuss CRISPR-Cas-based gene editing along with its classifications and mechanism of action. Furthermore, the therapeutic application of the CRISPR-Cas9 system in mutational disorders, delivery systems, as well as its advantages and limitations with a special emphasis on cancer treatment will be discussed.

Endosomal escape is a major bottleneck for efficient intracellular delivery of therapeutic cargoes, particularly for macromolecular biological cargoes such as peptides, proteins and nucleic acids. pH-responsive polymeric nanoparticles that can respond to changes in the pH of intracellular microenvironments have generated substantial interest in navigating the endosomal barrier. In this study, we applied the highly sensitive split luciferase endosomal escape quantification (SLEEQ) assay to better understand the endosomal escape efficiency of dual component pH-responsive nanoparticles based on poly(2-(diethylamino) ethyl methacrylate) (PDEAEMA) and poly(2-(diisopropylamino) ethyl methacrylate) (PDPAEMA). Previous work investigated the use of a disulfide-linked HiBiT peptide conjugate encapsulated within the nanoparticle core, which upon meeting the LgBiT protein in the cytosol demonstrated luminescence which could be quantified to assess endosomal escape. However, we were interested in understanding whether this assay could be tuned to understand the endosomal escape of both a therapeutic cargo and a larger carrier. To achieve this, we designed two different HiBiT conjugates by applying a carbonylacrylic-functionalized thioether (non-cleavable) linker, which is more stable in endosomes, and a less stable disulfide (cleavable) linker to attach HiBiT to the nanoparticle core. Nanoparticles with disulfide-linked HiBiT demonstrated a higher endosomal escape efficiency of 6-7%, whereas thioether-linked HiBiT demonstrated <3% endosomal escape efficiency with a twofold decrease in cytosolic delivery. This suggests that degradation of the disulfide linker in endosomes leads to cytosolic delivery of a free HiBiT cargo, while thioether-linked HiBiT polymers are larger and thus fewer HiBiT-carrier conjugates can escape the endosomes. Overall, this work demonstrates that the SLEEQ assay can be tuned to understand the cytosolic delivery of different components based on the use of different linker chemistries and thus it is an important tool for designing therapeutic delivery systems in the future.

Peptide-assisted lipofection enables efficient non-viral delivery of large CRISPR/Cas9 constructs for genome editing applications.

Lipid nanoparticles (LNPs) are among the most successful classes of nonviral delivery systems for nucleic acid-based therapeutics in treating human diseases. One of the key challenges in achieving efficient cytosolic delivery of nucleic acids is overcoming endosomal entrapment within cells. Conventional lipid bilayer-forming cationic and amino lipids mediate endosomal escape via the mechanism of lamellar-to-inverted hexagonal phase transition, resulting in suboptimal cytosolic cargo delivery. pH-sensitive amphiphilic cell membrane disruption and endosomal escape have emerged as a strategy for designing protonatable or ionizable lipids, especially nonlamellar lipids, for efficient cytosolic nucleic acid delivery. Nonlamellar amino lipids possess a large wedge-shaped tail structure and do not form stable lipid bilayers. These lipids and their corresponding LNPs remain neutral, non-amphiphilic, or minimally amphiphilic at physiological pH (7.4). They become amphiphilic upon protonation or ionization in acidic endosomes (pH 6.5–5.4). The electrostatic interaction of ionized nonlamellar lipids with the negatively charged endosome membrane, combined with their large wedge-like structures, disrupts the lipid bilayer, facilitating efficient endosomal escape. Additionally, the nonlamellar ionizable lipids can be fine-tuned by altering the structure of amino head groups and lipid tails to achieve the precisely controlled pH-sensitive amphiphilic membrane disruption at endosomal pH. Therefore, these lipids exhibit excellent safety profiles and high efficiency for in vivo delivery of various therapeutic nucleic acids. pH-sensitive amphiphilic membrane disruption and endosomal escape provide a feasible and effective mechanism for designing ionizable lipids for safe and efficient in vivo nucleic acid delivery.

Introduction: The field of RNA interference technology has been researched extensively in recent years. However, the development of clinically suitable, safe and effective drug delivery vehicles is still required.Areas covered: This paper reviews the recent advances of non-viral delivery of small interfering RNA (siRNA) by nanoparticles, including biodegradable nanoparticles, liposomes, polyplex, lipoplex and dendrimers. The characteristics, composition, preparation, applications and advantages of different nanoparticle delivery strategies are also discussed in detail, along with the recent progress of non-viral nanoparticle carrier systems for siRNA delivery in preclinical and clinical studies.Expert opinion: Non-viral carrier systems, especially nanoparticles, have been investigated extensively for siRNA delivery, and may be utilized in clinical applications in the future. So far, a few preliminary clinical trials of nanoparticles have produced promising results. However, further research is still required to pave the way to successful clinical applications. The most important issues that need to be focused on include encapsulation efficiency, formulation stability of siRNA, degradation in circulation, endosomal escape and delivery efficiency, targeting, toxicity and off-target effects. Pharmacology and pharmacokinetic studies also present another great challenge for nanoparticle delivery systems, owing to the unique nature of siRNA oligonucleotides compared with small molecules.

Toward CRISPR Therapies for Cardiomyopathies.