Biology subjects

Gustafsson, O.

Publications and source records attributed to Gustafsson, O..

7 recordsLinked to original sources

bioRxiv2026

Efficient delivery of gene editors using intein-engineered virus-like particles

Virus-like particles (VLPs) represent a promising next-generation drug delivery platform. However, conventional VLPs rely on multiple viral components for effective cargo encapsulation and delivery, raising safety concerns. Here, we present a novel strategy to engineer immature VLPs using a self-cleaving intein system. We employed viral Gag proteins as sorting domains, linking cargo proteins to Gag through inteins, thereby eliminating the need for the conventional protease cleavage typically mediated by the gag-pol protein. During VLP biogenesis, intein-mediated cleavage released cargo proteins into the lumen, enabling efficient intracellular delivery when VLP surfaces are pseudotyped with VSV-G. Optimal candidates for delivering Cre recombinase and gene editing tools (Cas9, Cas12a and base editors) were identified by screening various Gag proteins. Notably, these VLPs achieved robust gene editing in primary cells, including naive and activated T cells, as well as hematopoietic stem and progenitor cells (HSPCs). A single local intracerebroventricular (ICV) infusion of optimized particles induced up to 60% tdTomato expression in the brain regions of reporter mice, while intravenous injection resulted in significant recombination (up to 70%) of a variety of cell types across organs. Collectively, we developed a simplified, efficient VLP platform for intracellular cargo delivery with broad therapeutic potential for gene editing and treatment of human diseases.

molecular biology↗

bioRxiv2026

Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

Extracellular vesicles (EVs) are nano-scale structures produced by cells that transport biological substances for intercellular communication. The tetraspanins CD9, CD81, and CD63 are crucial to EV biogenesis and function. This study uses CRISPR-Cas9 system to knock out (KO) CD9, CD63, and CD81 in HEK293T cells. The goal is to investigate the role of these tetraspanins in EV bioengineering with the hypothesis that repressing endogenous production may increase the availability of exogenously introduced tetraspanin-fusion constructs and increase engineered EV production. Firstly, it is observed that individually knocking out a tetraspanin does not significantly affect EV formation. However, when all three tetraspanins are simultaneously knocked out, there is a marked decrease in EV production, as measured by nanoparticle tracking analysis (NTA). Secondly, upon reintroduction of the corresponding tetraspanins fused to firefly ThermoLuc (Tluc) or neon green (mNG) into the PanKO-, CD9KO, CD63KO-, and CD81KO-cells, the engineered EVs display a significant increase in production by 50% to 70% compared to transduction of wild-type (WT) cells, as measured by luminometer and imaging flow cytometry. These findings emphasize the potential of tetraspanin KO in the bioengineering of EVs, paving the way for new therapeutic applications by enhancing production and potentially modifying their cargo.

bioengineering↗

bioRxiv2025

Peptide nanoparticles for systemic mRNA delivery in rodents and non-human primates

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.

pharmacology and toxicology↗

bioRxiv2024

Advanced Peptide Nanoparticles Enable Robust and Efficient delivery of gene editors across cell types

Efficient delivery of the CRISPR/Cas9 system and its larger derivatives, base editors, and prime editors remain a significant 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 systems 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 a significant advancement in the feasibility and scalability of gene-editing delivery technologies.

bioengineering↗

bioRxiv2024

Surface adhesion and membrane fluctuations influence the elastic modulus of extracellular vesicles

Elastic properties of nanoscale extracellular vesicles (EVs) are believed to influence their cellular interactions, thus having a profound implication in intercellular communication. Yet, an accurate quantification of the elasticity of such small lipid vesicles is difficult even with AFM-based nanoindentation experiments as it crucially depends on the reliability of the theoretical interpretation of such measurements. Here we describe a complete method composed of theoretical framework, experimental procedure, and appropriate statistical approach for an accurate determination of bending modulus and effective elastic modulus of EVs. Further, we experimentally demonstrate that the quantification of EVs by the elastic modulus from AFM-based force spectroscopy measurement is marred by the interplay of their compositionally inhomogeneous fluid membrane with the adhesion forces from the substrate and thermal effects - two exquisite phenomena that could thus far only be theoretically predicted. The effects result in a large spreading of elastic modulus even for a single EV. Our unified model is then applied to genetically engineered classes of EVs to understand how the alterations in tetraspanin expression may influence their elastic modulus.

biophysics↗

bioRxiv2022

SARS-CoV-2 and HSV-1 Induce Amyloid Aggregation in Human CSF

The corona virus (SARS-CoV-2) pandemic and the resulting long-term neurological complications in patients, known as long COVID, have renewed the interest in the correlation between viral infections and neurodegenerative brain disorders. While many viruses can reach the central nervous system (CNS) causing acute or chronic infections (such as herpes simplex virus 1, HSV-1), the lack of a clear mechanistic link between viruses and protein aggregation into amyloids, a characteristic of several neurodegenerative diseases, has rendered such a connection elusive. Recently, we showed that viruses can induce aggregation of purified amyloidogenic proteins via the direct physicochemical mechanism of heterogenous nucleation (HEN). In the current study, we show that the incubation of HSV-1 and SARS-CoV-2 with human cerebrospinal fluid (CSF) leads to the amyloid aggregation of several proteins known to be involved in neurodegenerative diseases, such as: APLP1 (amyloid beta precursor like protein 1), ApoE, clusterin, 2-macroglobulin, PGK-1 (phosphoglycerate kinase 1), ceruloplasmin, nucleolin, 14-3-3, transthyretin and vitronectin. Importantly, UV-inactivation of SARS-CoV-2 does not affect its ability to induce amyloid aggregation, as amyloid formation is dependent on viral surface catalysis via HEN and not its ability to replicate. Additionally, viral amyloid induction led to a dramatic drop in the soluble protein concentration in the CSF. Our results show that viruses can physically induce amyloid aggregation of proteins in human CSF and result in soluble protein depletion, and thus providing a potential mechanism that may account for the association between persistent and latent/reactivating brain infections and neurodegenerative diseases. Significance StatementViruses have generally been excluded from the etiology of amyloid pathologies based on the assumption that amyloid formation requires a proteinaceous conformational template (a prion) to form. Here we show that neuroinvasive viruses induce amyloid aggregation of a plethora of proteins in human CSF even after UV inactivation. Our work illustrates that viruses can induce amyloid aggregation of endogenous human proteins in their native environment by acting as physical catalysts of amyloid nucleation and phase transition. Demonstrating this direct mechanistic link, which is independent of templating, can help better understand the link between viruses and neurodegenerative disorders, especially in the post-COVID-19 era.

microbiology↗

bioRxiv2019

The visual system of the genetically tractable crustacean Parhyale hawaiensis: diversification of eyes and visual circuits associated with low-resolution vision

BackgroundArthropod eyes have diversified during evolution to serve multiple needs, such as finding mates, hunting prey, and navigating in complex surroundings under varying light conditions. This diversity is reflected in the optical apparatus, photoreceptors and neural circuits that underpin vision. While this diversity has been extensively documented, our ability to genetically manipulate the visual system to investigate its function is largely limited to a single species, the fruitfly Drosophila melanogaster. Here, we describe the visual system of Parhyale hawaiensis, an amphipod crustacean for which we have established tailored genetic tools. ResultsAdult Parhyale have apposition-type compound eyes made up of [~]50 ommatidia. Each ommatidium contains four photoreceptor cells with large rhabdomeres (R1-4), expected to be sensitive to the polarisation of light, and one photoreceptor cell with a smaller rhabdomere (R5). The two types of photoreceptors express different opsins, belonging to families with distinct wavelength sensitivities. Using the cis.-regulatory regions of opsin genes, we established transgenic reporters expressed in each photoreceptor cell type. Based on these reporters, we show that R1-4 and R5 photoreceptors extend axons to the first optic lobe neuropil, revealing striking differences compared with the photoreceptor projections found in related crustaceans and insects. Investigating visual function, we show that Parhyale has a positive phototactic response and is capable of adapting its eyes to different levels of light intensity. ConclusionsWe propose that the visual system of Parhyale serves low-resolution visual tasks, such as orientation and navigation, based on broad gradients of light intensity and polarisation. Optic lobe structure and photoreceptor projections point to significant divergence from the conserved visual circuits found in other malacostracan crustaceans and insects, which could be associated with a shift to low-resolution vision. Our study provides the foundation for research in the visual system of this genetically tractable species.

zoology↗