bims-engexo 2024-10-06 papers

bims-engexo

Biomed News

on Engineered exosomes

Issue of 2024–10–06
six papers selected by
Ravindran Jaganathan, Universiti Kuala Lumpur

Enhanced spinal cord repair using bioengineered induced pluripotent stem cell-derived exosomes loaded with miRNA.
Engineering small extracellular vesicles with multivalent DNA probes for precise tumor targeting and enhanced synergistic therapy.
High specificity of engineered T cells with third generation CAR (CD28-4-1BB-CD3-ζ) based on biotin-bound monomeric streptavidin for potential tumor immunotherapy.
De novo design of mini-protein binders broadly neutralizing Clostridioides difficile toxin B variants.
Genetic engineering of megakaryocytes from blood progenitor cells using mRNA lipid nanoparticles.
Developing an enhanced chimeric permuted intron-exon system for circular RNA therapeutics.

Mol Med. 2024 Oct 01. 30(1): 168

Enhanced spinal cord repair using bioengineered induced pluripotent stem cell-derived exosomes loaded with miRNA.

Azar Abbas, Xiaosheng Huang, Aftab Ullah, Lishi Luo, Wenqun Xi, Yuanjiao Qiao, Kun Zeng.

   BACKGROUND: A spinal cord injury (SCI) can result in severe impairment and fatality as well as significant motor and sensory abnormalities. Exosomes produced from IPSCs have demonstrated therapeutic promise for accelerating spinal cord injury recovery, according to a recent study.
OBJECTIVE: This study aims to develop engineered IPSCs-derived exosomes (iPSCs-Exo) capable of targeting and supporting neurons, and to assess their therapeutic potential in accelerating recovery from spinal cord injury (SCI).
METHODS: iPSCs-Exo were characterized using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western blot. To enhance neuronal targeting, iPSCs-Exo were bioengineered, and their uptake by neurons was visualized using PKH26 labeling and fluorescence microscopy. In vitro, the anti-inflammatory effects of miRNA-loaded engineered iPSCs-Exo were evaluated by exposing neurons to LPS and IFN-γ. In vivo, biodistribution of engineered iPSC-Exo was monitored using a vivo imaging system. The therapeutic efficacy of miRNA-loaded engineered iPSC-Exo in a SCI mouse model was assessed by Basso Mouse Scale (BMS) scores, H&E, and Luxol Fast Blue (LFB) staining.
RESULTS: The results showed that engineered iPSC-Exo loaded with miRNA promoted the spinal cord injure recovery. Thorough safety assessments using H&E staining on major organs revealed no evidence of systemic toxicity, with normal organ histology and biochemistry profiles following engineered iPSC-Exo administration.
CONCLUSION: These results suggest that modified iPSC-derived exosomes loaded with miRNA have great potential as a cutting-edge therapeutic approach to improve spinal cord injury recovery. The observed negligible systemic toxicity further underscores their potential safety and efficacy in clinical applications.

Keywords:  Etc; Induced pluripotent stem cells-derived exosomes (iPSCs-Exo); Spinal cord injury (SCI); miR-199b-5p; miR-21-5p; miR-23b

DOI:  https://doi.org/10.1186/s10020-024-00940-6
J Colloid Interface Sci. 2024 Sep 28. pii: S0021-9797(24)02289-6. [Epub ahead of print]679(Pt A): 335-348

Engineering small extracellular vesicles with multivalent DNA probes for precise tumor targeting and enhanced synergistic therapy.

Qi Zhang, Ruo-Fei Ma, Ting-Ju Ren, Xiu-Yan Ren, Zhang-Run Xu.

  Small extracellular vesicles (sEVs) have gained wide attention as efficient carriers for disease treatment. However, the proclivity of sEVs to be ingested by source cells is insufficient to accurately target specific sites, posing a challenge in realizing controlled targeting treatment. Here, we developed an engineered sEV nanocarrier capable of precise tumor targeting and enhanced synergistic therapy. Multivalent DNA probes, comprising abundant AS1411 aptamers and telomerase primers, were innovatively modified on the sEV membrane (M-D-sEV) for precise tumor targeting. To achieve synergistic therapy, gold nanorod-cerium oxide nanostructures (Au NRs-CeO2) and manganese dioxide nanosheets-doxorubicin (MnO2 NSs-DOX) were encapsulated into liposomes (Lip-Mat). Then M-D-sEV and Lip-Mat were fused together through membrane fusion to obtain nanocarriers. Owing to the multivalence of the probes, the surface of the nanocarriers was loaded with numerous aptamers, which greatly enhances their targeting ability and promotes the accumulation of drugs. When nanocarriers were ingested by tumor cells, telomerase and multivalent DNA probes triggered their aggregation, enhancing the therapeutic effect. Furthermore, under laser irradiation, Au NRs-CeO2 converted light into hyperthermia, thereby inducing the destruction of nanocarriers membrane. This process initiated a series of reactions involving glutathione and H2O2 consumption, as well as DOX release, ultimately achieving synergistic tumor therapy. In vitro and in vivo studies demonstrated the remarkable targeting ability of multivalent DNA probes and excellent therapeutic effect of this strategy. The engineered strategy of sEVs provide a promising approach for precise tumor therapy and hold great potential for the development of efficient, safe, and personalized drug delivery systems.

Keywords:  Liposome; Membrane fusion; Multivalent DNA probe; Precise targeting; Small extracellular vesicle; Synergistic therapy

DOI:  https://doi.org/10.1016/j.jcis.2024.09.224
Front Immunol. 2024 ;15 1448752

High specificity of engineered T cells with third generation CAR (CD28-4-1BB-CD3-ζ) based on biotin-bound monomeric streptavidin for potential tumor immunotherapy.

Jorge Gallego-Valle, Verónica Astrid Pérez-Fernández, Jesús Rosales-Magallares, Sergio Gil-Manso, María Castellá, Europa Azucena Gonzalez-Navarro, Rafael Correa-Rocha, Manel Juan, Marjorie Pion.

   Introduction: Immunotherapy has revolutionized cancer treatment, and Chimeric Antigen Receptor T cell therapy (CAR-T) is a groundbreaking approach. Traditional second-generation CAR-T therapies have achieved remarkable success in hematological malignancies, but there is still room for improvement, particularly in developing new targeting strategies. To address this limitation, engineering T cells with multi-target universal CARs (UniCARs) based on monomeric streptavidin has emerged as a versatile approach in the field of anti-tumor immunotherapy. However, no studies have been conducted on the importance of the intracellular signaling domains of such CARs and their impact on efficiency and specificity.
Method: Here, we developed second-generation and third-generation UniCARs based on an extracellular domain comprising an affinity-enhanced monomeric streptavidin, in addition to CD28 and 4-1BB co-stimulatory intracellular domains. These UniCAR structures rely on a biotinylated intermediary, such as an antibody, for recognizing target antigens. In co-culture assays, we performed a functional comparison between the third-generation UniCAR construct and two second-generation UniCAR variants, each incorporating either the CD28 or 4-1BB as co-stimulatory domain.
Results: We observed that components in culture media could inhibit the binding of biotinylated antibodies to monomeric streptavidin-CARs, potentially compromising their efficacy. Furthermore, third-generation UniCAR-T cells showed robust cytolytic activity against cancer cell lines upon exposure to specific biotinylated antibodies like anti-CD19 and anti-CD20, underscoring their capability for multi-targeting. Importantly, when assessing engineered UniCAR-T cell activation upon encountering their target cells, third-generation UniCAR-T cells exhibited significantly enhanced specificity compared to second-generation CAR-T cells.
Discussion: First, optimizing culture conditions would be essential before deploying UniCAR-T cells clinically. Moreover, we propose that third-generation UniCAR-T cells are excellent candidates for preclinical research due to their high specificity and multi-target anti-tumor cytotoxicity.

Keywords:  Chimeric Antigen Receptor; Treg; engineered cells; immunosuppression; streptavidin-based CAR

DOI:  https://doi.org/10.3389/fimmu.2024.1448752
Nat Commun. 2024 Oct 02. 15(1): 8521

De novo design of mini-protein binders broadly neutralizing Clostridioides difficile toxin B variants.

Xinchen Lv, Yuanyuan Zhang, Ke Sun, Qi Yang, Jianhua Luo, Liang Tao, Peilong Lu.

Clostridioides difficile toxin B (TcdB) is the key virulence factor accounting for C. difficile infection-associated symptoms. Effectively neutralizing different TcdB variants with a universal solution poses a significant challenge. Here we present the de novo design and characterization of pan-specific mini-protein binders against major TcdB subtypes. Our design successfully binds to the first receptor binding interface (RBI-1) of the varied TcdB subtypes, exhibiting affinities ranging from 20 pM to 10 nM. The cryo-electron microscopy (cryo-EM) structures of the mini protein binder in complex with TcdB1 and TcdB4 are consistent with the computational design models. The engineered and evolved variants of the mini-protein binder and chondroitin sulfate proteoglycan 4 (CSPG4), another natural receptor that binds to the second RBI (RBI-2) of TcdB, better neutralize major TcdB variants both in cells and in vivo, as demonstrated by the colon-loop assay using female mice. Our findings provide valuable starting points for the development of therapeutics targeting C. difficile infections (CDI).

DOI: https://doi.org/10.1038/s41467-024-52582-1
J Thromb Haemost. 2024 Sep 26. pii: S1538-7836(24)00553-1. [Epub ahead of print]

Genetic engineering of megakaryocytes from blood progenitor cells using mRNA lipid nanoparticles.

Jerry Leung, Asel Primbetova, Colton Strong, Brenna N Hay, Han Hsuan Hsu, Andrew Hagner, Leonard J Foster, Dana Devine, Pieter R Cullis, Peter W Zandstra, Christian J Kastrup.

   BACKGROUND: Platelets are an essential component of hemorrhage control and management, and engineering platelets to express therapeutic proteins could expand their use as a cell therapy. Genetically engineered platelets can be achieved by modifying the platelet precursor cells, megakaryocytes (MKs). Current strategies include transfecting MK progenitors ex vivo with viral vectors harbouring lineage-driven transgenes and inducing the production of "in vitro" modified platelets. The use of viruses, however, poses challenges in clinical implementation, and no methods currently exist to genetically modify MKs with non-viral techniques. Lipid nanoparticles (LNP) are a non-viral delivery system that could enable a facile strategy to modify MKs with a variety of nucleic acid payloads.
OBJECTIVE: To investigate whether LNP can transfect cultured hematopoietic stem/progenitor cell (HSPC)-derived MKs to express exogenous proteins and induce functional changes.
METHOD: MK and MK progenitors differentiated from cord-blood derived HSPCs were treated with lipid nanoparticle formulations containing mRNA and resembling the clinically approved LNP formulations. Transfection efficiency was assessed through flow cytometry by expression of enhanced green fluorescent protein. Functional changes to the MKs were assessed through rotational thromboelastometry by expression of exogenous coagulation factor VII (FVII), a representative physiologically relevant protein.
RESULT: LNP enabled transfection efficiencies of 99% in MKs, and did not impair MK maturation, viability, and morphology. MKs engineered to express exogenous FVII decreased clotting time in FVII-deficient plasma following clot initiation.
CONCLUSION: This approach provides an easy-to-use modular platform to genetically modify MK and MK progenitors, which can be potentially extended to producing genetically modified cultured platelets.

Keywords:  cell therapy; genetic engineering; mRNA; megakaryocytes; nanoparticle drug delivery system

DOI:  https://doi.org/10.1016/j.jtha.2024.09.008
Theranostics. 2024 ;14(15): 5869-5882

Developing an enhanced chimeric permuted intron-exon system for circular RNA therapeutics.

Lei Wang, Chunbo Dong, Weibing Zhang, Xu Ma, Wei Rou, Kai Yang, Tong Cui, Shaolong Qi, Lijun Yang, Jun Xie, Guocan Yu, Lianqing Wang, Xiaoyuan Chen, Zhida Liu.

  Rationale: Circular RNA (circRNA) therapeutics hold great promise as an iteration strategy in messenger RNA (mRNA) therapeutics due to their inherent stability and durable protein translation capability. Nevertheless, the efficiency of RNA circularization remains a significant constraint, particularly in establishing large-scale manufacturing processes for producing highly purified circRNAs. Hence, it is imperative to develop a universal and more efficient RNA circularization system when considering synthetic circRNAs as therapeutic agents with prospective clinical applications. Methods: We initially developed a chimeric RNA circularization system based on the original permuted intron-exon (PIE) and subsequently established a high-performance liquid chromatography (HPLC) method to obtain highly purified circRNAs. We then evaluated their translational ability and immunogenicity. The circRNAs expressing human papillomavirus (HPV) E7 peptide (43-62aa) and dimerized receptor binding domain (dRBD) from SARS-CoV-2 were encapsulated within lipid nanoparticles (LNPs) as vaccines, followed by an assessment of the in vivo efficacy through determination of antigen-specific T and B cell responses, respectively. Results: We have successfully developed a universal chimeric permuted intron-exon system (CPIE) through engineering of group I self-splicing introns derived from Anabaena pre-tRNALeu or T4 phage thymidylate (Td) synthase gene. Within CPIE, we have effectively enhanced RNA circularization efficiency. By utilizing size exclusion chromatography, circRNAs were effectively separated, which exhibit low immunogenicity and sustained potent protein expression property. In vivo data demonstrate that the constructed circRNA vaccines can elicit robust immune activation (B cell and/or T cell responses) against tumor or SARS-CoV-2 and its variants in mouse models. Conclusions: Overall, we provide an efficient and universal system to synthesize circRNA in vitro, which has extensive application prospect for circRNA therapeutics.

Keywords:  chimeric permuted intron-exon system (CPIE); circular RNA (circRNA); mRNA therapeutics; ribozyme; size exclusion chromatography

DOI:  https://doi.org/10.7150/thno.98214