Thermostable Protein Engineering In article number 2404680, Trung Thanh Thach, Jaekyung Hyun Yong Ho Kim, and co-workers introduce a structure-guided engineered SaCas9 variant with enhanced thermostability and compactness, enabling efficient gene suppression through nonviral delivery. The engineered sdCas9-KRAB-R system demonstrates superior nuclear localization and in vivo knockdown efficiency, showcasing its potential as a robust tool for CRISPR interference and targeted genomic regulation.

Introduction: Danon Disease is a life-threatening Lysosomal storage disorder (LSD) caused by mutations in the Lysosome-associated membrane protein 2 (LAMP2) gene, leading to autophagy dysfunction and severe cardiomyopathy. We used patient-derived Danon iPSC-Cardiomyocytes (iPSC-CMs) and delivered lipid nanoparticle (LNP)-encapsulated LAMP2 mRNA to restore LAMP2 protein expression and lysosomal function, leading to phenotypic recovery. This approach directly addresses LAMP2 deficiency, offering a novel therapeutic strategy for Danon disease and establishing the LNP-mRNA platform as a promising protein replacement therapy. Method: LAMP2 mRNA was synthesized via in vitro transcription (IVT) and encapsulated into lipid nanoparticles (LNPs). LNP-mRNA complexes were characterized for particle size, encapsulation efficiency, and stability, with morphology analyzed using cryogenic transmission electron microscopy (Cryo-TEM). LAMP2 expression and functionality were evaluated in HEK293T cells and iPSC-CMs via Western blot and immunofluorescence. Subcellular localization of LAMP2 was confirmed by confocal microscopy with LysoTracker staining. Autophagy flux and autolysosomal function were assessed through LC3-I/II ratio, p62 levels, and pH-sensitive markers. Result: LNP-mRNA delivery conditions in iPSC-CMs were optimized using LNP-FLuc mRNA, determining optimal uptake conditions including ApoE and FBS concentrations. LAMP2 protein expression was confirmed in iPSC-CMs via Western blot and immunocytochemistry. Confocal microscopy with LysoTracker co-staining verified lysosomal localization of newly synthesized LAMP2 from LNP-mRNA with Pearson correlation analysis. Autophagic function was evaluated through autolysosome formation and autophagosome differentiation using pH-responsive EGFP markers, assessing functional restoration in Danon iPSC-CMs. Conclusion: Our study demonstrates that LNP-mRNA therapy restores LAMP2 protein levels in cardiac cells, presenting a novel therapeutic strategy for Danon disease. These findings suggest LNP-mediated mRNA delivery as a potential treatment for LSDs with cardiomyopathy. Further studies on long-term efficacy and safety may position this approach as a promising strategy for diverse genetic disorders requiring protein replacement therapy.

Thermostable Protein Engineering In article number 2404680, Trung Thanh Thach, Jaekyung Hyun Yong Ho Kim, and co-workers introduce a structure-guided engineered SaCas9 variant with enhanced thermostability and compactness, enabling efficient gene suppression through nonviral delivery. The engineered sdCas9-KRAB-R system demonstrates superior nuclear localization and in vivo knockdown efficiency, showcasing its potential as a robust tool for CRISPR interference and targeted genomic regulation.

Introduction: Danon Disease is a life-threatening Lysosomal storage disorder (LSD) caused by mutations in the Lysosome-associated membrane protein 2 (LAMP2) gene, leading to autophagy dysfunction and severe cardiomyopathy. We used patient-derived Danon iPSC-Cardiomyocytes (iPSC-CMs) and delivered lipid nanoparticle (LNP)-encapsulated LAMP2 mRNA to restore LAMP2 protein expression and lysosomal function, leading to phenotypic recovery. This approach directly addresses LAMP2 deficiency, offering a novel therapeutic strategy for Danon disease and establishing the LNP-mRNA platform as a promising protein replacement therapy. Method: LAMP2 mRNA was synthesized via in vitro transcription (IVT) and encapsulated into lipid nanoparticles (LNPs). LNP-mRNA complexes were characterized for particle size, encapsulation efficiency, and stability, with morphology analyzed using cryogenic transmission electron microscopy (Cryo-TEM). LAMP2 expression and functionality were evaluated in HEK293T cells and iPSC-CMs via Western blot and immunofluorescence. Subcellular localization of LAMP2 was confirmed by confocal microscopy with LysoTracker staining. Autophagy flux and autolysosomal function were assessed through LC3-I/II ratio, p62 levels, and pH-sensitive markers. Result: LNP-mRNA delivery conditions in iPSC-CMs were optimized using LNP-FLuc mRNA, determining optimal uptake conditions including ApoE and FBS concentrations. LAMP2 protein expression was confirmed in iPSC-CMs via Western blot and immunocytochemistry. Confocal microscopy with LysoTracker co-staining verified lysosomal localization of newly synthesized LAMP2 from LNP-mRNA with Pearson correlation analysis. Autophagic function was evaluated through autolysosome formation and autophagosome differentiation using pH-responsive EGFP markers, assessing functional restoration in Danon iPSC-CMs. Conclusion: Our study demonstrates that LNP-mRNA therapy restores LAMP2 protein levels in cardiac cells, presenting a novel therapeutic strategy for Danon disease. These findings suggest LNP-mediated mRNA delivery as a potential treatment for LSDs with cardiomyopathy. Further studies on long-term efficacy and safety may position this approach as a promising strategy for diverse genetic disorders requiring protein replacement therapy.

Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.

Cell-based artificial platelet production has made remarkable progress over the past three decades, driven by the need for safe and stable platelet sources in the face of donor limitations and transfusion-related risks. This review provides a chronological overview of the evolution of in vitro platelet production from various cell sources (CD34+ hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), and others) and highlights key advances in the field. We outline developments from the foundational experiments of the 1990s, through the introduction of iPSCs in the mid-2000s, to the adoption of three-dimensional culture and bioreactor technologies in the late 2010s and the emergence of clinical trials in the 2020s. In addition, we discuss future perspectives, including the role of advanced gene editing and scalable biomanufacturing technologies in accelerating clinical translation. This comprehensive review underscores the promise of artificial platelet production technologies for clinical applications and discusses the remaining challenges, such as scalability, cost-effectiveness, and regulatory hurdles. The recent completion of the first human clinical trials using iPSC-derived platelets marks a significant milestone, pointing to a future in which patient-specific or human leukocyte antigen-universal platelets may be transformed into transfusion medicine and regenerative therapies.

Depletion of S6K1 signaling in the early stage of human brain development drives the formation of retinal cells distinct from cortical neurons. Our findings demonstrate that S6K1 signaling fine-tunes neuronal and retinal lineage specification during brain development.

Based on the results, ID0085 appears to be the most promising therapeutic candidate for the treatment of hypercholesterolemia.

Our findings demonstrate that cell-cell contact inhibits hiPSC-CM proliferation through adherens junction formation, sarcomeric assembly, and reduced IGFBP2 secretion. Importantly, exogenous supplementation of IGFBP2 can overcome cell contact-mediated inhibition of hiPSC-CM proliferation and facilitate the growth of 3-dimensional cardiac tissue. These insights provide valuable implications for advancing cardiac tissue engineering and regenerative therapies.

Human induced pluripotent stem cells (hiPSCs) generate multiple clones with inherent heterogeneity, leading to variations in their differentiation capacity. Previous studies have primarily addressed line-to-line variations in differentiation capacity, leaving a gap in the comprehensive understanding of clonal heterogeneity. Here, we aimed to profile the heterogeneity of hiPSC clones and identify predictive biomarkers for cardiomyocyte (CM) differentiation capacity by integrating transcriptomic, epigenomic, endogenous retroelement, and protein kinase phosphorylation profiles. We generated multiple clones from a single donor and validated that these clones exhibited comparable levels of pluripotency markers. The clones were classified into two groups based on their differentiation efficiency to CMs-productive clone (PC) and non-productive clone (NPC). We performed RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with sequencing (ATAC-seq). NPC was enriched in vasculogenesis and cell adhesion, accompanied by elevated levels of phosphorylated ERK1/2. Conversely, PC exhibited enrichment in embryonic organ development and transcription factor activation, accompanied by increased chromatin accessibility near transcription start site (TSS) regions. Integrative analysis of RNA-seq and ATAC-seq revealed 14 candidate genes correlated with cardiac differentiation potential. Notably, TEK and SDR42E1 were upregulated in NPC. Our integrative profiles enhance the understanding of clonal heterogeneity and highlight two novel biomarkers associated with CM differentiation. This insight may facilitate the identification of suboptimal hiPSC clones, thereby mitigating adverse outcomes in clinical applications.