技术资料

Viral vector delivery of gene therapy represents a promising approach for the treatment of numerous retinal diseases. Adeno-associated viral vectors (AAV) constitute the primary gene delivery platform; however,their limited cargo capacity restricts the delivery of several clinically relevant retinal genes. In this study,we explore the feasibility of employing high-capacity adenoviral vectors (HC-AdVs) as alternative delivery vehicles,which,with a capacity of up to 36 kb,can potentially accommodate all known retinal gene coding sequences. We utilized HC-AdVs based on the classical adenoviral type 5 (AdV5) and on a fiber-modified AdV5.F50 version,both engineered to deliver a 29.6 kb vector genome encoding a fluorescent reporter construct. The tropism of these HC-AdVs was evaluated in an induced pluripotent stem cell (iPSC)-derived human retinal organoid model. Both vector types demonstrated robust transduction efficiency,with sustained transgene expression observed for up to 110 days post-transduction. Moreover,we found efficient transduction of photoreceptors and Müller glial cells,without evidence of reactive gliosis or loss of photoreceptor cell nuclei. However,an increase in the thickness of the photoreceptor outer nuclear layer was observed at 110 days post-transduction,suggesting potential unfavorable effects on Müller glial or photoreceptor cells associated with HC-AdV transduction and/or long-term reporter overexpression. These findings suggest that while HC-AdVs show promise for large retinal gene delivery,further investigations are required to assess their long-term safety and efficacy. View Publication

A distinctive feature of both type 1 and type 2 diabetes is the waning of insulin-secreting beta cells in the pancreas. New methods for direct and specific targeting of the beta cells could provide platforms for delivery of pharmaceutical reagents. Imaging techniques such as Positron Emission Tomography (PET) rely on the efficient and specific delivery of imaging reagents,and could greatly improve our understanding of diabetes etiology as well as providing biomarkers for viable beta-cell mass in tissue,in both pancreas and in islet grafts.The DiGeorge Syndrome Critical Region Gene 2 (DGCR2) protein has been suggested as a beta-cell specific protein in the pancreas,but so far there has been a lack of available high-affinity binders suitable for targeted drug delivery or molecular imaging. Affibody molecules belong to a class of small affinity proteins with excellent properties for molecular imaging. Here,we further validate the presence of DGCR2 in pancreatic and stem cell (SC)-derived beta cells,and then describe the generation and selection of several Affibody molecules candidates that target human DGCR2. Using an in-house developed directed evolution method,new DGCR2-binding Affibody molecules were generated and evaluated for thermal stability and affinity. The Affibody molecules variants were further developed as targeting agents for delivering imaging reagents to beta cell. The Affibody molecule ZDGCR2:AM106 displayed nanomolar affinity,suitable stability and biodistribution,with negligible toxicity to islets,qualifying it as a suitable lead candidate for further development as a tool for specific delivery of drugs and imaging reagents to beta cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-84574-y. View Publication

Fellows et al. report that individual dynein motors can move the entire axon length during retrograde transport. They find factors LIS1 and NDEL1,needed for transport initiation,also move with cargos. In the anterograde direction,dynein and its cofactor dynactin are transported separately,keeping them apart until required. Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein,which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level,it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here,we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction,we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore,LIS1 and NDEL1 also undergo long-distance movement,despite being mainly implicated with the initiation of dynein transport. Intriguingly,in the anterograde direction,dynein/LIS1 moves faster than dynactin/NDEL1,consistent with transport on different cargos. Therefore,neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required. View Publication

AbstractThe generation of chimeric antigen receptor?modified natural killer (CAR?NK) cells using induced pluripotent stem cells (iPSCs) has emerged as one of the paradigms for manufacturing off?the?shelf universal immunotherapy. However,there are still some challenges in enhancing the potency,safety,and multiple actions of CAR?NK cells. Here,iPSCs were site?specifically integrated at the ribosomal DNA (rDNA) locus with interleukin 24 (IL24) and CD19?specific chimeric antigen receptor (CAR19),and successfully differentiated into iPSC?derived NK (iNK) cells,followed by expansion using magnetic beads in vitro. Compared with the CAR19?iNK cells,IL24 armored CAR19?iNK (CAR19?IL24?iNK) cells showed higher cytotoxic capacity and amplification ability in vitro and inhibited tumor progression more effectively with better survival in a B?cell acute lymphoblastic leukaemia (B?ALL) (Nalm?6 (Luc1))?bearing mouse model. Interestingly,RNA?sequencing analysis showed that IL24 may enhance iNK cell function through nuclear factor kappa B (NF?B) pathway?related genes while exerting a direct effect on tumor cells. This study proved the feasibility and potential of combining IL24 with CAR?iNK cell therapy,suggesting a novel and promising off?the?shelf immunotherapy strategy. Zhang et al. successfully regenerated iNK cells from human iPSCs with rDNA locus gene editing. IL24 enhances the antitumor activity and proliferation of armored CAR?iNK cells,which may be involved in cellular?positive upregulation and adhesion pathways. View Publication

Simple SummaryHuman induced pluripotent stem cells hold great promise for treating neurological diseases. One of the biggest challenges,however,is the immune system: if transplanted cells are not a perfect match,the body may reject them. To overcome this,we aimed to create “off-the-shelf”,universal cells that could be safely used in anyone,without needing a matched donor. Using CRISPR-mediated gene editing tool,we deleted two key genes,B2M and CIITA,that are responsible for making proteins recognized by the immune system. Additionally,we engineered the cells to produce MIF,which helps protect against natural killer cell attacks. Overall,our study shows that combining MIF overexpression with the removal of B2M and CIITA can produce universal cells that avoid rejection by the immune system. This approach could help make stem cell therapies more widely available and effective for spinal cord injuries and other diseases. AbstractHuman induced pluripotent stem cells (iPSCs) offer immense potential as a source for cell therapy in spinal cord injury (SCI) and other diseases. The development of hypoimmunogenic,universal cells that could be transplanted to any recipient without requiring a matching donor could significantly enhance their therapeutic potential and accelerate clinical translation. To create off-the-shelf hypoimmunogenic cells,we used CRISPR-Cas9 to delete B2M (HLA class I) and CIITA (master regulator of HLA class II). Double-knockout (DKO) iPSC-derived neural progenitor cells (NPCs) evaded T-cell-mediated immune rejection in vitro and after grafting into the injured spinal cord of athymic rats and humanized mice. However,loss of HLA class I heightened susceptibility to host natural killer (NK) cell attack,limiting graft survival. To counter this negative effect,we engineered DKO NPCs to overexpress macrophage migration inhibitory factor (MIF),an NK cell checkpoint ligand. MIF expression markedly reduced NK cell-mediated cytotoxicity and improved long-term engraftment and integration of NPCs in the animal models for spinal cord injury. These findings demonstrate that MIF overexpression,combined with concurrent B2M and CIITA deletion,generates hiPSC neural derivatives that escape both T- and NK-cell surveillance. This strategy provides a scalable route to universal donor cells for regenerative therapies in SCI and potentially other disorders. View Publication

The advancement of Long-Read Sequencing (LRS) techniques has significantly increased the length of sequencing to several kilobases,thereby facilitating the identification of alternative splicing events and isoform expressions. Recently,numerous computational tools for isoform detection using long-read sequencing data have been developed. Nevertheless,there remains a deficiency in comparative studies that systemically evaluate the performance of these tools,which are implemented with different algorithms,under various simulations that encompass potential influencing factors. In this study,we conducted a benchmark analysis of thirteen methods implemented in nine tools capable of identifying isoform structures from long-read RNA-seq data. We evaluated their performances using simulated data,which represented diverse sequencing platforms generated by an in-house simulator,RNA sequins (sequencing spike-ins) data,as well as experimental data. Our findings demonstrate IsoQuant as a highly effective tool for isoform detection with LRS,with Bambu and StringTie2 also exhibiting strong performance. These results offer valuable guidance for future research on alternative splicing analysis and the ongoing improvement of tools for isoform detection using LRS data. Recently,various computational tools have emerged for detecting mRNA isoforms using long-read sequencing data. Here,the authors systemically evaluate and compare the performance of these tools. View Publication

Oxidative phosphorylation (OXPHOS) in the mitochondrial inner membrane is a therapeutic target in many diseases. Neural stem cells (NSCs) show progress in improving mitochondrial dysfunction in the central nervous system (CNS). However,translating neural stem cell-based therapies to the clinic is challenged by uncontrollable biological variability or heterogeneity,hindering uniform clinical safety and efficacy evaluations. We propose a systematic top-down design based on membrane self-assembly to develop neural stem cell-derived oxidative phosphorylating artificial organelles (SAOs) for targeting the central nervous system as an alternative to NSCs. We construct human conditionally immortal clone neural stem cells (iNSCs) as parent cells and use a streamlined closed operation system to prepare neural stem cell-derived highly homogenous oxidative phosphorylating artificial organelles. These artificial organelles act as biomimetic organelles to mimic respiration chain function and perform oxidative phosphorylation,thus improving ATP synthesis deficiency and rectifying excessive mitochondrial reactive oxygen species production. Conclusively,we provide a framework for a generalizable manufacturing procedure that opens promising prospects for disease treatment. Regulating oxidative phosphorylation and restoring redox homeostasis are crucial in neurological disorders. Here,the authors develop a top-down membrane self-assembly strategy to develop stem cell-derived artificial organelles (SAOs) that mimic mitochondrial oxidative phosphorylation without the risks associated with stem cell therapy. View Publication

Significance: Heart disease is the leading cause of death in the United States,yet research is limited by the inability to culture primary cardiac cells. Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) are a promising solution for drug screening and disease modeling. Aim: Induced pluripotent stem cell-derived CM (iPSC-CM) differentiation and maturation studies typically use heterogeneous substrates for growth and destructive verification methods. Reproducible,tunable substrates and touch-free monitoring are needed to identify ideal conditions to produce homogenous,functional CMs. Approach: We generated synthetic polyethylene glycol-based hydrogels for iPSC-CM differentiation and maturation. Peptide concentrations,combinations,and gel stiffness were tuned independently. Label-free optical redox imaging (ORI) was performed on a widefield microscope in a 96-well screen of gel formulations. We performed live-cell imaging throughout differentiation and early to late maturation to identify key metabolic shifts. Results: Label-free ORI confirmed the expected metabolic shifts toward oxidative phosphorylation throughout the differentiation and maturation processes of iPSC-CMs on synthetic hydrogels. Furthermore,ORI distinguished high and low differentiation efficiency cell batches in the cardiac progenitor stage. Conclusions: We established a workflow for medium throughput screening of synthetic hydrogel conditions with the ability to perform repeated live-cell measurements and confirm expected metabolic shifts. These methods have implications for reproducible iPSC-CM generation in biomanufacturing. View Publication

ABSTRACTThe paradoxical exacerbation of cellular injury and death during reperfusion remains a problem in the treatment of myocardial infarction. Mitochondrial dysfunction plays a key role in the pathogenesis of myocardial ischemia and reperfusion injury. Dysfunctional mitochondria can be removed by mitophagy,culminating in their degradation within acidic lysosomes. Mitophagy is pivotal in maintaining cardiac homeostasis and emerges as a potential therapeutic target. Here,we employed beating human engineered heart tissue (EHT) to assess mitochondrial dysfunction and mitophagy during ischemia and reperfusion simulation. Our data indicate adverse ultrastructural changes in mitochondrial morphology and impairment of mitochondrial respiration. Furthermore,our pH-sensitive mitophagy reporter EHTs,generated by a CRISPR/Cas9 endogenous knock-in strategy,revealed induced mitophagy flux in EHTs after ischemia and reperfusion simulation. The induced flux required the activity of the protein kinase ULK1,a member of the core autophagy machinery. Our results demonstrate the applicability of the reporter EHTs for mitophagy assessment in a clinically relevant setting. Deciphering mitophagy in the human heart will facilitate development of novel therapeutic strategies. Summary: Mitochondrial dysfunction and lysosomal degradation of mitochondria (mitophagy) is induced after ischemia and reperfusion simulation in human engineered heart tissue,as shown with an endogenous pH-sensitive mitophagy reporter. View Publication

Acute liver failure (ALF) is a hepatology emergency with rapid hepatic destruction,multiple organ failures,and high mortality. Despite decades of research,established ALF has minimal therapeutic options. Here,we report that the small bioactive compound SCM-198 increases the survival of male ALF mice to 100%,even administered 24?hours after ALF establishment. We identify adiponectin receptor 2 (AdipoR2) as a selective target of SCM-198,with the AdipoR2 R335 residue being critical for the binding and signaling of SCM-198-AdipoR2 and AdipoR2 Y274 residue serving as a molecular switch for Ca2+ influx. SCM-198-AdipoR2 binding causes Ca2+ influx and elevates the phosphorylation levels of CaMKII and NOS3 in the AdipoR2-CaM-CaMKII-NOS3 complex identified in this study,rapidly inducing nitric oxide production for liver protection in murine ALF. SCM-198 also protects human ESC-derived liver organoids from APAP/TAA injuries. Thus,selectively targeting the AdipoR2-CaM-CaMKII-NOS3 axis by SCM-198 is a rapid-acting therapeutic strategy for advanced ALF. Late-stage acute liver failure (ALF) has limited therapies. The authors show that the bioactive compound SCM-198 extends the ALF treatment window from 3 to 24?hours in mice by selectively targeting the identified AdipoR2-CaM-CaMKII-NOS3-NO axis. View Publication

ABSTRACTObjectivesAutism spectrum disorder (ASD) is a neurodevelopmental condition that affects social communication and behaviors. While previous studies using animal models have substantially expanded our knowledge about ASD,the lack of an appropriate human model system that accurately recapitulates the human?specific pathophysiology of ASD hinders the precise understanding of its etiology and the development of effective therapies. This study aims to replicate pathological phenotypes in cerebral organoids derived from idiopathic ASD patients and to conduct proof?of?concept research for the development of ASD therapeutics.MethodsWe conducted an in vitro disease modeling study using cerebral organoids derived from three idiopathic ASD patients. Additionally,we performed organoid?based phenotypic drug screening to identify potential therapeutic compounds that could ameliorate the phenotypes observed in cerebral organoids derived from idiopathic ASD patients.ResultsHere we show that cerebral organoids derived from idiopathic ASD patients display malformation of the ventricular zones and impaired early neuronal differentiation. Through organoid?based phenotypic drug screening,we successfully generated cerebral organoids with normal tissue architecture in which the delayed neuronal differentiation could also be accelerated. Notably,cerebral organoids from ASD patients exhibited a reduced number of GABAergic neurons compared to healthy controls,resulting in an imbalance in the excitatory and inhibitory neuron ratio. The differentiation defects into GABAergic neurons in patient?derived cerebral organoids could be rescued by treating with either IGF1 or Gabapentin,a GABA agonist.ConclusionsOur findings provide a framework for utilizing patient?derived cerebral organoids in the development of personalized pharmaceutical treatment for ASD. Summary of in vitro disease modeling and drug screening using ASD patient?derived COs. This figure highlights the major phenotypes observed in COASD and the therapeutic effects of each compound screened in this study. View Publication

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants,including genetic and non-genetic factors. A key pathological signature of ALS is the cytoplasmic mislocalization and aggregation of TDP-43 in affected motor neurons,which is found in 97% of cases. Recent reports have shown that mitochondrial dysfunction plays a significant role in motor neuron degeneration in ALS,and TDP-43 modulates several mitochondrial transcripts. In this study,we used induced pluripotent stem cell-derived motor neurons from ALS patients with TDP-43 mutations and a transgenic TDP-43M337V mouse model to determine how TDP-43 mutations alter mitochondrial function and axonal transport. We detected significantly reduced mitochondrial respiration and ATP production in patient induced pluripotent stem cell-derived motor neurons,linked to an interaction between TDP-43M337V with ATPB and COX5A. A downstream reduction in speed of retrograde axonal transport in patient induced pluripotent stem cell-derived motor neurons was detected,which correlated with downregulation of the motor protein complex,DCTN1/dynein. Overexpression of DCTN1 in patient induced pluripotent stem cell-derived motor neurons significantly increased the percentage of retrograde travelling mitochondria and reduced the percentage of stationary mitochondria. This study shows that ALS induced pluripotent stem cell-derived motor neurons with mutations in TDP-43 have deficiencies in essential mitochondrial functions with downstream effects on retrograde axonal transport,which can be partially rescued by DCTN1 overexpression. Dafinca et al. show that mutations in TDP-43 lead to decreased mitochondrial oxidative phosphorylation,partially due to interactions with the ATP production machinery and COX5A. These have direct effects on axonal transport,which is reduced in amyotrophic lateral sclerosis motor neurons,and overexpression of dynactin-1 significantly increases retrograde mitochondrial dynamics. View Publication