Immune cell infiltration into the central nervous system is increasingly recognized as a driver of neurodegeneration, yet its role in Huntington's disease remains unresolved. Addressing this question requires models that replicate the selective vulnerability of striatal neurons observed in patients, a challenge unmet by rodent systems. Here we use a previously established and genetically engineered pig model carrying the human huntingtin mutation with an expanded cytosine-adenine-guanine repeat, enabling investigation of immune-neural interactions in a physiologically relevant context. Using single-nucleus and spatial transcriptomics, integrated with immunohistochemistry and T cell receptor sequencing, we constructed a cellular map of the striatum. We identified an interferon-responsive microglial state that secretes chemokine ligand eight, recruiting cytotoxic CD8-positive T cells that release perforin and granzyme, thereby accelerating neuronal loss. Functional experiments confirmed the pathogenic role of chemokine ligand eight and demonstrated that its neutralization mitigates neurodegeneration. These findings uncover a species-dependent immune mechanism in Huntington's disease and nominate chemokine ligand eight-mediated T cell infiltration as a therapeutic target.

Abnormal cytoplasmic accumulation of TAR DNA-binding protein 43 (TDP-43) is a common pathological feature of TDP-43 proteinopathies. Since non-human primate models can better recapitulate this neuropathology than rodents, we used a monkey model to evaluate the therapeutic potential of stem cells for TDP-43-mediated neuropathology. We established a cynomolgus monkey model by expressing mutant TDP-43 (M337V) in the monkey striatum through AAV injection. This model exhibited motor dysfunction and abnormal cytoplasmic TDP-43 accumulation. Using multi-gene modified stem cells (NILB-hiPSCs) that can be induced to differentiate in vivo with doxycycline treatment, we found that transplanted NILB-hiPSCs improved the limb movements of the TDP-43-injected monkeys, differentiated into mature neurons, and were integrated with neural circuit activity in the monkey brain. Furthermore, NILB-hiPSC therapy reduced reactive gliosis and diminished the abnormal cytoplasmic localization of mutant TDP-43. These results highlight the potential of in vivo inducible stem cells for the treatment of TDP-43 proteinopathies.

The accumulation of mitochondrial DNA (mtDNA) mutations is a primary driver of mitochondrial dysfunction, which is intrinsically linked to aging and various pathologies. POLG, the catalytic subunit of DNA polymerase gamma, is essential for mtDNA replication; notably, a deficiency in its proofreading function precipitates the accumulation of mtDNA mutations. In this study, by combining prime editing with somatic cell nuclear transfer technology, we successfully generated a mitochondrial mutator pig model expressing proofreading-deficient POLG. These pigs exhibited elevated somatic mtDNA mutation loads and recapitulated key premature aging phenotypes, including weight loss, rough hair coat, anemia, structural alterations in the skin and testicular interstitium, increased apoptosis, and the up-regulation of senescence-associated markers, culminating in shortened life span. Given the physiological and metabolic similarities between pigs and humans, this mitochondrial mutator pig model represents an ideal preclinical tool for dissecting the mechanistic role of mtDNA mutations in aging and age-related pathologies and for accelerating the translation of therapeutic strategies.

Base editors (BEs) enable precise base substitutions, but their size exceeds the packaging capacity of adeno-associated virus (AAV), impeding in vivo applications. Here we design a split BE system that recruits deaminases to Cas9 nickase via coiled-coil heterodimers, resulting in various coiled-coil heterodimers-mediated base editors (CC-BEs), including cytidine base editor (CC-CBE), adenine base editor (CC-ABE), and their derivatives. We reveal that CC-BEs maintain and even improve the editing efficiency of the original unsplit BEs across various cell types and editing scopes, achieving maximum enhancements of 9.6-fold in human immortalized cells and 12.4-fold in primary somatic cells for CC-CBE. Using CC-ABE, we validate in vivo editing efficiency and successfully achieve A-to-G conversion in the Pcsk9 and Dmd genes via dual-AAV vectors in mice. Altogether, we develop a simple and universal strategy to address the challenges posed by the large size of BEs without compromising editing efficiency for base substitutions in vivo. Base editors (BEs) enable precise base substitutions but are limited by their large size. Here, the authors engineer a split BE system utilizing coiled-coil heterodimers (CC-BE) and demonstrate that CC-BEs maintain or even enhance efficiency, enabling gene therapy applications via dual AAV.

The insufficient contribution of human cells is a key obstacle to interspecies chimera. In this issue of Developmental Cell, He et al. harnessed the RNA collateral cleavage activity of Cas13 to diminish the competitive advantage of host cells, increasing integration ratio of human cells to 1% in host mice.

IntroductionXenotransplantation holds promise for addressing the organ shortage crisis. Multi-genetic modification of pigs, such as knockout of three carbohydrate antigen-related genes and expression of immunoprotective proteins, can significantly improve xenograft survival. However, existing multi-gene modification strategies face challenges: transposon-based transgenic technology may lead to unstable expression, while exogenous promoters used in site-specific integration strategies are susceptible to epigenetic silencing, making it difficult to maintain long-term, stable expression levels. Therefore, developing a donor pig model capable of achieving stable and long-lasting multi-gene expression is a critical need in the field.MethodsCRISPR-Cas9 technology was used to knockout three major glycan antigen genes (GGTA1, CMAH, β4GalNT2) to eliminate hyperacute rejection. Subsequently, four human protective genes (hCD55, hCD46, hTHBD, hEPCR) were site-specifically integrated into the porcine Rosa26 safe-harbor locus. Their expression was driven by the porcine endogenous Rosa26 promoter and the THBD core promoter, respectively, to ensure long-term stable and tissue-specific expression. Furthermore, the selection marker gene was efficiently removed using the Cre/loxP system.ResultsThe three glycan antigens were completely absent at both cellular and tissue levels in BM7G genetically modified pigs. What’s more, four protective proteins were stably expressed in vascular endothelial cells and major organs such as the heart, liver, and kidneys. Among them, hCD55 and hCD46 were widely expressed, while hTHBD and hEPCR were specifically expressed in the vascular region. In-vitro functional assays confirmed that BM7G porcine vascular endothelial cells significantly reduced the binding of human antibodies, effectively inhibited complement-dependent cytotoxicity, and decreased the formation of thrombin-antithrombin (TAT) complexes.ConclusionIn summary, by combining the knockout of xenoantigens with the use of endogenous promoters to drive the expression of multiple human protective genes, we successfully constructed a seven-gene modified pig model with low immunogenicity and synergistic protective functions. This provides an important donor resource for preclinical research in xenotransplantation.

Background & Aims: The shortage of liver donors remains a critical challenge in the treatment of end-stage liver failure. Previous studies have explored ectopic transplantation of hepatocyte organoids or spheroids to support unstable liver function. We hypothesized that the addition of non-parenchymal liver cells to hepatocyte organoids would improve overall survival in acute liver failure. Here, we aimed to engineer DNA origami-mediated multi-lineage liver microtissue (NAC-liver microtissue) and evaluate its therapeutic efficacy in a mouse model of acute liver failure. Methods: We employed self-assembling nucleic acid nanostructures (NAC) to construct two types of liver microtissues: one composed exclusively of hepatocytes (NAC-hepatocyte microtissue) and another incorporating both hepatocytes and non-parenchymal cells (NAC-liver microtissue). We then investigated their hepatic functional properties in vitro and evaluated therapeutic efficacy in vivo through intraperitoneal transplantation into mice undergoing extended hepatectomy. To explore the underlying mechanisms, we performed histological analysis and RNA sequencing of the remnant liver. Results: NAC-liver microtissue exhibited superior scalability, uniformity, and stable, enhanced hepatic functional activity compared to NAC-hepatocyte microtissue in vitro (n = 4, p <0.001). In vivo, transplantation of NAC-liver microtissue markedly improved survival in mice undergoing extended hepatectomy compared with untreated controls (70% [n = 10] vs. 8.3% [n = 12], p <0.001), whereas NAC-hepatocyte microtissue demonstrated moderate efficacy (25%, n = 8). Furthermore, NAC-liver microtissue transplantation improved liver function, lipid oxidation, and sinusoidal vascular network formation in the remnant liver of mice following extended hepatectomy. Conclusions: These findings provide therapeutic evidence supporting NAC-liver microtissue transplantation as a potential treatment for acute liver failure and highlight a novel strategy for extrahepatic cellular support in severe liver injury. Impact and implications: Although hepatocyte organoid or spheroid transplantation has long been explored as a potential therapy for liver disease, these approaches fail to replicate the cellular complexity of the liver, resulting in suboptimal functionality and limited therapeutic benefit. The therapeutic potential of multi-lineage liver microtissue for liver failure has not previously been reported. Here, we demonstrate that NAC-liver microtissue, composed of hepatocytes and non-parenchymal cells, provides superior functional outcomes both in vitro and in vivo compared with NAC-hepatocyte microtissue. These findings highlight the indispensable role of non-parenchymal cells in maintaining hepatic functionality and support the therapeutic potential of multi-lineage liver microtissue for patients with severe liver disease.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection has transformed molecular diagnostics through its speed and accuracy; however, one-pot formats are often limited by sensitivity and field suitability. Herein, we developed a universal light-controlled high-sensitivity one-pot CRISPR/Cas12a testing (ULTRAt) platform based on structure-engineered CRISPR RNA (crRNA) scaffolds. By incorporating photocaged 6-nitropiperonyloxymethyl groups into the crRNA stem-loop, Cas12a activity is transiently suppressed during isothermal amplification via structural modulation, enabling efficient target enrichment. Subsequent UV irradiation removes the protecting groups, restoring the native conformation and activating robust trans-cleavage. ULTRAt achieves a limit of detection of two copies of monkeypox virus per reaction with a 15-min time-to-result, representing a 100-fold sensitivity improvement over conventional assays. The platform further supports single-nucleotide polymorphism discrimination and human papillomavirus 16/18 genotyping. Analysis of 91 clinical samples demonstrates strong concordance between ULTRAt and reference qPCR and sequencing assays. Collectively, ULTRAt enables rapid, ultra-sensitive, and versatile one-pot detection, supporting near-patient diagnostics and genotyping.

Abstract Background HD is a devastating neurodegenerative disorder caused by the expansion of CAG repeats in the HTT. Silencing the expression of mutated proteins is a therapeutic direction to rescue HD patients, and recent advances in gene editing technology such as CRISPR/CasRx have opened up new avenues for therapeutic intervention. Methods The CRISPR/CasRx system was employed to target human HTT exon 1, resulting in an efficient knockdown of HTT mRNA. This therapeutic effect was substantiated in various models: HEK 293 T cell, the HD 140Q-KI mouse, and the HD-KI pig model. The efficiency of the knockdown was analyzed through Western blot and RT-qPCR. Additionally, neuropathological changes were examined using Western blot, immunostaining, and RNA sequencing. The impact on motor abilities was assessed via behavioral experiments, providing a comprehensive evaluation of the treatment's effectiveness. Results CRISPR/CasRx system can significantly reduce HTT mRNA levels across various models, including HEK 293 T cells, HD 140Q-KI mice at various disease stages, and HD-KI pigs, and resulted in decreased expression of mHTT. Utilizing the CRISPR/CasRx system to knock down HTT RNA has shown to ameliorate gliosis in HD 140Q-KI mice and delay neurodegeneration in HD pigs. Conclusions These findings highlight the effectiveness of the RNA-targeting CRISPR/CasRx as a potential therapeutic strategy for HD. Furthermore, the success of this approach provides valuable insights and novel avenues for the treatment of other genetic disorders caused by gene mutations.

During X chromosome inactivation (XCI), Xist RNA establishes silencing by coating the chromosome in cis and binding diverse proteins to promote formation of a heterochromatic domain. However, Xist repeat B role beyond initiation of XCI remains unclear. Here, we find that loss of Xist repeat B in female mice allows survival and leads to a small body size persisting throughout life. Epigenetic and transcriptomic analyses reveal low levels of H3K27me3 and H2AK119ub occupancy on the X chromosome, except in certain CpG island regions, and partial reactivation of X-linked genes on the inactive X across multiple tissues. Notably, overdosage of Usp9x promotes centrosome amplification and chromosome instability. We further demonstrate that Usp9x overdosage alters asymmetric cell division, thereby affecting the process of cell differentiation. Thus, Xist repeat B is necessary for gene-specific silencing during XCI maintenance and impacts cell proliferation and differentiation during development. This provides insights into repeat B importance in maintaining XCI.

The rabbit owns commercial importance in meat and fur production, and also has long served as a valuable animal model in biomedical research. Yet, the complete assembly of a high-quality rabbit reference genome has not been established. Here, we present a telomere-to-telomere (T2T) genome assembly of the New Zealand White (NZW) rabbit, the most complete and accurate rabbit genome to date. Using a haploid embryonic stem cell line (haESC) to overcame the challenges of homologous sequence interference and structural variation, we generate a genome with a contig N50 of 137.71 Mb, anchoring all chromosomes with only three gaps, through integrating long-read sequencing (PacBio HiFi, Oxford Nanopore), Hi-C, and Illumina data. Quality assessments reveal near-complete coverage of the BUSCO mammalian gene set, with 99.61% completeness. Notably, this complete assembly allowed for the first comprehensive annotation of the rabbit immunoglobulin loci and major histocompatibility complex region, which were previously unresolved in earlier genome versions. The NZW-T2T assembly based on haESC not only provides a critical resource for advancing rabbit-based research, but also set a new benchmark for de novo genome assembly for other animals with complex genome. ### Competing Interest Statement The authors have declared no competing interest.

Chronic pancreatitis (CP) is a progressive and irreversible fibroinflammatory disease that markedly increases susceptibility to pancreatic cancer and remains without effective targeted therapies. Among the genetic contributors to CP, the carboxypeptidase A1 p.Ser282Pro ( CPA1 S282P ) variant has been proposed to promote disease through misfolding-induced endoplasmic reticulum stress (ERS), although the broader pathogenic landscape remains incompletely defined. This study generated a rabbit model mimicking the human CPA1 S282P mutation using the SpRY-ABE-8.17 system. Homozygous CPA1 S282P rabbits exhibited characteristic human CP phenotypes following alcohol induction, including visceral pain, elevated serum lipase and amylase, inflammatory cell infiltration, and extensive pancreatic fibrosis. Biochemical analyses confirmed that the p.S282P mutation induced CPA1 misfolding and elevated the expression of ERS markers GRP78 and CHOP in both transfected HEK293T cells and homozygous mutant rabbits. Notably, the CPA1 S282P mutation markedly disrupted intra-pancreatic lipid homeostasis, contributing to the development of CP in mutant rabbits. This study successfully established the first rabbit model of CP that accurately recapitulates CP caused by a defined human point mutation. Additionally, this study provides insights into a previously unrecognized link between CPA1 and intra-pancreatic lipid metabolism, offering a foundation for identifying novel therapeutic targets for human CP.

The regenerative capacity of the central nervous system is extremely limited, posing significant challenges for repairing brain injuries. Human induced pluripotent stem cells (hiPSCs) offer a promising cell source for neural regeneration, but their clinical application is hindered by inefficient neuronal differentiation, and poor post-transplant survival. To overcome these challenges, we prepared electrospun fibrous membranes composed of acetylated glucomannan, gelatin, polycaprolactone, and carbon nanotubes. These scaffolds exhibited anti-inflammatory properties in vitro and in vivo. Their excellent adhesion and support properties mimic the extracellular matrix, facilitating the 3D culture of our previous engineered NILB-hiPSCs in vitro. After loading with doxycycline (differentiation inducer) and cyclosporin A (immunosuppressant), the scaffolds achieved sustained release of these agents, ensuring timely neuronal differentiation and maintaining localized immunosuppression, thereby circumventing the need for high systemic doses. Moreover, the composites improved the survival and differentiation of NILB-hiPSCs, promoting the neural repairment in controlled cortex injury mice. Our study highlights the importance of customized multifunctional biomaterials in stem cell transplantation therapy.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that lacks ideal models to comprehensively recapitulate its pathological features. TDP-43 pathology, a hallmark of neurodegenerative diseases, plays a critical role in disease progression. Given the anatomical and physiological similarities between pig and human brains, large animal models offer a unique advantage in more accurately simulating patient-specific disease characteristics. In this study, we rapidly established a TDP-43-induced neurodegenerative disease model in pigs through ear vein injection of the TDP-43M337V virus. Disease progression was systematically evaluated using behavioral assessments and pathological analyses. This porcine model produced extremely severe motor dysfunction accompanied by significant muscle atrophy and fibrosis. Additionally, characteristic TDP-43 pathological phenotypes were observed, including degeneration of spinal motor neurons and proliferation of glial cells in both the brain and spinal cord. Notably, TDP-43M337V induction led to a significant upregulation of TMEM106B, SOD1, and APOE4 levels. This TDP-43 porcine model recapitulates multiple key features of ALS and serves as a valuable complement to existing animal models, providing a robust platform for investigating TDP-43-related pathogenic mechanisms of TDP-43 and developing effective therapeutics.

5-Methylcytosine (m 5 C) is one of the posttranscriptional modifications in mRNA and is involved in the pathogenesis of various diseases. However, the capacity of existing assays for accurately and comprehensively transcriptome-wide m 5 C mapping still needs improvement. Here, we develop a detection method named DRAM (deaminase and reader protein assisted RNA methylation analysis), in which deaminases (APOBEC1 and TadA-8e) are fused with m 5 C reader proteins (ALYREF and YBX1) to identify the m 5 C sites through deamination events neighboring the methylation sites. This antibody-free and bisulfite-free approach provides transcriptome-wide editing regions which are highly overlapped with the publicly available bisulfite-sequencing (BS-seq) datasets and allows for a more stable and comprehensive identification of the m 5 C loci. In addition, DRAM system even supports ultralow input RNA (10 ng). We anticipate that the DRAM system could pave the way for uncovering further biological functions of m 5 C modifications.

Haploid embryonic stem cells (haESCs), which contain a single set of chromosomes, provide powerful systems for investigating gene function and creating gene-edited animal models. haESCs have been derived from several mammalian species including human, mouse and rats, yet not from rabbits. Here, we report the derivation of parthenogenetic haESCs of rabbit (rbPhESCs) using an optimized culture medium. These cells maintain a stable haploid karyotype during long-term culture and own pluripotency features comparable to diploid embryonic stem cells derived from rabbit fertilized embryos. By integrating gene trapping cassette into ROSA26 locus, rbPhESCs offer an ideal platform for high-throughput functional genomic screening, as shown in haESCs from other mammals. Consistent with their parthenogenetic origin, rbPhESCs durably preserve maternal-specific imprinting patterns even after extended culture in serum-containing conditions, providing a valuable platform for generating disease models deficient in maternally expressed imprinted genes. Therefore, our findings expand the repertoire of mammalian haESCs and establish rbPhESCs as a valuable platform for genetic studies and biotechnological applications in rabbits. ### Competing Interest Statement The authors have declared no competing interest.

To date, generating viable and functional hepatocytes in large scale remains challenge. By employing 3D suspension condition with the support of low concentration Matrigel, a novel culture system was developed to generate expandable hepatoblast organoids (HB-orgs) and mature polarised hepatocyte organoids (P-hep-orgs) from human embryonic stem cells (hESCs) in both dishes and bioreactors. scRNA-seq and functional assays were used to characterise HB-orgs and P-hep-orgs. hESC-derived HB-orgs could proliferate at least for 15 passages, leading to 1012 in total cells in 4 weeks. P-hep-orgs differentiated from HB-orgs displayed characteristics of mature hepatocytes with polarisation. Moreover, single-cell RNA sequencing exhibited that over 40% of cells in P-hep-orgs were highly fidelity with human primary hepatocytes. Eventually, large-scale production of P-hep-orgs could be generated from massively expanded HB-orgs within 1 week with similar number in bioreactors, which were achieved by the enhancements in energy metabolism contribute to the expansion of HB-orgs and maturation of P-hep-orgs in bioreactors. By providing a cost-efficient and robust platform, our study represents a significant step toward manufacturing large-scale functioning hESC-derived hepatocytes for cell-based therapeutics, disease modelling, pharmacology and toxicology studies.

Stem cell-based therapy holds great potential for substituting degenerated motor neurons (MNs) in amyotrophic lateral sclerosis (ALS). Missing protocols for advanced differentiation of transplanted cells into MNs, immune rejection, and the lack of suitable ALS models for preclinical trials have slowed the development of effective therapies. Here, we employed multiplex genetic-editing to generate a novel human pluripotent stem cell line containing doxycycline (Dox)-inducible MNs-specific transcription factors and comprehensively modified immunomodulatory genes. We transplanted these cells into the spinal cord of ALS large animal models (SOD1G93A pigs and TIA1P362L rabbits), which faithfully recapitulate pathologies and symptoms observed in ALS patients. The transplanted cells could efficiently differentiate into functional MNs upon Dox treatment in vivo, distribute throughout the spinal cord and motor cortex via extensive migration, survive long-term without the need for immunosuppression. Notably, these MNs integrated into host neural circuits, as evidenced by their long projection of peripheral axons to target muscle and reformation of neuromuscular junctions. As result, pathologies and motor deficits were substantially ameliorated in both animal models. One Sentence Summary Hypoimmunogenic human motor neurons induced from iPSCs in vivo reform neuromuscular junctions and ameliorate ALS disease in pig and rabbit models. ### Competing Interest Statement The authors have declared no competing interest.