Abstracts

1. A case of catastrophic visceral venous thromboembolism – digenic mutations of SERPINC1 and PROC in familial inheritance

Songkang LIU¹, Weihong YANG², Yonghua LI¹, Huanyan PENG¹, Dongye YANG¹*

1. Division of Gastroenterology & Hepatology, The University of Hongkong-Shenzhen Hospital, No.1 Haiyuan 1^st Road, Futian District, Shenzhen Guangdong, P.R. of China 2. Division of interventional radiology, The University of Hongkong-Shenzhen Hospital, No.1 Haiyuan 1^st Road, Futian District, Shenzhen Guangdong, P.R. of China

*Corresponding author: yangdy@hku-szh.org

Extensive visceral venous thromboembolism (EVVTE) is a complex and potentially fatal condition which has been found to have a familial genetic component as a risk factor. We identified a 24-year-old male patient with severe visceral thrombosis involved with portal, spleen and superior mesenteric veins. The patient’s coagulation function was impaired, with an elevated D-dimer of 14.77 μg/ml fibrinogen equivalent units and decreased levels of protein C, protein S, and Antithrombin III. The patient underwent transvascular thrombectomy via an aspiration catheter with continuous heparin infusion, but unfortunately required small bowel resection with double enterostomies.

Whole-exomic sequencing was conducted on this patient and his family to investigate the underlying etiology and revealed that the patient has mutations in the SERPINC1 (c.1219-12T>A) and PROC genes (c.565C>T). Sanger sequencing within the family suggests that the proband inherited one mutation from each of his parents. The proband is one of seven children, making the disease occurrence among family members particularly noteworthy. Currently, only the proband and his eldest sister demonstrate EVVTE symptoms, and both carry the digenic mutations. This indicates that the digenic mutations may significantly contribute to EVVTE symptom occurrence, posing a greater risk than each individual mutation. The youngest brother of the proband is also a carrier of the digenic mutations but is currently asymptomatic. The contributions of non-inherited factors to EVVTE symptoms is not well-understood, so continued follow-up for this family will be crucial. Additionally, further elucidation of the molecular mechanisms underlying how these digenic mutations cause EVVTE symptoms is imperative.

2. A distinct immune landscape in anti-synthetase syndrome profiled by a single-cell genomic study

Jiayu Ding,^3,4,5,6# Yanmei Li,^1,2# Zhiqin Wang,^3,4,5,6 Zhigang Cai,^{1,3,4,5,6 *} Wei Wei^{1,2 *}

1. Department of Rheumatology and Immunology, Tianjin Medical University General Hospital, Tianjin, China 2. Tianjin Clinical Research Center for Rheumatic and Immune Diseases, Tianjin, China 3. The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics; Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University, Tianjin, China 4. National Key Laboratory of Experimental Hematology, Tianjin, China 5. Tianjin Key Laboratory of Inflammatory Biology, Tianjin, China 6. Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China

^{*Corresponding authors: Zhigang Cai, Professor, Ph.D., us36zcai@tmu.edu.cn and Wei Wei, M.D., tjweiwei2003@163.com}

Anti-synthetase syndrome (ASS) is an uncommon idiopathic inflammatory myopathy characterized by the presence of at least one of three primary symptoms: myositis, interstitial lung disease, and arthritis. We examined the transcriptional profiles of peripheral blood mononuclear cells (PBMCs) and their immune repertoires from ASS patients to examine effects of the syndrome at the single-cell level. We performed single-cell RNA sequencing (scRNA-seq) analysis of PBMCs and bulk RNA sequencing data from patients with ASS, patients with anti-melanoma differentiation-associated gene 5-positive dermatomyositis (MDA5⁺ DM), and healthy controls (HCs). MDA5⁺ DM was used as a disease control because it has similar organ involvement as ASS. We performed flow cytometry to validate the results from the scRNA-seq analysis.After meticulous annotation of PBMCs, we noticed a significant decrease in the proportion of mucosal-associated invariant T cells in ASS patients compared to HCs along with a notable increase in the proportion of proliferative natural killer T cells. Compared with MDA5⁺ DM patients, ASS patients exhibited substantial enrichment of interferon pathways in their PBMCs, which were primarily mediated by IFN-II, and displayed a weak immune response. Furthermore, ASS patients exhibited more pronounced metabolic abnormalities, which may affect oxidative phosphorylation pathways. Immunophenotyping analysis of PBMCs from ASS patients revealed an increasing trend for the clone CQQSYSTPWTF. Using single-cell genomic datasets of ASS PBMCs, we revealed a distinctive profile in the immune system of individuals with ASS compared to MDA5⁺ DM or HCs.

3. A novel in situ mitochondrial gene therapy for the treatment of mitochondrial diseases with mitochondrial gene mutations

Min Zhao¹, Yi Wang¹, Hu-Lin Jiang^{1,2 *}

1. State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, China. 2. College of Pharmacy, Yanbian University, Yanji, 133002, China.

^{*Corresponding author: jianghulin3@cpu.edu.cn or jianghulin3@163.com}

Mitochondrial diseases are the most common group of hereditary metabolic disorders among rare diseases accompanied by mitochondrial dysfunctions, with an estimated prevalence of 1 in 4300, causing serious disability and a reduced life expectancy. Mutations have been identified in approximately 350 gene sites, including mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), associated with pathological manifestations. Direct delivery of wild-type mtDNA or artificial DNA encoding a therapeutic gene into the mitochondria matrix to complement endogenous mtDNA is a promising strategy to restore mitochondrial function and alleviate clinical symptoms of mitochondrial diseases. However, in situ mitochondrial gene therapy remains a challenge due to the severe pathological microenvironment of diseased mitochondria. Here, we report a novel pathologically responsive polymer-based mitochondrial gene delivery system with relatively high in situ mitochondrial gene transfection efficiency. In a mouse model of Leber’s hereditary optic neuropathy (LHON), a typical mitochondrial disease caused by mutations in mtDNA, we demonstrate that mitochondrial delivery of the therapeutic ND4 gene by the polymer-based system resulted in excellent therapeutic efficacy in vivo. This included restoration of ocular ND4 protein expression, repairing mitochondrial complex I, and recovery of retinal function. This establishes proof-of-concept for a novel pathologically responsive polymer-based in situ mitochondrial gene delivery system as a versatile strategy to deliver DNA into the mitochondria. This can be applied to the treatment of LHON and other mitochondrial diseases caused by mtDNA mutations.

4. A novel mutation of DSCAML1 modeling human rare disease

Zengjin Huang^{1,2,3,4 *}, Mieke Degreef^3,4, Dietmar Schmucker^3,4,5

1. Shenzhen Key Laboratory of Gene Regulation and Systems Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China 2. Department of Systems Biology, School of Life Science, Southern University of Science and Technology, Shenzhen, Guangdong 518005, China 3. VIB Center for Brain & Disease Research, Neuronal Wiring Laboratory, Leuven, 3000, Belgium 4. KU Leuven, Department of Neurosciences, Leuven research Institute for Neuroscience and Disease (LIND), Leuven, Belgium 5. The Life and Medical Sciences Institute (LIMES), University of Bonn, Bonn, Germany.

^{*Corresponding author: huangzj@mail.sustech.edu.cn}

The Down Syndrome Cell Adhesion Molecule-Like 1 (DSCAML1) gene is located on human chromosome 11 (11q 23.3). Individuals with Tourette and Jacobsen syndromes often have a terminal deletion at the end of the long arm of chromosome 11 (11q) containing this gene, making DSCAML1 a top candidate gene that may contribute to these syndromes. Studies using DSCAML1 mutant frogs (Xenopus tropicalis) have shown that loss-of-function DSCAML1 alleles caused froglets to phenocopy aspects of Tourette and Jacobsen syndromes in humans but result in lethality during the metamorphosis stage. DSCAML1 plays a critical role in differentiating and tiling photoreceptor cells. Additionally, it regulates the homeostatic synthesis of serotonin neurotransmitters. Investigation of novel hypomorphic DSCAML1 alleles in frogs provides a potential model for studying the progression of these syndromes and developing therapeutic approaches in vivo.

5. A novel rat model of Dravet syndrome recapitulates clinical hallmarks

Miao Li^a,b,1, Lixin Yang^a,b,1, Weixin Qian^a,c, Saikat Ray^d, Zhonghua Lu^a,b, Tao Liu^e, Ying-Ying Zou^f, Robert K. Naumann^a,b, Hong Wang^a,b,g*

^aThe Institute of Biomedical and Health Engineering, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences; Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen, 1068 Xueyuan Avenue, Shenzhen University Town, Nanshan District, 518055 Shenzhen, China ^bCAS Key Laboratory of Brain Connectome and Manipulation, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China ^cDivision of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China ^dDepartment of Brain Sciences, Weizmann Institute of Science, Rehovot, Israel ^eState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. ^fDepartment of Pathology and Pathophysiology, Faculty of Basic Medical Sciences, Kunming Medical University, Kunming, China. ^gShenzhen Key Laboratory of Drug Addiction, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

*Corresponding author: hong.wang@siat.ac.cn

Dravet syndrome (DS) is an infantile epileptic encephalopathy caused primarily by haploinsufficiency of the SCN1A gene. Although several mouse models of DS have been generated, they do not recapitulate the full spectrum of DS manifestations. In this study, we generated a novel rat model of DS by oviductal nucleic acid delivery (i-GONAD) of the CRISPR-Cas9 machinery to the zygote. We successfully introduced a frameshift mutation in the Scn1a gene, which results in a premature stop codon and reduces the expression level Scn1a by half. Interestingly, Scn1a ^+/- rats are indistinguishable from wild-type littermates prior to environmental stress. However, upon hyperthermia challenge, all P15 Scn1a ^+/- rats suffered severe heat-induced seizures, while wild-type littermates did not. During seizures, Scn1a ^+/- rats showed high delta and theta power by electroencephalogram. Fenfluramine, which has been approved by the U.S. Food and Drug Administration for the treatment of DS, reduced the severity of hyperthermia-induced seizures in Scn1a ^+/- rats. Taken together, this novel rat model of DS fulfills the three criteria (construct validity, face validity, and predictive validity) of a reliable preclinical model, suggesting that Scn1a ^+/- rats have great potential for dissecting the disease mechanisms and testing new therapeutic interventions to improve medicine for DS patients.

6. A spontaneous infection model of chronic granulomatous disease (CGD) decoded by single-cell transcriptional RNA-sequencing with spatial information (SeekSpace^TM)

Hanzhi Yu^1,2,3, Ge Dong^1,2,3, Yunxi Ma^1,2,3, Jiayu Ding^1,2,3, Jingjing Liu^1,2,3, Zhigang Zhao⁴, Zhiliang Zhou⁵, Shaozhuo Jiao⁶, Zhigang Cai^1,2,3,7,8

¹The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics; Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University, Tianjin, China ²National Key Laboratory of Experimental Hematology, Tianjin, China ³Tianjin Key Laboratory of Inflammatory Biology, Tianjin, China ⁴Medical Oncology Department of Tianjin First Central Hospital, Tianjin, China ⁵GeneMind Biosciences Co., Ltd., Shenzhen, China ⁶SeekGene BioSciences Co., Ltd., Beijing, China ⁷Department of Hematology, Tianjin Medical University Tianjin General Hospital, Tianjin, China ⁸Department of Rheumatology and Immunology, Tianjin Medical University Tianjin General Hospital, Tianjin, China

*Corresponding author: Zhigang Cai, Professor, Ph.D., us36zcai@tmu.edu.cn

Chronic granulomatous disease (CGD) is a rare and hereditary immunodeficiency disorder attributed to a malfunction in the NADPH oxidase 2 (NOX2) in phagocytes. Individuals with CGD exhibit increased susceptibility to pathogens such as bacteria and fungi because of a deficiency of NOX2-derived reactive oxygen species. This defect also contributes to an excessive inflammatory response, leading to tissue damage.

In this study, we established a CGD infection model in the Ncf2^-/- genetic background through controlled environmental exposure. Within Specific Pathogen-Free (SPF) facilities, Ncf2^-/- mice showed no signs of infection. However, under clean grade conditions, these mice spontaneously developed fibro-encapsulated necrotizing lung granulomas. Neutrophils and monocyte-derived macrophages were significantly increased in the lung tissues of CGD mice, developing a NOS2^high neutrophil subset with inflammatory transcriptional characteristics and a unique monocyte-derived macrophage subset with both M1 and M2 polarization. Moreover, the distinct macrophage subset was predominantly localized to the peripheral zone of the granuloma in CGD mice, actively remodeling the extracellular matrix by expressing Fn1 and Mmp12. Treatment of CGD mice with the migration inhibitory factor (MIF) 4-IPP effectively blocked MIF activity as well as inhibited NLRP3 expression and IL-1β production, thereby reducing neutrophil infiltration in lung tissue and correcting myeloid-skewed hematopoiesis. Knocking out the myeloid-specific pro-survival gene Morrbid enhanced apoptosis of myeloid cells in CGD, leading to an effective reduction in the progression of systemic inflammation.

In summary, we established an environmentally-dependent model of CGD infection and revealed Morrbid as a key driver of CGD hyperinflammation.

7. AAV-mediated GALC gene delivery iPSCs-derived brain organoids from a patient with Krabbe disease

Ya-feng Lv^{1 *}, Hongbo Li¹, Yang Li¹, Yuan-lang Hu¹, Chun-yu Cao¹, Xiao-fei Huang¹, Wei Wang^{2 *}

¹Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, College of Basic Medical Sciences, China Three Gorges University, Yichang 443000, Hubei, China ²China-Japan Friendship Hospital, Beijing 100029, China

*Corresponding authors: lvyafeng@ctgu.edu.cn (Ya-feng Lv) and stevanwei@163.com (Wei Wang)

Krabbe disease is a rare lysosomal storage disorder caused by mutations in the GALC gene. Loss of GALC results in accumulation of the toxic substance psychosine which contributes to neurological deterioration. Currently, hematopoietic stem cell transplantation is the only available treatment, but fails to address the disease’s underlying genetic cause. Gene therapy, particularly through delivery of recombinant adeno-associated virus (rAAV), has emerged as a promising alternative owing to its low immunogenicity and capacity for sustained gene expression. We used induced pluripotent stem cells (iPSCs) from a Krabbe disease patient to generate brain organoids that closely replicate the pathology and physiology of the human brain to establish an innovative model for investigating the disease. We investigated the transduction efficiencies of 11 adeno-associated virus (AAV) serotypes: AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV-PHP.eB, AAV-Anc80, and AAV-DJ within brain organoids. Our findings revealed AAV5 as the most efficacious vector for delivering the GALC gene. We then further characterized organoids transduced with AAV5 containing the GALC gene and found a marked enhancement of GALC enzyme activity and a significant reduction in psychosine levels. This study presents a novel model for the study of Krabbe disease and establishes the potential of AAV5 as an effective vector for targeted gene therapy.

8. AIdit301 is an ex vivo CRISPR therapy to effectively treat X-linked Severe Combined Immunodeficiency

Margaret Deng¹*, Heng Zhang¹*, Ke Ni¹*, Lihong Ding¹*, Lina Wei¹, Liyan Wen¹, Keyi Yao², Sujing Zong¹, Xixi Wang¹, Xiangyun Ma¹, Shikang Shi¹, Zhike Lu^{1 *}, Xiaodong Zhao^{3 *}, Yunfei An^{3 *}, Lijia Ma^{1,2,4 *}

1. Westlake Genetech, Hangzhou, China, 310024 2. School of Life Sciences, Westlake University, Hangzhou China, 310024 3. Department of Rheumatology & Immunology, Children’s Hospital of Chongqing Medical University; National Clinical Research Center for Child Health and Disorders, Chongqing, China, 400014 4. Westlake Laboratory, Hangzhou, China, 310024

*Corresponding authors: Zhike Lu, luzhike@westlakegenetech.com; Xiaodong Zhao: zhaoxd530@aliyun.com; Yunfei An: anyf82@aliyun.com; Lijia Ma: malijia@westlake.edu.cn

X-linked Severe Combined Immunodeficiency (X-SCID) is a life-threatening Primary Immune Deficiency (PID) caused by IL2RG mutations. The standard treatment allogeneic hematopoietic cell transplantation (allo-HCT) poses significant risks due to potential immune complications after transplantation and is limited by the scarcity of bone marrow donors who match the patient. We developed a novel ex vivo CRISPR therapy, AIdit301, which has the potential to cure >97% of PID patients caused by IL2RG mutations through homology-directed repair (HDR). AIdit301 employs AAV6 to deliver precise genome editing by Cas9/gRNA and a site-specific IL2RG cDNA template insertion. Preclinical studies demonstrate a 33.4±2.1% successful template insertion at the endogenous IL2RG genomic loci in human HSC cells, with cell viability of 84.9±4.3% (n=6). The edited HSCs express IL2RG robustly and maintain normal polyclonal differentiation capability. After infusion into a mouse model of X-SCID, the edited HSCs successfully engrafted and differentiated at the four-month checkpoint post-transplantation, with similar IL2RG expression and immune cell populations compared to controls. Tumorigenicity and off-target effects were thoroughly evaluated following industry standards. AIdit301 has been registered for an Investigator-Initiated Trial and is initiating patient recruitment in 2024. This HDR-based gene correction establishes proof-of-concept for developing ex vivo CRISPR therapies to treat both genetic diseases with identified causes as well as those that do not have definitively causative mutations.

9. AL01211 as a novel glucosylceramide synthase (GCS) inhibitor for treatment of Fabry disease

Jianxing Xiang^{1 *}, Michael Babcock¹, Pedro Huertas¹, Zhangqi Cao¹, Li Li¹, Jianhong Zheng¹, Jerry Shen¹

1. AceLink Therapeutics

*Corresponding author: Kevin.Xiang@acelinktherapeutics.com

Fabry disease is characterized by lysosomal accumulation of globotriaosylceramide (GL3) and globotriaosylsphingosine (lyso-GL3) resulting in a wide range of symptoms including pain, gastrointestinal issues, kidney failure, heart disease, and stroke. The current standard of care is enzyme replacement therapy (ERT) to reduce GL3 and lyso-GL3 levels, which improves many symptoms but ultimately fails to prevent patients from developing progressive renal and cardiac disease. A glucosylceramide synthase inhibitor (GCSi) can reduce glycosphingolipid (GSL) production, including GL3 and lyso-GL3, which has the potential to become a new treatment paradigm for Fabry disease as substrate reduction therapy (SRT). AL01211 is a novel orally-delivered non-brain penetrant GCSi being developed for the treatment of Fabry disease. Phase I clinical trials in healthy human subjects consisting of single ascending doses and 14-day multiple ascending doses demonstrated that AL01211 was overall safe, well-tolerated, exhibited dose-dependent pharmacokinetic and pharmacodynamic properties. Currently, AL01211 is in a Phase II clinical study to evaluate the safety, pharmacological activity, and initial treatment benefit of AL01211 in male participants with classic Fabry disease. The increased potency and low brain penetration make AL01211 a potentially safer and more efficacious therapy for Fabry disease patients, especially in younger patients seeking a convenient and life-long treatment option.

10. Analysis of Wilson’s family with SAPHO syndrome associated with vas deferens loss and repeated embryo implantation failure

Lizhen Lin¹, Yujia Guo², Ling Wang³, Xiaoting Huang⁴, Qicai Liu^{2,5,6 *}

1. Laboratory Department of Fuzhou Second General Hospital 2. First Affiliated Hospital of Fujian Medical University, reproductive medicine center 3. First Affiliated Hospital of Fujian Medical University, department of Pathology 4. College Student Innovation Team of Fujian Medical University 5. Key Laboratory of Clinical Laboratory Technology for Precision Medicine (Fujian Medical University), Fujian Province University 6. Vanke School of Public Health, National Graduate College for Engineers, Tsinghua University

*Corresponding author: lqc673673673@163.com

Wilson’s disease is an autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene with genetic and clinical heterogeneity. Reproductive system phenotypes have rarely been reported, but here we present a proband who showed loss of vas deferens, left renal vein nutcracker syndrome, decreased serum ceruloplasmin, SAPHO syndrome, and chronic recurrent multiple osteomyelitis and dermatitis. Full exon and Sanger sequencing found heterozygous mutations in the ATP7B gene, c.3316G>A (p.Val1106lle) and c.588c>a (p.Asp196Glu). Through pedigree investigation, we found a similar reproductive phenotype of the proband’s cousin (his aunt’s son), but with no presentation of SAPHO syndrome. Spermatography was performed for in vitro fertilization and embryo transfer (IVF-ET)-assisted pregnancy, and the proband successfully conceived a child. However, the proband also performed IVF-ET-assisted pregnancy by semen aspiration, but the embryo test showed that the embryo implantation potential was low (the chromosome chimerism rate was higher than 30%), and the implantation failed after four frozen embryo transfers. Therefore, we speculate that there is clinical heterogeneity in presentation of phenotypes caused by mutations in the ATP7B gene, and that these can include absence of the vas deferens and SAPHO syndrome, which may lead to low embryo implantation potential.

11. Association analysis of blood lipids, clinical characteristics, and cytokines in patients with systemic sclerosis

Hao Cheng¹, Hui-Dan Yang¹, Meng-Hua Xu¹, Xiao-Ying Zhang¹, Ting-Ting Zhang¹, Hong-Yan Wen^{1 *}

1. Research Center of Multi-omics Technology and Autoimmune Disease, Shanxi Medical University, Taiyuan, Shanxi, China; Medical Center of Rare Diseases, The Second Hospital of Shanxi Medical University, Taiyuan, Shanxi, China; Department of Rheumatology, The Second Hospital of Shanxi Medical University, Taiyuan, Shanxi, China.

*Corresponding author: wenhongyan0509@aliyun.com or wenhongyan@sxmu.edu.cn

Microvascular and endothelial cell lesions may be the early triggers of systemic sclerosis (SSc). This can lead to dyslipidemia, which activates inflammation and acts on vascular endothelial cells, leading to many vascular-related diseases. Therefore, we compared lipid levels in SSc patients with healthy controls (HCs) and analyzed their association with clinical features and inflammatory cytokines.

We found that SSc patients had higher triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels than HCs, but lower total cholesterol (TC) levels (P <0.05). SSc patients were divided into elevated TG (dyslipidemia) and normal TG (normal blood lipid) groups. Erythrocyte sedimentation rate (ESR), mean risk severity scores (mRSS), and risk of cardiac and renal involvement were higher in the dyslipidemia group (P <0.05) and TG was positively correlated with mRSS. SSc patients were then divided into decreased and normal high-density lipoprotein cholesterol (HDL-C) groups. ESR was increased, cardiac involvement was higher, and the anti-Scl-70 positive rate was lower in the HDL-C decreased group compared to the normal group (P <0.05). HDL-C was negatively correlated with the ESR. We then compared inflammatory cytokines in these comparison groups. IL-2, IL-6, IL-17, IFN-γ and TNF-α were higher in the dyslipidemia group than in normal blood lipid group (P <0.05). TG was positively associated with IL-2, IL-6, IL-10, IL-17, and IFN-γ and HDL-C was negatively associated with IL-2, IL-6, IL-10, and TNF-α. Taken together, these findings suggest that dyslipidemia plays an important role in triggering the early pathogenesis of systemic sclerosis.

Key Words: SSc, dyslipidemia, endothelial cell, trigger, cytokines

12. Blood-brain barrier-penetrating recombinant hARSA enzyme for treatment of metachromatic leukodystrophy

Hongya Pan, Haiyang Li, Lingna Zeng, Changjian Wang, Ximei Gu, Shiqun Liao, Xin Ma, Zhaozhong Han*

Linno Pharmaceauticals, Inc., 720 Cailun Road, Bldg#2 Suite 503, Zhangjiang Hi-Tech Park, Pudong New District, Shanghai, China

*Corresponding author: jack.han@linnopharma.com

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease characterized by damage of the myelin sheath of nerve fibers in the central and peripheral nervous systems. This occurs due to deficiency of lysosomal enzyme arylsulfatase A (ARSA) or sphingolipid activator protein B (SapB) and manifests as progressive motor and cognitive deficiency. Enzyme replacement therapy (ERT) is an effective, safe and affordable treatment option. However, delivering a recombinant enzyme to brain tissue is a challenge because of the need to cross the blood-brain barrier (BBB). To overcome this, we produced a BBB-penetrating recombinant human ARSA enzyme in CHO cells as a fusion protein. The construct contained a humanized single domain antibody fragment VHH which specifically binds to human transferrin and utilizes transferrin receptor on cerebral endothelial cells for crossing the BBB. The VHH fusion did not interfere with enzymatic activity of hARSA and enabled it to cross the BBB in a dose-dependent manner in mice and non-human primates. Intravenous dosing (450 nmol/kg) resulted in 10–15-fold higher levels of ARSA activity within brain tissue in mice at 4 hours post-administration. PET-CT imaging with I-124 labeled recombinant protein also indicated that intravenous dosing resulted in accumulation of the recombinant enzyme within brain tissue. In Arsa knock-out mice, intravenous dosing of the fusion protein for one month led to dose-dependent substrate reduction across the brain tissue as well as significant improvement in motor functions. This provides proof-of-concept for effectively treating MLD with a BBB-penetrating recombinant hARSA ERT.

13. Clinical characteristics and induced pluripotent stem cells disease model of Fabry Disease caused by a novel GLA mutation

Langping Gao^1,2,#, Zhihong Lu^1,#, Ying Zhang^2,3, Lexin Liu^1,2, Jingmiao Sun^1,2, Haidong Fu¹, Jianhua Mao^{1, *}, Lidan Hu^{1, *}

1. Department of Nephrology, Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, 310003 2. Zhejiang University School of Medicine, Hangzhou, China, 310058 3. Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China, 310058

*Corresponding authors: hulidan@zju.edu.cn and maojh88@zju.edu.cn

Fabry disease (FD) is a rare X-linked inherited disorder caused by mutations in the GLA gene. We established a cohort of FD patients and performed whole-exome sequencing (WES) to identify novel mutations. We investigated the etiology of the novel mutation (c.72G > A, p.Trp24*) using induced pluripotent stem cells (iPSCs) derived from a patient. We performed bioinformatics and molecular analyses to examine the impact of this mutation. We collected the peripheral blood mononuclear cells (PBMCs) from the affected patient and reprogrammed them into iPSCs. We assessed enzymatic activity to assess the function of lysosomal enzyme α-galactosidase A (α-Gal A). The patient demonstrated a classical FD presentation, including neuropathic pain, gastrointestinal disorders, and deficiency of α-Gal A activity, which were caused by a truncation of the protein after only 24 amino acids. The α-Gal A activity of the patient-specific iPSC (iPS-FD) was significantly lower (60%) than that of iPSCs derived from healthy donors (iPS-B1). This work establishes the etiology of a novel mutation in an affected patient and the utility of iPSCs as a valuable tool for investigating the molecular mechanisms of FD.

14. Clinical characteristics of 21-hydroxylase deficiency in 22 cases

Han Wang¹, Yuan Chen^{2 *}

1. Graduate School of Xinjiang Medical University, Urumqi 830001, Xinjiang Uygur Autonomous Region, China 2. Department of Endocrinology, People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi 830001, Xinjiang Uygur Autonomous Region, China

*Corresponding author: 1609904469@qq.com

We investigated the clinical characteristics, diagnosis, and treatment experience of 21-hydroxylase deficiency with the goal of enhancing clinical understanding, diagnosis, and treatment of this disease. A total of 22 patients (seven males and 15 females; average age: 13.0 (4.8, 23.0) years) with 21-hydroxylase deficiency who received treatment in the People’s Hospital of Xinjiang Uygur Autonomous Region from 2014 to 2022 were included in this study. We evaluated the clinical data and treatment outcomes of this cohort. Female patients exhibited clitoral enlargement, pigmentation, infrequent menstruation, amenorrhea, hirsutism, acne, accelerated growth, infertility, and vomiting. Male patients exhibited precocious puberty, pigmentation, infertility, nausea, and vomiting. Children with salt deficiency exhibited electrolyte disorders and acid-base imbalance changes, including hyponatremia, hyperkalemia, hypotension, and metabolic acidosis. All patients were confirmed to have 21-hydroxylase deficiency and were treated with glucocorticoid/mineralocorticoid replacement therapy. Five female patients also underwent external genital correction surgery. Among the 10 patients who were followed up with, five female patients experienced regular or gradual resumption of menstruation, but the improvement in sex hormone levels was not significant. We advise that newborns with gastrointestinal symptoms accompanied by electrolyte disorders, women with hyperandrogenism, male children with precocious puberty, and adult men with infertility should be screened for 21-hydroxylase deficiency. Upon diagnosis, hormone replacement therapy should be conducted to improve prognosis. Genetic testing should be performed as much as possible and regular follow-ups should be conducted to ensure normal adolescent development and good fertility.

15. Commercial development of gene therapy product for X-SCID in China

Qiling Xu¹,Yunfei An¹*

1. Children’s Hospital of Chongqing Medical University

X-linked severe combined immunodeficiency disease (X-SCID) is a rare inherited disease characterized by profound defects in T and B cell function. Although allogeneic hematopoietic stem-cell transplantation is curative, it is not available to all patients. Gene therapy provides an alternative. However, optimized vectors and improved procedures for gene therapy are still required in China.

We performed a preclinical study using a mouse model to examine the efficacy and safety of a self-inactivating lentiviral vector gene therapy for X-SCID. For clinical application, we performed large scale transduction using Good Manufacturing Practice standards for virus production. We observed corrected CD132 expression in CD34+ cells derived from the bone marrow of a patient with X-SCID. An investigator-initiated trial of this gene therapy for X-SCID has been completed and patients will soon be enrolled in a clinical trial at our institution. This has the potential to expand the available therapeutic options for patients with X-SCID in China.

16. Computing cell state discriminates the aberrant hematopoiesis and activated microenvironment in Myelodysplastic Syndrome through a single cell genomic study

Wenyan Jin^1,2,3,#, Xinyu Guo^4,#, Yuchen Wen^1,2,3, Zhiqin Wang^1,2,3, Xiaotong Ren⁴, Zhaoyun Liu⁴, Rong Fu⁴, Zhigang Cai^{1,2,3,4,5, *}, Lijuan Li^{4, *}

¹The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics; Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University, Tianjin, China ²National Key Laboratory of Experimental Hematology, Tianjin, China ³Tianjin Key Laboratory of Inflammatory Biology, Tianjin, China ⁴Department of Hematology, Tianjin Medical University Tianjin General Hospital, Tianjin, China ⁵Department of Rheumatology and Immunology, Tianjin Medical University Tianjin General Hospital, Tianjin, China ^#Equal contributors

*Corresponding authors: Zhigang Cai, Professor, Ph.D., us36zcai@tmu.edu.cn and Lijuan Li, Ph.D., MD., lli@tmu.edu.cn

Myelodysplastic syndromes (MDS) are a highly heterogeneous group of blood disorders which are characterized by ineffective hematopoiesis. Approximately 30% of MDS patients will proceed to acute myeloid leukemia (AML). The risk of MDS increases with age as somatic mutations accumulate in hematopoietic stem cells and progenitors (HSPC). Epidemiological data indicate that MDS incidence is increasing in resource-rich regions. We report preliminary results from an ongoing large cohort MDS study. Standard single cell transcriptome analysis of unsorted bone marrow (BM) cells identified deficient hematopoiesis in all five MDS patients examined. Three individuals were categorized as high-risk and two were categorized as low-risk. No clear increase in mutation burden was detected in the MDS patients, but the high-risk MDS group showed T cell activation and aberrant myelogenesis at the developmental stages between HSPCs and granulocyte/monocyte progenitor cells. Transcriptional factor analysis on the aberrant myelogenesis suggests that the epigenetic regulator chromatin structural protein-encoding gene HMGA1 is highly overactivated in the high-risk MDS group and moderately activated in the low-risk MDS group. Perturbation of HMGA1 by CellOracle simulated deficient hematopoiesis in mouse Lin-negative BM cells. Comparing MDS and AML cells to a common reference revealed a connection between myeloid leukemia development and abnormalities in hematopoietic hierarchy. We suggest that it is technically feasible to compare diseased bone marrow cells to a common reference to identify immune cell development abnormalities even for cohort sizes up to 1,000 patients.

17. Cooperative progression of colitis and leukemia modulated by clonal hematopoiesis via PTX3/IL-1β pro-inflammatory signaling

Yuchen Wen^1,2,#, Hang He^1,2,#, Hanzhi Yu^1,2, Jingjing Liu^1,2, Qingran Huo^1,2, Wenyan Jin^1,2, Zhiqin Wang^1,2, Guohui Du³, Jun Du⁴, Huaquan Wang⁵, Zhigang Zhao⁶, Zhigang Cai^{1,2,4,5, *}

1 The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics; Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University, Tianjin, China 2 National Key Laboratory of Blood Science, Tianjin, China 3 Department of Research and Development, Beijing SeekGene BioSciences Co. Ltd, Beijing, China 4 Department of Rheumatology and Immunology, Tianjin Medical University Tianjin General Hospital, Tianjin, China 5 Department of Hematology, Tianjin Medical University Tianjin General Hospital, Tianjin, China 6 Department of Medical Oncology, Tianjin First Central Hospital, School of Medicine, Nankai University, Tianjin, China

*Corresponding author: Prof. Zhigang Cai: us36zcai@tmu.edu.cn

Clonal hematopoiesis (CH) is a pre-pathological condition where an acquired somatic mutation in a gene is detected with a variant allele frequency (VAF) of at least 2% and is commonly associated with multiple chronic diseases. Mutations in TET2 are prevalent genetic drivers of CH. However, cooperative factors and mechanisms underlying TET2-deficiency related CH (TedCH) remain largely unknown. Recently, it has been suggested that diseases can occur before TedCH manifests and promote its development. This is an instance where diseases in non-hematopoietic organs may act as environmental non-genetic drivers of CH. To clarify the relationships between diseases involving immune dysfunction and CH, we tested the impact of various challenges on TedCH. We found that an inflammatory environment expedited TedCH development. Primary or chimeric Tet2-mutant mice spontaneously developed co-symptoms reminiscent of human chronic colitis and myeloid leukemia, which was exacerbated by inducing ulcerative colitis (UC) by feeding mice dextran sodium sulfate (DSS). Single cell RNA-seq (scRNA-seq) analysis revealed damage to the colon tissue in the Tet2-mutant mice in both normal physiological conditions and in the UC DSS-fed condition, causing dysbacteriosis of the gut microbiome. Our colon scRNA-seq analyses of humans and mice identified PTX3/IL-1β pro-inflammatory signaling in promoting colitis or leukemia. Finally, treatment with the IL-1R1 inhibitor Anakinra ameliorated TedCH trajectory and inflammation in both the colon and bone marrow. Our study suggests that PTX3/IL-1β signaling and clonal hematopoiesis cooperate to play important roles in a gut-bone marrow axis, and disruption of this relationship can cause colitis and leukemia.

18. Desloratadine alleviates ALS-like pathology in hSOD1^G93A mice via targeting 5HTR_2A on activated spinal astrocytes

Jian Lu, Lin Ma, Jiaying Wang ^{*, Xu Shen *}

Jiangsu Key Laboratory of Drug Target and Drug for Degenerative Diseases, School of Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China

*Corresponding authors: wangjy@njucm.edu.cn (J. Wang) and xshen@njucm.edu.cn (X. Shen)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with progressive loss of motor neurons in the spinal cord, cerebral cortex and brain stem. Previous studies have shown that autophagy and astrocyte-mediated neuroinflammation are involved in the pathogenesis of ALS, that the receptor 5HTR_2A participates in the early stage of astrocyte activation, and that 5HTR_2A antagonism may suppress astrocyte activation. In this study we evaluated the therapeutic effects of desloratadine (DLT), a selective 5HTR_2A antagonist, in human SOD1^G93A (hSOD1^G93A ) ALS model mice and elucidated the underlying mechanisms. ALS onset and progression were determined using rotarod and righting reflex tests\. We found that astrocyte activation accompanied serotonin receptor 2A (5HTR_2A) upregulation in the spinal cord, which were tightly associated with ALS-like pathology and effectively attenuated by DLT administration. We show that DLT administration significantly delayed ALS symptom onset time, prolonged lifespan, and ameliorated movement disorders, gastrocnemius injury, and spinal motor neuronal loss in hSOD1^G93A mice. Spinal cord-specific knockdown of 5HTR_2A by intrathecal injection of adeno-associated virus 9 (AAV9)-si-5Htr2a also ameliorated ALS pathology in hSOD1^G93A mice and occluded the therapeutic effects of DLT administration. Furthermore, we demonstrate that DLT administration promoted autophagy to reduce mutant hSOD1 levels through a 5HTR_2A/cAMP/AMPK pathway, suppressed oxidative stress through a 5HTR_2A/cAMP/AMPK/Nrf2-HO-1/NQO-1 pathway, and inhibited astrocyte neuroinflammation through a 5HTR_2A/cAMP/AMPK/NF-κB/NLRP3 pathway in the spinal cord of hSOD1^G93A mice. In summary, we found that 5HTR_2A antagonism by treatment with shows promise as a therapeutic strategy for ALS.

19. Diffusion-relaxation correlation spectroscopic imaging for evaluating change of white matter of X-linked adrenoleukodystrophy

Xingang Wang^1,#, Ru Wen^1,#, Shaoxin Xiang^{2, *} ,Wang Zhe³, Yuxin Yang², Liang Tan⁴, Chen Liu^{1, *}

1. Department of Radiology, Southwest Hospital, Army Medical University (Third Military Medical University), Chongqing 400038, China 2. United Imaging Research Institute of Intelligent Imaging, Beijing, China 3. United Imaging Healthcare Group, Shanghai, China 4. Neurosurgery, Southwest Hospital, Army Medical University (Third Military Medical University), Chongqing 400038, China ^#These authors have contributed equally to this work.

*Corresponding author: Chen Liu (liuchen@aifmri.com)

X-linked adrenoleukodystrophy (ALD) is a rare genetic disorder caused by mutations in the ABCD1 gene, leading to very long chain fatty acids (VLCFAs) accumulation and subsequent damage to various tissues. Diffusion-relaxation correlation spectroscopic imaging (DR-CSI) offers a comprehensive approach to assess tissue microenvironment by concurrently evaluating diffusion and T2 relaxation effects. This study aimed to investigate white matter changes in ALD using DR-CSI. Eight participants, including health controls (HCs) and different ALD phenotypes, underwent MRI with DR-CSI. Spectra were analyzed to derive compartment volume fractions, which reflect tissue characteristics. We observed distinctive peaks and variations in compartment volume fractions among ALD phenotypes. Statistical analysis revealed that the HC group had the highest f_A compared to the other groups in both the anterior and medial white matter. In the medial white matter, the carrier group achieved the highest f_C and the adrenomyeloneuropathy (AMN) group achieved the highest f_B. In the posterior white matter, the HC group again had the highest f_A and the AMN group achevied highest f_B. These findings illustrate the potential of DR-CSI in detecting white matter changes in ALD, which could provide valuable insights into disease pathophysiology. The ability of DR-CSI to differentiate signal contributions from diverse tissue compartments offers promise for enhancing our understanding of ALD and may inform the development of personalized treatment strategies. Further research utilizing DR-CSI could contribute to improved diagnosis, prognosis, and management of ALD.

20. Discoveries in ALD patients and carriers: familial-specific neurological abnormalities and blood marker changes

Qiuyu Su^1,2, Liang Tan^{2 *}

1.Yongchuan Hospital, Chongqing Medical University, Chongqing 402160, China 2.Chongqing Clinical Research Center for Neurosurgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing 400038, China

*Corresponding author: tracy200712@hotmail.com

Adrenoleukodystrophy (ALD) is a metabolic disorder caused by mutations in the ABCD1 gene, resulting in demyelination of the central nervous system and adrenal dysfunction. We aimed to explore the relationship between evoked potential (EP) abnormalities and behavioral impairments in ALD patients and carriers as well as to investigate related risk factors for the disorder.

Comprehensive assessments were conducted on ALD patients and their family members to collect clinical data. We used auditory, somatosensory potentials, and visual (EPs) as neurophysiological assessments. We assessed the functionality and extent of neural pathway damage by analyzing EP waveform, latency, and amplitude. Additionally, specific markers in the blood, such as S100β protein and serum very long-chain fatty acids, were measured to explore their association with EP abnormalities. Our results reveal EP abnormalities are present in the families of ALD patients and carriers exhibited more severe clinical symptoms. Consistency in the location of EP abnormalities was observed among families. Furthermore, ALD patients with EP abnormalities showed significant increases in specific blood markers, which are candidate biomarkers that could be associated with EP abnormalities.

These findings highlight the characteristics of EP abnormalities in ALD patients and carriers, which need to be further investigated to understand their connection with behavioral impairments. We identified specific blood markers which could serve as diagnostic indicators for predicting and screening ALD. Further research is necessary to understand the pathological mechanisms related to the neurological and metabolic aspects of ALD.

21. Disruption of TSGA10 causes male infertility due to acephalic spermatozoa syndrome in humans and mice

Feng Wan^1,2,3 Cuilian Zhang^{1,2,3 *} Haibin Guo^{1,2,3 *}

1. The Reproductive Medicine Center, Henan Provincial People’s Hospital, 7 Weiwu Rd, Zhengzhou City 450003, Henan, China 2. The Reproductive Medicine Center, People’s Hospital of Zhengzhou University, 7 Weiwu Rd, Zhengzhou City 450003, Henan, China 3. The Reproductive Medicine Center, Henan Provincial People’s Hospital of Henan University, 7 Weiwu Rd, Zhengzhou City 450003, Henan, China

*Corresponding authors: luckyzcl@qq.com and gordon820@zzu.edu.cn.

Acephalic spermatozoa syndrome (ASS) is a rare but severe type of teratozoospermia associated with male infertility associated with loss of TSGA10. However, the pathogenic mechanisms of ASS caused by TSGA10 deficiency remain unclear. We performed whole-exome sequencing of infertile patients with ASS and generated Tsga10^−/− mice using CRISPR/Cas9 technology. We examined TSGA10 protein in the sperm from affected individuals and mice as well as observed the sperm ultrastructure with transmission electron microscopy. Intracytoplasmic sperm injection (ICSI) was carried out using sperm from affected individuals and Tsga10^−/− male mice. Two novel mutations in TSGA10 were identified in two infertile patients with ASS. Tsga10^−/− male mice are infertile and displayed acephalic spermatozoa and oligospermia, replicating the sperm phenotype of the patients. TSGA10 was predominantly located at the head–tail junction of sperm in the control samples but was not detected in the sperm from affected individuals and Tsga10^−/− male mice. The head–tail coupling apparatus (HTCA) and mitochondrial sheaths were disorganized or missing in spermatozoa from affected individuals and Tsga10^−/− male mice, which suggesting that TSGA10 is essential for the stability and integrity of the HTCA and the mitochondrial sheath of spermatozoa. Furthermore, mature spermatozoa were significantly decreased at stages VII and VIII in the testes of Tsga10^−/− male mice. ICSI failed to result in live births using sperm from affected individuals and Tsga10^−/− male mice. These findings indicate that TSAG10 deficiency is responsible for male infertility with ASS and provide insights into the pathogenesis of ASS caused by TSAG10 deficiency.

22. Efficacy and prognostic assessment of chemotherapy-bridged transplantation in pediatric patients with advanced myelodysplastic syndromes

Xingchen Wang, Chenmeng Liu, Yunlong Chen, Yang Wan, Wenbin An, Xiaolan Li, Lipeng Liu, Fang Liu, Li Zhang, Yao Zou, Xiaojuan Chen, Yumei Chen, Ye Guo, Xiaofan Zhu, Wenyu Yang*

1. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China. 2. Tianjin Institute of Health Science, Tianjin 301600, China.

*Corresponding author: yangwenyu@ihcams.ac.cn

We aimed to evaluate the efficacy of different chemotherapy regimens prior to hematopoietic stem cell transplantation (HSCT) and assess the prognosis of pediatric patients with advanced myelodysplastic syndromes (MDS). We conducted a retrospective analysis of 30 patients who were treated from Dec 2007–Apr 2022. All patients underwent demethylating treatment or chemotherapy, with regimens including CAG (cytarabine + aclarubicin + granulocyte colony-stimulating factor (G-CSF))/HAG (homoharringtonine + Ara-C + G-CSF) (n=7), decitabine monotherapy (n=9), a combination of decitabine and chemotherapy (n=10), or AML-like chemotherapy/other regimens (n=4). Chromosomal analysis at diagnosis was possible in 32 (96.7%) patients, with 69% showing cytogenetic abnormalities, mainly monosomy 7 (37.9%). We were able to follow-up with 25 of the 30 patients receiving chemotherapy. The overall response (OR) rate to treatment was 60%. Complete remission (CR) and CR with incomplete blood count recovery (CRi) were achieved in 16% and 44% of patients, respectively. Other outcomes included partial remission (16%), disease progression (8%), and no remission (32%). The response rates across different treatments were not significantly different (p=0.18). With a median follow-up of 27.5 months (range 1–172), 23 patients underwent HSCT and showed significantly better 3-year overall survival and event-free survival than those who did not (83% vs 14% and 78% vs 14%, respectively, p<0.001 for both). Multivariate analysis indicated that HSCT and achieving an OR to chemotherapy impact the prognosis of pediatric patients. Thus, chemotherapy bridging to HSCT and achieving remission before transplantation can significantly improve the prognosis of children with advanced MDS.

23. Endoplasmic reticulum-targeted nuclear gene delivery system for the treatment of rare diseases

Na-Hui Liu¹, Yi Wang¹, Hu-Lin Jiang^1,2,3*

1. State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, Nanjing, 210009, China. 2. College of Pharmacy, Yanbian University, Yanji, 133002, China. 3. Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, China Pharmaceutical University, Nanjing, 210009, China.

*Corresponding author: jianghulin3@gmail.com or jianghulin3@cpu.edu.cn

Delivering gene therapy with nonviral vectors has notable advantages, such superior biosecurity and ease of large-scale production, and can be applied in the treatment of rare diseases. However, one challenge with this approach is being able to deliver genes to the nucleus. Here, we evaluate a novel nonviral vector which contains an endoplasmic reticulum (ER)-targeting group connected to an arginine-rich (AR) group through a glutathione (GSH)-responsive linker, which was termed ERAG. ERAG-containing complexes can enter target cells via caveolin-mediated endocytosis and achieve lysosomal escape through the action of its AR group. The construct is then aided by ER-targeting motifs, which facilitate the targeted delivery of its genes to the ER, enabling rapid access to the perinuclear region. ERAG then undergoes degradation because of high GSH levels in the perinuclear region, releasing the exogenous gene into the nucleus. Our results demonstrate that the ERAG fusion significantly enhanced the nuclear uptake of delivered genes and increased the efficiency of gene transfection. This presents proof-of-concept for a highly efficient and low-toxicity ER-targeted non-viral gene delivery system as a tool for gene therapy strategies to treat rare diseases.

24. Establishment of brain organoid models derived from patient-specific iPSCs with Perrault syndrome

Ya-feng Lv¹, Wei Wang^{2 *}

¹Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, College of Basic Medical Sciences, China Three Gorges University, Yichang 443000, Hubei, China. ²Department of Neurology, China-Japan Friendship Hospital, Beijing 100029, China

*Corresponding author: stevanwei@163.com (Wei Wang)

Perrault syndrome is a rare autosomal recessive genetic disorder characterized by progressive sensorineural hearing loss, ataxia, and peripheral neuropathy. In female patients, ovarian dysfunction may also occur. The lack of clarity regarding the correlation between specific genotypes and phenotypes significantly hinders the diagnosis and treatment of Perrault syndrome. In our study, we successfully established patient-derived induced pluripotent stem cells (iPSCs) harboring mutations in the TWNK gene by introducing reprogramming factors (Oct3/4, Sox2, Klf4, c-Myc) into skin fibroblast cells obtained from patients using Sendai virus. Subsequently, these iPSCs were induced to differentiate into brain organoids in vitro. Sanger sequencing confirmed that the TWNK gene mutation sites (c.794G > A, c.1181G > A) were retained in the brain organoids. Immunofluorescence staining showed that the patient-specific iPSCs could form brain organoids normally. Compared to iPSCs from healthy individuals, the brain organoids derived from patient iPSCs showed significantly reduced mitochondrial DNA copy numbers and ATP levels. This study establishes the first brain organoid model derived from iPSCs of a Perrault syndrome patient with TWNK gene mutations, providing a model for research into the pathogenesis of the disorder and the development of new therapies.

25. Family analysis and literature study of hereditary hypophosphatemic rickets with hypercalciuria

Lufeng Wang¹, Yanying Guo^{1 *}

1.Department of Endocrinology, People′s Hospital of Xinjiang Uygur Autonomous Region, Xinjiang Clinical Research Center for Diabetes, Urumqi 830000, China.

^{*Corresponding author: guozeyang@126.com}

Hereditary hypophosphatemia rickets with hypercalciuria is a rare autosomal recessive disorder characterized by reduced renal phosphate reabsorption leading to hypophosphataemia, rickets and bone pain. Here, we report an 11-year-old female proband, who was admitted to our hospital with bilateral genu varum deformity and short stature. Computed tomography showed kidney stones and blood tests showed hypophosphatemia. For a clear diagnosis, we employed high-throughput sequencing technology to screen for genetic abnormalities. The raw sequence data were compared to the known gene sequence data in publicly available sequence databases using Burrows-Wheeler Aligner software (BWA, 0.7.12-r1039) and pathogenic variant sites were annotated using Annovar. The proband and her affected sister were found to carry pathogenic homozygous alleles of the gene SLC34A3 (exon 13, c.1402C>T; p.R468W). Their parents were both heterozygous carriers of the variant. Genetic testing revealed that the patient also has a rare homozygous LRP5 (exon 18, c.3917C>T; p.A1306V) variant of uncertain significance. We examined the characteristics of this unusual case involving variants in both the SLC34A3 and LRP5 genes, which are independently inherited in autosomal recessive manners and the combination of which has not been previously reported in the literature. It is uncertain whether the presence of these two mutated genes in the same individual will result in more severe clinical symptoms. This report highlights that an accurate diagnosis is critical for early detection and treatment of genetic disease but can be difficult due to confounding genetic factors.

26. Fenfluramine attenuates seizures and behavioral abnormalities in a rat model of Dravet syndrome

Weixin Qian^1,2, Miao Li¹, Hong Wang^{1 *}

1. Research Center for Primate Neuromodulation and Neuroimaging, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. 2. Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230031, China.

^{*hong.wang@siat.ac.cn}

Dravet syndrome (DS) is a severe developmental epileptic encephalopathy. Patients suffer from recurrent seizures and have a high risk of sudden unexpected death. The most common cause of DS is haploinsufficiency of the SCN1A gene encoding the voltage-gated sodium channel Nav1.1. We have previously established a rat model of DS by disrupting the Scn1a gene. The antiepileptic drug fenfluramine (FFA) has reliably reduced seizure frequency in DS patients in several randomized clinical trials and has been approved by the U.S. Food and Drug Administration for the treatment of DS. However, the molecular mechanism and cell type target underlying the protective effect of FFA is not known. Here, we developed a new paradigm to mimic the progression of DS patients using a novel rat model of DS. We show that a low dose of FFA has a therapeutic effect in reducing the seizure frequency and reversing the behavioral abnormalities in DS rats. In addition, we have identified the key brain regions and cell types that correlate with the therapeutic effect of FFA. These findings shed light on the mechanics of DS and provide an effective preclinical platform for the development of novel therapies.

27. Gene Editing of Activated Phosphoinositide 3-Kinase δ Syndrome

Anle Zeng^1,2, Shuyu Fang^1,2, Yelei Gao^1,2, Lina Zhou^1,2, Xiaodong Zhao^1,2, Yunfei An^1,2

1.Children’s Hospital of Chongqing Medical University, Chongqing Key Laboratory of Child Infection and Immunity, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing, China 2.Division of Rheumatology and Immunology, Children’s Hospital of Chongqing Medical University, Chongqing, China

^{*Corresponding author: anyf82@aliyun.com}

Activated phosphoinositide 3-kinaseδ syndrome (APDS) is a rare autosomal dominant primary immunodeficiency disease (PID). Several causative mutations had been identified, with the most common being c.3061G>A which accounts for approximately 85% of all PIK3CD gene mutations. We generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) of an APDS patient carrying the c.3061G>A mutation and designed constructs for genetic editing of this mutation. We then transfected iPSCs with these constructs and used flow cytometry to isolate single cell clones. We found that iPSCs were successfully reprogrammed in 5–30% of cells. These cells could be successfully differentiated into hematopoietic stem-like cells, which can be implanted in vivo with a chimeric rate of ∼2%. This establishes a theoretical approach for treating patients with APDS which will require further investigation to evaluate its safety, efficacy, and feasibility in preclinical development.

28. Genetic analysis and gonadal dysfunction in adult females with non-classic 21-hydroxylase deficiency in a Chinese cohort

Ping Pan^†, Mei MEI^†, Linlin Jiang, Jia Huang, Dongzi Yang, Ping Yuan*, Yu Li*

Department of Obstetrics and Gynecology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangdong Guangzhou 510120, China ^†Ping PAN and Mei MEI contributed equally to this work and share first authorship.

*Corresponding authors: yuanp5@mail.sysu.edu.cn (YUAN Ping) and liyu7@mail.sysu.edu.cn (LI Yu)

We sought to characterize genetic features and gonadal dysfunction of non-classic congenital adrenal hyperplasia caused by 21-hydroxylase deficiency (21-OHD) in Chinese adult females. The clinical and CYP21A2 genetic data of 62 females with non-classic 21-OHD were retrospectively analyzed in a single-center cohort. Common signs of gonadal dysfunction were: history of prior miscarriage (62.9%, n=39), menstrual disorder (51.6%, n=32), and infertility (27.4%, n=17).Forty-nine patients (79.0%) had genetic variants, of which two patients had unexpected CYP17A1 gene variants. Of the 47 patients with CYP21A2 gene variants, nine had one variant and 38 had at least two variants, among which seven patients showed gene deletion or rearrangement. A total of 21 point mutations were detected, and the most variants were p.Ile173Asn (27.3%), c.293-13 C>G (15.9%), and c.-113 G>A (11.4%). This study presents an appreciable degree of gonadal dysfunction in non-classic 21-OHD females and illustrates the significance of genotyping for definite diagnosis.

29. Genetic imaging study of the glymphatic system and motor dysfunction in Spinocerebellar Ataxia Type 3

Peiling Ou¹, Linfeng Shi¹, Jian Wang¹, Chen Liu^{1 *}

1. Department of Radiology, Southwest Hospital, Army Medical University (Third Military Medical University), Chongqing 400038, China

^{*Corresponding author: Chen Liu, liuchen@aifmri.com}

We quantitatively assessed magnetic resonance imaging (MRI) in patients with Spinocerebellar Ataxia Type 3 (SCA3) to investigate alterations in the glymphatic system and identify associations with genetic information and motor dysfunction scores. This prospective study recruited 41 confirmed SCA3 patients and 41 matched healthy controls (HCs) for 3.0T head MRI scans and clinical assessments. The automatic segmentation and volume measurement of the choroid plexus was performed with Freesurfer 6.0. Analysis of perivascular spaces (PVS) in different brain regions was performed on T2-weighted imaging sequences using the deep learning model VB-Net. Independent sample rank sum tests were used to analyze the differences in the glymphatic system of SCA3. Partial correlation analyses were conducted within the SCA3 group between CAG repeats, the glymphatic system, and motor dysfunction. Compared to HC group, SCA3 patients showed a significant increase in choroid plexus and PVS volumes. In SCA3 patients, CAG repeats were positively correlated with Scale for the Assessment and Rating of Ataxia (SARA) and International Cooperative Ataxia Rating Scale (ICARS) scores as well as basal ganglia PVS volumes. Cerebellar and temporal lobe PVS volumes were positively correlated with SARA and ICARS scores. Choroid plexus volume was positively correlated with cerebellar and basal ganglia PVS volumes. We observed significant changes in the glymphatic system of SCA3 patients which were closely associated with genetic information and motor dysfunction. This discovery offers a new perspective for studying the pathophysiological mechanisms of SCA3 and suggests that targeting the glymphatic system may be a potential therapeutic strategy.

30. Inhibition of PTTG1 suppresses proliferation and promotes differentiation of neuroblastoma cells by inducing autophagy

Lihua Yuan

Department of Pediatric Surgery, University of Hong Kong-Shenzhen Hospital, Shenzhen, China

Corresponding author: yuanlh@hku-szh.org

PTTG1 is an oncogene that is highly expressed in various cancers and is involved in regulating the cell cycle in neuroblastoma (NB) cells. However, the specific role of PTTG1 in NB has not been extensively studied. We used cell proliferation, wound healing, and Transwell assays to assess proliferation, migration, and invasion abilities of NB cells. We used western blotting, PCR, and immunofluorescence assays to detect the expression of migration-related proteins, differentiation-related proteins, and autophagy-related proteins in NB cells. We modulated autophagy levels using a range of doses of the autophagy inhibitor 3-methyladenine (3-MA). PTTG1 is highly expressed in three NB cell lines, with the highest levels being in SK-N-SH cells. Disrupting PTTG1 significantly inhibited the activity of SK-N-SH cells, reducing their proliferation, migration, and invasion abilities, and was accompanied by a decrease in MMP2 and MMP9 protein expressions. In addition, we found that this increased levels of the differentiation marker TUBB3 and the autophagy marker LC3II. We found upregulated protein and mRNA levels of GAP43, TH, MEG, TUBB3, LC3II/LC3I, and BECLIN1, while downregulating the expression levels of P62 and mTOR. Treating cells with 3-MA partially reversed the effects of disrupting PTTG1. Thus, we suggest that PTTG1 is highly expressed in various NB cells and targeting it can have therapeutic effects on cellular functions by inducing autophagy.

31. Innovative non-viral delivery of CRISPR/Cas9 gene editing for treatment of neurogenetic disorder of Angelman syndrome

Xiaona Lu¹, Ying Xie², Youmei Bao², Gretchen Long², Wendy Sheu², Jiangbing Zhou^{2,5 *}, Yong-Hui Jiang^{1,3,4 *}

¹Departments of Genetics, ² Neurosurgery, ³Neurosciece, ⁴Pediatrics,and ⁵Biomedical Engineering Yale University School of Medicine

*Corresponding authors: jiangbing.zhou@yale.edu and yong-hui.jiang@yale.edu

CRISPR/Cas9 gene editing faces numerous challenges to treat neurogenetic disorders. Adeno-associated viruses (AAVs) overcome some of these barriers, but their immunogenicity and high risk of off-target effects due to the long-term presence of Cas9 protein limits their translational value. We developed an innovative non-viral delivery tool using chemically modified ribonucleoproteins (RNPs) conjugated with Cas9 protein and sgRNA (cRNP-Cas9/sgRNA, cRNPcg). cRNPcg achieved effective delivery of CRISPR to neuronal cells and transient expression of Cas9 protein. We tested cRNPcg using in vitro and in vivo models of Angelman syndrome (AS), a neurodevelopmental disorder caused by the deficiency of neuron and maternal-specific UBE3A gene expression. UBE3A is repressed in the paternal chromosome by non-coding UBE3A antisense (UBE3A-ATS) transcripts. Inactivation of UBE3A-ATS with antisense oligonucleotides (ASOs) is being evaluated in Phase 1/2 clinical trials. The transient nature of ASOs requires monthly intrathecal delivery, but cRNPcg could selectively inactivate Ube3a-ATS expression to achieve permanent therapeutic effects. We observed high gene editing efficiency (>75%) with widespread brain penetration, resulting in significant reduction of Ube3a-ATS and up to 30% reactivation of Ube3a across the cortex, hippocampus, and cerebellum. This led to significant improvement in multiple behavioral assessments and extended the latency for chemically-induced myoclonic and tonic seizures in an adult AS Ube3a^m-/p+ model. Importantly, we did not observe acute toxicity associated with cRNPcg. cRNPcg effectively reactivated UBE3A expression in the paternal chromosome of AS patient-derived induced neuronal progenitor cells. Our results demonstrate that cRNPcg can deliver CRISPR/Cas9 to the brain to treat neurogenetic disorders.

32. Integrative profiling untreated primary membranous nephropathy at the single cell transcriptome level using blood, kidney, and urine samples

Yuchen Wen^1,2,#, Qiuhua Gu^3,#, Xi Cheng³, Yan Qi³, Xin Cao⁴, Xiqian Gao⁴, Xiaoming Mao³, Wenya Shang³, Li Wei³, Junya Jia³, Tiekun Yan^{3, *}, Zhigang Cai^{1,2, *}

1 National Laboratory of Blood Science, Tianjin, China 2 The Province and Ministry Co-Sponsored Collaborative Innovation Center for Medical Epigenetics, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University, Tianjin, China 3 Department of Nephrology, Tianjin Medical University General Hospital, Tianjin, China 4 Department of Nephrology, Tianjin Medical University General Hospital-Airport Hospital, Tianjin, China

*Corresponding authors: Dr. Tiekun Yan, tjtiekun@163.com and Prof. Zhigang Cai, us36zcai@tmu.edu.cn

Membranous nephropathy (MN) is a glomerular disease and one of the main causes of adult nephrotic syndrome, which has an incidence of approximately 10–12 people per million in North America and 2–17 people per million in Europe. The disease is characterized by proteinuria, decreased serum albumin, systemic edema, immune complex deposition, and podocyte exfoliation. Approximately 80% of patients have unknown causes and are classified as primary membranous nephropathy (PMN). Although phospholipase A2 receptor (PLA2R) has been identified as a characteristic antigen, further research on the pathogenesis and treatment of this disease is still needed. Using single-cell transcriptome sequencing, we explored the characteristics of peripheral blood, kidney tissue, and urine in patients with PMN. In peripheral blood mononuclear cells (PBMCs), the portions of B cells and plasma cells were increased, with APRIL identified as a potential molecule regulating the activity of plasma cells. In the kidney samples, single-cell RNA sequencing (scRNA-seq) analysis showed abundant infiltration of T and myeloid cells, with enhanced interactions with podocytes. scRNA-seq analysis of urinary samples can be informative of the kidney cell landscape, and monitoring urinary samples is a promising method to monitor PMN development. Integrative analysis across the blood, kidney, and urine identified five molecules as common regulators of PMN. Finally, elevated diversity of clonal types in PBMC suggested the existence of autoreactive T and/or B cell receptors. The alterations of cell compositions and cell-cell communication signals offer important information on the pathogenesis of and efficacy of interventions on PMN progression.

33. Microglial M3 Muscarinic Receptors Mediates Multiple Sclerosis Alleviation: a research based on the Experimental Autoimmune Encephalomyelitis (EAE) mouse Model

Kaichun Wang¹, Yuanyuan Xie¹, Hongzhuan Chen¹, Jianrong Xu^{1, *}

1. Shanghai University of Traditional Chinese Medicine

*Corresponding author: janker.xu@gmail.com

Multiple sclerosis (MS) is an autoimmune disease characterized by the inflammatory demyelinating of neurons in the central nervous system (CNS). Due to its complex pathogenesis, the clinical effects of steroid pulse and disease-modifying treatments are limited and have been documented to have various adverse reactions. Therefore, it is urgent to explore new strategies for amelioration of MS. Microglia-mediated central inflammatory responses play crucial roles in the pathogenesis of MS, and inhibiting these inflammatory signals is a promising strategy for treating MS and improving neurological function. We previously found that microglial M3 muscarinic receptors (M3mAChRs) could exert anti-neuroinflammation effects by decreasing cytokine secretion. To assess how targeting these receptors could affect the progression of MS, we established an autoimmune encephalomyelitis (EAE) mouse model by crossing Chrm3 ^fl/fl C × 3cr1 ^iCre mice to create a conditional knockout strain (M3mAChRs ^CKO). These mice showed remarkably accelerated disease onset as assessed by clinical score of neurological function. Moreover, the demyelination area and T lymphocyte infiltration of spinal cords were exacerbated, along with more intense microglia activation. Multi-omics analysis including high-throughput sequencing, multiplex assay, and TMT protein quantification indicated that loss of M3mAChRs had a significant impact on the transcription and secretion of inflammation-associated factors. Chemotaxis, phagocytosis, immune stress response, and synthesis of interleukins were also inhibited. In summary, the M3mAChR-mediates anti-inflammatory effects in microglia and loss produces pathological phenotypes in EAE mice, which provides new insights for developments of clinical therapies for MS.

34. Molecular Mechanism underlying Lesch-Nyhan Syndrome in Female Patients

Zhewei Yang¹, Yiwei Chen^2,3, Qian Huang², Yu An^{1,2 *}

1. Human Phenome Institute, Zhangjiang Fudan International Innovation Center, MOE Key Laboratory of Contemporary Anthropology, Fudan University, Shanghai 201203, China 2. Chinese Medical & Research Network of Lesch-Nyhan Syndrome 3. Wonder Sir

^{*Corresponding author: Yu An, anyu@fudan.edu.cn}

Lesch-Nyhan syndrome (LNS) is a rare X-linked recessive neurogenetic disorder with a prevalence of approximately 1 in 380,000 which results from a deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Classic LNS phenotypes include uric acid overproduction, motor dysfunction, mental retardation, and recurrent self-mutilation. It is almost exclusively manifested in males, with only 10 female cases of LNS with complete biochemical and clinical manifestations having been reported. Heterozygous females are typically asymptomatic, but carriers pass the disorder to affected sons. Genetic testing has revealed eight distinct mutations of HPRT1 in female patients, which are accompanied by non-random X chromosome inactivation (XCI) to suppress expression of the wild-type HPRT1 allele. In X-linked recessive genetic disorders, females may be affected due to skewed X inactivation, translocation, homozygosity, loss of one copy of the X chromosome, or epigenetic abnormalities. One female affected by translocation (46,XX,t(X:2)(q26:p25)) involving the HPRT1 locus has been reported, but the genetic mechanism of female LNS patients is typically a skewed inactivation of the wild-type HPRT1 allele. We identified one female patient with a genetic HPRT defect from 75 LNS patients (1/75, 1.3%) collected by the Chinese Medical & Research Network of LNS demonstrating this genetic mechanism. Because there is typically negative selection against HPRT-deficient blood cells in heterozygous females, the HPRT activity in blood samples of female patients may appear normal, which can make screening for the disorder difficult. Thus, early detection of LNS, ideally prenatally, remains a significant challenge in clinical practice.

35. Morphological and functional retinal impairment is a reliable marker of disease severity in spinocerebellar ataxia

Feixue Liu^{1 *}

1. Southwest Hospital, Third Military Medical University (Army Military Medical University), Chongqing, China

*Corresponding author: 550689837@qq.com

We aimed to examine possible latency and amplitude changes in pattern visual evoked potentials (PVEP) and pattern electroretinogram (PERG) readings along with retinal and macular changes in optical coherence tomography (OCT) in spinocerebellar ataxia (SCA) to identify potential biomarkers. A cohort of 65 patients with SCA and 65 healthy control subjects underwent neurological and ophthalmological examination and investigations of OCT to measure the retinal and macular thickness and visual electrophysiology to assess visual pathways. Linear regression analysis was conducted to look for associations between retinal parameters and age, CAG repeats, disease duration, and scores for International Cooperative Ataxia Rating Scale (ICARS) and Scale for the Assessment and Rating of Ataxia (SARA). The average peripapillary retinal nerve fiber layer (pRNFL) was thicker in SCA than in controls in all sectors and the cup-to-disc ratio was significantly increased. Average ganglion cell layer (GCL) and minimum GCL thicknesses were significantly decreased in SCA3 patients in comparison to controls. The average thicknesses of RNFL and GCL negatively correlated with ICARS scores, whereas the minimum GCL thicknesses negatively correlated with SARA scores. Moreover, PERG and PVEP showed a significant (P < 0.01) increased latency of P100-wave and reduction in amplitude. In summary, we found that retinal abnormalities are highly specific for SCA. These findings highlight the potential for visual electrophysiology and OCT as non-invasive tools for assessing disease severity and progression in SCA3 patients.

36. mtDNA mutation at mt.G7755 locus in rat neurons causes ALS-like phenotype

Xiaolong Qi¹, Keru Li¹, Yuanwu Ma^{1 *}

1. Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Peking Union Medicine College, Beijing, China.

*Corresponding author: Yuanwu Ma, mayuanwu@cnilas.org

The mt.G8363A mutation is associated with brain atrophy, neuropathy, ataxia, and retinitis pigmentosa-like phenotype. In order to reveal the pathogenic mechanism of mt.G8363A mutation, we introduced this mutation into rat mitochondrial genome at the corresponding site (mt.G7755) using the DdCBE system. Because this mutation causes embryonic lethality, we successfully developed a novel conditional mutant rat model, allowing for precise spatiotemporal control of the mutation with the Cre/loxP system. We crossed this line with a neuron-specific Cre-expressing (NeuN-Cre) line to generate a novel rat model (mt.G7755A^NeuN-Cre) with this mutation present in the mitochondria of neuronal cells. These rats manifested an ALS-like phenotype including motor neuron loss, increased microglia presence, TDP-43 aggregation, muscle atrophy, and paralysis. Our findings suggest that the mt.G7755A^NeuN-Cre rat model could be a valuable system for investigating the underlying mechanisms driving the progression of ALS.

37. Novel compound heterozygous mutations in DNAH9 caused cilia loss sensitizes cells to transformation in hepatobiliary cancers

Siming Kong^1#, Wenyan Song^1,2#, Ziye Chen¹, Lingling Hou¹, Yadi Geng^1,2, Xinlong Yan², Hui Bai¹, and Yunfang Wang^{1,2,3 *}

1. Center for Clinical and Translational Science, Beijing Tsinghua Changgung Hospital, Tsinghua Univer-sity, Beijing, China 2. Beijing Key Laboratory of Environmental and Viral Oncology, College of Chemistry and Life Science, Beijing University of Technology, Beijing, China 3. Hepatopancreatobiliary Center, School of Clinical Medicine, Beijing Tsinghua Changgung Hospital, Tsinghua University, Beijing, China 4. Precision Hepatobiliary Center, Beijing Tsinghua Changgung Hospital, Tsinghua University, Beijing, China ^#Equal contributors

*Corresponding author: wyfa02717@btch.edu.cn

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by ciliary structural or functional abnormalities, leading to laterality defects in thoracoabdominal organs and respiratory tract disorders. Single-cell transcriptome analysis has identified cilium-related genes that show altered expression in cancer cells. Cilium loss has also been shown to sensitize cells to malignant transformation in some types of cancer. However, targeting ciliary defects has not been a major avenue of drug development. We identified a proband who had situs ambiguous with hepatobiliary cancer whose sister had asymptomatic situs inversus heterotaxy syndrome. We identified novel compound heterozygous mutations in DNAH9, which caused truncated DNAH9 protein and loss of function in cilia, leading to a final diagnosis of PCD. Genetic testing of the tumor tissue from the patient identified somatic mutations in CCND1, FGF3, FGF4, and FGF19, which led to increased proliferation and malignant transformation of hepatobiliary cells. We found that loss of Dnah9 in mice resulted in significantly higher expression of Ccnd1 and increased p53 signaling pathway in the liver. Furthermore, we observed significantly reduced expression of Sult2a, which resulted in accumulation of bile acid/cholesterol-related DNA adducts, thereby increasing the cellular susceptibility to carcinogenesis. These results reveal a relationship between the rare genetic disorder PCD and the development of tumors. Additionally, this work provides new insights into the understanding of DNAH9 function in cilia as a potential target for cancer prevention and gene therapy.

38. Pathological study and new intervention strategies of chronic multifocal osteomyelitis (CMO) in a murine model at single cell level

Qingran Huo^1,2,3,#, Jiayu Ding^1,2,3,#, Hang He^1,2,3, Zhigang Cai^{1,2,3,4,5, *}

¹The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics; Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University, Tianjin, China ²National Key Laboratory of Experimental Hematology, Tianjin, China ³Tianjin Key Laboratory of Inflammatory Biology, Tianjin, China ⁴Department of Hematology, Tianjin Medical University Tianjin General Hospital, Tianjin, China ⁵Department of Rheumatology and Immunology, Tianjin Medical University Tianjin General Hospital, Tianjin, China

*Corresponding author: Zhigang Cai, Professor, Ph.D., us36zcai@tmu.edu.cn

Autoinflammatory diseases (AIDs) are defined as abnormal activation of the innate immune system leading to spontaneous inflammation. Chronic recurrent multifocal osteomyelitis (CRMO) is an AID where inflammation causes bone damage. However, understanding the etiology of CRMO remain elusive and there is no effective treatment for it. A mouse model of the disease with a Pstpip2 mutation exhibits phenotypic characteristics similar to human CRMO. We found that Pstpip2 is co-expressed with the long non-coding RNA (lncRNA) Morrbid in mouse myeloid cells to regulate cellular lifespan. We generated a new frameshift mutant mouse strain with 5 bp deletion in Pstpip2 and observed a CRMO-like phenotype similar to the widely used Pstpip2 mutant CRMO model. We then created a Morrbid/Pstpip2 double-mutant strain and found that the loss of Morrbid effectively inhibited the symptoms and progression of the disease. Finally, single-cell RNA sequencing and flow cytometry indicated that the Pstpip2 mutation dysregulated activation of myeloid cells and caused excessive release of inflammatory cytokines to mediate the development of CRMO, and loss of Morrbid significantly ameliorated these abnormalities. In brief, our study provides new insights into the etiology of CRMO and identifies Morrbid as a therapeutic target to treat the disorder.

39. Pre-clinical evaluation of AGT mRNA replacement therapy for Primary Hyperoxaluria type I disease

Jiahao Ge¹, Xinye Zhu¹, Siyuan Tang¹, Mei Long¹, Xiaochen Bo¹, Jie Li¹, Yiqing Zhang¹, Ping Wan^{1, *}, Taihua Yang^{1, *}, Qiang Xia^{1,2,3, *}

1. Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China. 2. Shanghai Institute of Organ Transplantation, Shanghai 200127, China; 3. Shanghai Organ Transplantation and Immune Engineering Technology Research Center, Shanghai 200127, China.

*Corresponding authors: Ping Wan (gufeng182@126.com), Taihua Yang (yangtaihua@renji.com) and Qiang Xia (xiaqiang@shsmu.edu.cn)

Primary Hyperoxaluria type 1 (PH1) is a rare inherited liver disease resulting from the loss of alanine glyoxylate aminotransferase (AGT) function. AGT catalyzes the conversion of glyoxylate to glycine, and loss of AGT causes glyoxylate to accumulate and be converted to oxalate. Excess oxalate leads to the deposition of oxalate crystals in the kidney, causing organ failure and leading to half of patients dying of end-stage renal disease. We attempted a protein replacement therapy for AGT by delivering mRNA to cells. We optimized the human AGT mRNA sequence and encapsulated it in a lipopolyplex (LPP) for delivery. The human AGT mRNA/LPP produced functional AGT enzyme, which significantly colocalized with the peroxisome in vitro. We performed single dosing in wild-type (WT) mice and AgxtQ84^-/- rats and repeat dosing in WT cynomolgus monkeys, which produced high expression levels of AGT within 48 hours. Single dosing of 2 mg/kg human AGT mRNA/LPP in AgxtQ84^-/- rats resulted in a 69% reduction in urinary oxalate, suggesting therapeutic benefit. Toxicology of in the mice, rats and monkeys indicates that the highest non-serious toxic dose (HNSTD) in animals is 2 mg/kg. These findings indicate that delivery of human AGT mRNA/LPP in multiple animal models shows promising safety and efficacy to support clinical translation of mRNA-based protein replacement therapy for PH1.

40. Reduced Expression of PPP2R3A Shed Light on a Protective Strategy for Avoiding the Neurofibromas Malignancy

Yanan Yu^{1 *}, Yixiao Wang¹, Minghui Yue², Jianfeng Wang¹, Bingjie Xie¹, You Zhou¹, Kwangmin Choi³, Jianqiang Wu³, Zhichao Wang⁴ and Nancy Ratner^{3 *}

1. Department of Genetics, School of Life Science 2. School of Stomatology, Xuzhou Medical University, Jiangsu Province, China 3. Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio State, USA 4. Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

*Correspondence to Nancy.Ratner@cchmc.org, ynyu@xzhmu.edu.cn

NF1 gene mutations in Schwann cells (SC) drive benign neurofibroma formation in Neurofibromatosis type 1. However, NF1 gene mutations alone cannot fully explain the variable clinical presentation of the disorder, such as whether plexiform neurofibromas (PNF) or malignant tumors appear. SC-specific non-somatic NF1 mutations may contribute to or inhibit PNF development and/or transformation, which could affect the clinical presentation. We previously conducted whole exome sequencing (WES) of SC and fibroblast (FB) DNA from the same resected PNFs and identified multiple somatic SC variants. Mutations in the PPP2R3A gene was identified in three out of the nine examined neurofibromas. We introduced a point mutation into the PPP2R3A gene using CRISPR/Cas9 and found that it caused a significant reduction in the expression of its encoded protein, PR130. We performed microarray and immunoblotting assays and found that PR130 expression was significantly lower in both mouse and human neurofibroma tumors than in neurons. Interestingly, knockdown of PPP2R3A in mouse embryonic SCs only slightly increased sphere formation but expressing PR130 in tumor SC progenitor cells promoted tumor sphere formation. Clonal cells harboring the PPP2R3A point mutation showed reduced cell proliferation and migration. These results indicate that mutations in and reduced expression of the PPP2R3A gene in neurofibromas may be a protective strategy to prevent malignant transformation.

41. Repetitive transcranial magnetic stimulation modulates cortical-cerebellar circuit connectivity in spinocerebellar ataxia type 3

Yonghua Huang¹; Lihua Deng¹; Peiling Ou¹; Jian Wang*¹; Chen Liu^*1

¹7T Magnetic Resonance Translational Medicine Research Center, Department of Radiology, Southwest Hospital, Army Medical University (Third Military Medical University), Chongqing, China

*Corresponding Authors: Jian Wang (wangjian@aifmri.com) and Chen Liu (liuchen@aifmri.com)

Alterations in the structure and functionality of the cortical-cerebellar pathway are evident in individuals diagnosed with spinocerebellar ataxia type 3 (SCA3). Repetitive transcranial magnetic stimulation (rTMS), a method of modulating brain activity, is being increasingly recognized for its potential in treating neurodegenerative conditions. However, the applicability and efficacy of rTMS therapy, particularly targeting the cerebellar circuitry in SCA3, requires further investigation.

In this study, 25 individuals with SCA3 and 33 age- and sex-matched healthy controls were recruited. The severity of SCA3 symptoms was quantified using the Scale for the Assessment and Rating of Ataxia (SARA) and the International Cooperative Ataxia Rating Scale (ICARS), which measure motor abilities, and Montreal Cognitive Assessment (MoCA), which measures cognitive function. All subjects consented to participate in neuropsychological assessments and brain magnetic resonance imaging (MRI) scans.

Following the administration of multi-target rTMS to the cortical-cerebellar circuit in SCA3 patients, SARA and ICARS scores were significantly reduced (p<0.001) and MoCA scores were significantly increased (p<0.001). Additionally, a reduction and normalization in the functional connectivity strength within the cortical-cerebellar circuit were noted post-treatment.

These findings suggest that multi-target rTMS therapy, focused on the midline of the cortical-cerebellar circuit, not only ameliorates motor and cognitive deficits in SCA3 patients but also modulates the functional connectivity within the cortical-cerebellar circuit towards normalization.

42. TadA reprogramming to generate potent miniature base editors with high precision

Shuqian Zhang^1,2, Liting Song³, Bo Yuan⁴, Cheng Zhang¹, Jixin Cao³, Aihua Cao², Tian-Lin Cheng^{1 *}

1. Institutes for Translational Brain Research, Fudan University, Shanghai 200032, China 2. Department of Pediatrics, Qilu Hospital of Shandong University, Ji’nan 250012, China 3. Institute of Science and Technology for Brain inspired Intelligence, Key Laboratory of Computational Neuroscience and Brain Inspired Intelligence, Fudan University, Shanghai, 200433, China 4. Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China

^{*Corresponding author: chengtianlin@fudan.edu.cn}

Although miniature CRISPR Cas12f systems were recently developed, the editing efficacy and targeting range of derived miniature cytosine and adenine base editors (miniCBEs and miniABEs) have not been comprehensively studied. Moreover, functional miniCBEs have not yet been established. Here, we generated various Cas12f-derived miniCBEs and miniABEs with improved editing activities and diversified targeting scopes. We found that miniCBEs generated with traditional cytidine deaminases exhibited wide editing windows and high off-target effects. To improve the editing signatures of classical CBEs and derived miniCBEs, we engineered a TadA deaminase with mutagenesis screening to generate potent miniCBEs with high precision and minimized off-target effects. We show that the newly designed miniCBEs and miniABEs are able to correct pathogenic mutations in cell lines and introduce genetic mutations efficiently via adeno associated virus delivery in the brain in vivo. Together, this study provides alternative strategies for CBE development, expands the toolkits of miniCBEs and miniABEs and offers promising therapeutic tools for clinical applications.

43. Tandem CD19/CD20 CAR T cells successfully cured a central nervous system involvement in Epstein Barr virus associated post-transplant lymphoproliferative disorder of a patient with GATA2 deficiency

Yuanyuan Ren^1,2, Xia Chen^1,2, Fang Liu^1,2, Yang Wan^1,2, Wenyu Yang^1,2, Xiaofan Zhu^1,2, Ye Guo^{1,2 *}

1. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China. 2. Tianjin Institutes of Health Science, Tianjin 301600, China

*Corresponding Author: guoye@ihcams.ac.cn

GATA2 deficiency is a rare disorder of hematopoiesis, lymphatics, and immunity, and allo-geneic hematopoietic stem cell transplantation (allo-HCT) is the only curative therapy option. EBV-associated post-transplant lymphoproliferative disorder (EBV-PTLD) is a rare complication involving the central nervous system (CNS) after allo-HCT with poor outcomes. We report a successfully cured case of CNS-PTLD after umbilical cord blood transplantation of a patient with GATA2 deficiency. A 9-year-old girl underwent mismatched unrelated cord blood transplantation when she was identified with myelodysplastic syndrome secondary to GATA2 deficiency. Two months after transplant, she developed CNS-PTLD. This complication was resistant to initial front-line treatments including immunopressive therapy withdrawal, Rituximab, chemotherapy, and intrathecal injection of Rituximab or methotrexate as the disease progressed rapidly. We prepared tandem CD19/CD20 chimeric antigen receptor T (CART) cells derived from her peripheral lymphocytes with complete donor chimerism. CART cells were infused and no adverse events was recorded. Expansion of the CART cell population was detected in the patient’s peripheral blood and cerebrospinal fluid. The intracranial multifocal lesions were monitored by magnetic resonance imaging and found to have shrunk gradually. The patient was determined to be in remission and disease free at the most recent follow-up (12 months). These results suggest that this treatment is an effective and safe therapy for refractory CNS-PTLD after cord blood transplantation.

44. TARDBP mutations mediate mitochondrial autophagy in spinal muscular atrophy

Yong LIAN¹, Zhen FENG^{1 *}

1. College of Information and Engineering, First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China.

^{*Corresponding author: fengzhen@wmu.edu.cn}

We found that many spinal muscular atrophy (SMA) patients have mutations in the TARDBP gene. Mutations in TARDBP result in abnormal translocation of the TDP-43 protein to the mitochondria, activating mitochondrial degradation through the macroautophagy pathway. This leads to increased expression of PINK, Parkin, and p62, causing impaired mitochondrial function, particularly in the respiratory chain. Increased mitochondrial accumulation corresponds with decreased autophagic activity and is accompanied by downregulation of genes related to mitochondrial and lysosomal function. This contributes to the accumulation of autophagosomes and drives defects in autophagosome-lysosome fusion. This causes decreased expression of anti-apoptotic proteins, leading to more cell death and resulting in neuronal and axonal damage. Our study reveals that TARDBP gene mutations cause mitochondrial dysfunction, autophagic abnormalities, and cellular apoptosis as a mechanism to cause disease pathology. Further research is needed to elucidate the molecular mechanisms underlying these changes and develop new therapeutic strategies for patients harboring mutations in TARDBP.

45. Targeting diverse glycosylation moiety of mucus with mucinases for mucolysis therapy and upgrade drug delivery of mucinous cancers

Jun Liu¹; Zhenglong Sun^{1 *}

Shenzhen Bay Laboratory

Corresponding author: sunzl@szbl.ac.cn

Mucinous cancers are usually accompanied by tumor gland cells secreting large amounts of mucus, forming biogels. Biologically inert mucus can coat tumor cells and promote colonization, immune escape, and drug resistance. Large amounts of mucus accumulate and cannot be removed, eventually causing severe damage to patients. Currently, there is no effective targeting method that can quickly, efficiently, and safely remove the massive amount of mucus produced in mucinous cancers without invasive cytoreductive surgery. We screened proteases from gut probiotics for ability to degrade gel-like mucus from peritoneal pseudomyxoma rapidly and efficiently in vitro and identified two uncharacterized proteases as well as a mucinase from Bacteroides thetaiotaomicron. These enzymes target identical mucin-specific peptide sequences while exhibiting selectivity towards o-glycosylation moieties of mucus proteins. We then attempted to engineer these enzymes to recognize abnormal tumor-specific glycosylation in mucus to target the mucus produced by gland mucinous cancers. Liquefaction and removal of tumor mucus in vivo can lessen the severity of the disease and make targeting the affected cells more effective with conventional therapies while reducing the need for invasive surgery.

46. The deeper, The better? Efficacy of deep transcranial magnetic stimulation in spinocerebellar ataxia type 3

Peiling Ou¹, Linfeng Shi¹, Jian Wang¹, Chen Liu ^{1 *}

1. Department of Radiology, Southwest Hospital, Army Medical University (Third Military Medical University), Chongqing 400038, China

*Corresponding Author: Chen Liu, liuchen@aifmri.com

We aimed to investigate the efficacy of deep transcranial magnetic stimulation (dTMS) in improving motor symptoms in patients with spinocerebellar ataxia type 3 (SCA3). In this prospective study, we recruited 47 SCA3 patients who underwent 20 sessions of dTMS targeting the primary motor cortex (M1) and cerebellum. The treatment was delivered for 5 consecutive days per week for 4 weeks, with a total of 1200 pulses per session. Clinical outcomes were evaluated using the Scale for the Assessment and Rating of Ataxia (SARA) and the International Cooperative Ataxia Rating Scale (ICARS). Outcome assessments were performed at baseline and on the last day of dTMS intervention. Our findings suggest that patients receiving dTMS exhibited improvement in mobility, with a 2.0-point decrease in SARA scores (95% confidence interval (CI) –2.43 to –1.54, p < 0.001) and a 3.8-point decrease in ICARS scores (95% CI –4.91 to –2.65, p < 0.001) compared to baseline data. Importantly, we found no severe adverse events from dTMS therapy. This study indicates that dTMS can be a safe and effective intervention for the alleviation of ataxia in SCA3 and may provide an improved rehabilitative approach compared to traditional TMS for SCA3 therapy.

47. The establishment of cerebral cavernous malformation models

Xi Yang¹, Zhiming Han^{1, *}, Xiangjian Zheng^{2, *}

1. State Key laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China 2. Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China

*Corresponding Authors: hanzm@ioz.ac.cn and xzheng@tmu.edu.cn

Cerebral cavernous malformation (CCM) is a brain vascular disease which can cause stroke, cerebral hemorrhage and neurological deficits in affected individuals with no available treatment. CCM is caused by loss-of-function mutations in one of three genes (CCM1, CCM2 and CCM3). Multiple mouse models for CCM have been developed, although each faces limitations. Ponatinib, which has been approved by the U.S. Food and Drug Administration as a medication for certain types of cancer, has been reported to prevent CCM formation and progression. Here, we employed the Dre-Cre dual recombinase system to specifically delete Ccm genes in brain endothelial cells (ECs) and observed robust CCM lesions in the cerebrum. The survival curve and lesion burden analysis revealed that Ccm2 deletion causes modest CCM lesions with a median life expectancy of ∼10 months and Ccm3 gene deletion leads to severe CCM lesions with median life expectancy of ∼2 months. This inducible system allows for behavioral analyses to assess neurologic deficits to be conducted in adult mice in order to evaluate the effectiveness of treatments.

In addition, we demonstrate that deletion of the CCM3-interacting kinases STK24/25 in ECs also causes CCM lesion formation during postnatal life. We hypothesize that STK24/25 directly regulates MEKK3 activity through phosphorylation to inhibit its function, and so loss of the kinases result in excessive MEKK3 signaling. Thus, allosteric compounds that inhibit MEKK3 are a promising therapeutic strategy for preventing and treating CCM.

48. The PGT-M strategy using gametes and/or affected embryos as probands for linkage analysis in families without complete genetic information

Chen Xi¹, Jie Qiao¹*

1. Peking University Third Hospital

*Corresponidng author: jie.qiao@263.net

Preimplantation genetic testing for monogenic disorders (PGT-M) has been widely used to screen embryos from high-risk families. The rapid development of biopsy technology and bioinformatics, especially the emergence of Mutated Allele Revealed by Sequencing with Aneuploidy and Linkage Analyses (MARSALA) and single nucleotide polymorphism (SNP)-based linkage analysis, has further broadened the application of PGT-M. However, some monogenic disorders have a high rate of de novo variants, which are not detected by PGT-M. For these families with incomplete genetic information, the use of gametes and/or affected embryos as probands for linkage analysis can help complete haplotype construction and analysis.

We examined whole genome amplification (WGA) products from single sperm or polar body and biopsied trophectoderm cells, followed by low-throughput sequencing to a depth of approximately 2×. Next-generation sequencing (NGS) data can be used for calculating chromosomal ploidy and mosaic rate as well as for SNP-based linkage analysis. Appropriate SNP loci were selected through aligning NGS data of the parent and gamete. By using scHaplotyper software, wild-type and mutant haplotypes were constructed and chromosomal ploidy of the blastocysts were analyzed. MARSALA can complete the detection of chromosomal abnormalities and the variants at the single-cell level simultaneously.

For families with incomplete genetic information, linkage analysis using gametes and/or affected embryos as probands can greatly improve the accuracy and applicability of PGT-M.

49. The Quality of Placebos Used in Clinical Trials of Herbal Medicine for Rare Diseases

Yixian Li¹, Peipei Du¹, Ziwen Xu¹, Wenyue Hu², Tao Xiong^{2 *} Chi Zhang^{1,3 *}

1. Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China 2. Shenzhen Hospital, Beijing University of Chinese Medicine, Shenzhen, Guangdong, China 3. Institute for Brain Disorders, Beijing University of Chinese Medicine, Beijing, China

*Corresponding Authors: Chi Zhang, saga618@126.com and Tao Xiong, xt123abc@sohu.com

Specific herbal medicines (HMs) have been widely used to treat individual rare diseases. Reliable evaluation of HM efficacy depends on well-conducted trials, with particular attention being placed on the importance of placebo controls. We aimed to characterize qualities of placebo control procedures used in controlled trials of rare diseases being treated with HMs. We conducted a cross-sectional study by searching nine databases from their inception to April 15, 2024. Rare diseases were identified based on China’s First List of Rare Diseases. Data collection focused on the ethical practices and characteristics of placebo use. Two researchers selected studies and extracted data. Of the 62 placebo-controlled trials that were identified, multiple sclerosis accounted for the largest share at 32.3%. A total of 2,519 patients were assigned to the placebo group and 46.8% of trials included standard therapy. Our findings show that 53.2% of trials lacked ethical approvals, informed consent details were scarce, and none fully reported placebo components or evaluated their similarity. Additionally, 19.4% of placebos had active ingredients without inertness testing, while 20.0% of trials did not show a better safety profile in the placebo group. Flawed placebos are pervasive in HM clinical trials for rare diseases. Low-quality placebo control undermines the production of high-quality evidence required for using HMs in treating rare diseases. Our study emphasizes the urgent necessity of creating strict guidelines and standards for quality and application of placebos in HM trials for rare diseases, ensuring ethical practices and credible clinical evidence.

50. Tracing genetic diversity captures the molecular basis of misfolding disease

Pei Zhao^1#, Chao Wang^{1,2 * #}, Shuhong Sun^1,3,4, Xi Wang^1,5, and William E. Balch^{1 *}

¹Department of Molecular Medicine, Scripps Research, La Jolla, California, 92037, USA. ²Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen, China. ³Department of Nutrition and Food Hygiene, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing 211166, China; ⁴Institute for Brain Tumors, Collaborative Innovation Center for Cancer Personalized Medicine, and Center for Global Health, Nanjing Medical University, Nanjing 211166, China; ⁵National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. ^#Contributed equally

*Corresponding authors: chaowang@szbl.ac.cn and webalch@scripps.edu

Genetic variation in human populations can result in the misfolding and aggregation of proteins, giving rise to systemic and neurodegenerative diseases that require management by proteostasis. Here, we investigated the role of GRP94, a paralog of the endoplasmic reticulum chaperone Hsp90, in managing alpha-1-antitrypsin (A1AT) deficiency on a residue-by-residue basis using Gaussian process regression-based machine learning to profile the spatial covariance relationships that dictate protein folding arising from sequence variants in the population. Covariance analysis suggests a role for the ATPase activity of GRP94 in controlling the N- to C-terminal cooperative folding of A1AT responsible for the correction of liver aggregation and lung-disease phenotypes of A1AT deficiency. Gaussian process-based spatial covariance profiling provides a standard model built on covariant principles to evaluate the role of proteostasis components in guiding information flow from genome to proteome in response to genetic variation, potentially allowing us to intervene in the onset and progression of complex multi-system human diseases.

51. Transcript Analysis of Prader-Willi Syndrome Critical Region from NDN to SNRPN in Human and Mice

Hanshu Yu¹, Yong-hui Jiang^{2 *}

1. School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310008 China 2. Department of Genetics, Neuroscience and Pediatrics, Yale University School of Medicine, New Haven, CT, 06520 USA

Corresponding Author: yong-hui.jiang@yale.edu

Prader-Willi Syndrome (PWS) is a genomic imprinting disorder caused by deficiency of paternally expressed genes on chromosome 15q11.2–q13, which are regulated bidirectionally by an imprinting center (Prader-Willi syndrome-imprinting center, PWS-IC) upstream of the SNURF-SNRPN gene. Based on the findings to date, the PWS-IC drives paternal expression of small nucleolar RNA host gene 14 (SNHG14), a polycistronic locus which extends approximately 600 kb from SNURF-SNRPN to UBE3A-ATS. However, by analyzing the aligned sequence reads and assembled transcripts of strand-specific human brain RNA sequencing on total RNA (toRNA-seq) and polyA-enriched RNA (mRNA-seq), we found that SNHG14 in the human brain is a single transcript that extends approximately 2 Mb from a site 200 kb downstream of NDN to UBE3A-ATS, which is longer than the current annotations. Comparisons between the toRNA-seq and mRNA-seq datasets indicate co-transcriptional processing of the nascent transcript. Open reading frame (ORF) prediction indicates that the transcripts upstream of the PWS-IC do not have significant coding potential. We performed a similar RNA sequencing analysis strategy in mice. We found that the region from Ndn to Snrpn in the mouse genome is poorly-annotated and the actual Snhg14 transcript is also longer than the current annotations. We hypothesize that the long non-coding RNAs in this region are involved in imprinted regulation of MAGEL2, NDN, MKRNS in regions proximal to the PWS-IC in human and mice. This supports an epigenetic therapy strategy for treating PWS.

52. Unraveling the potential of autophagy in Spinal Muscular Atrophy therapy: investigating types, flux, and regulatory factors

Mengting ZHOU¹, Yong LIAN^{1 *}

1. College of Information and Engineering, First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China.

^{*Corresponding author: haerily@wmu.edu.cn}

Spinal Muscular Atrophy (SMA) is a catastrophic autosomal recessive neuromuscular disorder triggered by a decrease in the survival motor neuron (SMN) protein. While the genetic underpinnings of SMA have been extensively elucidated, the mechanisms by which reduced SMN contributes to the disease progression remain obscure. Here, we establish that cellular autophagy is disrupted in SMA. We found that aberrant autophagy occurs during the degradation phase of autophagy, which is caused by a failure in the docking and fusion process between lysosomes and autophagosomes. Consequently, targeting the dysfunction of autophagy may hold promise for improving the prognosis of SMA. However, precise understanding of the molecular mechanisms of how loss of SMN causes reduced autophagy and a strategy for how to restore autophagic function in SMA are needed. Further research is needed to understand the particular types of autophagy and rate of autophagic flux that are altered in SMA as a strategy to develop stand-alone or complementary treatments for patients.

53. Useful biomarkers and trigger factors for early pathogenic process of systemic sclerosis

Hao Cheng¹, Zhang-Xiao Ying¹, Meng-Hua Xu¹, Ting-Ting Zhang¹, Hui-Dan Yang¹, Hong-Yan Wen^{1 *}

1.Research Center of Multi-omics Technology and Autoimmune Disease, Shanxi Medical University, Taiyuan, Shanxi, China; Medical Center of Rare Diseases, The Second Hospital of Shanxi Medical University, Taiyuan, Shanxi, China; Department of Rheumatology, The Second Hospital of Shanxi Medical University, Taiyuan, Shanxi, China.

*Corresponding Author: wenhongyan0509@aliyun.com or wenhongyan@sxmu.edu.cn.

An imbalance between Treg and Th17 cell subsets may trigger the early pathogenesis of systemic sclerosis (SSc) by altering the production of the cytokines mediating regulatory T (Treg) cell plasticity. Using an improved absolute counting method of flow cytometry, we explored the changes in the absolute numbers of T-cell subsets. We found that the absolute counts of peripheral T, CD4+ T, CD8+ T, natural killer (NK), Treg, and Th2 cells in SSc patients were significantly lower than those in healthy controls (HCs), leading to significantly higher ratios of Th1/Th2 and Th17/Treg cells and resulting in immune dysfunction.

Additionally, SSc patients had significantly elevated serum levels of IL-2, IL-6, IL-10, and INFγ compared with HCs. We next evaluated whether the patients showed affected organs. We found a reduction in Treg cells in patients with Raynaud’s phenomenon and a decrease in Th17 cells with a lower Th17/Treg ratio in patients with joint pain. Receiver operating characteristic (ROC) curve analysis identified the optimal critical value of IL-2 for the presence of joint pain (2.67 pg/ml). Similarly, levels of IL-10 and INFγ were also elevated in patients with joint pain. We further identified a threshold for all three cytokines for predicting the occurrence of joint pain. In conclusion, the levels of T-cell subsets and cytokines may serve as useful biomarkers for predicting organ involvement, especially joint pain, in early SSc. Furthermore, they also are the potentially triggering factors for early pathogenic process of the disease.

54. Validation of therapeutic effect of circular-mRNA in human liver organoids with hereditary tyrosinemia type 1

Xin-ye ZHU¹, Si-yuan TANG¹, Jia-hao GE¹, Yi-xiao PAN¹, Xiao-chen BO¹, Yi-qing ZHANG¹, Jie LI¹, Mei LONG¹, Ping WAN^{1 *}, Tai-hua YANG¹, Qiang XIA^{1,2,3 *}

1. Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 1630 Dongfang Road, Shanghai 200127, China; 2. Shanghai Institute of Organ Transplantation, 1630 Dongfang Road, Shanghai 200127, China; 3. Shanghai Organ Transplantation and Immune Engineering Technology Research Center, 1630 Dongfang Road, Shanghai 200127, China.

*Corresponidng authors: gufeng182@126.com, yangtaihua@renji.com, and xiaqiang@shsmu.edu.cn

Hereditary tyrosinemia type 1 (HT1) is an inherited metabolic disease caused by deficiency of fumarylacetoacetate hydrolase (FAH). Medications such as nitisinone and strictly limiting protein consumption can mitigate symptoms of the disease, but cannot prevent its fatal complications, including progressive neurocognitive decline and hepatocyte carcinoma. Liver transplantation is the only curative treatment, and so there is a need for innovative therapies that can stabilize metabolite levels to allow for normal development. We developed a novel mRNA therapy and validated it in human FAH^‐/‐ liver organoids. Primary hepatocytes and nonparenchymal cells were isolated from HT1 patients’ livers after liver transplantation and used to establish three-dimension in vitro pathological models. mRNAs encoding eGFP and FAH were circularized and delivered into FAH^‐/‐ human liver organoids by lipid nanoparticle (LNP) and lipopolyplex (LPP) vehicles. Compared to linear-mRNA or LNP delivery, circular-mRNA eGFP/LPP produced a lower expression peak and a longer expression duration, which is closer to the physiological state. The circular-mRNA FAH/LPP produced expression at a compliable level as wild-type cells up to 7 days after transfection, and the protein co-localized with the functional hepatocytes that preserved albumin secretion. Transfected FAH^‐/‐ hepatocytes maintained expression of FAH and survived up to 30 days after transfection. These results indicate that circularization of mRNAs could weaken the expression and prolong the half-life, which is a promising strategy for replacing FAH expression to treat patients with HT1.

55. xNA: Precision ASO Design for Personalized Medicine and N-of-1 Trials in Rare Genetic Disorders in China

Shengpei Chen^{1 *}, Wanting Chen¹, Xiaoyu Pan¹, Yuxin Xie¹

¹xNA Biotechnology LLC, Shenzhen, China

*shengpei.chen@xna-tech.com

Rare genetic diseases present formidable challenges due to complexity and limited treatment options. Antisense oligonucleotides (ASOs) hold promise for personalized medicine, particularly in N-of-1 trials, yet their lack of tissue-specificity hinders widespread use and escalates research costs. To address this, we have developed xNA, a precision ASO designing platform, achieving remarkable tissue-specific targeting across various organs, including muscle, lung, liver, and CNS. In lung-specific targeting, our non-invasive ASO delivery method in mice yielded a 30-fold increase in therapeutic concentration, substantially enhancing efficacy while mitigating off-target effects. Moreover, our CNS-specific targeting ASOs have successfully traversed the blood-brain barrier via subcutaneous and intravenous injections in mice, eliciting a significant 20% change in gene expression within the cerebrum. Acknowledging the distinct challenges associated with rare genetic diseases, we have optimized ASO target selection for specific scenarios such as allele-specific knockdown and exon-skipping. In allele-specific knockdown, our customized ASOs reduced mutant mRNA levels by 63% without affecting wild-type mRNA. In conclusion, xNA represents a leap forward in precision medicine, offering tailored ASO therapies for rare genetic disorders. By leveraging tissue-specific targeting and personalized approaches, our platform aims to redefine rare genetic disease management, enhancing patient outcomes and quality of life.