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BackgroundGene therapy is gradually becoming recognized as a possibly curative therapeutic strategy for transfusion-dependent β-thalassemia (TDT). Gene therapy addresses the problem of donor scarcity through the application of autologous hematopoietic stem cells (HSCs), which also can reduce the risks that accompany allogeneic HSC transplantation. When using gene addition strategy, lentiviral vector is critical for the efficacy and safety of β-thalassemia gene therapy. In our preclinical studies, LentiHBBT87Qvector with optimized backbone was developed to efficiently restore β–globin expression in HSCs-derived erythroblasts of TDT patients with minimal risk of tumorigenesis. Here, we presented the clinical trial results of gene therapy using LentiHBBT87Qvector in two TDT patients.MethodIn an ongoing phase 1/2 trial (NCT05745532), auto-HSCs were mobilized from two TDT patients, and then transduced with LentiHBBT87Qvector. The gene-modified auto-HSCs is called HGI-001 injection. After four-day consecutive myeloablative conditioning, these two patients were administrated with HGI-001 injection via intravenous infusion. Medical examinations were performed in the transplantation unit to monitor patients’ status till the patients were clinically stable. Then, 24-month following-up visits are conducted to assess the safety and efficacy of HGI-001 injection. The safety endpoints of this clinical study include the incident and severity of adverse events (AEs); transplant-related mortality or disability events within 100 days post drug product infusion; vector-related replication competent lentivirus (RCL) and clonal variations containing specific viral integration sites; overall survival during this clinical trial. The major efficacy endpoint is the percentage of subjects with average vector copy number (VCN) in peripheral blood mononuclear cells (PBMCs) >0.1, and average expression of exogenous HbAT87Q>2.0g/dL at the 24thmonth after reinfusion of HGI-001 injectionResultsThe rapid neutrophil and platelet engraftment successfully happened after reinfusion of HGI-001 injection. The two patients with non-β0/β0genotype have been transfusion-independent for 24 months and 21 months post-treatment. At the last visit, the levels of HbAT87Qare 7.3 and 6.9g/dL, and the levels of total hemoglobin are 9.8 and 10.1 g/dL. After the two subjects stopped transfusions, the iron overload has been alleviated without iron chelation treatment. Most AEs are myeloablative conditioning related, and can be controlled through clinically standard therapeutic managements. No clone dominance related to vector integration nor RCL has been observed.ConclusionGene therapy with optimized LentiHBBT87Qvector (HGI-001 injection) assist two TDT patients become transfusion-independent without serious adverse events related to the product.

Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long-time ex vivo culture. However, the underlying mechanisms of the stemness differences because of the cell sources and the culture stimulation are not fully understood. Here, we applied single-cell RNA-seq (scRNA-seq) to analyze the naïve and stimulated human CD34+ cells from cord blood (CB) and (mPB). We collected over 16,000 single-cell data to construct a comprehensive trajectory inference map and characterized the HSC population on the hierarchy top, which is under quiescent state. Then we compared HSCs in CB to those in mPB and HSCs of naïve samples to those of cultured samples, and identified stemness-related genes (SRGs) associated with culture time (CT-SRGs) and cell source (CS-SRGs), respectively. Interestingly, CT-SRGs and CS-SRGs share genes enriched in the signaling pathways such as mRNA catabolic process, Translational initiation, Ribonucleoprotein complex biogenesis and Cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT-SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS-SRGs specifically contain genes related purine and ATP metabolic process which is important to initiate hematopoiesis. Finally, we presented an application through a small-scale drug screening using Connectivity Map (CMap) against CT-SRGs and found a small molecule cucurbitacin I, targeting STAT3/JAK2, can efficiently expand HSCs ex vivo while maintaining its stemness. These results indicate SRGs revealed by scRNA-seq can provide helpful insights to understand the stemness differences under diverse circumstances, and CT-SRGs can be a valuable database to identify candidates enhancing functional HSC expansion during ex vivo culture.

Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long-time ex vivo culture. However, the underlying mechanisms of the stemness differences because of the cell sources and the culture stimulation are not fully understood. Here, we applied single-cell RNA-seq (scRNA-seq) to analyze the naïve and stimulated human CD34+ cells from cord blood (CB) and mobilized peripheral blood (mPB). We collected over 16,000 single-cell data to construct a comprehensive trajectory inference map and characterized the HSCs population on the hierarchy top, which is under quiescent state. Then we compared HSCs in CB to those in mPB and HSCs of naïve samples to those of cultured samples, and identified stemness-related genes (SRGs) associated with culture time (CT-SRGs) and cell source (CS-SRGs), respectively. Interestingly, CT-SRGs and CS-SRGs share genes enriched in the signaling pathways such as mRNA catabolic process, Translational initiation, Ribonucleoprotein complex biogenesis and Cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT-SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS-SRGs specifically contain genes related purine and ATP metabolic process which is important to initiate hematopoiesis. Finally, we presented an application through a small-scale drug screening using Connectivity Map (CMap) against CT-SRGs and found a small molecule cucurbitacin I, targeting STAT3/JAK2, can efficiently expand HSCs ex vivo while maintaining its stemness. These results indicate SRGs revealed by scRNA-seq can provide helpful insights to understand the stemness differences under diverse circumstances, and CT-SRGs can be a valuable database to identify candidates enhancing functional HSCs expansion during ex vivo culture.

Rationale: Sjogren's syndrome(SS) is a chronic autoimmune disease, which damages exocrine glands especially salivary and lacrimal glands, with xerostomia and xerophthalmia as common symptoms. Patient concerns: We report a case of a 49-year-old woman presented with pancytopenia. Her laboratory examinations lead us diagnose her as Sjogren's syndrome complicating pancytopenia. She had neurological symptoms during her treatment, which represent only 4.5% of Sjogren's syndrome complicating damage in nervous system. Diagnoses: Sjogren's syndrome complicating pancytopenia. Interventions: Dexamethasone (40mg QD for 4 days) and immunoglobulin (25g QD for 2 days) were administered for intensive treatment followed by oral methylprednisolone 40mg QD as maintenance treatment. Total glucosides of paeony 0.6g TID and danazol 0.2g BID per os were given. We also gave her Piperacillin-tazobactam and moxifloxacin for anti-infection and Fluconazole for anti-fungal therapy, as well as other supportive treatments. Outcomes: Follow-up of the patient observed the normalization of peripheral blood cell count, immunity indices and neurological examinations 6 months after discharge. Lessons: For patients presented with blood system abnormalities unilineage or multiple-lineage cytopenia in particular, history investigations and relevant examinations should be considered to exclude the existence of autoimmune diseases like Sjogren's syndrome.

In this paper, researches have been carried out on how to improve microprocessor system reliability using fault tolerant theory and technique. A kind of three module redundant fault-tolerant microprocessor system structure has been proposed which can mask both transient fault and permanent fault. This fault-tolerant system has been realized in hardware using microcontroller 80c552 as core. The fault-tolerant system consists of three functional modules with the same hardware and a fault-masking network. The three modules are alternative in function and the fault-masking network is used for fault-detection and fault-tolerance. The fault-tolerant microprocessor system has been applied in robotization automatic drilling machine and as the main control unit which executes key tasks. Its powerful fault-tolerance and high reliability have been proved by practice.