Erratum: CircRNA_100395 inhibits cell proliferation and metastasis in ovarian cancer via regulating miR-1228/p53/epithelial-mesenchymal transition (EMT) axis: Erratum.

[This corrects the article DOI: 10.7150/jca.35041.].

C-reactive protein impairs immune response of CD8+ T cells via FcγRIIb-p38MAPK-ROS axis in multiple myeloma.

BACKGROUND: C-reactive protein (CRP) is a prototypical acute phase protein in humans with the function of regulating immune cells. Serum CRP levels are elevated in multiple myeloma (MM), associated with MM cell proliferation and bone destruction. However, its direct effects on T lymphocytes in MM have not been elucidated. METHODS: Public data sets were used to explore the correlation of CRP levels with immune cell infiltration and cytotoxicity score of CD8+ T cells in MM. In vitro, repeated freeze-thaw myeloma cell lines were taken as tumor antigens to load dendritic cells (DCs) derived from HLA-A*0201-positive healthy donors. MM-specific cytotoxic T cells (MM-CTL) were obtained from T lymphocytes of the corresponding donors pulsed with these DCs. B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR)-T cells were manipulated by transfecting with lentivirus encoding an anti-BCMA single-chain variable fragment. Then T cells from healthy controls, MM-CTLs and BCMA CAR-T cells were exposed to CRP and analyzed for cell proliferation, cytotoxicity, immunophenotypes. CRP binding capacity to T cells before and after Fc gamma receptors IIb (FcγRIIb) blockage, p38 mitogen-activated protein kinase (MAPK) pathway and the downstream molecules were also detected. In vivo, both normal C57BL/6J mice and the Vk*MYC myeloma mouse models were applied to confirm the impact of CRP on T cells. RESULTS: CRP levels were negatively correlated with cell-infiltration and cytotoxicity score of CD8+ T cells in MM. In vitro experiments showed that CRP inhibited T-cell proliferation in a dose-dependent manner, impaired the cytotoxic activity and upregulated expression of senescent markers in CD8+ T cells. In vivo results validated the suppressive role of CRP in CD8+ T cells. CRP could bind to CD8+ T cells, mainly to the naïve T subset, while the binding was dramatically decreased by FcγRIIb blockage. Furthermore, CRP resulted in increased phosphorylation of p38 MAPK, elevated levels of reactive oxygen species and oxidized glutathione in CD8+ T cells. CONCLUSIONS: We found that CRP impaired immune response of CD8+ T cells via FcγRIIb-p38MAPK-ROS signaling pathway. The study casted new insights into the role of CRP in anti-myeloma immunity, providing implications for future immunotherapy in MM.

Unravelling the imbalanced Th17-like cell differentiation by single-cell RNA sequencing in multiple myeloma.

Multiple myeloma (MM) is a bone marrow resident hematological malignancy. T helper (Th) cells play an essential role in maladjustment of immune function and promotion of myeloma cell proliferation and survival, which has not been fully elucidated. Here, we compared transcriptome profiles of CD4+ T cells in bone marrow samples of 3 healthy individuals and 10 MM patients before and after treatment using single-cell RNA sequencing. CD4+ T cells were divided into 7 clusters. Imbalanced Th17-like cell differentiation was indicated in MM based on bioinformation analyses, which involved IL2-STAT5 pathways and transcription factors NKFB1, RELA, STAT3, and GTF2A2. Pseudotime trajectory analysis of CD4+ T cell clusters further uncovered the enhanced transition of Th17-like to regulatory T (Treg) cells in MM, which was featured by expression changes of PLAC8, NKFB1, RELA, STAT3, and STAT1 along with the developmental path. Reduced cell-cell interaction between MM cells and CD4+ naïve/recently activated naïve T cells via CD74-APP might lead to imbalanced Th17-like cell differentiation. Checkpoints via TIGIT-NECTIN3 and LGALS9-CD47 in Treg and MM cells were also identified. Our study reveals imbalanced differentiation pattern of Th17-like cells and the immunosuppressive profiles in connection with MM cells, which might help to shed light on CD4+ T cell function in MM.

EBV-encoded miRNAs BHRF1-1 and BART2-5p aggravate post- transplant lymphoproliferative disorder via LZTS2-PI3K-AKT axis.

Post-transplant lymphoproliferative disorder (PTLD) is one of the most serious complications after transplantation. Epstein-Barr virus (EBV) is a key pathogenic driver of PTLD. About 80% of PTLD patients are EBV positive. However, the accuracy of preventing and diagnosing EBV-PTLD by monitoring EBV DNA load is limited. Therefore, new diagnostic molecular markers are urgently needed. EBV-encoded miRNAs can regulate a variety of EBV-associated tumors and are expected to be potential diagnostic markers and therapeutic targets. We found BHRF1-1 and BART2-5p were significantly elevated in EBV-PTLD patients, functionally promoting proliferation and inhibiting apoptosis in EBV-PTLD. Mechanistically, we first found that LZTS2 acts as a tumor suppressor gene in EBV-PTLD, and BHRF1-1 and BART2-5p can simultaneously inhibit LZTS2 and activate PI3K-AKT pathway. This study shows that BHRF1-1 and BART2-5p can simultaneously inhibit the expression of tumor suppressor LZTS2, and activate the PI3K-AKT pathway, leading to the occurrence and development of EBV-PTLD. Therefore, BHRF1-1 and BART2-5p are expected to be potential diagnostic markers and therapeutic targets for EBV-PTLD patients.

Single-cell technologies in multiple myeloma: new insights into disease pathogenesis and translational implications.

Multiple myeloma (MM) is a hematological malignancy characterized by clonal proliferation of plasma cells. Although therapeutic advances have been made to improve clinical outcomes and to prolong patients' survival in the past two decades, MM remains largely incurable. Single-cell sequencing (SCS) is a powerful method to dissect the cellular and molecular landscape at single-cell resolution, instead of providing averaged results. The application of single-cell technologies promises to address outstanding questions in myeloma biology and has revolutionized our understanding of the inter- and intra-tumor heterogeneity, tumor microenvironment, and mechanisms of therapeutic resistance in MM. In this review, we summarize the recently developed SCS methodologies and latest MM research progress achieved by single-cell profiling, including information regarding the cancer and immune cell landscapes, tumor heterogeneities, underlying mechanisms and biomarkers associated with therapeutic response and resistance. We also discuss future directions of applying transformative SCS approaches with contribution to clinical translation.

Single-cell RNA sequencing reveals XBP1-SLC38A2 axis as a metabolic regulator in cytotoxic T lymphocytes in multiple myeloma.

The mechanisms underlying the functional impairment and metabolic reprogramming of T lymphocytes in multiple myeloma (MM) have not been fully elucidated. In this study, single-cell RNA sequencing was used to compare gene expression profiles in T cells in bone marrow and peripheral blood of 10 newly diagnosed MM patients versus 3 healthy donors. Unbiased bioinformatics analysis revealed 9 cytotoxic T cell clusters. All 9 clusters in MM had higher expression of senescence markers (e.g., KLRG1 and CTSW) than the healthy control; some had higher expression of exhaustion-related markers (e.g., LAG3 and TNFRSF14). Pathway enrichment analyses showed downregulated amino acid metabolism and upregulated unfolded protein response (UPR) pathways, along with absent expression of glutamine transporter SLC38A2 and increased expression of UPR hallmark XBP1 in cytotoxic T cells in MM. In vitro studies revealed that XBP1 inhibited SLC38A2 by directly binding to its promoter, and silencing SLC38A2 resulted in decreased glutamine uptake and immune dysfunction of T cells. This study provided a landscape description of the immunosuppressive and metabolic features in T lymphocytes in MM, and suggested an important role of XBP1-SLC38A2 axis in T cell function.

Distinct mechanisms of dysfunctional antigen-presenting DCs and monocytes by single-cell sequencing in multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy with the hallmark of immunodeficiency, including dysfunction of T cells, NK cells, and APCs. Dysfunctional APCs have been reported to play a key role in promoting MM progression. However, the molecular mechanisms remain elusive. Here, single-cell transcriptome analysis of dendritic cells (DC) and monocytes from 10 MM patients and three healthy volunteers was performed. Both DCs and monocytes were divided into five distinct clusters, respectively. Among them, monocyte-derived DCs (mono-DC) were shown to develop from intermediate monocytes (IM) via trajectory analysis. Functional analysis showed that, compared with healthy controls, conventional DC2 (cDC2), mono-DC, and IM of MM patients exhibited impaired antigen processing and presentation capacity. Moreover, reduced regulon activity of interferon regulatory factor 1 (IRF1) was found in cDC2, mono-DC and IM of MM patients according to single-cell regulatory network inference and clustering (SCENIC) analysis, while the downstream mechanisms were distinct. Specifically in MM patients, cathepsin S (CTSS) was markedly downregulated in cDC2, major histocompatibility complex (MHC) class II transactivator (CIITA) was significantly decreased in IM, in addition both CTSS and CIITA were downregulated in mono-DC based on differentially expressed genes analysis. In vitro study validated that knockdown of Irf1 downregulated Ctss and Ciita respectively in mouse DC cell line DC2.4 and mouse monocyte/macrophage cell line RAW264.7, which ultimately inhibited proliferation of CD4+ T cells after being cocultured with DC2.4 or RAW264.7 cells. This current study unveils the distinct mechanisms of cDC2, IM, and mono-DC function impairment in MM, offering new insight into the pathogenesis of immunodeficiency.

Dynamic single-cell RNA-seq analysis reveals distinct tumor program associated with microenvironmental remodeling and drug sensitivity in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a hematological malignancy characterized by clonal proliferation of malignant plasma cells. Despite extensive research, molecular mechanisms in MM that drive drug sensitivity and clinic outcome remain elusive. RESULTS: Single-cell RNA sequencing was applied to study tumor heterogeneity and molecular dynamics in 10 MM individuals before and after 2 cycles of bortezomib-cyclophosphamide-dexamethasone (VCD) treatment, with 3 healthy volunteers as controls. We identified that unfolded protein response and metabolic-related program were decreased, whereas stress-associated and immune reactive programs were increased after 2 cycles of VCD treatment. Interestingly, low expression of the immune reactive program by tumor cells was associated with unfavorable drug response and poor survival in MM, which probably due to downregulation of MHC class I mediated antigen presentation and immune surveillance, and upregulation of markers related to immune escape. Furthermore, combined with immune cells profiling, we uncovered a link between tumor intrinsic immune reactive program and immunosuppressive phenotype in microenvironment, evidenced by exhausted states and expression of checkpoint molecules and suppressive genes in T cells, NK cells and monocytes. Notably, expression of YBX1 was associated with downregulation of immune activation signaling in myeloma and reduced immune cells infiltration, thereby contributed to poor prognosis. CONCLUSIONS: We dissected the tumor and immune reprogramming in MM during targeted therapy at the single-cell resolution, and identified a tumor program that integrated tumoral signaling and changes in immune microenvironment, which provided insights into understanding drug sensitivity in MM.

Rapid generation of genetically engineered T cells for the treatment of virus-related cancers.

Adoptive transfer of T cell receptor (TCR)-engineered T cells targeting viral epitopes represents a promising approach for treating virus-related cancers. However, the efficient identification of epitopes for T cells and the corresponding TCR remains challenging. Here, we report a workflow permitting the rapid generation of human papillomavirus (HPV)-specific TCR-T cells. Six epitopes of viral proteins belonged to HPV16 or HPV18 were predicted to have high affinity to A11:01 according to bioinformatic analysis. Subsequently, CTL induction were performed with these six antigen peptides separately, and antigen-specific T cells were sorted by FACS. TCR clonotypes of these virus-specific T cells were determined using next-generation sequencing. To improve the efficiency of TCRαß pair validation, a lentiviral vector library containing 116 TCR constructs was generated that consisted of predominant TCRs according to TCR repertoire analysis. Later, TCR library transduced T cells were simulated with peptide pool-pulsed antigen-presenting cells, then CD137-positive cells were sorted and subjected to TCR repertoire analysis. The top-hit TCRs and corresponding antigen peptides were deduced and validated. Through this workflow, a TCR targeting the E692-101 of HPV16 was identified. These HPV16-specific TCR-T cells showed high activity towards HPV16-positive human cervical cancer cells in vitro and efficiently repressed tumor growth in a murine model. This study provides a HPV16-specific TCR fitted to the HLA-A11:01 population, and exemplifies an efficient approach that can be applied in large-scale screening of virus-specific TCRs, further encouraging researchers to exploit the therapeutic potential of the TCR-T cell technique in treating virus-related cancers.

A Five-Gene Risk Score Model for Predicting the Prognosis of Multiple Myeloma Patients Based on Gene Expression Profiles.

Multiple myeloma is a heterogeneous plasma cell malignancy that remains incurable because of the tendency of relapse for most patients. Survival outcomes may vary widely due to patient and disease variables; therefore, it is necessary to establish a more accurate prognostic model to improve prognostic precision and guide clinical therapy. Here, we developed a risk score model based on myeloma gene expression profiles from three independent datasets: GSE6477, GSE13591, and GSE24080. In this model, highly survival-associated five genes, including EPAS1, ERC2, PRC1, CSGALNACT1, and CCND1, are selected by using the least absolute shrinkage and selection operator (Lasso) regression and univariate and multivariate Cox regression analyses. At last, we analyzed three validation datasets (including GSE2658, GSE136337, and MMRF datasets) to examine the prognostic efficacy of this model by dividing patients into high-risk and low-risk groups based on the median risk score. The results indicated that the survival of patients in low-risk group was greatly prolonged compared with their counterparts in the high-risk group. Therefore, the five-gene risk score model could increase the accuracy of risk stratification and provide effective prediction for the prognosis of patients and instruction for individualized clinical treatment.

Generation of neoantigen-specific T cells for adoptive cell transfer for treating head and neck squamous cell carcinoma.

Adoptive cell therapy using TCR-engineered T cells (TCR-T cells) represents a promising strategy for treating relapsed and metastatic cancers. We previously established methods to identify neoantigen-specific TCRs based on patients' PBMCs. However, in clinical practice isolation of PBMCs from advanced-stage cancer patients proves to be difficult. In this study, we substituted blood-derived T cells for tumor-infiltrating lymphocytes (TILs) and used an HLA-matched cell line of antigen-presenting cells (APCs) to replace autologous dendritic cells. Somatic mutations were determined in head and neck squamous cell carcinoma resected from two patients. HLA-A*02:01-restricted neoantigen libraries were constructed and transferred into HLA-matched APCs for stimulation of patient TILs. TCRs were isolated from reactive TIL cultures and functionality was tested using TCR- T cells in vitro and in vivo. To exemplify the screening approach, we identified the targeted neoantigen leading to recognition of the minigene construct that stimulated the strongest TIL response. Neoantigen peptides were used to load MHC-tetramers for T cell isolation and a TCR was identified targeting the KIAA1429D1358E mutation. TCR-T cells were activated, exhibited cytotoxicity, and secreted cytokines in a dose-dependent manner, and only when stimulated with the mutant peptide. Furthermore, comparable to a neoantigen-specific TCR that was isolated from the patient's PBMCs, KIAA1429D1358E-specific TCR T cells destroyed human tumors in mice. The established protocol provides the required flexibility to methods striving to identify neoantigen-specific TCRs. By using an MHC-matched APC cell line and neoantigen-encoding minigene libraries, autologous TILs can be stimulated and screened when patient PBMCs and/or tumor material are not available anymore. Abbreviations: Head and neck squamous cell carcinoma (HNSCC); adoptive T cell therapy (ACT); T cell receptor (TCR); tumor-infiltrating lymphocytes (TIL); cytotoxic T lymphocyte (CTL); peripheral blood mononuclear cell (PBMC); dendritic cell (DC); antigen-presenting cells (APC).

Breast cancer-derived exosomes transmit lncRNA SNHG16 to induce CD73+γδ1 Treg cells.

γδT cells have been reported to exert immunosuppressive functions in multiple solid malignant diseases, but their immunosuppressive functional subpopulation in breast cancer (BC) is still undetermined. Here, we collected 40 paired BC and normal tissue samples from Chinese patients for analysis. First, we showed that γδT1 cells comprise the majority of CD3+ T cells in BC; next, we found that CD73+γδT1 cells were the predominant regulatory T-cell (Treg) population in BC, and that their prevalence in peripheral blood was also related to tumour burden. In addition, CD73+γδT1 cells exert an immunosuppressive effect via adenosine generation. We also found that BC could modulate CD73 expression on γδT cells in a non-contact manner. The microarray analysis and functional experiments indicated that breast tumour cell-derived exosomes (TDEs) could transmit lncRNA SNHG16, which upregulates CD73 expression, to Vδ1 T cells. Regarding the mechanism, SNHG16 served as a ceRNA by sponging miR-16-5p, which led to the derepression of its target gene SMAD5 and resulted in potentiation of the TGF-ß1/SMAD5 pathway to upregulate CD73 expression in Vδ1 T cells. Our results showed that the BC-derived exosomal SNHG16/miR-16-5p/SMAD5-regulatory axis potentiates TGF-ß1/SMAD5 pathway activation, thus inducing CD73 expression in Vδ1 T cells. Our results first identify the significance of CD73+Vδ1 Tregs in BC, and therapy targeting this subpopulation or blocking TDEs might have potential for BC treatment in the future.

CircRNA_100395 inhibits cell proliferation and metastasis in ovarian cancer via regulating miR-1228/p53/epithelial-mesenchymal transition (EMT) axis.

Purpose: Circular RNAs (circRNAs) have been reported to regulate the incidence of tumor by regulating the transcriptional level and post-transcriptional level of tumor-related genes, and are significantly correlated with tumor metastasis and progression. CircRNA_100395 (circ_100395) has been reported to suppress lung cancer cell proliferation, and might act as an oncogene in deveopment of various cancers. However, the expression and function of circ_100395 in ovarian cancer has not been systematically researched. Methods: The expression of circ_100395 in ovarian cancer tissues was detected by Real-time Quantitative polymerase chain reaction (RT-qPCR), while the relationship between circ_100395 expression and clinicopathological characteristics was further analyzed. After increasing the expression of circ_100395 by plasmid transfection in ovarian cancer cells, we further investigated the cell proliferation, invasion and migration by cell counting kit-8 (CCK-8), and Transwell assays. Epithelial-mesenchymal transition (EMT) pathway was also measured by western blotting. In addition, the relationship among circ_100395, miR-1228 and p53 in ovarian cancer, was explored by luciferase reporter assay. Results: The expression of circ_100395 was found to be significantly down-regulated in ovarian cancer, while low expression of circ_100395 was highly correlated with the poor outcomes. In addition, upregulation of circ_100395 could significantly inhibit tumor growth, metastasis and EMT signaling pathway in ovarian cancer. Furthermore, the expression level of circ_100395 was negatively correlated with the expression of miR-1228, and with the addition of miR-1228 could reverse anti-cell proliferation effect induced by circ_100395 in ovarian cancer cells. In addition, p53 might be the key target of circ_100395 / miR-1228 axis in ovarian cancer. Conclusion: CircRNA_100395 could inhibit cell growth and metastasis of ovarian cancer cells via regulating the miR-1228/p53/EMT axis.

The Therapeutic Potential of Small Activating RNAs for Colorectal Carcinoma.

Small double-strand RNAs have been recognized as master regulators of gene expression. In contrast to the evolutionary conserved RNA interference machinery, which degrades or inhibits the translation of target mRNAs, small activating RNA (saRNA) activates the specific gene in a target dependent manner through a similar mechanism as RNAi. Recently, saRNA mediated expression regulation of specific genes has been extensively studied in cancer researches. Of particular interest is the application of the RNA mediated gene activation within colorectal cancer (CRC) development, due to the high incidence of the CRC. In this review, we summarize the current knowledge of saRNA mediated genetic activation and its underlying mechanisms. Furthermore, we highlight the advantages of the utilization of saRNAs induced gene expression as an investigating tool in colorectal cancer research. Finally, the possibility and the challenge of the saRNA application as a potential therapy for colorectal cancer are addressed.

[Feasibility of bone marrow mesenchymal stem cells differentiation in diabetic pancreatic microenvironment].

OBJECTIVE: The bone marrow mesenchymal stem cells (BMSCs) have the capacity to differentiate into insulin-producing cells (IPCs) in vitro. However, low differentiation efficiency and poor maturity are the main obstacles. To investigate the feasibility of BMSCs differentiation into IPCs in diabetic pancreatic microenvironment of pigs. METHODS: BMSCs were isolated and purified from the bone marrow of a 4-week-old male pig. Fifteen female pigs (aged 8 to 10 weeks, weighing 8 to 10 kg) were randomly divided into 3 groups: normal control group (group A, n=5), diabetic control group (group B, n=5), and BMSCs transplanted group (group C, n=5). The pigs of groups B and C were treated by auris vein injections of streptozocin and alloxan for 3 days to induce diabetes mellitus (DM) model, whose blood glucose level 2 days all greater than 17 mmol/L was successful DM model. A total of 1.1 mL of the 3rd passage BMSCs labeled with enhanced green fluorescent protein (EGFP), with cell density of 5 x 10(7)/ mL, were injected into subcapsular pancreas of group C at multiple points, normal saline at the same dosage into those of groups A and B. After 30 days of monitoring blood glucose, the histological analysis of islet number and size were done; the immunofluorescence staining was used to detect the protein expression of insulin in the new-formed islets. The EGFP+ cells were collected from the sections using laser-capture microdissection; RT-PCR was used to detect insulin mRNA and pancreatic and duodenal homeobox factor 1 (PDX1) mRNA expressions from EGFP+ cells, and the insulin and sex determining region of the Y chromosome (SRY) genes were detected by fluorescence in situ hybridization (FISH). RESULTS: The blood glucose level decreased significantly in group C when compared with that in group B from 18 days and gradually decreased with time (P < 0.05). The histological observation showed that the number of islets was increased significantly in group C when compared with that in group B (10.9 +/- 2.2 vs. 4.6 +/- 1.4, P < 0.05), and there was no significant difference when compared with that in group A (10.9 +/- 2.2 vs.12.6 +/- 2.6, P > 0.05). The size of new-formed islets in group C was significantly smaller than that in group A [(47.2 +/- 19.6) microm vs. (119.6 +/- 27.7) microm, P < 0.05]. The immunofluorescence staining showed that new-formed islets of group C expressed insulin protein. RT-PCR showed that the microdissected EGFP+ cells of group C expressed insulin mRNA and PDX-1 mRNA. FISH showed that the new-formed islet cells of group C contained SRY gene in Y chromosome and insulin double positive cells. CONCLUSION: BMSCs can differentiate into IPCs in diabetic pancreatic microenvironment of pigs.

Pyoderma gangrenosum related to a new granulocyte colony-stimulating factor.

A 23-year-old Caucasian man diagnosed with stage IVB Hodgkin's disease was referred to a university oncology section after completing 1.5 cycles of chemotherapy. His chemotherapy consisted of doxorubicin HCL, bleomycin, dacarbazine, and vinblastine, with prophylactic administration of a granulocyte colony stimulating factor. He had developed postchemotherapy complications of possible cellulitis and necrotizing fasciitis that required wound debridement. The wound and tissue cultures were negative. Biopsies taken at the time revealed a dense inflammatory infiltrate consistent with an abscess. Over the course of 2 months, the wound healed with systemic antibiotics. The patient was reluctant to resume chemotherapy for his Hodgkin's disease because of his previous presumed skin infections. However, positive emission tomographic scanning revealed disease progression. Doxorubicin, bleomycin, dacarbazine, and prophylactic pegfilgrastim (a granulocyte colony-stimulating factor), were administered. Vinblastine was excluded from the new regimen. Shortly after chemotherapy and an injection of pegfilgrastim, the patient developed poorly defined, rapidly progressive erythema, edema, and pain in his right forearm. He presented to the emergency room, was evaluated by the orthopedics service, and taken to the operating room for debridement of suspected necrotizing fasciitis. When the dermatology service consulted the following day, the patient had developed an erythematous, edematous, tender plaque on his chest. After developing two additional lesions that began to ulcerate despite treatment with imipenem, vancomycin, clindamycin, rifampin, and gentamicin, the patient consented to a skin biopsy. His wound cultures continued to be negative.

Transgene produces massive overexpression of human beta -glucuronidase in mice, lysosomal storage of enzyme, and strain-dependent tumors.

beta-Glucuronidase (GUSB) is a lysosomal enzyme important in the normal step-wise degradation of glycosaminoglycans. Deficiency of GUSB causes the lysosomal storage disease mucopolysaccharidosis VII (MPS VII, Sly disease). Affected patients have widespread progressive accumulation of beta-glucuronide-containing glycosaminoglycans in lysosomes. Enzyme replacement, bone marrow transplantation, and gene therapy can correct lysosomal storage in the MPS VII mouse model. Gene therapy in MPS VII patients and animals may result in massive overexpression of GUSB in individual tissues, and the toxicity of such overexpression is incompletely investigated. To gain insight into the effect of massive overexpression of GUSB, we established 19 transgenic mouse lines, two of which expressed very high levels of human GUSB in many tissues. The founder overexpressing mice had from >100- to several thousand-fold increases in tissue and serum GUSB. The enzyme expression in most tissues decreased in subsequent generations in one line, and expression in liver and marrow fell in subsequent generations of the other. Both lines had morphologically similar widespread lysosomal storage of GUSB and secondary elevations of other lysosomal enzymes, a finding characteristic of lysosomal storage disease. One line developed tumors, and one did not. These transgenic models show that massive overexpression of a lysosomal enzyme can be associated with dramatic morphological alterations, which, at least in one of the two lines, had little clinical consequence. For the other transgenic line, the high frequency of tumor development in F(2) FVB progeny suggests that the vector used to generate the transgenic lines has an integration site-dependent potential to be oncogenic, at least in this strain background.