Regulated control of gene therapies by drug-induced splicing.

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote Xon, does not require the co-expression of any regulatory proteins. Using Xon, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of Xon to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.

Clinicopathological characteristics and outcomes of gastrointestinal stromal tumors with high progranulin expression.

BACKGROUND & AIMS: Progranulin (PGRN) is known to promote tumorigenesis and proliferation of several types of cancer cells. However, little is known about the clinicopathological features of patients with gastrointestinal stromal tumors (GISTs) with regard to PGRN expression. METHODS: A retrospective analysis was performed on patients with GISTs who underwent curative surgical resection between 2007 and 2017. PGRN expression was evaluated by immunohistochemical (IHC) analysis and semi-quantitatively categorized (no expression, 0; weak, 1+; moderate, 2+; strong, 3+). Tumors with a staining intensity of 2+ or 3+ were considered high PGRN expression. RESULTS: Fifty-four patients were analyzed; 31 patients (57%) were male. The median age at surgery was 60 years (range, 33-79), and the most common primary site was the stomach (67%). Thirty-five patients (65%) had spindle histology; 42 patients (78%) were separated as a high-risk group according to the modified National Institutes of Health (NIH) classification. High PGRN-expressing tumors were observed in 27 patients (50%), had more epithelioid/mixed histology (68% vs. 32%; p = 0.046), and KIT exon 11 mutations (76% vs. 24%; p = 0.037). Patients with high PGRN-expressing tumors had a worse recurrence-free survival (RFS) (36% of 5-year RFS) compared to those with low PGRN-expressing tumors (96%; p<0.001). Multivariate analysis showed that high PGRN expression and old age (>60 years) were independent prognostic factors for poor RFS. CONCLUSIONS: High PGRN-expressing GISTs showed more epithelioid/mixed histology and KIT exon 11 mutations. PGRN overexpression was significantly associated with poor RFS in patients with GISTs who underwent curative resection.

Progranulin expression induced by follicle-stimulating hormone in ovarian cancer cell lines depends on the histological subtype.

Epithelial ovarian cancer (EOC) is a heterogeneous disease that can be categorized into four major histological subtypes. Its etiology remains poorly understood due mainly to this heterogeneity. Follicle-stimulating hormone (FSH) has been implicated as a risk factor in EOC and has been suggested that may influence the development of specific subtypes. In addition, FSH regulates different aspects of ovarian cancer tumorigenesis. FSH downstream target genes in EOC have not been fully identified. Progranulin (PGRN) overexpression is associated with cell proliferation, invasion, chemoresistance, and shortened overall survival in ovarian cancer. Recently, we demonstrated that PGRN expression is regulated through the PI3K signaling pathway in clear cell ovarian carcinoma (CCOC) cells. In contrast, we also demonstrated that PGRN synthesis in serous ovarian cancer (SOC) cells is regulated via PKC but not by the PI3K signaling pathway. Several studies have demonstrated that FSH induces PKC and PI3K activation. Thus, this study was to investigate the effect of FSH on PGRN production in the CCOC cell line TOV-21G as compared to the SOC cell lines SKOV3 and OVCAR3. Cultured TOV-21G, SKOV3, and OVCAR3 cells were incubated with different concentrations of FSH for 48 h. PGRN mRNA and protein expression were assessed by RT-PCR and Western blotting, while PGRN secretion was measured by ELISA. PGRN mRNA and protein expression, as well as PGRN secretion, significantly increased after FSH stimulation in TOV-21G but not in SKOV3 and OVCAR3 cells. These data indicate that FSH induces PGRN expression and secretion only in CCOC cells. Establishing specific features for CCOC could reveal potential diagnostic and therapeutic targets.

Clinical significance of progranulin correlated with serum soluble Oxford 40 ligand in primary Sjögren's syndrome.

The present study aimed to investigate the association between the expressions of serum progranulin (PGRN) and serum soluble Oxford 40 ligand (sOX40L) and determine their clinical significances in primary Sjögren's syndrome (pSS).The present study included a total of 68 patients with pSS and 50 healthy controls. Demographic data and clinical basic information were collected. Enzyme-linked immunosorbent assay (ELISA) was performed to determine serum levels of PGRN, sOX40L and interleukins. Spearman's correlation coefficient and Mann-Whitney U test were used to determine the correlation between PGRN, and sOX40L and the association between PGRN and sOX40L and disease activity and disease severity.Serum interleukin (IL)-4, IL-6, IL-10, PGRN, and sOX40L levels were significantly higher in pSS patients as compared to the healthy controls. A positive correlation was observed between PGRN and sOX40L. Patients with elevated levels of PGRN or sOX40L exhibited higher disease activity compared to those with lower levels. Patients with III to IV stages of pSS or multiple system damage showed higher serum levels of PGRN and sOX40L.Elevated serum PGRN, and sOX40L levels were relevant with disease activity and severity in patients with pSS.

Premature termination codon readthrough upregulates progranulin expression and improves lysosomal function in preclinical models of GRN deficiency.

BACKGROUND: Frontotemporal lobar degeneration (FTLD) is a devastating and progressive disorder, and a common cause of early onset dementia. Progranulin (PGRN) haploinsufficiency due to autosomal dominant mutations in the progranulin gene (GRN) is an important cause of FTLD (FTLD-GRN), and nearly a quarter of these genetic cases are due to a nonsense mutation. Premature termination codons (PTC) can be therapeutically targeted by compounds allowing readthrough, and aminoglycoside antibiotics are known to be potent PTC readthrough drugs. Restoring endogenous PGRN through PTC readthrough has not previously been explored as a therapeutic intervention in FTLD. METHODS: We studied whether the aminoglycoside G418 could increase PGRN expression in HEK293 and human induced pluripotent stem cell (hiPSC)-derived neurons bearing the heterozygous S116X, R418X, and R493X pathogenic GRN nonsense mutations. We further tested a novel substituted phthalimide PTC readthrough enhancer in combination with G418 in our cellular models. We next generated a homozygous R493X knock-in hiPSC isogenic line (R493X-/- KI), assessing whether combination treatment in hiPSC-derived neurons and astrocytes could increase PGRN and ameliorate lysosomal dysfunction relevant to FTLD-GRN. To provide in vivo proof-of-concept of our approach, we measured brain PGRN after intracerebroventricular administration of G418 in mice expressing the V5-tagged GRN nonsense mutation R493X. RESULTS: The R418X and R493X mutant GRN cell lines responded to PTC readthrough with G418, and treatments increased PGRN levels in R493X-/- KI hiPSC-derived neurons and astrocytes. Combining G418 with a PTC readthrough enhancer increased PGRN levels over G418 treatment alone in vitro. PGRN deficiency has been shown to impair lysosomal function, and the mature form of the lysosomal protease cathepsin D is overexpressed in R493X-/- KI neurons. Increasing PGRN through G418-mediated PTC readthrough normalized this abnormal lysosomal phenotype in R493X-/- KI neuronal cultures. A single intracerebroventricular injection of G418 induced GRN PTC readthrough in 6-week-old AAV-GRN-R493X-V5 mice. CONCLUSIONS: Taken together, our findings suggest that PTC readthrough may be a potential therapeutic strategy for FTLD caused by GRN nonsense mutations.

Prognostic Value of Progranulin in Patients with Colorectal Cancer Treated with Curative Resection.

Progranulin (PGRN) has been characterized as an autocrine growth and survival factor and is known to stimulate tumorigenesis and proliferation of several types of cancer cell. However, little is known about the prognostic role of PGRN in colorectal cancer (CRC). A retrospective analysis was performed for patients with colorectal cancer who underwent curative resection between May 2013 and June 2015. PGRN expression in tumor cells was semi-quantitatively categorized (no expression, 0; weak/focal, 1+; moderate/focal or diffuse, 2+; strong/diffuse, 3+), and high expression was considered for tumors graded ≥2+ staining intensity. A total of 109 patients (28 stage I, 32 stage II, and 49 stage III) were analyzed. Thirty-eight patients (35%) had tumors with high PGRN expression, and there was a trend of elevated pre-operative CEA and CA19-9 levels in patients with high PGRN-expressing tumors compared to those with low PGRN-expressing tumors (CEA, 49% vs. 21%; CA19-9, 21% vs. 7%). The 3-year recurrence-free survival (3Y-RFS) and overall survival rates were 83.7% (95% CI, 76.8-90.6) and 96.0% (95% CI, 92.3-99.7), respectively. Patients with high PGRN-expressing tumors had a worse rate of 3Y-RFS (66.8%) compared to those with low PGRN-expressing tumors (92.4%; p = 0.010). Multivariate analysis showed that high PGRN expression, age (>66 years), stage (III), and perineural invasion (+) were independent prognostic factors associated with poor RFS after adjusting for confounding factors including sex, MSI, tumor location, KRAS, and lympho-vascular invasion. PGRN overexpression was significantly associated with poor RFS in patients with CRC who have undergone curative resection.

Role of progranulin in adipose tissue innate immunity.

Regulation of progranulin in adipocytes and its role in inflammation is poorly understood. AIM: (i) to investigate regulation of progranulin in adipocyte differentiation and adipose tissue compartments, (ii) to address progranulin expression in two murine (C57BL/6) models of inflammation. RESULTS: Progranulin expression was induced during adipocyte differentiation. Neither estradiol nor testosterone or metabolic stimuli such as glucose and insulin modified progranulin synthesis. Fatty acids, bile acids and incretins GLP-1 and GIP-1 exerted potent and differential effects on progranulin secretion. LPS, TNF and IL6 significantly increased progranulin secretion. TLR9 agonists decreased and TLR1/2, TLR3, TLR5, and TLR2/6 ligands increased progranulin expression. TLR3-mediated progranulin induction was abrogated by inhibitors of NF-κB and PI3K pathways. Progranulin expression between murine epididymal and subcutaneous adipose tissue did not differ in total adipose tissue, in isolated adipocytes or in the stromal-vascular cell fraction (SVC). However, SVC expressed significantly higher levels of progranulin than adipocytes at all sites. In adipocytes, female mice had significantly higher progranulin expression at all sites. An intra-peritoneal LPS challenge in mice did not affect adipose tissue progranulin expression, whereas peritoneal infection by S. aureus increased progranulin expression after 24 h. CONCLUSIONS: There are relevant sex-, site- and cell-specific effects on progranulin gene expression that is induced during adipocyte differentiation and modulated by various inflammatory and metabolic factors. Most importantly, ligands for TLR1/2 and TLR2/6 (recognizing S. aureus) in vitro and infection by S. aureus in vivo induce progranulin expression suggesting a role of adipocytes in protection against infection by gram-positive bacteria.

Extensive intraspecies cryptic variation in an ancient embryonic gene regulatory network.

Innovations in metazoan development arise from evolutionary modification of gene regulatory networks (GRNs). We report widespread cryptic variation in the requirement for two key regulatory inputs, SKN-1/Nrf2 and MOM-2/Wnt, into the C. elegans endoderm GRN. While some natural isolates show a nearly absolute requirement for these two regulators, in others, most embryos differentiate endoderm in their absence. GWAS and analysis of recombinant inbred lines reveal multiple genetic regions underlying this broad phenotypic variation. We observe a reciprocal trend, in which genomic variants, or knockdown of endoderm regulatory genes, that result in a high SKN-1 requirement often show low MOM-2/Wnt requirement and vice-versa, suggesting that cryptic variation in the endoderm GRN may be tuned by opposing requirements for these two key regulatory inputs. These findings reveal that while the downstream components in the endoderm GRN are common across metazoan phylogeny, initiating regulatory inputs are remarkably plastic even within a single species.

Progranulin Promotes Bleomycin-Induced Skin Sclerosis by Enhancing Transforming Growth Factor-ß/Smad3 Signaling through Up-Regulation of Transforming Growth Factor-ß Type I Receptor.

Progranulin (PGRN) is an autocrine growth factor with numerous physiological and pathologic roles. Previous reports demonstrated PGRN could increase dermal fibroblasts in wound healing and activate cancer-associated fibroblasts in some cancers. Because systemic sclerosis (SSc) is a prototypical fibrosis-related disorder, here, the aim was to clarify the role and mechanism of PGRN in bleomycin (BLM)-induced model of SSc for the first time. It was observed that the serum PGRN levels were increased in SSc patients compared with healthy controls. Immunohistology and quantitative RT-PCR demonstrated that PGRN was also elevated in the lesion from the mice model of BLM-induced dermal fibrosis. In addition, in BLM-treated mice, PGRN deficiency not only attenuated dermal fibrosis but also decreased the differentiation of myofibroblasts. The reduced progression of skin sclerosis in PGRN-deficient mice was associated with down-regulation of transforming growth factor (TGF)-ß receptor I (TßR I) and decreased level of phosphorylated Smad3, with correspondingly impaired expression of its downstream target gene connective tissue growth factor (CTGF) in skin lesion. In contrast, exogenous PGRN significantly increased the level of TßR I and phosphorylated Smad3 in cultured mouse fibroblasts. This study demonstrates that PGRN plays a promoting role in the development of dermal fibrosis through the activation of the TGF-ß/Smad3 signaling via up-regulation of TßR I. PGRN may be a new therapeutic target in SSc.

TDP-43 accelerates deadenylation of target mRNAs by recruiting Caf1 deadenylase.

TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein, whose loss-of-function mutation causes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. Recent studies demonstrated that TDP-43 binds to the 3' untranslated region (UTR) of target mRNAs to promote mRNA instability. Here, we show that TDP-43 recruits Caf1 deadenylase to mRNA targets and accelerates their deadenylation. Tethering TDP-43 to the mRNA 3'UTR recapitulates destabilization of the mRNA, and TDP-43 accelerates their deadenylation. This accelerated deadenylation is inhibited by a dominant negative mutant of Caf1. We find that TDP-43 physically interacts with Caf1. In addition, we provide evidence that TDP-43 regulates poly(A) tail length of endogenous Progranulin (GRN) mRNA. These results may shed light on the link between dysregulation of TDP-43-mediated mRNA deadenylation and pathogenesis of neurodegenerative diseases.

Peripheral GRN mRNA and Serum Progranulin Levels as a Potential Indicator for Both the Presence of Splice Site Mutations and Individuals at Risk for Frontotemporal Dementia.

Progranulin (GRN) gene mutations are a major cause of frontotemporal dementia (FTD). Most mutations identified to date are null mutations, which are predicted to cause the pathology via haploinsufficiency. Decreased peripheral progranulin protein (PGRN) levels are associated with the presence of GRN null mutations and are accepted as reliable biomarkers. In this study, our aim was to test whether the presence of specific GRN splice site mutations (c.- 8+2T>G and c.708+6_9del), could be predicted by peripheral mRNA or protein GRN levels, by studying affected and asymptomatic individuals from FTD families. We also tested four missense GRN variants to assess if altered GRN levels depended on the type of mutation.Our results confirmed a reduction in both mRNA and protein PGRN levels in the splice site mutation carriers, which is consistent with previous reports for null mutations. Our results also suggested that both decreased peripheral GRN mRNA and serum PGRN levels indicate the presence of pathogenic mutations in affected individuals, and identify the asymptomatic individuals at risk, without previous knowledge of genetic status. Both inferences suggest a potential use of peripheral GRN mRNA or serum PGRN levels as biomarkers for families with FTD.