Efficient gene transduction in pigs and macaques with the engineered AAV vector AAV.GT5 for hemophilia B gene therapy.

Gene therapy using adeno-associated virus (AAV)-based vectors has become a realistic therapeutic option for hemophilia. We examined the potential of a novel engineered liver-tropic AAV3B-based vector, AAV.GT5, for hemophilia B gene therapy. In vitro transduction with AAV.GT5 in human hepatocytes was more than 100 times higher than with AAV-Spark100, another bioengineered vector used in a clinical trial. However, liver transduction following intravenous injection of these vectors was similar in mice with a humanized liver and in macaques. This discrepancy was due to the low recovery and short half-life of AAV.GT5 in blood, depending on the positive charge of the heparin-binding site in the capsid. Bypassing systemic clearance with the intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced liver transduction in pigs and macaques. AAV.GT5 did not develop neutralizing antibodies (NAbs) in two of four animals, while AAV-Spark100 induced serotype-specific NAbs in all macaques tested (4 of 4). The NAbs produced after AAV-Spark100 administration were relatively serotype specific, and challenge with AAV.GT5 through the hepatic artery successfully boosted liver transduction in one animal previously administered AAV-Spark100. In summary, AAV.GT5 showed different vector kinetics and NAb induction compared with AAV-Spark100, and intra-hepatic vascular administration may minimize the vector dose required and vector dissemination.

PAM-flexible Cas9-mediated base editing of a hemophilia B mutation in induced pluripotent stem cells.

BACKGROUND: Base editing via CRISPR-Cas9 has garnered attention as a method for correcting disease-specific mutations without causing double-strand breaks, thereby avoiding large deletions and translocations in the host chromosome. However, its reliance on the protospacer adjacent motif (PAM) can limit its use. We aimed to restore a disease mutation in a patient with severe hemophilia B using base editing with SpCas9-NG, a modified Cas9 with the board PAM flexibility. METHODS: We generated induced pluripotent stem cells (iPSCs) from a patient with hemophilia B (c.947T>C; I316T) and established HEK293 cells and knock-in mice expressing the patient's F9 cDNA. We transduced the cytidine base editor (C>T), including the nickase version of Cas9 (wild-type SpCas9 or SpCas9-NG), into the HEK293 cells and knock-in mice through plasmid transfection and an adeno-associated virus vector, respectively. RESULTS: Here we demonstrate the broad PAM flexibility of SpCas9-NG near the mutation site. The base-editing approach using SpCas9-NG but not wild-type SpCas9 successfully converts C to T at the mutation in the iPSCs. Gene-corrected iPSCs differentiate into hepatocyte-like cells in vitro and express substantial levels of F9 mRNA after subrenal capsule transplantation into immunodeficient mice. Additionally, SpCas9-NG-mediated base editing corrects the mutation in both HEK293 cells and knock-in mice, thereby restoring the production of the coagulation factor. CONCLUSION: A base-editing approach utilizing the broad PAM flexibility of SpCas9-NG can provide a solution for the treatment of genetic diseases, including hemophilia B.

In patients with hemophilia B, the blood does not clot properly, leading to excessive bruising and bleeding. Hemophilia B is caused by an error in a gene called coagulation factor IX (F9). To treat patients with hemophilia B, we might be able to use a technology called CRISPR-Cas9 to edit and correct this genetic error, restoring factor IX function and improving clotting. Here, we test a specific CRISPR-Cas9 approach in cells and animals. We show that we are able to correct the genetic error in F9 in cells isolated from a patient with severe hemophilia B. We also show that we can fix the error in mice and that this increases levels of factor IX in the blood of the mice. With further testing, this gene-editing approach may be a viable therapy for patients with hemophilia B or similar genetic disorders.

Successful liver transduction by re-administration of different adeno-associated virus vector serotypes in mice.

BACKGROUND: Intravenous administration of adeno-associated virus (AAV) vectors is a promising gene therapy approach for monogenic diseases. However, re-administration of the same AAV serotype is impossible because of the induction of anti-AAV neutralizing antibodies (NAbs). Here, we examined the feasibility of re-administrating AAV vector serotypes different from the initial AAV vector serotype. METHODS: Liver-targeting AAV3B, AAV5, and AAV8 vectors were intravenously injected in C57BL/6 mice, and the emergence of NAbs and the transduction efficacy following re-administration were evaluated. RESULTS: For all serotypes, re-administration of the same serotype was not possible. Although the highest neutralizing activity of NAb was induced by AAV5, anti-AAV5 NAbs did not react with other serotypes, resulting in successful re-administration with the other serotypes. AAV5 re-administration was also successful in all mice treated with AAV3B and AAV8. Effective secondary administration of AAV3B and AAV8 was observed in most mice initially administrated AAV8 and AAV3B, respectively. However, few mice developed NAbs cross-reacting with the other serotypes, especially those with close sequence homology. CONCLUSIONS: In summary, AAV vector administration induced NAbs relatively specific to the administrated serotype. Secondary administration of AAVs targeting liver transduction could be successfully achieved by switching AAV serotypes in mice.

The seroprevalence of neutralizing antibodies against the adeno-associated virus capsids in Japanese hemophiliacs.

Adeno-associated virus (AAV) vectors are promising modalities of gene therapy to address unmet medical needs. However, anti-AAV neutralizing antibodies (NAbs) hamper the vector-mediated therapeutic effect. Therefore, NAb prevalence in the target population is vital in designing clinical trials with AAV vectors. Hence, updating the seroprevalence of anti-AAV NAbs, herein we analyzed sera from 100 healthy individuals and 216 hemophiliacs in Japan. In both groups, the overall seroprevalence against various AAV serotypes was 20%-30%, and the ratio of the NAb-positive population increased with age. The seroprevalence did not differ between healthy participants and hemophiliacs and was not biased by the concomitant blood-borne viral infections. The high neutralizing activity, which strongly inhibits the transduction with all serotypes in vitro, was mostly found in people in their 60s or of older age. The multivariate analysis suggested that "60s or older age" was the only independent factor related to the high titer of NAbs. Conversely, a large proportion of younger hemophiliacs was seronegative, rendering them eligible for AAV-mediated gene therapy in Japan. Compared with our previous study, the peak of seroprevalences has shifted to older populations, indicating that natural AAV exposure in the elderly occurred in their youth but not during the last decade.

IκBζ regulates the development of nonalcoholic fatty liver disease through the attenuation of hepatic steatosis in mice.

IκBζ is a transcriptional regulator that augments inflammatory responses from the Toll-like receptor or interleukin signaling. These innate immune responses contribute to the progression of nonalcoholic fatty liver disease (NAFLD); however, the role of IκBζ in the pathogenesis of NAFLD remains elusive. We investigated whether IκBζ was involved in the progression of NAFLD in mice. We generated hepatocyte-specific IκBζ-deficient mice (Alb-Cre; Nfkbizfl/fl) by crossing Nfkbizfl/fl mice with Alb-Cre transgenic mice. NAFLD was induced by feeding the mice a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). CDAHFD-induced IκBζ expression in the liver was observed in Nfkbizfl/fl mice, but not in Alb-Cre; Nfkbizfl/fl mice. Contrary to our initial expectation, IκBζ deletion in hepatocytes accelerated the progression of NAFLD after CDAHFD treatment. Although the increased expression of inflammatory cytokines and apoptosis-related proteins by CDAHFD remained unchanged between Nfkbizfl/fl and Alb-Cre; Nfkbizfl/fl mice, early-stage steatosis of the liver was significantly augmented in Alb-Cre; Nfkbizfl/fl mice. Overexpression of IκBζ in hepatocytes via the adeno-associated virus vector attenuated liver steatosis caused by the CDAHFD in wild-type C57BL/6 mice. This preventive effect of IκBζ overexpression on steatosis was not observed without transcriptional activity. Microarray analysis revealed a correlation between IκBζ expression and the changes of factors related to triglyceride biosynthesis and lipoprotein uptake. Our data suggest that hepatic IκBζ attenuates the progression of NAFLD possibly through the regulation of the factors related to triglyceride metabolism.

A sensitive and reproducible cell-based assay via secNanoLuc to detect neutralizing antibody against adeno-associated virus vector capsid.

Most gene therapy clinical trials that systemically administered adeno-associated virus (AAV) vector enrolled only patients without anti-AAV-neutralizing antibodies. However, laboratory tests to measure neutralizing antibodies varied among clinical trials and have not been standardized. In this study, we attempted to improve the sensitivity and reproducibility of a cell-based assay to detect neutralizing antibodies and to determine the detection threshold to predict treatment efficacy. Application of the secreted type of NanoLuc and AAV receptor-expressing cells reduced the multiplicity of infection (MOI) for AAV transduction and improved the sensitivity to detect neutralizing antibodies with a low coefficient of variation, whereas the detection threshold could not be improved by the reduction of MOI to <100. After human immunoglobulin administration into mice at various doses, treatment with high-dose AAV8 vector enabled evasion of the inhibitory effect of neutralizing antibodies. Conversely, gene transduction was slightly influenced in the mice treated with low-dose AAV8 vector, even when neutralizing antibodies were determined to be negative in the assay. In conclusion, we developed a reliable and sensitive cell-based assay to measure neutralizing antibodies against AAV and found that the appropriate MOI to detect marginal neutralizing antibodies was 100. Other factors, including noninhibitory antibodies, marginally influence in vivo transduction at low vector doses.

Characterization and visualization of murine coagulation factor VIII-producing cells in vivo.

Coagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of the F8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31high, CD146high, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)+. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin- and C-type lectin-like receptor-2 (CLEC-2)+. In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31high, CD146high, Lyve1+, CLEC-2+, and podoplanin- in liver sinusoidal endothelial cells.

Alloreactive T Cells Display a Distinct Chemokine Profile in Response to Conditioning in Xenogeneic GVHD Models.

BACKGROUND: Chemokines and chemokine receptors are potential targets for the prevention and treatment of graft-versus-host disease (GVHD). The objective of the current study is to determine the clinical relevance of xenogeneic transplantation models in terms of host and donor chemokine profiles and, if this is the case, to assess the clinical efficacy of C-C chemokine receptor (CCR) 5 antagonist maraviroc for the prevention of GVHD using this model. METHODS: Xenogeneic GVHD was induced by intravenous injection of 5 × 10 human pan T cells into NOD/Shi-scid-IL2rγ (NOG) mice or MHC class I/II-deficient NOG mice in the presence or absence of total body irradiation before transplantation. RESULTS: Extensive tissue destruction with human T-cell infiltration was observed throughout the body, particularly in lungs and liver, but relatively mild in gut. Consistent with this finding, quantitative polymerase chain reaction confirmed the upregulation of mouse CXC chemokine ligand (CXCL) 9 and CXCL10 in lungs and CCL4 in lungs and liver but not in gut. The addition of total body irradiation (1) led to the early release of mouse CCL4 and CXCL10, (2) upregulated a number of chemokine-related genes in human T cells, (3) induced higher expression of CCR5 on human CD4 and CD8 T cells and CXCR3 on human CD4 T cells, and (4) promoted their migration and proliferation in organs, resulting in more severe tissue damage. In this context, pharmacological CCR5 blockade neither ameliorated GVHD nor prolonged survival in NOG mice. CONCLUSIONS: Our experimental data do not demonstrate clinical benefit of CCR5 antagonist for the prevention of GVHD in a myeloablative setting.

Interleukin-4-induced ß-catenin regulates the conversion of macrophages to multinucleated giant cells.

The cytokine interleukin-4 (IL-4) exerts pleiotropic effects on macrophages as it plays a key role in the immune response to infectious agents, allergens, and vaccines. Macrophages exposed to IL-4 drastically change their gene expression and metabolic state to adjust to new functional requirements. IL-4 also induces macrophages to fuse together and form multinucleated giant cells (MGCs). MGC formation is associated with chronic inflammation resulting from persistence of pathogenic microorganisms or foreign materials in tissues. Very little is known, however, about the mechanisms regulating IL-4-induced macrophage-to-MGC conversion. We observed a dramatic increase in ß-catenin protein but not mRNA amount in mouse macrophages following exposure to IL-4. To investigate the role of ß-catenin in macrophages, we generated mice with a myeloid cell-specific deletion of the ß-catenin gene. Ablation of ß-catenin expression did not affect the viability of macrophages or impair expression of known IL-4-inducible genes. Intriguingly, ß-catenin-deficient macrophages incubated with IL-4 formed MGCs with markedly greater efficiency than wild-type macrophages. Similar increases in multinucleated cell formation were detected in the peritoneal cavity of myeloid cell-specific ß-catenin knockout mice injected with chitin, which is known to induce endogenous IL-4 production. Our findings reveal ß-catenin as a novel regulator of macrophage responses to IL-4, and suggest that therapeutic modulation of its expression or function may help enhance the effectiveness or ameliorate the pathology of IL-4-driven immune responses.

YM-175 induces apoptosis of human native monocyte-lineage cells via inhibition of prenylation.

Nitrogen-containing bisphosphonates (NCBPs) have been widely used as standard supportive therapy to reduce skeletal-related events (SREs) in myeloma patients through suppression of osteoclast activity. In various prospective randomized trials that were performed following preliminary reports concerning efficacy, NCBPs have shown a significant beneficial effect on myeloma bone disease through both suppression of bone resorption and direct antimyeloma activity. Thus, NCBPs have an influence on many types of human cells. In this study, we examined the effect of an NCBP (YM-175) on an apoptosis of a monocytic cell line and of human native monocytes/macrophages and dendritic cells (DCs). We confirmed that monocytes, monocyte-derived macrophages, DCs, and a monoblastic cell line (THP-1) showed dose-dependent and time-dependent apoptosis related to the activation of caspases after exposure to YM-175 at concentrations below that at which the apoptosis of myeloma cell lines was induced. Such apoptosis of monocytic cells was suppressed by the addition of farnesol or geranylgeraniol. These findings suggest that the inhibition of monocyte-lineage cells or DCs by NCBPs might interfere with phagocytic activity or pathogen-presenting activity.

Increased levels of interleukin 33 in sera and synovial fluid from patients with active rheumatoid arthritis.

OBJECTIVE: To determine levels of interleukin 33 (IL-33) in serum and synovial fluid (SF) and their clinical associations in patients with rheumatoid arthritis (RA). To evaluate the ability of activated peripheral blood mononuclear cells (PBMC) and fibroblast-like synoviocytes (FLS) from RA patients to release IL-33. METHODS: Sera were obtained from 59 patients with RA, 10 patients with infectious diseases, and 42 healthy volunteers. SF samples were obtained from 15 patients with RA and 13 with osteoarthritis. IL-33 levels were measured using a sandwich ELISA after removal of rheumatoid factor with protein A-Sepharose beads. FLS were stimulated with IL-1beta and tumor necrosis factor, and treated with or without chemical damage. PBMC were stimulated with anti-CD3/CD28 antibodies. The levels of IL-33 were measured in the culture supernatants and cell lysates by ELISA or immunoblotting. RESULTS: Serum IL-33 levels were significantly higher in RA patients, especially in the high disease activity group compared to the moderate or low activity group. IL-33 levels in SF were elevated in all 15 RA patients measured. IL-33 levels were higher in SF samples than in sera in 7 RA patients measured simultaneously. The 30-kDa IL-33 precursor was detected in the culture supernatants of damaged FLS but was not detected in those of activated PBMC and non-damaged FLS. CONCLUSION: IL-33 levels were elevated in sera and SF samples from patients with RA, and correlated with disease activity. IL-33 was produced mainly in inflamed joints; IL-33/ST2L signaling might play an important role in joint inflammation of human RA.

Mature interleukin-33 is produced by calpain-mediated cleavage in vivo.

Interleukin (IL)-33 is a novel member of the IL-1 family. IL-33 is primarily synthesized as a 30-kDa precursor (pro-IL-33). Pro-IL-33 is cleaved by caspase-1 into an 18-kDa mature form (mature IL-33) in vitro. Recombinant mature IL-33 has been known to induce T-helper type-2 (Th2)-associated cytokines and inflammatory cytokines via its receptor, ST2L. However, processing of pro-IL-33 in vivo has not been clarified yet. Here, we report that calpain mediates pro-IL-33 processing in vivo. Pro-IL-33 was expressed by stimulating human epithelial cells with phorbol 12-myristate 13-acetate. Calcium ionophore induced pro-IL-33 cleavage and mature IL-33 production. This cleavage was inhibited by treatment with a calcium chelator and calpain inhibitors. Moreover, short interfering RNA-mediated knockdown of calpains suppressed pro-IL-33 cleavage. These results indicate that calpains play a critical role in pro-IL-33 processing in vivo.

TRAF6 is a critical signal transducer in IL-33 signaling pathway.

IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L. However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38, JNK and Nuclear factor-kappaB (NF-kappaB) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappaB in TRAF6 deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAF6 and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappaB activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity.

Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation.

The ST2 gene produces a soluble secreted form and a transmembrane form, referred to as soluble ST2 and ST2L, respectively. A recent study has reported that interleukin (IL)-33 is a specific ligand of ST2L and induces production of T helper type 2 (Th2) cytokines. Although soluble ST2 is highly produced in sera of asthmatic patients and plays a critical role for production of Th2 cytokines, the function of soluble ST2 in relation to IL-33 signaling remains unclear. Here we show antagonistic effects of soluble ST2 on IL-33 signaling using a murine thymoma EL-4 cells stably expressing ST2L and a murine model of asthma. Soluble ST2 directly bound to IL-33 and suppressed activation of NF-kappaB in EL-4 cells stably expressing ST2L, suggesting that the complex of soluble ST2 and IL-33 fails to bind to ST2L. In a murine model of asthma, pretreatment with soluble ST2 reduced production of IL-4, IL-5, and IL-13 from IL-33-stimulated splenocytes. These results indicate that soluble ST2 acts as a negative regulator of Th2 cytokine production by the IL-33 signaling. Our study provides a molecular mechanism wherein soluble ST2 modulates the biological activity of IL-33 in allergic airway inflammation.

SNIP1 is a candidate modifier of the transcriptional activity of c-Myc on E box-dependent target genes.

Using a yeast two-hybrid screen, we found that SNIP1 (Smad nuclear-interacting protein 1) associates with c-Myc, a key regulator of cell proliferation and transformation. We demonstrate that SNIP1 functions as an important regulator of c-Myc activity, binding the N terminus of c-Myc through its own C terminus, and that SNIP1 enhances the transcriptional activity of c-Myc both by stabilizing it against proteosomal degradation and by bridging the c-Myc/p300 complex. These effects of SNIP1 on c-Myc likely contribute to synergistic effects of SNIP1, c-Myc, and H-Ras in inducing formation of foci in an in vitro transformation assay and also in supporting anchorage-independent growth. The significant association of SNIP1 and c-Myc staining in a non-small cell lung cancer tissue array is further evidence that their activities might be linked and suggests that SNIP1 might be an important modulator of c-Myc activity in carcinogenesis.

ST2 suppresses IL-6 production via the inhibition of IkappaB degradation induced by the LPS signal in THP-1 cells.

LPS induces the production of inflammatory cytokines via the stimulation of Toll-like receptors. In this study, we demonstrated that a soluble secreted form of the ST2 gene product (ST2), a member of the interleukin-1 receptor family, suppressed the production of IL-6 in an LPS-stimulated human monocytic leukemia cell line, THP-1. Immunofluorescence confocal microscopy revealed the binding of ST2 to the surface of the THP-1 cells, in which ST2 led to decreased binding of nuclear factor-kappaB to the IL-6 promoter. Furthermore, the degradation of IkappaB in the cytoplasm after LPS stimulation was reduced by pretreatment with ST2. These results demonstrated that ST2 negatively regulates LPS-induced IL-6 production via the inhibition of IkappaB degradation in THP-1 cells.

T-helper type 2 cell-specific expression of the ST2 gene is regulated by transcription factor GATA-3.

ST2 is a member of the interleukin-1 receptor family and is expressed in type-2 T helper (Th2) cells. Here, we have studied the molecular mechanism responsible for the transcriptional regulation of the ST2 gene in Th2 cells using a mouse thymoma cell line, EL-4. The ST2 gene has distal and proximal promoters. ST2 mRNA was produced from the distal promoter in EL-4 cells stimulated with both phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (Bt2cAMP). The region of approximately 100 bp upstream of transcription start site, containing two GATA consensus sites, was indispensable for the activation of the distal promoter in reporter gene analysis. An electrophoretic mobility shift assay showed that transcription factor GATA-3 bound one of the GATA consensus sites (from -84 to -79) with nuclear extracts from PMA plus Bt2cAMP-stimulated EL-4 cells. The overexpression of GATA-3 enhanced the activity of the distal promoter. On the other hand, mutations of the GATA consensus site canceled out the enhancement by GATA-3. These data suggest that GATA-3 is an important transcription factor for the expression of the ST2 gene in Th2 cells.

A novel splice variant of mouse interleukin-1-receptor-associated kinase-1 (IRAK-1) activates nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK).

Interleukin-1 (IL-1)-receptor-associated kinase (IRAK) is an indispensable signalling molecule for host-defence responses initiated by a variety of ligands that bind to members of the Toll/IL-1 receptor family. Here we report a novel splice variant of mouse IRAK-1, IRAK-1-S, which is generated by utilizing a new splicing acceptor site within exon 12. IRAK-1-S cDNA is shorter than the originally reported IRAK-1 (IRAK-1-W) cDNA by 271 nucleotides, and the subsequent frameshift causes a premature termination of translation after 23 amino acids, which are unique to the IRAK-1-S protein. To elucidate the physiological function of IRAK-1-S, we overexpressed it in 293T cells and studied the effects on the IL-1 signalling cascade. As it lacks the C-terminal region of IRAK-1-W that has been reported to contain the TRAF6 (tumour necrosis factor receptor-associated factor 6) binding domain, IRAK-1-S was unable to bind TRAF6 protein, which is a proposed downstream signalling molecule. However, IRAK-1-S overexpressed in 293T cells induced constitutive activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK) independent of stimulation by IL-1, as did IRAK-1-W. To clarify the mechanism of NF-kappaB activation by IRAK-1-S in the absence of binding to TRAF6, we demonstrated that IRAK-1-S binds to IRAK-1-W through its death domain; the findings suggested that overexpressed IRAK-1-S may bind endogenous IRAK-1-W and activate TRAF6 through IRAK-1-W. These results also indicate that this novel variant may play roles in the activation of NF-kappaB and JNK by IL-1 and other ligands whose signal transduction is dependent on IRAK-1 under physiological conditions.

Muscle-specific exonic splicing silencer for exon exclusion in human ATP synthase gamma-subunit pre-mRNA.

Mitochondrial ATP synthase gamma-subunit (F(1)gamma) pre-mRNA undergoes alternative splicing in a tissue- or cell type-specific manner. Exon 9 of F(1)gamma pre-mRNA is specifically excluded in heart and skeletal muscle tissues and in acid-stimulated human fibrosarcoma HT1080 cells, rhabdomyosarcoma KYM-1 cells, and mouse myoblast C2C12 cells. Recently, we found a purine-rich exonic splicing enhancer (ESE) element on exon 9 via transgenic mice bearing F(1)gamma mutant minigenes and demonstrated that this ESE functions ubiquitously with exception of muscle tissue (Ichida, M., Hakamata, Y., Hayakawa, M., Ueno E., Ikeda, U., Shimada, K., Hamamoto, T., Kagawa, Y., Endo, H. (2000) J. Biol. Chem. 275, 15992-16001). Here, we identified an exonic negative regulatory element responsible for muscle-specific exclusion of exon 9 using both in vitro and in vivo splicing systems. A supplementation assay with nuclear extracts from HeLa cells and acid-stimulated HT1080 cells was performed for an in vitro reaction of muscle-specific alternative splicing of F(1)gamma minigene and revealed that the splicing reaction between exons 8 and 9 was the key step for regulation of muscle-specific exon exclusion. Polypyrimidine tract in intron 8 requires ESE on exon 9 for constitutive splice site selection. Mutation analyses on the F(1)gammaEx8-9 minigene using a supplementation assay demonstrated that the muscle-specific negative regulatory element is positioned in the middle region of exon 9, immediately downstream from ESE. Detailed mutation analyses identified seven nucleotides (5'-AGUUCCA-3') as a negative regulatory element responsible for muscle-specific exon exclusion. This element was shown to cause exon skipping in in vivo splicing systems using acid-stimulated HT1080 cells after transient transfection of several mutant F(1)gammaEx8-9-10 minigenes. These results demonstrated that the 5'-AGUUCCA-3' immediately downstream from ESE is a muscle-specific exonic splicing silencer (MS-ESS) responsible for exclusion of exon 9 in vivo and in vitro.