Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice.

Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, however, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues. In general, AAV vectors are used for in vivo delivery, but AAV injections cannot be repeated because of neutralization antibodies. Here we report a chemically defined lipid nanoparticle (LNP) system which is able to deliver Cas9 mRNA and sgRNA into skeletal muscle by repeated intramuscular injections. Although the expressions of Cas9 protein and sgRNA were transient, our LNP system could induce stable genomic exon skipping and restore dystrophin protein in a DMD mouse model that harbors a humanized exon sequence. Furthermore, administration of our LNP via limb perfusion method enables to target multiple muscle groups. The repeated administration and low immunogenicity of our LNP system are promising features for a delivery vehicle of CRISPR-Cas9 to treat skeletal muscle disorders.

Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses.

BACKGROUND: High-temperature fermentation technology with thermotolerant microbes has been expected to reduce the cost of bioconversion of cellulosic biomass to fuels or chemicals. Thermotolerant Kluyveromyces marxianus possesses intrinsic abilities to ferment and assimilate a wide variety of substrates including xylose and to efficiently produce proteins. These capabilities have been found to exceed those of the traditional ethanol producer Saccharomyces cerevisiae or lignocellulose-bioconvertible ethanologenic Scheffersomyces stipitis. RESULTS: The complete genome sequence of K. marxianus DMKU 3-1042 as one of the most thermotolerant strains in the same species has been determined. A comparison of its genomic information with those of other yeasts and transcriptome analysis revealed that the yeast bears beneficial properties of temperature resistance, wide-range bioconversion ability, and production of recombinant proteins. The transcriptome analysis clarified distinctive metabolic pathways under three different growth conditions, static culture, high temperature, and xylose medium, in comparison to the control condition of glucose medium under a shaking condition at 30°C. Interestingly, the yeast appears to overcome the issue of reactive oxygen species, which tend to accumulate under all three conditions. CONCLUSIONS: This study reveals many gene resources for the ability to assimilate various sugars in addition to species-specific genes in K. marxianus, and the molecular basis of its attractive traits for industrial applications including high-temperature fermentation. Especially, the thermotolerance trait may be achieved by an integrated mechanism consisting of various strategies. Gene resources and transcriptome data of the yeast are particularly useful for fundamental and applied researches for innovative applications.

Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9.

Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases.

Delivery of full-length factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A.

Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice. piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

Biochemical features of a catalytic antibody light chain, 22F6, prepared from human lymphocytes.

Human antibody light chains belonging to subgroup II of germ line genes were amplified by a seminested PCR technique using B-lymphocytes taken from a human adult infected with influenza virus. Each gene of the human light chains was transferred into the Escherichia coli system. The recovered light chain was highly purified using a two-step purification system. Light chain 22F6 showed interesting catalytic features. The light chain cleaved a peptide bond of synthetic peptidyl-4-methyl-coumaryl-7-amide (MCA) substrates, such as QAR-MCA and EAR-MCA, indicating amidase activity. It also hydrolyzed a phosphodiester bond of both DNA and RNA. From the analysis of amino acid sequences and molecular modeling, the 22F6 light chain possesses two kinds of active sites as amidase and nuclease in close distances. The 22F6 catalytic light chain could suppress the infection of influenza virus type A (H1N1) of Madin-Darby canine kidney cells in an in vitro assay. In addition, the catalytic light chain clearly inhibited the infection of the influenza virus of BALB/c mice via nasal administration in an in vivo assay. In the experiment, the titer in the serum of the mice coinfected with the 22F6 light chain and H1N1 virus became considerably lowered compared with that of 22F6-non-coinfected mice. Note that the catalytic light chain was prepared from human peripheral lymphocyte and plays an important role in preventing infection by influenza virus. Considering the fact that the human light chain did not show any acute toxicity for mice, our procedure developed in this study must be unique and noteworthy for developing new drugs.

Negative feedback by IRE1ß optimizes mucin production in goblet cells.

In mammals, the prototypical endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme 1 (IRE1) has diverged into two paralogs. IRE1α is broadly expressed and mediates the unconventional splicing of X-box binding protein 1 (XBP1) mRNA during ER stress. By contrast, IRE1ß is expressed selectively in the digestive tract, and its function remains unclear. Here, we report that IRE1ß plays a distinctive role in mucin-secreting goblet cells. In IRE1ß(-/-) mice, aberrant mucin 2 (MUC2) accumulated in the ER of goblet cells, accompanied by ER distension and elevated ER stress signaling such as increased XBP1 mRNA splicing. In contrast, conditional IRE1α(-/-) mice showed no such ER distension but a marked decrease in spliced XBP1 mRNA. mRNA stability assay revealed that MUC2 mRNA was greatly stabilized in IRE1ß(-/-) mice. These findings suggest that in goblet cells, IRE1ß, but not IRE1α, promotes efficient protein folding and secretion in the ER by optimizing the level of mRNA encoding their major secretory product, MUC2.

Highly efficient method of preparing human catalytic antibody light chains and their biological characteristics.

The ultimate goal of catalytic antibody research is to develop new patient therapies that use the advantages offered by human catalytic antibodies. The establishment of a high-throughput method for obtaining valuable candidate catalytic antibodies must be accelerated to achieve this objective. In this study, based on our concept that we can find antibody light chains with a high probability of success if they include a serine protease-like catalytic triad composed of Ser, His, and Asp on a variable region of the antibody structure, we amplified and cloned DNAs encoding human antibody light chains from germline genes of subgroup II by seminested PCR using two primer sets designed for this purpose. Seven DNA fragments encoding light chains in 17 clones were derived from germline gene A18b, 6 DNA fragments from A3/A19, 2 DNA fragments from A17, and a clone DNA fragment from A5 and O11/O1. All light chains expressed in Escherichia coli and highly purified under nondenaturing conditions exhibited amidolytic activity against synthetic peptides. Some of the light chains exhibited unique features that suppressed the infectious activity of the rabies virus. Furthermore, the survival rate of mice in which a lethal level of the rabies virus was coinoculated directly into the brain with light chain 18 was significantly improved. In the case of humans, these results demonstrate that high-throughput selection of light chains possessing catalytic functions and specificity for a target molecule can be attained from a light-chain DNA library amplified from germline genes belonging to subgroup II.

Catalytic and biochemical features of a monoclonal antibody heavy chain, JN1-2, raised against a synthetic peptide with a hemagglutinin molecule of influenza virus.

It has long been an important issue to produce a catalytic antibody that possesses the ability to lose the infectivity of a bacteria or virus. The monoclonal antibody JN1-2 was generated using a synthetic peptide (TGLRNGITNKVNSVIEKAA) conjugated with human IgG. The peptide sequence includes the conserved region of the hemagglutinin molecule (HA(1) and HA(2) domains), which locates on the envelope of the influenza virus and plays an important role in influenza A virus infection. The monoclonal antibody specifically reacted with the HA2 domain, not only of H2 but also of an H1 strain of the H1N1 subtype (H1 strain). The heavy chain (JN1-2-H) isolated from the parent antibody showed catalytic activity cleaving the above antigenic peptide with very high turnover (kcat = 26 min(-1)), and it could slowly degrade the recombinant HA(2) domain by the catalytic function. Interestingly, the heavy chain exhibited the ability to reduce the infectivity of type A H1N1 but not type B, indicating specificity to type A. This characteristic monoclonal catalytic antibody heavy chain could suppress the infection of the influenza virus in vitro assays.

Mammalian ER stress sensor IRE1ß specifically down-regulates the synthesis of secretory pathway proteins.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress. The ER stress sensor inositol requiring enzyme-1beta (IRE1ß), which is specifically expressed in intestinal epithelial cells, is thought to be involved in translational repression. However, its mechanism of action is not fully understood. Using a reporter that can evaluate and distinguish between translation efficiency in the cytosol and on the ER membrane, we show here that IRE1ß represses translation on the ER membrane but not in the cytosol, and that this selective repression depends on the RNase activity of IRE1ß.

Characteristic features of InfA-15 monoclonal antibody recognizing H1, H3, and H5 subtypes of hemagglutinin of influenza virus A type.

Hemagglutinin molecule is an envelope protein of influenza virus and plays an important role in the infection to human cells. Many mutations are observed in the molecule, which generates sixteen subtypes (H1-H16) of the hemagglutinin molecule for influenza virus A type. The subtypes such as H1, H2, H3, and H5 out of the sixteen are underlined molecules, which are responsible to Spain, Asia, Hong Kong, and Avian Flu, respectively. Based on the sequence analysis, three short sequences, which are highly conserved in the subtypes of influenza virus A type, were extracted. The sequence peptides were chemically synthesized and conjugated with BSA for immunization into Balb/c mice. A sequence GMVDGWYG located at the domain of fusion protein in the hemagglutinin molecule exhibited a high immuno-response, resulting in the production of a monoclonal antibody (mAb; InfA-15). The unique features of InfA-15 mAb were investigated from the viewpoint of immunological reaction, the binding affinity, the steric conformation, etc. The InfA-15 mAb could react with the H1, H3, and H5 subtype of hemagglutinin molecule of influenza virus A type. ELISAs using InfA-15 mAb suggested a wide reaction spectrum for the hemagglutinin of many important influenza viruses A type.

Acrylamide degradation by filamentous fungi used in food and beverage industries.

Filamentous fungi (24 strains) used in food and beverage industries were investigated for acrylamide-degradation ability: Aspergillus oryzae KBN1010 showed the highest ability. Little acrylic acid was produced but no glycidamide was detected during AA degradation in roasted green tea; therefore, A. oryzae could be used for reducing the AA concentration.

Radiotherapy quality assurance review in a multi-center randomized trial of limited-disease small cell lung cancer: the Japan Clinical Oncology Group (JCOG) trial 0202.

BACKGROUND: The purpose of this study was to analyze the radiotherapy (RT) quality assurance (QA) assessment in Japan Clinical Oncology Group (JCOG) 0202, which was the first trial that required on-going RT QA review in the JCOG. METHODS: JCOG 0202 was a multi-center phase III trial comparing two types of consolidation chemotherapy after concurrent chemoradiotherapy for limited-disease small cell lung cancer. RT requirements included a total dose of 45 Gy/30 fx (bis in die, BID/twice a day) without heterogeneity correction; elective nodal irradiation (ENI) of 30 Gy; at least 1 cm margin around the clinical target volume (CTV); and interfraction interval of 6 hours or longer. Dose constraints were defined in regards to the spinal cord and the lung. The QA assessment was classed as per protocol (PP), deviation acceptable (DA), violation unacceptable (VU), and incomplete/not evaluable (I/NE). RESULTS: A total of 283 cases were accrued, of which 204 were fully evaluable, excluding 79 I/NE cases. There were 18 VU in gross tumor volume (GTV) coverage (8% of 238 evaluated); 4 VU and 23 DA in elective nodal irradiation (ENI) (2% and 9% of 243 evaluated, respectively). Some VU were observed in organs at risk (1 VU in the lung and 5 VU in the spinal cord). Overall RT compliance (PP + DA) was 92% (187 of 204 fully evaluable). Comparison between the former and latter halves of the accrued cases revealed that the number of VU and DA had decreased. CONCLUSION: The results of the RT QA assessment in JCOG 0202 seemed to be acceptable, providing reliable results.

Relation between elective nodal failure and irradiated volume in non-small-cell lung cancer (NSCLC) treated with radiotherapy using conventional fields and doses.

INTRODUCTION: The role of elective nodal irradiation of non-small-cell lung cancer (NSCLC) patients treated with radiotherapy remains unclear. We investigated the significance of treating clinically uninvolved lymph nodes by retrospectively analyzing the relationship between loco-regional failure and the irradiated volume. METHODS: Between 1998 and 2003, patients with IA-IIIB NSCLC were treated with radiotherapy. The eligibility criteria for this study were an irradiation dose of 60Gy or more and a clinical response better than stable disease. Typical radiotherapy consisted of 40 Gy/20 fr to the tumor volumes (clinical target volume of the primary tumor [CTVp], of the metastatic lymph nodes [CTVn], and of the subclinical nodal region [CTVs]), followed by off-cord boost to CTVp+n to a total dose 60-68 Gy/30-34 fr. The relationship between the sites of recurrence and irradiated volumes was analyzed. RESULTS: A total of 127 patients fulfilled the eligibility criteria. Their median overall and progression-free survival times were 23.5 (range, 4.2-109.7) and 9.0 months (2.2-109.7), respectively. At a median follow-up time of 50.5 months (range, 14.2-83.0) for the surviving patients, the first treatment failure was observed in 95 patients (loco-regional; 41, distant; 42, both; 12). Among the patients with loco-regional failure, in-field recurrence occurred in 38 patients, and four CTVs recurrences associated with CTVp+n failure were observed. No isolated recurrence in CTVs was observed. CONCLUSIONS: In-field loco-regional failure, as well as distant metastasis, was a major type of failure, and there was no isolated elective nodal failure. Radiation volume adequacy did not seem to affect elective nodal failure.

Pattern of tumor recurrence in initially nonmetastatic breast cancer patients: distribution and frequency of metastases at unusual sites.

BACKGROUND: Breast cancer is likely to have systemic involvement. However, to the authors' knowledge there are few reports to date regarding clinically detected patterns of metastasis, meticulously studied in regard to the natural history of breast cancer, including unusual sites of metastasis. METHODS: Patients treated for invasive breast cancer from April 1983 to May 2007 were retrospectively analyzed. Patterns of clinically apparent tumor recurrence, focusing especially on unusual metastases, were studied as well as possible risk factors for unusual metastases and their influence on survival. RESULTS: Overall, 3783 patients were eligible for the current analysis. The median duration of follow-up was 5.0 years (range, 0.6 years-20.4 years). Cumulative 5-year and 10-year survival rates were 89.7% and 81.5%, respectively. "Unusual metastasis" was defined as systemic failure with a frequency of < or =1%; in the current series; it was observed in 85 (2.2%) patients %. Of those, 70 (82%) had preceding metastasis in the usual sites. The median duration until the development of usual and unusual metastasis was 2.3 years and 3.6 years, respectively (P < .0001). Among 764 patients with distant metastasis, the 5-year cumulative overall survival rate in those with or without unusual metastasis was 53.5% and 53.4 years, respectively (P = .33). No risk factors for unusual metastasis were identified. CONCLUSIONS: This retrospective study examined the frequency of unusual metastases in a large number of Japanese patients with initially nonmetastatic breast cancer. The prognosis of patients with unusual metastases was found to be similar to that of patients with metastasis only at more usual sites.

Evaluation of acute intestinal toxicity in relation to the volume of irradiated small bowel in patients treated with concurrent weekly gemcitabine and radiotherapy for locally advanced pancreatic cancer.

BACKGROUND: Treatment of concurrent gemcitabine and radiotherapy for pancreatic cancer was reported to have a higher rate of severe acute intestinal toxicity. This study evaluated the acute intestinal toxicity in relation to the volume of irradiated small bowel and other factors using dosimetric analyses in pancreatic cancer patients treated with gemcitabine-based chemoradiotherapy. MATERIALS AND METHODS: The patient population was derived from a phase II trial of concurrent weekly gemcitabine and radiotherapy for locally advanced pancreatic cancer. Gemcitabine was administered weekly at a dose of 250 mg/m2. The total dose was 50.4 Gy in 28 fractions using a four-field conformal technique. A dose-volume histogram was generated for the small bowel, colon and planning target volume (PTV) and dosimetric parameters were recorded. Correlations between the acute intestinal toxicity and the volume of irradiated small bowel and other factors were evaluated. RESULTS: Forty-two patients enrolled between July 2001 and July 2002 were analyzed. Grade 3+ acute intestinal toxicities were observed in twenty-four (62%) patients. There was no correlation between the acute intestinal toxicity and the volume of irradiated small bowel. However, the total volume of PTV was shown to be significantly correlated with the development of Grade 3+ acute intestinal toxicity (p = 0.021). CONCLUSION: The volume of irradiated small bowel did not directly influence the acute intestinal toxicity, but only the volume of PTV significantly correlated with severe acute intestinal toxicity.

Significance of coronary artery calcification score (CACS) for the detection of coronary artery disease (CAD) in chronic dialysis patients.

BACKGROUND: Coronary artery disease (CAD) is a principal cause of death in patients with end-stage renal disease (ESRD). The coronary artery calcification score (CACS), determined by electron-beam computed tomography (EBCT), is useful for the detection of CAD in non-ESRD patients. There are few reports on the usefulness of EBCT for the detection of CAD, however, in ESRD patients. We examined the relation between CACS and CAD in ESRD patients. METHODS: Coronary angiography (CAG) was used to diagnose CAD in patients with significant coronary artery stenosis (>or=50%). We examined 76 ESRD patients on chronic dialysis therapy from 1997 to 2005, of which 51 are men, 25 are women, mean (S.D.) age of 57.9 (12.1) years and mean (S.D.) HD duration of 7.7 (6.6) years. There were 50 (35 men, 15 women) patients with CAD and 26 (16 men, 10 women) without CAD. RESULTS: The median CACS was 1290 in all patients, 1689 in the CAD group and 527 in the non-CAD group; the mean (S.D.) CACS was 1833 (2003) in all patients, 2338 (2209) in the CAD group and 861 (991) in the non-CAD group. CACS was significantly higher in the CAD group than in the non-CAD group. The CACS cutoff values for predicting CAD were calculated at intervals of 100. At the cutoff values of >or=100, >or=500, >or=1000, >or=2000, and >or=3000, the sensitivity was 98%, 90%, 68%, 42%, and 32% and the specificity was 35%, 50%, 69%, 85%, and 96%, respectively. CONCLUSIONS: EBCT is not adequate for screening asymptomatic ESRD patients. Because EBCT is less invasive than CAG, further study is necessary to determine whether CAG should be performed in all high-risk ESRD patients on chronic dialysis.

Pro-gastrin-releasing peptide as a factor predicting the incidence of brain metastasis in patients with small cell lung carcinoma with limited disease receiving prophylactic cranial irradiation.

BACKGROUND: Prophylactic cranial irradiation (PCI) reduces the incidence of brain metastasis with an effect on overall survival in patients with small cell lung carcinoma (SCLC). In spite of multidisciplinary intensive treatment approaches, many patients still experience brain metastasis. The authors retrospectively analyzed the characteristics of the first failure event due to brain metastasis (FBM) in patients treated with PCI. METHODS: Between January 1990 and April 2004, 71 patients with limited disease SCLC were treated with PCI after completing systemic treatment at the National Cancer Center Hospital (Tokyo, Japan). Univariate and multivariate analyses were used to identify factors related to FBM and survival. RESULTS: The FBM and overall incidence of brain metastasis (OBM) were 16.9 % (12 of 71) and 26.8% (19 of 71), respectively. Median time to progressive disease and median survival were 8.4 months and 21.6 months, respectively. Elevation of pro-gastrin-releasing peptide (Pro GRP) level before PCI was found to be a significant predictive and prognostic factor for FBM, OBM, and survival on multivariate analysis (P = 0.007, P = 0.025, and P = 0.009, respectively). CONCLUSIONS: An elevated Pro GRP level before PCI was found to be significantly related to FBM and survival, and should be considered before PCI is performed.

[Evaluation and related factors in coronary artery calcification in chronic dialysis patients].

Electron beam CT (EBCT) has been used to measure coronary artery calcification score (CACS). We have been studied CACS on chronic dialysis patients and examined the relationship between CACS and laboratory variables, incidence of ischemic heart disease, and survival. High CACS is often observed in patients with high serum phosphate, high calcium phosphate product, and dyslipidemia. Several factors for calcification both stimulating and suppressing have been playing a role in chronic dialysis patients. CACS is a surrogate marker of adequate control of uremia.