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Branched polyethylenimine (bPEI) (M w =25 k) conjugated with catechol groups (PEI-C) was crosslinked to produce PEI-catechol nanogels (PCNs), which were used as a new class of small interfering RNA (siRNA) delivery carriers. The conjugated catechol groups were crosslinked with the primary amines of PEI under basic conditions. The size and morphology of the PCNs were analyzed by dynamic light scattering (DLS) and atomic force microscopy (AFM). The resulting PCNs were quite stable and had an average diameter of 111.4 ± 14.8 nm in an aqueous solution. Compared to bPEI, the PCNs exhibited much lower cytotoxicity, and formed more stable complexes with siRNA. The PCNs/siRNA complexes exhibited enhanced cellular uptake and promoted gene silencing efficiency, suggesting that they can be used potentially as a less cytotoxic siRNA carrier.

In this study, dimerized siRNAs linked by a cleavable disulfide bond were synthesized for efficient intracellular delivery and gene silencing. The reducible dimerized siRNAs showed far enhanced complexation behaviors with cationic polymers as compared to monomeric siRNA at the same N/P ratio, as demonstrated by microscopic techniques and gel characterization. Dimerized siRNAs targeting green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) were complexed with linear polyethylenimine (LPEI), and treated to various cell lines to examine gene transfection efficiencies. In comparison to monomer siRNA/LPEI complexes, dimeric siRNA/LPEI complexes showed greatly enhanced cellular uptake and gene silencing effects in vitro. These results were mainly due to the higher charge density and promoted chain flexibility of the dimerized siRNAs, providing more compact and stable siRNA complexes. In addition, the conjugation strategy of reducible siRNA dimers was further extended: poly(ethylene glycol) (PEG)-modified dimerized siRNAs and heterodimers of siRNAs targeting two different genes (e.g., GFP and VEGF) were synthesized, and their gene silencing efficiencies were characterized. The dimerized siRNA complex system holds great potential for in vivo systemic gene therapy.

Di- and triblock siRNA/PEG copolymers were synthesized and complexed with cationic SLN for assessing their gene silencing efficiency as a function of PEG density. A sssiRNA and a sassiRNA were separately conjugated with PEG via a disulfide linkage. AB-type diblock and ABA-type triblock copolymers were successfully prepared by stoichiometric hybridization of sssiRNA-PEG conjugate with sassiRNA and sassiRNA-PEG conjugate, respectively. The resultant di- and triblock copolymers were characterized by means of GPC and gel electrophoresis. The serum stability of siRNA in the copolymers was enhanced as compared to that of naked siRNA. Using cationic SLN as a model carrier, the PEGylation density effect of the siRNA-PEG/SLN complexes on gene silencing and cellular uptake was analyzed.

Pluronic/polyethylenimine shell crosslinked nanocapsules with embedded magnetite nanocrystals (PPMCs) were developed for magnetically triggered delivery of siRNA. The positively charged PPMCs formed stable nanosized polyelectrolyte complexes via electrostatic interactions with negatively charged siRNA-polyethylene glycol conjugate (siRNA-s-s-PEG) that was linked via a cleavable disulfide linkage. PPMC/siRNA-s-s-PEG polyelectrolyte complexes were efficiently taken up by cancer cells upon exposure to a magnet, thereby enhancing intracellular uptake and silencing effect of siRNA. The present study suggests that these novel nanomaterials could be potentially utilized for magnetically triggered delivery of various nucleic acid-based therapeutic agents.

Small interfering RNA (siRNA) with 19-21 base pairs has been recently recognized as a new therapeutic agent for effectively silencing a specific gene on a post-transcription level. For siRNA therapeutics, safe and efficient delivery issues are significant hurdles to clinical applications. Here we present a new class of biologically active siRNA structure based on chemically self-crosslinked and multimerized siRNA through cleavable disulphide linkages. The multimerized siRNA can produce more stable and compact polyelectrolyte complexes with less cytotoxic cationic carriers than naked siRNA because of substantially increased charge densities and the presence of flexible chemical linkers in the backbone. The cleavable and multimerized siRNA shows greatly enhanced gene-silencing efficiencies in vitro and in vivo through a target-messenger-RNA-specific RNA interference processing without significantly eliciting immune induction. This study demonstrates that the multimerized siRNA structure complexed with selected cationic condensing agents can serve as potential gene-silencing therapeutics for treating various diseases.

Background: The involvement of certain organs such as the adrenal gland and ovaries is rare in non-Hodgkin’s lymphoma (NHL). There are few studies comparing clinical features and prognosis based on the extranodal organ involved. Methods: We selected patients presenting with predominantly extranodal involvement among patients diagnosed with NHL from 1998 to 2009 at the Samsung Medical Center. Results: Forty-eight patients with NHL involving rare extranodal sites were analyzed. The extranodal sites were as follows: adrenal gland (n = 14), ovary (n = 13), pancreas (n = 11), uterus (n = 4), esophagus (n = 4) and prostate (n = 2). Diffuse large B-cell lymphoma (DLBCL) was the most common (n = 39), and the median overall survival (OS) was 16.63 months. There was no significant difference in OS according to the involved sites. Rituximab plus CHOP failed to provide an additive survival benefit over CHOP alone in 39 DLBCL patients. The OS of DLBCL in rare extranodal sites was worse than that in common sites when compared based on tumor stage. Conclusions: NHL involving rare extranodal sites had a poor prognosis, and the impact of rituximab on survival was negligible. Thus, more intensive therapeutic strategies should be considered for NHL involving rare extranodal sites.

This study was carried out to evaluate the clinical significance of ascitic fluid carcinoembryonic antigen (CEA) in advanced gastric cancer patients with ascites. From November 2001 to February 2008, 119 gastric cancer patients with concurrent ascites who were clinically diagnosed with carcinomatosis, were retrospectively reviewed with regard to ascitic fluid cytology and clinicopathological parameters. Serum CEA (sCEA) and ascitic fluid CEA (aCEA) were measured using a chemiluminescent enzyme immunoassay. The patients’ median age was 50 years (range 23–80 years). The median value of aCEA was significantly higher than sCEA [130.5 ng/ml (range 0.2–12.211 ng/ml) vs. 2.1 ng/ml (range 0.02–8.152 ng/ml), p

Background A variety of synthetic carriers, such as cationic polymers and lipids, have been used as nonviral carriers for small interfering RNA (siRNA) delivery. Although siRNA polyplexes and lipoplexes exhibited good gene silencing efficiencies, they often showed serious cytotoxicities, which are not useful for clinical applications. A double-stranded RNA binding cellular protein with highly specific siRNA binding property and noncytotoxicity was used for siRNA delivery. Methods A double-stranded RNA binding domain (dsRBD) of human double-stranded RNA activated protein kinase R was genetically produced and utilized to complex siRNA for intracellular delivery. For characterization of the siRNA/dsRBD complexes, decomplexation assay and RNase protection assay were performed. Cytotoxicity and target gene inhibition ability were also examined using human carcinoma cell lines. Results The recombinantly produced polypeptide dsRBD exhibited its inherent binding activity for siRNA without sequence specificity, and the siRNA/dsRBD complexes protected siRNA from degradation by ribonucleases. Green fluorescent protein (GFP) siRNA/dsRBD complexes showed prominent down-regulation of a target GFP gene, when an endosomal escape function was supplemented by addition of a fusogenic peptide, KALA, in the formulation. Conclusions The results suggest that dsRBD-based protein carriers could be successfully applied for a wide range of therapeutic siRNAs for intracellular gene inhibition without showing any cytotoxicity. Copyright © 2009 John Wiley & Sons, Ltd.

A polyelectrolyte complex(PEC) micelle-based siRNA delivery system has been developed for vascular endothelial growth factor (VEGF), and its antitumor efficacy has been demonstrated using in-vivo animal models. Penetration and distribution through the avascular regions of human solid tumors after extravasation are important issues for antitumor efficacy, especially for macromolecules such as VEGF siRNA PEC micelles. Using an in-vitro solid tumor model, multicellular layers(MCL) culture of human colorectal cancer cells, we evaluated the penetration kinetics and efficacy of VEGF siRNA PEC micelles(PEC-siRNA) in comparison to unmodified siRNA(N-siRNA). The PEC-siRNA showed full penetration (15-17 layers of cells) with a unique punctuated distribution pattern at 48 h following initial accumulation in the top layers and a significant suppression of mRNA and protein expression in a dose-dependent manner after 72 h exposure. Although the initial penetration of N-siRNA was faster than that of PEC-siRNA, N-siRNA showed complete loss of activity due to its instability within 24 h. Our data support the idea that PEC micelle formulation may provide stable penetration tool through the multilayers of cancer cells and ensure the gene silencing effect of VEGF. This study also demonstrated that MCL could serve as a useful in-vitro model to evaluate the dose- and time-dependent profiles of penetration and efficacy of macromolecular delivery systems in human solid tumor avascular regions.

A polymeric gene carrier was developed to deliver vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) for prostate cancer cells in a target-specific manner. Prostate cancer-binding peptide (PCP) was conjugated with polyethylenimine (PEI) via a poly(ethylene glycol) (PEG) linker (PEI-PEG-PCP). The PEI-PEG-PCP conjugate could effectively condense siRNA to form stable polyelectrolyte complexes (polyplexes) with an average diameter of approximately 150 nm in an aqueous solution. VEGF siRNA/PEI-PEG-PCP polyplexes exhibited significantly higher VEGF inhibition efficiency than PCP-unmodified polycationic carriers (PEI-PEG or PEI) in human prostate carcinoma cells (PC-3 cells). The enhanced gene silencing activity of VEGF siRNA/PEI-PEG-PCP was maintained even under serum conditions, owing to the steric stabilization of the polyplexes with hydrophilic PEG grafts. Confocal microscopic studies revealed that the siRNA/PEI-PEG-PCP polyplexes were delivered into PC-3 cells in a PCP ligand-specific manner.

Polyelectrolyte complex (PEC) micelles modified with cancer cell targeting moieties were prepared for intracellular delivery of vascular endothelial growth factor (VEGF) small interfering RNA (siRNA). A luteinizing hormone-releasing hormone (LHRH) peptide analogue was coupled as a cancer targeting ligand to the distal end of the poly(ethylene glycol) (PEG)-siRNA conjugate. The siRNA-PEG-LHRH conjugate self-assembled to form nanosized PEC micelles upon mixing with poly(ethylenimine) (PEI) via ionic interactions. The PEC micelles showed spherical morphology with a hydrodynamic diameter of ca. 150 nm. For LHRH receptor overexpressing ovarian cancer cells (A2780), the PEC micelles with LHRH exhibited enhanced cellular uptake compared to those without LHRH, resulting in increased VEGF gene silencing efficiency via receptor-mediated endocytosis. This study showed that PEC micelles decorated with specific cell-recognizable targeting ligands could be used for targeted delivery of siRNA.

For efficient cancer therapy, small interfering RNA (siRNA) should be stably and efficiently delivered into the target tissue and readily taken up by cancer cells. To address these needs, a polyelectrolyte complex (PEC) micelle-based siRNA delivery system was developed for anti-angiogenic gene therapy. The interaction between poly(ethylene glycol) (PEG)-conjugated vascular endothelial growth factor siRNA (VEGF siRNA-PEG) and polyethylenimine (PEI) led to the spontaneous formation of nanoscale polyelectrolyte complex micelles (VEGF siRNA-PEG/PEI PEC micelles), having a characteristic siRNA/PEI PEC inner core with a surrounding PEG shell layer. Intravenous as well as intratumoral administration of the PEC micelles significantly inhibited VEGF expression at the tumor tissue and suppressed tumor growth in an animal tumor model without showing any detectable inflammatory responses in mice. Upon examination of the PEC micelle distribution and in vivo optical imaging following intravenously injection, enhanced accumulation of the PEC micelles was also observed in the tumor region. This study demonstrates the feasibility of using PEC micelles as a potential carrier for therapeutic siRNAs in local and systemic treatment of cancer.

Pluronic/poly(ethylenimine) (PEI2K) nanocapsules (NCs) exhibiting a thermally reversible swelling/deswelling volume expansion behavior were synthesized and used for an siRNA delivery nanocarrier as well as an effective endosome breaking agent. Pluronic/PEI2K nanocapsules were in a collapsed state with an average size of 118.9+/-15.3 nm at 37 degrees C, but in a swollen state with that of 412.3+/-83.2 nm at 15 degrees C. The collapsed Pluronic/PEI2K NCs having a highly positive surface zeta potential value were utilized to load siRNA-PEG conjugate on the surface via electrostatic interactions. The siRNA-PEG/NCs nanocomplexes were transfected to the cells at 37 degrees C for efficient cellular uptake. The transfected cells were treated with a brief cold shock to induce an abrupt volume expansion of the NCs within an endosome compartment to physically burst out the endosomal membrane. Far efficient gene silencing effects for both green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF) were observed after the cold shock treatment to the transfected cells.

Breast cancers (BC) in younger, premenopausal patients (YBC) tend to be more aggressive with worse prognosis, higher chance of relapse and poorer response to endocrine therapies compared to breast cancers in older patients. The proportion of YBC (age ≤ 40) among BC in East Asia is estimated to be 16-32%, significantly higher than the 7% reported in Western countries. In addition, approximately half of the Asian BC patients were premenopausal compared to 15-30% in the West. To characterize the molecular bases of Asian YBC, we have performed whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) on tumor and matched normal samples from 168 Korean BC patients consisting of 106 YBC cases (age ≤ 40) and 62 OBC cases (age > 40). We then performed comparison analyses with the TCGA BC cohort consisting of 1,116 tumors from primarily Caucasian patients, also grouped by age into YBC (age ≤ 40), IBC (40 < age ≤ 60) and OBC (age > 60). We performed logistic regression analyses to identify differentially expressed (DE) genes and pathways among age-based cohorts while controlling for the confounding effects of molecular subtype, tumor purity and stage. Within the Asian cohort, we found that estrogen response, endocrine therapy resistance, and various metabolism pathways are up-regulated in YBCs while cell cycle, proliferation and inflammatory pathways are up-regulated in OBCs. To separately examine molecular signatures from tumor, stroma and normal compartments, we used non-negative matrix factorization (NMF) analyses to virtually dissect bulk tumor expression data and identified 14 factors including 3 factors associated with normal tissues, 1 factor associated with stroma and 1 factor associated with tumor infiltrating leukocytes (TILs). By examining the correlation between pathway gene expression and NMF factors, we inferred that DE pathways such as fatty acid metabolism, bile acid biosynthesis, and epithelial-to-mesenchymal transition (EMT) were mainly active in stromal and normal tissue compartments. The TIL factor was significantly enriched in Asian BCs relative to Caucasian BCs with the highest TIL factor weight observed in Asian OBCs. Using gene expression signatures representing distinct types of TILs, we classified the combined cohort into three subtypes of varying TIL activities. Consistent with results from the NMF analysis, the TIL-high subtype is also significantly enriched in Asian BCs relative to Caucasian BCs. To our knowledge, this is the first large-scale multi-omics study of Asian breast cancer. Comparative analyses of multi-omics profiles from Asian and primarily Caucasian BC cohorts identified distinguishing molecular signatures associated with Asian BCs. Further, many signatures appeared to be specific to non-tumor compartments within bulk tumor, indicating that young Asian BCs may harbor distinctive tumor microenvironment. Citation Format: Yeon Hee Park, Ying Ding, Soo-Hyeon Lee, Hae Hyun Jung, Woosung Chung, Soonweng Cho, Jin-Ho Kim, Shibing Deng, Yoon-la Choi, Julio Fernandez, Se Kyung Lee, Seok Won Kim, Jeong Eon Lee, Ji-Yeon Kim, Jin Seok Ahn, Young-Hyuck Im, Seok Jin Nam, Woong-Yang Park, Zhengyan Kan. Comparative analyses of multi-omics profiles reveal distinctive molecular signatures of young Asian breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3370. doi:10.1158/1538-7445.AM2017-3370

10108 Background: Onycholysis and other nail toxicities occur in approximately 20-30% of breast cancer(BC) patients receiving docetaxel(D) chemotherapy. Onycholysis, the separation of the nail plate from nail bed, is also often associated with painful paronychia decreasing patients’ the efficacy of a hydrating nail solution (EVONAIL solution, Evaux Laboratories, France) for the prevention and treatment of D-induced onycholysis and nail toxicities. Methods: This study is a prospective randomized controlled study of hydrating nail solution for prevention or treatment of onycholysis in patients with BC receiving neo/adjuvant 3-weekly D after doxorubicin plus cyclophosphamide. In experimental arm, each patient painted hydrating nail solution on nails and periungual areas once a day till developing onycholysis grade 2 or more. After Gr 2 onycholysis development, patients painted EVONAIL twice a day regardless of treatment arms. The primary endpoint is the incidence of onycholysis Gr 2 or more and recovery rate from Gr2 onycholysis. The secondary endpoints include: the incidence of all grade onycholysis; duration from first docetaxel treatment until onycholysis symptom appearance; degree of pain from nail toxicities; the incidence of other nail toxicities. Results: Since Aug 2015 to May 2016, 103 patients were enrolled and completed this study (Experimental arm(E) : 51, control arm(C): 52). Of 103 patients, 25 cases of Gr1 and 22 of Gr2 onycholysis were observed (Gr1 and 2(n,(%)) : 8(15.7%) and 7(13.7%) in E, 17(32.7%) and 15(21.4%)in C, respectively). Hydrating nail solution resulted in statistically significant reduction of Gr2 onycholysis compared to C (HR = 0.39, 95% CI 0.16, 0.96; P = 0.041) and all grade onycholysis decreased in experimental arm with statistical significance (HR = 0.37, 95% CI 0.20-0.69, P = 0.002). However, the effect of onycholysis treatment was not observed. Conclusions: Hydrating nail solution significantly reduced the incidence of D induced onycholysis in BC patients (NCT02670603). Clinical trial information: NCT02670603.

Background The use of chemotherapy in advanced cancer patients has increased with the development of novel, high-efficacy anticancer therapeutic agents. In the current study, we analyzed the 10-year trends in patients receiving chemotherapy at the end of life. Method We retrospectively reviewed mortality data for advanced cancer patients who died in 2000, 2005, and 2010 at a single institution. The trends of receiving palliative chemotherapy at the end of life were assessed for each year. In addition, logistic regression analysis was performed to determine the factors associated with receiving chemotherapy. Results We analyzed the records of 2,345 patients who died of cancer. Patients with less responsive tumors were less likely to receive chemotherapy than patients with responsive tumors at the time of death. Patients who were ≥ 65 years were less likely to receive chemotherapy compared with patients who were

In this study, we systematically explore the influence of the lipophilic group on the siRNA transfection properties of the polycationic-based delivery vectors. For this, a novel and modular synthetic strategy was developed for the preparation of polymers carrying a cationic site and a lipophilic group at each polymer repeat unit. These bifunctional polymers could form a complex with siRNA and deliver it to human colon carcinoma cells (HT-29-luc). In general, transfection capability increased with an increase in the chain length of the lipophilic moiety. The best transfection agent, a polymer containing ammonium groups and pentyl side chains, exhibited lower toxicity and higher transfection efficiency than branched and linear polyethylenimines (PEI). Moreover, as opposed to PEI, the transfection efficiency of polymer/siRNA complexes remained unchanged in the presence of bafilomycin A1, a proton pump inhibitor, suggesting that the present system did not rely on the “proton sponge” effect for siRNA delivery.

Cell-penetrating peptides have been widely investigated as delivery vehicles for oligonucleotides (e.g., siRNA and antisense oligonucleotides). Different delivery strategies can be used, such as co-incubation, direct conjugation, non-covalent complex, and modification on the surface of liposome or polymer complexes. However, several challenges remain for their preclinical and clinical development. Endosomal escape, lack of cell/tissue specificity, and toxicity are major concerns in the design of cell-penetrating peptide-mediated delivery systems. In this commentary, we highlight recent reports of cell-penetrating peptide incorporation into oligonucleotide delivery systems and underline the remaining challenges, particularly for preclinical and clinical applications.

Background Several studies have suggested a cancer risk reduction in statin users although the evidence remains weak for stomach cancer. The purpose of this study was to use an exact-matching case–control design to examine the risk of gastric cancer associated with the use of statins in a cohort of patients with diabetes. Methods Cases were defined as patients with incident gastric cancer identified by International Classification of Diseases 16.0 ~ 16.9 recorded at Samsung Medical Center database during the period of 1999 to 2008, at least 6 months after the entry date of diabetes code. Each gastric cancer case patient was matched with one control patient from the diabetes patient registry in a 1:1 fashion, blinded to patient outcomes. Results A total of 983 cases with gastric cancer and 983 controls without gastric cancer, matched by age and sex, were included in the analysis. The presence of prescription for any statin was inversely associated with gastric cancer risk in the unadjusted conditional logistic regression model (OR: 0.18; 95% CI: 0.14 – 0.24; P < .0001). Multivariate analysis using conditional logistic regression with Bonferroni’s correction against aspirin indicated a significant reduction in the risk of gastric cancer in diabetes patients with statin prescriptions (OR: 0.21; 95% CI: 0.16 – 0.28; P < .0001). After adjustment for aspirin use, a longer duration of statin use was associated with reduced risk of gastric cancer, with statistical significance (P Conclusions A strong inverse association was found between the risk of gastric adenocarcinoma and statin use in diabetic patients.

Breast cancers (BC) in younger, premenopausal patients (YBC) tend to be more aggressive with worse prognosis, higher chance of relapse and poorer response to endocrine therapies compared to breast cancers in older patients (OBC). The proportion of YBC (age ? 40) among BC in East Asia is estimated to be 16-32%, significantly higher than the 7% reported in Western countries. Genomic and molecular characterizations have deepened our understanding of breast cancer biology in areas ranging from intrinsic subtypes to treatment responses, however, the molecular bases of Asian YBC remains poorly characterized. We have performed whole-exome sequencing (WES), whole-transcriptome sequencing (WTS) and high coverage targeted sequencing on tumor and matched normal samples from 133 Korean BC patients consisting of 74 YBC cases (age ? 40). We further performed immunohistochemistry (IHC) analyses to characterize tumor-infiltrating lymphocytes (TILs) in 46 tumors using four markers (CD45, CD4, CD8 and CD163). We found that BRCA1/2 germline deleterious mutations are enriched in YBC and the ER+/HER2- subtype, indicating that Asian ER+ YBC has a significant germline contribution. MutSig analysis4 identified ARID1A as a significantly mutated gene, implicating chromatin modeling as a cancer driver in Asian BC. Differential expression analyses suggested that Asian YBC differ in energy metabolism and are more active in protein synthesis than OBC tumors, whereas OBC is more proliferative than YBC. Using gene expression signatures representing distinct immune cell types and immunohistochemistry, we classified our cohort into four subtypes of varying TIL activities: high, medium, low and quiet. The majority of immunogenic cases with high TIL levels lie in ER+ or HER2+ subtypes although higher proportion is seen in TNBC. Moreover, YBC tumors appear to harbor lower levels of TIL activities than OBC, suggesting that younger patients may be less likely to benefit from immunomodulatory therapies than older patients. To our knowledge, this is the first large-scale multi-omics study of Asian breast cancer and would significantly contribute to the compendium of molecular data available for young, premenopausal breast cancer. While the major landmarks in the molecular and immune landscape of Asian BC look similar to that of the predominantly Caucasian BC cohorts, we have identified a number of distinguishing characteristics pointing to distinctive oncogenic mechanisms underlying Asian BC. Citation Format: Yeon Hee Park, Ying Ding, Soonweng Cho, Soo-Hyeon Lee, Hae Hyun Jung, Woosung Chung, Jinho Kim, Woong-Yang Park, Eric Powell, Pamela Vizcarra, Shibing Deng, Se Kyung Lee, Seok Won Kim, Jeong Eon Lee, Ji-Yeon Kim, Jin Seok Ahn, Young-Hyuck Im, Seok Jin Nam, Zhengyan Kan. Multi-omics and immuno-oncology profiling of an Asian breast cancer cohort enriched in young and premenopausal patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-325.

The first attempt to prepare biologically active siRNA-based microhydrogels is reported. The self-assembled microhydrogels were fabricated using sense/antisense complementary hybridization between single-stranded linear and Y-shaped trimeric siRNAs. The siRNA microhydrogels were condensed using a popular cationic polymer such as LPEI to form compact, stable siRNA/polymeric nanoparticles that exhibited superb cellular uptake efficiency and gene silencing activity.

Introduction: For patients with refractory bone and soft tissue sarcoma (STS), treatment options have been limited. Ifosfamide is an alkylating agent with well-demonstrated efficacy against STS, and dose-dependent activity. The aim of this retrospective study was to evaluate the response rate, progression-free survival (PFS), progression-free rate (PFR), and median duration of response to high-dose ifosfamide (HDI) as at least second-line chemotherapy for patients with advanced bone sarcoma and STS. Patients and Methods: Thirty metastatic, unresectable sarcoma patients who were treated with HDI chemotherapy between May 1999 and November 2007 were included in the analysis. In total, 106 cycles (median 3 cycles; range 1–8 cycles) were administered. Twenty-one patients received treatment as second-line chemotherapy, and 9 patients as third-line treatment. HDI was given at a dose of 2 g/m2 over 3 h, and at a dose of 2 g/m2 per day; continuous infusion was administered on 6 consecutive days (2 g/m2/6 days) every 3 weeks. Results: After a median follow-up of 49 months (range 10–114), median PFS was 2.9 months (range 0.4–9.3) and median overall survival 8.7 months (range 0.4–57.8). The 3- and 6-month PFR were 47% (SE 9.1%) and 20% (SE 7.3%), respectively. Median response duration of HDI was 2.9 months (range 0.7–7.6). Of the 28 evaluable patients, 2 (7%) achieved complete response, 5 (18%) partial response, and 4 (14%) stable disease, and overall disease control was 39%. Two responders out of 7 (28.5%) and 4 patients out of 11 (36%) with controlled disease by HDI had a synovial sarcoma. Two patients were not evaluable because they were switched to another treatment due to ifosfamide-induced encephalopathy. Grade 3–4 neutropenia was seen in 13 (43%) patients, and treatment-related death was observed in one patient. Conclusion: HDI at a total dose of 14 g/m2 with mesna is still an active salvage regimen, particularly in patients with synovial sarcomas.

Efficient gene transfer into mammalian cells mediated by small molecular amphiphile-polymer conjugates, bile acid-polyethylenimine (BA-PEI), is demonstrated, opening an efficient transport route for genetic materials across the cell membrane. This process occurs without the aid of endocytosis or other energy-consuming processes, thus mimicking macromolecular transduction by cell-penetrating peptides. The exposure of a hydrophilic face of the amphiphilic BA moiety on the surface of BA-PEI/DNA complex that mediates direct contact of the BA molecules to the cell surface seems to play an important role in the endocytosis- and energy-independent internalization process. The new modality of the polymeric biomimetics can be applied to enhanced delivery of macromolecular therapeutics.

Di- and triblock siRNA/PEG copolymers were synthesized and complexed with cationic SLN for assessing their gene silencing efficiency as a function of PEG density. A sssiRNA and a sassiRNA were separately conjugated with PEG via a disulfide linkage. AB-type diblock and ABA-type triblock copolymers were successfully prepared by stoichiometric hybridization of sssiRNA-PEG conjugate with sassiRNA and sassiRNA-PEG conjugate, respectively. The resultant di- and triblock copolymers were characterized by means of GPC and gel electrophoresis. The serum stability of siRNA in the copolymers was enhanced as compared to that of naked siRNA. Using cationic SLN as a model carrier, the PEGylation density effect of the siRNA-PEG/SLN complexes on gene silencing and cellular uptake was analyzed.

Objective: The aims of this study were to identify prognostic factors for overall survival (OS) in patients with small cell lung cancer (SCLC) and to construct a prognostic index on the basis of their expected OS. Long-term survivors were also given special attention. Methods: We retrospectively analyzed 295 patients diagnosed with SCLC from January 2002 to September 2007 at the Severance Hospital, Seoul, Republic of Korea. Patient history was reviewed regarding both clinical and tumor-related markers, and treatment-related factors were also evaluated. Results: There were 131 (44.4%) patients with limited disease (LD) and 164 (55.6%) with extensive disease (ED). Median follow-up lasted 9.4 months. Objective response to chemotherapy was 66.8%. The median survival time (MST) was 11.2 months in all patients, 20.4 months in LD patients, and 7.7 months in ED patients. The median progression-free survival was 9.4 months. Independent prognostic factors for overall survival (OS) were extent of disease, performance status, and CYFRA 21-1 level. We classified all patients into four groups based on the results of multivariate analysis using classification and regression tree analysis. Median survival times were 22.7, 13.7, 8.5, and 3.2 months, respectively. A total of 51 (17.3%) patients from the entire study population were evaluated for long-term survival, which was defined as survival >2 years. Their MST was 43.1 months, and extent of disease, performance status, and CYFRA 21-1 were independent prognostic factors for long-term survival. Conclusions: We confirmed the well-known prognosticators, disease extent and performance status, in our patients, but further identified CYFRA 21-1 as independent prognostic factor. A prognostic index was constructed to create four classifications of SCLC considering these variables.

Biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) was conjugated to the 3′ end of small interfering RNA (siRNA) via a disulfide bond to synthesize siRNA–PLGA hybrid conjugates. siRNA–PLGA conjugates were spontaneously self-assembled to form a spherical core/shell type micellar structure of ~ 20 nm in an aqueous environment, probably by hydrophobic interaction of PLGA blocks in the core surrounded by an siRNA shell layer. When linear polyethylenimine was added to the siRNA–PLGA micelles in aqueous solution, stable siRNA–PLGA/LPEI micelles with a size of ~ 30 nm were produced via ionic complexation between siRNA and LPEI in the outer shell. The cationic siRNA–PLGA/LPEI micelles showed superior intracellular uptake and enhanced gene silencing effect, compared to naked siRNA/LPEI complexes. The hybrid micelle structure based on siRNA and PLGA can be potentially used as an efficient siRNA delivery system for gene silencing.

Development of nano-sized gene delivery vehicles for small interfering RNA (siRNA) delivery is of great importance for their clinical applications such as cancer therapy. Herein, we demonstrate the controlled synthesis of polyethyleneimine (PEI)-coated gold nanoparticles (AuNPs) using catechol-conjugated PEI (PEI-C) for siRNA delivery. Since the conjugated catechol groups are reductive and moderately hydrophobic, PEI-C formed spherical multi-cored micelles in aqueous solution and served as reductive templates for the growth and synthesis of spherical AuNPs with tunable sizes and surface charges. PEI-C was stably anchored on the surface of growing crystal gold seeds with crosslinking, resulting in robust cationic AuNPs. The fabricated PEI-coated AuNPs formed stable complexes with siRNA, and the complexes showed an excellent gene silencing effect in cancer cells. Size and surface charge values of the synthesized AuNPs had a great influence on intracellular uptake and unpacking of siRNA, and the resultant gene silencing efficiency. The PEI-coated AuNPs exhibited an extremely low cytotoxicity due to the reduced density of primary amine groups and the absence of uncomplexed PEI fraction in aqueous solution.

Small interfering RNA (siRNA) was conjugated with poly(ethylene glycol) (PEG) at four different terminal ends (sense 3', sense 5', antisense 3', and antisense 5') via cleavable disulfide and noncleavable thioether linkages to evaluate their gene silencing efficiencies upon complexation with Lipofectamine2000. The PEGylation site at the four siRNA termini and PEG molecular weight were not critical factors to significantly affect gene silencing activities. Cleavable siRNA-PEG conjugates showed comparable gene silencing activities to naked siRNA, and exhibited sequence-specific degradation of a target mRNA. Interestingly, noncleavable siRNA-PEG conjugates were processed by Dicer, enabling to exert RNAi effect without showing a target sequence-specific manner. However, only cleavable siRNA-PEG conjugates significantly reduced the extent of INF-alpha release as compared to noncleavable siRNA-PEG conjugates, suggesting that they can be potentially used for therapeutic siRNA applications.

Purpose: The aim of this study was to determine whether the ERCC1 expression is effective to predict the clinical outcomes of patients with advanced gastric cancer (AGC) and who were treated with cisplatin-based first-line chemotherapy. Materials and Methods: A total of 89 measurable AGC patients received cisplatin and capecitabine, with or without epirubicin, as a part of a randomized phase II study. Patients were included for the current molecular analysis if they had received two or more cycles of chemotherapy, their objective tumor responses were measured and if their paraffin-embedded tumor samples were available. The ERCC1 expression was examined by performing immunohistochemical (IHC) staining, and the patients were divided into two groups (positive or negative) according to the presence of IHC staining of the tumor cell nuclei. Results: Of the 32 eligible patients, 21 patients (66%) had tumor with a positive expression of ERCC1 and the remaining 11 patients had tumor with a negative ERCC1-expression. The ERCC1-negative patients achieved a higher response rate than that of the ERCC1-positive patients (44% vs. 28%, respectively), although the difference was not statistically significant (p=0.42). The median survival time for the all patients was 14.6 months (95% CI: 13.6 to 15.6 months). The one-year survival rate was similar for the ERCC1-negative patients (61%) and the ERCC-1-positive patients (70%). Conclusion: In the current study, the tumor ERCC1 expression by IHC staining could not predict the clinical response or survival of AGC patients who were treated with cisplatin-based first-line chemotherapy. The ERCC1 protein expression does not appear to be a useful tool for the selection of tailored chemotherapy for these patients.

Purpose: We investigated the efficacy and safety of weekly paclitaxel monotherapy in previously treated patients with advanced gastric cancer (AGC) and poor performance status (PS) according to the Eastern Cooperative Oncology Group (ECOG). Patients and Methods: Patients with evaluable disease who failed at least one previous chemotherapy and had a PS of 2–3, received paclitaxel 70 mg/m2 on days 1, 8 and 15 every 4 weeks. Results: The median overall survival (OS) was 5.5 months (95% confidence interval, CI, 3.3–7.8) and progression-free survival was 2.1 months (95% CI, 1.2–3.0). The overall response rate was 3.8% and the disease control rate was 25.0%. Treatment-related toxicities were tolerable. OS was 7.1 (95% CI, 5.4–9.5) and 3.7 months (95% CI, 2.1–5.3) for patients with PS-ECOG 2 and 3, respectively (p < 0.001). When evaluated according to the previous treatment, OS was 5.1 (95% CI, 3.3–7.0) and 6.5 months (95% CI, 3.8–9.3) for patients receiving two and three or more lines of treatment, respectively (p = 0.815). With multivariate analysis, PS was a significant factor for OS. Conclusion: Survival in patients treated with weekly paclitaxel monotherapy was comparable to other second- or third-line chemotherapies for AGC, with acceptable toxicities in previously treated patients with poor PS.

For therapeutic applications of small interfering RNA (siRNA), serum stability, enhanced cellular uptake, and facile endosome escape are key issues for designing carriers. In this study, green fluorescent protein (GFP) siRNA was conjugated to a six-arm polyethylene glycol (PEG) derivative via a reducible disulfide linkage (6PEG-siRNA). The 6PEG-siRNA conjugate was also functionalized with a cell penetrating peptide, Hph1 to enhance its cellular uptake property (6PEG-siRNA-Hph1). The 6PEG-siRNA-Hph1 conjugate was electrostatically complexed with cationic self-crosslinked fusogenic KALA peptide (cl-KALA) to form multifunctional polyelectrolyte complex micelles for gene silencing. The resultant siRNA complex formulation with multiple PEG chains showed superior physical stability and resistance to enzymatic degradation. The 6PEG-siRNA-Hph1/cl-KALA complexes exhibited enhanced GFP gene silencing efficiency for MDA-MB-435 cells in the serum containing condition. The current reducible and multifunctional polyelectrolyte complex micelles are expected to have high potential for efficient delivery of therapeutic siRNA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010

Cationic solid lipid nanoparticles (SLN), reconstituted from natural components of protein-free low-density lipoprotein, were used to deliver small interfering RNA (siRNA). The cationic SLN was prepared using a modified solvent-emulsification method. The composition was 45% (w/w) cholesteryl ester, 3% (w/w) triglyceride, 10% (w/w) cholesterol, 14% (w/w) dioleoylphosphatidylethanolamine (DOPE), and 28% (w/w) 3beta-[ N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-chol). The SLN had a mean diameter of 117+/-12 nm and a surface zeta potential value of +41.76+/-2.63 mV. A reducible conjugate of siRNA and polyethylene glycol (PEG) (siRNA-PEG) was anchored onto the surface of SLN via electrostatic interactions, resulting in stable complexes in buffer solution and in even 10% serum. Under an optimal weight ratio of DC-chol of SLN and siRNA-PEG conjugate, the complexes exhibited higher gene silencing efficiency of GFP and VEGF than that of polyethylenimine (PEI) 25K with showing much reduced cell cytotoxicity. Flow cytometry results also showed that siRNA-PEG/SLN complexes were efficiently taken up by cells. Surface-modified and reconstituted protein-free LDL mimicking SLN could be utilized as noncytotoxic, serum-stable, and highly effective carriers for delivery of siRNA.

Gold nanoparticles chemically modified with primary amine groups were developed as intracellular delivery vehicles for therapeutic small interfering RNA (siRNA). The positively charged gold nanoparticles could form stable polyelectrolyte complexes through electrostatic interactions with negatively charged siRNA-polyethylene glycol (PEG) conjugates having a cleavable di-sulfide linkage under reductive cytosol condition. The resultant core/shell type polyelectrolyte complexes surrounded by a protective PEG shell layer had a well-dispersed nanostructure with a hydrodynamic diameter of 96.3+/-25.9 nm, as determined by dynamic light scattering and transmission electron microscopy. Confocal laser scanning microscopy revealed that the nanosized polyelectrolyte complexes were efficiently internalized in human prostate carcinoma cells, and thus enhanced intracellular uptake of siRNA. Furthermore, the siRNA/gold complexes significantly inhibited the expression of a target gene within the cells without showing severe cytotoxicity. The current study demonstrated that positively charged gold nanoparticles could be potentially applied for intracellular delivery of siRNA.

Endoscopic mucosal resection (EMR) is widely accepted as a standard treatment for early gastric cancer or gastric adenoma. However, EMR inevitably results in the formation of large iatrogenic ulcer at the resected area. Although the characteristics of EMR-induced ulceration are not fully understood, this type of ulcer is thought to heal faster and to recur less often than non-iatrogenic gastric ulcer. Current available evidences have suggested that EMR-induced ulcers heal within 2-3 months. Herein, we report two cases of non-healing persistent gastric ulcers after EMR. One is a case of gastric carcinoma which developed at the same site of previous EMR site for the low grade dysplasia. The other is a case in which persistent EMR-induced ulcer was healed in the long run after Helicobacter pylori eradication therapy.

To develop a small interfering RNA (siRNA) delivery system with low cytotoxicity and high transfection efficiency, siRNA was conjugated to poly(ethylene glycol) via a disulfide linkage (siRNA-PEG) to prepare polyelectrolyte complex micelles (PECMs) by condensing with a cationic fusogenic peptide (KALA). The siRNA-PEG conjugate exhibited enhanced resistance to degradation from nucleases. Anionic siRNA-PEG conjugate and cationic KALA, when mixed in an aqueous phase, spontaneously formed nano-sized PECMs (200nm) that have an inner core of charge neutralized siRNA/KALA complex surrounded by a PEG corona. Vascular endothelial growth factor (VEGF) siRNA was used to demonstrate VEGF sequence-specific gene inhibition in prostate carcinoma cells (PC-3 cells). The extent of gene silencing was gradually increased with increasing nitrogen to phosphate (N/P) ratio and the concentration of siRNA-PEG/KALA PECMs. These results suggest that the formulation of siRNA-PEG/KALA PECMs could be widely applied for intracellular delivery of various therapeutic siRNAs.

Purpose: We evaluated the safety and effectiveness of the Magnetic Resonanceguided Focused Ultrasound (MRgFUS) with the ExAblate Conformal Bone System for the palliation of painful bone metastases. Materials and Methods: Our Institutional Review Board approved this study, and all patients gave informed consent prior to enrollment. A total of six painful metastatic bone lesions in five patients were treated using MRgFUS with the ExAblate Conformal Bone System for pain palliation. The follow-up sessions were at 3 days, 2 weeks, 1, 2, and 3 months, and 1 year after treatment. Efficacy was evaluated by the changes in visual analog scale (VAS) scores. At 3-months and 1-year follow-ups, unenhanced computed tomography and contrast-enhanced MR imaging examinations were performed. All adverse events were assessed to evaluate treatment safety. Results: All patients showed significant pain relief within 2 weeks. Two patients experienced complete pain reduc tion that lasted for 1 year. Two other patients showed pain relief measured as VAS scores of 2 and 4 on their last follow-up. Although the remaining patient had experienced significant pain relief in two lesions, the VAS score re-increased on his last follow-up. The size of the enhancing soft tissue mass in metastatic lesions decreased, and new bone formation was seen on follow-up images. Although adverse events were not serious, non-specific leg pain and second degree skin burn were noted. Conclusion: MRgFUS was demonstrated to be effective palliative treatment within 2 weeks in selected patients with painful bone metastases.

Pluronic hydrogel nanoparticles cross-linked with poly(ethylenimine) (PEI) were synthesized by a modified emulsification/solvent evaporation method. Pluronic F-127 preactivated at the terminal group with p-nitrophenyl chloroformate was dissolved in dichloromethane, and the organic solution was emulsified in deionized water containing PEI by sonication. Primary amine groups of PEI in the aqueous phase were conjugated and/or cross-linked with activated Pluronic F-127 in the vicinity of the water/dichloromethane interface, resulting in the formation of shell-cross-linked Pluronic/PEI nanocapsules. Pluronic/PEI nanocapsules exhibited a volume transition behavior over a temperature range of 24-33 degrees C. The thermally reversible swelling/deswelling of Pluronic/PEI nanocapsules was caused by temperature-dependent hydrophobic interaction of cross-linked and/or grafted Pluronic polymer chains in the nanocapsules. Pluronic/PEI nanocapsules were utilized to break up intracellular endosomal compartments by swelling-induced destabilization of the endosomal membrane triggered by a cold-shock treatment.

Introduction The hyperpolarized 13C-pyruvate is a robust imaging biomarker to evaluate the metabolic status of cancer to monitor the anti-cancer therapeutic effect and to predict the prognosis as earliest as possible by 10,000 fold increase of MRS sensitivity. According to the clinical epidemiological research, metformin reduces cancer incidence or mortality in type II diabetes patients. In this study, we investigated the anti-cancer metabolic effect of metformin as an adjuvant for the radiation therapy to treat brain metastasis from triple negative breast cancer in nude mouse model by measuring the metabolic flux through the hyperpolarized 13C-pyruvate MRS. Methods Six weeks old BALB/C nude mice were stereotactically implanted 2×105 MDA-MB-231-luciferase breast cancer cells in the striatum of brain. After 1 week from tumor implantation, animals were randomly divided into 4 groups: vehicle (distilled water), oral metformin alone (MET), radiation treatment (RT), and radiation combined with adjuvant oral metformin treatment (METRT). Mice were treated with 300 mg/kg of oral metformin for 5 days in a week from week 1 until when the animals die. MRI and 13C MRS was performed at week 2 and week 3, before and after treatment. Brain tumors were irradiated at week 2 with 3Gy for 5 consecutive days. MRI and MRS was performed at 9.4T Bruker MRI scanner using 20mm surface dual tuned 1H-13C coil. 75mmole of hyperpolarized [1-13C] pyruvate was injected via tail vein in all mice. Metabolic flux was calculated as lactate/pyruvate ratio. The progression and the response to treatment was also monitored by bioluminescence imaging. Paraffin sections were stained with H&E for histological evaluation. Results There was no difference in tumor growth and metabolic flux rate between vehicle and MET alone groups. Vehicle group showed 260% increase in tumor mass, whereas RT group showed only 39% increase in tumor volume. 13C MRS showed 60% decrease in lactate/pyruvate flux ratio in RT group, whereas vehicle group showed 60% increase in metabolic flux ratio. Interestingly, the tumor volume was significantly regressed and 13C-lactate flux was significantly decreased in METRT group after treatment. Bioluminescence imaging showed time-dependent increasing flux in vehicle group, but RT and METRT groups showed decreased photon flux activity. In comparison of METRT with RT alone group, bioluminescence activity was significantly decreased in METRT group. H&E staining of histological sections showed significant tumor necrosis and decreased cellularity in METRT group. Conclusion Hyperpolarized 13C-pyruvate MRS informs the adjuvant therapeutic effect of metformin and RT in brain metastasis from triple negative breast cancer. Citation Format: Young-suk Choi, Han-Sol Lee, Jung-Sung Lee, Hyun-Jin Park, Ju-Hyun Lee, Eun-Kyung Wang, Seung-Wook Yang, Eunhae Joe, Soo Hyeon Lee, Dong-Hyun Kim, Ho-Taek Song. Hyperpolarized 13C-pyruvate MRS quantitatively monitor the therapeutic efficacy of adjuvant metformin combined RT in brain metastasis from triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2067. doi:10.1158/1538-7445.AM2014-2067

A 43-year-old woman with breast cancer who was on neoadjuvant chemotherapy presented with cough, sputum and mild fever. High-resolution computed tomography showed diffuse ground glass opacities in bilateral lungs and subpleural patchy consolidations. Initially, she was thought to have pneumonia or interstitial lung diseases such as drug-induced pneumonitis and treated with antibiotics and steroids. She subsequently got breast cancer surgery because of disease progression, and concurrent thoracoscopic lung biopsy revealed metastatic carcinoma of the lung from breast cancer. The diagnosis of suspected interstitial lung disease can be made without lung biopsy, but malignancy should always be considered and lung biopsy should be performed in the absence of a definitive clinical diagnosis.

Galactose-α1,3-galactose (α1,3-gal) epitope is synthesised at a high concentration on the surface of pig cells by α1,3-galactosyltransferase gene (GGTA1). The α1,3-gal is responsible for hyperacute rejection in pig-to-human xenotransplantation. The generation of transgenic pigs as organ donors for humans is necessary to eliminate the GGTA1 gene that synthesise Galα(1,3)Gal. Knock-in system refers to the integration a foreign gene at a specific target, disrupting the target gene by inserting the transgene. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as complement inhibitor to protect self cells from complement attack. The generation of GGTA1 knockout pigs expressing DAF is necessary to use as organ donors for humans. In this study, we established cell lines of GGTA1 knockout expressing DAF from pig ear fibroblasts to apply somatic cell nuclear transfer. A GGTA1-DAF knock-in vector for positive-negative selection (PNS) consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the diphtheria toxin A fragment (DT-A) gene as a negative selectable marker gene. The DAF gene was subcloned in the GGTA1 gene exon 4 and the endogenous start codon was replaced by that of DAF. The ear fibroblast cell lines were isolated from Chicago miniature pig and used for transfection. Linearized targeting vector DNA was transfected into these cells by electroporation. After selection of G-418 during 11 days, we analysed 242 colonies using PCR and long PCR. We isolated 2 positive colonies for GGTA1 gene disruption. To confirm expression of the hDAF and GGTA1 genes, we isolated total RNA from normal pig cells and the knock-in somatic cell line. A cDNA synthesised using reverse transcriptase progressed to RT-PCR by a pair of DAF-specific primers and α1,3-GT specific primer, respectively. Expression of DAF was detected in DAF knock-in somatic cells, butnot in normal pig cell. Expression of GGTA1 gene was decreased in the knock-in somatic cell line compared with normal pig cell. These cell lines may be used production of α1,3-GT knockout and DAF expression pig for xenotransplantation.

Gene targeting is a genetic technique that utilises homologous recombination between an engineered exogenous DNA fragment and the endogenous genome of an animal. In domestic animals, gene targeting has provided an important tool for producing knockout pigs for the α1,3-galactosyltransferase gene (GGTA1) to use in xenotransplantation. The frequency of homologous recombination is a critical parameter for the success of gene targeting. The efficiency of homologous recombination in somatic cells is lower than that in mouse embryonic stem cells. The application of gene targeting to somatic cells has been limited by its low efficiency. Recently, knockout rat and mouse were generated by introducing nonhomologus end joining (NHE)-mediated deletion or insertion at the target site using zinc-finger nucleases (ZFN). Therefore, the development of effective knockout and knock-in techniques in domestic animals is very important in biomedical research. In this study, we investigated homologous recombination events at the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene locus using ZFN in porcine primary fibroblast. The CMAH-targeted ZFN plasmid and mRNA were purchased from Sigma-Aldrich (St Louis, MO, USA). Porcine ear fibroblasts cells were obtained from a 10-day-old male Chicago miniature pigs. The fibroblasts were cultured in DMEM containing 15% fetal bovine serum, 1 × nonessential amino acids, 1 × sodium pyruvate, 10–4 M β-mercaptoethanol, 100 unit mL–1 penicillin and 100 μg mL–1 streptomycin. The cells were trypsinized and resuspended at a concentration of 1.25 × 107 cells mL–1 in F10 nutrient mixture. Four hundred microliters of the cell suspension was electroporated in a 4-mm cuvette with 4 pulses of 1 ms duration using 400V capacitive discharges using the CMAH neo targeting vector and ZFN plasmid or RNA. The CMAH neo targeting vector consists of the neomycin resistance gene (neo) as a positive selectable marker gene, 789-bp 5′ arm and 763-bp 3′ arm from exon 8 of CMAH gene. After selection of G-418, PCR analysis was performed using 64 colonies transfected with ZFN plasmid and 48 colonies transfected with ZFN RNA. As a result, 19 positive colonies were identified in colonies transfected with ZFN plasmid and 15 colonies were identified in colonies transfected with ZFN RNA. The targeting efficiency was 29.7 and 31.6% in the colonies transfected with ZFN plasmid and ZFN RNA, respectively. To our knowledge, this study provides the first evidence that the efficiency of gene targeting using ZFN was higher than that of conventional gene targeting in the porcine fibroblast. These cell lines may be used in production of CMAH knockouts for xenotransplantation.

A speech recognition system that is insensitive to external noise and applicable to actual life includes an A/D converter that converts analog voice signals to digital signals. An FIR filtering section employs powers-of-two conversion to filter the digital signals converted at the A/D converter into numbers of channels. A characteristic extraction section immediately extracts speech characteristics having strong noise-resistance from the output signals of the FIR filtering section without using additional memories. A word boundary detection section discriminates the information of the start-point and the end-point of a voice signal on the basis of the characteristics extracted by the characteristic extraction section. Finally, a normalization/recognition section codes and outputs the final result by carrying out a timing normalization and a classifying process using a radial basis function (RBF) neural network on the basis of the voice characteristics provided by the characteristic extraction section and the information for the start-point and the end-point of the voice signal from the word boundary detection section.