SEARCH
Search Details
MASUMI Koizumi KyokoGraduate School of Science, Technology and Innovation / Department of Science, Technology and InnovationAssociate Professor
Research activity information
■ Award- Sep. 2017 化学工学会, バイオ部会優秀ポスター賞, 昆虫細胞による2Aペプチドを用いた抗体タンパク質の生産Japan society
- Jun. 2005 日本乳癌学会, 研究奨励賞, 「網羅的遺伝子発現解析による乳癌のドセタキセル効果予測診断法の開発」
- Mar. 2005 大阪対がん協会, 研究奨励賞, 「網羅的遺伝子発現解析による乳癌のドセタキセル効果予測診断法の開発」
- Mar. 2002 大阪対がん協会, 研究奨励賞, 「乳癌の予後を決定する遺伝子群の同定」
- Recombinant adeno-associated virus (rAAV) is a prominent viral vector currently available for human gene therapy. The diameter of the rAAV capsid is ∼25 nm, and a positive or negative single-stranded DNA is packaged within the vector capsid. In this report, we describe a concise method to examine the extracted rAAV genome using an automated electrophoresis system. The rAAV genome, prepared from vector particles through either heat treatment at 95°C for 10 min or the phenol-chloroform extraction method, was analyzed using an automated electrophoresis system under denaturation conditions. The heat treatment protocol demonstrated a comparable yield with the phenol-chloroform extraction protocol, and the quantified amounts of the rAAV genome obtained using the automated electrophoresis system were consistent with those quantitated by quantitative PCR. Additionally, crude rAAV extractions could also be analyzed by the automated electrophoresis system after DNase I treatment. These results indicated that this simple and quick analysis using automated electrophoresis is highly useful for confirming the purity and integrity of the rAAV genome.Feb. 2024, Human gene therapy, 35(3-4) (3-4), 104 - 113, English, International magazine[Refereed]Scientific journal
- Adeno-associated virus (AAV) vectors are produced as a mixture of the desired particle (full particle, FP), which is filled with the designed DNA, product-related impurities such as particle without DNA (empty particle, EP), and aggregates. Cesium chloride or iodixanol equilibrium density gradient ultracentrifugation (DGE-UC) has been used for the purification of AAV vectors. DGE-UC can separate FP from impurities based on the difference in their buoyant densities. Here, we report the applications and limitations of equilibrium density gradient analytical ultracentrifugation (DGE-AUC) using a modern AUC instrument that employs DGE-UC principles for the characterization and quantitation of AAV vectors. We evaluated the quantitative ability of DGE-AUC in comparison with sedimentation velocity AUC (SV-AUC) or band sedimentation AUC (BS-AUC) using AAVs with different DNA lengths and different serotypes. DGE-AUC enabled the accurate quantification of the ratio of FP to EP when the AAV vector primarily contains these particles. Furthermore, we developed a new workflow to identify the components of separated peaks in addition to FP and EP. Ultraviolet absorption spectra obtained by multiwavelength detection can also support peak assignment following component identification. DGE-AUC experiments for AAV vectors have limitations with regard to minor components with low absorption at the detected wavelength or those with a density similar to that of major components of AAV vectors. DGE-AUC is the only analytical method that can evaluate particle density heterogeneity; therefore, SV-AUC or BS-AUC and DGE-AUC are complementary methods for reliable assessment of the purity of AAV vectors.Jan. 2024, Analytical chemistry, 96(2) (2), 642 - 651, English, International magazine[Refereed]Scientific journal
- Abstract The recombinant adeno‐associated viral (rAAV) vector is one of the most effective viral vectors in gene therapy because of its low immunogenicity, high transduction efficiency, broad tissue specificity, and long‐term transgene expression ability. HEK293T cells are widely used to produce rAAV vectors, and acquisition of high rAAV‐producing cells is one of the essential processes for higher rAAV vector production. To acquire such cells, a method to identify cells with high expression of transgene protein was developed; however, the relationship between high expression of transgene protein and increased production of rAAV has not been sufficiently studied to date. We used the fluorescent protein ZsGreen1 as a transgene, subdivided the adherent cells according to the intensity of transgene expression protein, and reconfirmed the relationship between rAAV vector production and the amount of intracellular plasmid. We found that the amount of rAAV produced was not correlated with the intensity of expression of the transgene protein but may be correlated with the amount of intracellular plasmid. We also found that cells with high expression of transgene protein might not necessarily produce large amounts of rAAV vector. Based on these results, it was suggested that intracellular plasmid copy number could be used as a new marker for cells producing high levels of rAAV vector.Lead, Wiley, Jun. 2023, Engineering Reports, 6(1) (1)[Refereed]Scientific journal
- Recombinant adeno-associated virus (rAAV) is a viral vector commonly used in gene therapy. Residual host cell DNA is an impurity that has been associated with the risk of infection and oncogenicity. Thus, it needs to be monitored for quality control. We aimed to develop a droplet digital polymerase chain reaction (ddPCR) method targeting 18S ribosomal RNA (rRNA) genes to quantitate residual host cell DNA. The copy number of the 18S rRNA gene was determined using two sets of primer pairs for 116- and 247-bp amplicons sharing the C-terminus. For conversion of the copy number of the 18S rRNA gene into the mass concentration of genomic DNA, the accurate copy number of 18S rRNA genes in HEK293 genomic DNA was determined by comparison with copy numbers of three reference genes (EIF5B, DCK, and HBB). Results showed that 88.6-97.9% of HEK293 genomic DNA spiked into rAAV preparations was recovered. The ddPCR-based assay was applied to rAAV preparations to quantitate residual host cell DNA as an impurity. Our findings indicate that the assay can be used for the quantitation and size distribution of residual host cell DNA in rAAV products.Jun. 2023, Human gene therapy, 34(11-12) (11-12), 578 - 585, English, International magazine[Refereed]Scientific journal
- Virus-like particles (VLPs) are hollow nanoparticles composed of recombinant viral surface proteins without a virus genome. In the present study, we investigated the production of influenza VLPs using recombinant insect cells. DNA fragments encoding influenza A virus hemagglutinin (HA) and matrix protein 1 (M1) were cloned with the Drosophila BiP signal sequence in plasmid vectors containing a blasticidin and a neomycin resistance gene, respectively. After Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with a pair of constructed plasmid vectors, stably transformed cells were established via incubation with blasticidin and G418. Western blot analyses showed that recombinant High Five cells secreted HA and M1 proteins into the culture supernatant. Immunoprecipitation of the culture supernatant with an anti-HA antibody and transmission electron microscopy suggested that secreted HA and M1 proteins were in a particulate structure with a morphology similar to that of an influenza virus. Hemagglutination assay indicated that expressed HA molecules retained hemagglutination activity. In a shake-flask culture, recombinant cells achieved a high HA yield (≈ 10 μg/ml) comparable to the yields obtained using the baculovirus-insect cell system. Recombinant insect cells may serve as excellent platforms for the efficient production of influenza VLPs for use as safe and effective vaccines and diagnostic antigens.Elsevier BV, Nov. 2020, Biochemical Engineering Journal, 163, 107757 - 107757, English, International magazine[Refereed]Scientific journal
- Insect cells have recently proven to be an excellent platform for the high-level production of functional recombinant proteins. Autophagy is an important mechanism that promotes cell survival by eliminating damaged organelles and protein aggregates, and it also may influence recombinant protein production. In the present study, we compared the effects that autophagy inducers rapamycin, everolimus, and lithium chloride exert on recombinant lepidopteran insect cells that secrete an engineered antibody molecule. Compared with nontreatment, treatment with either rapamycin or everolimus prolonged cell growth to allow high cell density, improved viability in the declining phase, and then increased the yield of secreted antibodies. These positive effects appeared to be induced via autophagy since autophagosomes were clearly detected, particularly in cells treated with rapamycin or everolimus. Unlike rapamycin, another autophagy inducer, FK506, was ineffective in insect cells. The addition of an appropriate autophagy inducer may be effective in increasing the productivity of recombinant proteins in insect cells.Sep. 2020, Cytotechnology, 73(3) (3), 299 - 305, English, International magazine[Refereed]Scientific journal
- Antibody Fab fragments consist of heavy chain (Hc) and light chain (Lc) polypeptides assembled with a disulphide bond. The production of a recombinant Fab fragment requires the simultaneous expression of two genes encoding both an Hc and an Lc in the same host cell. In the present study, we investigated the production of Fab fragments in lepidopteran insect cells using a bicistronic plasmid vector carrying the Hc and Lc genes linked with a 2A self-cleaving peptide sequence from the porcine teschovirus-1. We also examined the arrangement of a GSG spacer sequence and a furin cleavage site sequence with the 2A sequence. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of culture supernatants showed that Trichoplusia ni BTI-TN-5B1-4 (High Five) cells transfected with a plasmid in which the Hc and Lc genes were joined by the 2A sequence successfully secreted Fab fragments with antigen-binding activity after self-cleavage of the 2A peptide. The GSG linker enhanced 2A cleavage efficiency, and the furin recognition site was useful for removal of 2A residues from the Hc. Transfection with a single plasmid that contained sequences for GSG, the furin cleavage site, GSG, and the 2A peptide between the Hc and Lc genes exhibited a higher productivity than co-transfection with a set of plasmids separately carrying the Hc or Lc gene. These results demonstrate that bicistronic expression with the appropriate combination of a furin recognition site, GSG linkers, and a 2A peptide may be an effective way to efficiently produce recombinant antibody molecules in insect cells.Aug. 2020, Journal of bioscience and bioengineering, 130(2) (2), 205 - 211, English, Domestic magazine[Refereed]Scientific journal
- Springer Science and Business Media LLC, Aug. 2008, Nature Medicine, 14(9) (9), 939 - 948[Refereed]Scientific journal
- Purpose Docetaxel is one of the most effective anticancer drugs available in the treatment of breast cancer. Nearly half of the treated patients, however, do not respond to chemotherapy and suffer from side effects. The ability to reliably predict a patient's response based on tumor gene expression will improve therapeutic decision making and save patients from unnecessary side effects. Patients and Methods A total of 44 breast tumor tissues were sampled by biopsy before treatment with docetaxel, and the response to therapy was clinically evaluated by the degree of reduction in tumor size. Gene expression profiling of the biopsy samples was performed with 2,453 genes using a high-throughput reverse transcriptase polymerase chain reaction technique. Using genes differentially expressed between responders and nonresponders, a diagnostic system based on the weighted-voting algorithm was constructed. Results This system predicted the clinical response of 26 previously unanalyzed samples with over 80% accuracy, a level promising for clinical applications. Diagnostic profiles in nonresponders were characterized by elevated expression of genes controlling the cellular redox environment (ie, redox genes, such as thioredoxin, glutathione-S-transferase, and peroxiredoxin). Overexpression of these genes protected cultured mammary tumor cells from docetaxel-induced cell death, suggesting that enhancement of the redox system plays a major role in docetaxel resistance. Conclusion These results suggest that the clinical response to docetaxel can be predicted by gene expression patterns in biopsy samples. The results also suggest that one of the molecular mechanisms of the resistance is activation of a group of redox genes.Lead, American Society of Clinical Oncology (ASCO), Jan. 2005, Journal of Clinical Oncology, 23(3) (3), 422 - 431[Refereed]Scientific journal
- Molecular classification of primary breast tumors possessing distinct prognostic properties.The natural progression of breast cancer differs greatly between patients; the precise prediction of this disease course will improve the efficacy of therapeutics. Gene expression profiling may elucidate the undiscovered biological variations between seemingly similar cancers, leading to a new cancer classification system valuable in accurate diagnosis. The expression levels of 2412 genes, derived from 98 cancer samples, were precisely recorded by a high throughput RT-PCR technique, adapter-tagged competitive PCR. Subsequent cluster analysis revealed a molecular profile, correlating with estrogen receptor levels and the presence of lymph node metastases. We analyzed 301 cancer samples for the expression patterns of 21 genes critical in this categorization. The classification of the samples into three major groups was verified utilizing principal component analysis. This molecular classification system correlated significantly with early recurrence, independent of lymph node status. This malignant potential is associated with the expression levels of a group of genes, which comprise a set of candidates potentially useful in diagnostic prediction. These genes and the associated control mechanisms may also be effective therapeutic targets.Lead, Jan. 2002, Human molecular genetics, 11(2) (2), 199 - 206, English, International magazine[Refereed]Scientific journal
- 第70回日本生物工学会大会, Sep. 2018, Japanese, 日本生物工学会大会, 吹田, Domestic conference昆虫細胞における2Aペプチドを用いた抗体生産Oral presentation
- 化学工学会第50回秋季大会, Sep. 2018, English, 化学工学会, 鹿児島, Domestic conferenceゲノム編集技術を用いた組換え昆虫細胞の作製Poster presentation
- 化学工学会第83年会, Mar. 2018, Japanese, 化学工学会, 吹田, Domestic conference昆虫細胞によるインフルエンザウイルス抗原タンパク質の生産Poster presentation
- 化学工学会第49回秋季大会, Sep. 2017, Japanese, 化学工学会, 名古屋, Domestic conference昆虫細胞による2Aペプチドを用いた抗体タンパク質の生産Poster presentation
- 日本学術振興会, 科学研究費助成事業, 奨励研究, 兵庫医科大学, 01 Apr. 2016 - 31 Mar. 2017胆管細胞癌におけるＢ型肝炎ウイルスゲノム組み込み解析研究
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Mukogawa Women's University, 01 Apr. 2012 - 31 Mar. 2015The ACTN3 gene is a potential biomarker for the risk of non-contact sports injury in female athletesSports injuries can become serious impairments for all athletes. Most notably, female athletes are at higher risk than men for sports injury, for example, anterior cruciate ligament (ACL) disorder. However, there is currently no genetic marker to determine if a female athlete harbors a predisposition for muscle trauma. Hence, we performed single nucleotide polymorphism genotyping of the α-actinin-3 (ACTN3) in 99 young female athletes who had been injured during a sports activity, and we compared the occurrence of muscle traumas with the genotypes using the chi-square test. For the ACTN3 577R allele, the subjects who had non-contact muscle injury had a marked increase in frequency (p-value = 0.0015; odds ratio = 2.52). The significant increase in non-contact muscle injury related to ACTN3 577R alleles suggests that ACTN3 is likely to be involved in muscle strain and that non-contact muscle injury might occur due to the presence of this allele.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Mukogawa Women's University, 2011 - 2013Educational research for genetics and gene analysis to the next generation.We performed lectures and practices at high schools and universities as described in the application, and then extensively at elementary schools (including parents), drink-suppliers, pharmacists. For 3rd grade students in university, we designed a questionnaire to investigate changes in attitude of alcohol drinking and in quantity of daily alcohol intake, because all the students have reached a legal age for alcohol drinking, 20 or over. We developed and optimized the experimental protocol for high school students and also basic technologies for advanced lectures, which consist of the personal genome information and and its medical application, to pharmacology students.
- 日本学術振興会, 科学研究費助成事業, 若手研究(B), 地方独立行政法人大阪府立病院機構大阪府立成人病センター(研究所), 2005 - 2006染色体の増幅・欠失の網羅的検出解析による多発及び再発肝細胞癌の遺伝子診断の確立肝細胞癌では、外科的な根治的切除後や局所治療後も、5年以内に約80%で再発が見られる。これは微少肝内転移(IM)や異時性多中心性発癌(MC)が原因と考えられるが、IMとMCでは治療方針が異なるために、この両者を見分けることは癌のオーダーメイドな治療を行う上で臨床上重要であると考えられる。本研究では、染色体におけるゲノムコピー数を測定する手法を開発し、その手法を用いてIMとMCの染色体異常を同定し、その違いを解明することを目的とする。 まず、今年度はPCRベースでの染色体ゲノムコピー数の変化を同定する手法(Competitive Genome PCR法;CGP法と命名)の開発を行った。CGP法はPCRで行うため、簡便で臨床的にも使いやすいと考えられる。現在までに本手法を開発し確立したが、本手法の感度を検定するため、以下の解析を行った。まず、X染色体のコピー数の異なるcell line(46XY(1X)、46XX(2X)、47XXX(3X)、48XXXX(4X)、49XXXXX(5X)の5種類の核型をもつcell line)からのgenomic DNAを用いて、46XXをreferenceとし、本手法によりX染色体のコピー数の比較を行ったところ、微小なゲノムコピー数変化も検出でき、高感度で解析が行えることが判明した。また12種類のneuroblastoma cell lineからのgenomic DNAを用いた、MYCN遺伝子近傍の増幅領域の同定をするなど、実用的な解析が可能であることも明らかとした。この手法は、昨年11月に国内で特許申請を行ったほか、現在、論文の投稿も完了している。現在は肝細胞癌を用いて、IM,MCの遺伝子診断を行うべく、プライマーを設計し、順次解析を行っているところである。