1996 Fiscal Year Annual Research Report

遺伝子治療実用化にむけての技術開発及びそのシステムの検討

Research Project

Project/Area Number	07557169
Research Institution	Kumamoto University
Principal Investigator	松田一郎熊本大学, 医学部, 教授 (10000986)
Co-Investigator(Kenkyū-buntansha)	緒方嘉貴テルモ(株)研究開発センター, 研究員内山秀樹テルモ(株)研究開発センター, 専門研究員島田隆日本医科大学, 教授 (20125074) 遠藤文夫熊本大学, 医学部附属病院, 講師 (00176801) 山本哲郎熊本大学, 医学部, 教授 (60112405)
Keywords	遺伝子治療 / ベクター / リポソーム / AAVベクター / ウイルソン病 / OTC欠損症
Research Abstract	1.a)表面修飾剤を含む多重膜リポソーム(MLV),TRXリポソームを新たに2種開発した。この中に目的とする遺伝子(GAGプロモーターに組み込まれたプラスミド)をトランスフェクトして導入効率を検討した。標的細胞にはCosl,HepG2及びウィルソン病のモデルラットであるLECラット肝細胞由来の培養細胞を用い、市販のリポソームであるリポフェクチン(DNAを外側につけるタイプ)、ジーントランスファ(内封型)と比較した。トランスジーンは、βガラクトシダーゼ,オルニチントランスカルバミラーゼ(OTC),ウイルソン病遺伝子(Wdeu)を用いた。少数例ではあるが、OTC欠損症モデル動物(Spfash)及びウイルソン病モデルラット(LECラット)を用いてin vitro導入も試みた。導入効率の判定には、組織染色(βガラクトシダーゼ),酵素活性測定(OTC),免疫抗体(Wdeu)を用いた。 b)TRXシリーズはいずれも市販のリポフェクチン、トランスジーンに比べて3〜10倍の効率で遺伝子導入を達成することが確認された。しかも、リポフェクチンは血清存在下では導入効率が低下するのは対し、TRXシリーズは、血清存在下で導入効率は劣化せず、むしろ上昇する結果も得られた。このことはin vitroでの応用の可能性を示唆している。 c)TRX9と遺伝子の経静脈投与、肝臓への直接注射を試みた。経静脈投与では肝組織の目的遺伝子発現はほとんど得られなかったが、肝臓直接注射では肝組織に目的遺伝子の発現がみられた。但し、グッパー細胞での発現が多く、肝細胞での発現はほとんどないか、あっても少数であった。今後、表面修飾にさちに工夫(抗レセプター抗体など)を加える必要がある。 2.他にAAVベクターについても検討した。

Research Products
(24 results)

All Other

All Publications (24 results)

[Publications] Kiwaki,K.: "Correction of ornithine transcarbamylase deficiiency in adult spf-ash mice and OTC-deficient human hepatocyutes with recombinant adenoviorus bearting the CAG promoter." Human Gene Teherapy. 7. 821-830 (1996)
[Publications] Hoshide,R.: "Prenatal monitoring in a family at high risk for omithine transcarbamylase (OTC) deficienc ; a new mutation of an A-to-C transversion in position +4 of intron of the OTC gene that is likely abokish enzyme activity." Am J Med Genet. 64. 459-464 (1996)
[Publications] Matsuda,I.: "Phenotype variability in male patients carrying the mutant ornithine transcarbamylase (OTC) allele,Arg40His,ranging from a child with an unfavorable prognosis to an asymptomatic older adult." J Med Genet. 33. 645-648 (1996)
[Publications] Uchino,T.: "Three brothers with progressive hepatoc dysfucntion and severe hepaticsteatosis due to a patent ductus venosus." Ganseroentenolgy.110. 1964-1966 (1996)
[Publications] Hoshide R.: "Molecular cloning,tissue distribution and chromosomal localization of human cationic amino acid transporter(HCAT2)." Genomics. 38. 174-178 (1996)
[Publications] Kubo S.: "In Vivo correction of 4-hydroxyplenylpyruvic acid dioxygenase deficiencies in III mice with recombinant adenovirus." Human Gene Therapy. 8. (1997)
[Publications] Komaki S.: "Familsal lethal inheritance of a mutated paternal gene in females causing X-linked ornithine transcarbamylase (OTC) gene." Am J Med Genet. (in press). 65-71 (1997)
[Publications] Shimadzu,M.: "Ten novel mutations of ornithine transcarbamylase (OTC) deficiency" Human Mutation. (in press). (1997)
[Publications] Hoshino A.: "Complete sequence analysis of rat tranaferrin and its expression in the digestive glands." Comp.Biochem.Biophys.113B. 491-497 (1996)
[Publications] Shimada T.: "Targeted gene transfer into CD4 positive cells by HIV based retrovital vectors." Molecular Biology of Hematopoiesis. (Abraham,N.G.,Asano,S.,Brittinger,G.,and Shadduck,R,eds.). 323-329 (1996)
[Publications] Watanabe A.: "Genomic organization and expression of the human NSH3 gene." Genomics. 31. 311-318 (1996)
[Publications] Taniguchi T.: "Stability of p53 tumor suppressor gene mutations during the process of metastasis and during chemotherapy." Lung Cancer. 14. 219-228 (1996)
[Publications] Tamatose K.: "A new strategy for large scale preparation of high titer recombinant adeno-associated virus (AAV) vectors by using packaging cell lines and sulfonated column." Hum.Gene Ther.7. 507-513 (1996)
[Publications] Shimada T.: "Current status and future prospects of human gene therapy." Acta Ped.Japonica. 38. 176-181 (1996)
[Publications] Orimo H.: "First trimester prenatal molecular diagnosis of infantile hypophosphatasia in a Japanese family." Prenatal Diag.16. 559-563 (1996)
[Publications] HuH.: "Infectivities of human and other primate lentiviruses are activated by desialylation of the virion surface." J.Virol.70. 7462-7470 (1996)
[Publications] Palombo F.: "HMutsβ,a heterodimer of hMSH2 and hMSH3,binds to insertion/deletion loops in DNA." Furrent Biol.6. 1181-1184 (1996)
[Publications] Obaru K.: "Gene therapy for adult T cell leukemia using human immunodeficiency virus vector carrying the thymidine kinase gene of herpes simplex virus type 1." Human Gene Ther.7. 2203-2208 (1996)
[Publications] Miyake K.: "Two step gene transfer using adenovirus vector containing the CD4 gene." Hum. Gene Ther.18. 2281-2287 (1996)
[Publications] Yasukawa M.: "Human herpesvirus 7 infection of lymphoid and myeloid cell lines transduced with an adnovirus vector containing the CD4 gene." J.Viol.71. 1708-1712 (1997)
[Publications] Hayashi Z.: "Prenatal diagnosis of steroid 21-hydroxylase deficiency by analysis of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) profiles." Prenatal Diag.(in press). (1997)
[Publications] Goseki-Sone M.: "Hypophosphatasia : identification of five novel missense mutations in the tissue-nonspecific alkaline phosphatase gene among Japanese patients." Jum.Mo.Genet.(in press). (1997)
[Publications] Yamamoto S.: "HSV-TK/GCV mediated killing of tumor cells induces tumor-specific cytotoxic T cells in mice." Hum.Mol.Genet.(in press). (1997)
[Publications] Orimo H.: "Detection of deletion 1154-1156 hypophosphatasia mutation using TNSALP exon amplification." Genomics. (in press). (1997)

1996 Fiscal Year Annual Research Report

遺伝子治療実用化にむけての技術開発及びそのシステムの検討

Principal Investigator

松田 一郎 熊本大学, 医学部, 教授 (10000986)

Research Products

[Publications] Kiwaki,K.: "Correction of ornithine transcarbamylase deficiiency in adult spf-ash mice and OTC-deficient human hepatocyutes with recombinant adenoviorus bearting the CAG promoter." Human Gene Teherapy. 7. 821-830 (1996)

[Publications] Hoshide,R.: "Prenatal monitoring in a family at high risk for omithine transcarbamylase (OTC) deficienc ; a new mutation of an A-to-C transversion in position +4 of intron of the OTC gene that is likely abokish enzyme activity." Am J Med Genet. 64. 459-464 (1996)

[Publications] Matsuda,I.: "Phenotype variability in male patients carrying the mutant ornithine transcarbamylase (OTC) allele,Arg40His,ranging from a child with an unfavorable prognosis to an asymptomatic older adult." J Med Genet. 33. 645-648 (1996)

[Publications] Uchino,T.: "Three brothers with progressive hepatoc dysfucntion and severe hepaticsteatosis due to a patent ductus venosus." Ganseroentenolgy.110. 1964-1966 (1996)

[Publications] Hoshide R.: "Molecular cloning,tissue distribution and chromosomal localization of human cationic amino acid transporter(HCAT2)." Genomics. 38. 174-178 (1996)

[Publications] Kubo S.: "In Vivo correction of 4-hydroxyplenylpyruvic acid dioxygenase deficiencies in III mice with recombinant adenovirus." Human Gene Therapy. 8. (1997)

[Publications] Komaki S.: "Familsal lethal inheritance of a mutated paternal gene in females causing X-linked ornithine transcarbamylase (OTC) gene." Am J Med Genet. (in press). 65-71 (1997)

[Publications] Shimadzu,M.: "Ten novel mutations of ornithine transcarbamylase (OTC) deficiency" Human Mutation. (in press). (1997)

[Publications] Hoshino A.: "Complete sequence analysis of rat tranaferrin and its expression in the digestive glands." Comp.Biochem.Biophys.113B. 491-497 (1996)

[Publications] Shimada T.: "Targeted gene transfer into CD4 positive cells by HIV based retrovital vectors." Molecular Biology of Hematopoiesis. (Abraham,N.G.,Asano,S.,Brittinger,G.,and Shadduck,R,eds.). 323-329 (1996)

[Publications] Watanabe A.: "Genomic organization and expression of the human NSH3 gene." Genomics. 31. 311-318 (1996)

[Publications] Taniguchi T.: "Stability of p53 tumor suppressor gene mutations during the process of metastasis and during chemotherapy." Lung Cancer. 14. 219-228 (1996)

[Publications] Tamatose K.: "A new strategy for large scale preparation of high titer recombinant adeno-associated virus (AAV) vectors by using packaging cell lines and sulfonated column." Hum.Gene Ther.7. 507-513 (1996)

[Publications] Shimada T.: "Current status and future prospects of human gene therapy." Acta Ped.Japonica. 38. 176-181 (1996)

[Publications] Orimo H.: "First trimester prenatal molecular diagnosis of infantile hypophosphatasia in a Japanese family." Prenatal Diag.16. 559-563 (1996)

[Publications] HuH.: "Infectivities of human and other primate lentiviruses are activated by desialylation of the virion surface." J.Virol.70. 7462-7470 (1996)

[Publications] Palombo F.: "HMutsβ,a heterodimer of hMSH2 and hMSH3,binds to insertion/deletion loops in DNA." Furrent Biol.6. 1181-1184 (1996)

[Publications] Obaru K.: "Gene therapy for adult T cell leukemia using human immunodeficiency virus vector carrying the thymidine kinase gene of herpes simplex virus type 1." Human Gene Ther.7. 2203-2208 (1996)

[Publications] Miyake K.: "Two step gene transfer using adenovirus vector containing the CD4 gene." Hum. Gene Ther.18. 2281-2287 (1996)

[Publications] Yasukawa M.: "Human herpesvirus 7 infection of lymphoid and myeloid cell lines transduced with an adnovirus vector containing the CD4 gene." J.Viol.71. 1708-1712 (1997)

[Publications] Hayashi Z.: "Prenatal diagnosis of steroid 21-hydroxylase deficiency by analysis of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) profiles." Prenatal Diag.(in press). (1997)

[Publications] Goseki-Sone M.: "Hypophosphatasia : identification of five novel missense mutations in the tissue-nonspecific alkaline phosphatase gene among Japanese patients." Jum.Mo.Genet.(in press). (1997)

[Publications] Yamamoto S.: "HSV-TK/GCV mediated killing of tumor cells induces tumor-specific cytotoxic T cells in mice." Hum.Mol.Genet.(in press). (1997)

[Publications] Orimo H.: "Detection of deletion 1154-1156 hypophosphatasia mutation using TNSALP exon amplification." Genomics. (in press). (1997)

松田一郎熊本大学, 医学部, 教授 (10000986)