JP4791651B2 - Paramyxovirus vector for foreign gene transfer - Google Patents

Description

【００５６】
＜レポーター遺伝子発現の比較＞
LLC-MK2細胞を6well プレートに1wellあたり1〜5×10⁵ cellsずつ蒔き、24時間培養した後、各ウイルスベクターをmoi=2で感染させ、24時間後培養上清を100μl 回収し、SEAPアッセイを行った。アッセイはReporter Assay Kit -SEAP-（東洋紡）で行い、ルミノイメージアナライザーLAS1000（富士フィルム）で測定した。測定値はSeV18+/SEAPの値を100としてそれぞれ相対値として表した。その結果、図５に示したいずれの位置にSEAP遺伝子を挿入した場合でもSEAP活性が検出された。SEAP活性はゲノムの下流に位置するに従って下がり、すなわち発現量が下がっていることがわかった。SEAP遺伝子の発現量は、挿入位置がゲノムの上流（NP側）から下流（L側）に行くに従い単調減少した。例えば、NP遺伝子とP遺伝子の間にSEAP遺伝子を挿入した場合には、NP遺伝子の上流にSEAP遺伝子を挿入したベクターと、P遺伝子とM遺伝子の間にSEAP遺伝子を挿入したベクターの中間の発現量が検出された。
【００５７】
＜マルチクローニングサイトの作製＞
マルチクローニングサイトをSeVベクターに付加させた。方法は以下の二種類。
1）センダイウイルス（SeV）全長ゲノムcDNA、pSeV18⁺ b(+)（Hasan, M. K. et al., 1997, J. General Virology 78: 2813-2820）（「pSeV18⁺ b(+)」は「pSeV18⁺」ともいう）cDNAのゲノム中のいくつかの制限酵素サイトを壊し、つぶした制限酵素サイトを含む新たな制限酵素サイトを各遺伝子のスタートシグナルとATG翻訳開始シグナルの間に導入した（図６）。
2）すでに構築したSeVベクターcDNAにマルチクローニングサイト配列と転写開始シグナル-介在配列-終結シグナルを付加させてNotIサイトへ組み込む（図７）。
【００５８】
1）の場合、導入方法としてはまず、図６(A)のように pSeV18⁺ をEag Iで消化した断片（2644bp）、Cla Iで消化した断片（3246bp）、ClaI/Eco RIで消化した断片（5146bp）、及びEco RIで消化した断片（5010bp）をそれぞれアガロース電気泳動で分離、該当するバンドを切り出し、QIAEXII Gel Extraction System（QIAGEN社製）で回収・精製した。Eag Iで消化した断片はLITMUS38（NEW ENGLAND BIOLABS社製）、Cla Iで消化した断片、ClaI/Eco RIで消化した断片、及びEco RIで消化した断片はpBluescriptII KS+（STRATAGENE社製）にライゲーションし、サブクローニングした。続いて制限酵素サイトの破壊、導入にはQuickchange Site-Directed Mutagenesis kit（STRATAGENE社製）を使った。
【００５９】
制限酵素サイトの破壊にはSal I:（センス鎖）5'-ggagaagtctcaacaccgtccacccaagataatcgatcag-3'（配列番号：１７）、（アンチセンス鎖）5'-ctgatcgattatcttgggtggacggtgttgagacttctcc-3'（配列番号：１８）、Nhe I:（センス鎖）5'-gtatatgtgttcagttgagcttgctgtcggtctaaggc-3'（配列番号：１９）、（アンチセンス鎖）5'-gccttagaccgacagcaagctcaactgaacacatatac-3'（配列番号：２０）、Xho I: （センス鎖）5'-caatgaactctctagagaggctggagtcactaaagagttacctgg-3'（配列番号：２１）、（アンチセンス鎖）5'-ccaggtaactctttagtgactccagcctctctagagagttcattg-3'（配列番号：２２）、また制限酵素導入にはNP-P間：（センス鎖）5'-gtgaaagttcatccaccgatcggctcactcgaggccacacccaaccccaccg-3'（配列番号：２３）、（アンチセンス鎖）5'-cggtggggttgggtgtggcctcgagtgagccgatcggtggatgaactttcac-3'（配列番号：２４）、P-M間：（センス鎖）5'-cttagggtgaaagaaatttcagctagcacggcgcaatggcagatatc-3'（配列番号：２５）、（アンチセンス鎖）5'-gatatctgccattgcgccgtgctagctgaaatttctttcaccctaag-3'（配列番号：２６）、M-F間：（センス鎖）5'-cttagggataaagtcccttgtgcgcgcttggttgcaaaactctcccc-3'（配列番号：２７）、（アンチセンス鎖）5'-ggggagagttttgcaaccaagcgcgcacaagggactttatccctaag-3'（配列番号：２８）、F-HN間：（センス鎖）5'-ggtcgcgcggtactttagtcgacacctcaaacaagcacagatcatgg-3'（配列番号：２９）、（アンチセンス鎖）5'-ccatgatctgtgcttgtttgaggtgtcgactaaagtaccgcgcgacc-3'（配列番号：３０）、HN-L間：（センス鎖）5'-cccagggtgaatgggaagggccggccaggtcatggatgggcaggagtcc-3'（配列番号：３１）、（アンチセンス鎖）5'-ggactcctgcccatccatgacctggccggcccttcccattcaccctggg-3'（配列番号：３２）をそれぞれ合成し反応に用いた。導入後、それぞれの断片を上記同様に回収・精製し、cDNAをアセンブリした（図６(B)）。
【００６０】
2）の場合、（センス鎖）5'-ggccgcttaattaacggtttaaacgcgcgccaacagtgttgataagaaaaacttagggtgaaagttcatcac-3'（配列番号：３３）、（アンチセンス鎖）5'-ggccgtgatgaactttcaccctaagtttttcttatcaacactgttggcgcgcgtttaaaccgttaattaagc-3'（配列番号：３４）を合成し、それぞれの合成DNAをリン酸化し、85℃ 2分、65℃ 15分、37℃ 15分、室温 15分でアニーリングさせ、SeV cDNAへ組み込む。あるいはpUC18またはpBluescriptII等のマルチクローニングサイトを終結シグナル-介在配列-開始シグナル含むプライマーでPCRしてサブクローニングし、これをSeV cDNAへ組み込む。得られたcDNAでのウイルス再構成は上記の通り行う。
【００６１】
[実施例２] センダイウイルスにおける極性効果を利用した遺伝子発現量の制御 II
＜SeVゲノムcDNAの構築とウイルスの回収＞
センダイウイルス（SeV）全長ゲノムcDNA、pSeV(+)のcDNAにNot IサイトをL遺伝子の翻訳終止コドンと転写終結シグナルの間に導入した。導入方法としてはまず、図８のように、pSeV(+)をEco RIで消化した断片（5010bp）をアガロース電気泳動で分離、該当するバンドを切り出し、QIAEXII Gel Extraction System（QIAGEN社製）で回収・精製した。回収した断片はpBluescriptII SK+（STRATAGENE社製）にライゲーションし、サブクローニングした。続いて制限酵素 Not I サイトの導入にはQuickchange Site-Directed Mutagenesis kit（STRATAGENE社製）を使った。プライマーはセンス側：5'-cgtgcagaacgatcgaagctccgcggccgctggaagtcttggacttgtcc-3'（配列番号：３５）、アンチセンス側：5'-ggacaagtccaagacttccagcggccgcggagcttcgatcgttctgcacg-3'（配列番号：３６）をそれぞれ合成し反応に用いた。導入後、Xho I-Mlu Iで消化した断片（2010bp）を上記同様に回収・精製し、cDNAをアセンブリした（図８）。
【００６２】
遺伝子発現量を見るための遺伝子としてヒトVascular Endothelial Growth Factor (VEGF)をPCRでサブクローニングした。プライマーには 制限酵素Asc Iサイトを付加した5'プライマー：5'-gcggcgcgccaaccatgaactttctgctgtcttgggtgcattgg-3'（配列番号：３７）、3'プライマー：5'-gcggcgcgcctcaccgcctcggcttgtcacatctgcaagt-3'（配列番号：３８）を合成し、PCRを行った。酵素にはKOD - plus - DNAポリメラーゼ（TOYOBO社製）を用いた。PCR後、産物をAsc Iで消化し、電気泳動により精製・回収した。これにセンダイウイルスの転写終結シグナル-介在配列-転写開始シグナルを付加させるためのプラスミドを構築した（図９）。pSeV18+のMlu I - Sph I断片（3317bp）を組み込んだLITMUS38（pAC114とする）に終結シグナル-介在配列-開始シグナルとクローニングサイト（Asc I - Pme I）含む合成二本鎖DNA（センス鎖：5'-ggccgctaagaaaaacttagggtgaaagttcacttcacgagggcgcgccgtttaaactgc-3'（配列番号：３９）、アンチセンス鎖：5'-ggccgcagtttaaacggcgcgccctcgtgaagtgaactttcaccctaagtttttcttagc-3'（配列番号：４０））を組み込んだものであるpAG180を作製した（図９）。このプラスミドのAsc Iサイトに、精製・回収したVEGF PCR産物をライゲーションし、クローニングした。これをNot Iで消化してVEGF遺伝子断片を電気泳動で回収・精製し、センダイウイルスゲノムcDNAのNot Iサイトにライゲーションし組み込んだ。ウイルスベクターcDNAをpSeV+L/VEGFとした。また実施例１記載の方法で作成した終結シグナル-介在配列-開始シグナル（図２参照）を、VEGF cDNAの下流につなぎ、pSeV18+、pSeV(+)HNLに組み込んだもの（それぞれをpSeV18+/VEGF、pSeV(+)HNL/VEGFとする）も発現量の比較のため構築した。それぞれのcDNAからウイルスを従来の方法通り回収し、それぞれSeV18+/VEGF、SeVHNL/VEGF、SeVL/VEGFとした。
【００６３】
＜発現量の比較＞
LLC-MK2細胞を6well プレートに1wellあたり5×10⁵cellsずつ蒔き、24時間培養した後、各ウイルスベクターをmoi=5で感染させ、24時間後培養上清を200μl 回収し、ELISAにより培養上清中のVEGFの定量を行った。その結果を図１０に示す。これによりL遺伝子の下流に組み込むことでVEGFの発現量がNP遺伝子の上流に組み込んだときより1/10ほど低下することがわかった。本発明者らは、転写開始シグナルを変更することで発現量を変化させることができることを開示（WO01/18223）しており、これを組み合わせることによりさらに発現量の制御の幅が広がることが予想される。
【００６４】
【発明の効果】
本発明により、外来遺伝子を導入することができる、複製能を有するパラミクソウイルスベクターが提供された。本発明のベクターは、外来遺伝子の挿入位置を調節することにより、発現レベルを制御することが可能である。特に、外来遺伝子をネガティブ鎖ゲノムに挿入することにより、外来遺伝子の発現レベルを低く抑えることができる。また、２種類以上の部位に外来遺伝子が挿入されたウイルスベクターの作製も考えられる。特にセンダイウイルスベクターは遺伝子導入効率も広範な細胞種に対して極めて高く、さらに本発明により外来遺伝子の発現レベルを制御することができるため、遺伝子治療などの生体における遺伝子導入への利用が期待される。
【００６５】
【配列表】

【図面の簡単な説明】
【図１】センダイウイルスゲノムcDNA断片のサブクローニング（A）と新たにNotIサイトを導入し構築した5種類のセンダイウイルスゲノムcDNAの構造（B）を示す図である。
【図２】 SEAPにNotIサイト、転写開始シグナル、介在配列、転写終結シグナルを付加するためのクローニング用プラスミドの構造を示す図である。
【図３】 L遺伝子の下流のセンダイウイルスゲノムの構造（A）、およびL遺伝子の下流へ外来遺伝子を挿入するためのクローニング用DNAの構造（B）を示す図である。
【図４】各センダイウイルスベクターのプラークアッセイの結果を示す図である。LAS1000で取り込んだプラークアッセイの蛍光画像の一部を示す。
【図５】各センダイウイルスベクター間におけるレポーター遺伝子（SEAP）の発現量の違いを比較した結果を示す図である。SeV18+/SEAPのデータを100としてそれぞれ相対値を表した。SEAP遺伝子が下流に位置するに従ってその活性すなわち発現量が低下していくことがわかった。
【図６】センダイウイルスゲノムcDNAに構築したマルチクローニングサイトの構造を示す図である。
【図７】マルチクローニングサイトの構造を示す図である。塩基配列は、PacI-PmeI-BssHII-TspRIの制限酵素サイトとE/I/S配列を有するマルチクローニングサイトの例を示す。
【図８】センダイウイルスゲノムcDNA断片のサブクローニングと新たにNot Iサイトを導入し構築したセンダイウイルスゲノムcDNAの構造を示す図である。
【図９】 VEGFにNot Iサイト、転写開始シグナル、介在配列、転写終結シグナルを付加するためのクローニング用プラスミドの構造および付加後のVEGFの構造を示す図である。
【図１０】 ELISAによるVEGF搭載のSeVベクターの発現量の比較を示す図である。L遺伝子の後にVEGFを搭載したものが最も発現量が低かった。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a replication-friendly paramyxovirus vector into which a foreign gene can be introduced.
[0002]
[Prior art]
Many conventional gene therapy clinical research approaches have utilized viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses. These gene therapy vectors have significant medical application problems such as limited introduction efficiency and sustained expression and cytotoxicity and immunogenicity of the vector itself (Lamb, RA & Kolakofsky, D., Paramyxoviridae: the viruses and their replication. In Fields Virology, 3rd edn, (Edited by BN Fields, DM Knipe & PP Howley) pp.1177-1204 (Philadelphia, Lippincott-Raven. (1996)). Vectors have been proposed based on lentiviruses and HSV, and improvements in existing vectors have been energetically carried out, but all these vectors exist in the form of DNA in the nucleus in the life cycle. Therefore, it is difficult to completely avoid safety concerns related to random interactions with patient chromosomes.
[0003]
Recent rapid advances in reverse genetics technology have made it possible to develop vectors based on RNA viruses that have been delayed in development. Recombinant RNA viruses show high gene transfer efficiency and expression ability, suggesting high potency as gene therapy vectors (Roberts, A. & Rose, JK, Virology 247,1-6 (1998); Rose, JK, Natl. Acad. Sci. USA 93, 14998-15000 (1996); Palese, P. et al., Proc. Natl. Acad. Sci. USA 93, 11354-11358 (1996)). Paramyxovirus vectors having negative-strand RNA in the genome have several characteristics that are significantly different from those of retrovirus, DNA virus, or plus-strand RNA virus vectors. Its genome or antigenome does not function directly as mRNA and cannot initiate viral protein synthesis or genome replication. Both the viral RNA genome and the antigenome always exist in the form of ribonucleoprotein (RNP) complexes, and hybridize to naked genomic RNA complementary to mRNAs, as seen in positive-stranded RNA viruses. Antisense problems such as interfering with RNP assembly are rare. These viruses have their own RNA polymerase, which uses the RNP complex as a template to transcribe viral mRNA or replicate viral genomes. Notably, negative-strand RNA (nsRNA) viruses grow only in the cytoplasm of the host cell and do not have a DNA phase, so there is no chromosomal integration. Furthermore, homologous recombination between RNAs has not been recognized. These properties seem to contribute greatly to the stability and safety of negative-strand RNA viruses as gene expression vectors.
[0004]
The present inventors have paid attention to Sendai virus (SeV) which is not pathogenic to humans among negative-strand RNA viruses, particularly Z strain which is particularly attenuated. SeV is a non-segmented negative-strand RNA virus, belonging to paramyxovirus and a kind of murine parainfluenza virus. This virus adheres to the host cell membrane via two envelope glycoproteins, hemagglutinin-neuraminidase (HN) and fusion protein (F), and causes membrane fusion, effectively with its own RNA polymerase. Releases the RNA genome present in the form of a ribonucleoprotein (RNP) complex into the cytoplasm, where it transcribes viral mRNA and replicates the genome (Bitzer, M. et al., J. Virol. 71 (7) : 5481-5486, 1997). Virus envelope protein F is an inactive precursor protein (F₀) And cleaved into F1 and F2 by proteolytic cleavage by trypsin (Kido, H. et al., Biopolymers (Peptide Science) 51 (1): 79-86, 1999). It causes membrane fusion. This virus is said to be nonpathogenic to humans. Also, a laboratory attenuated strain (Z strain) has been isolated, and only induces mild pneumonia in rodents as natural hosts. This strain has been widely used as a research model at the molecular level, such as the transcriptional replication mechanism of paramyxovirus, and has also been used to produce hybridomas. In addition to this high level of safety, the virus can be used in cell lines or eggs.^{9 ~ 11}High production titer of pfu / ml. Among the recent successful collection systems of negative-strand RNA viral vectors from cDNA, Sendai virus shows particularly high reconstitution efficiency. Recombinant wild viruses introduced with a foreign gene have attracted attention for their ability to efficiently and stably express the introduced foreign gene.
[0005]
So far, Sendai virus vectors with foreign genes inserted upstream of the NP gene are known, but when foreign genes are inserted into other sites, what kind of virus reconstitution and expression of foreign genes is required? It was not known whether it would have an impact.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a paramyxovirus vector having replication ability capable of introducing a foreign gene.
[0007]
[Means for Solving the Problems]
The present inventors constructed a viral vector DNA in which a foreign gene is inserted in the site before and after each gene encoding the Sendai virus viral protein, and examined the reconstitution of the virus and the expression level of the foreign gene. That is, a new restriction enzyme site for introducing a foreign gene was introduced into Sendai virus (SeV) full-length genomic cDNA between the start signal of each gene encoding the viral protein protein and the ATG translation start signal. A foreign gene (human secreted alkaline phosphatase gene) was inserted into this restriction enzyme site, and when Sendai virus was reconstructed using LLC-MK2 cells, it was confirmed that Sendai virus with replication ability was reconstructed. It was done. Each of these viruses was amplified in chicken eggs to prepare a virus stock solution. The virus was infected with LLC-MK2 cells with the titer, and the expression level of the foreign gene was measured. When the foreign gene was inserted at any of the examined positions, the expression of the foreign gene was observed. When a foreign gene is inserted upstream of the genome (3 ′ side of the negative strand), that is, before the NP gene or between the NP gene and the P gene, the expression level of the foreign gene is relatively high, and the foreign gene is inserted. It was found that the expression of the foreign gene decreases as the position approaches the downstream of the genome (5 ′ side of the negative strand).
[0008]
These results show that if the foreign gene is located upstream of the NP gene or downstream of the NP gene (between the NP gene and the P gene), it is possible to obtain a relatively high expression of the foreign gene. It is shown that the expression level can be reduced if it is located downstream of the genome. Based on this knowledge, in order to obtain high expression of foreign genes, foreign genes are inserted upstream of the genome, that is, 3 'side of the negative strand genome. If this is not preferable, it is possible to control the expression level of the foreign gene in the vector by inserting the foreign gene downstream of the genome, that is, 5 ′ of the negative strand genome. Thus, the paramyxovirus vector of the present invention is extremely useful as a paramyxovirus vector for attenuating and expressing foreign genes. The paramyxovirus vector of the present invention is useful for the expression of foreign genes in vivo and in vitro, and is particularly expected to be applied as a gene therapy vector taking advantage of the excellent characteristics of paramyxovirus.
[0009]
RNA viruses may point to genome stability problems, but the results of heterologous gene expression with the SeV vector show almost no base mutations even after successive passages of the virus. It has been shown to be stably expressed over time (Yu, D. et al. Genes Cells 2, 457-466 (1997)). This negative-strand RNA virus replicon-based vector is based on a successful positive-strand RNA virus, Semliki forest virus or Sindbis viruses replicon. Compared to the virus vector, there are several advantages in terms of properties such as the stability of the genome, the size of the transgene due to the absence of the capsid structural protein, or packaging flexibility. A Sendai virus vector having replication ability can introduce foreign DNA up to at least 4 kbp, and by adding a transcription unit, it may be possible to express two or more genes simultaneously. This Sendai virus replicon-based vector re-infects surrounding cells with the replicated virus, and multiple copies of the RNP replicated in the cytoplasm of the infected cell are distributed to daughter cells as the cell divides. Therefore, sustained expression is expected. Furthermore, the present inventors have found that Sendai virus vectors are gene-transferred with high efficiency into blood cells, particularly granulocytes, and also into c-kit positive primitive cells. This suggests that this vector can be a highly applicable vector with a very wide tissue application range.
[0010]
That is, the present invention relates to a replication-friendly paramyxovirus vector into which a foreign gene can be introduced, more specifically,
(1) A paramyxovirus vector having a foreign gene and having replication ability, wherein the foreign gene is located downstream of the gene encoding the viral protein in the negative strand genomic RNA contained in the vector. ,
(2) A paramyxovirus vector having a foreign gene and having replication ability, the vector according to any one of (a) to (f) below,
(A) a vector in which a foreign gene is inserted between the gene encoding the viral protein located first from the 3 ′ end of the negative-strand genomic RNA contained in the vector and the gene encoding the second viral protein;
(B) a vector in which a foreign gene is inserted between a gene encoding a viral protein located second from the 3 ′ end of the negative-strand genomic RNA contained in the vector and a gene encoding the third viral protein;
(C) a vector in which a foreign gene is inserted between the gene encoding the viral protein located third from the 3 ′ end of the negative-strand genomic RNA contained in the vector and the gene encoding the fourth viral protein,
(D) a vector in which a foreign gene is inserted between the gene encoding the viral protein located fourth from the 3 ′ end of the negative-strand genomic RNA contained in the vector and the gene encoding the fifth viral protein;
(E) a vector in which a foreign gene is inserted between the gene encoding the viral protein located fifth from the 3 ′ end of the negative-strand genomic RNA contained in the vector and the gene encoding the sixth viral protein;
(F) a vector in which a foreign gene is inserted between a gene encoding a viral protein located at the sixth position from the 3 ′ end of negative-strand genomic RNA contained in the vector and a trailer sequence;
(3) Genes encoding viral proteins located in the first to sixth positions from the 3 ′ end of negative-strand genomic RNA contained in the vector are, in order, NP gene, P gene, M gene, F gene, HN gene, and L The vector according to (2), which is a gene;
(4) DNA encoding negative strand genomic RNA or its complementary strand contained in the paramyxovirus vector according to any one of (1) to (3),
(5) DNA encoding negative-strand genomic RNA or its complementary strand contained in a paramyxovirus vector having replication ability, downstream of the gene encoding viral protein in the negative-strand genomic RNA or its complementary strand DNA holding a cloning site for inserting foreign genes,
(6) A DNA encoding a negative-strand genomic RNA contained in a replication-competent paramyxovirus vector or a complementary strand thereof, the DNA according to any one of (a) to (f) below,
(A) DNA having a cloning site for inserting a foreign gene between the gene encoding the viral protein located first from the 3 ′ end of the negative-strand genomic RNA and the gene encoding the second viral protein;
(B) A cloning site for inserting a foreign gene between the gene encoding the viral protein located second from the site corresponding to the 3 ′ end of the negative-strand genomic RNA and the gene encoding the third viral protein. DNA to hold,
(C) A cloning site for inserting a foreign gene between the gene encoding the viral protein located third from the site corresponding to the 3 ′ end of the negative-strand genomic RNA and the gene encoding the fourth viral protein. DNA to hold,
(D) a cloning site for inserting a foreign gene between the gene encoding the viral protein located fourth from the site corresponding to the 3 ′ end of the negative-strand genomic RNA and the gene encoding the fifth viral protein. DNA to hold,
(E) a cloning site for inserting a foreign gene between the gene encoding the viral protein located fifth from the site corresponding to the 3 ′ end of the negative-strand genomic RNA and the gene encoding the sixth viral protein. DNA to hold,
(F) DNA having a cloning site for inserting a foreign gene between a gene encoding a viral protein located sixth from the site corresponding to the 3 ′ end of negative-strand genomic RNA and a trailer sequence;
(7) Genes encoding viral proteins located first to sixth from the site corresponding to the 3 ′ end of the negative-strand genomic RNA are NP gene, P gene, M gene, F gene, HN gene, and L in this order. The DNA according to (6), which is a gene;
(8) A vector DNA that holds the DNA according to any one of (4) to (7) in a transcriptional manner,
(9) The vector DNA according to (8), which holds positive-strand genomic RNA in a transcribable manner,
(10) A method for controlling the expression level of a foreign gene in a paramyxovirus vector,
(A) a method of positioning a foreign gene between a gene encoding a viral protein located first from the 3 ′ end of the negative-strand genomic RNA contained in the vector and a gene encoding the second viral protein,
(B) a method of positioning a foreign gene between a gene encoding a viral protein located second from the 3 ′ end of the negative-strand genomic RNA contained in the vector and a gene encoding the third viral protein,
(C) a method of positioning a foreign gene between a gene encoding a viral protein located third from the 3 ′ end of the negative-strand genomic RNA contained in the vector and a gene encoding the fourth viral protein,
(D) a method of positioning a foreign gene between a gene encoding a viral protein located fourth from the 3 ′ end of the negative-strand genomic RNA contained in the vector and a gene encoding the fifth viral protein,
(E) a method of positioning a foreign gene between a gene encoding a viral protein located fifth from the 3 ′ end of the negative-strand genomic RNA contained in the vector and a gene encoding the sixth viral protein,
(F) a method of positioning a foreign gene between a gene encoding a viral protein located sixth from the 3 ′ end of negative-strand genomic RNA contained in a vector and a trailer sequence;
About.
[0011]
In the present invention, the “viral vector” refers to a viral particle having the ability to introduce a nucleic acid molecule into a host. In the present invention, the paramyxovirus refers to a virus belonging to the genus Paramyxovirus or a derivative thereof. Examples of the paramyxovirus to which the present invention can be applied include human parainfluenza virus type 1 (HPIV-1), human parainfluenza virus type 3 (HPIV-3), bovine parainfluenza virus type 3 (BPIV-3), Sendai Virus (Sendai virus; also called mouse parainfluenza virus type 1), simian parainfluenza virus type 10 (SPIV-10) and the like are included. More preferably, the virus of the present invention is Sendai virus. These viruses can be natural strains, wild strains, mutant strains, laboratory passage strains, artificially constructed strains, and the like. Incomplete viruses such as DI particles (J. Virol. 68, 8413-8417 (1994)), synthesized oligonucleotides, and the like can also be used as materials for producing the virus vector of the present invention.
[0012]
The genes encoding the paramyxovirus viral proteins include the NP, P, M, F, HN, and L genes. “NP, P, M, F, HN, and L genes” refer to genes encoding nucleocapsid, phospho, matrix, fusion, hemagglutinin-neuraminidase, and large protein, respectively. Each gene in each virus belonging to the Paramyxovirus subfamily is generally expressed as follows. In general, the NP gene is sometimes referred to as “N gene”.
Paramyxovirus NP P / C / V M F HN-L
Rubravirus NP P / V M F HN (SH) L
Mobilivirus NP P / C / V M F H-L
[0013]
For example, the accession number of the nucleotide sequence database of each gene of Sendai virus that is also classified in the genus Respirovirus of the Paramyxoviridae family is M29343, M30202, M30203, M30204, M51331 for the NP gene. , M55565, M69046, X17218, P gene is M30202, M30203, M30204, M55565, M69046, X00583, X17007, X17008, M gene is D11446, K02742, M30202, M30203, M30204, M69046, U31956, X00584, X53056, F For genes D00152, D11446, D17334, D17335, M30202, M30203, M30204, M69046, X00152, X02131, for HN genes D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, X56131, and L0005 for D00053 , M30202, M30203, M30204, M69040, X00587, X58886.
[0014]
“Having replication ability” means that when a viral vector infects a host cell such as LLC-MK2 or CV-1 cell, the virus replicates in the cell and infectious virus particles are produced. In the present invention, “DNA” includes single-stranded DNA and double-stranded DNA.
[0015]
In the present invention, “gene” refers to genetic material, and includes nucleic acids such as RNA and DNA. There is no restriction | limiting in the origin of a gene, It can derive from the sequence | arrangement designed naturally or artificially. Artificial proteins include, for example, fusion proteins with other proteins, dominant negative proteins (including receptor soluble molecules or membrane-bound dominant negative receptors), deletion-type cell adhesion molecules, and soluble cells It may be in the form of a surface molecule or the like. Alternatively, it may be a gene encoding a partial peptide of a bacterial or viral antigen protein related to an infectious disease. Further, it may be a nucleic acid that does not encode a protein such as an antisense nucleic acid or a ribozyme.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Paramyxoviruses generally contain a complex of RNA and protein (ribonucleoprotein; RNP) inside the envelope. RNA contained in RNP is a single strand RNA of negative strand (minus strand) that is the genome of paramyxovirus, and NP protein, P protein, and L protein bind to this RNA to form a complex . RNA contained in this RNP serves as a template for transcription and replication of the viral genome (Lamb, RA, and D. Kolakofsky, 1996, Paramyxoviridae: The viruses and their replication. Pp.1177-1204. In Fields Virology, 3rd edn. Fields, BN, DM Knipe, and PM Howley et al. (ed.), Raven Press, New York, NY). The RNP complex autonomously replicates in the cell and increases the copy number of the gene (RNA contained in the complex). This results in high expression of the foreign gene from the vector with the foreign gene.
[0017]
The viral vector of the present invention usually comprises (a) a vector DNA encoding a negative-strand single-stranded RNA derived from paramyxovirus or its complementary strand (positive strand), a cell expressing NP, P, and L proteins. It can be prepared by transcription with (helper cells), (b) culturing the cells, and collecting virus particles from the culture supernatant. RNA transcribed from vector DNA forms an RNP complex with NP, L, and P proteins, and further, viral particles encased in an outer shell containing an envelope protein.
[0018]
The DNA (vector DNA) encoding the viral genome, which is expressed in helper cells, encodes the negative strand (negative strand RNA) of the genome or its complementary strand (positive strand RNA). For example, a negative-strand single-stranded RNA or a DNA encoding a complementary strand thereof is ligated downstream of the T7 promoter and transcribed into RNA by T7 RNA polymerase. As a promoter, a desired promoter can be used in addition to a promoter containing a T7 polymerase recognition sequence. Alternatively, helper cells may be transfected with RNA transcribed in vitro. The strand to be transcribed in the cell may be a positive strand or a negative strand of the viral genome, but it is preferable to increase the efficiency of reconstruction by allowing the positive strand to be transcribed.
[0019]
In the case of Sendai virus (Sendai virus), the native virus has a genome size of about 15,000 bases, followed by a short 3 'leader region in the negative strand, followed by NP (nucleocapsid), P (phospho), M (matrix). ), F (fusion), HN (hemagglutinin-neuraminidase), and 6 genes encoding L (large) proteins are aligned, and has a short 5 ′ trailer region at the other end. In the present invention, some genes may be deficient as long as they have replication ability, and the arrangement of these genes on the viral genome may not be the same as that of the wild type. Since M, HN, and F proteins are not required for RNP formation, RNP is constructed by transcribing this genomic RNA (positive or negative strand) in the presence of NP, P, and L proteins. Infectious viral particles are constructed from RNP. The vector can be reconstructed using, for example, LLC-MK2 cells. The supply of NP, P, and L proteins can be performed by introducing an expression vector encoding each gene into a cell. Each gene may be integrated into the host cell chromosome. The NP, P, and L genes that are expressed to form the RNP need not be exactly the same as the NP, P, and L genes encoded in the vector genome. That is, the amino acid sequences of the proteins encoded by these genes are not limited to the amino acid sequences of the proteins encoded by the RNP genome, as long as they have the activity of forming RNP with genomic RNA and inducing gene expression from this RNP. Mutations may be introduced, or homologous genes of other viruses may be substituted. When RNP is formed, NP, P, and L genes are expressed from this RNP, and RNP autonomously replicates in the cell, and a viral vector is produced together with the envelope protein.
[0020]
The produced virus can be amplified or passaged by reinfection of cultured cells, chicken eggs, individuals (eg, mammals such as mice) and the like. In addition, the viral vector of the present invention can be amplified by reintroducing RNP formed by reconstitution of the viral vector into a host cell such as LLC-MK2 cell and culturing. This process includes (a) a step of introducing a negative strand single-stranded RNA derived from paramyxovirus and a complex consisting of NP, P / C, and L proteins into a cell, and (b) culturing the cell. Recovering virus particles from the culture supernatant.
[0021]
In order to introduce RNP into a cell, it is possible to introduce it by forming a complex with, for example, lipofectamine or polycationic liposome. Specifically, various transfection reagents can be used. For example, DOTMA (Boehringer), Superfect (QIAGEN # 301305), DOTAP, DOPE, DOSPER (Boehringer # 1811169) and the like can be mentioned. Chloroquine can also be added to prevent degradation in endosomes (Calos, M.P., 1983, Proc. Natl. Acad. Sci. USA 80: 3015).
[0022]
During virus reconstitution, envelope proteins other than the envelope protein encoded by the genomic RNA may be expressed in the cells. Examples of such proteins include envelope proteins of other viruses, such as the G protein (VSV-G) of vesicular stomatitis virus (VSV). The paramyxovirus vector of the present invention may be a vector containing an envelope protein derived from a virus other than the virus from which the genome is derived, such as a VSV-G protein. In addition to the viral envelope protein, for example, it has a polypeptide derived from an adhesion factor, a ligand, a receptor, etc. that can adhere to a specific cell in the extracellular region, and the polypeptide derived from the viral envelope It is possible to use a chimeric protein or the like possessed in the inner region. Thereby, a vector targeting a specific tissue can also be created. These may be encoded in the viral genome, or may be supplied by expression of a gene other than the genome (for example, another expression vector or a gene on the host chromosome) when the viral vector is reconstructed.
[0023]
In addition, the viral vector of the present invention is one in which a viral gene contained in the vector is modified in order to reduce immunogenicity due to SeV protein or to increase RNA transcription efficiency or replication efficiency, for example. May be. Specifically, for example, it is considered that at least one of NP gene, P / C gene, and L gene, which are replication factors, is modified to enhance the function of transcription or replication. HN protein, which is one of the structural proteins, has both hemagglutinin activity and neuraminidase activity, which are hemagglutinins. For example, if the former activity can be weakened, blood It may be possible to improve the stability of the virus in it, and it is also possible to regulate the infectivity, for example by modifying the activity of the latter. In addition, the fusion ability of membrane-fused liposomes can be regulated by modifying the F protein involved in membrane fusion. In addition, for example, it has become possible to analyze antigen-presenting epitopes of F protein and HN protein that can be antigen molecules on the cell surface by establishing a reconstitution system. Weakened Sendai virus can also be made.
[0024]
The viral vector of the present invention encodes a foreign gene in a negative-strand single-stranded RNA or has a site for inserting a foreign gene. As the foreign gene, a desired gene desired to be expressed in the target cell can be used. For example, for the purpose of gene therapy, a gene for treating a target disease is inserted into the viral vector DNA. The foreign gene can be inserted downstream of each gene of the virus (NP, P, M, F, HN, and L genes) (see Examples). Here, “downstream” refers to a site adjacent to the 3 ′ side in the sense strand encoding the protein. That is, in the case of negative strand RNA (or DNA), the downstream of the gene is the 5 ′ adjacent site of the gene, and in the case of positive strand RNA (or DNA), it is the 3 ′ adjacent site of the gene. .
[0025]
The present invention is particularly useful when it is desired to limit the expression of a foreign gene. As the foreign gene is inserted downstream of the negative strand genome, the expression level of the foreign gene can be reduced. In this case, the vector of the present invention is a paramyxovirus vector having a foreign gene and having a replication ability, and a virus located second from the 3 ′ end in the negative-strand genomic RNA contained in the vector. A vector in which a foreign gene is located at least downstream from a gene encoding a protein is preferable. More preferably, the foreign gene is at least downstream from the gene encoding the viral protein located at the third, further preferably the fourth, more preferably the fifth, and even more preferably the sixth from the 3 ′ end in the negative strand genomic RNA. Is located. The present invention also relates to a method for controlling the expression level of a foreign gene using the paramyxovirus vector of the present invention. The expression level can be relatively increased as the foreign gene is positioned on the 3 ′ side of the negative strand genome, and the expression level can be decreased as the foreign gene is positioned on the 5 ′ side. When it is desired to relatively reduce the expression level, for example, a gene encoding a viral protein located first from the 3 ′ end of the negative-strand genomic RNA contained in the paramyxovirus vector and a second viral protein. More preferably between the second and third, more preferably between the third and fourth, more preferably between the fourth and fifth, more preferably between the second and third. Between the fifth and sixth, more preferably between the sixth and the trailer arrangement. Thereby, it is possible to control the expression of a foreign gene to a desired level.
[0026]
For example, in wild-type paramyxoviruses, the viral genes are arranged in the order of NP, P, M, F, HN, and L in order from 3 ′ of the negative genome. It may be an arrangement. In order not to disturb the expression of the preceding and succeeding genes, an E-I-S sequence (transcription termination sequence-intervening sequence-transcription initiation sequence) or a portion thereof is appropriately inserted before and / or after the foreign gene. For example, when a foreign gene is introduced into DNA encoding the Sendai virus genome, it is desirable to insert a sequence having a multiple of 6 bases between genes encoding viral proteins (Journal of Virology, Vol. 67 No. 8, 1993, p. 4822-4830). The expression level of the inserted foreign gene can be controlled by the type of transcription initiation sequence added to the 5 ′ side (first) of the foreign gene. It can also be controlled by the position of gene insertion and the base sequence before and after the gene.
[0027]
As shown in the examples, in Sendai virus, the closer the insertion position is to the 3 ′ end of the negative-strand RNA (in the gene arrangement on the wild-type virus genome, the closer to the NP gene), High expression level. In order to obtain high expression of a foreign gene, a foreign gene is located downstream (ie, between the first gene and the second gene) of the most upstream (3 ′ side of the negative strand) gene encoding the viral protein. Insert. Specifically, in the gene arrangement of the wild type genome, a foreign gene is inserted downstream of the NP gene (in the negative strand, 5 ′ adjacent site of the NP gene), that is, between the NP gene and the P gene. When a foreign gene is inserted between the second and third from the upstream (3 'side of the negative strand) of the gene encoding the viral protein, the expression is attenuated more than when inserted between the first and second. It is done. In this case, in the gene arrangement of the wild type genome, it is preferable to insert a foreign gene downstream of the P gene (in the negative strand, 5 ′ adjacent site of the P gene), that is, between the P gene and the M gene. The closer the insertion position is to the 5 ′ end of the negative-strand RNA (in the gene arrangement on the wild-type virus genome, the closer to the L gene), the lower the expression level of the inserted gene. Inserting a foreign gene between the third and fourth from the upstream (3 'side of the negative strand) of the gene encoding the viral protein results in a more attenuated expression, and the foreign gene is between the fourth and fifth. Is inserted, the expression level can be further reduced. When inserting a foreign gene between the 3rd and 4th, in the gene arrangement of the wild-type genome, it is downstream of the M gene (in the negative strand, 5 'adjacent site of the M gene), that is, between the M gene and the F gene. It is preferable to insert a foreign gene. When a foreign gene is inserted between the 4th and 5th gene, the gene arrangement of the wild-type genome is downstream of the F gene (in the negative strand, 5 'adjacent site of the F gene), that is, between the F gene and the HN gene. It is preferable to insert a foreign gene. Furthermore, in order to suppress the expression of foreign genes to a low level, the gene downstream of the gene encoding the viral protein that is the most downstream (5 ′ side of the negative strand) among the genes encoding the viral protein (that is, the first gene from the 5 ′ side) Between the first and second genes; in the wild-type viral genome, between the 5th gene and the 6th gene from the 3 'side, to obtain lower expression (ie 1 from the 5' end of the negative strand) The foreign gene is inserted between the first gene and the trailer sequence, or between the 6th gene and the trailer sequence from the 3 ′ side in the wild type genome. Specifically, in the gene arrangement of the wild type genome, upstream of the L gene (3 ′ adjacent site of the L gene in the negative strand) or downstream (5 ′ adjacent site of the L gene in the negative strand), that is, HN, respectively. A foreign gene is inserted between the gene and the L gene or between the L gene and the trailer sequence. The vector of the present invention may hold another foreign gene at a position other than the insertion as described above.
The insertion position of the foreign gene can be appropriately adjusted so as to obtain a desired expression level of the gene and so that the combination with the genes encoding the preceding and subsequent viral proteins is optimal.
[0028]
In order to allow easy insertion of foreign genes, a cloning site can be designed at the insertion site. The cloning site can typically be a restriction enzyme recognition sequence. Preferably, a restriction enzyme site is designed that is unlikely to exist in the foreign gene to be inserted. As such a restriction enzyme, those having a long recognition sequence such as a restriction enzyme capable of recognizing 8 bp are preferable. Examples of restriction enzymes for 8 bp recognition include Asc I (GG ↓ CGCGCC), Fse I (GGCCGG ↓ CC), Not I (GC ↓ GGCCGC), Pac I (TTAAT ↓ TAA), Pme I (GTTT ↓ AAAC), Sfi I (GGCCNNNN ↓ NGGCC), Sgf I (GCGAT ↓ CGC), Srf I (GCCC ↓ GGGC), Sse232 I (CG ↓ CCGGCG), Sse8387 I (CCTGCA ↓ GG), and Swa I (ATTT ↓ AAAT) However, it is not limited to these. The cloning site may be a so-called multicloning site having a plurality of restriction enzyme recognition sequences. Further, it may be a sequence cleaved by an endonuclease other than a restriction enzyme. It is also possible to insert a foreign gene by recombination using the cloning site as a recognition sequence for the recombinant enzyme. Designing these sequences in the DNA encoding the viral genome can be performed by known mutagenesis methods. Furthermore, it may be possible to divide the foreign gene insertion site in advance to obtain a cloning site. If the 5 ′ end of the fragmented vector DNA is dephosphorylated, a clone having a foreign gene inserted can be preferentially generated. In addition, if the 3 'end of the fragmented vector DNA is made to be a single base protruding end of T, it is possible to easily clone a foreign gene amplified by PCR (the end is the protruding end of A). . If the vector DNA is a circular DNA such as a plasmid, both ends are not released even if the cloning site is divided, so that high ligation efficiency can be obtained.
[0029]
Insertion of a foreign gene into DNA encoding a viral genome (vector DNA) can be performed by, for example, Hasan, MK et al., 1997, J. General Virology 78: 2813-2820, Kato, A. et al., 1997, EMBO. According to the description of J. 16: 578-587 and Yu, D. et al., 1997, Genes Cells 2: 457-466, it can be constructed as follows.
[0030]
First, a DNA sample containing the desired foreign gene cDNA base sequence is prepared. It is preferable that the DNA sample can be confirmed as a single plasmid by electrophoresis at a concentration of 25 ng / μl or more. Hereinafter, a case where a foreign gene is inserted into a DNA encoding a viral genome using a NotI site will be described as an example. If the target cDNA base sequence contains a NotI recognition site, use a site-specific mutagenesis method or the like to modify the base sequence so that the encoded amino acid sequence is not changed, and remove the NotI site in advance. It is preferable to keep it. A desired gene fragment is amplified and recovered from this sample by PCR. In order to add NotI sites at both ends of the amplified fragment and to add copies of Sendai virus transcription termination sequence (E), intervening sequence (I) and transcription initiation sequence (S) (EIS sequence) at one end As a primer pair including an enzyme cleavage site sequence, a transcription termination sequence (E), an intervening sequence (I), a transcription initiation sequence (S) and a partial sequence of the target gene, a forward synthetic DNA sequence and a reverse synthetic DNA sequence ( Antisense strand) is created.
[0031]
For example, the forward-side synthetic DNA sequence is 5 ′ to ensure cleavage by NotI, preferably any two or more nucleotides (preferably 4 bases that do not contain sequences derived from NotI recognition sites such as GCG and GCC, more preferably Is selected, and the NotI recognition site gcggccgc is added to the 3 ′ side, and any 9 bases or a multiple of 6 to 9 is added to the 3 ′ side as a spacer sequence. A form corresponding to about 25 bases of ORF is added to the 3 ′ side from the start codon ATG of the desired cDNA. It is preferable to select about 25 bases from the desired cDNA so that the last base is G or C, and use it as the 3 ′ end of the forward-side synthetic oligo DNA.
[0032]
The reverse synthetic DNA sequence is selected from the 5 'side to any 2 or more nucleotides (preferably 4 bases not containing sequences derived from NotI recognition sites such as GCG and GCC, more preferably ACTT), and the 3' side. Is added with a NotI recognition site gcggccgc, and an oligo DNA of an inserted fragment for adjusting the length is added to the 3 ′ side thereof. The length of this oligo DNA, including the NotI recognition site gcggccgc, is designed so that the total of the complementary strand base sequence of cDNA and the EIS base sequence of Sendai virus genome derived from Sendai virus described later is a multiple of 6. (So-called “rule of six”; Kolakofski, D. et al., J. Virol. 72: 891-899, 1998). Further, on the 3 ′ side of the inserted fragment, a complementary strand sequence of Sendai virus S sequence, preferably 5′-CTTTCACCCT-3 ′ (SEQ ID NO: 1), I sequence, preferably 5′-AAG-3 ′, E sequence Complementary strand sequence, preferably 5'-TTTTTCTTACTACGG-3 '(SEQ ID NO: 2), and the last base of the complementary strand corresponding to about 25 bases counted backward from the start codon of the desired cDNA sequence on the 3' side The length is selected so as to be G or C, and the sequence is added to obtain the 3 ′ end of the reverse synthetic oligo DNA.
[0033]
For PCR, for example, a usual method using ExTaq polymerase (Takara Shuzo) can be used. Vent polymerase (NEB) is preferably used. The amplified fragment of interest is digested with NotI and then inserted into the NotI site of the plasmid vector pBluescript. The base sequence of the obtained PCR product is confirmed with a sequencer, and a plasmid with the correct sequence is selected. The insert is excised from this plasmid with NotI and cloned into the NotI site of the plasmid containing the genomic cDNA. It is also possible to obtain a recombinant Sendai virus cDNA by directly inserting into the NotI site without using the plasmid vector pBluescript.
[0034]
The DNA encoding the viral genome is constructed by linking an appropriate transcription promoter to construct a vector DNA, which is transcribed in vitro or in a cell, and reconstituted in the presence of viral L, P, and NP proteins. And a viral vector containing this RNP can be generated. The present invention provides a method for producing the vector, comprising the step of transcribing the DNA encoding the genome of the paramyxovirus vector of the present invention in the presence of paramyxovirus L, P and NP proteins. The present invention also provides DNA for producing the paramyxovirus vector of the present invention comprising the DNA. The present invention also relates to the use of DNA encoding the genome of the vector for producing the paramyxovirus vector of the present invention. Virus reconstitution from viral vector DNA can be performed according to known methods (WO 97/16539; WO 97/16538; Durbin, AP et al., 1997, Virology 235: 323-332; Whelan , SP et al., 1995, Proc. Natl. Acad. Sci. USA 92: 8388-8392; Schnell. MJ et al., 1994, EMBO J. 13: 4195-4203; Radecke, F. et al., 1995 , EMBO J. 14: 5773-5784; Lawson, ND et al., Proc. Natl. Acad. Sci. USA 92: 4477-4481; Garcin, D. et al., 1995, EMBO J. 14: 6087-6094 ; Kato, A. et al., 1996, Genes Cells 1: 569-579; Baron, MD and Barrett, T., 1997, J. Virol. 71: 1265-1271; Bridgen, A. and Elliott, RM, 1996 , Proc. Natl. Acad. Sci. USA 93: 15400-15404). By these methods, virus vectors of the Paramyxoviridae family including parainfluenza, vesicular stomatitis virus, rabies virus, measles virus, Linder pest virus, Sendai virus and the like can be reconstituted from DNA.
[0035]
For example, the method for introducing vector DNA into a cell is as follows: (1) a method for preparing a DNA precipitate that can be taken up by the target cell; (2) suitable for uptake by the target cell; There are a method of making a complex containing DNA having a positive charge characteristic with little toxicity, and a method of instantaneously opening a hole enough for DNA molecules to pass through a target cell membrane by an electric pulse.
[0036]
As (2), various transfection reagents can be used. For example, DOTMA (Boehringer), Superfect (QIAGEN # 301305), DOTAP, DOPE, DOSPER (Boehringer # 1811169) and the like can be mentioned. (1) includes, for example, a transfection method using calcium phosphate. DNA that has entered cells by this method is taken up by phagocytic vesicles, but it is known that a sufficient amount of DNA also enters the nucleus. (Graham, FL and Van Der Eb, J., 1973, Virology 52: 456; Wigler, M. and Silverstein, S., 1977, Cell 11: 223). Chen and Okayama looked at optimizing the transfer technology: 1) Incubate cells with coprecipitate incubation conditions 2-4% CO₂ 3) Reported that optimal precipitation is obtained when the DNA concentration in the precipitation mixture is 20-30 μg / ml. (Chen, C. and Okayama, H., 1987, Mol. Cell. Biol. 7: 2745). Method (2) is suitable for transient transfection. In the old days DEAE-dextran (Sigma # D-9885 M.W. 5 × 10^Five ) A method of preparing a mixed solution at a desired DNA concentration ratio and performing transfection is known. Since many of the complexes are degraded in endosomes, chloroquine can be added to enhance the effect (Calos, M.P., 1983, Proc. Natl. Acad. Sci. USA 80: 3015). The method (3) is a method called electroporation and is more versatile than the methods (1) and (2) in that there is no cell selectivity. Efficiency is said to be good under the optimum conditions of pulse current duration, pulse shape, electric field (gap between electrodes, voltage) strength, buffer conductivity, DNA concentration, and cell density.
[0037]
As described above, the method (2) among the three categories is easy to operate and can examine a large number of specimens using a large amount of cells. Therefore, a transfection reagent is suitable in the present invention. Preferably, Superfect Transfection Ragent (QIAGEN, Cat No. 301305) or DOSPER Liposomal Transfection Reagent (Boehringer Mannheim, Cat No. 1811169) is used.
[0038]
Specifically, reconstitution from cDNA can be performed as follows.
Use a minimum essential medium (MEM) containing 10% fetal bovine serum (FCS) and antibiotics (100 units / ml penicillin G and 100 μg / ml streptomycin) on a plastic plate of 24 to 6 holes or a 100 mm Petri dish. Monkey kidney cell line LLC-MK2 is 70-80% confluent (1 × 10⁶ Cell) and, for example, recombinant vaccinia virus expressing T7 polymerase vTF7-3 (Fuerst, TR et al., Inactivated by UV irradiation treatment for 20 minutes in the presence of 1 μg / ml psoralen). Proc. Natl. Acad. Sci. USA 83: 8122-8126, 1986, Kato, A. et al., Genes Cells 1: 569-579, 1996) is infected with 2 PFU / cell. The amount of psoralen added and the UV irradiation time can be appropriately adjusted. One hour after infection, 2 to 60 μg, more preferably 3 to 5 μg of the above recombinant Sendai virus cDNA is transformed into a plasmid (24-0.5 μg of a plasmid expressing a viral protein acting in trans essential for the generation of the full-length Sendai virus genome. pGEM-N, 12-0.25 μg pGEM-P, and 24-0.5 μg pGEM-L, more preferably 1 μg pGEM-N, 0.5 μg pGEM-P, and 1 μg pGEM-L) (Kato, A et al., Genes Cells 1: 569-579, 1996) and transfection by a lipofection method using Superfect (QIAGEN). Transfected cells are optionally serum-free containing only 100 μg / ml rifampicin (Sigma) and cytosine arabinoside (AraC), more preferably only 40 μg / ml cytosine arabinoside (AraC) (Sigma). In order to minimize the cytotoxicity of vaccinia virus and maximize the recovery rate of the virus, the optimal concentration of the drug is set (Kato, A. et al., 1996, Genes Cells 1: 569- 579). After culturing for 48 to 72 hours after transfection, the cells are collected, and freeze-thaw is repeated three times to disrupt the cells. Then, the cells are transfected and cultured in LLC-MK2 cells. Alternatively, the culture supernatant is collected, added to the culture medium of LLC-MK2 cells, infected and cultured. The culture solution is collected after 3 to 7 days of culture. Alternatively, the collected cells may be co-cultured by overlaying other cells. Alternatively, the above-mentioned freeze-thawed cell disruption may be inoculated into the allantoic membrane of a 10-day-old embryonated chicken egg, and the urine fluid may be collected after about 3 days. The virus titer contained in the culture supernatant or urine fluid can be determined by measuring the hemagglutination activity (HA). HA is an “endo-point dilution method” (Kato, A. et al., 1996, Genes Cells 1: 569-579; Yonemitsu, Y. & Kaneda, Y., Hemaggulutinating virus of Japan-liposome-mediated gene delivery to vascular. Ed. by Baker AH. Molecular Biology of Vascular Diseases. Method in Molecular Medicine: Humana Press: pp. 295-306, 1999). To remove contaminating vaccinia virus vTF7-3, the obtained urine fluid sample is diluted appropriately (for example, 10⁶ And can be reamplified with chicken eggs. The reamplification can be repeated three times, for example. The resulting virus stock can be stored at -80 ° C.
[0039]
The recovered paramyxovirus can be purified to be substantially pure. The purification method can be performed by a known purification / separation method including filtration, centrifugation, column purification and the like. “Substantially pure” means that the isolated material (such as a compound, polypeptide, or virus) accounts for a major proportion as a component in the sample in which it is present. Typically, the substantially pure component present in the sample is 50% or more of the total of the other components in the sample, preferably 70% or more, more preferably 80% or more, and even more preferably 90%. Occupy more than 50%. The ratio is calculated by a procedure known to those skilled in the art, for example, by weight ratio [w / w]. Of course, it should be calculated excluding solvents, salts, added compounds and the like. As a specific method for purifying paramyxovirus, for example, a method using cellulose sulfate ester or crosslinked polysaccharide sulfate ester (Japanese Patent Publication No. 62-30752, Japanese Patent Publication No. 62-33879, and Japanese Patent Publication No. 62-30753). And a method of adsorbing to a sulfated-fucose-containing polysaccharide and / or a degradation product thereof (WO97 / 32010).
[0040]
The recombinant Sendai virus vector of the present invention can be appropriately diluted with, for example, physiological saline or phosphate buffered saline (PBS) to obtain a composition. When the recombinant Sendai virus vector of the present invention is propagated in chicken eggs, urine can also be included. The recombinant Sendai virus vector-containing composition of the present invention may contain a physiologically acceptable carrier or medium such as deionized water or 5% dextrose aqueous solution. A “physiologically acceptable carrier or vehicle” is a material that can be administered with a vector and does not significantly inhibit gene transfer by the vector. In addition, vegetable oils, suspending agents, surfactants, stabilizers, biocides and the like may be contained. Preservatives and other additives can also be added.
[0041]
As long as the viral vector is reconstituted, the host cell used for reconstitution is not particularly limited. For example, in reconstitution of Sendai virus vector, cultured cells such as monkey kidney-derived CV-I cells, LLC-MK2 cells, hamster kidney-derived BHK cells, human-derived cells, and the like can be used. By expressing an appropriate envelope protein in these cells, infectious virus particles having the envelope can also be obtained. In addition, in order to obtain a large amount of Sendai virus vector, it is possible to infect a chicken egg with a virus vector obtained from the above host and amplify the vector. A method for producing viral vectors using eggs has already been developed (Nakanishi et al., (1993), "Advanced Protocol III for Neuroscience Research, Molecular Neuronal Physiology", Koseisha, Osaka, pp.153-172. ). Specifically, for example, fertilized eggs are placed in an incubator and cultured at 37-38 ° C. for 9-12 days to grow embryos. The Sendai virus vector is inoculated into the allantoic cavity, and eggs are cultured for several days to propagate the virus vector. Conditions such as the culture period may vary depending on the recombinant Sendai virus used. Thereafter, the urine fluid containing the virus is collected. Separation and purification of Sendai virus vector from urine can be performed according to a conventional method (Yasuhito Tashiro, “Virus Experiment Protocol”, supervised by Nagai and Ishihama, Medical View, pp.68-73, (1995)).
[0042]
If a viral vector is prepared using a disease therapeutic gene as a foreign gene, this vector can be administered to perform gene therapy. As an application of the viral vector of the present invention to gene therapy, a foreign gene that can be expected to have a therapeutic effect can be supplied in a patient's body by either gene expression by direct administration or gene expression by indirect (ex vivo) administration. It is possible to express a deficient endogenous gene or the like. Since the viral vector of the present invention is highly safe and can control the expression level of foreign genes, it is expected to be compatible with a wide range of clinical applications. The foreign gene is not particularly limited, and may be a nucleic acid that does not encode a protein such as antisense or ribozyme in addition to a nucleic acid encoding a protein. Further, if a gene encoding a bacterial or viral antigen related to an infectious disease is used as a foreign gene, immunity can be induced in the animal by administering it to the animal. That is, it can be used as a vaccine. The present invention can also be applied to viruses having a high need for vaccines such as pathogenic paramyxoviruses such as measles virus and mumps virus.
[0043]
When used as a vaccine, it is conceivable to apply the virus vector of the present invention to, for example, tumors, infectious diseases, and other common diseases. For example, for tumor therapy, a gene having a therapeutic effect can be expressed in tumor cells or antigen-presenting cells (APC) such as DC cells using the vector of the present invention. Examples of such a gene include cancer antigen Muc-1 or Muc-1-like mucin tandem repeat peptide (US Pat. No. 5,744,144), melanoma gp100 antigen and the like. Such gene therapy has been widely applied to breast cancer, colon cancer, pancreatic cancer, prostate cancer, lung cancer and the like. It is also effective to combine cytokines that enhance the adjuvant effect. Examples of such genes include i) a combination of IL-2 and single-chain IL-12 (Proc. Natl. Acad. Sci. USA 96 (15): 8591-8596, 1999), ii) IL-2 And interferon-γ (US Pat. No. 5,798,100), iii) granulocyte colony-stimulating factor (GM-CSF) used alone, iv) combination of GM-CSF and IL-4 for treatment of brain tumors (J. Neurosurgery 90 (6), 1115-1124 (1999)).
[0044]
As for the treatment of infectious diseases, in influenza, for example, the highly toxic strain H5N1 type envelope, in Japanese encephalitis, for example, envelope chimera (Vaccine, vol. 17, No. 15-16, 1869-1882 (1999)), AIDS For example, vaccine treatment by oral administration of HIV gag or SIV gag protein (J. Immunology (2000) vol. 164, 4968-4978), HIV envelope protein, administration in polylactic acid-glycol copolymer (Kaneko, H. et al., Virology 267: 8-16 (2000)), in cholera, for example, the B subunit (CTB) of cholera toxin (Arakawa, T., et al., Nature Biotechnology (1998) 16 (10) : 934-8, Arakawa, T., et al., Nature Biotechnology (1998) 16 (3): 292-7). In rabies, for example, glycoproteins of rabies virus (Lodmell, DL et al., 1998, Nature Medicine 4 (8): 949-52), capsid of human papillomavirus type 6 in cervical cancer Npaku L1 (J. Med. Virol, 60, 200-204 (2000)) and the like.
[0045]
When the vector of the present invention is used as a vaccine, an immunostimulant such as cytokine, cholera toxin, salmonella toxin or the like can be combined to increase immunogenicity. The vaccine can also be combined with adjuvants such as alum, incomplete Freund's adjuvant, MF59 (oil emulsion), MTP-PE (muramyl tripeptide derived from mycobacterial cell wall), and QS-21 (derived from soapbark tree Quilaja saponaria) .
[0046]
Application to general diseases is also conceivable. In diabetes, for example, a peptide of an insulin fragment is expressed in a type I diabetes model animal (Coon, B. et al., J. Clin. Invest., 1999, 104 (2): 189-94).
[0047]
The dose of the vector varies depending on the disease, patient weight, age, sex, symptom, administration purpose, administration form, administration method, etc., but can be determined as appropriate by those skilled in the art. Preferably, the amount of vector contained in the administration composition is about 10^FiveAbout 10 from pfu / ml¹¹It should be within the range of pfu / ml. More preferably, the amount of vector contained in the administration composition is about 10⁷About 10 from pfu / ml⁹It should be within the range of pfu / ml. Most preferably, about 1 × 10⁸About 5 x 10 from pfu / ml⁸It is preferred to administer an amount in the pfu / ml range in a pharmaceutically acceptable carrier.
[0048]
In addition, when treatment is completed and it is necessary to inhibit the growth of the viral vector, or during treatment, an RNA-dependent RNA polymerase inhibitor can be administered to specifically propagate the viral vector without damaging the host. Can be deterred.
[0049]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.
[Example 1] Control of gene expression using polarity effect in Sendai virus I
<Construction of SeV genomic cDNA>
Sendai virus (SeV) full-length genomic cDNA, pSeV (+) (Kato, A. et al., Genes to Cells 1: 569-579, 1996) And ATG translation initiation signal. First, as shown in Fig. 1 (A), pSeV (+) was digested with Sph I / Sal I (2645 bp), Cla I digested (3246 bp), and Cla I / Eco RI. The fragments (5146 bp) were separated by agarose electrophoresis, the corresponding bands were excised, collected and purified by QIAEXII Gel Extraction System (manufactured by QIAGEN). The fragment digested with Sph I / Sal I was ligated to LITMUS38 (manufactured by NEW ENGLAND BIOLABS), the fragment digested with Cla I and the fragment digested with Cla I / Eco RI were sub-cloned into pBluescriptII KS + (manufactured by STRATAGENE). Next, the Quickchange Site-Directed Mutagenesis kit (manufactured by STRATAGENE) was used to introduce the Not I site. The primer used for each introduction is sense strand: 5'-ccaccgaccacacccagcggccgcgacagccacggcttcgg-3 '(SEQ ID NO: 3), antisense strand: 5'-ccgaagccgtggctgtcgcggccgctgggtgtggtcggtgg-3' (SEQ ID NO: 4), between PM Sense strand: 5'-gaaatttcacctaagcggccgcaatggcagatatctatag-3 '(SEQ ID NO: 5) Antisense strand: 5'-ctatagatatctgccattgcggccgcttaggtgaaatttc-3' (SEQ ID NO: 6), MF sense strand: 5'-gggataaagtccctctgttcccctct SEQ ID NO: 7), antisense strand: 5'-ggggagagttttgcaaccaagcggccgcaagggactttatccc-3 '(SEQ ID NO: 8), sense strand between F-HN: 5'-ggtcgcgcggtactttagcggccgcctcaaacaagcacagatcatgg-3' (SEQ ID NO: 9), antisense strand 5'-ccatgatctgtgcttgtttgaggcggccgctaaagtaccgcgcgacc-3 '(SEQ ID NO: 10), sense strand between HN-L: 5'-cctgcccatccatgacctagcggccgcttcccattcaccctggg-3' (SEQ ID NO: 11), antisense strand: 5 ' -cccagggtgaatgggaagcggccgctaggtcatggatgggcagg-3 '(SEQ ID NO: 12) was synthesized and used.
[0050]
Using the above-mentioned subcloned SalI / SphI fragments between NP-P, ClaI fragments between PM, MF, ClaI fragments, F-HN, and HN-L between the NP-P as templates, Quickchange Site- Introduction was performed according to the protocol of Directed Mutagenesis kit. The introduced product was digested with the subcloned enzyme again, recovered and purified in the same manner, and assembled into the original Sendai genomic cDNA. As a result, as shown in FIG. 1 (B), 5 types (pSeV (+) NPP, pSeV (+) PM, pSeV (+) MF, pSeV (+) FHN and pSeV ( +) HNL) Sendai virus genomic cDNA was constructed.
To insert a foreign gene, a DNA fragment having a termination signal-intervening sequence-start signal (E-I-S) added downstream of the foreign gene is inserted into the NotI site of the genomic cDNA. For this purpose, for example, a sequence (see FIG. 2) in which a multicloning site and a termination signal-intervening sequence-start signal are sandwiched between two NotI sites is prepared in advance, and a foreign gene is inserted into the multicloning site. Is simple.
[0051]
To insert a foreign gene downstream of the L gene, for example, pSeV (+) or the desired SeV cDNA (pSeV18⁺, PSeV (+) NPP, pSeV (+) PM, pSeV (+) MF, pSeV (+) FHN and pSeV (+) HNL) etc., and the restriction enzyme present between the L gene stop codon and transcription termination signal A gene can be inserted into the Kpn I site (FIG. 3 (A)). In this case, a KpnI site is added to both ends of the foreign gene to be inserted, and a termination signal-intervening sequence-start signal is added to the 5 ′ side by PCR or the like (FIG. 3B). The added DNA is incorporated into SeV cDNA, and cDNA with a foreign gene inserted downstream of the L gene is obtained.
[0052]
Human secreted alkaline phosphatase (SEAP) was subcloned by PCR as a reporter gene for observing gene expression level. As primers, an Asc I restriction enzyme site was added. PCR was performed. PSEAP-Basic (manufactured by CLONTECH) was used as a template, and Pfu tourbo DNA polymerase (manufactured by STRATAGENE) was used as an enzyme. After PCR, the product was digested with Asc I and purified and recovered by electrophoresis. Synthetic double-stranded DNA including multicloning site (Pme I-Asc I-Swa I) and termination signal-intervening sequence-initiation signal at the Not I site of pBluescriptII KS + as a subcloning plasmid [sense strand: 5'-gcggccgcgtttaaacggcgcgccatttaaatccgtagtaagaaaaacttagggtgaaagttcatc ' (SEQ ID NO: 15) and antisense strand: 5′-gcggccgcgatgaactttcaccctaagtttttcttactacggatttaaatggcgcgccgtttaaacgcggccgc-3 ′ (SEQ ID NO: 16)] were prepared (FIG. 2). The purified and recovered PCR product was ligated to the Asc I site of this plasmid and cloned. This was digested with Not I, and the SEAP gene fragment was recovered and purified by electrophoresis, and ligated and incorporated into the above five Sendai virus genomic cDNAs and the Not I site of pSeV18 +, respectively. Respective viral vectors are pSeV (+) NPP / SEAP, pSeV (+) PM / SEAP, pSeV (+) MF / SEAP, pSeV (+) FHN / SEAP, pSeV (+) HNL / SEAP and pSeV18 (+) / SEAP.
[0053]
<Reconstruction of virus>
LLC-MK2 cells 2 × 10⁶ Recombinant vaccinia virus (PLWUV-VacT7) expressing T7 polymerase treated with psoralen and UV treatment (PLWUV-VacT7) after seeding in a 100 mm dish in cells / dish and cultured for 24 hours (Fuerst, TR et al., Proc. Natl. Acad. Sci. USA) 83: 8122-8126, 1986, Kato, A. et al., Genes Cells 1: 569-579, 1996) at room temperature at moi = 2 for 1 hour. OptiMEM (produced by GIBOCO BRL) in the amount ratio of 12μg, 4μg, 2μg, and 4μg / dish for each Sendai virus cDNA, pGEM / NP, pGEM / P, and pGEM / L incorporating SEAP after washing the cells Suspend in 100 μl of SuperFect transfection reagent (QIAGEN), mix, leave at room temperature for 15 minutes, finally add 3 ml of OptiMEM containing 3% FBS, add to cells and incubate for 3-5 hours did. After culturing, the cells were washed twice with MEM not containing serum and cultured for 72 hours in MEM containing cytosine β-D-arabinofuranoside (AraC). These cells were collected, the pellet was suspended in 1 ml of PBS, and freeze-thawing was repeated three times. After inoculating 100 μl of these eggs that had been incubated for 10 days and incubating at 35 ° C. for 3 days, the urine fluid was collected. In order to make vaccinia virus free, another 10^-Five~Ten^-7 The eggs were diluted and then re-inoculated into eggs, collected in the same manner, dispensed, and stocked at -80 ° C. The virus vector names are SeVNPP / SEAP, SeVPM / SEAP, SeVMF / SEAP, SeVFHN / SEAP, SeVHNL / SEAP, and SeV18 / SEAP.
[0054]
<Measurement of titer by plaque assay>
CV-1 cells in a 6-well plate at 5 x 10 per well^FiveCells were seeded and cultured for 24 hours. After washing with PBS, 10% with BSA / PBS (1% BSA in PBS)^-3,Ten^-Four,Ten^-Five,Ten^-6,Ten^-7 Incubate the recombinant SeV diluted in 1 hour, wash with PBS, and overlay 3 ml per well with BSA / MEM / agarose (mixed with 0.2% BSA + 2 × MEM and an equal amount of 2% agarose) 6 days at 37 ℃, 5% CO₂ In culture. After culturing, 3 ml of ethanol / acetic acid (ethanol: acetic acid = 1: 5) was added, left for 3 hours, and removed together with agarose. After washing three times with PBS, the cells were incubated with a rabbit anti-Sendai virus antibody diluted 100 times at room temperature for 1 hour. Alexa Flour diluted 200 times after washing 3 times with PBS^TM Labeled goat anti-rabbit Ig (G + H) (Molecular Probe) was added and incubated at room temperature for 1 hour. After washing with PBS three times, fluorescence images were captured with a lumino image analyzer LAS1000 (Fuji Film), and plaques were measured. The results are shown in FIG. The results of the titer obtained from this are shown in Table 1.
[0055]
[Table 1]
Titer results for each recombinant Sendai virus measured from plaque assay results

[0056]
<Comparison of reporter gene expression>
LLC-MK2 cells in a 6-well plate 1 to 5 x 10 per well^Five Cells were seeded and cultured for 24 hours, each virus vector was infected with moi = 2, and after 24 hours, 100 μl of the culture supernatant was collected and subjected to SEAP assay. The assay was performed with Reporter Assay Kit -SEAP- (Toyobo) and measured with a lumino image analyzer LAS1000 (Fuji Film). The measured values were expressed as relative values with the SeV18 + / SEAP value being 100. As a result, SEAP activity was detected when the SEAP gene was inserted at any position shown in FIG. It was found that the SEAP activity decreased as it was located downstream of the genome, that is, the expression level decreased. The expression level of SEAP gene decreased monotonously as the insertion position went from upstream (NP side) to downstream (L side) of the genome. For example, when the SEAP gene is inserted between the NP gene and the P gene, expression between the vector in which the SEAP gene is inserted upstream of the NP gene and the vector in which the SEAP gene is inserted between the P gene and the M gene A quantity was detected.
[0057]
<Production of multi-cloning site>
Multiple cloning sites were added to the SeV vector. There are the following two methods.
1) Sendai virus (SeV) full-length genomic cDNA, pSeV18⁺ b (+) (Hasan, M. K. et al., 1997, J. General Virology 78: 2813-2820) ("pSeV18⁺ b (+) ”is` `pSeV18⁺(Also referred to as “). Some of the restriction enzyme sites in the cDNA genome were broken, and a new restriction enzyme site including a crushed restriction enzyme site was introduced between the start signal of each gene and the ATG translation start signal (FIG. 6). .
2) A multicloning site sequence and a transcription initiation signal-intervening sequence-termination signal are added to the already constructed SeV vector cDNA and incorporated into the NotI site (FIG. 7).
[0058]
In the case of 1), the introduction method is pSeV18 as shown in Fig. 6 (A).⁺ The fragment digested with Eag I (2644 bp), the fragment digested with Cla I (3246 bp), the fragment digested with ClaI / Eco RI (5146 bp), and the fragment digested with Eco RI (5010 bp) were separated by agarose electrophoresis. The corresponding band was cut out and collected and purified by QIAEXII Gel Extraction System (manufactured by QIAGEN). Fragments digested with Eag I were ligated to LITMUS38 (NEW ENGLAND BIOLABS), Cla I digested fragments, ClaI / Eco RI digested fragments, and Eco RI digested fragments to pBluescriptII KS + (STRATAGENE). Subcloned. Subsequently, a Quickchange Site-Directed Mutagenesis kit (manufactured by STRATAGENE) was used to destroy and introduce the restriction enzyme site.
[0059]
For disruption of restriction enzyme sites, Sal I: (sense strand) 5′-ggagaagtctcaacaccgtccacccaagataatcgatcag-3 ′ (SEQ ID NO: 17), (antisense strand) 5′-ctgatcgattatcttgggtggacggtgttgagacttctcc-3 ′ (SEQ ID NO: 18), Nhe I: (Sense strand) 5'-gtatatgtgttcagttgagcttgctgtcggtctaaggc-3 '(SEQ ID NO: 19), (antisense strand) 5'-gccttagaccgacagcaagctcaactgaacacatatac-3' (SEQ ID NO: 20), Xho I: (sense strand) 5'-caatgaactctctagagaggctggt '(SEQ ID NO: 21), (antisense strand) 5'-ccaggtaactctttagtgactccagcctctctagagagttcattg-3' (SEQ ID NO: 22), or between NP-P for restriction enzyme introduction: (sense strand) 5'-gtgaaagttcatccaccgatcggctcactcgaggccacacccaaccccaccgcc-3 SEQ ID NO: 23), (antisense strand) 5'-cggtggggttgggtgtggcctcgagtgagccgatcggtggatgaactttcac-3 '(SEQ ID NO: 24), PM: (sense strand) 5'-cttagggtgaaagaaatttcagctagcacggcgcaatggcagatatc-3' Number: 25), (antisense strand) 5'-gatatctgccattgcgccgtgctagctgaaatttctttcaccctaag-3 '(SEQ ID NO: 26), between MF: (sense strand) 5'-cttagggataaagtcccttgtgcgccctctggttgcaaaactctcccc-3' (sequence number: 27), (antisense strand) 5'-ggggagagttttgcaaccaagcgcgcacaagggactttatccctaag-3 '(SEQ ID NO: 28), F-HN: (sense strand) 5'-ggtcgcgcggtactttagtcgacacctcaaacaagcacagatcatgg-3' (SEQ ID NO: 29), (antisense strand) 5'ctgctctctct SEQ ID NO: 30), HN-L: (sense strand) 5′-cccagggtgaatgggaagggccggccaggtcatggatgggcaggagtcc-3 ′ (SEQ ID NO: 31), (antisense strand) 5′-ggactcctgcccatccatgacctggccggcccttcccattcaccctggg-3 ′ (SEQ ID NO: 32) Used for the reaction. After the introduction, each fragment was recovered and purified in the same manner as described above to assemble cDNA (FIG. 6 (B)).
[0060]
In the case of 2), (sense strand) 5'-ggccgcttaattaacggtttaaacgcgcgccaacagtgttgataagaaaaacttagggtgaaagttcatcac-3 '(SEQ ID NO: 33), (antisense strand) 5'-ggccgtgatgaactttcaccctaagtttttcttatcaacactgttttaggaccgcttatta Phosphorylated, annealed at 85 ° C for 2 minutes, 65 ° C for 15 minutes, 37 ° C for 15 minutes, and room temperature for 15 minutes and incorporated into SeV cDNA. Alternatively, a multicloning site such as pUC18 or pBluescriptII is subcloned by PCR with a primer containing a termination signal-intervening sequence-start signal, and this is incorporated into SeV cDNA. Virus reconstitution with the resulting cDNA is performed as described above.
[0061]
[Example 2] Control of gene expression using polarity effect in Sendai virus II
<Construction of SeV genomic cDNA and virus recovery>
A Not I site was introduced into the Sendai virus (SeV) full-length genomic cDNA, pSeV (+) cDNA, between the translation termination codon of the L gene and the transcription termination signal. As the introduction method, first, as shown in Fig. 8, a fragment (5010 bp) obtained by digesting pSeV (+) with Eco RI was separated by agarose electrophoresis, the corresponding band was cut out, and recovered with QIAEXII Gel Extraction System (QIAGEN). -Purified. The recovered fragment was ligated into pBluescriptII SK + (manufactured by STRATAGENE) and subcloned. Subsequently, a Quickchange Site-Directed Mutagenesis kit (manufactured by STRATAGENE) was used for introduction of the restriction enzyme Not I site. The primers were synthesized using the sense side: 5′-cgtgcagaacgatcgaagctccgcggccgctggaagtcttggacttgtcc-3 ′ (SEQ ID NO: 35) and the antisense side: 5′-ggacaagtccaagacttccagcggccgcggagcttcgatcgttctgcacg-3 ′ (SEQ ID NO: 36). After the introduction, a fragment (2010 bp) digested with Xho I-Mlu I was recovered and purified in the same manner as above to assemble cDNA (FIG. 8).
[0062]
Human Vascular Endothelial Growth Factor (VEGF) was subcloned by PCR as a gene for observing the gene expression level. As a primer, 5 'primer with restriction enzyme Asc I site added: 5'-gcggcgcgccaaccatgaactttctgctgtcttgggtgcattgg-3' (SEQ ID NO: 37), 3 'primer: 5'-gcggcgcgcctcaccgcctcggcttgtcacatctgcaagt-3' (SEQ ID NO: 38) PCR was performed. For the enzyme, KOD-plus-DNA polymerase (manufactured by TOYOBO) was used. After PCR, the product was digested with Asc I and purified and recovered by electrophoresis. A plasmid for adding a Sendai virus transcription termination signal-intervening sequence-transcription initiation signal was constructed (FIG. 9). Synthetic double-stranded DNA (sense strand: 5) containing termination signal-intervening sequence-initiation signal and cloning site (Asc I-Pme I) in LITMUS38 (referred to as pAC114) incorporating the Mlu I-Sph I fragment (3317 bp) of pSeV18 + '-ggccgctaagaaaaacttagggtgaaagttcacttcacgagggcgcgccgtttaaactgc-3' (SEQ ID NO: 39), antisense strand: 5'-ggccgcagtttaaacggcgcgccctc (prepared from the figure that includes AG, which is 9) (peg, which is created by AG, which is 9). The purified and recovered VEGF PCR product was ligated into the Asc I site of this plasmid and cloned. This was digested with Not I, the VEGF gene fragment was recovered and purified by electrophoresis, and ligated into the Not I site of Sendai virus genomic cDNA. The viral vector cDNA was pSeV + L / VEGF. In addition, the termination signal-intervening sequence-initiation signal (see FIG. 2) prepared by the method described in Example 1 was linked downstream of the VEGF cDNA and incorporated into pSeV18 + and pSeV (+) HNL (each of which was pSeV18 + / VEGF, pSeV (+) HNL / VEGF) was also constructed for comparison of expression levels. Viruses were collected from each cDNA as in the conventional method, and designated as SeV18 + / VEGF, SeVHNL / VEGF, and SeVL / VEGF, respectively.
[0063]
<Comparison of expression level>
LLC-MK2 cells in a 6-well plate 5 x 10 per well^FiveCells were seeded and cultured for 24 hours, and then each virus vector was infected at moi = 5. After 24 hours, 200 μl of the culture supernatant was collected, and VEGF in the culture supernatant was quantified by ELISA. The result is shown in FIG. As a result, it was found that the expression level of VEGF decreased by about 1/10 by incorporating it downstream of the L gene compared to when incorporating it upstream of the NP gene. The present inventors have disclosed that the expression level can be changed by changing the transcription initiation signal (WO01 / 18223), and by combining this, the range of control of the expression level is expected to be further expanded. Is done.
[0064]
【The invention's effect】
According to the present invention, a replication-friendly paramyxovirus vector capable of introducing a foreign gene has been provided. The expression level of the vector of the present invention can be controlled by adjusting the insertion position of a foreign gene. In particular, by inserting a foreign gene into the negative strand genome, the expression level of the foreign gene can be kept low. It is also conceivable to produce a viral vector in which a foreign gene has been inserted into two or more sites. In particular, Sendai virus vectors have extremely high gene transfer efficiency for a wide range of cell types, and the present invention can control the expression level of a foreign gene. Therefore, it is expected to be used for gene transfer in living organisms such as gene therapy. The
[0065]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the structure of subcloning (A) of Sendai virus genomic cDNA fragments and the structures of five types of Sendai virus genomic cDNA constructed by newly introducing NotI sites (B).
FIG. 2 is a diagram showing the structure of a cloning plasmid for adding a NotI site, transcription initiation signal, intervening sequence, and transcription termination signal to SEAP.
FIG. 3 is a diagram showing the structure of a Sendai virus genome downstream of the L gene (A) and the structure of the cloning DNA for inserting a foreign gene downstream of the L gene (B).
FIG. 4 shows the results of plaque assay for each Sendai virus vector. A portion of the fluorescence image of a plaque assay captured with LAS1000 is shown.
FIG. 5 is a graph showing the results of comparing the difference in the expression level of reporter gene (SEAP) between Sendai virus vectors. The relative value was expressed with SeV18 + / SEAP data as 100. It was found that as the SEAP gene is located downstream, its activity, that is, the expression level decreases.
FIG. 6 is a diagram showing the structure of a multiple cloning site constructed in Sendai virus genomic cDNA.
FIG. 7 is a diagram showing the structure of a multiple cloning site. The base sequence shows an example of a multicloning site having a restriction enzyme site of PacI-PmeI-BssHII-TspRI and an E / I / S sequence.
FIG. 8 is a diagram showing the structure of Sendai virus genomic cDNA constructed by subcloning a Sendai virus genomic cDNA fragment and newly introducing a Not I site.
FIG. 9 shows the structure of a cloning plasmid for adding a Not I site, a transcription initiation signal, an intervening sequence, and a transcription termination signal to VEGF and the structure of VEGF after the addition.
FIG. 10 is a diagram showing a comparison of expression levels of SeV vectors loaded with VEGF by ELISA. The expression level was lowest when VEGF was loaded after the L gene.

Claims (6)

It holds a foreign gene, and a Sendai virus vector having a replication ability, a gene encoding a viral protein that is located from the 3 'end of negative-strand genomic RNA contained in base compactors in the first 6 th, sequentially NP A gene, a P gene, an M gene, an F gene, an HN gene, and an L gene, which code a gene encoding a viral protein located third from the 3 ′ end of the negative-strand genomic RNA and a fourth viral protein A vector in which a foreign gene is inserted between genes. A DNA encoding a negative strand genomic RNA or a complementary strand thereof contained in the Sendai virus vector according to claim 1 . A DNA encoding negative-strand genomic RNA or the complementary strand thereof contained in the Sendai virus vector having a replication ability, a gene encoding a viral protein which is located in the first 6 position from the 3 'end of the Ne Gatibu strand genomic RNA Are NP gene, P gene, M gene, F gene, HN gene, and L gene in this order, and a gene encoding a viral protein located third from the site corresponding to the 3 ′ end of the negative-strand genomic RNA ; DNA having a cloning site for inserting a foreign gene between genes encoding the fourth viral protein. A vector DNA which holds the DNA according to claim 2 or 3 in a transcribable manner. The vector DNA according to claim 4 , which holds positive-strand genomic RNA in a transcribable manner. A method for producing a Sendai virus vector, comprising a step of transcribing the DNA according to claim 2 in the presence of Sendai virus NP protein, P protein, and L protein .

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