JPH07507689A - Specific tissue targeting methods and compositions - Google Patents

Specific tissue targeting methods and compositions

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JPH07507689A
JPH07507689A JP6501565A JP50156594A JPH07507689A JP H07507689 A JPH07507689 A JP H07507689A JP 6501565 A JP6501565 A JP 6501565A JP 50156594 A JP50156594 A JP 50156594A JP H07507689 A JPH07507689 A JP H07507689A
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カン、ユイット　ワイ
カサハラ、ノリユキ
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ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors

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Abstract

(57)【要約】本公報は電子出願前の出願データであるため要約のデータは記録されません。 (57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

【発明の詳細な説明】 特定組織のターゲツテング方法及び組成物本発明は、米国国立衛生研究所により 授与された登録番号第ＡＭ−１６６６６に基づく政府の支援により行われた。合 衆国政府が本発明において一定の権利を有する。[Detailed description of the invention] Methods and Compositions for Targeting Specific Tissues The present invention was developed by the National Institutes of Health in the United States. This work was supported by the Government under the grant number AM-16666. If The United States Government has certain rights in this invention.

発明の分野 本発明は、物質例えば核酸の送達（ｄｅｌ　１ｖｅｒｙ）を特定の組織へ優先的 にターゲティノグする（向かわせる）ための方法及び組成物に関する。field of invention The present invention enables preferential delivery of substances such as nucleic acids to specific tissues. Methods and compositions for targeting.

発明の背景 遺伝子治療の分野では、ウィルスベクター及びリポソームが細胞へ遺伝子を導入 するために提唱されている（ウィリアムス（Ｗｉｌｌｉａｍｓ）、Ｄ、Ａ、　（ ＋９８８）　ＨｅｍａｔｏｌｏｇｖＯｎｃｏｌｏｇｙ　Ｃｌ１ｎｉｃｓ　ｏｆ　 Ｎｏｒｔｈ　Ａｍｅｒｉｃａ、２：２７７−２８７　；及びマニツＱｌａｎｎｉ ｎｏ）、@Ｒ。Background of the invention In the field of gene therapy, viral vectors and liposomes are used to introduce genes into cells. It has been proposed to do so (Williams, D.A., ( +988) HematologvOncology Cl1nics of North America, 2:277-287; and Manitz Qlanni. no), @R.

Ｊ　ら、（１９８８）ＢｉｏＴｅｃｈｎｉｑｕｅｓ、６：６８２−６９０）　ｏ このような手段が、ある場合ニハ・ベクター又はリポソームの局所的な投与、例 えば、肺組織への局所的送達のための吸入によるウィルスベクターの投与をもた らすが、このような手段の主な欠点は、組織特異的な向性かないことである。従 って、ウィルスベクター又はリポソーム中に包含されたＤＮＡは多くの異なった 型の細胞へ特異的に送達されることかできない。J et al. (1988) BioTechniques, 6:682-690) Such means may include local administration of Niha vectors or liposomes, e.g. For example, by administering viral vectors by inhalation for local delivery to lung tissue. However, the main drawback of such tools is the lack of tissue-specific tropism. subordinate Therefore, the DNA encapsulated in a viral vector or liposome can contain many different It cannot be delivered specifically to cells of this type.

多くの遺伝子疾…は、１つの組織型において発症が認められ、遺伝子を多くの異 なる細胞型−＼非特異的に挿入することは必要ないだけでなく、特定の環境下に より傷つけら第１るかもしれない（セルデン（Ｓｅｌｄｅｎ）、　Ｒ，Ｆ、ら、 （１９８７）　Ｎｅｗ　Ｅｎｇｌ。Many genetic diseases are found to occur in one histological type, and many genetic diseases cell types - ＼Not only is it unnecessary to insert non-specifically, but also under certain circumstances (Selden, R.F., et al.) (1987) New Engl.

Ｊ、ｊｌｅｄ、、３１７：　１０６７−１０７６）。J, Jled, 317: 1067-1076).

更に、殆との癌は１つの細胞型から派生すると考えられている。現在では、化学 療法か癌を制御するための最も広く使用される手段である。しかし、このような 化学療法は化学治療剤の送達において組織特異的ではない。むしろ急速に分裂し ている癌細胞に与えられる逆の効果は、正常細胞にも課される。Furthermore, most cancers are thought to derive from a single cell type. Currently, chemistry It is the most widely used therapy or means for controlling cancer. But something like this Chemotherapy is not tissue specific in the delivery of chemotherapeutic agents. rather rapidly splitting The opposite effect exerted on cancerous cells is also imposed on normal cells.

最近のある刊行物は、肝細胞を標的化するためにＤＮＡに共有結合したタンパク 質の使用を￥紐告しているが、この手段はウィルスベクターによって達成される −［高い効率とは対照的に、ＤＮＡの取り込みにおいて低い効率であった（ウー （１〜’ｕｌＧ、Ｙ、ら、（＋９８８）　Ｊ、Ｂｉｏｌ、Ｃｈｅｍ、　２６３： １４６２１−１４６２４）。A recent publication describes a protein covalently attached to DNA to target liver cells. This method is achieved by means of a viral vector. -[low efficiency in DNA uptake, in contrast to high efficiency (woo (1~'ulG, Y, et al., (+988) J, Biol, Chem, 263: 14621-14624).

この技術の現状では、種々の物質の送達を特定の細胞型ヘターゲティングするた めの方法及び組成物に対する要求が存在することは明らかである。従って、本発 明の目的は、ここに、物資の送達を特定の細胞型にターゲティングするためのビ ヒクル（伝達体）を提供することである。The current state of the technology is limited to targeting the delivery of various substances to specific cell types. Clearly, a need exists for methods and compositions for this purpose. Therefore, the original Our goal here is to develop strategies for targeting the delivery of goods to specific cell types. It is to provide a vehicle (transmitter).

より詳細には、本発明の目的は、核酸又は治療剤のような物質の送達を、生物内 部に含ま第１る哺乳細胞の亜集団（サブポピユレーション）ヘタ−ゲットするた めの方法及び組成物を提供することである。More particularly, it is an object of the present invention to facilitate the delivery of substances such as nucleic acids or therapeutic agents into living organisms. In order to target the first subpopulation of mammalian cells contained in the It is an object of the present invention to provide methods and compositions for achieving this goal.

発明の概要 前述の目的に従って、本発明は、物質の送達を哺乳細胞の亜集団に優先的にター ゲティングするためのビヒクルが含まれる。細胞並集団は、該細胞の表面におけ る結合対の亜集団特異的な第１の構成物の存在によって特徴付けられる。該ビヒ クルは、小区分（コンパートメント）及び該小区分に含まれる物質を画定する組 換えターゲティング部を含むエンベロープから構成される。組換えターゲティン グ部は、第」及び第２ドメインを含む。第１ドメインは、ビヒクルのエンベロー プを形成する又はこれに結合（会合）することができ、一方、第２ドメインは、 細胞の亜集団の表面における第１の結合構成物と相互作用することができる。Summary of the invention In accordance with the foregoing objectives, the present invention provides for targeting the delivery of substances preferentially to subpopulations of mammalian cells. A targeting vehicle is included. A parallel population of cells is located on the surface of the cell. characterized by the presence of a subpopulation-specific first member of the binding pair. The bihi A system is a collection that defines a subdivision (compartment) and the substances contained in the subdivision. It consists of an envelope containing a retargeting section. recombinant targeting The domain includes a second domain. The first domain is the envelope of the vehicle. can form or bind (associate) with the second domain, while the second domain can interact with a first binding member on the surface of a subpopulation of cells.

本発明は、物質の送達を上記哺乳類細胞の亜集団に優先的にターゲティングする ための方法を含み、これは、亜集団に含まれる哺乳類細胞の集団と上記ビヒクル とを接触することを含む。The present invention preferentially targets the delivery of substances to subpopulations of mammalian cells as described above. a population of mammalian cells comprised in a subpopulation and said vehicle. including contact with.

図面の簡単な説明 図１は、混成エリスロボイエチンーウィルスエンベローブ遺伝子の構築を示して いる。Brief description of the drawing Figure 1 shows the construction of a hybrid erythroboietin-viral envelope gene. There is.

図２は、エリスロボイエチン配列の検出を含むツク・ソケージング細胞サブクロ ーンのウェスタンプロットを示している。Figure 2 shows the tsukusocaging cell subcrop containing the detection of erythroboietin sequences. Western plot of the period is shown.

図３は、ポリクローナル抗ＥＰＯ抗体による細胞表面ＥＰＯエピトープの検出に 基づくパンケージング細胞株のフローサイトメトリー解析を示している。Figure 3 shows the detection of cell surface EPO epitopes using polyclonal anti-EPO antibodies. Flow cytometry analysis of a pancased cell line based on Figure 1 is shown.

図４は、ネオマイシンに暴露したもの又はＥＰＯ−ｅｎｖタンノ（り質を含むネ オマイノン耐性遺伝子をコートするレトロウィルスベクターに曝した後にネオマ イシンで処理したＨｅＬａ細胞（野性型又はエリスロボイエチンリセブターを発 現している）の生存能を示している。Figure 4. neoma after exposure to a retroviral vector that coats the omynon resistance gene. HeLa cells treated with icin (wild type or expressing erythroboietin receptors) This shows the viability of the cells.

図５は、ＮＩＨ３Ｔ３細胞による類似実験を示している。Figure 5 shows a similar experiment with NIH3T3 cells.

本発明は、エリスロボイエチンの配列の部分に想到するアミノ酸配列を含む混成 ウィルスエンベロープタンパク質を含むウィルスが、細胞表面上にエリスロポイ エ千〉リセブターを提示する細胞に優先的に感染することができるということの 発見に基づく。The present invention provides a hybrid compound containing an amino acid sequence that corresponds to a portion of the sequence of erythroboietin. Viruses containing viral envelope proteins deposit erythropoiets on the cell surface. E: The fact that cells presenting receptors can be preferentially infected Based on findings.

本文において使用される場合、本発明の“ビヒクル”とは、小区分及びその中に 含ま第１る物質を画定するエンベロープを含み、該物質の送達を哺乳類細胞の特 定亜集団に優先的にタープテイングすることができる全ての組成物として定義す る。哺乳類細胞の亜集団は、該亜集団の表面における結合対の第１の構成物の存 在によって特徴付けられる。ビヒクルは、更に、エンベロープを形成する又は二 Ａ１に結合することかてきる°組換えターゲテイング部”を含み、該ビヒクルに 対して細胞特異性を与えるエンベロープによって定義される。この点で、該ター ゲティング部は、少なくとも２つのドメインを含む。第１ドメインはビヒクルを 形成する又はこれに結合することができ、一方、第２ドメインは哺乳類細胞の亜 集団の表面上の結合対の第１の構成物と相互作用することができる。As used herein, the term “vehicle” of the present invention refers to the subdivision and its contents. comprising an envelope defining a first substance contained therein, which facilitates the delivery of said substance to mammalian cells; Defined as all compositions that can preferentially tarp a fixed subpopulation. Ru. A subpopulation of mammalian cells is characterized by the presence of the first member of a binding pair on the surface of the subpopulation. Characterized by presence. The vehicle may further form an envelope or double The vehicle contains a recombinant targeting moiety capable of binding to A1. defined by the envelope that confers cell specificity to the cell. In this respect, the target The targeting portion includes at least two domains. The first domain is the vehicle the second domain can form or bind to mammalian cell subtypes, while the second domain can interact with the first member of the binding pair on the surface of the population.

本発明の１具体例では、ビヒクルは“ウィルスビヒクル１を構成する。他の具１ １例では、ビヒクルは゛°リポソームビヒクル”を構成する。本発明のウィルス ビヒクルは、一般に天然発生ウィルスにより形成され、特にこれらのウィルスＩ ま真核細胞、例えば哺乳類細胞を感染可能なウィルスである。よく知られている ように、ウィルスは、ウィルスのトランスフェクション及び生殖に必要な種々の 遺伝子をコードするＲＮＡ又はＤＮＡから構成されるゲノムを含む。ウィルスゲ ノム内に包含さｉｌするものは、種々の酵素例えばＤＮＡ又はＲＮＡポリメラー ゼ等並びに“ウィルスコートタンパク質”と呼ばれるウィルスゲノムを包む構造 タンパク質をコートする核酸配列である。In one embodiment of the invention, the vehicle constitutes a “viral vehicle 1. Other ingredients 1 In one example, the vehicle constitutes a "liposomal vehicle." Vehicles are generally formed by naturally occurring viruses and in particular these viruses I It is a virus that can infect eukaryotic cells, such as mammalian cells. well known As such, viruses have a variety of functions necessary for viral transfection and reproduction. It includes a genome consisting of RNA or DNA that encodes a gene. virus game Encompassed within the genome are various enzymes such as DNA or RNA polymers. and the structure that wraps the virus genome called “viral coat protein.” A nucleic acid sequence that coats a protein.

あるウィルスコートタンパク質は、カプシドと知られているウィルス粒子の一部 を形成する。これに加えて、あるウィルスは、しばしばウィルスエンベロープタ ンパク質を含むカプシドを取り囲む脂質二重膜を更に含む。用いたウィルス及び そのエンベロープ構造、即ちカプシドタンパク質のみなのか、膜及びエンベロー プタンパク質と組み合わせたカプシドタンパク質なのかに依存して、カプシドタ ンパク質又はエンベロープタンパク質が、本発明のウィルスビヒクルのためのタ ーゲティング部を形成するために用いられる。A virus coat protein is part of the virus particle known as the capsid. form. In addition to this, certain viruses often contain It further includes a lipid bilayer membrane surrounding the protein-containing capsid. The virus used and Its envelope structure, i.e., is it only capsid protein, membrane and envelope structure? Depending on the capsid protein in combination with the capsid protein, The protein or envelope protein is a protein for the viral vehicle of the invention. used to form a targeting section.

ターゲティング部がウィルスビヒクルのエンベロープを形成する又はこれに結合 する二とができるドメインを含むので、全部又は一部のウィルスコートタンパク 質が組換えターゲティング部を構築するために用いられる。コートタンパク質の 一部のみを用いた場合には、エンベロープ形成に必要且つ十分なコートタンパク 質の部分が用いられる。ウィルスエンベロープタンパク質のために、このような ぜ・要且つ必須な特性には、アンカリング（固着）領域及び膜通過領域が含まれ 、これらはエンベロープタンパク質に利用されて、感染細胞にエンベロープタン パク質を提示する。エンベロープタンパク質を含むウィルスが使用される場合に は、ターゲティング部はこのようなアンカー及び膜通過型領域を含むことが好ま しい。The targeting moiety forms or binds to the envelope of the viral vehicle. All or part of the viral coat protein contains two domains that can quality is used to construct the recombinant targeting moiety. coat protein If only a portion is used, sufficient coat protein is necessary and sufficient for envelope formation. The quality part is used. Due to viral envelope proteins, such Important and essential properties include anchoring regions and membrane passage regions. , these are used by envelope proteins to transfer envelope proteins to infected cells. Presenting quality. When viruses containing envelope proteins are used The targeting portion preferably includes such an anchor and a transmembrane region. Yes.

ターゲティング部の第２ドメインは、哺乳類細胞の亜集団の表面に存在する結合 対の第１の構成物と相互作用することができる。本文において用いられる場合゛ 結合対゛には、リセブターーリガンド複合体、抗原−抗体複合体、酵素−基質複 合体等が含まれる。従って、細胞の亜集団は、該集団の細胞の表面上の１つの結 合対の存在によって定義できる。例えば、エリスロポイエチンリセブターを有す る細胞は、エリスロポイエチンリセブターと相互作用可能な第２ドメインとして 本発明のターゲティング部にエリスロポイエチン（リガンド）を利用することに よって標的化され得る細胞性並集団を定義する。或ｕｎｉｔ、該１Ｊセブターを ターゲティング部に使用して、リガンドを亜集団の細胞の表面上（こ提示するこ と力くできる。The second domain of the targeting moiety targets binding molecules present on the surface of a subpopulation of mammalian cells. can interact with the first member of the pair. When used in the main text゛ Binding partners include receptor-ligand complexes, antigen-antibody complexes, and enzyme-substrate complexes. Includes merging, etc. Therefore, a subpopulation of cells is defined by one bond on the surface of the cells of the population. It can be defined by the existence of a conjunction. For example, with erythropoietin receptor cells that have erythropoietin receptors as a second domain capable of interacting with erythropoietin receptors. Using erythropoietin (ligand) as the targeting moiety of the present invention thus defining a subpopulation of cells that can be targeted. A unit, the 1J Seventer The targeting moiety allows the ligand to be displayed on the surface of a subpopulation of cells. I can do it forcefully.

同様に、細胞の亜集団を、この集団内に含まれる細胞の表面上の抗原又（よ＃冗 ｆ本の存在によって定義することもてきる。例えば、細胞の亜集団の表面上（こ 含まれる表面マーブＪ−を、当業者によく知られて０る方法を二よってモノクロ ーナル抗体を作製するために用いることがてきる。モノクローナル抗体（特（こ アンカー及び膜通過型領域を含むもの並びに、ｃＤＮＡのクローニング及び改変 を介して設ｉｔしてこのような配列を含めたもの）を、ターゲテイング部のみ（ 例え１ｆ、１ノボ′ノームヒヒクル）又はウィルス表面と結合可能なタンノくり 質との組み合わせとして使用する二とができる。後者の場合の例には、ターゲテ イング部（＝（よ、（九体力Ａらの軽鎖とし結合する１ｇ分子の重鎮の可変域に 少なくとも結合するウィルスエンへロープタンパク質からのアンカリング及び膜 通過領域を含むキメラ抗体力（本質的に含まれる。或いは、本発明のビヒクルは 免疫グロブリンの膜結合形態を提示する１ルバ細胞の亜集団をタープ・？卜する ことができる。このような場合（こ（よ、膜結合抗体に対する抗原を用いて、タ ーゲテイング部の第２ドメインを形成する。Similarly, a subpopulation of cells can be defined as a subpopulation of cells containing antigens or It can also be defined by the existence of f books. For example, on the surface of a subpopulation of cells (this The contained surface marbs are monochromated by two methods well known to those skilled in the art. It can be used to produce null antibodies. Monoclonal antibodies (special) Containing anchors and transmembrane regions, and cloning and modification of cDNAs containing such an array), and only the targeting part ( (e.g. 1f, 1novo'norm vehicle) or tannokuri that can bind to the virus surface. Can be used in combination with quality. An example of the latter case is the target Ing part Anchoring from at least the viral envelope protein that binds and the membrane A chimeric antibody force containing a transit region (essentially included; alternatively, the vehicle of the invention A subpopulation of 1 Luba cells that presents membrane-bound forms of immunoglobulins is tarp? to watch be able to. In such cases, using an antigen against a membrane-bound antibody, - form the second domain of the gating section.

一般に、本発明のビヒクルを形成するために用し１られる組換えターゲテインク ゛部は、結合に１の第２の構成物の全部又は一部を含む。ある場合；こ（よ、結 合対間の相互作用は、連続エピトープに関連した相互作用に基づし）て０る。あ る場合（こ１よ、ター￥テインク部の形成に用いることができる結合対の第２の 構成物の最ｚｌｚ成分は、二のようなエピトープから成る。しかし、多くの場合 １こ（よ、相互（乍用（よ不連続エピトープに関連している。例えは、タンパク 質の場合（二１よ、該タンノ（り質の一部アミノ酸配列内の２以上の領域は、結 合エピトープを形成するための該タンパク質の三次溝道において物理的に近い隣 接部にもたらされる。このようなタンパク質の部分のみを用いてターゲテイング 部を形成する場合、ｇｉＩ′ｇｔ類細胞の亜集団に存在する結合対の他の構成物 と最も強く相互作用すること力（できるよう（こ、二のような結合領域を包含す る一部アミノ酸配列の部分がターゲテイング部（二含ま第１る二とか好ましい。Generally, recombinant target inks used to form the vehicles of the invention The first part includes all or part of the second component in the bond. If there is; The interactions between pairs are 0 (based on interactions associated with consecutive epitopes). a (In this case, the second of the bonding pair that can be used to form the tartar The most zlz component of the construct consists of two similar epitopes. But in many cases 1. They are related to mutually discontinuous epitopes. For example, proteins In the case of protein (21), two or more regions within the partial amino acid sequence of protein physically close neighbors in the tertiary groove of the protein to form a synergistic epitope. brought to the junction. Targeting using only this part of the protein other members of the binding pair present in a subpopulation of giI'gt cells. The force that interacts most strongly with the A part of the amino acid sequence is preferably a targeting part (including two, first, first, second, etc.).

本発明のビヒクルは、哺乳類細胞の亜集団に物質の送達を優先的（こタープ・ソ トする。このような優先的なタープティングは、結合対の第１の構成物を提示し ていない同種の哺乳類細胞の異なる亜集団に対して、結合対の第１の構成物を含 む哺乳類細胞の亜集団−＼の物質の送達を比較することによって定義される。亜 集団に対する該ビヒクルの結合又は亜集団細胞へのビヒクルの物質の転移（ｔｒ ａｎｓｆｅｒｒａｌ）によって測定される送達の増加は、一般に２倍より大きい 。しかし、実施例において示されるように、本文中で開示されたウィルスビヒク ルによる感染における効率の増加は、この実験に用いられたりセブターを有しな い野性型細胞と比較して、１０倍から約３０〜４０倍の範囲であった。The vehicles of the invention preferentially deliver substances to subpopulations of mammalian cells. to Such preferential tarpting presents the first member of the binding pair. containing the first member of the binding pair to different subpopulations of homogeneous mammalian cells that are not Defined by comparing the delivery of substances to subpopulations of mammalian cells. sub Binding of the vehicle to the population or transfer of vehicle material to subpopulation cells (tr The increase in delivery measured by ansferral is generally greater than 2-fold . However, as shown in the Examples, the virus vehicles disclosed in the text The increase in efficiency in infection by the bacteria used in this experiment and ranged from 10-fold to approximately 30-40-fold compared to wild-type cells.

本文中の実施例では、組換えターゲティング部はモロニーマウス白血病ウィルス （ＭｏＬｆＬＶ）のエンベロープタンパク質のカルボキシ末端を含む。第２ドメ イ肩よエリスロポイエチンのアミノ末端を含む。このコンストラクト（構築物） をコートするプラスミドが、ウィルス粒子の形成がもたらされる条件下において 全長ＭｏＭＬＶエンベロープタンパク質も発現可能なパッケージング細胞株中で 定現さ第１る場合、本発明のウィルスビヒクルが形成される。この特別の場合に は、１１１換えターゲティング部は、ウィルス粒子の残りを含む小区分を画定し ているエンベロープ（第２及びＭｏＭＬＶエンベロープタンパク質に対応してい る）と結合する。従って形成されると、ウィルス小区分内に含まれる物質はウィ ルスゲノムを包囲するタンパク質又はゲノム自身を含むことができる。前者の場 合には、ウィルスカプシドタンパク質によりパッケージング可能な、予め形成さ れたタンパク質は、この小区分に含まれる物質を含むことができる。しかし殆ど の具体例では、このようなウィルスビヒクル内部に含まれる対象物質は、ウィル スのゲノムに導入された組換えＤＮＡ配列である。ウィルスビヒクルは、細胞の 亜集団に送達される・＼き物質が核酸の場合に好ましい。In the examples herein, the recombinant targeting moiety is Moloney murine leukemia virus (MoLfLV) contains the carboxy terminus of the envelope protein. 2nd dome It contains the amino terminus of erythropoietin. This construct under conditions that result in the formation of viral particles. in a packaging cell line that can also express the full-length MoMLV envelope protein. When first expressed, a viral vehicle of the invention is formed. in this particular case The 111 recombinant targeting portion delimits the subsection containing the remainder of the virus particle. envelope (corresponding to the second and MoMLV envelope proteins) ). Thus, once formed, the material contained within the viral subdivision is can include proteins surrounding the virus genome or the genome itself. former place In some cases, a preformed protein that can be packaged by the viral capsid protein. The proteins listed may include substances included in this subdivision. But mostly In a specific example, the substance of interest contained within such a virus vehicle is This is a recombinant DNA sequence introduced into the genome of a plant. The viral vehicle is a cell It is preferred when the substance delivered to the subpopulation is a nucleic acid.

従って、遺伝子疾患の治療に用いられた場合、熟練技術者は欠陥遺伝子及び正常 な野性型遺伝子を発現する細胞型を認識する。その後、表面マーカーを、野性ヤ 遺伝子を正常に発現している該細胞タイプ上に同定する。その後、本発明のウィ ルスビヒクルは、改変されたウィルスが選択された表面マーカーを認識し、結合 することができるように、選択されたウィルスのエンベロープを形成又は結合可 能な組換えターゲティング部を含むように設計される。本発明の実行に用いられ るウィルスの選択と共にウィルスゲノムに組み込まれるための組換え核酸のサイ ズは、用いるためのウィルスの選択を容易にするために決定される。ウィルスの ゲノムは、選択された組換え核酸を収納することができるか、又はこのような組 換え核酸がウィルスゲノムへ取り込まれて、更に組換えターゲティング部を含有 する完全なウィルスゲノムを形成するためにパッケージされ得るように、非必須 配列をｆＪＦ除して改変したようなものでなければならない。この観点から、組 み込まれる組換え核酸を収納するだけでなく、改変ウィルスの菌内を弱毒化する ように改変さオ］た野性型ウィルスのゲノムが好ましい。このような改変は、１ 以上のＤＮＡ又はＲＮＡをコードするウィルス遺伝子の除去が含まれるが、これ に限定さねない。本発明の実行に用いることができるウィルスの例には、レトロ ウィルス、アデノウィルス及びアデノ関連ウィルスが含まれる（バーフナ−（Ｂ ｅｒｋｎｅｒ）。Therefore, when used in the treatment of genetic diseases, skilled technicians are able to distinguish between defective and normal genes. Recognize cell types that express wild-type genes. Then, apply surface markers to wild animals. The gene is identified on the cell type in which it is normally expressed. After that, the invention The virus vehicle allows the modified virus to recognize and bind to selected surface markers. Envelopes of selected viruses can be formed or combined to It is designed to contain a recombinant targeting moiety capable of recombination. used in carrying out the invention The size of the recombinant nucleic acid to be integrated into the viral genome along with the selection of viruses The virus size is determined to facilitate the selection of viruses for use. of the virus The genome may contain selected recombinant nucleic acids or The recombinant nucleic acid is incorporated into the viral genome and further contains the recombinant targeting moiety. non-essential so that they can be packaged to form a complete viral genome The sequence must be modified by dividing the sequence by fJF. From this point of view, the group Not only does it contain the recombinant nucleic acid to be ingested, but it also attenuates the inside of the modified virus. Preferred is the genome of a wild-type virus that has been modified in this way. Such modification is 1 This includes the removal of viral genes encoding the above DNA or RNA, but this may be limited to. Examples of viruses that can be used to carry out the invention include viruses, adenoviruses and adeno-associated viruses (Berfner (B)). erkner).

Ｋ、Ｌ、　（１９８８）　ＢｉｏＴｅｃｎｉｑｕｅｓ　６：６１６−６２９）。K, L. (1988) BioTechniques 6:616-629).

エリスロボイエチンリセブターを発現する培養哺乳類細胞に対する優先的なター ゲティングを証明するウィルスビヒクルを実施例は開示する。従って、ｉｎ　ｖ ｉｖＯて用いら第１る場合、ウィルスビヒクルはエリスロボイエチンリセブター を発現する生物内のこのような細胞を標的とする。このようなビヒクルの特定の 用途は、鋳状赤血球貧血、β−サラセミアのような異常血色素症の治療である。A preferential target for cultured mammalian cells expressing the erythroboietin receptor. The examples disclose viral vehicles that demonstrate targeting. Therefore, inv In the first case used in ivO, the viral vehicle is erythroboietin receptor. target such cells within an organism that express the . Certain of such vehicles Its use is in the treatment of hemochromatosis such as cast cell anemia and β-thalassemia.

これらの疾患は遺伝的欠陥に関連し、異常なグミビン鎖が作られるか、β−グロ ビン鎖があったとしても極僅かしか生成されない。実施例において、β−グロビ ン遺伝子をコートするゲノムクローンがウィルスビヒクルのゲノムへ挿入される 場合には、得られたウィルスビヒクルは春型細胞だけてなくエリスロボイエチン リセブターの提示によって証拠付けられる赤血球分化に拘束された造血幹細胞由 来の春型細胞の前駆体を優先的に認識可能である。ウィルスがこのようなエリス ロボイエチン前駆体をトランスフェクトする場合、β−グロビン遺伝子は先祖細 胞のゲノム・＼組み込まれる。結果として、正常β−グロビン遺伝子はゲノムへ 組み込まれ、正常な量のβ−グロビン又は野性型のβ−グロビンタンパク質を生 成することによって、この疾患を改変することができる。These diseases are associated with genetic defects that result in the production of abnormal gummybin chains or Even if there are bottle chains, very few are generated. In an example, β-globin A genomic clone encoding the protein gene is inserted into the genome of the viral vehicle. In some cases, the resulting viral vehicle contains not only spring-form cells but also erythroboietin. Hematopoietic stem cell origin committed to erythroid differentiation evidenced by the presentation of receptors. Precursors of future spring-type cells can be preferentially recognized. The virus is like this When transfecting the loboietin precursor, the β-globin gene is The genome of the cell is integrated. As a result, the normal β-globin gene is transferred to the genome. and produce normal amounts of β-globin or wild-type β-globin protein. This disease can be modified by making a difference.

本発明のウィルスビヒクルは、治療剤の送達を癌細胞のような疾病細胞のターゲ ティングのために用いることもてきる。例えば、表面マーカーが癌細胞上に優先 的に発現されている場合（即ち、癌細胞の表面上のみ又は、非癌細胞型に認めら れるよりも高いレベルで）、適当なターゲティング部を用いて、このような優先 的な発現マーカーに結合するためにウィルスビヒクルをターゲットすることがて きる。ウィルスビヒクルのゲノムは、例えば毒性ポリペプチド例えばりシン、ジ フテリア毒素等を発現することができる核酸発現ユニットと組み換えられる。The viral vehicles of the present invention target the delivery of therapeutic agents to diseased cells, such as cancer cells. It can also be used for tinging. For example, surface markers are prioritized on cancer cells. (i.e., only on the surface of cancer cells or found in non-cancerous cell types) (at a higher level than It is possible to target viral vehicles to bind to specific expression markers. Wear. The genome of the viral vehicle contains, for example, toxic polypeptides such as lysine, di- It is recombined with a nucleic acid expression unit capable of expressing phtheria toxin and the like.

癌細胞の認識、結合及びトランスフェクションの際に、発現ユニットは毒性ポリ ペプチドを生成して、優先的に癌細胞を死滅させる。この点に関して、発現ユニ ットは、毒性ポリペプチドをコードする核酸と、毒性ポリペプチドを生成するよ うにトランスフェクトされた癌細胞で核酸が発現されるように核酸に操作可能に 連結された１以上の発現調節配列とを含む。このような発現調節配列は、好まし くは分化した癌細胞からの組織特異的な細胞タイプに対する特異性を証明するよ うなものであり、より好ましくは癌細胞によって専ら発現される発現調節配列、 更に好ましくは優先的に発現される表面マーカーの発現を制御している発現調節 配列である。Upon recognition, binding and transfection of cancer cells, the expression unit contains toxic polypeptides. Generate peptides to preferentially kill cancer cells. In this regard, the expression unit The kit contains a nucleic acid encoding a toxic polypeptide and a protein that produces the toxic polypeptide. Nucleic acids can be manipulated so that they are expressed in cancer cells transfected with sea urchins and one or more linked expression control sequences. Such expression control sequences are preferably or to demonstrate specificity for tissue-specific cell types from differentiated cancer cells. expression regulatory sequences, more preferably expressed exclusively by cancer cells; More preferably, an expression regulation controlling the expression of a preferentially expressed surface marker. It is an array.

二のような具体例では、好ましく選択されるウィルスはレトロウィルス科から選 択される。レトロウィルスは、宿主のゲノムへ効率よく組み込むために活発に分 裂している細胞を要求するので、好ましい。非癌性細胞が癌細胞上に優先的に提 示された表面マーカーを発現する場合、本発明のウィルスビヒクルは、このよう な細胞を認識し、結合し、トランスフェクトすることもできる。このバックグラ ウントレヘルは、このような正常細胞上に見出される表面マーカーの量及び該マ ーカーか提示されている環境に依存する。更に、ウィルスビヒクルが、組換えタ ーゲティング部と共に存在している正常ウィルスコートタンパク質例えばエンベ ロープタンパク質を含む場合、ウィルスビヒクルは、ターゲティング部による１ ｈｌｋのために用いられる表面マーカーの存在とは独立して、非特異的な細胞の トランスフェクト能を存する。しかし、レトロウィルスを本発明を実施するため に用いて癌細胞を処理する場合、このような非癌性細胞における感染のパックグ ラウンドは、ウィルスの組み込み及び毒性遺伝子の発現が起きそうもないので、 急速に分裂しない正常細胞に対して傷害を起こしそうもない。In embodiments such as 2, the preferably selected virus is a virus selected from the Retroviridae family. selected. Retroviruses actively segregate in order to efficiently integrate into the host genome. This is preferred because it requires cells that are cleft. Non-cancerous cells are preferentially presented on cancer cells. When expressing the indicated surface markers, the viral vehicle of the invention It can also recognize, bind to, and transfect other cells. This background graph Untreher describes the amount of surface markers found on such normal cells and the depending on the environment being presented. Furthermore, the viral vehicle is a recombinant protein. The normal viral coat proteins present with the targeting moiety, e.g. When containing a rope protein, the viral vehicle can be Independent of the presence of surface markers used for hlk, non-specific cellular Possesses transfection ability. However, in order to carry out the present invention, retroviruses When treating cancer cells using round, since viral integration and expression of virulence genes are unlikely to occur. It is unlikely to cause damage to normal cells that do not divide rapidly.

オンコシン（癌遺伝子）の過剰発現は、しばしば癌のオンセット及び発達に関連 している。多くの場合、発現されたオンコシンは癌細胞の表面上に位置している リセブターをコードしている。例えば、プロトオンコシン（癌原遺伝子）ＨＥＲ ２か膜通過型チロシンキナーゼをコードし、この過剰発現は幾つかのヒト悪性疾 患に関連し、これには胸、卵巣、胃及び内皮細胞癌と非小細胞腫アデノ腫瘍が含 ま１１ることが近年開示されている。ホルメス（Ｈｏｌｍｅｓ）ら、（１９９２ ）　５ｃｉｅｎｃｅ　２５６を参照のこと。更に記述されているように、タンパ ク質へレグリン−αは、ＨＥＲ２オンコシンを発現している腫瘍細胞株に対する 単一の親和性結合部位を有する。従って、ヘレグリンーαタンパク質は、本文に おいて開示される技術による」−記の同定さ第１た癌の治療における１つの候補 である。Overexpression of oncosins (oncogenes) is often associated with cancer onset and development are doing. Expressed oncosins are often located on the surface of cancer cells Codes a resetter. For example, proto-oncosin (proto-oncogene) HER It encodes two transmembrane tyrosine kinases, and its overexpression is associated with several human malignant diseases. related to cancer, including breast, ovarian, gastric and endothelial cell carcinomas and non-small cell adenotumours. Eleven things have been disclosed in recent years. Holmes et al. (1992 ) See 5science 256. As further described, the tamper HER2 oncosin-expressing tumor cell lines Has a single affinity binding site. Therefore, heregulin-α protein is included in the main text. A candidate for the treatment of the first cancer identified by the technique disclosed in It is.

癌細胞に加えて、本発明を自己免疫疾患の治療に用いることができる。例えば、 Ｔ細胞仲介自己免疫疾患は、自己抗原を認識し、これと相互作用して、自己免疫 ｔｊ＜答を引き出してしまうＴ細胞抗原リセブター（ＴＣＡＲ）を含むＴ細胞の 亜集団によって特徴はけられる。実験的自己免疫能を髄炎（ＥＡＥ）は、マウス におけるミニリン主体タンパク質誘導脱鞘疾患である。近年、この疾患が一部分 、Ｔｍ１ｌｉｆｆ抗原リセブターレパトアからの特定セグメントサブセットを含 有するよく定義さ１１た可変域を含むＴ細胞の亜集団の存在に基づいていること が示された。また、自己免疫疾患に寄与するＴ細胞の亜集団のＴＣＡＲのβ鎖内 部に含まれるエピトープに特異的なモノクローナル抗体で処理されたマウスは、 ミニリン主体タノバク質てチャレンジされた場合に該疾患の開始を防止すること ができるということも報告された。In addition to cancer cells, the invention can be used to treat autoimmune diseases. for example, T cell-mediated autoimmune diseases recognize and interact with self-antigens, leading to autoimmunity. T cell antigen receptor (TCAR) that elicits the answer Characteristics are differentiated by subpopulations. Experimental autoimmune myelitis (EAE) in mice This is a miniphosphorus-based protein-induced sheathing disease. In recent years, this disease has become , containing a specific subset of segments from the Tm1liff antigen reservoir repertoire. It is based on the existence of subpopulations of T cells that contain a well-defined variable range of It has been shown. In addition, within the β chain of TCAR of T cell subpopulations that contribute to autoimmune diseases, Mice treated with monoclonal antibodies specific for epitopes contained in Preventing the onset of the disease when challenged with minirin-based tanobacteria It was also reported that it is possible.

特にヒトにおいて、他のＴ細胞仲介自己免疫疾患が種々の自己免疫疾患の基礎を 形成すると考えられているので、本発明のビヒクルは、このような自己免疫疾患 の治療又は予防に対する違う手段として用いることができる。このような場合、 自己免疫疾患に寄与するＴＣＡＲに対して特異的なモノクローナル抗体は、本発 明のビヒクルを形成するために用いら第１る。ウィルスビヒクルを用いる場合、 ウィルス内部に含ま１１る物質は、好ましくは自己免疫誘導ＴＣＡＲを含むこの ようなＴ細胞・＼ｌ・ランスフエクトしたときに発現することができる毒性遺伝 子である。Other T cell-mediated autoimmune diseases may underlie various autoimmune diseases, especially in humans. The vehicle of the present invention is believed to be effective in treating such autoimmune diseases. can be used as a different means for the treatment or prevention of In such a case, Monoclonal antibodies specific for TCAR, which contributes to autoimmune diseases, are The first is used to form a bright vehicle. When using a viral vehicle, The 11 substances contained within the virus preferably include autoimmune-inducing TCARs. Toxic genes that can be expressed when transfected with T cells such as It is a child.

或いは、リポソームを用いる場合、そこに含まれる物質は、例えばリシン又はジ フテリア毒素タンパク質のような細胞を殺すことができる毒性ポリペプチド又は 化学治療剤のような治療剤をコードする発現可能な遺伝子を含む核酸とすること ができる。Alternatively, when using liposomes, the substance contained therein may be, for example, lysine or di- Toxic polypeptides that can kill cells, such as phtheria toxin protein or A nucleic acid containing an expressible gene encoding a therapeutic agent such as a chemotherapeutic agent Can be done.

ウィルスビヒクルに加えて、リポソームビヒクルを本発明の実施に用いることか できる。リポソームは当業者によく知られており、一般に、種々の薬物又は他の 化学物質を被包化することができる脂質二重膜を含む膜状小胞から構成されてい る。例えは、米国特許第４．０５３．５８５号：第４．３９７．８４６号：第４ ．４１１．８９４号；第４．４２７、６４９号並びに、パパハジョボロス（Ｐａ ｐａｈａｄｊｏｐｏｌｏｕｓ）ら、（１９６７）　Ｂｉｏｃｈｅｍ、Ｂｉ。In addition to viral vehicles, liposomal vehicles may be used in the practice of this invention. can. Liposomes are well known to those skilled in the art and generally contain various drugs or other It consists of membranous vesicles containing a lipid bilayer membrane that can encapsulate chemicals. Ru. For example, U.S. Patent No. 4.053.585:4.397.846:4 ．． 411.894; 4.427, 649 and Papahajoboros (Papa Hajoboros) (1967) Biochem, Bi.

ｐｈｙｓ、　Ａｃｔａ、　、　＋３５：６３９　；パンガム（Ｂａｎｇｈａｍ） 、　Ｊ　、ら、（１９６５）　Ｊ、Ｍｏ１．Ｂｉｏｌ、１２F２３８゜ ２５２、バッリ（Ｂａｐｚｒｊ　＞とコーン（Ｋｏｒｎ）、（１９７３）　８ｉ ｏｃｈｅｍ、　Ｂｉｏｐｈｓ、　Ａｃｔａ、　、　２９８：P０１５　； 及び、ミャモトら、（１９７１）　”Ｐｒｅｐａｒａｔｉｏｎ　ａｎｄ　Ｃｈａ ｒａｃｔｅｒｉｓｔｉｃｓ　ｏｆ　Ｌｉｐｉｄ　ＶｅｓｉｃＩｅｓ”、Ｊ、λＩ ｅｍｂｒａｎｅ　Ｂｉｏｌ、　４：２５２−２６９を参照のこと。phys, Acta, +35:639; Bangham , J, et al. (1965) J, Mo1. Biol, 12F238° 252, Bapzrj and Korn, (1973) 8i ochem, Biophs, Acta, 298:P015; and Miyamoto et al. (1971) “Preparation and Cha racteristics of Lipid VesicIes”, J, λI See Embrane Biol, 4:252-269.

薬物又は他の化合物がリポソームへ取り込まれた場合、この物質は一般にリポ′ ノームを合成するために用いられる反応混合物に含まれる。従って、リポソーム ビヒクルを形成するために本発明の実施では、リポソームビヒクルの小区分中に 含まれる・＼き物質例えは、核酸、薬物又は治療剤は、リポソーム合成の間に包 含さイする。更に、本発明のターゲティング部もリポソームの形成の間に包含さ れる。When drugs or other compounds are incorporated into liposomes, this material is generally Included in the reaction mixture used to synthesize gnomes. Therefore, liposomes In the practice of the invention to form a vehicle, during subdivision of the liposome vehicle, Examples include nucleic acids, drugs, or therapeutic agents that are included during liposome synthesis. Contains. Additionally, the targeting moieties of the present invention can also be included during liposome formation. It will be done.

リポ゛ノームを用いる場合、ターゲティング部は、好ましくは、リポソームの脂 質二重膜と結合するように例えば免疫グロブリン又は他の膜結合タンパク質から のアンカリング領域及び膜通過領域を含む。勿論、哺乳類細胞の亜集団に含まれ る結合対の第１構成物と相互作用することができるドメインも含む。好ましいタ ーゲティング部には、細胞表面抗原に特異的なモノクローナル抗体（好ましくは ＩｇＭ）又はクローニング及び改変によってアンカリング又は膜通過領域を含む ように設計された抗体を含む。When using liposomes, the targeting moiety is preferably a liposomal lipid. e.g. from immunoglobulins or other membrane-bound proteins so as to bind to the plasma bilayer membrane. including an anchoring region and a membrane passage region. Of course, it is included in a subpopulation of mammalian cells. It also includes a domain capable of interacting with the first member of the binding pair. Preferred Ta The targeting portion contains a monoclonal antibody (preferably IgM) or contain anchoring or transmembrane regions by cloning and modification. Contains antibodies designed to

ターゲティング部かリポソーム内部にもたらされず、むしろリポソーム形成の間 にリポソーム膜に取り込まれるので、ここで形成されたリポソームはターゲティ ング部を含み、ここで第１の結合膜と相互作用可能なドメインはリポソームの内 膜及び外膜に提示される。The targeting moiety is not brought inside the liposome, but rather during liposome formation. The liposomes formed here are incorporated into the liposome membrane. a binding region, where a domain capable of interacting with the first binding membrane is located inside the liposome. Presented in the membrane and adventitia.

下記に実施例によって開示されるか、これは添付の請求項の範囲を制限するため に構成さ才またものではない。Disclosed below by way of example, which limits the scope of the appended claims. It's not just a composition.

実施例１ 本実施例は、真核発現ベクターにおける混成エリスロポイエチン＋ウィルスエン ベロープ（ＥＰＯ−ｅｎｖ’）遺伝子の構築を説明する。Example 1 This example shows the hybrid erythropoietin + viral enzyme in a eukaryotic expression vector. The construction of the envelope (EPO-env') gene will be explained.

野性型モロニーマウス白血病ウィルス（ＭｏＭＬＶ）のエンベロープ遺伝子全体 （ｅ　ｎ　ｖ遺伝子；レトロウィルスゲノムの位置５７６０〜７８２０　；シニ ック（ＳｈｉｎｎｉｃｋｌＴ、Ｍ、ら、（＋９８１）　Ｎａｔｕｒｅ　２９３＋ ５４３−５４８）をｐＢＲ３２２主体プラスミドベクターでクローン化した。ｅ ｎｖ遺伝子の転写は、５’−ＭｏＭＬＶ長末端反復（ＬＴＲ）で誘導する。ｐｓ ｉ（パッケージングシグナル）配列を除去したが、ベクターはｅ　ｎ　Ｎ’のｍ ＲＮＡのための５°及び３゛スプライシングシグナルを維持している。間のｇａ ｇ及びｐｏｌ横築遺伝子は、ｅｎｖメツセージの外側で正常にスプライスされる ものであり（ワイズ（Ｗｅｉｓｓ）、　Ｒ，、Ｎ、チック（Ｔｅｉｃｈ）　、　 Ｈ−パルマス（Ｖａｒｍｕｓ）　、Ｊ、　コフィン（Ｃｏｆｆｉｎ）編、（１９ ８４）　”ＲＮＡ　Ｔｕｍｏｒ　Ｖｉｒｕｓｅｓ：Ｍｏ１ｅｃｕｆａｒ　Ｂｉｏ ｌｏｇｙ　ｏｆ　Ｔｕｍｏｒ　Ｖｉｒｕｓｅｓ”第２版、コールド・スプリング ・ハーバ−・ラホラトリー、コールド・スプリング・ハーバ−、ニューヨーク化 ）、これらは除去さイ］、従って、スプライスされるために残されたスプライシ ングシグナルの間に小配列のみか維持されている。Entire envelope gene of wild-type Moloney murine leukemia virus (MoMLV) (env gene; position 5760-7820 of retrovirus genome; (ShinnicklT, M, et al., (+981) Nature 293+ 543-548) was cloned into a pBR322-based plasmid vector. e Transcription of the nv gene is induced at the 5'-MoMLV long terminal repeat (LTR). ps Although the i (packaging signal) sequence was removed, the vector Maintains 5° and 3° splicing signals for RNA. ga between The g and pol transverse genes are normally spliced outside the env message. (Weiss), R,, N, Teich, Edited by H-Varmus, J. Coffin, (19) 84) “RNA Tumor Viruses: Mo1ecufar Bio "logy of Tumor Viruses" 2nd edition, Cold Spring ・Harbor Lahore, Cold Spring Harbor, New Yorkization ), these are removed], so the splices left to be spliced are Only a small array is maintained between the switching signals.

その後、エンベロープ部分は除去のために選択されてＥＰＯコード配列と置換さ 第１る。エンベロープの外側タンパク質サブユニット（ｇｐ　７０）の中央に、 プロリン富裕領域かあり、これは、細胞リセブター認識の為の有望な候補ドメイ ンである（コツホ（Ｋｏｃｈ）、Ｗ、ら、（＋９８３）　Ｊ、Ｖｉｒｏｌ、、４ ５：１−９）。他の部位には、この分子のアミノ末端部分に相同性がある領域と 隣接している２つの領域（マーク（λ（ａｒｋ）、　Ｇ、　Ｅ、ら、（１９８４ ）Ｊ、Ｖｉｒｏｌ、、４９：５３０−５３９）と、ＭｏＭＬＶのパラリトジエニ ソク変異体をもたらす点突然変異の部位（ツレツク（Ｓｚｕｒｅｋ）、　Ｐ、　 Ｆ、ら、（１９８８）Ｊ、　Ｖｉｒｏｌ、　６２：３５７−３６０’）とを含む 。これら領域はＥＰＯとの置換のターゲット部位である。同時に、アミノ末端で ｅｎｖシグナルペプチドとカルボキシ末端領域で内部エンヘローブサブユニット （ｐＩ　５）及びスルフヒドリル結合に寄与するシスティン残基とをコードする ｇｐ７０配列の部分は、無傷のままで維持される（ソニック（Ｓｈｉｎｎｉｃｋ ）、　Ｔ、　Ｍ、ら、９８１　）　Ｎａｔｕｒｅ　２９３：５４３−５４８）。The envelope portion is then selected for removal and replaced with the EPO coding sequence. First. In the center of the outer envelope protein subunit (gp 70), There is a proline-rich region, which is a promising candidate domain for cellular receptor recognition. (Koch, W. et al., (+983) J. Virol, 4 5:1-9). Other sites include regions of homology to the amino-terminal portion of this molecule. Two adjacent areas (mark (λ(ark), G, E, et al. (1984 ) J, Virol, 49:530-539) and the MoMLV paralithogiene. Site of point mutation resulting in sok mutant (Szurek, P, F. et al. (1988) J. Virol, 62:357-360'). . These regions are target sites for substitution with EPO. At the same time, at the amino terminus internal enherobe subunit with env signal peptide and carboxy-terminal region (pI 5) and cysteine residues that contribute to sulfhydryl bonding. Portions of the gp70 sequence remain intact (Sonic ), T, M, et al., 981) Nature 293:543-548).

特徴的な制限部位は５９２３（ＢｓｔＥＩＩ’）及び６５３７ＣＢａｍＨＩ　） の位置に存在する。これら２つの部位の間のエンベロープ配列の除去は、ｅｎｖ シグナル配列を維持し、ＥＰＯ配列（約５００塩基対の長さ）をｇｐ７ｏのアミ ノ末端て挿入可能にする。Characteristic restriction sites are 5923 (BstEII') and 6537CBamHI) It exists in the position of Removal of the envelope sequence between these two sites results in env The signal sequence is maintained and the EPO sequence (approximately 500 base pairs long) is inserted into the amino acid of gp7o. It can be inserted at the end.

第２のＥＰＯ−ｅｎｖ混成物は、更に中央位置にＥＰＯ配列を挿入するように構 築され、プロリン富裕領域と直接重複する。この領域に都合のいい制限部位が存 在しないので、特徴部位はＰＣＲ仲介突然変異誘発によって６２３０と６７５０ の位置に作り出される。これらの新しい制限部位を用いて介在するｅｎｖ配列を 除去してＥＰＯ配列を挿入する。The second EPO-env hybrid is further configured to insert the EPO sequence in a central position. built up and directly overlaps with the proline-rich region. Convenient restriction sites exist in this region. Since there are no is created at the position of Use these new restriction sites to modify the intervening env sequence. Remove and insert EPO sequence.

２７アミノ酸シグナルペプチドがない成熟１６６アミノ酸ペプチドホルモンをコ ートするＥＰＯのｃＤＮＡ配列（ジャコブ（Ｊａｃｏｂｓ）、　Ｋ、ら、（１９ ８５）　Ｎａｔｕｒｅ　３１３：８０６−８１０　、リン（Ｌｉｎ’）、　Ｆ、 　Ｋ、ら、（＋９８５）　Ｐｒｏｃ、Ｎａｔｌ、Ａｃａｄ、Ｓｃｉ、、ＵＳＡ、 ８２ニアT８０−７ ５８４）を用いて、ＭｏＭＬＶのｅ　ｎ　ｖ配列（シニック、Ｔ、Ｍ、ら、（１ ９８１）　Ｎａｔｕｒｅ　２９３・５４３−５４８）と置換し、これはエンベロ ープタンパク質のアミノ末端と中央領域が叶去さイ１ている。ＰＣＲ仲介突然変 移誘発（イエス（Ｉｎｎｊｓ）、　Ｍ、　Ａ、、ゲルファン１”（Ｇｅｌ　ｆａ ｎｄｌ　Ｄ、　Ｈ，、スニンスキ（Ｓｎｉｎｎｓｋｙ）、　Ｊ、　Ｊ、、ホワイ ト（Ｗｈｉｔｅ）、Ｔ、Ｊ、編、（１９９０’）　”ＰＣＲＰｒｏｔｏｃｏｌｓ ：　Ａ　Ｇｕｉｄｅ　ｔｏ　Ａｌｅｔｈｏｄｓ　ａｎｄ　Ａｐｐｌｊｃａｔｉｏ ｎｓ”　、アJデミツ クブレス社、サンディエゴ、カリフォルニア化）を用いて、適当な制限部位を通 して挿入することができるようにＥＰＯ配列の互換性末端を作りだし、一方、好 適なリーディングフレームは維持されている（図１）。Coating a mature 166 amino acid peptide hormone with no 27 amino acid signal peptide EPO cDNA sequence (Jacobs, K. et al., (1999)) 85) Nature 313:806-810, Lin', F. K, et al. (+985) Proc, Natl, Acad, Sci, USA, 82 Near T80-7 584) was used to construct the MoMLV e nv sequence (Shinnick, T, M, et al., (1 981) Nature 293, 543-548), which is The amino terminus and central region of the protein are removed. PCR-mediated sudden changes Transfer induction (Jesus (Innjs), M, A,, Gel fa ndl D, H, Sninnsky, J, J, Why White, T.J., eds. (1990') "PCR Protocols :　A Guide to Alethods and Appljcatio ns”, AJ Demitsu (Kubles, San Diego, CA) through appropriate restriction sites. create a compatible end of the EPO sequence so that it can be inserted with The proper reading frame is maintained (Figure 1).

ＥＰＯ−ｅｎｖ横築物を細胞株ＰＡ３１７（７ンＱｌａｎｎ）、　Ｒ１ら、（１ ９８３）　Ｃｅ１１３３：１５３−１５９　；及びコーン（Ｃｏｎｅ）、　Ｒ， Ｄ、ら、（１９８４）　Ｐｒｏｃ、　Ｎａｔ　１．　Ａｃａｄ、　Ｓｃｉ、　、 @ＵＳＡ、　８１　： ６３ａ−６３５３）にトランスフェクトした。ＰＡ３１７Ａ３１７細胞Ｈ３Ｔ３ 細胞の誘導体であり、これはＭｏＭＬＶ遺伝子を安定的に組み込まれている；Ｐ Ａ３１７細胞はｇａｇ及びｐｏ＋遺伝子と、両種性ｅｎｖ遺伝子とを有するが、 ｐｓｉパノケージングングナル配列は除去されている。従って、これらの細胞は 、ウィルスタンパク質を野性型（内因性）ウィルスへ、アッセンブリすることな くもたらし、百踵性（即ち、マウスと非マウス細胞の両方を感染することができ る）と共にしりオン・＼、ｐｓｉ配列を含有する外因性ウィルスベクターパッケ ージングするために用いられ得る。EPO-env transverse constructs were grown in cell lines PA317 (7Qlann), R1 et al. 983) Ce1133:153-159; and Cone, R. D. et al. (1984) Proc, Nat 1. Acad, Sci, @USA, 81: 63a-6353). PA317A317 cells H3T3 a derivative of cells that have stably integrated the MoMLV gene; A317 cells have gag and po+ genes and an amphipathic env gene, but The psi panocasing signal sequence has been removed. Therefore, these cells , without assembly of viral proteins into wild-type (endogenous) virus. and is capable of infecting both mouse and non-mouse cells. Exogenous viral vector package containing the psi sequence together with can be used for

ＥＰＯ−ｅｎＸ・プラスミドをＰＡ３１７Ａ３１７細胞ン酸カルシウム沈殿法（ ウィグラー（Ｗｉｇｌｅｒ）、　Ｍ、ら、（１９７９）　Ｃｅ１ｌ　１６：７７ ）を用いて、選択可能マーカーとしてメトトレキサート耐性ジヒドロ葉酸レダク ターゼ（ＤＨＰＲ）遺伝子（シモ〉セン（Ｓｉｍｏｎｓｅｎｌ　Ｃ，Ｒ，ら、（ １９８１Ｄ　Ｎｕｃｋｅｉｃ　Ａｃ１ｄｓ　Ｒｅｓ、、１６二２２３５−２２４ ６）と共に同時トランスフェクトした。トランスフェクション後、細胞をメトト レキサーｌ〜を含有する培地中で、ｅ　ｎ　Ｎ’配列の安定した組み込みについ て選別した。そのゲノム・＼該配列を安定して組み込まれた細胞はメトトレキサ ート耐性であり、従って、この培地中で生き残り、プレート密度が十分に低い場 合には独立コロニーとして生育する。安定なりローンをこの方法で拾い上げ、別 のウェルに生育させｔこ。EPO-enX plasmid was transferred to PA317A317 cells by calcium phosphate precipitation method ( Wigler, M. et al. (1979) Ce1l 16:77 ) and methotrexate resistance dihydrofolate reductase as a selectable marker. (DHPR) gene (Simonsen C, R, et al. 1981D Nuckeic Ac1ds Res, 1622235-224 6). After transfection, cells were Regarding the stable incorporation of en N' sequences in medium containing Lexar l~ It was selected by The genome/cells that have stably integrated this sequence are methotrexa is resistant to this medium and therefore survives in this medium if the plating density is low enough. In some cases, they grow as independent colonies. Pick up stable loans in this way and Grow it in a well.

安定的にトランスフェクトされたサブクローンを単離した後、ウェスタンプロッ ト及びフローサイトメトリーを実施して、組換えエンベロープタンパク質を発現 ると共にパンケージング細胞の表面に到達しているものを確保した。モノクロー ナル抗ＥＰＯ抗体及びヒオチン化二次抗体を用いて、化学蛍光主体技術を用いて ウェスタンプロットでの発現を検出しくリンスコツト化１ｎｓｃｏｔ）、　Ｗ、 　Ｄ、、　（１９９０）Ｌｉｎｓｃｏｌｊ’　ｓ　ｄｉｒｅｃｔｏｒｙ　ｏｆ　 Ｉｍｍｕｎｏｌｏｇｉｃａｌ　ａｎｄ　Ｂｉｏｌｏｇｉｃａｌ　Ｒｅａｇｅｎｔ 刀A第６版、 ミルハレイ、カリフォルニア化）、ＥＰＯ−ｅｎｖ混成タンパク質が完全な７０ ｋＤ（キロダルトン）のバンドとして幾つかのクローンにおいて検出されたが、 他では除去され又は再編成されているよってあった（図２）。親細胞株ＰＡ３１ ７を陰性対照物として用いて、ポリクローナル抗ＥＰＯ抗血清及びフルオレセイ 〉漂識化二次抗体を用いて、混成ＥＰＯ−ｅｎｖ含有細胞株の細胞表面上におけ るＥＰＯ発現をフローサイトメトリーによって検出した（図３）。After isolating stably transfected subclones, Western Express the recombinant envelope protein by performing test and flow cytometry. At the same time, we ensured that the cells reached the surface of the pancaged cells. monochrome Using a chemifluorescence-based technique using a null anti-EPO antibody and a hyotinated secondary antibody. To detect expression in Western blot, rinse 1nscott), W, D. (1990) Lincolj's directory of Immunological and Biological Reagent Katana A 6th edition, Milharei, Californian), EPO-env hybrid protein is complete 70 It was detected as a kD (kilodalton) band in some clones, but In others they have been removed or reorganized (Figure 2). Parent cell line PA31 Polyclonal anti-EPO antiserum and fluorescein 〉On the cell surface of a hybrid EPO-env-containing cell line using a drifted secondary antibody, EPO expression was detected by flow cytometry (Fig. 3).

レトロウィルスベクターのパッケージング本実施例は、ネオマイシン耐性（ｎｅ ｏ”）遺伝子及びβ−ガラクトシダーゼ（β−ｇａ　Ｉ）遺伝子を含有する複製 欠損レトロウィルスベクターをパフケージするための実施例２の細胞株の用途を 開示する。Packaging of retroviral vectors This example demonstrates neomycin resistance (ne o”) gene and the β-galactosidase (β-ga I) gene. Use of the cell line of Example 2 for puffaging defective retroviral vectors Disclose.

高レベルの組換えエンベロープタンパク質を生成する安定的なトランスフェクシ ントを、ｐｓｉパッケージングシグナルを含むが、そのｇａｇ、ｐｏｌ及びｅ、 ｎｖ遺伝子は除去されネオマイシン耐性（ｎｅｏ’）及びβ−ガラクトシダーゼ （β−ｇａ　Ｉ）をコードしている遺伝子と置換している複製欠損レトロウィル スベクターで、リン酸カルシウム沈殿法によりトランスフェクトした。更に、こ れらのベクターを対照物として親の野性型両種性パッケージング細胞株ＰＡ３１ ７−＼トランスフェクトした。これらの一過性発現ウィルスを含む細胞培養上清 を回収して、細胞性小塊を除去するために濾過して、ＥＰＯリセブターを有する 又は有しないターゲット細胞を感染するために用いた。Stable transfection that produces high levels of recombinant envelope protein components, including the psi packaging signals, such as gag, pol and e, The nv gene was removed and neomycin resistance (neo') and β-galactosidase Replication-deficient retrovirus that replaces the gene encoding (β-ga I) Transfection was performed using the vector by calcium phosphate precipitation method. Furthermore, this These vectors were used as a control in the parental wild-type amphipathic packaging cell line PA31. 7-\transfected. Cell culture supernatant containing these transiently expressed viruses is collected and filtered to remove cellular nodules and has an EPO receptor. used to infect target cells with or without.

ターゲット細胞は４つのタイプから構成される；　（Ｉ）野性型ＨｅＬａ細胞、 これは非マウスてありＥＰＯリセブターを発現していない（ドアンドレア（Ｄ’ 　Ａｎｄｒｅａ’ｉ、Ａ、ら、（１９８９）　Ｃｅ１ｌ　５７：２７７−２８５ ）、（２）安定的にトランスフェクトされているＨｅＬａ細胞てあってＥＰＯリ セブターを有するもの、（３）野性型ＮＩＨ３Ｔ３’ａ胞、これはマウス繊維芽 細胞でありＥＰＯリセブターを発現していない、（４）安定的にトランスフェク トされているＨｅＬａ細胞であってＥＰＯリセブターを有するもの。Target cells are composed of four types; (I) wild-type HeLa cells; This is a non-mouse and does not express the EPO receptor (D'Andrea (D' Andrea'i, A. et al. (1989) Ce1l 57:277-285 ), (2) stably transfected HeLa cells with EPO (3) wild-type NIH3T3'a cells, which are mouse fibroblasts; (4) stably transfected cells that do not express EPO receptors. HeLa cells that have been treated with EPO receptors.

ＨｅＬａ及びＮＩＨ３Ｔ３細胞を、メトトレキサート耐性ＤＨＰＲ遺伝子を含有 する適当な真核発現ベクター中でクローン化されたＥＰＯリセブターｃＤＮＡを 同時トランスフェクトした（シモンセン、Ｃ，Ｓ、ら、（１９８８）　Ｎｕｖｌ ｅｉｃ　Ａｃ１ｄｓ　Ｒｅｓ、　、　１６：２２３５−２２４６）。メトトレキ サートを用いた選別の後、生き残ったコロニーを単離して、ＥＰＯリセプターの 発現を放射性リガンド（”’Ｉ−ＥＰＯ）結合アッセイによって測定した。最も 高レベルのＥＰＯリセブターの発現を示すサブクローンを選別して、ＥＰＯリセ ブター〇発現を更に増やすために更にメトトレキサートの濃度を上げて含む培地 中で生育させた。これらの細胞のりセブターのレベルは、細胞当たり約１０００ リセブターの単位として計算した。感染実験の前に追加陽性対照群としてｎｅｏ ’／β−ｇａｌベクターのみ（未パッケージ）をリン酸カルシウム法によって両 方の細胞型へトランスフェクトして、Ｍ　ｏ　Ｍ　ＬＶのＬＴＲによって発現が 向けられているネオマイシン耐性遺伝子がこれらの細胞において効率よく発現さ れているかを確認した。HeLa and NIH3T3 cells containing the methotrexate resistance DHPR gene EPO receptor cDNA cloned into an appropriate eukaryotic expression vector to co-transfected (Simonsen, C.S., et al. (1988)) eic Ac1ds Res, 16:2235-2246). Methotreki After selection with SERT, surviving colonies were isolated and EPO receptor Expression was measured by radioligand (''I-EPO) binding assay. Subclones showing high levels of EPO receptor expression were selected and Medium containing increased concentrations of methotrexate to further increase pig〇 expression I grew it inside. The level of cell glue septa in these cells is approximately 1000 per cell. Calculated as units of receiver. neo as an additional positive control group before infection experiments. ’/β-gal vector alone (unpackaged) was purified by the calcium phosphate method. expression is induced by the M LV LTR. The targeted neomycin resistance gene is efficiently expressed in these cells. I checked to see if it was.

ウィルスの暴露した全て細胞をネオマイシンを用いた選別に付して、生存コロニ ー試験してギムサ染色の後に計測した。組換えＥＰＯ−ｅｎｖタンパク質を発現 しているウィルスをＥＰＯリセブターを有する細胞と接触させた場合に、誼すセ ブターを有しない細胞と比較して、感染の効率の向上が得られた。この効率の向 」二は、ＥＰＯリセブターを有しない対応する野性型の細胞と比較して、ＥＰＯ リセブターを有するＨｅＬａ細胞の場合で約１０倍（図４）、ＥＰＯリセブター を有するＮＩＨ３Ｔ３細胞の場合で３０〜４０倍であった。All cells exposed to the virus were subjected to selection using neomycin to identify viable colonies. - tested and measured after Giemsa staining. Expressing recombinant EPO-env protein If a virus containing EPO receptors is brought into contact with cells containing EPO receptors, An improved efficiency of infection was obtained compared to cells without pigs. This efficiency '2 compared to corresponding wild-type cells without EPO receptors. Approximately 10 times more EPO receptors (Figure 4) than in HeLa cells with receptors. It was 30 to 40 times higher in the case of NIH3T3 cells with .

ＤＴ適具体例を説明してきたが、種々の変更がここで開示された具体例に対して 行わ４］ｉ’Ｊること並びに、このような変更が本発明の範囲内であると意図さ れていることは当業者にとって明らかである。Although specific examples of DT suitable have been explained, various changes may be made to the specific examples disclosed here. 4] i'J and that such modifications are intended to be within the scope of this invention. It is clear to those skilled in the art that

ＥＰＯ／ｅｎｖ　（Ｎ末端５９２３部（２）ＥＰＯ／ｅｎｖ（中央ｍυ フロントページの続き （５１）　Ｉｎｔ、　Ｃ１，６識別記号　庁内整理番号Ａ６１　Ｋ　４８１００ 　８３１４−４ＣＣＯ７Ｈ２１１０４Ｂ　８６１５−４Ｃ（７２）発明者　カサ ハラ、ノリュキ アメリカ合衆国　９４１３１　カリフォルニア州　サンフランシスコ　ニー、１ ０５　ダイアモンドハイツ　プルバード　５１４０ＩEPO/env (N-terminal 5923 parts (2) EPO/env (center mυ Continuation of front page (51) Int, C1, 6 identification symbol Internal office reference number A61 K 48100 8314-4CCO7H21104B 8615-4C (72) Inventor Casa Hara, Noryuki United States of America 94131 California State San Francisco Nee, 1 05 Diamond Heights Pull Bird 5140I

Claims (1)

【特許請求の範囲】 １．哺乳類細胞の亜集団の表面における結合対の第１の構成物の存在によって特 徴付けられる亜集団に物質の送達を優先的にターゲッティングするためのビヒク ルであって、該ビヒクルが小区分及び該小区分に含まれる物質を画定する組換え ターゲティング部を含むエンベロープを含み、該ターゲティング部が互いに天然 ては結合していない第１及び第２ドメインを含み、該第１ドメインが該小区分を 画定するエンベロープを形成又はこれと結合し、該第２ドメインが該ビヒクルを 哺乳類細胞の該亜集団ヘターゲットさせる該結合対の第２の構成物又はその部分 を含む当該ビヒクル。 ２．前記ビヒクルが前記細胞へ前記物質を移送することができる請求の範囲第１ 項記載のビヒクル。 ３．前記物質が治療剤である請求の範囲第１項記載のビヒクル。 ４．前記治療剤が核酸である請求の範囲第３項記載のビヒクル。 ５．前記物質が組換えウィルスゲノムを含む請求の範囲第２項記載のビヒクル。 ６．前記エンベロープが膜を含む請求の範囲第１項記載のビヒクル。 ７．前記エンベロープがウィルスコートタンパク質を含む請求の範囲第１項記載 のビヒクル。 ８．前記ウィルスコートタンパク質がウィルスエンベロープタンパク質を含む請 求の範囲第７項記載のビヒクル。 ９．前記ターゲティンケ部の前記第１ドメインがウィルスコートタンパク質の部 分を含む請求の範囲第７項記載のビヒクル。 １０．前記ウィルスコートタンパク質がウィルスエンベロープタンパク質を含む 請求の範囲第９項記載のビヒクル。 ｌｌ．リガンドに対する特異的な表面リセプターの存在によって特徴付けられる 哺乳類細胞の亜集団に対して物質の送達を優先的にターゲッティングするための 方法てあって、該亜集団に含まれる哺乳類細胞の集団と請求の範囲第１〜１０及 び１２項のいずれか１項に記載のビヒクルとを接触することを含み、該リセプタ ー及び該リガンドが前記結合対の前記第１及び第２の構成物を含む当該方法。 １２．前記結合対がエリスロポイエチン及びエリスロポイエチンリセプターを含 む請求の範囲第１項記載のビヒクル。[Claims] 1. characterized by the presence of the first member of the binding pair on the surface of a subpopulation of mammalian cells. Vehicles for preferentially targeting the delivery of substances to subpopulations to be recruited. a recombinant vehicle, wherein the vehicle defines a subcategory and a substance contained in the subcategory. an envelope containing a targeting moiety, wherein the targeting moieties are native to each other; comprises unbound first and second domains, the first domain defining the subdivision. forming or binding to an envelope that defines the second domain, which binds to the vehicle; a second member of the binding pair or a portion thereof targeted to the subpopulation of mammalian cells; The vehicle containing. 2. Claim 1, wherein said vehicle is capable of transporting said substance to said cells. Vehicles as described in Section. 3. A vehicle according to claim 1, wherein said substance is a therapeutic agent. 4. 4. The vehicle of claim 3, wherein said therapeutic agent is a nucleic acid. 5. 3. The vehicle of claim 2, wherein said material comprises a recombinant viral genome. 6. 2. The vehicle of claim 1, wherein said envelope comprises a membrane. 7. Claim 1, wherein the envelope comprises a virus coat protein. vehicle. 8. The virus coat protein may include a virus envelope protein. A vehicle according to claim 7. 9. The first domain of the targeting part is a part of a virus coat protein. 8. A vehicle according to claim 7, comprising minutes. 10. the viral coat protein includes a viral envelope protein A vehicle according to claim 9. ll. characterized by the presence of specific surface receptors for the ligand for preferentially targeting the delivery of substances to subpopulations of mammalian cells. a population of mammalian cells included in the subpopulation and claims 1 to 10; contacting the receptor with a vehicle according to any one of paragraphs 12 and 12. - and the method wherein said ligand comprises said first and second members of said binding pair. 12. the binding pair comprises erythropoietin and an erythropoietin receptor; A vehicle according to claim 1.
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