アデノ随伴ウイルス

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アデノ随伴ウイルス(: Adeno-associated virus:AAV)とはヒトや霊長目の動物に感染する小型(20nm程度)の、パルボウィルス科ティンペンドウィルス属に分類されるヘルパー依存型のエンベロープを持たないウイルス。非常に弱い免疫反応しか引き起こさないず、病原性は現在の所確認されていない。分裂期にある細胞にもそうでない細胞にもゲノムを送り込むことができ、宿主細胞にゲノムを送り込まずとも染色体外で生存することができる [1]。そのような特色があるためにベクターウイルスを用いた遺伝子治療やヒトの疾患モデル細胞の作成などに用いられる[2]

遺伝子治療ベクター[編集]

利点と欠点[編集]

野生型のAAVは、病原性の欠落を主とする様々な特徴のために、遺伝子治療の研究者から多くの興味を集めてきた。このウィルスは、分裂していない細胞へと感染し、宿主細胞の19番染色体上のAAVS1と呼ばれる領域に挿入される。[3][4]この特徴は、ランダムな遺伝子挿入とそれによる突然変異や細胞の癌化を引き起こすレトロウイルスに比べて優れたものである。AAVの遺伝子の挿入はAAAVS1において発生するものがほとんどであり、ランダムな挿入は無視できる割合でしか発生しない。しかし、ウィルスベクターとしてのAAVの研究が進むにつれて、rep遺伝子とcap遺伝子を取り除くことで、この遺伝子の挿入能力も取り除かれている。 遺伝子治療のために作られた遺伝子配列は、プライマーの働きをする末端逆位配列(ITR)の間に挿入される。ウィルスによって作られた一本鎖DNAは、宿主細胞のDNAポリメラーゼによって二本鎖DNA になり、コンカテマーを形成する。分裂していない細胞においては、コンカテマーはそのままの状態で保存される。一方で分裂する細胞においては、エピソームDNAは宿主細胞によって複製されてないのでAAVのDNAは失われる。また、AAVは免疫原性をほとんど持っていないために抗体があまり産生されい。そのために、抗体依存性細胞傷害が明確には見られない[5][6][7] 。 一方でこのウィルスベクターを持ちることによるデメリットも存在する。AAVに搭載できる遺伝子配列は比較的限られており、多くの場合この遺伝子の4.8 kbpの配列を完全に置き換える必要がある。したがって大きな遺伝子はAAVベクターの使用には適していない。また、ウィルスの感染による液性免疫系の活性が発生することもデメリットとなる。免疫系によりAAVが無力化されるため、いくらかの用途ではAAVのメリットが制約される。そのため、多くの臨床実験では、免疫系の活性が弱い脳で行われている。 以上のように、AAVは、ウィルスベクターとして優れた特徴を持つために変種もつくられている。例えば自己相補型AAV(scAAV)は、二本鎖DNAを最初から形成するAAVの変種であり、通常のAAVと違い二本鎖DNAを形成する時間がかからないために効率に優れている。しかしながら、scAAVは二本鎖DNAを形成するため二本分の遺伝子配列を搭載するために通常のAAVにくらべて搭載できる遺伝子の大きさが半減する[8] 。またscAAVは通常のAAVより高い免疫原性を保有しており細胞傷害性T細胞をより強く活性化させてしまう[9]


臨床試験[編集]

現在までに、AAVは117の臨床試験に用いられている(遺伝子治療全体の5.6%)。[10] レーバー先天性黒内障,[11][12][13]血友病,[14]うっ血性心不全,[15]パーキンソン病.[16]などの疾患においてPhase1あるいは Phaseでの臨床試験における有望な結果が報告されている。

Selected Clinical Trials using AAV-Based Vectors[17]
症候 遺伝子 投与法 フェイズ 被験者数 進行状況
奉納性線維症 CFTR エアロゾル吸入 I 12 完了
CFTR エアロゾル吸入 II 38 完了
CFTR エアロゾル吸入 II 100 完了
血友病 B FIX 筋肉内投与 I 9 完了
FIX 肝動脈内投与 I 6 終了
関節炎 TNFR:Fc Intraarticular I 1 進行中
遺伝性肺気腫 AAT 筋肉内投与 I 12 進行中
レーバー先天性黒内障 網膜下l I-II Multiple Several 進行中 and 完了
加齢黄斑変性 sFlt-1 網膜下l I-II 24 進行中
筋ジストロフィー Sarcoglycan 筋肉内投与 I 10 進行中
パーキンソン病 GAD65, GAD67 頭蓋内 I 12 完了[18]
カナバン病 AAC 頭蓋内 I 21 進行中
バッテン病 CLN2 頭蓋内 I 10 進行中
アルツハイマー病 NGF 頭蓋内 I 6 進行中
うっ血性心不全 SERCA2a Intra-coronary IIb 250 進行中

前立腺がんにたいする臨床試験はphase IIIに達している[17]が、ex vivoでの研究はAAVのりようには含めていない。

病理学[編集]

AAVはいかなる疾患も引き起こさないと考えれている。しかし、AAVのDNAが変異した精子を含む精液においてよくみられることから、男性不妊症との関与が示唆されている。しかしAAVの感染と男性不妊症との因果関係今のところ見つかっていない[19]

ウィルスの構造[編集]

ゲノムの構造[編集]

AAVのゲノムは、4.7 kbpの一本鎖DNS(ssDNA)から成る。DNA鎖の末端には末端逆位配列(ITR)をもち、rep遺伝子とcap遺伝子の2つのオープンリーディングフレーム(ORF)を持つ。rep遺伝子は、AAVの生存に必要なRepタンパクをコードしており、cap遺伝子は、VP1,VP2,VP3という3つのカプシドタンパクをコードしている。cap遺伝子がコードするカプシドタンパクにより正二十面体のカプシドを形成する。[20]

ITR配列[編集]

ITRは両末端に存在する145 bpの配列である。ITRはAAVの増殖に必要とされる。[21] ITRはヘアピン構造をとり、この領域が複製開始点となるために、DNAプライメラーゼ無しでDNA合成を行なう事ができる(セルフプライミング)。また、ITRは野生型AAVにおける宿主細胞への遺伝子挿入や、[22][23] DNAase耐性を持ったカプシドの形成にも関与している[24]

遺伝子治療に用いる範囲では、ITR配列唯一のシスエレメントであり、capとrepがトランスエレメントであるように見える。しかし、ITRはウィルスの複製及びカプシド形成の過程での唯一のシスエレメントでない可能性が示唆されている。一部の研究においては、rep遺伝子の配列の中にRep依存型シスエレメント(CARE)が存在することが示さておりCAREはシスエレメントとして増殖とカプシド形成を調整するとされる。 [25][26][27][28]

rep配列[編集]

ゲノムの左末端にはp5及びp19プロモーターと、イントロンを含むmRNA領域が存在する。したがってスプライシングの結果分子量の異なるRep78,Rep68,Rep52,Rep40の4つの転写産物が合成される。 [29] Rep78とRep68はITRのヘアピン構造に結合する。またRepタンパクはすべてATPと結びついてヘリカーゼ活性を発揮する。またp40プロモーターによって転写がアップレギュレーションされ、p5,p19プロモーターによって転写がダウンレギュレーションされる [23][29][30][31][32][33]

cap配列[編集]

The right side of a positive-sensed AAV genome encodes overlapping sequences of three capsid proteins, VP1, VP2 and VP3, which start from one promoter, designated p40. The molecular weights of these proteins are 87, 72 and 62 kiloDaltons, respectively.[34] All three of them are translated from one mRNA. After this mRNA is synthesized, it can be spliced in two different manners: either a longer or shorter intron can be excised resulting in the formation of two pools of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool. Usually, especially in the presence of adenovirus, the longer intron is preferred, so the 2.3-kb-long mRNA represents the so-called "major splice". In this form the first AUG codon, from which the synthesis of VP1 protein starts, is cut out, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon, which remains in the major splice, is the initiation codon for VP3 protein. However, upstream of that codon in the same open reading frame lies an ACG sequence (encoding threonine) which is surrounded by an optimal Kozak context. This contributes to a low level of synthesis of VP2 protein, which is actually VP3 protein with additional N terminal residues, as is VP1.[35][36][37][38]

Since the bigger intron is preferred to be spliced out, and since in the major splice the ACG codon is a much weaker translation initiation signal, the ratio at which the AAV structural proteins are synthesized in vivo is about 1:1:20, which is the same as in the mature virus particle.[39] The unique fragment at the N terminus of VP1 protein was shown to possess the phospholipase A2 (PLA2) activity, which is probably required for the releasing of AAV particles from late endosomes.[40] Muralidhar et al. reported that VP2 and VP3 are crucial for correct virion assembly.[37] More recently, however, Warrington et al. showed VP2 to be unnecessary for the complete virus particle formation and an efficient infectivity, and also presented that VP2 can tolerate large insertions in its N terminus, while VP1 can not, probably because of the PLA2 domain presence.[41]

The crystal structure of the VP3 protein was determined by Xie, Bue, et al..[42]

AAVの血清型[編集]

As of 2006 there have been 11 AAV serotypes described, the 11th in 2004.[43] All of the known serotypes can infect cells from multiple diverse tissue types. Tissue specificity is determined by the capsid serotype and pseudotyping of AAV vectors to alter their tropism range will likely be important to their use in therapy.

AAV2[編集]

Serotype 2 (AAV2) has been the most extensively examined so far.[44][45][46][47][48][49] AAV2 presents natural tropism towards e.g. skeletal muscles,[50] neurons,[44] vascular smooth muscle cells[51] and hepatocytes.[52]

Three cell receptors have been described for AAV2: heparan sulfate proteoglican (HSPG), aVβ5 integrin and fibroblast growth factor receptor 1 (FGFR-1). The first functions as a primary receptor, while the latter two have a co-receptor activity and enable AAV to enter the cell by receptor-mediated endocytosis.[53][54][55]) These study results have been disputed by Qiu, Handa, et al..[56] HSPG functions as the primary receptor, though its abundance in the extracellular matrix can scavenge AAV particles and impair the infection efficiency.[57]

AAV-2とガン[編集]

Studies have shown that serotype 2 of the virus (AAV-2) apparently kills cancer cells without harming healthy ones. "Our results suggest that adeno-associated virus type 2, which infects the majority of the population but has no known ill effects, kills multiple types of cancer cells yet has no effect on healthy cells," said Craig Meyers,[58] a professor of immunology and microbiology at the Penn State College of Medicine in Pennsylvania.[59] This could lead to a new anti-cancer agent.

他の血清型[編集]

Although AAV2 is the most popular serotype in various AAV-based research, it has been shown that other serotypes can be more effective as gene delivery vectors. For instance AAV6 appears much better in infecting airway epithelial cells, AAV7 presents very high transduction rate of murine skeletal muscle cells (similarly to AAV1 and AAV5), AAV8 is superb in transducing hepatocytes[60][61][62] and AAV1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells.[63] AAV6, a hybrid of AAV1 and AAV2,[62] also shows lower immunogenicity than AAV2.[61]

Serotypes can differ with the respect to the receptors they are bound to. For example AAV4 and AAV5 transduction can be inhibited by soluble sialic acids (of different form for each of these serotypes),[64] and AAV5 was shown to enter cells via the platelet-derived growth factor receptor.[65]

免疫学[編集]

AAV is of particular interest to gene therapists due to its apparent limited capacity to induce immune responses in humans, a factor which should positively influence vector transduction efficiency while reducing the risk of any immune-associated pathology.

生得免疫[編集]

The innate immune response to the AAV vectors has been characterised in animal models. Intravenous administration in mice causes transient production of pro-inflammatory cytokines and some infiltration of neutrophils and other leukocytes into the liver, which seems to sequester a large percentage of the injected viral particles. Both soluble factor levels and cell infiltration appear to return to baseline within six hours. By contrast, more aggressive viruses produce innate responses lasting 24 hours or longer.[66]

液性免疫[編集]

The virus is known to instigate robust humoral immunity in animal models and in the human population where up to 80% of individuals are thought to be seropositive for AAV2. Antibodies are known to be neutralising and for gene therapy applications these do impact on vector transduction efficiency via some routes of administration. As well as persistent AAV specific antibody levels, it appears from both prime-boost studies in animals and from clinical trials that the B-cell memory is also strong.[67] In seropositive humans, circulating IgG antibodies for AAV2 appear to be primarily composed of the IgG1 and IgG2 subclasses, with little or no IgG3 or IgG4 present.[68]

細胞免疫[編集]

The cell-mediated response to the virus and to vectors is poorly characterised and has been largely ignored in the literature as recently as 2005.[67] Clinical trials using an AAV2-based vector to treat haemophilia B seem to indicate that targeted destruction of transduced cells may be occurring.[69] Combined with data that shows that CD8+ T-cells can recognise elements of the AAV capsid in vitro[70], it appears that there may be a cytotoxic T lymphocyte response to AAV vectors. Cytotoxic responses would imply the involvement of CD4+ T helper cells in the response to AAV and in vitro data from human studies suggests that the virus may indeed induce such responses including both Th1 and Th2 memory responses.[68] A number of candidate T cell stimulating epitopes have been identified within the AAV capsid protein VP1, which may be attractive targets for modification of the capsid if the virus is to be used as a vector for gene therapy.[69][68]

AAV infection cycle[編集]

There are several steps in the AAV infection cycle, from infecting a cell to producing new infectious particles:

  1. attachment to the cell membrane
  2. endocytosis
  3. endosomal trafficking
  4. escape from the late endosome or lysosome
  5. translocation to the nucleus
  6. formation of double-stranded DNA replicative form of the AAV genome
  7. rep genes expression
  8. genome replication
  9. cap genes expression, synthesis of progeny ssDNA particles
  10. assembly of complete virions, and
  11. release from the infected cell.

Some of these steps may look different in various types of cells, which, in part, contributes to the defined and quite limited native tropism of AAV. Replication of the virus can also vary in one cell type, depending on the cell's current cell cycle phase.[71]

The characteristic feature of the adeno-associated virus is a deficiency in replication and thus its inability to multiply in unaffected cells. The first factor that was described as providing successful generation of new AAV particles, was the adenovirus, from which the AAV name originated. It was then shown that AAV replication can be facilitated by selected proteins derived from the adenovirus genome,[72][73] by other viruses such as HSV,[74] or by genotoxic agents, such as UV irradiation or hydroxyurea.[75][76][77]

The minimal set of the adenoviral genes required for efficient generation of progeny AAV particles, was discovered by Matsushita, Ellinger et al..[72] This discovery allowed for new production methods of recombinant AAV, which do not require adenoviral co-infection of the AAV-producing cells. In the absence of helper virus or genotoxic factors, AAV DNA can either integrate into the host genome or persist in episomal form. In the former case integration is mediated by Rep78 and Rep68 proteins and requires the presence of ITRs flanking the region being integrated. In mice, the AAV genome has been observed persisting for long periods of time in quiescent tissues, such as skeletal muscles, in episomal form (a circular head-to-tail conformation).[78]

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