ピコルナウイルス向性のマイクロRNAベースの規制
Summary
We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.
Abstract
Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3′ non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.
Introduction
制限された親和性を有するベクターを設計するために広く、適用可能な簡単かつ効果的な方法の開発は、安全性、生物学的理解とウイルスの治療的有用性を高めるための主要な機会を提供します。いくつかのメカニズムは、形質導入、転写、および翻訳ベースの技術を含む、ウイルス親和性を標的とするために存在します。しかし、これらの方法は、標的細胞内の欠陥シグナル伝達経路を必要とするか、またはウイルスゲノムに大規模なコード配列の挿入を必要とするかもしれない、すべてのベクター系に一般的に適用されません。さらに、これらの方法は、有意にその治療活性を阻害し、修飾されていないシステムへの洞察を制限する、ウイルスの弱毒化をもたらすことができます。
マイクロRNAは、真核細胞において遺伝子サイレンシングを媒介する非コードRNA小さな（22~25ヌクレオチド）です。メッセンジャーRNA（mRNAの）に相補的な標的配列（応答要素）を結合することによりマイクロRNAの機能resulti転写産物の不安定化、分解又は翻訳抑制でNG。マイクロRNAは、通常、部分的な相補性を有する応答エレメントに結合し、遺伝子発現を1、2、3、4、5に小さな修正をもたらします。遺伝子発現のより有意な変化は、応答エレメント6の相補性を増加させることによって達成することができます。成熟マイクロRNAの何千もの細胞や組織の種類7、8、9のさまざまな種と多数出品差分発現パターンの様々な同定されています。これらのマイクロRNAシグネチャーは、ウイルスゲノム10内に完全に相補的な応答エレメントを組み込むことによって、ウイルス増幅の細胞特異的な制限のために利用することができます= "外部参照"> 11、12、13。このマイクロRNA標的化技術の全体的な目標は、追加の減衰することなくベクターゲノムの指向性を制御することです。
ウイルスの親和性を調節するためのこの方法の有用性は、もともと特定の組織14、15、16における導入遺伝子の発現を制限するレンチウイルスベクターで実証されました。この技術は、その後、正常組織10に望ましくない毒性を排除することによって、多くの腫瘍溶解性ウイルスの安全性プロファイルを改善するために複製および非複製増強遺伝子療法のためのウイルスベクター、ならびに膨大な配列、11、12、13、17に印加されています。また、安全かつ電子を生成するために利用されていますffective生弱毒化ワクチン、ならびにウイルスおよびワクチンの製造18を処理し 、19、20、21を改善します。生産システムにおける野生型の成長レベルを維持しながら、マイクロRNA-ターゲティングベクターのは、ワクチン接種のホストまたはターゲット・システムの減衰を可能にすることができます。マイクロRNA標的はまた、他のホスト22の送信を維持しながら、一つの種（ 例えばヒト）の送信を制限することによって、研究目的のためにウイルスの生物学的安全性を向上させるために使用することができます。最後に、中・深ウイルスの増殖23、24、25、26を分離することにより、ウイルスのライフサイクルと病因と免疫における細胞型の特定の役割の分析を可能にすることができるマイクロRNAは、標的とします。
この技術は代替を提供していますすべてのウイルス系のために簡単に実装された方法および適用をターゲットernative。さらに、特定の細胞型で異なる発現パターンを有する成熟マイクロRNAの拡大を続けるコレクションは、この技術は汎用性の高いことができます。マイクロRNAに基づく標的化は、システムの機能を損なうことなく、ウイルスの様々なシステムに有効であることが証明されています。この技術の主要な制限は、試行錯誤の最適化、エスケープ変異のための潜在的な、および内因性転写産物に対する潜在的なオフターゲット効果が含まれます。しかし、これらの制限は、一般的に最適化され、合理的な応答エレメントの設計で克服することができます。プラスセンスRNAウイルスは、そのゲノムのプラスセンスの向きと完全に細胞質複製サイクル中にマイクロRNAの機械への転写物の利用可能性にマイクロRNAを標的に特に応答する傾向にあります。ここでは、マイクロRNA標的ピコルナウイルスを生成するためのプロトコルとexperimを記述します内部の方法は、in vitroでのターゲティングの効率性と特異性を検証します。
Protocol
Representative Results
Discussion
ウイルスゲノム内のマイクロRNA応答エレメントの設計、組成およびローカリゼーションは、有効性及び特異性を標的に決定するであろう。これらの最適化は、試行錯誤が必要になります。しかし、合理的な設計RNA構造解析と最小最適化10、11、12、13、38と、この技術の実?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Al and Mary Agnes McQuinn, the Richard M. Schulze Foundation, and an NIH Relief Grant from the Mayo Clinic funded representative work described here.
Materials
| RE encoding Oligonucleotides | IDT | PAGE-Purified Ultramer | Sequence Designed by Investigator |
| Oligonucleotides encoding unique restriction site | IDT | 25nM | Sequence Designed by Investigator |
| Expand High Fidelity PCR Kit | Sigma Aldrich | 11732641001 | Many other High Fidelity Polymerase PCR kits available |
| T4 DNA Ligase System | NEB | M0202S | |
| MEGAscript Kit | ThermoFisher Scientific | AM1333 | |
| MEGAclear Kit | ThermoFisher Scientific | AM1908 | |
| 0.5 M EDTA | ThermoFisher Scientific | AM9260G | RNase-free |
| 5 M NH4 Acetate | ThermoFisher Scientific | N/A | Comes in MEGAclear Kit |
| Ethanol | ThermoFisher Scientific | BP2818100 | |
| Nuclease-free Water | Fisher Scientific | AM9938 | |
| TransIT-2020 Transfection Reagent | Mirus | MIR 5404 | |
| TransIT-mRNA Transfection Reagent | Mirus | MIR 2225 | |
| 0.2 μm syringe filter | Millipore | SLGP033RS | |
| 2mL Screw-Cap Tubes | Sarstedt | 72.694.005 | |
| Cell Scrapers | Fisher Scientific | 08-100-241 | |
| MicroRNA Mimics | Dharmacon | Varied | |
| MTT Cell Proliferation Assay | ATCC | 30-1010K | |
| Subcloning Efficiency DH5α Competent Cells | ThermoFisher Scientific | 18265017 | |
| pBlueScript II Vectors | Agilent Technologies | Variable (e.g. 212205) | There are different plasmids with T7 or T3 promoters and variable cloning sites to enable cloning and RNA transcription. |
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