Functional interrogation of paramyxovirus genomes with efficient reverse genetics

通过有效的反向遗传学对副粘病毒基因组进行功能询问

• 批准号: 8973532
• 负责人: Benhur Lee
• 金额: $ 20.88万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8973532
• 关键词: Address; Adherence; Attenuated; Attenuated Live Virus Vaccine; Attenuated Vaccines; Biology; Cell Line; Cells; Clinical; Codon Nucleotides; Complementary DNA; Coupled; Custom; Development; Electroporation; Engineering; Ensure; Event; Exhibits; Generations; Genes; Genetic; Genetic Recombination; Genome; Genomics; Glycine decarboxylase; Health; Human; Insertional Mutagenesis; Libraries; Life; Mediating; Methodology; Methods; Molecular; Morbidity - disease rate; Mutagenesis; Nucleotides; Paramyxoviridae; Paramyxovirinae; Paramyxovirus; Pathogenesis; Plasmids; Polymerase; Production; Proteins; RNA Viruses; Rabies virus; Reagent; Recombinants; Reporting; Research; Research Personnel; Sendai virus; System; T7 RNA polymerase; Transcript; Transfection; Vaccinia virus; Vaccinium; Viral; Viral Genes; Viral Genome; Viral Vaccines; Virus; Virus Diseases; Work; attenuation; base; burden of illness; cytotoxic; cytotoxicity; design; gene function; genome-wide; hammerhead ribozyme; high throughput analysis; improved; mortality; paramyxovirus vaccine; pathogen; pressure; promoter; research study; reverse genetics; tissue culture; tool; vaccine candidate; vaccine development; vector

DESCRIPTION (provided by applicant): Reverse genetics or de novo generation of infectious virus entirely from cloned cDNA is an invaluable tool for rescuing recombinant live-attenuated vaccine strains for many paramyxoviruses (PVs) that cause significant morbidity and mortality, and is indispensable for basic studies on PV biology. Despite these advances, reverse genetics systems for most paramyxoviruses (PVs) have remained notoriously inefficient for the past 15 years. Rescue efficiency, when documented, is ~1 in 106-107 transfected cells. This is a limiting barrier. Reverse genetics for paramyxoviruses require an exacting combination of viral support plasmids, a T7-promoter driven viral antigenome, and an abundant supply of T7RNAP, usually provided by recombinant vaccinia expressing T7RNAP (rVV-T7RNAP), or in some cases, by cell lines stably expressing T7RNAP. Neither strategy is compatible with making recombinant live-attenuated virus vaccine candidates for human trials, which requires GMP compliant cell lines, conditions and reagents. To overcome these many issues, we have custom- designed a codon-optimized T7 polymerase (T7opt) gene that enables the rescue of rPVs entirely from cloned cDNAs using a single-step transfection methodology. This method was used to rescue representative viruses from all five major Paramyxovirinae genera. The use of T7opt makes efficient rescue of PVs possible without rVV-T7RNAP. Rescue efficiency was further enhanced, up to 3-4 logs in some cases (~1 in 103 transfected cells), with the addition of a hammerhead ribozyme (Hh-Rbz) sequence in the transcribed antigenome. We engineered the activity and placement of this Hh-Rbz to ensure adherence to the "rule of six" by pre-designed autocatalytic cleavage of the transcript-initiating 3Gs derived from the full T7 promoter. Our T7opt-HhRbz methodology is faster, safer, more efficient, and allows the possibility of easy adaptation to GMP compliant conditions. Our objective is to leverage the transformative improvement in rescue efficiencies provided by our highly efficient T7opt-HhRbz system to functionally interrogate and explore a previously inaccessible phenotypic and genetic landscape in PV biology. In so doing, we also hope to broaden the range of attenuation options and molecular tools available to the broader field of researchers working on developing live-attenuated paramyxovirus vaccines. We proposed the following Specific Aims to fulfill our stated objective: AIM 1. Explore additional strategies to further enhance the rescue efficiency and robustness of our T7opt- HhRbz system in order to ensure the broadest applicability of our reverse genetics system. Additional improvements will facilitate attenuated vaccine development, and enable higher throughput analysis of the experiments in AIM 2, which is to functionally interrogate select Paramyxovirinae genomes using insertional mutagenesis. Our strategy utilizes the rule of six to our advantage while our high efficiency T7opt- HhRbz based rescue makes this aim feasible. We will interrogate the phenotypic landscape of our insertional mutagenesis library with a set of biologically relevant selective pressures.

描述(申请人提供)：完全从克隆的cDNA反向遗传学或从头产生传染性病毒是拯救许多引起严重发病率和死亡率的副粘病毒(PV)的重组减毒活疫苗株的宝贵工具，并且对于PV生物学的基础研究是不可或缺的。尽管取得了这些进展，但在过去的15年里，大多数副粘病毒(PV)的反向遗传学系统仍然效率低下。当记录在案时，106-107转基因细胞的拯救效率为~1。这是一个限制障碍。副粘病毒的反向遗传学需要病毒支持质粒、T7启动子驱动的病毒抗基因组和充足的T7RNAP供应的严格组合，通常由表达T7RNAP的重组痘苗(rVV-T7RNAP)提供，在某些情况下，由稳定表达T7RNAP的细胞系提供。这两种策略都不适用于制造用于人体试验的重组减毒活疫苗候选疫苗，这需要符合GMP的细胞系、条件和试剂。为了克服这些问题，我们定制了一种密码子优化的T7聚合酶(T7opt)基因，它能够使用一步转染法将RPV完全从克隆的cDNA中拯救出来。这种方法被用来从所有五个主要的副粘病毒属中拯救具有代表性的病毒。T7opt的使用使得在没有rVV-T7RNAP的情况下有效地抢救静脉曲张成为可能。在转录的抗基因组中加入锤头状核酶(HH-RBZ)序列，进一步提高了拯救效率，在某些情况下可达到3-4logs(103个转基因细胞中~1个)。我们设计了这个HH-RBZ的活性和位置，以确保通过预先设计的自动催化切割来自完整的T7启动子的转录启动3GS来遵守“六规则”。我们的T7opt-HhRbz方法更快、更安全、更高效，并允许轻松适应符合GMP的条件。我们的目标是利用我们的高效T7opt-HhRbz系统在救援效率方面的革命性改进，从功能上询问和探索光伏生物学中以前无法进入的表型和遗传景观。通过这样做，我们还希望为致力于开发减毒副粘病毒活疫苗的更广泛领域的研究人员提供更广泛的减毒选择和分子工具。为了实现我们的既定目标，我们提出了以下具体目标：目标1.探索其他策略，以进一步提高我们的T7opt-HhRbz系统的救援效率和健壮性，以确保我们的反向遗传学系统的最广泛适用性。额外的改进将促进减毒疫苗的开发，并使AIM 2中的实验能够进行更高的吞吐量分析，该实验将使用插入突变从功能上询问选定的副粘病毒基因组。我们的战略利用六规则对我们有利，而我们基于T7opt-HhRbz的高效率救援使这一目标变得可行。我们将用一组生物学上相关的选择压力来询问我们的插入突变文库的表型图景。

项目成果

Crystal structure and solution state of the C-terminal head region of the narmovirus receptor binding protein.

• DOI: 10.1128/mbio.01391-23
• 发表时间: 2023-10-31
• 期刊: mBio
• 影响因子: 6.4

Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing.

• DOI: 10.1038/mtm.2016.57
• 发表时间: 2016
• 期刊: Molecular therapy. Methods & clinical development
• 作者: Park A;Hong P;Won ST;Thibault PA;Vigant F;Oguntuyo KY;Taft JD;Lee B
• 通讯作者: Lee B

Fitness selection of hyperfusogenic measles virus F proteins associated with neuropathogenic phenotypes

• DOI: 10.1073/pnas.2026027118
• 发表时间: 2021-05-04
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Ikegame, Satoshi;Hashiguchi, Takao;Lee, Benhur
• 通讯作者: Lee, Benhur