Identification of host cell components essential for the SARS-CoV-2 life cycle
Lead Research Organisation:
University of Cambridge
Department Name: Medicine
Abstract
All viruses are dependent on host cell machinery for aspects of their lifecycle. We plan to identify non-essential host (human) cell components that are required for the life cycle of the SARS-CoV-2 virus. If we are able to identify them, these will provide critical intervention points for developing drugs that could prevent virus infection or hamper/stop viral replication.
We will used a genetic approach, mutating each of the 20,000 genes in the genome and testing whether the virus is able to infect the cell, replicate and produce new infectious particles. The results will provide insights into molecular host-pathogen interactions at various steps of the virus life cycle including entrance, replication, biosynthesis, and release.
Experimentally, we will try to identify mutant cells which do not support aspects of the viral life cycle. We will then seek to identify the underlying mutant gene(s) which are able provide this protection. Technically this will be achieved by killing/removing cells which have been successfully infected, allowing the "resistant" ones to survive.
The initial stage of the project will be to establish sensitive and specific assays in human cell lines which can be successfully infected with SARS-CoV-2. Genetic screens would then be conducted with CRISPR libraries that target all the gene in the genome as well as a library specialised on "drug targetable" host genes. If hits are identified in a "druggable" library, the results could be rapidly translated. For instance by taking the corresponding drug and testing this on cells in culture.
Hits identified by genome wide screens will take longer to translate, depending on the gene that is identified and prior work on the gene.
The reporter cell lines we are developing to support this project as well as any targets we identify should support collaborative efforts with other laboratories and industrial partners, to translate these findings into host-directed therapies.
We will used a genetic approach, mutating each of the 20,000 genes in the genome and testing whether the virus is able to infect the cell, replicate and produce new infectious particles. The results will provide insights into molecular host-pathogen interactions at various steps of the virus life cycle including entrance, replication, biosynthesis, and release.
Experimentally, we will try to identify mutant cells which do not support aspects of the viral life cycle. We will then seek to identify the underlying mutant gene(s) which are able provide this protection. Technically this will be achieved by killing/removing cells which have been successfully infected, allowing the "resistant" ones to survive.
The initial stage of the project will be to establish sensitive and specific assays in human cell lines which can be successfully infected with SARS-CoV-2. Genetic screens would then be conducted with CRISPR libraries that target all the gene in the genome as well as a library specialised on "drug targetable" host genes. If hits are identified in a "druggable" library, the results could be rapidly translated. For instance by taking the corresponding drug and testing this on cells in culture.
Hits identified by genome wide screens will take longer to translate, depending on the gene that is identified and prior work on the gene.
The reporter cell lines we are developing to support this project as well as any targets we identify should support collaborative efforts with other laboratories and industrial partners, to translate these findings into host-directed therapies.
Organisations
People |
ORCID iD |
| Allan Bradley (Principal Investigator) | |
| Kart Tomberg (Co-Investigator) |
Publications
| Description | To date, the findings from the award are mostly technical, but are stated here in view of the intense public interest. To conduct genetic screens with SARS-Cov2, it is necessary to build a cell-based system consisting of a cell line expressing Cas9, ACE2 and TMRPSS2. A key technical requirement is that the cell needs to highly infectible, ideally close to 100%, with a lentivirus in which the envelope has been changed to become SARS-Cov-2 spike protein (pseudo-typed). We have now established these conditions which has required serial sub-cloning of cells expressing the various transgenes and assessment of the resultant clones. One key finding has been our investigation of methods to express SARS-Cov-2 S protein to achieve efficient pseudo-typing. We have found remarkable differences between various codon-optimised versions of the S-protein from many sources. While they all express S-protein to some degree, the levels of expression are generally poor compared to other viruses and consequently the titres achieved comparatively low. The manner in which this has been solved by many in the field has been to increase ACE2 expression to higher levels which facilitates virus binding and entry. However, the problem encountered in pseudo-typing tells us that S-protein expression is far from optimal. We have therefore been working on trying to understand what is limiting and exploring approaches to optimise this. We have recently completed the screen and are validating host target genes. |
| Exploitation Route | The genes identified bt our screens will be of commercial interest if they can be druged We are exploring ways to improve S-protein expression, if we are successful this may be useful for S-protein use in some vaccine formats. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | We successfully raised funding for a startup company based on results generated with this award. This company is called Expression Edits limited and employs three members of staff from the laboratory two of whom were supported by the grant. |
| First Year Of Impact | 2022 |
| Sector | Pharmaceuticals and Medical Biotechnology |
| Impact Types | Economic |
| Company Name | EXPRESSIONEDITS LTD |
| Description | The company intends to commercialise the discovery of the importance of introns in transgenes. |
| Year Established | 2021 |
| Impact | The company started operations mid 2022. It has established a small team of wet-lab (3) and bioinformatics personnel (3) who are building a pipeline that is designed to improve gene expression in mammalian cells. |
| Website | https://www.expressionedits.com |