Production of viruses in cell culture
Inventors
Bean, Andrew • Lowenthal, John William • Malaver-Ortega, Luis Fernando • Tripp, Ralph A.
Assignees
Commonwealth Scientific and Industrial Research Organization CSIRO • University of Georgia Research Foundation Inc
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12054750-B2
Publication Date
2024-08-06
Expiration Date
Abstract
The present invention relates to methods of replicating viruses in vitro. In particular, the invention relates to a genetically modified population of cells, and/or a population of cells treated with an exogenous compound, wherein the cells are capable of producing more virus than cells lacking the genetic modification and/or lacking treatment with the exogenous compound. The invention also relates to methods of producing populations of such cells, as well as the use of the viruses obtained to prepare vaccine compositions.
Core Innovation
The invention provides in vitro virus replication by using a population of cells in which antiviral activity is reduced. A genetic modification is introduced into the genome of the cells by a programmable nuclease that knocks-out the expression of interferon alpha/beta receptor 1 (IFNAR1) when compared to isogenic cells lacking the genetic modification. Cells are inoculated in vitro with a Paramyxoviridae virus and cultured for a predetermined period of time to replicate the virus.
The core concept centers on reducing interferon signaling through IFNAR1 knock-out to increase virus replication relative to isogenic controls. The programmable nuclease includes RNA-guided engineered nucleases and other programmable nuclease classes such as TALEN and ZFN. The approach is extended by reducing expression of one or more selected antiviral genes from a specified list, beyond IFNAR1.
The invention further includes generating and using an in vitro population of cells comprising a Paramyxoviridae virus together with the IFNAR1 gene knock-out. A production workflow is described in which cells are provided with the genetic modification, screened for increased virus production relative to isogenic cells lacking the genetic modification, and higher-producing cells are selected, with optional clonal expansion. The document also links the replication process to vaccine production by harvesting replicated virus or virus particles and preparing a vaccine composition, including optional conversion into split or subunit virus particles and optional inactivation steps.
Claims Coverage
The partial content identifies two independent claims. Both share a common inventive concept of increasing replication of a Paramyxoviridae virus in vitro by using a programmed-nuclease-mediated knock-out of IFNAR1 expression in cells, relative to isogenic cells lacking the genetic modification.
Programmable nuclease-mediated IFNAR1 knock-out for in vitro replication of Paramyxoviridae
Obtaining a population of cells having a genetic modification in the genome introduced by a programmable nuclease that knocks-out the expression of interferon alpha/beta receptor 1 (IFNAR1) in the cells when compared to isogenic cells lacking the genetic modification; inoculating the cells in vitro with the virus; and culturing the cells for a predetermined period of time to replicate the virus.
In vitro cell population comprising Paramyxoviridae virus with IFNAR1 knock-out
An in vitro population of cells comprising a Paramyxoviridae virus and a genetic modification in the genome of the cells introduced by a programmable nuclease that knocks-out the expression of interferon alpha/beta receptor 1 (IFNAR1) gene in the cells.
Overall, the claims coverage centers on using programmable-nuclease genome modifications that knock-out IFNAR1 expression to reduce interferon alpha/beta receptor activity in cells, thereby enabling higher in vitro replication of a Paramyxoviridae virus compared with isogenic unmodified cells.
Stated Advantages
Increases virus replication relative to isogenic controls lacking the genetic modification.
Allows in vitro replication suitable for vaccine production workflows by enabling harvesting replicated virus or particles for preparing a vaccine composition.
Documented Applications
Vaccine production workflows using harvested replicated virus or particles, with preparation of a vaccine composition, including optional inactivation and optional disruption into split or subunit virus particles.
Interested in licensing this patent?