Cell hybrids as host cells for high efficiency production of gene therapy vectors and viral vaccines

Abstract

This disclosure provides a technology for adapting host cells to maximize production and improve quality of viral vectors and particles. Cell hybrids are formed from parental cell lines, and divided or cloned into multiple aliquots for testing. Aliquots are chosen that have high production capacity and phenotypic features for virus production, such as an optimal level of intracellular organelles, and used to establish producer cell lines. The producer cells can be genetically altered to express a transgene that encodes viral elements for production of the viral vectors or particles with a therapeutic payload. The hybrid producer cells generate more viral vectors or particles per cell with higher functional titer, thereby lowering the cost of production of pharmaceutical agents for use in gene therapy and immunization.

The invention claimed is: 
     
         1 . A process for establishing a producer cell line for high efficiency production of viral vectors or particles, the process comprising:
 (a) providing a starter population of cultured cells;   (b) forming cell hybrids from the starter population, each comprising two or more cells;   (c) dividing the cell hybrids into a plurality of aliquots;   (d) taking a sample of cells from each of the aliquots;   (e) genetically altering cells in each of the samples to express elements of a virus and a reporter gene;   (f) measuring production of viral vectors or particles containing a product of the reporter gene by each of the samples, thereby identifying which aliquots contain cell hybrids produce a higher functional titer of viral vectors or particles than cell hybrids in other aliquots;   (g) growing cells from one or more of the aliquots identified in step (f) to establish said producer cell line.   
     
     
         2 . The process of  claim 1 , wherein the cell hybrids formed in step (b) are cloned in step (c), or wherein the cells measured in step (f) are cloned during or following step (g). 
     
     
         3 . The process of  claim 1 , wherein the reporter gene encodes a protein that emits a detectable signal, such as enhanced green fluorescent protein (EGFP). 
     
     
         4 . The process of  claim 1 , wherein step (c) comprises sorting or separating cell hybrids according to cell phenotype. 
     
     
         5 . The process of  claim 4 , wherein the cells grown in step (g) have high levels of mitochondria and reactive oxygen species (ROS) per cell compared with other hybrids formed in step (b). 
     
     
         6 . A process for establishing a producer cell line for high efficiency production of viral vectors or particles, the process comprising:
 (a) providing a starter population of cultured cells;   (b) forming cell hybrids from the starter population, each comprising two or more cells;   (c) dividing the cell hybrids into a plurality of aliquots;   (d) testing the aliquots or a subpopulation thereof to identify which aliquots have higher amounts of mitochondria and/or reactive oxygen species (ROS) per cell;   (e) growing cells from one or more of the aliquots identified in step (d) to establish said producer cell line.   
     
     
         7 . The process of  claim 1 , wherein step (f) comprises identifying which aliquots contain cell hybrids produce more viral capsids per cell. 
     
     
         8 . The process of  claim 1 , wherein step (f) comprises identifying which aliquots produce a greater proportion of capsids that are filled with the reporter gene. 
     
     
         9 . The process of  claim 1 , further comprising selecting cell hybrids or cell aliquots that have a higher growth rate than other cell hybrids. 
     
     
         10 . The process of  claim 1 , wherein the starter population is selected from:
 a single cell line selected from CHO cells, mouse myeloma NSO cells, mouse myeloma SP2/0 cells, human embryonic kidney 293 (HEK 293) cells, baby hamster kidney 21 (BHK-21) cells, VERO cells, PER.C6 cells, and HeLa cells;   autotypic hybrids thereof; and   hybrids of such cells with cells from another cell line or with primary cells.   
     
     
         11 . The process of  claim 1 , further comprising genetically altering cells from the producer cell line to express a transgene encoding one or more elements of a virus, thereby adapting the cells to produce a viral vector or particle containing said elements. 
     
     
         12 . The process of  claim 10 , further comprising genetically altering said cells to express a transgene encoding a payload, thereby adapting the cells to produce a viral vector or particle encapsulating said payload. 
     
     
         13 . A producer cell line that has been adapted for high efficiency production of viral vectors or particles according to the following process:
 (a) providing a starter population of cultured cells;   (b) forming cell hybrids from the starter population, each comprising two or more cells;   (c) dividing the cell hybrids into a plurality of aliquots;   (d) taking a sample of cells from each of the aliquots;   (e) genetically altering cells in each of the samples to express elements of a virus and a reporter gene;   (f) measuring production of viral vectors or particles containing a product of the reporter gene by each of the samples, thereby identifying which aliquots contain cell hybrids produce a higher functional titer of viral vectors or particles than cell hybrids in other aliquots;   (g) growing cells from one or more of the aliquots identified in step (f) to establish said producer cell line.   
     
     
         14 . The producer cell line of  claim 12 , which has been genetically altered to express a transgene encoding one or more elements of a virus and a transgene constituting or encoding a payload, thereby adapting the cells to produce a viral vector or particle encapsulating said payload. 
     
     
         15 . A line of producer cells wherein each cell is a hybrid of two or more parental cells from a starter cell population, wherein the producer cells comprise an expressible transgene that encodes one or more elements of a virus, whereby the cells are configured to produce a viral vector or particle containing said elements. 
     
     
         16 . The producer cell line of  claim 15 , wherein the cells further comprise a transgene constituting or encoding a payload, thereby adapting the cells to produce a viral vector or particle encapsulating said payload. 
     
     
         17 . The producer cell line of  claim 14 , wherein the payload includes a nucleic acid configured for expression in a human subject in vivo upon administration to the subject. 
     
     
         18 . The producer cell line of  claim 14 , wherein the payload includes a protein configured for delivery into cells of the subject upon administration of the particle to the subject. 
     
     
         19 . The producer cell line of  claim 14 , which compared with cells from the starter cell population that have been genetically altered with the same transgenes, produces more viral vectors or particles, produces viral vectors or particles with higher functional titer, or produces viral vectors or particles with a higher proportion containing said payload. 
     
     
         20 . A method of manufacture, comprising culturing cells from the producer cell line of  claim 14  to produce viral vectors or particles encapsulating said payload. 
     
     
         21 . The method of  claim 20 , wherein the payload is a nucleic acid that encodes an antigen of SARS-CoV-2, the virus that causes COVID-19.