A team of Portuguese scientists has reduced the size of helper plasmids for recombinant adeno-associated viral (rAAV) production.
The team, from Instituto de Biologia Experimental e Tecnológica (iBET), says the research could simplify rAAV manufacturing by improving plasmid DNA production, reducing costs, and the amounts of adenoviral genes expressed in cells.
“Since adenoviral genes are toxic when constitutively expressed in cells, having fewer genes to integrate and regulate simplifies the cell line development process,” says Sofia Fernandes, PhD, a senior scientist in the animal cell technology unit at iBET.
According to Fernandes, understanding which helper components are necessary for rAAV production could allow for stable cell lines that don’t require helper components. It also allows for smaller plasmids that are easier to produce and transfect, she says.
The team tested the impact on eight novel helper plasmids with different deletions in the E2 and E4 genes on four rAAV serotypes. They discovered that some deletions affected productivity, reducing viral genome packaging by ten times in some cases. However, by reincorporating a set of regions into the genes, they could improve productivity again.
“One of the best combinations we found involved a smaller helper plasmid than the ones we [typically] use [in-house], that restored the levels of rAAV productivity,” Fernandes explains.
She goes on to explain that other combinations of deletions produce reasonable quality and viral titers and could be helpful if components of a typical plasmid caused toxicity.
“A smaller plasmid is generally easier and more cost-effective to produce,” she says. “And, moreover, having fewer toxic genes expressed in the helper plasmid can improve viability and potentially enhance rAAV production.”
She adds, “Additionally, some of these genes may unintentionally integrate into the rAAV gene, contaminating the final product.”
According to Fernandes, the team has now moved on to comparing two of the smaller helper plasmids as they scale up production from milliliters to 20 L volumes.
“[Our plan is to] develop a stable cell line for producing rAAVs using this knowledge,” she says.
The team also investigated how rAAV promoter activity varied in the absence of proteins that regulate AAV transcription, as well as helper factors, in two different cell lines. They are in the process of writing up this research, she says.
The team’s research on adenoviral elements is due to be published in Human Gene Therapy. Fernandes presented on both projects at the recent Bioprocessing Summit Europe.
The post Researchers Test Minimal Helper Plasmids for AAV Production appeared first on GEN - Genetic Engineering and Biotechnology News.
The team, from Instituto de Biologia Experimental e Tecnológica (iBET), says the research could simplify rAAV manufacturing by improving plasmid DNA production, reducing costs, and the amounts of adenoviral genes expressed in cells.
“Since adenoviral genes are toxic when constitutively expressed in cells, having fewer genes to integrate and regulate simplifies the cell line development process,” says Sofia Fernandes, PhD, a senior scientist in the animal cell technology unit at iBET.
According to Fernandes, understanding which helper components are necessary for rAAV production could allow for stable cell lines that don’t require helper components. It also allows for smaller plasmids that are easier to produce and transfect, she says.
The team tested the impact on eight novel helper plasmids with different deletions in the E2 and E4 genes on four rAAV serotypes. They discovered that some deletions affected productivity, reducing viral genome packaging by ten times in some cases. However, by reincorporating a set of regions into the genes, they could improve productivity again.
“One of the best combinations we found involved a smaller helper plasmid than the ones we [typically] use [in-house], that restored the levels of rAAV productivity,” Fernandes explains.
She goes on to explain that other combinations of deletions produce reasonable quality and viral titers and could be helpful if components of a typical plasmid caused toxicity.
“A smaller plasmid is generally easier and more cost-effective to produce,” she says. “And, moreover, having fewer toxic genes expressed in the helper plasmid can improve viability and potentially enhance rAAV production.”
She adds, “Additionally, some of these genes may unintentionally integrate into the rAAV gene, contaminating the final product.”
According to Fernandes, the team has now moved on to comparing two of the smaller helper plasmids as they scale up production from milliliters to 20 L volumes.
“[Our plan is to] develop a stable cell line for producing rAAVs using this knowledge,” she says.
The team also investigated how rAAV promoter activity varied in the absence of proteins that regulate AAV transcription, as well as helper factors, in two different cell lines. They are in the process of writing up this research, she says.
The team’s research on adenoviral elements is due to be published in Human Gene Therapy. Fernandes presented on both projects at the recent Bioprocessing Summit Europe.
The post Researchers Test Minimal Helper Plasmids for AAV Production appeared first on GEN - Genetic Engineering and Biotechnology News.