Lu Guanda's team's new article: Synthetic gene control system for precise control of mammalian cells

Using a CRISPR protein-based approach, MIT researchers have developed a new method that can precisely control the amount of
a particular protein produced in mammalian cells.

This technology can be used to fine-tune the production of useful proteins, such as monoclonal antibodies used to treat cancer and other diseases, or for other aspects of
cell behavior.
In their new study, published in Nature Communications, the researchers show that the system can work in a wide variety of mammalian cells, with very consistent
results.

"It's a highly predictable system that we can pre-design and then get the expected results," said
William C.
W.
Chen, a former MIT research scientist.
"It's a very adjustable system for many different biomedical applications
in different cell types.
"

Other authors include former MIT research scientist Leonid Gaidukov and postdoc Yong Lai
.
Senior author Timothy Lu, an associate professor
of bioengineering, electrical engineering and computer science at MIT, led the study.

Gene control

Many therapeutic proteins, including monoclonal antibodies, are produced in large bioreactors containing mammalian cells engineered to produce the desired protein
.
A few years ago, researchers at MIT's Synthetic Biology Center, including Lu's lab, began working with Pfizer Inc.
Collaborate on the development of synthetic biology tools to facilitate the production of
these useful proteins.

To do this, the researchers targeted the promoter
of the gene they wanted to upregulate.
In all mammalian cells, genes have a promoter region that binds to transcription factors, which are proteins
that initiate the transcription of genes into messenger RNA.

In previous work, scientists designed synthetic transcription factors, including a protein called zinc fingers, to help activate the target gene
.
However, zinc fingers and most other types of synthetic transcription factors must be redesigned for each gene they target, making their development challenging and time-consuming
.

In 2013, researchers in Lu's lab developed a CRISPR-based transcription factor that allowed them to more easily control the transcription
of naturally occurring genes in mammalian and yeast cells.
In the new study, the researchers set out to build on this work to create a library of synthetic biological parts, allowing them to deliver a transgenic gene — a gene that is not normally expressed by cells — and precisely control its expression
.

"The idea is to have a full-spectrum synthetic promoter system that can be from very low to very high to accommodate different cellular applications," Chen said
.

The system designed by the researchers consists of several parts
.
One is the gene to be transcribed, and there is an "operator" sequence consisting of
a series of artificial transcription factor binding sites.
Another component is the guide RNA, which binds
to these operator sequences.
Finally, the system also includes a transcriptional activation domain attached to the
inactivated Cas9 protein.
When this inactivated Cas9 protein binds to the guide RNA that synthesizes the promoter site, the CRISPR-based transcription factor initiates gene expression
.

The promoter sites used for this synthesis system are designed to be different from naturally occurring promoter sites, so the system does not affect genes
in the cell's own genome.
Each operator contains between 2 and 16 copies of the leading RNA binding sites, and the researchers found that their system could initiate gene transcription at a rate that linearly corresponded to the number of binding sites, allowing them to precisely control the amount
of protein produced.

High consistency

The researchers tested their system in several types of mammalian cells, including Chinese hamster ovary (CHO) cells, which are commonly used to produce therapeutic proteins
in industrial bioreactors.
They found very similar results in CHO cells and other cells they tested, including myoblasts (precursors of muscle cells) from mice and rats, human embryonic kidney cells, and human induced pluripotent stem cells
.

"The system has very high consistency across different cell types and different target genes," Chen said
.
"This is a good starting point for thinking about how to modulate gene expression and cell behavior
with a highly tunable, predictable artificial system.
"

After demonstrating for the first time that they could use the new system to induce cells to produce the expected amount of fluorescent protein, the researchers said they could also program the system to produce the two main fragments
of a monoclonal antibody called JUG444.

The researchers also programmed CHO cells to produce different amounts of human antibodies
called anti-PD1.
When human T cells come into contact with these cells, they become stronger tumor cell killers if they produce large amounts of antibodies
.

Although the researchers were able to obtain high yields of the antibodies they needed, further work was needed to incorporate the system into industrial processes
, they said.
Unlike the cells used in industrial bioreactors, the cells used in this study were grown on a flat surface, not in
a liquid suspension.

"It's a system that promises to be used in industrial applications, but first we have to apply it to suspension cells to see if they make proteins
in the same way.
" I think it should be the same because there's no reason it shouldn't, but we still need to test it," Chen said
.

William C.
W.
Chen, Leonid Gaidukov, Yong Lai, Ming-Ru Wu, Jicong Cao, Michael J.
Gutbrod, Gigi C.
G.
Choi, Rachel P.
Utomo, Ying-Chou Chen, Liliana Wroblewska, Manolis Kellis, Lin Zhang, Ron Weiss , Timothy K.
Lu.
A synthetic transcription platform for programmable gene expression in mammalian cells.
Nature Communications, 2022; 13 (1)