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Researchers have developed a technique that can help fine-tune the production of
monoclonal antibodies and other useful proteins.
Using CRISPR-based proteins, MIT researchers have developed a new method
to precisely control the amount of specific proteins produced in mammalian cells.
This technology can be used to precisely regulate the production of useful proteins, including monoclonal antibodies
for the treatment of cancer and other diseases.
It can also precisely calibrate other aspects of
cell behavior.
In their new study, the researchers showed that the system can work in a variety of mammalian cells, with very consistent
results.
A paper describing this result was recently published in the journal Nature Communications
.
"It's a highly predictable system that we can design up front and then get the desired results," said
William C.
W.
Chen, a former MIT research scientist.
"It's a very tunable system for many different biomedical applications
with different cell types.
"
Chen is one of the lead authors of the new study, along with former MIT research scientist Leonid Gaidukov and postdoc Yong Lai
.
The study was led by Timothy Lu, an associate professor
of bioengineering, electrical engineering, and computer science at MIT.
Many therapeutic proteins, including monoclonal antibodies, are produced in large bioreactors containing engineered mammalian cells 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 promoters of the genes they wanted to upregulate
.
In all mammalian cells, genes have a promoter region that binds to transcription factors, which are proteins
that initiate genes to transcribe into messenger RNA.
In previous work, scientists engineered synthetic transcription factors, including a protein called zinc finger, to help activate target genes
.
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 a library of synthetic biological parts based on this work, allowing them to pass on 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 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 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 at the synthetic promoter site, CRISPR-based transcription factors can initiate gene expression
.
The promoter sites used for this synthetic 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 copies of 2 to 16 guide RNA binding sites, and the researchers found that their system can initiate gene transcription at a rate that corresponds linearly to the number of binding sites, allowing them to precisely control the amount
of protein produced.
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
.
"This system has very high consistency across different cell types and different target genes," Chen said
.
"It's a good starting point to think about how to regulate gene expression and cell behavior
with a highly tunable, predictable artificial system.
"
After first demonstrating that they could use the new system to induce cells to produce the expected amount of fluorescent protein, the researchers say they could also use the system to program the two main fragments
of a monoclonal antibody called JUG444.
The researchers also programmed CHO cells to produce varying amounts of human antibodies
called anti-PD-1.
When human T cells come into contact with these cells, they become stronger tumor cell killers
if they produce large amounts of antibodies.
They say that although the researchers were able to obtain high yields of the antibodies they needed, further work is needed to incorporate the system into industrial processes
.
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 why it shouldn't, but we still need to test it," Chen said
.
Reference: A synthetic transcription platform for programmable gene expression in mammalian cells
