University of Utah professor heads international team exploring how to avoid transgene silencing

A key problem for mammalian biotechnology research is that transgenes, genes transferred from one organism to cells in the genome of another, can degrade over time, thus decreasing the transgene's effectiveness.

This loss of expression is called transgene silencing. The silencing limits the applications of many engineered cells in research or therapy that require longer time periods of stable operation.

The mechanism of transgene silencing and some proposed solutions is the subject of an article in Cell Systems, Dec. 21, 2022, by an international group of researchers. The researchers term transgene silencing  a "bottleneck for many mammalian-cell-based biotechnology applications." 

The authors, including University of Utah biomedical engineering professor Tara Deans describe many forms of transgene silencing in mammalian cells. Sometimes only a few of the cells don't express the transgene, sometimes the level of expression in individual cells decreases and sometimes silencing becomes a heritable effect.

The mechanisms of silencing

The article presents an overview of the mechanisms potentially involved in transgene silencing and offer suggestions for how to get around it. The authors also call on other researchers to include information on the  transgene silencing they encounter in their scientific articles. Increasing information about the subject will lead to potential solutions.

Several silencing mechanisms are highlighted in detail.  The first is when proliferation-associated processes promote silencing, because of the "inherent antagonism" between transcription and the DNA replication process, for example in stem cell reprogramming.  

The authors also describe how DNA methylation contributes to epigenetic silencing, specifically cytosine methylation, viral vectors and transposon systems (transposons are chromosomal segments that work to integrate the transgenes).

Avoidance mechanisms

The authors propose some practical guidelines to avoid transgene silencing. The first, they suggest, is that the choice of the transgene delivery method can influence the probability of silencing occurring.  They recommend testing the effect of different promoters under controlled conditions.

Another strategy they suggest is inserting the transgenic DNA at a specific place in the genome that is considered to be a "safe harbor," instead of a random site within the genome. Several safe sites are elaborated. 

The cell type chosen for transgene expression also makes a difference, they write, and many examples are given of cells that are more prone to allow silencing. 

Finally, the authors advise paying attention to the nutrients used in fostering growth, because some are more apt to promote transgene silencing. 

Looking forward, the authors present methods that need to be further developed to minimize transgene silencing.  In conclusion they emphasize the need for sharing more research information on transgenes to help speed solutions to the problem. 

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Alan Cabrera et al. "The sound of silence: Transgene silencing in mammalian cell engineering." Cell Systems, Dec. 21, 2022.

DOI: https://doi.org/10.1016/j.cels.2022.11.005

An interview with Tara Deans

The importance of mitigating transgene silencing in mammalian cell engineering

Senior author Tara Deans is an associate professor in the Department of Biomedical Engineering at the University of Utah. She spoke with Current Science Daily about transgene silencing.

Please summarize the problem of transgene silencing for a non-technical audience.

Transgenes are genes that are inserted into the genome of cells for the purpose of adding their expression to the collection of other genes already present in the cell. For example a gene called Green Florescence Protein (or GFP) can be added to cells. When GFP is expressed (or on), the cells turn green. This gene has been extremely valuable in many studies because it allows scientists to watch its expression under the microscope over time.

In addition to GFP, various other transgenes can be added to the genome of cells for many purposes. A few examples include studying stem cell development and how stem cells are involved in the healing of tissues, the production of important biological molecules in industry, and for creating new therapeutic cells.

 In short, for these studies and applications to be successful, the transgenes need to be on. A significant challenge that can arise is the topic of this manuscript:  transgene silencing. This occurs when the transgenes are turned off, or silenced, by cells over time. 

Can you give some examples of biotech applications where gene silencing is an obstacle.

Some examples include recombinant protein production of important therapeutic molecules, and the creation of therapeutic cells. 

What are some of the solutions you propose to get around transgene silencing?

To get around this challenge, we discussed the importance of identifying effective places in the chromatin for putting in the transgenes and understanding the impact of this placement on both the chromatin and the function of the transgene in different cell types.

Some efforts to accomplish this include computational approaches by analyzing publicly available databases, and experimental approaches that include molecular and genetic screens to map potential causes of the silencing.

You call for scientists to include in their publications on transgenes the documentation describing silencing performance. Have you had any response to this?

Our manuscript was published at the end of December 2022, so there hasn’t been much time for the community to respond in publications. However, we hope that this piece will encourage scientists to include a discussion and data on the frequencies of transgene silencing that was observed in their studies.

Along these lines, we encourage publications to include a table of transgene performance of the multiple colonies screened in the study, especially those that displayed sub-optimal expression. Of particular importance is including cell types, promoters used, number of cell passages, the type of transfection/transduction used, and the observed timing of the sub-optimal cell behavior. 

I would like to emphasize the last point here. To have scientists include a discussion and data on the many cell clones screened, and making this data publicly available, will be an important step to improve approaches in cellular reprogramming that will help move the field forward.