Six Years for One Step Towards Unnatural Amino Acid–Controlled Gene Therapy

Stories behind the paper “Restoration of dystrophin expression in mice by suppressing a nonsense mutation through the incorporation of unnatural amino acids” that was recently published in Natural Biomedical Engineering.

Hello, my readers. I am Qing Xia, a professor in Peking University (China) and the corresponding author of the paper “Restoration of dystrophin expression in mice by suppressing a nonsense mutation through the incorporation of unnatural amino acids” that was recently published in Natural Biomedical Engineering. In this post, I will share with you some experiences behind the paper.

My scholar backgrounds include clinical medicine, immunology, neuroscience, chemical biology and protein engineering, and I became fascinated with the unnatural amino acid (UAA) incorporation technique since 2011. At the beginning, the main laboratory work was creating stable cell lines that could express the UAA incorporation systems, so that proteins mutated with premature termination codons (PTCs) can be site-specifically modified with UAAs in these cell lines. A series of stable cell lines were constructed to enable the incorporation of different UAAs, making it a mature protein engineering technique in our laboratory. One feature of the UAA incorporation system is that the system works only when all its 3 elements (i.e., the aminoacyl-tRNA synthase, the cognate tRNA, and the UAA) are present. The dependence on additional UAAs therefore serves as a lock of the system function.

In 2015, I took one step beyond protein engineering and started to investigate the potential of these UAA incorporation systems in treating nonsense mutation diseases (such as Duchenne muscular dystrophy, DMD), because I noticed that these diseases also contain PTCs that lead to truncated proteins and deficient protein expressions. If these disease-causing PTCs were read-through by the UAA incorporation systems, full-length proteins modified with UAAs at nonsense sites would be the product, the protein expression could be possibly restored, and thus nonsense mutation diseases might be treated. This idea seemed promising as a universal gene therapy to nonsense mutation diseases. While gene therapy has potential safety risks and has caused deaths in history1, the UAA dependence of the system may work as a switcher or controller of gene therapy (Figure 1), which may be another advantage of this strategy.

Figure 1. The strategy of UAA-controlled gene therapy for nonsense mutation diseases. The figure was cited and adapted from the aforementioned paper (

Qi Yang, one of my students and co-first author of this paper, piloted this idea and performed most cellular experiments. For preliminary animal experiments, we chose transient transfection of UAA incorporation system plasmids into the tibialis anterior muscle of mdx mice by in situ electroporation. However, the resulting dystrophin restoration was quite low (less than 10%) and attenuated with time. We attributed the low efficiency to the transient transfection of in situ electroporation, which was also invasive and only restricted to the short-term evaluations.

Then, I considered increasing the restoration efficiency by transgenic mice2 that stably express the UAA incorporation systems. Our earlier experiences with stable cell lines had demonstrated higher efficiencies over transient transfection in vitro, and transgenic mice might be the upgrade in vivo model similar to stable cell lines. Ningning Shi, another co-first author of this paper, took over the experiment and began to construct the transgenic mice harboring the UAA incorporation systems. After several trials, the UAA incorporation system (PylRS-tRNAPyl) were successfully introduced into mice, and the genotype could be passed on to offspring and easily crossed with other disease model mice. The success of such transgenic mice was partially due to that the UAA incorporation system remained silent in function and only started working at the presence of additional UAAs. We tested our therapeutic strategy on these transgenic mice and the dystrophin restoration was increased to around 30%, which could reach the reported therapeutic levels3.

In 2019, I planned to publish these results and started to write the paper. Qi yang, Jiaqi Lu, Aikedan Abulimiti, Jialu Cheng and Haoran Zhang helped in organizing the results, preparing the figures and writing the manuscript. Haoran Zhang and Ningning Shi told me that although the dystrophin restoration was promoted to a considerable level in transgenic mice, we still did not demonstrate an applicable administration route for therapy. I knew one available solution was to adopt the delivery technology like AAV just as a reviewer advised us3. Fortunately, the kindly NBME editors and reviewers judged the novelty of the paper and suggested a series of future improvements including performing necessary AAV delivery experiments to demonstrate the translational potential. Delivered by the AAV intraperitoneally or intramuscularly, the UAA incorporation system could restore the dystrophin expression in mdx mice in a UAA-dependent manner. Additional UAA doses to maintain the therapeutic activity is a constraint, but also an advantage of this approach, like the two sides of a coin. Do you think the strategy of UAA-controlled gene therapy interesting and useful? The implications of the findings may extend well beyond DMD into aspects like in vivo protein engineering. Treatment of DMD is a representative demonstration of this approach. Nevertheless, in vivo delivery to create novel protein functionality via UAA incorporation is a potentially exciting avenue for future protein engineering.

After submitting the final revisions and related materials, the paper was accepted by Nature Biomedical Engineering on 27 June 2021. I spent 6 years on this project along with my students and finally we made a step towards unnatural amino acid–controlled gene therapy. Thank you for reading.


  1. Marshall, E. Gene therapy death prompts review of adenovirus vector. Science 286, 2244-2245 (1999).
  2. Han, S. et al. Expanding the genetic code of Mus musculus. Nat. Commun. 8, 14568 (2017).
  3. Le Guiner, C. et al. Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy. Nat. Commun. 8, 16105 (2017).

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