Did Chinese scientists gene edit tardigrade DNA into human stem cells?

A recent article claims Chinese military scientists increased the radiation tolerance of human cells by inserting tardigrade DNA -- did this really happen?

Source: Midjourney

Last week, one of my undergrad students sent me an email with the header “Credibility?”

The email linked to an article in Popular Mechanics suggesting that Chinese military scientists are experimenting with “creating super soldiers able to withstand nuclear warfare.” And their plan? Using gene editing to insert tardigrade DNA into human cells.

Despite tardigrades being remarkably resilient creatures, this had the feel of a manufactured story. And so I started to dig a little further.

The journey was an interesting one, and unexpected!

The source of the Popular Mechanics article was a piece in the South China Morning Post with the headline “Chinese team behind extreme animal gene experiment says it may lead to super soldiers who survive nuclear fallout.”

This, in turn, was allegedly based on a paper in the Chinese-language journal Military Medical Sciences, published back in October.

Try as I might though, I could not find that original paper. I even asked one of my grad students (who’s Chinese) if she could track it down. She couldn’t.

This led me to suspect that the story was a fake. I even went so far as to see what ChatGPT would produce if asked to write a fake story about Chinese military scientists, tardigrades, gene editing, and radiation-resistant super soldiers, just to see how close it came to the Popular Mechanics piece.

It came scarily close!

What was nagging at me though was that the science here makes sense. Tardigrades are known to be resistant to radiation damage, and this is associated with a protein that is unique to them: the Dsup protein (for “damage suppressor”). Dsup suppresses damage from radicals and radiation at the cellular level, and there is growing interest in its therapeutic uses.

For some years now there’s been speculation that Dsup could be used to protect humans from the impacts of cosmic radiation while traveling in deep space. If this was the case, it could be a game changer for extending human space exploration. But that would require some pretty radical genetic engineering.

The same genetic engineering could also be used to protect against—or even treat—acute exposure to radiation here on Earth. This in itself could be transformative on many fronts.

Given growing interest in the protective properties of Dsup, the Popular Mechanics piece felt like it could be true. But without primary source validation, I wasn’t sure I could trust it—so I sent a note back to my undergrad student saying “looks like this is a made up story.”

But then Jieshu—my grad student— came up trumps and tracked down the original research paper! (It isn’t yet available on the main journal website, but the journalist who wrote the original story kindly passed it on to her).

It makes for fascinating reading.

I’ve included ChatGPT (using GPT4) translations of the beginning of the paper’s introduction and the concluding discussion below (which I checked with Jieshu — they are pretty accurate). The main takeaways are:

• The researchers were interested in novel treatments for acute radiation syndrome, and set out to explore whether the genetic sequence in tardigrades that leads to Dsup being produced could be inserted into human cells.

• Dsup protects against DNA damage from free radicals in tardigrades. As ionizing radiation is a significant source of free radicals within cells, the protein is also able to reduce the impacts of radiation exposure.

• Researchers used CRISPR/Cas9 gene editing to insert the genetic sequence for producing Dsup into a line of human embryonic stem cells that is widely used in biomedical research (hESC-H9).

• The researchers found that “the tardigrade Dsup protein provides some protection to cellular DNA against oxidative stress in the gene-edited stem cells.” They conclude “Since oxidative stress is a central link in the development of many diseases such as cancer, aging, diabetes, inflammation, and Parkinson's disease, studying the Dsup gene may provide a strategy for improving human cell tolerance to oxidative stress and have significant implications for applications in cell or organism protection, aging, and stem cell differentiation.” (Translations)

• They also recognized that, because they are inserting a tardigrade gene sequence into a human genome, this “may raise safety concerns that need further investigation.” I’d say this may be a slight understatement of the challenges in going from a proof of concept experiment to full-blown genetic enhancement!

• I could not find any references to “super soldiers” in the paper! It appears that this came up in the conversation between researchers and the South China Morning Post journalist, rather than being part of the original publication.

Interestingly, this isn’t the first time that the genetic sequence that leads to expression of Dsup has been inserted into human cells. In 2021 an Italian team experimented with immortalized human kidney cells (not using CRISPR in this case) and found that Dsup increased the expression of DNA damage repair genes. And in 2020, Chris Mason and colleagues published research on bioRxiv using the same cell line where they concluded “Our methods and tools provide evidence that the effects of the Dsup protein can be potentially utilized to mitigate such damage during spaceflight.” What is particularly relevant about this research is that Chris Mason is a strong proponent of genetically engineering humans to increase the safety of human space exploration.

Clearly, inserting tardigrade DNA into human-derived cells is more common than it might at first seem.

This research is fascinating—especially from the perspective of the future of being human. But it does raise ethical concerns—both as a potential gene therapy and as a route to future genetic augmentation. In fact, the South China Morning Post article quotes a Shanghai-based scientist as saying “The purpose of the gene editing experiment is to change human genes” … “First, it claims to cure diseases. Then capital will join and play. This game may not have a happy ending”.

Even from a therapeutic perspective there are ethical issues here. However, from the context of how advanced gene editing may one day be used to alter our bodies to be more resilient in harsh environments, it’s a technology worth watching.

Update:

(added 4/4/23) As Vincent Lynch points out on Twitter, “Unfortunately Dsup increases DNA damage in human neurons, which is one hell of a trade off if you’re out to make nuclear war resistant super soldiers!” — which just goes to show, if something looks too good to be true, it probably is … especially in biology!

https://twitter.com/DevoEvoMed/status/1646823170828128258

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ChatGPT (GPT4) translations from the original paper:

Introduction

Acute radiation sickness (ARS) is a medical challenge faced by military personnel, civilians, and emergency responders when dealing with nuclear accidents and nuclear terrorism. Based on clinical manifestations, ARS can be divided into bone marrow type (> 2 ~ 3 Gy), gastrointestinal type (5 ~ 12 Gy), and cerebral type (10 ~ 20 Gy) [1]. DNA damage is the main cause of radiation biological effects, including direct and indirect damage. The former refers to the damage caused by ionization or excitation of DNA molecules by charged particles, while the latter refers to the indirect damage to DNA molecules caused by a large number of free radicals generated by particle ionization of water molecules [2].

In recent years, research on extremophile organisms has revealed how they survive in such extreme environments, including radiation-tolerant tardigrades, such as Ramazzottius varieornatus. Also known as water bears, they are 0.1 ~ 1 mm long invertebrates that can tolerate high and low temperatures, vacuum, pressure, radiation, and other extreme environments [3 - 6]. In September 2007, European Space Agency researchers discovered that some water bears could survive after exposure to vacuum and outer space radiation [7]. This indicates that water bears hold the secret to radiation tolerance. In 2016, Hashimoto et al. [8] first reported the successful decoding of the extremophile radiation-tolerant R. varieornatus genome. Through gene expression analysis, researchers identified a potential key protein for water bears to resist radiation damage: damage suppressor (Dsup). This highly charged disordered protein is expressed in large amounts during the embryonic stage of water bears, and its carboxyl-terminal end is similar to the HGMN sequence in vertebrates. It can specifically bind to nucleosomes and form dispersed protein clusters with its >60% SAGK residues, protecting DNA from X-ray-induced hydroxyl radical damage [9]. This provides us with an insight: the radioprotective effect of Dsup protein might be an effective measure to improve and enhance DNA protection capacity, fundamentally enhance ARS treatment capabilities, and thereby increase the survival rate of ARS patients.

Discussion

Radiation can cause DNA double-strand breaks, leading to genomic instability and even cell death. Currently, there are two recognized mechanisms to improve radiation tolerance: antioxidant defense and effective DNA repair [13, 14]. Tardigrade Dsup protein binds to DNA to form a natural protective shield, reducing the damage to DNA caused by reactive oxygen species (ROS) produced during oxidative stress. Studies have shown that after X-ray irradiation, HEK293 cells stably transfected with Dsup still maintain a certain level of activity and proliferative capacity, with DNA damage reduced by approximately 40% compared to the control group [8]. Therefore, the tardigrade Dsup protein provides some protection to cellular DNA against oxidative stress. Since oxidative stress is a central link in the development of many diseases such as cancer, aging, diabetes, inflammation, and Parkinson's disease [15], studying the Dsup gene may provide a strategy for improving human cell tolerance to oxidative stress and have significant implications for applications in cell or organism protection, aging, and stem cell differentiation.

We hypothesize that human embryonic stem cells stably transfected with the Dsup gene may also improve DNA protection or damage inhibition, including oxidative stress tolerance. Since the Dsup protein is specific to tardigrades, its effects on human cells are still largely unknown. Therefore, this study focuses on preliminarily clarifying the impact of Dsup gene expression on the biological properties of human embryonic stem cells, providing help for understanding the effects of the Dsup gene on human embryonic stem cell development and differentiation and downstream application research, and laying a foundation for further studying tardigrade extreme environmental tolerance [16-18].

In this study, we successfully constructed the Dsup vector and enriched gene-modified human pluripotent stem cells using positive clone drug screening, PCR identification, and flow sorting technologies. Genomic identification confirmed that the gene modification integration frame was completely integrated into the AAVS1 site without important mutations, and the karyotype analysis results showed that all cell groups had a normal karyotype. Spurio et al. [19, 20] found that the overexpression of some DNA-binding proteins could cause severe DNA aggregation and loss of cell vitality. Since the Dsup protein is an exogenous DNA-binding protein, its expression in human embryonic stem cells may cause similar responses and affect their biological properties. We passaged the purified and enriched efficacy-enhanced human embryonic stem cells multiple times, and by examining the morphology, key pluripotency markers (OCT4, SOX2, NANOG, SSEA-4, TRA-1-60), and alkaline phosphatase, we found that Dsup gene-modified human embryonic stem cells maintained their cell morphology and pluripotency while stably passaging. Cell proliferation experiments showed that the proliferation rate of hESC-H9-Dsup cells was higher than that of hESC-H9-Control and hESC-WT groups, which is similar to Hashimoto's finding that Dsup promotes the proliferation of HEK293 cells [8]. Furthermore, apoptosis experiments showed no significant difference between the hESC-H9-Dsup group and the hESC-Control group, indicating that Dsup expression does not damage cell vitality, but rather promotes cell proliferation to a certain extent. The specific regulatory mechanism still needs further research. This also indicates that Dsup gene-modified embryonic stem cells can be further used to study the protective mechanisms for human cells and the effects on development and differentiation. In addition, radiation damage experiments showed that Dsup-modified hESC-H9 cells have radiation tolerance capabilities.

Although this study confirmed that precise knock-in of Dsup into hESC-H9 has no effect on its biological properties, the Dsup protein is a tardigrade-specific protein, and its immunogenicity after cross-species expression remains unknown. This may raise safety concerns that need further investigation. Moreover, pluripotent stem cell differentiation into hematopoietic lineage-specific cells is a complex, multi-stage process regulated by various factors. As Dsup is a nucleosome-binding protein, whether its overexpression affects the regulatory network and its binding to DNA is another area for further research.

In summary, Dsup-modified hESC-H9 cells can be used not only to study the effects of the Dsup gene on human embryonic stem cell development and differentiation but also to investigate the interference and DNA protection effects produced by the stable knock-in of DNA protective genes into the human chromosome 19 AAVS1 site on the transcription of other genes. These findings lay the foundation for further clarification of the molecular mechanisms of Dsup and exploration of its potential application value.

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