Team:SJTU-BioX-Shanghai/Human Practices
Integrated Human Practice
Why LUCAS?
We started by investigating public awareness of, as well as attitude towards gene-editing tools and methods. We had our expectancy before sending the questionnaire to the public, which is, acceptance of gene-editing tools would increase as people learn more about it. What is startling, however, is that we were only halfly right. Indeed, there is a growing awareness of CRISPR and other gene-editing tools, but the percentage of those people who accept gene-editing on the human body has shrunk.
One of our explanations is that people now focus more on the safety of biotechnology, which makes them more cautious about applying gene-editing techniques to the human body.
It then came to us that, what if the off-target gene-editing rate drops so low that human gene-editing is no other than minimally invasive surgery?
To Get Started
By talking to Sun Ruilin from Shanghai Model Organisms, we learnt that in support of current technology, gene-editing efficiency is able to meet most demands, especially for CRISPR-Cas9. In other words, it’s no longer necessary to pay too much attention to whether Cas9 protein binds to a DNA sequence. Instead, the binding accuracy should be of more concern.
The existence of the off-target effect is normal and has its reason since all guide RNAs we are using comes from prokaryotes, whose genome is smaller than eukaryotes, and with less similar sequences. Given the situation, if the enzyme that recognizes PAM has not evolved well enough during the evolution process, it will miss the target we want.
According to Ruilin Sun, current approaches to lower the off-target rate of CRISPR system mainly work on two problems: to design a smaller Cas9 protein (the common molecular weight of Cas9 protein is around 200kD, which is way too big), or to optimize gRNA sequence.
Shortly after the talk with Ruilin Sun, we read about present gene-editing techniques and realized that both working strategies mentioned above aimed to improve the universal off-target effect, in other words, the off-target effect might be generally improved, regardless of specific genetic loci.
However, there’s still much room when it comes to editing one given target as current technologies lack specificity. The off-target effect is not weak enough to support in vivo editing, especially in the human body. To sum up, if gene therapy is to mature from storytelling to sound clinical practice, the off-target effect in the CRISPR-Cas9 system needs to be further weakened, yet no such research had been done so far to our concern.
That’s how we conceived of the idea of designing a system that uses fluorescent signals to demonstrate whether Cas9 protein binds to target DNA sequence and to calculate the on-target ratio.
Selecting Editing Site
Data shows that lung cancer has the highest morbidity and mortality rate among all cancers now in China. According to Di Chen, a medical graduate of Shanghai Jiao Tong University School of Medicine, lung cancer can be divided into non-small cell lung cancer and small cell lung cancer. Among them, non-small cell lung cancer accounts for about 85% of lung cancer. He said that “ Gene editing therapy is of great potential. There has been a successful example of using CRISPR-Cas9 to edit the PD-1 gene in vitro T cells, and then transplanting to non-small cell lung cancer (NSCLC) patients. This method can significantly improve the mid-term survival time of patients.”
We somehow felt obliged to do something to improve the present situation in China and work on non-small cell lung cancer. But we weren’t sure whether we could actually do some good since thousands of excellent scientists had been worked on this problem for years, and we didn’t really know how to start.
At this point, an exciting new development came to our notice. The team of Professor Lu You from the Department of Thoracic Oncology published an online study titled "Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer" in Nature Medicine and reported the use of CRISPR-Cas9 gene-editing technology. Lu edited the PD-1 gene in T cells in vitro, cultured those T cells, and then transfused it back to patients with non-small cell lung cancer (NSCLC). They proved the safety and feasibility of this therapy in NSCLC for the first time. [2]
Our confidence was somehow boosted after reading this exciting paper, and we decided to choose PDCD1 to be our target editing-site. We had completely no idea whether we would succeed, and our goal seemed to be too grand to reach, but by starting small: focusing on only one editing site, we might actually solve a practical problem.
Directed Evolution
We persuaded ourselves that we had gathered enough information, either from science publications, experts, or from the general public. Our initial project embarked on improving the off-target effect of the CRISPR-Cas9 system, especially on the PDCD1, a gene critical in non-small cell lung cancer.
We’ve already learnt that current researches focused on weakening off-target editing effects on a wide range of editing sites and made limited improvements. Better refinement method was needed, and we had no fear of trying to research cutting-edge technologies.
Frances H. Arnold’s talked about her Nobel Prize-winning achievement on October 20, 2019, at the Institute of Biophysics, Chinese Academy of Sciences. The playback of her speech greatly inspired us. Directed evolution best suits our project design, as well as the value we first meant to deliver. As far as we’re concerned, no such work had been conducted at a specific editing site, the off-target rate of which had been low enough to practice in humans. While we were doing background research of our LUCAS design, we were assured by experts that LUCAS has certain innovation, since not much work had been done to employ directed evolution and optimize the CRISPR system.
We then interviewed Tong Xiajing, professor and researcher at Shanghai University of Science and Technology, to do a feasibility assessment for us. She cleared our thoughts by explaining in detail how directed evolution experiments should be conducted. She gave us an upbeat assessment of our general train of thought, yet she also pointed out that our project might require huge amounts of work.
Realizing Defects
Professor Tong was not the only one who worried about our workload. The feedback we received during CCIC 2020 suggested the same. We had an in-depth conversation with Professor Feng Gu, whom we happened to know that had published a study recently, using targeted selection to identify high-fidelity SaCas9 protein in human cells. Sadly, though our design was somehow a little bit similar to his work, he questioned the viability of LUCAS. We realized that excess workload mainly came from the method we used to select Cas9 protein. Different from a traditional selection that uses antibiotics, our design used fluorescent signals to weed out proteins.
Adjustments
However, it is a rather tricky problem. We were unable to solve it until we talked to Professor Yi Xiao, a researcher from the School of Life Sciences and Biotechnology, SJTU. His work focused on developing CRISPR-CAS biotechnology and biomolecular sensors. Therefore, we posed to him questions that had bothered us. It was kind of Professor Xiao and his students to help improve our experimental design, and to share with us their experience when conducting transcription activation experiments. More importantly, Professor Xiao suggested using a transcription repression loop to further verify the feasibility of the reporting system. On one hand, the effect of the transcription suppression system is more significant. The difference between the on-target and off-target effects can be more intuitively and clearly seen, compared with the transcription suppression system. On the other hand, a direct comparison of the two systems, transcription activation, and suppression could indicate which system is more suitable for the evolutionary circuit of the CRISPR system.
Being Encouraged by Latent Demand
There was a period when the whole team was quite depressed. Our design was criticized by many, and the results of some preliminary experiments were concerning. We started to question ourselves, did our project even deserve further implementation?
We must know better about our own project. Why did we start it in the first place? What goods could we do even if we wouldn’t be able to finish up before the competition due?
Before long, we noticed that the Cas9 optimization system that we constructed might be of greater use. It was most delightful that Professor Tianlin Cheng from Institute for Translational Brain Research at Fudan University agreed with us. Many thanks to his kindness, he told us that he himself was very interested in this kind of low-off-target gene-editing technique. This greatly inspired us, that our project might not be put into practice soon enough, however, LUCAS’s latent demand mainly comes from our peers. Different labs may be interested in different genes regarding various physiological status or pathological status. Some may demand animal models with accurately edited gene targets. Biotechnology companies might be interested in our system as well if we could commercialize our optimizing system after simplifying it. LUCAS system that we constructed can run at various gene targeting sites, thus improving the safety of CRISPR-Cas9 to the next level.
It was a pleasant interview with Professor Tianlin Cheng, he helped get our ideas into shape. He suggested that other than normal Cas9 protein, Cas9 variants are a research hotspot in recent years. By applying the same experimental processes of LUCAS to Cas9 variants, the editing efficiency might be further improved. Cheng also agreed with our assumption that LUCAS could be used as an evaluation system, judging Cas9 protein’s off-target effect as well as its editing efficiency.
Clinical and Market Prospects
If we managed to use LUCAS to select a Cas9 protein that has minimum off-target effect on PDCD1, the next thing of our concern is clinical studies. We might need to think of registering clinical trials in Chinese Clinical Trial Registry in the late future.
By asking our classmates, and even senior fellow students who only conduct basic research, we found out that most researchers are not familiar with certain policy on clinical studies. We decided to organize the process of applying for clinical trials.
All studies in humans or using specimens taken from humans, including controlled or uncontrolled trials of the efficacy and safety of various interventions (e.g., randomized controlled trials, case-control studies, cohort studies, and uncontrolled studies), prognostic studies, etiological studies, and diagnostic trials that include various diagnostic techniques, reagents, and devices, require registration and announcement.
If LUCAS is to apply for clinical trial, we first need to establish a project. All sorts of details are needed, including basic information, project abstract, studying background, main content, expected goals, innovations and study features, working conditions, expected accomplishments and judgements, years of project implementation and annual planning schedule, financial budget, research assistant commitment, commitment of the project statistician, project leader commitment, section comments and hospital approval comments.
Basic information: research topic, year of commencement, project leader and members and their titles, as well as division of work.
Project abstract
Studying background: research status and project significance Main content: purpose, researching strategy, volunteer-recruiting criteria, grouping method, pre-risk assessment, security measures and contingency, data analyzing methods, informed consent and ethics.
Working conditions
Expected outcomes: trial results and assessments.
Years of project implementation and annual planning schedule.
Financial budget
Letter of commitment: from research assistants, from project statistician, and from project leader, certifying truthfulness and accuracy of the statements above.
Comments from specific hospital section
Approval of the hospital.
The proposal will then be handed to two departments, Hospital Research Department and Hospital Ethics Department. If both departments approve of the project, then the proposal will be sent to Science and Technology Commission of Shanghai, before presenting to the judges there. They will decide whether the project is qualified to be put into clinical trials, and how much expenses is approved. If the whole process above is completed, we will be able to start recruiting volunteers! It goes without saying that all the experiments will be strictly inspected.
If LUCAS is to apply for a patent, or to packaged into a product and sold to commercial corporations, a different set of process should be delivered and will not be discussed here.
Afterthoughts
Modeling is better?
LUCAS also stands for ‘the Last Universal Common Ancestor’, indicating that we’re using directed evolution to optimize our Cas9 protein. We took two approaches to perform this task, modeling and wet lab experiments. Modeling used machine learning to predict off-target events, while wet lab experiments created mutations and then select the most suitable Cas9 proteins. Most of the results presented comes from wet lab experiments, however, it’s quite obvious that machines are more efficient, and that the most suitable protein picked out by modeling comes from the complete sequence collection, which is much larger than the mutation library obtained by experiments.
Utilitarianism
When we were gathering background information, we were surprised to find out that no study had been conducted that focused on only one editing site. It somehow reflects the microcosm of academical circles. If a project can’t be of great use immediately, or if it can only benefit a small group of people, it’s hard to request for project funding. And if the researcher feels that he can’t submit his article to influential journals, he might give it up in the first place!
We have gone so far from what we were taught at a young age. As an ancient civilization, Confucius had proposed two concepts thousands of years ago, Li and Yi. Yi stands for the highest principles to be respected and the highest ethics to be pursued in human social activity and human relations. Li stands for the interests and utilitarian interests that satisfy the needs of human life. The two principles should be equally valued; however, we have focused way too much on Li.
What’s interesting is that the dialectic relationship can also be applied to the process of directed evolution. We will never find the ‘best’ protein, only one that suits current situation best. One will never get satisfied if he focuses too much on Li.
References
[1] Ou, S. H., Zell, J. A., Ziogas, A., & Anton-Culver, H. (2007). Prognostic factors for survival
of stage I nonsmall cell lung cancer patients : a population-based analysis of 19,702 stage I
patients in the California Cancer Registry from 1989 to 2003. Cancer, 110(7), 1532–1541.
https://doi.org/10.1002/cncr.22938
[2] Lu, Y., Xue, J., Deng, T., Zhou, X., Yu, K., Deng, L., Huang, M., Yi, X., Liang, M., Wang, Y.,
Shen, H., Tong, R., Wang, W., Li, L., Song, J., Li, J., Su, X., Ding, Z., Gong, Y., Zhu, J., … Mok,
T. (2020). Safety and feasibility of CRISPR-edited T cells in patients with refractory
non-small-cell lung cancer. Nature medicine, 26(5), 732–740.
https://doi.org/10.1038/s41591-020-0840-5
SJTU-BioX-Shanghai
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