Overview

The goal of our project iPSCs Safeguard is to minimize the potential risks of iPSCs. However, with the suicide genes and the lentiviral vectors we used, our design may be argued to potentially bring about new safety risks. What we are delivering here is an organized outcome of the various discussions we had for each parts about their safety in this summer.

1. Lentivirus safety[1]

Lentivirus method is widely used in both biological research and gene therapy. It can intergrate genes into the genome of target cells. Though we cannot avoid the oncogenic risk of lentivirus transfection, however, for iPSCs and hepatoma cells, lentivirus method is nearly the only way for stable transfection. While most vectors don't have the ability to transfect to iPSCs and hapatoma cells, or the inserted genes are easy to be lost, lentivirus not only guarantee efficiency, but stability as well. In addition, in lentivirus vectors the transcription and translation of our target genes are more likely to those processes of other eukaryotic genes. So the product of the circuit can be easier to be predicted and controlled, because it is more likely to natural products in eukaryotic cells. Our circuit is aiming at stable and long guard against carcinogenesis. From this point of view, more stable the circuit is, more safety it guarantees.

A fact we must admit here is the potential risks of lentivirus for integrating into host cell genome and thus bringing about new safety threats . However, when comparing with the heavy fact that the intrinsic tumorigenesis risks cuased by the four reprogramming transcrption factors, the potential pressure of the delivery system seems to be much lighter. Anyway, before put into use, we must do more experiment to measure its risks and try additional methods to ensure its safety.

2. Suicide gene safety

RIP1 and RIP3[2] are effective suicide genes, as we tested, but it not only cause apoptosis, but also cause necrosis. This may be a potential risk of inflammatory response. However, the possibility for normal cells to become cancer cells is very low, so the amount of cell death caused by our circuit actually is little. The inflammatory response is a macroscopical phenomenon and it requires a certain amount of cell necrosis, not that little. Moreover, resent research has found some regulatory factors to decide RIP1/RIP3 to cause apoptosis or necrosis, and the inducement work from necrosis and apoptosis of RIP1 is done by some scientists. So we can expect this side effect of RIP1 and RIP3 can be overcome. In addition, apoptin[3], another suicide gene we have tested, does not have this side effect because it only cause apoptosis. Apoptin may become a better suicide gene because it specifically causes apoptosis in cancer cells. Though in our test it didn't show as stable effect as RIP1 and RIP3, by optimizing the circuit and transfection condition, we can expect that it will become the protagonist of our suicide gene.

RIP1 and RIP3 are anthropogenic genes and apoptin is a chicken source gene. They can only cause cell death in a specific condition of eukaryotic cell, so it's relatively safe. And because they only cause cell death, they can hardly be separated without control because the "uncontrollable" spread is automatically prevented because without the circuit the cell dies. Thus we think these suicide genes are safe and have little risk of wrongly spread. VP3 is an interesting protein with the ability to only lead to apoptosis in cancer cells. Its safety has been proved in various types of cell lines by many papers. However, since it was derived from chicken anemia virus,enough tests should be done before transfect this protein into mammalian cells, especially mouse and human iPSCs.

3. Laboratory safety

Researcher safety

All the experiments have been done according to laboratory safety rules of Sun Yat-sen University, and experimenters have been rigorously trained before practice to guarantee the safety and health of team members.The cell we used in every experiment is harmless to the experimenters. We also used lentivirus in some experiments. Lentivirus is risk group 2+ and may cause infection under improper usage. We are fully alert to it and operate carefully. Experimenters only use lentivirus vector on super clean bench in special room. We strictly follow the protocol and avoid careless operation.

Public safety

The circuit we designed cannot function outside certain cell lines in the laboratory, and the lentivirus we used cannot auto-replicated and only serves as a one-off vector. Experimenters is careful not to release any parts to the public. And even the worst situation happens as it is released, it will hardly bring risks.

Environment safety

We have designed the eukaryotic circuit, which cannot be expressed by procaryotic microorganisms. And the cell we used is very fragile and it will soon die leaving the culture medium. So it will not bring contamination to the environment.

[1]Tom Dull et al, J. Virol.,72,11,(1998)

[2] Duan-Wu Zhang et al, Science 325, 332 ,(2009)

[3] Claude Backendorf et al, Annu. Rev. Pharmacol. Toxicol,48,143,(2008)