Future work

Parts modification

As described in the project page, we have successfully constructed, assembled and tested our device and each of the parts works well as expected. Since our device is designed for clinical application, extra works must be done to reduce potential harm to the body and guarantee an optimal effect. Sensibility, specialty and safety should be together put into consideration. So each of the parts should be elaborated selected, combined and tested.

First, the suicide gene. We want to find out a suicide gene that can function without extra stimuli from the environment. Also, the killing effect should be stable among different cell lines and cell situations. So, we select the response signaling elements in pathways relative to cell death. We have already tested the RIP1 and RIP3 and both genes successfully lead to programmed cell death. However, there are three ways to induce cell death: apoptosis, nercrosis and autophagy. Theoretically, the apoptosis pathway is the safest one because cell fragments will be degraded within apoptosis body. We have tested for hBax, hBaxS184A and fragment caspase3 and none of them works out because of the wrong origin of plasmids. We should also test the immunity response when the cells go through suicide.

A special gene called apoptin is also within our consideration. According to published papers, apoptin is a 121 amino acids peptide which was first isolated from Chicken anemia virus(CAV )and can induce cell death in several cancer cell lines but not in normal cells. Apoptin possibly functions by recognizing some early signal of cancer and inducing apoptosis via a non-p53 dependent pathway. Preclinical in vivo models based on different human xenografts indicate that apoptin is a safe and efficient potential anticancer drug.

If the safety, sensibility and speciality of apoptin can be guaranteed, it would be a one-shot solution to prevent cancer in every kind of directional redifferentated cells.

Secondly, the microRNA targets. In our project, miR-122 target is selected as a sensor module in our design. In normal cells, microRNA-122 can control more than 200 genes by binding to 3' UTR of their mRNA and lead to degradation of the mRNA hence controlling gene expression. The naturally occurring miR-122 target has mismatch to microRNA-122 but can still function with knockdown efficiency. We have noticed that the team Heidelberg 2010 has used the target design of miRNA-122, or homogeneous two copies and heterogeneous four copies. But in our design, we need to best reduce leakage expression of suicide gene so we have tested for 1, 2 and 4 copies of targets rather than characterizing mismatch combination of miR122 targets.

Thirdly, the Tet system. Detailed principle of the Tet system refers to parts description page. The Tet-on system is turned on by adding doxycycline to the medium and the Tet-off system is on without doxycycline. If we use the Tet-on system, doxycycline is required to maintain selective pressure which is inconvenient for further clinical use as patient should take additional medicine all their lives. Thus if we preferred the Tet-off system, doxycycline should be added right after introducing the pathway to cells, most likely before or in the pluripotent stage. Whether the use of doxycycline will interfere cell pluripotency or directional differentiation is under investigation. Despite Tet system, there are other modules available as pathway switches. No matter which system we choose, this part should reach to following standard as: low leakage expression, high fold increase, less interfere to normal metabolism in human cells and easy to control.

Fourth, fine-tuning of parts. To achieve our initial goal, killing cancerous and wrong kinds of cells while leaving the normal hepatocytes alive, more rigid standards should be reached. Despite the well-functioning of independent parts, we need to think about the sensitivity and speciality of the whole pathway, the tolerance of FN or FP results. Two of our parts are responsible for it: the leakage expression and fold increase of Tet promoter and the knockdown efficiency of the miR-122 target. Since the expression of Tetpromoter varies among different cells due to the differences of integration site, such tests should be performed after our constant expression cell line is built.

Pluripotent test in iPSC

To confirm the availability of our pathway to iPS cell line, a perquisite requirement is that such pathway will not interfere iPSC pluripotency or at least its benefit outweights the costs. There are two main concerns for the application of our design: the use of doxycycline and the leakage expression of suicide gene. By controlling the dosage of doxycycline or even change a whole new switch system and prepare for alternative suicide gene, such concerns might be overcome. Actually, this is one of our on-going work to confirm the pluripotency of iPSCs with the pathway. We will perform the teratoma test in miPSCs and hiPSCs to examine the pluripotency of iPSCs.

Directional differentiation from iPSCs to hepatocytes

This test provides a direct way to prove that our pathway can successfully increase the efficiency of directional differentiation. Since the pathway responsible for hepatocytes differentiation remains a mystery, we do not know if the pathway might crosstalk with relative endogenous pathway although theoretically this would not happen because the Tetsystem is independent to the human signaling pathway. To perform this test, we need extra training in inducing directional differentiating cells from iPSCs which is still a technical barrier for us. However, if the above experiments go well, we can have a try.

Carcinogenesis test in liver cells

One of the shining points of our design is that it provides a new way to fight against cancer using strategies of self-surveillance, self-clearage and self-stabilizing. Previous tests have confirmed that microRNA-122 is specifically expressed in normal hepatocytes. And our device needs to prove that it induces cell death not only in cancer cell lines, but also in transformed cells and such effect can be maintained. So, we need to transfer our pathway into iPS-derived or primary cultured hepatocytes, and induce carcinogenesis and test for our device again.

Perspective

Target selection

Our project aims at providing a new strategy to increase the efficienct of iPS directional differentiation and prevent carcinogenesis in transplant tissue. Here, we choose the iPS-derived hepatocytes as a model to test our idea. We have selected the liver-specific miR-122 as a marker to distinguish normal, differentiated liver cells from undifferentiated or cancerous cells. Actually there are many tissue specific and cancer specific microRNAs as deciphered by microarray, Our design can be broadened into usage in other tissue types by altering microRNA targets. By using engineering regulatory pathway, the rigid standard for highly specific microRNA can be reached by applying several kinds of microRNAs.

Alternative strategies

There are two main goals of our project: (1) increasing the efficiency of transferring iPSCs into tissue cells with the process of cell kind division much simplified and (2) auto-clearing the cancerous cells if the iPSC-derived tissue is transplanted into human in the long term. MicroRNA is a nice one-shoot to both problems. However, there might be difficulties in finding single specific and reliable microRNA target for every desired cell kinds. Hence we seek for alternative way to solve this problem.

Combination of several microRNAs can increase the specific of devision. In our project, we let the cells with high level of desired microRNA alive, but with the introduction of toxin-antitoxin system, cells with specific high or low level of microRNAs can be alive. By engineering, our choice of microRNAs can be broadened from single, specific and high level of microRNA to cluster of microRNAs to stepwise choose the cells we want.

Instead of microRNA-based silencing, there are other ways to achieve the goals of our project. For example, placing the suicide gene under control of tissue specific promoter can also autonomously select desired kind of cells in a cell mass. By using tumor-specific suicide gene, apoptin under control of tissue specific promoter, same aim of our project can be achieved. If we just focus on eliminating cancerous cells, tumor specific promoter like hTERT can be utilized. Hence, we can intentionally select the functions independently and combine them together as we need.

Vector for gene therapy

Our iPS safeguard device is developed not only for tissue transplant but also for cell and genetic therapy. Patients with inherent metabolic disease would benefit from a safer and more reliable tissue from himself or herself with genetic information modified. There is a growing trend to inject immune cells back into patients' bodies to fight against cancer, but the dynamic development of immune cells remains elusive. Maybe we can insert an auto-adjust circuit into those cells and let them differentiate and migrate as we expected. Any transformed cells have the concern of origin or assistant of carcinogenesis, including the lymphoid cells previously defined as cancer fighter. In this regard, our device would be a safeguard to protect and care for cell therapy.