UPDATE 09/18/2013

Which suicide gene?

Why do we need a suicide gene?

In our project, we try to build a circuit that can prevent the iPSC-differentiated tissues from cancer formation. The best way to do that is by introducing a conditional expression system, which can kill the cells at proper time. For killing cells appropriately, we need suicide genes.

And the suicide genes should not introduce any harmful effect to normal tissues, when eliminating cancer cells. Naturally, the best genes that fit our requirements will be genes that can successfully induce apoptosis in cells. But many apoptosis pathways are blocked in cancer cells. However, only a little part of cells will become cancer cells at any certain time, so expressing necrosis gene in cancer cells will not lead to severe inflammation, provided that the necrosis gene can be tightly control under our device. For these reasons, we included certain kinds of necrosis gene in our finalist, after in searching of a lot of published work.

Our final candidates of suicide gene are listed below:

Table 1 suicide gene

hBax&hbax S184a

Delta TK

Caspase 3

RIP1

RIP3

apoptin

The reason that we consider these kinds of genes will be introduced in the following description.

Hbax&hbax mutant

Hbax is a member of the Bcl-2 related protein family from human. The Family contains pro-apoptotic and anti-apoptotic proteins, and the balance among them determines the cell survival. Hbax is the pro-apoptotic protein. During apoptosis, hbax will insert into the mitochondrial outer membrane and form permeable channels, release pro-apoptotic signals, finally lead to apoptosis[1].

Hbax S184a[3] is a mutant of hbax that can constantly insert into mitochondrial outer membrane. We guess that it may have stronger apoptosis-induced effect than normal hbax.

It have been reported that the overexpression of this gene can successfully induce apoptosis in Hela cell line and HEK-293 cell line[3], due to its generality, we determine to use it as one of the candidates that we will try.

However, when we express them in Hep G2 cell lines, they can not induce observable apoptosis. Probably because this pathway have been blocked in many kinds of cancers. So we eliminate this gene finally. But, although it can not successfully kill Hep G2 cell line(and probably most kinds of cancers), we discover that the pathway is conserved in yeast,so we also try to express the gene and its mutant in yeast and find the killing effect is dose-dependent. So they may be useful in designing safety device when using yeast as chassis. We finally submit it and its mutant form as an improvement of the pre-existing part of part registry.

(show results)

Delta TK:

TK is the abbreviation of thymidine kinase from HSV(Herpes simplex virus). It can convert the non-toxic prodrug ganciclovior into toxic product that can incorporate into replicating DNA strand and finally lead to apoptosis in cancer cells.[4]

Due to its bystander effect, TK expression in cancer cells under the ganciclovior treatment may also hurt the normal cells in neighborhood[5]. So that we use a truncated version that won’t lead to apoptosis when expressing in a low level.[6]

However, due to several reasons including its drug inducible property and the time limit, we haven’t try it yet. We may do it in the following days, and its drug inducible property may confer some advantages in some circuit design.

Caspase 3

Caspase 3 is the most downstream executer of apoptosis in mammalian cells. Almost every apoptosis process will need the execution of caspase 3. As a cysteine protease, it can directly cleavage proteins inside cells and take part in DNA fragmentation[7]. Caspase 3 contains two subunits,p17 and p12 ,which are translated in the same ORF. When cleavaged by caspase 9, another kind of protease involving in apoptosis, they will form a dimer that will act as an active form[8].

We split its gene into two parts, p17 and p12, and use leuzine zipper to direct the dimerization of the two subunits.[8](pitcture)

Although it is the most downstream executer of the apoptosis pathway, we finally do not try it for 3 reasons:

1. The apoptotic effect needs the two subunit to be expressed simultaneously, this increasing the complication of our circuit;
2. To overcome the anti-apoptotic protein XIAP[9], which is high-expressed in Hep G2 cell lines[10], we may need an extremely high expression of caspase 3 .
3. We have mistakenly clone the wrong ORF of two subunits from the plasmid, so it leave us no time to do the experiment of caspase 3 before regional jamboree=.=…;

We may also try it and another version of active caspase 3---the reconstitute caspase 3 [11]in following days.

A technology with many possibilities

The groundbreaking work that Yamakana and his group did in 2006 truly opened up a completely new research field to this world. It was a paradigm-shifting fundamental discovery, as it was the first demonstration that an intact differentiated somatic cell could be reprogrammed to become pluripotent. iPSCs technology, a new technology with many possibilities, attracts scientists and researchers all over the world, mainly for its three unique superiorities:

- No strong immunological reaction: Since iPS cells can be cultured from the patient's own cells, it is not quite possible to lead to immunological reaction when directionally differentiated iPSCs are transplanted after in vitro culture. While in 2011 several studies argued the weak immune reaction to transgene-free iPSCs, these arguments were subsided later by other scientists and Yamanaka himself, pointing out that "the most prominent study that reported the immunogenicity of the cells examined undifferentiated iPSCs, which will never be used in cell transplantation therapy"[6]. Recent work by Araki and Guha used syngeneic mouse models to demonstrate that transplanted iPSC-derived embryoid bodies, skin and bone marrow tissues engraft efficiently with almost no signs of rejection[6].

- "Thank god we don't need embryos anymore!": Simultaneously, the technology of iPSC was also hailed by many commentaries as a milestone advance that solved the ethical and political problems in stem cell research field. In the past, stem cell research relied heavily of embryonic stem cells harvested from embryos. Human embryos are not easy to come by, and many people consider research involving use of human embryonic stem cells to be ethically questionable[8]. With no further evidence, iPSC technology not only breaks the ethical barrier of relying on using eggs or earlier embryos for deriving stem cells, but also leads to a convenient way of obtaining patient-specific stem cells.

- Wide application in different fields: Nowadays, scientists are making progress toward applying iPSCs in many fields. One of the applications - which has already become reality - is to use iPS cells in both analysis of disease mechanisms and investigation of potential new treatments. In the future scientists hope to be able to use iPS cells to culture cells that can be transplanted into the body and replace diseased cells. Two examples are the dopamine-producing cells in the brain that degenerate in patients with Parkinson disease, and the insulin-producing cells that die off in patients with diabetes[13]. So far, more than 100 reports published in the past three years using disease-specific iPSCs[8].

These advances above together made the iPSC to be the "star cell" which offers immense potential as a source for regenerative therapies. However, the intrinsic qualities of self renewal and pluripotency that make these cells so therapeutically promising are also responsible for an equally fundamental tumorigenic potential, and the the intrinsic shortcomings of the current methods of inducing pluripotency even pose more uncontrollable risks on that. High rates of tumorigenicity has become a most crucial hurdle for large-scale clinical implementation of iPSC technology.

Challenges

Tumorigenicity as a clinical hurdle

The risks of iPSC tumorigenicity have been widely concerned over the past several years. In the study in 2009, Yamakana tested 55 mice transplanted with SNS (secondary neurospheres) from 11 TTP-iPS cell clones, 46 mice among them died or became weak within 9 weeks after transplantation because of tumors. Whereas in contrast group, the number of the tumor-showing mice transplanted with SNS derived from the ES cell clones was only 3 among 34[11]. This result indicated that the mice transplanted with iPSCs have a much higher rate of tumorigenicity than with ESCs.

- The "fantastic four"are double-edged swords: In the initial study in 2006, Yamanaka has pointed out that among the four transcriptional factors he used, two (Oct-4 and Myc) were oncogenes[3]. This led to the result that the iPSCs with the four exogenetic oncogenes were more prone to tumorigenesis. However, subsequent studies successively substantiated that both the Oct-4 and Myc are not essentially required for cell self-renewal. For example, in making human iPS cells, Yu et al. used a different set of four factors (Oct-4, Sox-2, Nanog, and Lin-28) to induce pluripotent stem cells from human somatic cells. These results began to raise the scientists' hopes to find the better combination of the cocktails to induce the safer iPSC. However, it turned out to be wishful thinking.

Scientists figure out that whatever the combination is, almost every inducing/reprogramming factors remaining in the cocktail are oncogenes by definition. Their over-expression has be associated with some forms of cancer. Of particular importance is the MYC transcription factor, which has emerged as one of the fundamental genes shared by iPSCs and cancer. Ectopic activation of OCT4 in somatic cells, induces dysplastic development and features of malignancy. NANOG has a role in the self renewal of CD24+ cancer stem cells in hepatocellular carcinoma. SOX2 has been shown to drive cancer-cell survival and oncogenic fate in several cancer types, including squamous cell carcinomas of the lung and esophagus. Klf-4 has been reported to promote DNA repair checkpoint uncoupling and cellular proliferation in breast cancers by p53 suppression[7].

- Risks from delivery methods: Compared to ESCs, iPSCs are exposed to a number of factors that could promote oncogenic transformation, such as genomic insertion of reprogramming vectors, over expression of oncogenic transcription factors and a global hypomethylation resembling that seen in cancers[7].

Methods of diminishing the tumorigenic transformation of iPSCs have mainly focused on a variety of gene delivery vectors that minimize genomic disruption. These strategies can be generally divided into two categories: integrating vectors that can be excised from the host genome and non-integrating vectors. However, all methods can not escape their shortcomings of low transduction efficiency, or even bring about new risks. Here is a table which concludes the oncogenic risks associated with methods of inducing pluripotency in somatic cells[7].

From the table above, we can generally realize that these promising strategies for teratoma prevention mostly improve the safety of iPSC generation at the expense of efficiency, which is still another remaining challenge in iPSC technology.

In summary, it wouldn't be more perfect if scientists found a method which guaranteed both the safety and efficiency in iPSCs technology. Just as Andrew S Lee wrote on Nature Medicine, "Ideally, the most stringent safety regimes would utilize a flexible, combinatorial approach that may require tailoring for specific PSC lines or graft types. Should these techniques fail to adequately remove enough residual PSCs, retrospective tumor treatments may also be used, including oncologic chemotherapy, radiation and surgery or the incorporation of suicide ablation genes[7]." Admittedly, just as the saying goes, "You can't have your cake and eat it!" With our knowledge of iPSC biology, it is not likable that such a perfect regime would emerge in several coming years. However, as a group of young pre-scientists like us, maybe, it is our time to look into this problem and try to figure it out in another way.