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.

RIP 1

RIP1 is the abbreviation of Receptor interacting protein kinase 1 in mammalian cells. It is an important regulator of cell survival and death, and takes part in several program cell death pathways[12]. It has been reported that overexpression of RIP 1 can induce both apoptosis and necrosis[13] in certain cell lines. So it is a potential suicide gene that we may use.

We express this gene in several cell lines, including HTC-75, Bosc, and Hep G2 cell line. We observed both apoptosis and necrosis(show picture below). Although it can induce necrosis in cancer cell lines, we still consider it as our choice of suicide gene, for several reasons:

1. Only a small number of cells will become cancerous at any certain time, so the necrosis of these cells will not lead to severe inflammation, and can be treated easily;
2. Many apoptosis pathways have been blocked in cancer cells, but there is some evidence revealing that when the apoptosis pathways have been blocked the necrosis pathway will be activated[14], so we also consider the necrosis pathway.
3. We have cloned another Receptor interacting protein kinase, the RIP 3, which has been proved that can interact with RIP 1 and lead to necrosis[15], so we think that even we can not successfully kill the cells by overexpression of RIP 1,we may co-express it with RIP 3.

Surely, we will try to find genes that only lead to apoptosis but not necrosis in the future work, the solution may be a combination of multiple apoptotic genes, even by expression of multiple miRNAs. The intricacy problem of cancer apoptosis makes us believe that synthetic biology, which can easily design complicate circuits[16], will play an important role in gene therapy of treating cancer, where the simple solution may not exist.

RIP 3

RIP3 , like RIP 1, is also a member of Receptor Interacting protein family. It works via the interaction with RIP 1, and induce the necrosis pathway that RIP 1 mediated, but not the apoptosis pathway.[17]

However, it has also been reported that overexpression of RIP 3 in certain cell lines can induce apoptosis[18].In our experiment, we discover that RIP 3 can lead to cell death in several cell lines, including HTC-75 cell lines and bosc, one kind of HEK-293 cell lines. And we observe that when we want to construct the lentivirus that expressing RIP 3 , the MOI we get are significantly lower than the lentivirus expressing RIP 1 or apoptotin, probably means RIP 3 are far more toxic in bosc than RIP 1 or apoptin. Another effect that we discovered is when overexpressing RIP 3, the cell dies mainly through necrosis pathway, while overexpression of RIP 1 is mainly inducing apoptosis in cells.

Apoptin

Apoptin, a protein first isolated from Chicken anemia virus, has been regarded as a potential drug for cancer treatment[19]. The protein can selectively kill cancer cells but not normal cells, which has been proved in multiple experiments involving over 70 cell lines[20]. And the in-vivo test in mice is very exciting: the intraperitoneal injection of vector carrying the apoptin seems not conferring any observable side effect on mice[21].

The mechanism of how apoptin works is not fully understood. It probably works via the non-p53 apoptosis pathway[22],hence is not easy to be blocked. We have once observed the apoptotic effect of apoptin in Hep G2 cell lines, but we still need to repeat the experiment and get more data. The localization of apoptin in cancer cells is in nuclear, which is different from nomal cells[4] .And we does observe the localization of apoptin is in nuclear, by expressing a fusion protein that fuse the apoptin and eGFP.

A special character of apoptin is that it works like a sensor, and probably by recognizing certain early signals of cancer formation[23]. These signals may be general, which explained that why the apoptin can kill such a broad spectrum of cancer cells . So the construct EF-1alpha-apoptin(show figure)

may provide a general circuit for safety issues of gene therapy and renew the concept of sensor. And making use of the by-stander effect, TAT-apoptin[24] or SP-TAT-apoptin[25] may be more powerful and provide a “safe environment” for the cells under genetic manipulation.

However we still need to warn that although Apoptin have been proved to be safe for many normal cell types, it still has not been proved in all normal cell types. So before using apoptin in your project, you still need to consider this point and do some experiment for the cell type that you don’t want to kill. Also, when express it in vivo, you should consider and test thoroughly about the immune reaction effect, make sure that it does not happen or can be controlled.

Future:

Combination of suicide genes? Or using the scalable miRNA circuit?

When our project is proceeding, we discover that apoptosis pathways are generally blocked in cancer cell lines, and that’s the reason why cancer is so hard to treat. However, Synthetic biology has a great advantage in designing complicated circuits, and hence, it will be possible to introduce several suitable suicide gene together and construct a circuit that can overcome the blocking problem of apoptosis pathway in cancer cells. And by screening the published work we also find that the shRNA and miRNA expression technique today can successfully knock-down gene expression, and the expression of shRNA and miRNA is controllable[25][ 26]ircuit design based on shRNA and miRNA is scalable, because the sequence of them are short enough so that the whole circuit can be much more complicated than the design base on suicide proteins. And the broader target of miRNA and shRNA may be harder to be blocked. For this reason, the short RNA -based circuit, including CRISPRi[27 may be a promising way for cancer treatment and gene therapy design.

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.