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[22] .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.

Which tet system?

Why do we need a tet-control system?

To achieve our goal, we need to ensure that the circuit we build into iPS Cells will not be harmful to the iPS cells when we are doing directed differentiation. This means, the circuit cannot be activated during in vitro differentiation. The easiest way will be to find a construct that can easily differentiated cancer cells from iPS cells. However, because all kinds of cancer cells can self renewal, so can iPS cells, we just cannot find any remarkable difference between iPS cells and cancer cells. More importantly, if the construct cannot successfully kill iPS cells, it can’t prevent teratoma formation, which is also one of our goals.

So how can we achieve our goal, which at first sight is impossible?

The answer is, the condition. We culture the iPS cells in vitro, and do the directed differentiation in vitro. This means, if we can find a way to control the expression of suicide genes in vitro, the construct won’t kill the iPS cells during these processes. But after the transplantation, both teratoma formation and cancer formation can be prevented. And what is the most robust and well-developing inducible system in mammalian cells?

It is the tet-control systems.

That’s why we need it.

What is tet control system?[1][28]

Tet control system is a beautiful combination of prokaryotic expression control systems and eukaryotic expression control system. TetR protein is a repressor from E,coli’s Tn10 transposon, which can repress the expression downstream of tet-operon by binding to tetO sequence when Tc(tetracycline) or Dox( Doxycline, a drug that similar to Tc) is absence. VP16 is an activation domain comes from Herpes simplex virus. By fusing them, we can get a protein which is named tTA. This protein will bind to tetO when dox is absence. By changing the four amino acids in TetR domain of tTA, we get rtTA, a fusion protein that will bind to tetO sequence when Dox is added. On the other side, by fusing 7 consecutive tetO sequences with a minimal CMV promoter, we will get a new kind of regulatory promoter---TRE. This promoter will be activated when tTA or rtTA bind to it.

It has been proved that tet-control system is low toxic, and can achieve high induction level and low leaky expression. More over, because the tet-inducible part is isolated from E.coli, the pleiotropic effect can be avoided..

Which kinds of tet-control system?

1.On or off, this is a question.

Basically, there are two kinds of tet control system. The first one, tet-off, is based on tTA, which will activate downstream expression when Tc or Dox is absence. The second one,tet-on, is based on rtTA, which will activate downstream expression when Dox is added.

So, which one should we use?

Our construction need the expression of suicide gene when it detects the cells become cancerous. So that means the tet part should be activated in vivo, only suppressed by the miRNA-122. After the transplantation, it will be inconvenient to intake dox constantly, because dox is somehow, a kind of antibiotics. Also, To intake dox after transplantation will need higher dose of dox to achieve the same level of expression, comparing to add dox into culture medium. So, the better choice will be using tet-off system, which can achieve constantly expression without adding dox. For preventing the expression of suicide gene in iPS cells, we only need to add dox during the in-vitro differentiation.

However, combining a special suicide gene apoptin, which will not kill normal cells when constantly expressing, with another suicide gene, it is possible to use tet-on system without constantly intake the dox in vivo. And the time of adding dox will be shorter.(show circuit design)

For this consideration, we also test the tet-on expression system in our experiment.

2. One, two, three: which is the golden generation?

There are 3 generations of tet-expression systems exist now.

The first generation is named tet-on&tet-off, the second generation is named tet-on advanced&tet-off advanced[28]and the developing 3 generation only contains tet on system now, which is named tet-on 3G[29].

The selection of tet-inducible system will depend on the suicide gene that we use. If the suicide gene is strong enough and induce cell death in a low expression level, leaky expression will be somehow, intolerable. But if the suicide gene is relatively weak, we need the one that can achieve highest expression.

We have tried to test the three generations of systems as well as we can, for determining which system we should used.

(show results)

Bonus: another tet-expression systems?

When we are proceeding in our project, we discover that the more reliable expression systems may exist. For example, we get a novel tet-control system which making use of KRAB[30], a transcription factor that can lead to reversible epigenetic modification of the promoter that we want to control. This can achieve a more robust and controllable expression of GOI(gene of interest. And the KRAB part have been used to fusion to TALE[31] or dCas9[32], which should be called “site-directed epigenetic modification”. Although this system may lead to irreversible silence of circuits in ES cells, which limit its use in our project, we still characterized this parts, for the future use(we may use it to construct safeguarding device for gene therapy).

(show figures)

Another tet- systems that we notice is the systems that induce the expression of miRNA[33] or shRNA[34]. We have discuss the possible use of miRNA and shRNA in construct gene therapy and safeguarding circuit, so we will pay attention to these systems in the future. (Show figures)