UPDATE 09/18/2013

why liver?

Liver cancer is the fifth deadly cancer in the world and 55% of the patients are from China. Every year, thousands of patients wait for liver transplant to save their lives. Recent advances in iPS-derived hepatocytes provides a new way to solve the problem of donor shortage and reduce immuno-response. iPS-derived hepatocytes can also be used for treatment of metabolic disease and scanning for hepatic toxicity. 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.

why miR-122?

MicroRNA-122(miR-122) is a liver-specific microRNA that is most abundantly expressed in the liver as it accounts for approximately 70% of all hepatic microRNAs. Interesting, it is specifically highly expressed in normal hepatocytes instead of undifferentiated or cancerous cells. According to this, we have designed a complementary miR-122 target as a sensor to select the healthy hepatocytes we want when high level of endogenous miR-122 bind to our target in 3'UTR and then degrade the mRNA of suicide gene.

Which suicide gene?

Why suicide gene?

In our project, we try to build a circuit that can prevent the iPSC-differentiated tissues from tumor formation. Instead of using some medicines to kill them, like the traditional gene therapies did, what we want to achieve here is to make the tumor cells kill themselves, in other word, automatically commit suicide. So we decided to use an exogenous suicide gene.

The ideal suicide genes that fit our design here will be genes that can successfully induce apoptosis in cells and do not introduce any harmful effect to normal tissues. However, many apoptosis pathways are reported to be blocked in tumor cells. From recent reports and papers, we found out that there have been many genes reported to have important roles in mammalian cell apoptosis. After a long time comparison and consideration, our final candidates of suicide gene are listed below:

- hBax & hBax mutant

- delta TK

- caspase3

- rip1

- rip3

- apoptin

The introduction of each genes about its suicide mechanisms and the reasons why we choose them are as follows:

Which suicide gene?

hBax & hBax mutant

hBax is a member of the Bcl-2 related protein family from human. This 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 be inserted into the mitochondrial outer membrane and form permeable channels, release pro-apoptotic signals, finally lead to apoptosis[1]. The pathway of apoptosis is showed in Figure 1.

Figure1. apoptosis pathway induced by hBax

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

It have been reported that over expression of this gene can successfully induce apoptosis in both Hela cell line and HEK-293 cell line[2]. Due to its generality, we finally determined to use it as one of our suicide genes candidates.

However, our experiments results finally showed to us that, when expressed in Hep G2 cell line, which is a typical hepatoma cell line, they can not induce observable apoptosis. However, although it can not successfully kill Hep G2 cell line, we figured out that the pathway is conserved in yeast[4] ,so we also tried to transfect the gene and its mutant in yeast and finally proved that the killing effect is observable and even dose-dependent (BBa_K1061006). So this extra result may be useful for other team in the future who want to apply safety device in yeast. We finally submitted the hBax as a biobrick and its mutant form as an improvement of the pre-existing part of part registry.

(show results)

Caspase 3

Caspase 3 is the last downstream executer of apoptosis in most mammalian cells. Almost every apoptosis process needs the execution of caspase 3. As a cysteine protease, it can directly cleavage proteins inside cells and take part in DNA fragmentation[4]. 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[5].

Actually we split its gene into two parts, p17 and p12, and we used leuzine zipper to direct the dimerization of the two subunits[5].

However, in our project we finally decided to drop trying this gene, mainly for 3 reasons:

1. The apoptotic effect needs two subunit to be expressed simultaneously, which would increase the complication of our circuit.
2. To overcome the anti-apoptotic protein XIAP[6], which is high-expressed in Hep G2 cell line[7], we may need an extremely high expression of caspase 3, which would also be a problem for our experiment.
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=.=.

Besides, we may try to test another version of active caspase 3---the reconstitute caspase 3 [8]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, taking part in several program cell death pathways[9]. It has been reported that over expression of RIP 1 can induce both apoptosis and necrosis[10] in certain cell lines. So it is a potential suitable suicide gene that we can use in our device.

We tested rip1 in several cell lines, including HTC-75, Bosc, and Hep G2 cell lines. What we have observed is a mix of both apoptosis and necrosis( result ). Although it can induce necrosis in cancer cell lines, which may potentially cause inflammation in tissues, we still consider it as our choice of suicide gene. The reasons are as follows:

1. We know that in iPSC differentiation period, only a small fraction of cells will become cancerous at a certain time, so the necrosis of these cells would not likely to lead to severe inflammation.
2. Many apoptosis pathways have been reported to be blocked in tumor cells, but there is some evidence revealing that when the apoptosis pathways are blocked the necrosis pathway will be activated[11]. Besides, some recent papers showed that RIP1 can trigger cell necrosis only with extra exogenous medicine. In other circumstances RIP1 could lead to apoptosis.
3. We have cloned another receptor interacting protein kinase, RIP 3, which has been proved to interact with RIP 1 and lead to necrosis[12], so we think that even the over expression of RIP 1 can not successfully kill the cells, it may also co-express with RIP 3.

Surely, we will keep trying to find genes that only lead to apoptosis but not necrosis in the future, the better solution, in our mind, may be a combination of multiple apoptotic genes.

RIP 3

　　 RIP3, like RIP 1, is also a member of Receptor Interacting Protein family. It works via the interaction with RIP 1, and thus induces the necrosis pathway which RIP 1 mediated.[1]

However, it have also been reported that over expression of RIP 3 in certain cell lines can induce apoptosis[15].In our experiment, we found out that RIP 3 can lead to cell death in several cell lines, including HTC-75 cell lines and HEK-293 cell lines.

Because RIP 3 mainly induces necrosis, we select it as our candidate of suicide genes for the same reasons as we select RIP 1. And we also suggest that when using RIP 3 to cure cancer, tight control expression system may be necessary.

Apoptin

Apoptin, a protein first isolated from Chicken anemia virus, has been regarded as a potential drug for cancer treatment[16]. It has been reported in over 70 cell lines that apoptin can selectively kill cancer cells but not normal cells[17]. The result of in vivo test in mice is very exciting: the intraperitoneal injection of vector carrying the apoptin seems not confer any observable side effect on mice[18].

The mechanism of how apoptin works is not fully understood. It probably works via the non-p53 apoptosis pathway[17],hence is not easy to be blocked. The localization of apoptin in cancer cells is in nuclear, while in normal cells it locates in cytoplasm[17] . This was proved by our experiment results.

A special character of apoptin is that it works like a sensor, probably by recognizing certain early signals of cancer formation[17]. These signals may be general, which explained the reason why the apoptin can kill such a broad spectrum of cancer cells. So the construct of EF-1alpha-apoptin may provide a general circuit for safety issues of gene therapy and renew the concept of sensor. Making use of the by-stander effect, TAT-apoptin[20] or SP-TAT-apoptin[21] may be powerful and provide a "safe environment" for the cells under genetic manipulation.

However, we still have to say that although Apoptin has been proved to be safe for many normal cell types, it has not been proved in all normal cell types. So we should consider and test thoroughly about the immune reaction effect, making sure that it does not happen or can be controlled.

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[22].

Due to its bystander effect, TK expression in cancer cells under the ganciclovior treatment may also hurt the normal cells in neighborhood[23]. So we use a truncated version that won't lead to apoptosis[24].

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

Future work: Combination of suicide genes? Or using the scalable miRNA circuit?

With our project proceeded, we found out that apoptosis pathways are generally blocked in cancer cell lines (maybe this's one of the reasons why cancers are so hard to deal with). However, Synthetic biology has a great advantage in designing complicated circuits, and hence, it will be possible to combine 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 publication work we also found out that the shRNA and miRNA expression technique, which is controllable[1][2], can successfully knock-down gene expression.

The circuit design based on shRNA and miRNA should be 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. The broader target of miRNA and shRNA is, the 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 differentiate cancer cells from iPS cells. However, the most common feature of cancer cells is self-renewal, which is also the property of iPS cells. For this reason, it is not easy to find a way to differentiate them. 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 this stage. 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 almost non-toxic to mammalian cells(we also get the data about this,see our result), 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 to express the suicide gene when detecting the cells are cancerous. So 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.

For this consideration, we also test the tet-on expression system in our experiment. Unforturnately, the latest result that we got shows that apoptin maybe harmful to iPS cells. But, this kind of construction may still be used in other kinds of stem cells, for instance, the adipose-derived stem cells.

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

There are 3 generations of tet-expression systems 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: other kinds of tet-expression systems?

When we are proceeding in our project, we discover that 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 fuse with TALE[31] or dCas9[32] which can achieve 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 are the systems that induce the expression of miRNA[33] or shRNA[34]. We have discuss the possible use of miRNA and shRNA in constructing gene therapy and safeguarding circuit, so we will pay attention to these systems in the future. (Show figures)

Why EF-1α promoter？

To consistently express the protein tTA of our circuit in a high level, we finally select a human elongation factor 1 alpha ( PEF1α ) to replace the original promoter pCMV to be our promoter in tet-off system.

Generous & strong

Since Elongation Factor 1α takes part in translation[1]，almost every types of mammalian cells need to express this factor. Hence its promoter, EF-1alpha, is constantly activated no matter what kind of state the cell is undergoing. In our design, the expression of our device is supposed to be stable and persistent during the whole differentiating process, so we choose EF-1alpha promoter to drive tTA transcription. Besides, EF-1alpha promoter is one of the strongest promoters that has been used in constructing mammalian circuit[2].

Without being silenced in PSCs

EF-1alpha promoter has been proved to be particularly useful in constructing stable cell lines[2]. Compared to another strong promoter, CMV promoter, which has also been widely used in constructing mammalian circuit, EF-1alpha promoter won't undergo transgenic silence in certain cell types, including the iPS cells in our project and hematopoietic stem cells et al[3].

Previous work[4] has showed that the expression level of gene driven by EF-1alpha promoter is more robust in several cell lines, which will provide convenience to construct general circuit used in different cell lines. The data that characterize in one type of cell lines will be reliable in other types.

(Figure from reference [4])

Why lentivral vector?

This year, we decided to use lentivirus vector to carry our circuits, deliver and long-term maintain of our circuit in our chassis genome. We will introduce the mechanism of lentiviral transfection system and the reasons why we choose it.

> What is lentiviral vector?

Lentiviral vectors (LV) are viral-based gene delivery systems that can stably deliver genes or RNAi into primary cells or cell lines with up to 100% efficiency. LVs bind to target cells using an envelope protein which allows for release of the LV RNA containing the gene or gene silencing sequence into the cell. The LVs RNA is then converted into DNA using an enzyme called reverse transcriptase by a process called reverse transcription. The DNA pre-integration complex then enters the nucleus and integrates into the target cell's chromosomal DNA. Gene delivery is stable because the target gene or gene silencing sequence is integrated in the chromosome and is copied along with the DNA of the cell every time the cell divides. One of the discriminating features of LVs is their ability to integrate into non-dividing cells, in contrast to other vectors that either don't integrate efficiently into chromosomal DNA (e.g. non-viral, Adenoviral and Adenoviral-Associated vectors) or can only integrate upon cell division (e.g. conventional Retroviral vectors).

> Why we choose it?

Because our device is supposed to be expressed stably in a long term and therefore persistently safeguard the cells under any condition, we finally choose lentivirus to be our delivery system into iPSCs, for its high transfection efficiency and the ability to integration into host genome. Though we can never escape the fact that the use of lentivirus could increase the oncogenic risks in cells at the same time, however, considering another fact that almost every inducing/reprogramming factors in iPSCs are oncogenes, maybe the better thing we could do here is to deliver a monitor device which would persistently safeguard the cells in a long term.

To sum up, we choose lentivirus as our delivery system mainly for the following reasons:


• Lentivector can integrate into human genome in a multi-copy way.

• Lentivector can stably expressed in wide types of cell lines, without being silenced or irreversible transgenic suppression in ESCs and iPSCs.

• Lentivector has a relatively higher efficiency compared with other integrating virus.

• Lentivector is relatively safer in all integrating virus.

The lentivector we used in our project is the 3rd generation inducible lentivirus pPRIME[1], whose packaging plasmids are psPAX2 and pMD2G. We packaged lentivirus mix in 293T cell lines.