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We show that the sensor can distinguish both p53 knockout and cells expressing a common TP53 cancer mutation from otherwise isogenic TP53 wild-type cells. Importantly, the system is sensitive enough to specifically target TP53 loss-of-function cells with the HSV-TK pro-drug Ganciclovir both in vitro and in vivo. Our work opens new ways to programming cell intrinsic transformation protection systems that rely on endogenous components.
Under normal physiological conditions, expression of p53 is kept at low intracellular levels 3but in response to cellular stresses such as DNA damage, oncogene activation, ribosomal stress and hypoxia, expression of p53 is rapidly induced and the protein is stabilized 456.
As a consequence, p53 exerts its Phonak tinnitus app as a transcription factor, upregulating and downregulating genes implicated in cell cycle control, DNA repair, senescence and apoptosis 789. One might have expected that such a vital cellular gatekeeper would be essential during embryogenesis, but it turned out that a normal p53 gene is not strictly required for mouse development. Because of its prominent role in cancer, ways to target the p53 pathway have been long sought after.
Although targeting transcription factors such as p53 remains challenging 13some progress in this regard has been described by the identification of drugs that activate or restore the function of p53 in cells that carry particular p53 mutations However, first-generation drugs have not yet shown hoped-for clinical responses Advances in synthetic biology have made it possible to engineer cellular circuits with broad therapeutic potential 16 However, the generation of genetic circuits that rely on endogenous eukaryotic proteins and that sense proteins at low abundance remain the exception 2324 In this study, we build a genetic p53 device, capable of sensing the p53 status in human cells.
We show that the sensor is capable of discriminating p53 wild-type WT from otherwise isogenic, p53 knockout KO cells. In order to design a p53 sensor, we initiated investigation of a collection of different genetic elements derived from pregulated genes.
To ensure broad-range sensitivity to p53 alterations, we opted to rely on elements Genetic system builder both pupregulated and pdownregulated genes 27 First, we commenced to sense p53 transcription repressing abilities. All three elements showed reduced luciferase expression when the cells were co-transfected with the plasmid encoding WT p53, whereas the mutant RH-version and the control failed to repress luciferase expression, unmasking direct or indirect repressive activity of p53 on these promoters Fig.
Recently, several regions outside of the SCD core promoter were also implicated in pmediated repression Development of a genetic p53 sensor. Relative luciferase expression is given Auto action service cotransfection of indicated constructs. Interestingly, this element was repressed by expression of the p53 RH-mutant, indicating that this mutation switches the protein from an activator to a repressor on this model promoter Fig.
Based on these auspicious features the pshort element was selected for further studies. We then proceeded to combine repressed and activated elements in a two-gene network in which the luciferase output is co-repressed by WT p This was Genetic system builder through the use of the SCD. F4 element upstream of the luciferase gene in combination with the pshort element driving the expression of a microRNA30a-imbedded short hairpin RNA shRNA targeting the luciferase transcript Fig.
When both elements were co-transfected together with the plasmid encoding WT p53, an enhanced effect in p53 suppression from 6. F4 element alone to In order to address whether the sensor can function in the context of endogenous levels of p53, we exchanged the luciferase gene with Herpes simplex virus thymidine kinase HSV-TKto allow targeting of cells with the pro-drug Ganciclovir GCV.
This data suggested that the sensor is sensitive to endogenous p53 levels. Importantly, a 25 times lower concentration of GCV could still eliminate GFP-positive cells, albeit at a longer time interval, revealing a wide range of effective drug concentrations Supplementary Fig. These data demonstrate that p53 KO cells can be successfully targeted using the 2G sensor in vitro. In addition to p53 deletions, many p53 alterations result in gain-of-function mutants that exhibit oncogenic properties 40 To investigate whether the sensor can differentiate between mutant and WT p53 Fig.
The sensor can discriminate between WT and a hot-spot mutant of p Employed Cas9-proteins and the utilized gRNAs are shown. Lower GCV concentrations eliminated RQ cells as well, although longer incubation was required for complete depletion of mutant cells Supplementary Fig. These data provide evidence of high sensitivity of the p53 sensor, whereby an endogenous single point mutation in the TP53 allele could be sensed and used to successfully target mutant cells.
To investigate whether non-transformed, primary cells can also be sensitized to the loss of p53 we made use of mouse embryonic fibroblasts MEF carrying conditional Trp53 alleles Trp53 floxed MEFs, Fig. Taken together, these data revealed responsiveness of the sensor in primary cells. The sensor discriminates p53 WT from p53 KO cells in primary embryonic fibroblasts. Primers used for PCR are depicted.
We finally assessed whether the 2G sensor can specifically target p53 KO cells i n vivo. In order to address this question and directly compare the effect of GCV on WT-2G and KO-2G cells, both types were injected subcutaneously into opposite flanks of immunodeficient nude mice: WT-2G cells were injected into the right back side and KO-2G cells were injected into the left back side of the same Bedeutung flaggenfarben. Fifty days post injection, WT-2G and KO-2G tumors had grown to similar sizes in Elsa round control group treated with water, demonstrating that both cell lines initiate tumors with similar growth kinetics as xenografts.
Remarkably, GCV had no effect on WT-2G tumor formation, and only a minute effect on the tumor growth, while having a strong effect on both tumor formation and tumor growth in the case of KO-2G cells Supplementary Fig. Kon tiki ganzer film deutsch, the 2G sensor was able to selectively eliminate only the cells lacking the TP53 gene in an in vivo model.
Several recent advances in synthetic biology have focused on the design of mammalian transgene control devices that form a basis for the assembly of therapeutic networks in mammalian cells This is specifically true for mammalian transcription factors that usually represent a small fraction of the total proteome and where noise to signal ratio is thus quite high With this work we present a genetic p53 sensor, capable of sensing both the absence of the WT protein and the presence of mutated versions of the protein.
Our system demonstrates that endogenous mammalian proteins can be sensed, even at low levels. Built as a two-module unit, the sensor is a flexible construct, which should be amenable to further improvements. For instance, improving the scaffolding and targeting capabilities of the shRNA module could possibly contribute to future designs One important and interesting aspect would be to test if the sensor could be optimized toward particular p53 mutants, since it is known that transcriptional profiles of different gain-of-function mutants are not the same 9 Finally, the overall strategy to build sensors should be applicable to other tumor suppressors.
We envision that the combination of such sensors would allow the creation of robust transformation protection systems that could block cancer formation. The modular sensing device presented here can be seen as an illustration of a rational synthetic biology approach to both interrogating and programming cellular function. Whether the described network acts Genetic system builder a sensor only or as a programming device is determined solely by the primary output and can be accordingly adjusted.
In the case of GFP or any other easily detectable marker, the sensor functions as a reporter to gauge activity of the endogenous protein. This feature of the sensor can be particularly useful in designing screens for probing modulators of p53 activity.
Since both of these events would result in reduction of primary output signal, one could either select for specific cells HSV-TK as a primary output or simply monitor the signal GFP as a primary output.
In addition, many novel approaches, including synthetic lethality and collateral Rail nation nicht erreichbar screens, are being developed to either target defects in p53 or elucidate further the function of the protein 50 Here too, we suspect, the sensor could serve a role.
Possible p53 sensor applications. Important steps are highlighted by arrows. A model to determine p53 activity in primary cancer cells is presented. Red and white dots illustrate leukemic p53 mutant cells and healthy donor cells after transplantation in the patient, respectively. Arrows highlight important steps. We also foresee that the p53 reporter could be usefully employed in clinical applications. Sequencing the p53 gene in tumor samples provides the mutation status of the gene, but this information says little about the specific p53 activity in the tumor cells due to dominant negative protein interactions, mislocalization or MDM2 overexpression 5253 Transferring the sensor into primary tumor cells followed by readout of the output would provide a measure to estimate p53 activity in the cells Fig.
Finally, we envision possible therapeutic potential for a genetic p53 sensor. Coupling the p53 status to cell survival outcome provides an opportunity to target cells with compromised p53 function.
Important hurdles, however, would need to be considered if the sensor would be used clinically. First, as is the case for any gene therapy approach, efficient and safe delivery of Five spiele sensor would need to be resolved.
Recent advances in the field of oncolytic viruses provide a particularly appealing route to dealing with this problem. Several oncolytic viruses have been either in clinical trials or already approved for therapeutic purposes 56and it has been shown that prodrug-activating enzymes armed oncolytic viruses promote synergy with prodrugs Alternatively, the sensor could be used to modify oncolytic viruses so that they are capable of replication only in pdeficient cells.
This could be accomplished by introducing genes necessary for viral replication as the primary output of the sensor Fig. Clinical application of this approach can be illustrated by the example of acute myeloid leukemia with complex karyotype CK-AML where the alteration of WT p53 is known to lead to exceedingly dismal outcomes 58 As a consequence, rapid relapse is frequently observed in patients receiving allogeneic bone marrow transplants.
All of the mentioned therapeutic possibilities face, however, the problem of variable p53 expression levels in different cell types and tissues. Whether the physiological p53 levels are high enough to suppress the sensor output in p53 WT cells would need to be addressed for each case separately. One initial step in this direction would be to generate a transgenic mouse that carries the sensor and then investigate the sensor output HSV-TK in different tissues, providing essential information about biosafety.
Overall, our work opens new ways for thinking about programming cells by designing genetic safeguards that protect organisms from cancer development. All cells were checked for mycoplasma contamination and none was positive. For all the other plate formats, the amount of plasmid DNA or siRNA was scaled according to the surface area of the wells. Other constructs used in the study are: For establishing p53 mutant-expressing cells, pCMV-neo-Bam pasmids were used addgene, and This new fragment F4 then served as a tamplate for F3 element.
Malvorlagen bibi und tina all cases, embedding was done via overlapping PCR reactions.