Retrovolution: HIV-driven evolution of cellular genes and improvement of anticancer drug activation.

PLoS Genet. 2012 Aug;8(8):e1002904.

Rossolillo P, Winter F, Simon-Loriere E, Gallois-Montbrun S, Negroni M.

Architecture et Reactivité de l’ARN, Université de Strasbourg, CNRS, IBMC, Strasbourg, France.

Abstract

In evolution strategies aimed at isolating molecules with new functions, screening for the desired phenotype is generally performed in vitro or in bacteria. When the final goal of the strategy is the modification of the human cell, the mutants selected with these preliminary screenings may fail to confer the desired phenotype, due to the complex networks that regulate gene expression in higher eukaryotes. We developed a system where, by mimicking successive infection cycles with HIV-1 derived vectors containing the gene target of the evolution in their genome, libraries of gene mutants are generated in the human cell, where they can be directly screened. As a proof of concept we created a library of mutants of the human deoxycytidine kinase (dCK) gene, involved in the activation of nucleoside analogues used in cancer treatment, with the aim of isolating a variant sensitizing cancer cells to the chemotherapy compound Gemcitabine, to be used in gene therapy for anti-cancer approaches or as a poorly immunogenic negative selection marker for cell transplantation approaches. We describe the isolation of a dCK mutant, G12, inducing a 300-fold sensitization to Gemcitabine in cells originally resistant to the prodrug (Messa 10K), an effect 60 times stronger than the one induced by the wt enzyme. The phenotype is observed in different tumour cell lines irrespective of the insertion site of the transgene and is due to a change in specificity of the mutated kinase in favour of the nucleoside analogue. The mutations characterizing G12 are distant from the active site of the enzyme and are unpredictable on a rational basis, fully validating the pragmatic approach followed. Besides the potential interest of the G12 dCK variant for therapeutic purposes, the methodology developed is of interest for a large panel of applications in biotechnology and basic research.

PMID: 22927829

 

Supplement:

Directed evolution strategies, aimed at the creation of proteins or sequences with improved or modified functions, have gained increasing interest in the last years. In classical evolution procedures, a library of mutated genes is generated in vitro by error-prone PCR or DNA shuffling and screened for the desired properties by biochemical assays or, when possible, by genetic screening in bacteria. When the aim of the evolution is the creation of a mutant modifying the phenotype of the human cell, these screening procedures often bring to the isolation of false positives that, once introduced in the cell, fail to generate the desired phenotype. This is due to the differences in protein folding, the lack of post-translational modifications and to the complex epistatic network that regulates the expression of phenotypes in the cells of higher eukaryotes.

To bypass this problem we developed a system that, by exploiting the error-prone nature of HIV-1 replication machinery and the ability of the virus to introduce its genetic material in the human cell, allows the generation and screening of libraries of mutated genes directly in the human cell. Briefly, the procedure consists in producing replication-defective HIV-1 derived vectors containing a copy of the gene target of the evolution in their genomic RNA and mimicking successive HIV-1 replication cycles with these vectors (figure 1). At each cycle, the viral polymerase will introduce mutations in the genomic RNA and these mutations will be re-shuffled by the mechanism of recombination (1), generating a library of mutated target genes directly inserted in the vectors for the delivery into target human cells, where it can be screened for the desired phenotype.

Présentation1As a proof of concept, we applied the Retrovolution system to evolve the gene of the human deoxycytidine kinase, a cellular kinase involved in the salvage pathway of the deoxyribonucleotide biosynthesis (natural substrate deoxycytidine), which also activates by phosphorylation the deoxycytidine analogues used as chemotherapic compounds (like AraC and Gemcitabine) in cancer treatment (2). The appearance of resistant forms among tumor cells and the toxicity of high concentrations of nucleoside analogues for non-tumor cells represent the major limits for the treatments with these compounds. The aim of the evolution of the dCK gene was the isolation of a mutated kinase that could induce tumor cell death at low doses of chemotherapics, to be used as suicide gene in cancer therapies.

We used the library of mutated dCK genes obtained after 16 cycles of Retrovolution to transduce the target cells (Messa10K cells, uterine sarcoma cells resistant to the treatment with Gemcitabine due to a lack of dCK activity) at low multiplicity of infection, to ensure that each cell stochastically did not receive more than one vector. We then isolated the separate transduced clones and grew them in the presence of increasing concentrations of Gemcitabine. An MTT assay (which allows to estimate the proportion of dead cells in a population) on the different clones allowed us to calculate the Gemcitabine IC50 for each clone and to compare it to the one of the cells containing the wt dCK gene. By this procedure we were able to isolate a dCK mutant, G12, inducing cell death at doses of Gemcitabine 300 times lower than the doses needed to induce death in untreated cells, an effect 60 times higher than the one induced by wt dCK (figure 2). The insertion of the G12 mutant induced a sensitization phenotype to Gemcitabine even in other tumor cell lines (HT-29, HEK-293T).

Matteo Negroni-3

Figure 2. Ratios of cell survival in the presence of increasing concentrations of Gemcitabine. Colored arrows indicate the IC50 for the different populations. Blue line: untransduced Messa 10K cells; red line: Messa 10KI+ wt dCK; green line: Messa 10K + G12.

 

As the G12 mutant was characterized by mutations far from the active site and unpredictable on a rational basis, we performed biochemical assays on the purified protein to unravel the mechanism underlying the sensitization phenotype conferred by the mutant in vivo. These assays revealed that G12, while phosphorylating Gemcitabine with an efficiency comparable to the wt dCK one, completely lost the ability to phosphorylate the natural substrate dC, becoming specific for the chemotherapic compound (figure 3). This results in a reduction of the competition between the two substrates in vivo likely resulting in the sensitization of the cells to the prodrug.

figure 5.pptImportance of the study: The pragmatic approach of Retrovolution allowed us to isolate a dCK mutant sensitizing tumor cells to the chemotherapic compound Gemcitabine, a task where previous in vitro studies failed (3). The mutant we isolated constitutes a valid candidate to be used as a suicide gene in cancer therapies or as a negative selection marker in transplantation medicine. G12 is characterized by mutations unpredictable on a rational basis. We showed that the property of producing the library and allowing its screening in the human cell, ensuring that each mutant emerging from the procedure will be relevant for modifying the phenotype of the cells, is central for this findings.

Retrovolution thus opens new avenues for the modification of genes conferring complex phenotypes of interest to human cells for a broad field of applications in basic and applied research.

 

References

Original reference of the work described:

Rossolillo P, Winter F, Simon-Loriere E, Gallois-Montbrun S, and Negroni M

Retrovolution: HIV-driven Evolution of Cellular Genes and Improvement of Anticancer Drug Activation

PLoS Genetics (2012) 8(8): e1002904. doi:10.1371/journal.pgen.1002904.s

 

References cited

1. Negroni M and Buc H. Mechanisms of retroviral recombination. Ann. Rev. Genet. (2001), 35, 275-302.

2. Van Rompay AR, Johansson M, Karlsson A (2003) Substrate specificity and phosphorylation of antiviral and anticancer nucleoside analogues by human deoxyribonucleoside kinases and ribonucleoside kinases. Pharmacol Ther 100: 119-139.

3. Sabini E, Hazra S, Ort S, Konrad M, Lavie A (2008) Structural basis for substrate promiscuity of dCK. J Mol Biol 378: 607-621.

 

Contact:

Matteo Negroni

Retroviruses and molecular evolution laboratory

UPR9002

Institut de Biologie Moleculaire et Cellulaire

15, rue René Descartes

67084 Strasbourg

France

Tel. +33-388-417-006

Email: m.negroni@ibmc-cnrs.unistra.fr

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