Retrovolution: HIV–Driven Evolution of Cellular Genes and Improvement of Anticancer Drug Activation

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.

Potent Sensitisation of Cancer Cells to Anticancer Drugs by a Quadruple Mutant of the Human Deoxycytidine Kinase

“…Since competition for the dCK enzyme between natural substrate and incoming drug molecules is crucial for the efficiency of cell death induction [ 31 , 32 ], this feature suggested that such mutants once introduced into cancer cells would not have induced a sensitisation to the treatment. For one of these (the triple mutant A100V, R104M, D133A) [ 28 ], it was indeed reported that its introduction into Messa10K cells, uterine sarcoma cells resistant to gemcitabine [ 33 ], resulted in no sensitisation to this drug [ 34 ].…”

“…We recently developed an experimental approach (retrovolution) where a gene of interest is inserted into conditional replication-defective VSV-pseudotyped HIV-1 derived vectors that are used to mimic multiple infectious cycles, during which the sequence of interest accumulates mutations due to the error-prone nature of the HIV-1 replication machinery [ 34 ]. This generates a library of variants of the gene of interest that are already inserted into a biological vector appropriate for an efficient delivery of the transgene to human cells, which can then be directly screened for the desired phenotype.…”

“…This generates a library of variants of the gene of interest that are already inserted into a biological vector appropriate for an efficient delivery of the transgene to human cells, which can then be directly screened for the desired phenotype. Using, as a sequence of interest, the cDNA coding for the human dCK, we isolated a mutant, named G12, which induces a 300-fold sensitisation to gemcitabine in cells originally resistant to this drug (Messa 10K), an effect 60 times stronger than that observed in controls cells where an extra copy of the wt dCK had been inserted [ 34 ]. This mutant is characterised by the presence of three mutations, located in positions of the protein distant from the active site and which are therefore unlikely to have been predicted on a rational basis.…”

“…The other two mutations (E247K and L249M) instead are located in the “base-sensing loop” that modulates the folding of the protein upon binding of the phosphate donor (ATP or UTP), ( S1 Fig ) [ 28 ]. No alteration of the degree of dimerization was however observed in vitro for the mutant, suggesting that the reason for the increased gemcitabine sensitivity that it induces is not attributable to a change in its expression expression level [ 34 ], but is possibly due to a modification of the overall structure of the enzyme.…”

“…G12 was isolated after screening of the library resulting from 17 successive cycles of retrovolution [ 34 ]. This way, mutations were introduced randomly in the gene with no external selection pressure applied during the procedure that would favour the isolation of the desired phenotype.…”