Long-term mitotic stability of integration-deficient lentiviral vector (IDLV) episomes

In genetic diseases, a mutated gene is the cause of the disorder, and gene replacement therapy with a functional transgene can be therapeutic. A vector, typically of viral origin, can deliver the relevant transgene to the target cells where required. Lentiviral vectors are a type of retroviral vector that integrates their proviral DNA into the cellular genome and can effectively transduce dividing and non-dividing cells, including stem cells. These vectors, however, have a finite risk of causing insertional mutagenesis. In contrast, integration deficient-lentiviral vectors (IDLVs), whose genome becomes a circular episome, can mediate stable transduction in quiescent cells, but the episomal genomes are rapidly lost during cell division. I have confirmed that IDLV episomes can be stabilised in Chinese hamster ovary (CHO) cells through the induction of a transient cell cycle arrest in G1 with a combination of methionine and serum depletion; it has been hypothesised that this arrest is required to allow sufficient time for interaction between the episome and the host nuclear components at the time of transduction. I have explored whether cell cycle arrest of CHO cells at other cell cycle stages can promote IDLV episome establishment. For this purpose, I have used nocodazole, nocodazole/thymidine, SAHA, RO-3306, mimosine and lovastatin and obtained positive results with nocodazole, SAHA, mimosine, and lovastatin. In an attempt to investigate the episomal status of the vector genomes, I have tried to use fluorescent in situ hybridisation (FISH). To further demonstrate clinical relevance, I have used nocodazole, nocodazole/thymidine and mimosine on induced pluripotent stem cells (iPSC), generated by reprogramming somatic cells to an embryonic state. These studies show the potential for manipulations of the cell cycle to affect the transduction efficiency of IDLVs, and perhaps their episomal status.