Development of viral & non-viral episomal vectors for gene therapy applications

Abstract

Gene therapy consists of methods which attempt to repair or replace defective genes responsible for disease, or to add genes to a therapeutic effect. To achieve this, two episomally maintained recombinant viral vectors have shown promising results: integration-deficient lentiviral vectors (IDLVs), and adeno-associated virus (AA V) vectors. The non-integrating nature of these vectors improves their safety profile but also limits transgene retention as nuclear episomes generally get lost during cell division. In the present study, the establishment of stable replicating episomes via transduction with AA V and IDL V gene therapy vectors was examined in CHO cells. Different DNA elements and cell culture conditions were evaluated, and in particular the effects of (i) DNA elements called S/MARs (scaffold/matrix attachment regions) which are involved in chromatin organisation, transcription and replication, and (ii) induction of transient cell cycle arrest in transfected and transduced cell populations. In the case of both AA V and IDL V vectors, the incorporation of S/MAR elements into vector transcription units had only marginal effects on the establishment of stable transgene- positive cell populations, either with or without induction oftransient cell cycle arrest. However, a striking general result was observed in cell populations transduced with IDL Vs and subjected to a transient cell cycle arrest soon after transduction. Under these conditions, following release from cell cycle arrest and in the absence of any selection pressure, substantial populations (10-25%) of proliferating and stably transduced cells emerged and were maintained over at least 100 population doublings. This establishment of stable transduction was seen only with IDLVs, was crucially . dependent on the induction of a period of transient cell cycle arrest, occurred independently of the presence of S/MAR elements, and resulted in transgene-positive cell populations which could be isolated and propagated as stable clonal cell lines. In these polyclonal and clonal IDL V -transduced cell lines, the existence of non-integrated vector genomes in the form of multi-copy nuclear episomes was confirmed by evidence from linear amplification -mediated PCR, deep sequencing, Southern blotting and FISH (fluorescent in situ hybridisation). 2 The cumulative evidence suggests that transduction of eHO cells with IDL Vs followed by a short period of induced cell cycle arrest leads to the establishment of stable IDL V- based nuclear episomes which are transcriptionally active and undergo replication and segregation during cell division without the need for antibiotic-based or other positive selection pressure. Preliminary investigations were also done to test the capacity of combined IDL V transduction and transient cell cycle arrest to establish stable episome Hel.a cells and murine haematopoietic stem cells. However, further experiments are required either to optimise the protocol in these cells or to find other clinically relevant cell types in which the protocol can be implemented. The transfer of this technology to a variety of clinically relevant human stem or progenitor cell populations could improve the safety profile of a range of gene therapy strategies currently under investigation. 3EThOS - Electronic Theses Online ServiceGBUnited Kingdo