Long-Term Episomal Transgene Expression from Mitotically Stable Integration-Deficient Lentiviral Vectors

Abstract

Nonintegrating gene delivery vectors have an improved safety profile compared with integrating vectors, but transgene retention is problematic as nonreplicating episomes are progressively and rapidly diluted out through cell division. We have developed an integration-deficient lentiviral vector (IDLV) system generating mitotically stable episomes capable of long-term transgene expression. We found that a transient cell cycle arrest at the time of transduction with IDLVs resulted in 13-45% of Chinese hamster ovary (CHO) cells expressing the transgene for over 100 cell generations in the absence of selection. The use of a scaffold/matrix attachment region did not result in improved episomal retention in this system, and episomes did not form after transduction with adeno-associated viral or minicircle vectors under the same conditions. Investigations into the episomal status of the vector genome using (1) linear amplification-mediated polymerase chain reaction followed by deep sequencing of vector-genome junctions, (2) Southern blotting, and (3) fluorescent in situ hybridization strongly suggest that the vector is not integrated in the vast majority of cells. In conclusion, we have developed an IDLV procedure generating mitotically stable episomes capable of long-term transgene expression. The application of this approach to stem cell populations could significantly improve the safety profile of a range of stem and progenitor cell gene therapies.

The role of hypoxia-inducible factors in neovascular age-related macular degeneration: a gene therapy perspective

“…Viral vectors are more sustainable and effective than non-viral vectors, and thus preferable. Commonly used viral vectors in gene therapy models include recombinant adeno-associated virus (rAAV) vectors, adenovirus (Ad), and integrating-deficient lentivirus (IDLV) [215]. rAAV vectors have been the most effective for retinal gene therapy due to sustainable transduction of photoreceptors and RPE.…”

Retrovirus-based vectors for transient and permanent cell modification

“…A transient cell cycle arrest during transduction sufficed to establish mitotically stable LV episomes in the absence of selection without requiring any additional genetic elements. However, this has so far only been demonstrated in Chinese hamster ovary (CHO) cells [54]. For application of these concepts, it has to be considered that only a subgroup of transduced cells establishes episomal maintenance, thus necessitating at least an enlarged starting cell population.…”

The β-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells

“…The development of extrachromosomal vectors has mainly been driven by the need to address the safety issue of gene therapy vectors, in particular, the problem of insertional mutagenesis1, and involves vectors such as self-replicating stable episomes2, pFARs-plasmids free of antibiotic resistance markers3, and minicircle DNA plasmid derivatives lacking a bacterial backbone4. The presence of the scaffold/matrix attachment region (S/MAR) also confers long-term mitotic stability to integration-deficient lentiviral, episomal vectors56; however, that of the truncated S/MAR does not improve episomal retention7.…”