Non-autonomous gene expression: causes and consequences

Abstract

Abstract
Since 1989, over 2,500 human gene therapy clinical trials have been conducted. A common, often implicit, assumption underlying these trials, and transgene experiments generally, is that genes are relatively autonomous in their expression. That is, the transcription of both the transgene and the endogenous genes mainly depends on the specific DNA sequence of the gene, along with promoter and enhancer elements. Therefore, if a transgene vector is successfully integrated into the target genome and escapes silencing, it should function regardless of the genomic location and, once integrated, it will not interfere with the transcription of the neighbouring host genes. However, several large-scale integrated transgene studies have shown that transgene expression can vary by many folds, depending on its genomic position, often adopting the expression profile of the surrounding area. On the other hand, less is known about whether transgenes commonly impact their neighbouring genes at a distance, a question of relevance not least for gene therapy safety. To investigate this issue, the clinically relevant Sleeping Beauty (SB) transposon gene vector system was adapted to facilitate a large-scale investigation of the hypothesis that transgenes can influence the transcription of their neighbouring genes from a distance. In total, 62 samples were generated from transfected and clonally expanded human induced pluripotent stem cells, with a total of 931 integrations. No evidence was found for the impact of a transgene on the transcription of neighbouring genes, even when the integration occurred only a few kilobases from the nearest host gene. This suggests that there are few safety concerns regarding transcriptional impact on neighbouring genes by SB, at least in a pluripotent context. However, due to the large genomic and transcriptomic datasets generated, several previously undescribed observations were made. For example, SB has a ~10% chance to cause a significant transcriptional mutation when randomly integrated, the standard method in gene therapy trials. Further, it was found that although considered one of the safest gene therapy vectors, SB is almost as likely to integrate next to an oncogene as in a genomic safety harbour (GSH). These are putative genomic sites where an integration is unlikely to cause secondary effects. Fourteen new strong GSH candidates were identified for potential future targeting methods. Moreover, despite being used for its assumed randomness concerning genomic features, SB was four times more likely than expected by chance to land within enhancer domains. The first SB integration hotspots in the human genome were also identified (regions with a significantly higher frequency of integrations than expected by chance), including ones located inside introns. Finally, despite earlier claims, SB does not necessarily require TATA sequences for integration, but only TA. These findings indicate that potentially mutagenic SB integrations may have been underestimated or previously excluded in earlier studies.

Date of Award	25 Mar 2026
Original language	English
Awarding Institution	University of Bath
Supervisor	Laurence Hurst (Supervisor) & Araxi O. Urrutia (Supervisor)