Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy

Abstract

Friedreich's ataxia (FRDA) is the most common inherited form of ataxia in humans. It is caused by severe downregulation of frataxin (FXN) expression instigated by hyperexpansion of the GAA repeats located in intron 1 of the FXN gene. Despite numerous studies focused on identifying compounds capable of stimulating FXN expression, current knowledge regarding cis-regulatory elements involved in FXN gene expression is lacking. Using a combination of episomal and genome-integrated constructs, we defined a minimal endogenous promoter sequence required to efficiently drive FXN expression in human cells. We generated 19 constructs varying in length of the DNA sequences upstream and downstream of the ATG start codon. Using transient transfection, we evaluated the capability of these constructs to drive FXN expression. These analyses allowed us to identify a region of the gene indispensable for FXN expression. Subsequently, selected constructs containing the FXN expression control regions of varying lengths were site specifically integrated into the genome of HEK293T and human-induced pluripotent stem cells (iPSCs). FXN expression was detected in iPSCs and persisted after differentiation to neuronal and cardiac cells, indicating lineage independent function of defined regulatory DNA sequences. Finally, based on these results, we generated AAV encoding miniFXN genes and demonstrated in vivo FXN expression in mice. Results of these studies identified FXN sequences necessary to express FXN in human and mouse cells and provided rationale for potential use of endogenous FXN sequence in gene therapy strategies for FRDA.

DNA methylation in Friedreich ataxia silences expression of frataxin isoform E

“…This is critical because gene and protein replacement therapies are actively being developed with the specific goal of replenishing frataxin-M 52 – 54 , i.e., inadvertently not addressing the deficiency of frataxin-E. It is noteworthy that gene therapy constructs containing varying lengths of intron 1 sequence have been designed 55 , and these could potentially deliver both isoforms of frataxin. Indeed, FXN gene reactivation strategies, also being developed 10 , 28 , 56 , 57 , may have an added advantage of utilizing the endogenous gene regulatory elements that could permit reactivation of both isoforms.…”

High Levels of Frataxin Overexpression Lead to Mitochondrial and Cardiac Toxicity in Mouse Models

“…To ensure the safety of future clinical trials, AAV vectors will need to be designed to manage appropriately their expression profile across these different tissues. This could be achieved with (1) synthetic weak promoter or the endogenous human frataxin promoter, 62 , 63 , 64 (2) vector de-targeting strategies, 65 , 66 and/or (3) the design of expression cassette responsive to negative cellular feedback loop. Leveraging our knowledge of the cellular response to toxic FXN overexpression might help achieve the self-regulation of the vector expression and avoid toxic overexpression.…”

Methylated and unmethylated epialleles support variegated epigenetic silencing in Friedreich ataxia

“…This heterochromatin plus an R-loop structure also known to form in the CpG island shore ( 38 ) are likely to pose a formidable block to transcriptional elongation, which is known to occur in FRDA ( 6 , 27–30 , 34 , 35 ). Additionally, CpG island shore methylation plays a role in regulating transcription ( 41–43 ), and there is some evidence that the FXN CpG island shore contains regulatory elements ( 44 ). Therefore, the hypermethylated FRDA-DMR could possibly interfere with FXN transcriptional regulation.…”