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

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Li, J; Li, Y; Wang, J; Gonzalez, TJ; Asokan, A; Napierala, JS; Napierala, M

Published Date

  • August 2020

Published In

Volume / Issue

  • 31 / 15-16

Start / End Page

  • 839 - 851

PubMed ID

  • 32527155

Pubmed Central ID

  • PMC7462031

Electronic International Standard Serial Number (EISSN)

  • 1557-7422

Digital Object Identifier (DOI)

  • 10.1089/hum.2020.053


  • eng

Conference Location

  • United States