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

Dr. Jixue Li, Dr. Yanjie Li, Dr. Jun Wang, Dr. Trevor J. Gonzalez, Dr. Aravind Asokan, Dr. Jill Napierala, and Dr. Marek Napierala; Hum Gene Ther. 2020;10.1089/hum.2020.053. doi:10.1089/hum.2020.053

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.


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