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.

中文翻译：

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定义人类 Frataxin 基因中的转录调控元件：对基因治疗的意义。

弗里德赖希共济失调 (FRDA) 是人类最常见的遗传性共济失调形式。它是由位于FXN基因的内含子 1 中的 GAA 重复序列的过度扩张引起的 frataxin (FXN) 表达的严重下调引起的。尽管有大量研究集中在鉴定能够刺激FXN表达的化合物，但目前缺乏关于FXN基因表达中涉及的顺式调节元件的知识。使用附加型和基因组整合结构的组合，我们定义了有效驱动FXN所需的最小内源启动子序列在人体细胞中表达。我们在 ATG 起始密码子上游和下游生成了 19 个长度不同的 DNA 序列的构建体。使用瞬时转染，我们评估了这些构建体驱动FXN表达的能力。这些分析使我们能够确定对FXN表达必不可少的基因区域。随后，选定的包含FXN的结构不同长度的表达控制区域被位点特异性整合到 HEK293T 和人类诱导的多能干细胞 (iPSC) 的基因组中。在 iPSC 中检测到 FXN 表达，并在分化为神经元和心脏细胞后持续存在，表明定义的调节 DNA 序列的谱系独立功能。最后，基于这些结果，我们生成了编码迷你FXN基因的 AAV，并在小鼠体内证明了FXN 的表达。这些研究的结果确定了在人和小鼠细胞中表达 FXN 所必需的FXN序列，并为在 FRDA 基因治疗策略中潜在使用内源FXN序列提供了基本原理。