Engineering Genetic Systems for Treating Mitochondrial Diseases

Abstract

Mitochondria are intracellular energy generators involved in various cellular processes. Therefore, mitochondrial dysfunction often leads to multiple serious diseases, including neurodegenerative and cardiovascular diseases. A better understanding of the underlying mitochondrial dysfunctions of the molecular mechanism will provide important hints on how to mitigate the symptoms of mitochondrial diseases and eventually cure them. In this review, we first summarize the key parts of the genetic processes that control the physiology and functions of mitochondria and discuss how alterations of the processes cause mitochondrial diseases. We then list up the relevant core genetic components involved in these processes and explore the mutations of the components that link to the diseases. Lastly, we discuss recent attempts to apply multiple genetic methods to alleviate and further reverse the adverse effects of the core component mutations on the physiology and functions of mitochondria.

Keywords: gene therapy; heteroplasmy; mitochondrial DNA; mitochondrial disease; mitochondrial gene delivery.

Figure 1

Methods of reducing mtDNAs harboring mutations to treat misregulation of mitochondrial gene expression. (A) Nuclease cleaves the target mutated sites. (B) ZFN: constructed by fusing the Fok I endonuclease with an array of zinc fingers, each having a recognition ability for a three-base DNA sequence. (C) TALEN: constructed by fusing Fok I with single base-recognizing domains, TALEs. (D) CRISPR: gRNA recognizes the mutation-including domain, and Cas9 cleaves mtDNA around the gRNA-bound site. (E) DdCBE: TALEs recognize the periphery of the mutation, and the two parts of DddA were fused to form the whole DddA toxin that can convert cytosine to thymidine.

Figure 2

Methods for delivery of genetic components into mitochondria. (A) Microinjection is one of the physical methods. (B) MTS-mediated translocation can deliver DNAs into the mitochondrial matrix. (C) MTS-modified AAV particles can import target genes into mitochondria. (D) TPP-dendrimer/DNA polyplex is a dendrimer-based carrier that cannot import DNA into mitochondria but target mitochondria. Liposome-based carriers are DQAsome and MITO-Porter. (E) Mitochondrial expression of the gene transferred using DQAsome-mediated transfection system was confirmed. (F) MITO-Porters are surface-functionalized liposome-based carriers that increase transporting efficiency of target substances to mitochondria. (G) PNPASE can mediate the import of both small and large RNAs into mitochondria.