Gene correction to treat mitochondrial associated diseases
Mitochondria are unique organelles in the cell that play a critical role in energy production, among other functions. They are responsible for creating a vast majority of the energy needed by the body to sustain life and organ function. Thus, when they fail, as in the case of mitochondrial diseases, a cell is not capable of generating sufficient energy and cell death follows. This leads to dysfunction in organs with the highest energy demands, such as the heart, brain and muscles. Despite decades of efforts, optimal treatments have not yet been established.
Mitochondrial proteins are encoded by both nuclear (nDNA) and mitochondrial (mtDNA) genomes. Mitochondria have their own translation system to synthesize mtDNA-encoded proteins essential for mtDNA replication, transcription, translation, and assembly of the oxidative phosphorylation system complexes. Mutations in either the nDNA or mtDNA as well as defects in the translation machinery can cause protein abnormalities that result in mitochondrial disease. Recent studies have identified homozygous mutations in the gfm1 gene, which encodes for the mitochondrial translation factor EFG1, in mitochondrial disease patients. EFG1 catalyzes the translocation of peptidyl tRNA from the ribosomal acceptor aminoacyl site to the peptidyl site following peptide bond formation, with the concomitant removal of the deacylated tRNA, advancement of the mRNA by one codon and exposure of the next codon. Thus, mutations in EFG1 may cause a deficiency in mitochondrial translation and function, thereby resulting in mitochondrial disease. Recent advances in genome editing technologies provide the possibility to target and correct the underlying genetic mutation in monogenic diseases, such as this one. These technologies do have limitations in that they have semi-random integration of the vectors, incomplete control over transgene copy number and expression level, a risk of insertional mutagenesis, as well as low efficiency. Recently, we have developed a gene editing strategy termed Homology-Independent Targeted Insertion (HITI) that is based on the CRISPR/Cas9 system, which harnesses elements of the NHEJ pathway to achieve efficient targeted knock-in in both proliferating and non-dividing cells. Our HITI method can specifically target the genetic locus associated with the disease with minimal insertion/deletion frequency. In addition, our HITI technology can be applied to gene correction in postmitotic cells in vivo.