ABSTRACT
The development of curative treatments for mitochondrial diseases, which are often caused by mutations in mitochondrial DNA (mtDNA) that impair energy metabolism and other aspects of cellular homoeostasis, is hindered by an incomplete understanding of the underlying biology and a scarcity of cellular and animal models. Here we report the design and application of a library of double-stranded-DNA deaminase-derived cytosine base editors optimized for the precise ablation of every mtDNA protein-coding gene in the mouse mitochondrial genome. We used the library, which we named MitoKO, to produce near-homoplasmic knockout cells in vitro and to generate a mouse knockout with high heteroplasmy levels and no off-target edits. MitoKO should facilitate systematic and comprehensive investigations of mtDNA-related pathways and their impact on organismal homoeostasis, and aid the generation of clinically meaningful in vivo models of mtDNA dysfunction.
Subject(s)
Gene Editing , Genome, Mitochondrial , Mice , Animals , Genome, Mitochondrial/genetics , DNA, Mitochondrial/genetics , Mutation , Gene LibraryABSTRACT
Mitochondria host key metabolic processes vital for cellular energy provision and are central to cell fate decisions. They are subjected to unique genetic control by both nuclear DNA and their own multi-copy genome - mitochondrial DNA (mtDNA). Mutations in mtDNA often lead to clinically heterogeneous, maternally inherited diseases that display different organ-specific presentation at any stage of life. For a long time, genetic manipulation of mammalian mtDNA has posed a major challenge, impeding our ability to understand the basic mitochondrial biology and mechanisms underpinning mitochondrial disease. However, an important new tool for mtDNA mutagenesis has emerged recently, namely double-stranded DNA deaminase (DddA)-derived cytosine base editor (DdCBE). Here, we test this emerging tool for in vivo use, by delivering DdCBEs into mouse heart using adeno-associated virus (AAV) vectors and show that it can install desired mtDNA edits in adult and neonatal mice. This work provides proof-of-concept for use of DdCBEs to mutagenize mtDNA in vivo in post-mitotic tissues and provides crucial insights into potential translation to human somatic gene correction therapies to treat primary mitochondrial disease phenotypes.
Subject(s)
DNA, Mitochondrial/genetics , Gene Editing/methods , Genes, Mitochondrial/genetics , Genetic Therapy/methods , Mitochondrial Diseases/therapy , Animals , Dependovirus/genetics , Female , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Humans , Male , Mice , Mitochondria/genetics , Mitochondrial Diseases/genetics , Models, Animal , Mutagenesis , Mutation , Proof of Concept StudyABSTRACT
BACKGROUND: The rat has served usefully as a model for fecal incontinence and exploration of the mechanism of action of sacral neuromodulation. However, there is a gap in knowledge concerning representation(s) on the primary sensory cortex of this anatomical region. METHODS: Multi-electrode array (32 channels) and intrinsic optical signal (IOS) processing were used to map cortical activation sites following anorectal electrical stimulation in the rat. A simple method for expanding a 32-electrode array to a virtual 2700 array was refined. KEY RESULTS: The IOS method identified activation of parietal cortex following anorectal or first sacral nerve root (S1) stimulation; however, the signal was poorly localized and large spontaneous vasomotion was observed in pial vessels. In contrast, the resulting high-density maps showed two anatomically distinct cortical activation sites to anorectal stimulation. CONCLUSIONS & INFERENCES: There are two distinct sites of activation on the parietal cortex following anorectal stimulation in the rat. The implications for sacral neuromodulation as a therapy for fecal incontinence are discussed.
Subject(s)
Anal Canal/innervation , Brain Mapping/methods , Evoked Potentials, Somatosensory , Rats/anatomy & histology , Rectum/innervation , Somatosensory Cortex/physiology , Afferent Pathways/anatomy & histology , Animals , Electric Stimulation , Electrodes , Female , Pia Mater/blood supply , Rats, Wistar , Spinal Nerve Roots/anatomy & histology , Spinal Nerve Roots/physiology , Vasomotor System/anatomy & histology , Vasomotor System/physiologyABSTRACT
BACKGROUND: The rat has served usefully as a model for fecal incontinence and exploration of the mechanism of action of sacral neuromodulation (SNM). There remains a deficit in information regarding the location and type of spinal neurons which receive anorectal input and the effect of SNM on those neurons. METHODS: Single neuronal extracellular recordings of neurons receiving anorectal input were made at the S1 level of the spinal cord using sharp glass electrodes. SNM at S1 was delivered at 2 Hz for 3 minutes and its effect on discharge was quantified. KEY RESULTS: In total, 31 units (n = 14 animals) receiving anorectal synaptic input were recorded at the first sacral (S1) segmental level in either lamina III or IV of the dorsal horn. The inputs were classified according to afferent fiber conduction speed (16 Aδ, 11 Aß, and 4 C-fiber). The baseline firing frequency (ie, the mean firing frequency before the application of SNM) was 0.48 Hz ± 0.49 (mean ± SD) and 58% of units responded to acute SNM with either an increase or decrease in mean firing frequency. CONCLUSIONS & INFERENCES: In this study, the majority of spinal neurons receiving anorectal input changed their activity in response to SNM. These findings provide the basis for future studies which aim to explore the precise cellular mechanism of action of SNM on this fecal continence pathway.
