Project Details
Description
Abstract
Mitochondria have critical normal roles in metabolism, organ homeostasis, apoptosis and aging. Mitochondria
play important but still largely mysterious roles in human physiology. Mutations in both nuclear DNA associated
with proteins imported into mitochondria as well as mitochondrial DNA (mtDNA) are pathogenic. Despite this
clear association of genotype with disease, there are no current treatments for patients with mitochondrial
disease.
Mitochondria represent a unique cellular compartment with different DNA and RNA repair and editing
rules. For example, DNA nucleases that introduce double strand breaks and subsequent repair in nuclear DNA
induce the degradation of mtDNA. Indeed, none of the common repair pathways found in the nucleus are
active in mitochondria.
This proposal uses the well-established TALE-based programmable DNA binding system for targeting
mtDNA and the single-tube FUSX TALE assembly system to rapidly generate any protein-based genome
engineering reagents. Similarly, we use a new, protein-based and programmable RNA binding system based
on PPR proteins, a class of naturally occurring, mitochondrially localized RNA editors from plants.
This application harnesses the unique environment of mitochondria to generate a new toolbox to
expand the repertoire of tools to edit the human genome (RFA-RM-18-017). To develop these new
molecular reagents for mtDNA and mtRNA editing of somatic cells, we will conduct the following aims:
I. Develop new classes of mtDNA editing tools. Enhanced approaches to the use of mitoTALENs for
preferential degradation of pathogenic mtDNA variants for MELAS and KSS will be developed, including
novel nuclearly encoded reporters to detect non-mitochondrial off-targeting gene editing events. A new
class of TALE mitochondrial base editors will be developed to directly edit mtDNA for pathogenic variants.
II. mtRNA editing tools will be generated through harnessing the PPR family of naturally occurring
programmable RNA editors. We will use our new FUSR assembly system to rapidly develop optimal RNA
binding reagents, including the fusion to a set of test RNA nuclease or editing protein domains. Errant
fusion transcripts in mtDNA deletion or single base variants in heteroplasmic cells will be used as the test
paradigm for potential RNA editing platform development with the potential use as a therapeutic.
Milestones for initial stages include the establishment of novel mtDNA heteroplasmy converting mitoTALENs
against MELAS and KSS followed by testing of the new mtDNA base editor. For mtRNA editing, establishing
PPR scaffolding rules for mtRNA binding followed by new programmable RNA nucleases and editors will be
established. Deliverables include these novel mtDNA and mtRNA editing systems as well as humans cells
with matched nuclearly encoded off-target reporter cassettes for use by any mitochondrial gene editing
therapeutic system.
Status | Finished |
---|---|
Effective start/end date | 8/21/18 → 7/31/23 |
Funding
- National Institute of Allergy and Infectious Diseases: $397,500.00
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