Abstract Details

B - Gene Targeting and Gene Correction -> B2 - Gene Targeting and Gene Correction – In Vitro Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

1061: Unconstrained Mitochondrial DNA Base Editing Enables Precise Disease Modeling

Type: Poster Session

Poster Board Number: 1061

Presentation Details

Session Title: Thursday Poster Session
Location:
Start Time: 5/18/2023 12:00
End Time: 5/18/2023 14:00

The human mitochondrial genome is a 16,569 bp long, multicopy, circular double-stranded DNA (dsDNA) molecule that encodes 37 genes essential for cellular energy metabolism. Pathogenic mitochondrial DNA (mtDNA) variants are prevalent in ~1 in 8,000 people and are causal in incurable metabolic disorders. Current mtDNA editing strategies rely on all-protein systems, such as transcription activator-like effector (TALE)-based technologies. In general, these technologies are composed of a TALE, which acts as a programmable DNA-binding domain, fused to an effector domain. In particular, dsDNA deaminase A (DddA)-derived cytosine base editors (DdCBEs) consist of TALE-DddA_tox fusions. Given its preference for dsDNA, DddA_tox, the deaminase domain of DddA, is split into two inactive halves to avoid toxicity. Thus, a DdCBE monomer incorporates either the N- or C-terminus of split DddA_tox downstream of a TALE. In the context of DdCBE pairs, binding of their respective DNA-binding domains to adjacent target sequences enables the reassembly of functional DddA_tox, followed by targeted cytidine deamination. Then, uracil glycosylase inhibitors positioned downstream of the split DddA_tox halves impede the excision of the resulting uracil residues. Subsequently, U•G intermediates are resolved into T•A base pairs during mtDNA replication, which occurs even in post-mitotic cells. This process results in programmed C•G-to-T•A conversions in mtDNA. Despite their flexibility and robustness, the versatility of canonical DdCBEs is limited by the requirement of a thymine immediately upstream of their respective TALE target sequences. This double 5’T constraint (one per DdCBE monomer) can be difficult or impractical in certain contexts. Based on our previous work on the FusX TALE Base Editor, a platform for the rapid design and assembly of mitochondrial base editors, we generated improved DdCBEs that bypass the 5’T requirement. First, seeking to characterize the activity and specificity profiles of improved DdCBEs relative to their canonical counterparts, we generated DdCBE pairs that target sequences preceded by 5’T or 5’V (V = A, C, G) in both canonical and improved formats. Hence, for a single locus, we tested four DdCBE pairs: a canonical or improved 5’T-compliant pair, and a canonical or improved 5’T-uncompliant pair. We chose four mtDNA loci to test this strategy: MT-ATP6, MT-CO1, MT-ND2, and MT-ND4. Thus far, we have not observed significant differences between the activities of 5’T-compliant canonical vs. improved DdCBEs. Additionally, utilizing improved DdCBEs, we installed the pathogenic m.3242G>A variant with no bystander editing at the MT-TL1 locus in HEK293T cells, demonstrating their potential for precise mitochondrial disease modeling. In summary, we are evaluating the efficiency and specificity of improved DdCBEs that bypass the 5’T requirement, which will support unconstrained mtDNA base editing as a strategy for disease modeling and gene therapy applications.

Santiago R. Castillo¹, Brandon W. Simone², Karl J. Clark³, Stephen C. Ekker<sup>3

1</sup>Department of Molecular Medicine, Mayo Clinic, Rochester, MN,²Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA,³Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN
 S.R. Castillo: None.