Therapeutic Genome Mutagenesis Using Synthetic Donor DNA and Triplex-Forming Molecules

Abstract

Genome mutagenesis can be achieved in a variety of ways, though a select few are suitable for therapeutic settings. Among them, the harnessing of intracellular homologous recombination affords the safety and efficacy profile suitable for such settings. Recombinagenic donor DNA and mutagenic triplex-forming molecules co-opt this natural recombination phenomenon to enable the specific, heritable editing and targeting of the genome. Editing the genome is achieved by designing the sequence-specific recombinagenic donor DNA to have base mismatches, insertions, and deletions that will be incorporated into the genome when it is used as a template for recombination. Targeting the genome is similarly achieved by designing the sequence-specific mutagenic triplex-forming molecules to further recruit the recombination machinery thereby upregulating its activity with the recombinagenic donor DNA. This combination of extracellularly introduced, designed synthetic molecules and intercellularly ubiquitous, evolved natural machinery enables the mutagenesis of chromosomes and engineering of whole genomes with great fidelity while limiting nonspecific interactions. Herein, we demonstrate the harnessing of recombinagenic donor DNA and mutagenic triplex-forming molecular technology for potential therapeutic applications. These demonstrations involve, among others, utilizing this technology to correct genes so that they become physiologically functional, to induce dormant yet functional genes in place of non-functional counterparts, to place induced genes under regulatory elements, and to disrupt genes to abrogate a cellular vulnerability. Ancillary demonstrations of the design and synthesis of this recombinagenic and mutagenic molecular technology as well as their delivery and assayed interaction with duplex DNA reveal a potent technological platform for engineering specific changes into the living genome.

Antiviral effects of hepatitis B virus S gene-specific anti-gene locked nucleic acid in transgenic mice

“…Therefore, treatment with anti-sense-LNA does not achieve satisfactory effects. TFOs have emerged as potential regulators of biological activity for direct modifications of genomic DNA at selected sites through mutagenesis or homologous recombination and changing the anti-gene therapeutic method[ 21 - 23 ]. In the present study, anti-gene-LNA was assessed for its antiviral effects in transgenic mice.…”

Modification of the aminopyridine unit of 2′-deoxyaminopyridinyl-pseudocytidine allowing triplex formation at CG interruptions in homopurine sequences

“…The antigene strategy is based on the sequence-specific recognition of duplex DNA by triplex-forming oligonucleotides (TFOs) at the major groove side, which can modulate gene expression at the transcriptional level. TFOs have been exploited in a wide range of biological activities ( 1–4 ), including gene expression regulation ( 5–7 ), site-specific DNA damage ( 8–11 ), DNA repair, recombination ( 12 , 13 ) and mutagenesis ( 14 , 15 ).…”

A genome editing primer for the hematologist

“…[1][2][3][4] The uses of triple helix forming oligonucleotides as triggers and viral genomes such as adeno-associated virus (AAV) as donors for homologous recombination in the absence of additional nucleases continue to demonstrate promise as effective means of targeted genome modification. 5,6 However, the relatively low rates of gene correction that can typically be realized by these approaches has both limited research and deterred clinical applications. 7 Following the introduction of a double-strand break (DSB) at a locus, rates of homologous recombination increase by roughly 3 orders of magnitude.…”