Rational design of base, sugar and backbone modifications improves ADAR-mediated RNA editing.

AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space.

Endogenous ADAR-mediated RNA editing in non-human primates using stereopure chemically modified oligonucleotides.

Technologies that recruit and direct the activity of endogenous RNA-editing enzymes to specific cellular RNAs have therapeutic potential, but translating them from cell culture into animal models has been challenging. Here we describe short, chemically modified oligonucleotides called AIMers that direct efficient and specific A-to-I editing of endogenous transcripts by endogenous adenosine deaminases acting on RNA (ADAR) enzymes, including the ubiquitously and constitutively expressed ADAR1 p110 isoform. We show that fully chemically modified AIMers with chimeric backbones containing stereopure phosphorothioate and nitrogen-containing linkages based on phosphoryl guanidine enhanced potency and editing efficiency 100-fold compared with those with uniformly phosphorothioate-modified backbones in vitro. In vivo, AIMers targeted to hepatocytes with N-acetylgalactosamine achieve up to 50% editing with no bystander editing of the endogenous ACTB transcript in non-human primate liver, with editing persisting for at least one month. These results support further investigation of the therapeutic potential of stereopure AIMers.

Stereochemistry Enhances Potency, Efficacy, and Durability of Malat1 Antisense Oligonucleotides In Vitro and In Vivo in Multiple Species.

Purpose: Antisense oligonucleotides have been under investigation as potential therapeutics for many diseases, including inherited retinal diseases. Chemical modifications, such as chiral phosphorothioate (PS) backbone modification, are often used to improve stability and pharmacokinetic properties of these molecules. We aimed to generate a stereopure MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) antisense oligonucleotide as a tool to assess the impact stereochemistry has on potency, efficacy, and durability of oligonucleotide activity when delivered by intravitreal injection to eye. Methods: We generated a stereopure oligonucleotide (MALAT1-200) and assessed the potency, efficacy, and durability of its MALAT1 RNA-depleting activity compared with a stereorandom mixture, MALAT1-181, and other controls in in vitro assays, in vivo mouse and nonhuman primate (NHP) eyes, and ex vivo human retina cultures. Results: The activity of the stereopure oligonucleotide is superior to its stereorandom mixture counterpart with the same sequence and chemical modification pattern in in vitro assays, in vivo mouse and NHP eyes, and ex vivo human retina cultures. Findings in NHPs showed durable activity of the stereopure oligonucleotide in the retina, with nearly 95% reduction of MALAT1 RNA maintained for 4 months postinjection. Conclusions: An optimized, stereopure antisense oligonucleotide shows enhanced potency, efficacy, and durability of MALAT1 RNA depletion in the eye compared with its stereorandom counterpart in multiple preclinical models. Translational Relevance: As novel therapeutics, stereopure oligonucleotides have the potential to enable infrequent administration and low-dose regimens for patients with genetic diseases of the eye.

Structure-based design, synthesis, and A-site rRNA cocrystal complexes of functionally novel aminoglycoside antibiotics: C2" ether analogues of paromomycin.

A series of 2"-O-substituted ether analogues of paromomycin were prepared based on new site-selective functionalizations. X-ray cocrystal complexes of several such analogues revealed a new mode of binding in the A-site rRNA, whereby rings I and II adopted the familiar orientation and position previously observed with paromomycin, but rings III and IV were oriented differently. With few exceptions, all of the new analogues showed potent inhibitory activity equal or better than paromomycin against a sensitive strain of S. aureus. Single digit microM MIC values were obtained against E. coli, with some of the ether appendages containing polar or basic end groups. Two analogues showed excellent survival rate in a mouse septicemia protection assay. Preliminary histopathological analysis of the kidney showed no overt signs of toxicity, while controls with neomycin and kanamycin were toxic at lower doses.