DNA/RNA heteroduplex oligonucleotides: An unanticipated twist in the delivery of ASOs (2022)

Delivery of RNA oligonucleotide therapeutics into tissues and cells remains the rate-limiting problem to solve before these therapeutic platforms can be utilized to treat wide-spread human disease.1Dowdy S.F. Overcoming cellular barriers for RNA therapeutics.Nat. Biotechnol. 2017; 35: 222-229https://doi.org/10.1038/nbt.3802Crossref PubMed Scopus (518) Google Scholar, 2Hammond S.M. Aartsma-Rus A. Alves S. Borgos S.E. Buijsen R.A.M. Collin R.W.J. Covello G. Denti M.A. Desviat L.R. Echevarría L. et al.Delivery of oligonucleotide-based therapeutics: challenges and opportunities.EMBO Mol. Med. 2021; 13: e13243https://doi.org/10.15252/emmm.202013243Crossref PubMed Scopus (72) Google Scholar, 3Corey D.R. Damha M.J. Manoharan M. Challenges and opportunities for nucleic acid therapeutics.Nucleic Acid Therapeut. 2022; 32: 8-13https://doi.org/10.1089/nat.2021.0085Crossref PubMed Scopus (5) Google Scholar, 4Dowdy S.F. Setten R.L. Cui X.-S. Jadhav S.G. Delivery of RNA therapeutics: the great endosomal escape.Nucleic Acid Therapeut. 2022; https://doi.org/10.1089/nat.2022.0004Crossref PubMed Google Scholar Typical antisense oligonucleotides (ASOs) are single-stranded RNAs that sterically block splicing or are, in the case of Gapmer ASOs, a combination of RNA on the ends with an intervening DNA segment in the middle that activates RNase H to cleave the target mRNA.1Dowdy S.F. Overcoming cellular barriers for RNA therapeutics.Nat. Biotechnol. 2017; 35: 222-229https://doi.org/10.1038/nbt.3802Crossref PubMed Scopus (518) Google Scholar,2Hammond S.M. Aartsma-Rus A. Alves S. Borgos S.E. Buijsen R.A.M. Collin R.W.J. Covello G. Denti M.A. Desviat L.R. Echevarría L. et al.Delivery of oligonucleotide-based therapeutics: challenges and opportunities.EMBO Mol. Med. 2021; 13: e13243https://doi.org/10.15252/emmm.202013243Crossref PubMed Scopus (72) Google Scholar In contrast, siRNAs are double-stranded RNAs that induce RNA interference (RNAi) responses to knock down the target mRNA.1Dowdy S.F. Overcoming cellular barriers for RNA therapeutics.Nat. Biotechnol. 2017; 35: 222-229https://doi.org/10.1038/nbt.3802Crossref PubMed Scopus (518) Google Scholar,2Hammond S.M. Aartsma-Rus A. Alves S. Borgos S.E. Buijsen R.A.M. Collin R.W.J. Covello G. Denti M.A. Desviat L.R. Echevarría L. et al.Delivery of oligonucleotide-based therapeutics: challenges and opportunities.EMBO Mol. Med. 2021; 13: e13243https://doi.org/10.15252/emmm.202013243Crossref PubMed Scopus (72) Google Scholar Compared with native RNA, ASOs are heavily modified and have a full phosphorothioate backbone, where one of the non-bridging oxygen atoms is replaced with a more "hydrophobic" sulfur atom, which results in both a significant increase in metabolic stability and an enhanced escape from endosomes.1Dowdy S.F. Overcoming cellular barriers for RNA therapeutics.Nat. Biotechnol. 2017; 35: 222-229https://doi.org/10.1038/nbt.3802Crossref PubMed Scopus (518) Google Scholar,2Hammond S.M. Aartsma-Rus A. Alves S. Borgos S.E. Buijsen R.A.M. Collin R.W.J. Covello G. Denti M.A. Desviat L.R. Echevarría L. et al.Delivery of oligonucleotide-based therapeutics: challenges and opportunities.EMBO Mol. Med. 2021; 13: e13243https://doi.org/10.15252/emmm.202013243Crossref PubMed Scopus (72) Google Scholar,4Dowdy S.F. Setten R.L. Cui X.-S. Jadhav S.G. Delivery of RNA therapeutics: the great endosomal escape.Nucleic Acid Therapeut. 2022; https://doi.org/10.1089/nat.2022.0004Crossref PubMed Google Scholar All of the ASO's 2′ hydroxyls have been replaced with 2′ O-methyl (OMe), 2′ methoxyethyl (MOE) or 2′–4′ locked nucleic acids (LNAs).1Dowdy S.F. Overcoming cellular barriers for RNA therapeutics.Nat. Biotechnol. 2017; 35: 222-229https://doi.org/10.1038/nbt.3802Crossref PubMed Scopus (518) Google Scholar,2Hammond S.M. Aartsma-Rus A. Alves S. Borgos S.E. Buijsen R.A.M. Collin R.W.J. Covello G. Denti M.A. Desviat L.R. Echevarría L. et al.Delivery of oligonucleotide-based therapeutics: challenges and opportunities.EMBO Mol. Med. 2021; 13: e13243https://doi.org/10.15252/emmm.202013243Crossref PubMed Scopus (72) Google Scholar ASOs need a targeting domain to concentrate the ASO on the diseased cell type of interest. N-acetylgalactosamine (GalNAc), cholesterol, and anti-transferrin receptor monoclonal antibodies (mAbs) are all examples of ASO targeting domains that are being tested in the clinic for delivery of ASOs and siRNAs.1Dowdy S.F. Overcoming cellular barriers for RNA therapeutics.Nat. Biotechnol. 2017; 35: 222-229https://doi.org/10.1038/nbt.3802Crossref PubMed Scopus (518) Google Scholar, 2Hammond S.M. Aartsma-Rus A. Alves S. Borgos S.E. Buijsen R.A.M. Collin R.W.J. Covello G. Denti M.A. Desviat L.R. Echevarría L. et al.Delivery of oligonucleotide-based therapeutics: challenges and opportunities.EMBO Mol. Med. 2021; 13: e13243https://doi.org/10.15252/emmm.202013243Crossref PubMed Scopus (72) Google Scholar, 3Corey D.R. Damha M.J. Manoharan M. Challenges and opportunities for nucleic acid therapeutics.Nucleic Acid Therapeut. 2022; 32: 8-13https://doi.org/10.1089/nat.2021.0085Crossref PubMed Scopus (5) Google Scholar, 4Dowdy S.F. Setten R.L. Cui X.-S. Jadhav S.G. Delivery of RNA therapeutics: the great endosomal escape.Nucleic Acid Therapeut. 2022; https://doi.org/10.1089/nat.2022.0004Crossref PubMed Google Scholar To obtain optimal ASO activity, the conjugated targeting domain needs to be separated or cleaved from the ASO in the cell. While there are several approaches to address this issue, Prof. Takanori Yokota's laboratory at the Tokyo Medical and Dental University approached it in an entirely unique manner that paid off in spades. Back in 2015, they designed a prodrug-like double-stranded DNA/RNA heteroduplex oligonucleotide (HDO) that contains a Gapmer ASO that is hybridized to a complementary RNA (cRNA) oligonucleotide that the targeting domain is conjugated to, which is similar to siRNA designs.5Nishina K. Piao W. Yoshida-Tanaka K. Sujino Y. Nishina T. Yamamoto T. Nitta K. Yoshioka K. Kuwahara H. Yasuhara H. et al.DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing.Nat. Commun. 2015; 6: 7969https://doi.org/10.1038/ncomms8969Crossref PubMed Scopus (78) Google Scholar The cRNA contains a central stretch of unmodified phosphodiester RNA nucleotides that are opposite the DNA gap of the hybridized ASO. Once inside the cell, the duplex is recognized by RNase H, and the cRNA strand is selectively cleaved, resulting in release of the fragments along with the conjugated targeting domain, and thereby activating the ASO. While this is a straightforward approach to activate the ASO, what was completely unexpected is that it also resulted in a significant increase in ASO delivery and activity compared with the same active ASO delivered as a single strand. Mechanistically, the HDO appears to result in a more rapid and efficient shuttling of the ASO into the nucleus compared with the same ASO as a single strand.6Ono D. Asada K. Yui D. Sakaue F. Yoshioka K. Nagata T. Yokota T. Separation-related rapid nuclear transport of DNA/RNA heteroduplex oligonucleotide: unveiling distinctive intracellular trafficking.Mol. Ther. Nucleic Acids. 2021; 23: 1360-1370https://doi.org/10.1016/j.omtn.2020.11.022Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar The Yokota lab has now published a series of studies investigating HDOs in preclinical models.5Nishina K. Piao W. Yoshida-Tanaka K. Sujino Y. Nishina T. Yamamoto T. Nitta K. Yoshioka K. Kuwahara H. Yasuhara H. et al.DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing.Nat. Commun. 2015; 6: 7969https://doi.org/10.1038/ncomms8969Crossref PubMed Scopus (78) Google Scholar,7Kuwahara H. Song J. Shimoura T. Yoshida-Tanaka K. Mizuno T. Mochizuki T. Zeniya S. Li F. Nishina K. Nagata T. et al.Modulation of blood-brain barrier function by a heteroduplex oligonucleotide in vivo.Sci. Rep. 2018; 8: 4377https://doi.org/10.1038/s41598-018-22577-2Crossref PubMed Scopus (17) Google Scholar, 8Suzuki M. Ishibashi S. Iwasawa E. Oguma T. Saito Y. Li F. Otsu S. Ichinose K. Yoshioka K. Nagata T. Yokota T. Effective silencing of miR-126 after ischemic stroke by means of intravenous α-tocopherol-conjugated heteroduplex oligonucleotide in mice.Sci. Rep. 2021; 11: 14237https://doi.org/10.1038/s41598-021-93666-yCrossref PubMed Scopus (3) Google Scholar, 9Nagata T. Dwyer C.A. Yoshida-Tanaka K. Ihara K. Ohyagi M. Kaburagi H. Miyata H. Ebihara S. Yoshioka K. Ishii T. et al.Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood-brain barrier and knock down genes in the rodent CNS.Nat. Biotechnol. 2021; 39: 1529-1536https://doi.org/10.1038/s41587-021-00972-xCrossref PubMed Scopus (19) Google Scholar, 10Ohyagi M. Nagata T. Ihara K. Yoshida-Tanaka K. Nishi R. Miyata H. Abe A. Mabuchi Y. Akazawa C. Yokota T. DNA/RNA heteroduplex oligonucleotide technology for regulating lymphocytes in vivo.Nat. Commun. 2021; 12: 7344https://doi.org/10.1038/s41467-021-26902-8Crossref PubMed Scopus (2) Google Scholar, 11Nishi R. Ohyagi M. Nagata T. Mabuchi Y. Yokota T. Regulation of activated microglia and macrophages by systemically administered DNA/RNA heteroduplex oligonucleotides.Mol. Ther. J. Am. Soc. Gene Ther. 2022; 30: 2210-2223https://doi.org/10.1016/j.ymthe.2022.02.019Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar, 12Miyashita A. Kobayashi M. Ishibashi S. Nagata T. Chandrasekhar A. Zochodne D.W. Yokota T. The role of long noncoding RNA MALAT1 in diabetic polyneuropathy and the impact of its silencing in the dorsal root ganglion by a DNA/RNA heteroduplex oligonucleotide.Diabetes. 2022; 71: 1299-1312https://doi.org/10.2337/db21-0918Crossref PubMed Scopus (1) Google Scholar In this issue of Molecular Therapy: Nucleic Acids, Kaburagi et al. utilized the Yokota lab's previously published tocopherol-conjugated HDO (Toc-HDO) to investigate the ability of systemically administered Toc-HDOs to efficiently enter dorsal root ganglia (DRG) neurons in the peripheral nervous system. The DRG lacks a sufficient neurovascular barrier, enabling access to the interstitium surrounding DRG neurons. Impressively, intravenously (i.v.) administration of Toc-HDO resulted in a 2-fold higher target gene knock down in murine DRG neurons from the cervical to lumbar cord and in the sciatic nerve compared with the same single-stranded ASO. However, there are a couple of caveats when thinking of converting these observations into the clinical setting. First, for ASOs, this was an exceedingly high dose of 50 mg/kg (see Figure 6). Second, the MALAT1 target gene is the ASO field's go-to target gene because it is by far the best ASO and responsive target gene identified. As such, it is not surprising that it shows the best activity here. Third, having said that, a single 50 mg/kg dose of Tco-HDO targeting Scarb1 showed a ∼70% knockdown (see Figure 6). Lastly, because the ASO does not have the hydrophobic Toc lipid conjugated to it, the TocHDO to ASO comparison here is apples to oranges. However, this group has previously published the direct comparison and observed a significant improvement in HDO delivery and activity.9Nagata T. Dwyer C.A. Yoshida-Tanaka K. Ihara K. Ohyagi M. Kaburagi H. Miyata H. Ebihara S. Yoshioka K. Ishii T. et al.Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood-brain barrier and knock down genes in the rodent CNS.Nat. Biotechnol. 2021; 39: 1529-1536https://doi.org/10.1038/s41587-021-00972-xCrossref PubMed Scopus (19) Google Scholar Overall, the HDO technology defies the field. The collective results from the Yokota lab place a solid foundation for further improvements as HDOs move toward clinical development for a variety of indications, including DRG pathophysiology, neuropathic pain, and peripheral nerve diseases. R.L.S. was supported by the NCI CBIO training grant ( T32CA067754-25 ).