{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T16:17:52Z","timestamp":1762273072960,"version":"build-2065373602"},"reference-count":20,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2023,1,21]],"date-time":"2023-01-21T00:00:00Z","timestamp":1674259200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001352","name":"National University of Singapore","doi-asserted-by":"publisher","award":["NUHSRO\/2017\/068\/Bridging\/06","NUHSRO\/2021\/089\/Kickstart\/10\/LOA","T1-2014Apr-02","T1-BSRG 2015-05","H19H0G1006"],"award-info":[{"award-number":["NUHSRO\/2017\/068\/Bridging\/06","NUHSRO\/2021\/089\/Kickstart\/10\/LOA","T1-2014Apr-02","T1-BSRG 2015-05","H19H0G1006"]}],"id":[{"id":"10.13039\/501100001352","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Kickstart Grant","award":["NUHSRO\/2017\/068\/Bridging\/06","NUHSRO\/2021\/089\/Kickstart\/10\/LOA","T1-2014Apr-02","T1-BSRG 2015-05","H19H0G1006"],"award-info":[{"award-number":["NUHSRO\/2017\/068\/Bridging\/06","NUHSRO\/2021\/089\/Kickstart\/10\/LOA","T1-2014Apr-02","T1-BSRG 2015-05","H19H0G1006"]}]},{"DOI":"10.13039\/501100001459","name":"Ministry of Education of Singapore","doi-asserted-by":"publisher","award":["NUHSRO\/2017\/068\/Bridging\/06","NUHSRO\/2021\/089\/Kickstart\/10\/LOA","T1-2014Apr-02","T1-BSRG 2015-05","H19H0G1006"],"award-info":[{"award-number":["NUHSRO\/2017\/068\/Bridging\/06","NUHSRO\/2021\/089\/Kickstart\/10\/LOA","T1-2014Apr-02","T1-BSRG 2015-05","H19H0G1006"]}],"id":[{"id":"10.13039\/501100001459","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001348","name":"Agency for Science, Technology and Research","doi-asserted-by":"publisher","award":["NUHSRO\/2017\/068\/Bridging\/06","NUHSRO\/2021\/089\/Kickstart\/10\/LOA","T1-2014Apr-02","T1-BSRG 2015-05","H19H0G1006"],"award-info":[{"award-number":["NUHSRO\/2017\/068\/Bridging\/06","NUHSRO\/2021\/089\/Kickstart\/10\/LOA","T1-2014Apr-02","T1-BSRG 2015-05","H19H0G1006"]}],"id":[{"id":"10.13039\/501100001348","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Covalently closed dumbbell-shaped DNA delivery vectors comprising the double-stranded gene(s) of interest and single-stranded hairpin loops on both ends represent a safe, stable and efficacious alternative to viral and other non-viral DNA-based vector systems. As opposed to plasmids and DNA minicircles, dumbbells can be conjugated via the loops with helper functions for targeted delivery or imaging. Here, we investigated the non-covalent linkage of tri-antennary N-acetylgalactosamine (GalNAc3) or a homodimer of a CD137\/4-1BB-binding aptamer (aptCD137-2) to extended dumbbell vector loops via complementary oligonucleotides for targeted delivery into hepatocytes or nasopharyngeal cancer cells. Enlarging the dumbbell loop size from 4 to 71 nucleotides for conjugation did not impair gene expression. GalNAc3 and aptCD137-2 residues were successfully attached to the extended dumbbell loop via complementary oligonucleotides. DNA and RNA oligonucleotide-based dumbbell-GalNAc3 conjugates were taken up from the cell culture medium by hepatoblastoma-derived human tissue culture cells (HepG2) with comparable efficiency. RNA oligonucleotide-linked conjugates triggered slightly higher levels of gene expression, presumably due to the RNaseH-mediated linker cleavage, the release of the dumbbell from the GalNAc3 residue and more efficient nuclear targeting of the unconjugated dumbbell DNA. The RNaseH-triggered RNA linker cleavage was confirmed in vitro. Finally, we featured dumbbell vectors expressing liver cancer cell-specific RNA trans-splicing-based suicide RNAs with GalNAc3 residues. Dumbbells conjugated with two GalNAc3 residues triggered significant levels of cell death when added to the cell culture medium. Dumbbell vector conjugates can be explored for targeted delivery and gene therapeutic applications.<\/jats:p>","DOI":"10.3390\/pharmaceutics15020370","type":"journal-article","created":{"date-parts":[[2023,1,23]],"date-time":"2023-01-23T03:26:41Z","timestamp":1674444401000},"page":"370","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Non-Covalent Linkage of Helper Functions to Dumbbell-Shaped DNA Vectors for Targeted Delivery"],"prefix":"10.3390","volume":"15","author":[{"given":"Pei She","family":"Loh","sequence":"first","affiliation":[{"name":"Healthy Longevity Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore"},{"name":"Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117597, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Volker","family":"Patzel","sequence":"additional","affiliation":[{"name":"Healthy Longevity Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore"},{"name":"Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117597, Singapore"},{"name":"Department of Medicine, Addenbrooke\u2019s Hospital, University of Cambridge, Cambridge CB2 0QQ, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1038\/nrg2971","article-title":"State-of-the-art gene-based therapies: The road ahead","volume":"12","author":"Kay","year":"2011","journal-title":"Nat. Rev. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1007\/s10616-011-9413-2","article-title":"Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells","volume":"64","author":"Mok","year":"2012","journal-title":"Cytotechnology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"434","DOI":"10.2174\/156652309790031148","article-title":"Addressing the Challenge: Current and Future Directions in Ovarian Cancer Therapy","volume":"9","author":"Kaur","year":"2009","journal-title":"Curr. Gene Ther."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1016\/S0264-410X(02)00450-4","article-title":"DNA vaccination with linear minimalistic (MIDGE) vectors confers protection against Leishmania major infection in mice","volume":"21","author":"Sack","year":"2002","journal-title":"Vaccine"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1006\/mthe.2001.0322","article-title":"A Novel Minimal-Size Vector (MIDGE) Improves Transgene Expression in Colon Carcinoma Cells and Avoids Transfection of Undesired DNA","volume":"3","author":"Schakowski","year":"2001","journal-title":"Mol. Ther."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1073\/pnas.96.1.91","article-title":"Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus","volume":"96","author":"Zanta","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1581","DOI":"10.1038\/mt.2016.138","article-title":"Advanced design of dumbbell-shaped genetic minimal vectors improves non-coding and coding RNA expression","volume":"24","author":"Jiang","year":"2016","journal-title":"Mol. Ther."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Zogg, H., Singh, R., and Ro, S. (2022). Current Advances in RNA Therapeutics for Human Diseases. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23052736"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1038\/nbt.1560","article-title":"Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors","volume":"27","author":"Dassie","year":"2009","journal-title":"Nat. Biotechnol."},{"key":"ref_10","first-page":"2954","article-title":"Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery","volume":"52","author":"Zhou","year":"2011","journal-title":"J. Vis. Exp."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1016\/0003-9861(80)90121-6","article-title":"Synthesis of some cluster galactosides and their effect on the hepatic galactose binding system","volume":"205","author":"Kawaguchi","year":"1980","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1878","DOI":"10.1038\/mt.2011.145","article-title":"Targeting 4-1BB costimulation to disseminated tumor lesions with bi-specific oligonucleotide aptamers","volume":"19","author":"Pasto","year":"2011","journal-title":"Mol. Therapy."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.omtn.2018.01.006","article-title":"RNA structure design improves activity and specificity of trans-splicing triggered cell death in a suicide gene therapy approach","volume":"11","author":"Poddar","year":"2018","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e120","DOI":"10.1093\/nar\/gkv583","article-title":"Efficient Production of Superior Dumbbell-Shaped DNA Minimal Vectors for Small Hairpin RNA Expression","volume":"43","author":"Yu","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2198","DOI":"10.1038\/nprot.2007.325","article-title":"Enzymatic ligation assisted by nucleases: Simultaneous ligation and digestion promote the ordered assembly of DNA","volume":"2","author":"Cost","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3160","DOI":"10.1002\/anie.200353445","article-title":"Small-Interfering-RNA Expression in Cells Based on an Efficiently Constructed Dumbbell-Shaped DNA","volume":"43","author":"Taki","year":"2004","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1093\/nass\/3.1.191","article-title":"Direct and efficient synthesis method for dumbell-shaped linear DNA using PCR in vitro","volume":"3","author":"Taki","year":"2003","journal-title":"Nucleic Acids Symp. Ser."},{"key":"ref_18","unstructured":"Loh, P.S., and Patzel, V. (Unpublished work, 2022). Fluorescence microscopy images, Unpublished work."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1705","DOI":"10.1038\/sj.gt.3300784","article-title":"The bystander effect in the HSVtk\/ganciclovir system and its relationship to gap junctional communication","volume":"5","author":"Touraine","year":"1998","journal-title":"Gene Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3406","DOI":"10.1093\/nar\/gkg595","article-title":"Mfold web server for nucleic acid folding and hybridization prediction","volume":"31","author":"Zuker","year":"2003","journal-title":"Nucleic Acids Res."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/15\/2\/370\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:13:07Z","timestamp":1760119987000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/15\/2\/370"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,1,21]]},"references-count":20,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2023,2]]}},"alternative-id":["pharmaceutics15020370"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics15020370","relation":{},"ISSN":["1999-4923"],"issn-type":[{"type":"electronic","value":"1999-4923"}],"subject":[],"published":{"date-parts":[[2023,1,21]]}}}