Improved specificity and efficiency of in vivo adenine base editing therapies with hybrid guide RNAs
Phenylketonuria (PKU), pseudoxanthoma elasticum (PXE) and hereditary tyrosinemia type 1 (HT1) are autosomal recessive disorders linked to the PAH, ABCC6, and FAH and HPD genes, respectively. Here we evaluate the off-target editing profiles of clinical lead guide RNAs (gRNAs) that, when combined with adenine base editors (ABEs), correct the recurrent PAH P281L variant, PAH R408W variant or ABCC6 R1164X variant, or disrupt either of two sites in the HPD gene (a modifier gene of HT1) in human hepatocytes. To mitigate off-target mutagenesis, we systematically screen hybrid gRNAs with DNA nucleotide substitutions. Comprehensive and variant-aware specificity profiling of these hybrid gRNAs reveals dramatically reduced off-target editing and reduced bystander editing in cells. In humanized PAH P281L and ABCC6 R1164X mouse models of PKU and PXE, we show that when formulated in lipid nanoparticles with ABE messenger RNA, selected hybrid gRNAs revert disease phenotypes, reduce off-target editing, increase on-target editing and reduce bystander editing in vivo. These studies highlight the use of hybrid gRNAs to improve the safety and efficiency of adenine base-editing therapies.
Authors
- Madelynn N. Whittaker (ORCID: https://orcid.org/0000-0002-3382-1134)
- Dominique L. Brooks
- Ishaan Jindal (ORCID: https://orcid.org/0000-0001-9568-3964)
- Julia Hacker (ORCID: https://orcid.org/0000-0001-5216-5016)
- Kiran Musunuru (ORCID: https://orcid.org/0000-0003-3298-0368)
- Garima Dwivedi (ORCID: https://orcid.org/0000-0002-6101-2481)
- Lauren Testa (ORCID: https://orcid.org/0000-0002-0169-0651)
- Michael A. Levine (ORCID: https://orcid.org/0000-0003-0036-7809)
- Josh Zhiyong Wang
- William H. Peranteau (ORCID: https://orcid.org/0000-0003-1608-861X)
- Mohamad‐Gabriel Alameh (ORCID: https://orcid.org/0000-0002-5672-6930)
- Xiao Wang (ORCID: https://orcid.org/0000-0001-7502-8292)
- Qiaoli Li (ORCID: https://orcid.org/0000-0002-8495-7103)
- Rebecca C. Ahrens‐Nicklas (ORCID: https://orcid.org/0000-0001-8243-7123)
- Ping Qü (ORCID: https://orcid.org/0009-0005-6442-6558)
- Daphne Volpp
- Aidan Quigley
- Sarah Grandinette
- Hooda Said (ORCID: https://orcid.org/0000-0002-2989-0209)
Institutions
- Children's Hospital of Philadelphia (US)
- Agilent Technologies (United States) (US)
- Thomas Jefferson University (US)
- George Mason University (US)
- Cardiovascular Institute of the South (US)
- Jefferson Institute (US)
- Penn Center for AIDS Research (US)
- University of Pennsylvania (US)
Publication Details
- Journal
- Nature Biomedical Engineering
- Published
- 2025-10-28
- DOI
- https://doi.org/10.1038/s41551-025-01545-y
- Citations
- 5
- Primary Topic
- RNA regulation and disease
- Type
- article
- Field-Weighted Citation Impact
- 4.03
Funders
- National Science Foundation
- American Heart Association
- National Institutes of Health
- Common Fund