Improved specificity and efficiency of in vivo adenine base editing therapies with hybrid guide RNAs

Madelynn N Whittaker^#^{1

2

3

4}, Lauren C Testa^#^{1

2

3}, Aidan Quigley^{1

2

3}, Dominique L Brooks^{1

2

3}, Sarah A Grandinette^{1

2

3}, Hooda Said^{5

6}, Garima Dwivedi^{7

8}, Ishaan Jindal^{1

2

3}, Daphne Volpp^{1

2

3}, Julia L Hacker^{1

2

3}, Ping Qu^{1

2

3}, Josh Zhiyong Wang⁹, Michael A Levine¹⁰, Rebecca C Ahrens-Nicklas^{3

11}, Qiaoli Li^{12

13}, Kiran Musunuru^{14

15

16

17}, Mohamad-Gabriel Alameh^{18

19

20

21}, William H Peranteau^{22

23}, Xiao Wang^{24

25

26}

Affiliations

¹ Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
² Division of Cardiovascular Medicine, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
³ Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
⁴ Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.
⁵ Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
⁶ Department of Bioengineering, George Mason University, Fairfax, VA, USA.
⁷ Division of Infectious Diseases, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁸ Penn Institute for RNA Innovation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁹ Agilent Technologies Inc., Santa Clara, CA, USA.
¹⁰ Division of Endocrinology and Diabetes, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
¹¹ Division of Human Genetics and Metabolism, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
¹² Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA, USA.
¹³ PXE International Center of Excellence in Research and Clinical Care, Thomas Jefferson University, Philadelphia, PA, USA.
¹⁴ Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. kiranmusunuru@gmail.com.
¹⁵ Division of Cardiovascular Medicine, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. kiranmusunuru@gmail.com.
¹⁶ Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. kiranmusunuru@gmail.com.
¹⁷ Division of Human Genetics and Metabolism, Children's Hospital of Philadelphia, Philadelphia, PA, USA. kiranmusunuru@gmail.com.
¹⁸ Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA. mg.alameh@pennmedicine.upenn.edu.
¹⁹ Department of Bioengineering, George Mason University, Fairfax, VA, USA. mg.alameh@pennmedicine.upenn.edu.
²⁰ Penn Institute for RNA Innovation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. mg.alameh@pennmedicine.upenn.edu.
²¹ Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. mg.alameh@pennmedicine.upenn.edu.
²² The Center for Fetal Research, Children's Hospital of Philadelphia, Philadelphia, PA, USA. peranteauw@chop.edu.
²³ Division of Pediatric General, Thoracic, and Fetal Surgery, Children's Hospital of Philadelphia, Philadelphia, PA, USA. peranteauw@chop.edu.
²⁴ Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. xiao8@pennmedicine.upenn.edu.
²⁵ Division of Cardiovascular Medicine, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. xiao8@pennmedicine.upenn.edu.
²⁶ Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. xiao8@pennmedicine.upenn.edu.

^# Contributed equally.

PMID: 41152569
DOI: 10.1038/s41551-025-01545-y

Improved specificity and efficiency of in vivo adenine base editing therapies with hybrid guide RNAs

Madelynn N Whittaker et al. Nat Biomed Eng. 2025.

. 2025 Oct 28.

doi: 10.1038/s41551-025-01545-y. Online ahead of print.

Authors

Affiliations

¹ Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
² Division of Cardiovascular Medicine, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
³ Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
⁴ Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.
⁵ Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
⁶ Department of Bioengineering, George Mason University, Fairfax, VA, USA.
⁷ Division of Infectious Diseases, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁸ Penn Institute for RNA Innovation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁹ Agilent Technologies Inc., Santa Clara, CA, USA.
¹⁰ Division of Endocrinology and Diabetes, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
¹¹ Division of Human Genetics and Metabolism, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
¹² Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA, USA.
¹³ PXE International Center of Excellence in Research and Clinical Care, Thomas Jefferson University, Philadelphia, PA, USA.
¹⁴ Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. kiranmusunuru@gmail.com.
¹⁵ Division of Cardiovascular Medicine, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. kiranmusunuru@gmail.com.
¹⁶ Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. kiranmusunuru@gmail.com.
¹⁷ Division of Human Genetics and Metabolism, Children's Hospital of Philadelphia, Philadelphia, PA, USA. kiranmusunuru@gmail.com.
¹⁸ Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA. mg.alameh@pennmedicine.upenn.edu.
¹⁹ Department of Bioengineering, George Mason University, Fairfax, VA, USA. mg.alameh@pennmedicine.upenn.edu.
²⁰ Penn Institute for RNA Innovation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. mg.alameh@pennmedicine.upenn.edu.
²¹ Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. mg.alameh@pennmedicine.upenn.edu.
²² The Center for Fetal Research, Children's Hospital of Philadelphia, Philadelphia, PA, USA. peranteauw@chop.edu.
²³ Division of Pediatric General, Thoracic, and Fetal Surgery, Children's Hospital of Philadelphia, Philadelphia, PA, USA. peranteauw@chop.edu.
²⁴ Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. xiao8@pennmedicine.upenn.edu.
²⁵ Division of Cardiovascular Medicine, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. xiao8@pennmedicine.upenn.edu.
²⁶ Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. xiao8@pennmedicine.upenn.edu.

^# Contributed equally.

PMID: 41152569
DOI: 10.1038/s41551-025-01545-y

Abstract

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.

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Conflict of interest statement

Competing interests: M.N.W. is a fellow with Artis Ventures. J.Z.W. is an employee of Agilent Technologies. R.C.A.-N. is an advisor to Latus Bio and AskBio. K.M. is an advisor to and holds equity in Verve Therapeutics and Variant Bio, is an advisor to LEXEO Therapeutics and Capstan Therapeutics, and receives research funding from Nava Therapeutics and Beam Therapeutics. M.-G.A. is an advisor to Afrigen Biologics. X.W. is an advisor to and receives research funding from Entact Bio, is an advisor to Pluvia Biotech, and receives research funding from Arbor Biotechnologies. The University of Pennsylvania and Children’s Hospital of Philadelphia have filed patent applications related to the use of base editing for the treatment of phenylketonuria and tyrosinemia (inventors include M.N.W., D.L.B., R.C.A.-N., K.M., W.H.P. and X.W.). The remaining authors declare no competing interests.

Update of

Improved specificity and efficiency of in vivo adenine base editing therapies with hybrid guide RNAs.
Whittaker MN, Testa LC, Brooks DL, Quigley A, Said H, Dwivedi G, Jindal I, Volpp D, Qu P, Wang JZ, Levine MA, Ahrens-Nicklas RC, Li Q, Musunuru K, Alameh MG, Peranteau WH, Wang X. Whittaker MN, et al. bioRxiv [Preprint]. 2025 Apr 9:2024.04.22.590531. doi: 10.1101/2024.04.22.590531. bioRxiv. 2025. Update in: Nat Biomed Eng. 2025 Oct 28. doi: 10.1038/s41551-025-01545-y. PMID: 38712058 Free PMC article. Updated. Preprint.

References

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1. Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017). - DOI - PubMed - PMC
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1. Brooks, D. L. et al. Rapid and definitive treatment of phenylketonuria in variant-humanized mice with corrective editing. Nat. Commun. 14, 3451 (2023). - DOI - PubMed - PMC
1. Brooks, D. L. et al. A base editing strategy using mRNA-LNPs for in vivo correction of the most frequent phenylketonuria variant. HGG Adv. 5, 100253 (2024). - PubMed

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Improved specificity and efficiency of in vivo adenine base editing therapies with hybrid guide RNAs

Affiliations

Improved specificity and efficiency of in vivo adenine base editing therapies with hybrid guide RNAs

Authors

Affiliations

Abstract

Conflict of interest statement

Update of

References

Grants and funding

LinkOut - more resources

Full Text Sources

Miscellaneous