Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

Raed Ibraheim; Phillip W L Tai; Aamir Mir; Nida Javeed; Jiaming Wang; Tomás C Rodríguez; Suk Namkung; Samantha Nelson; Eraj Shafiq Khokhar; Esther Mintzer; Stacy Maitland; Zexiang Chen; Yueying Cao; Emmanouela Tsagkaraki; Scot A Wolfe; Dan Wang; Athma A Pai; Wen Xue; Guangping Gao; Erik J Sontheimer

doi:10.1038/s41467-021-26518-y

Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

Nat Commun. 2021 Nov 1;12(1):6267. doi: 10.1038/s41467-021-26518-y.

Authors

Raed Ibraheim^{1

2}, Phillip W L Tai^{3

4}, Aamir Mir^{1

2}, Nida Javeed¹, Jiaming Wang³, Tomás C Rodríguez¹, Suk Namkung³, Samantha Nelson⁴, Eraj Shafiq Khokhar¹, Esther Mintzer⁵, Stacy Maitland⁵, Zexiang Chen¹, Yueying Cao¹, Emmanouela Tsagkaraki^{6

7}, Scot A Wolfe^{5

8}, Dan Wang^{1

3}, Athma A Pai¹, Wen Xue^{1

5

6

8}, Guangping Gao^{3

4

8

9}, Erik J Sontheimer^{10

11

12}

Affiliations

¹ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
² Tessera Therapeutics, Inc., 200 Sidney Street, Cambridge, MA, 02139, USA.
³ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
⁴ Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
⁵ Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
⁶ Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
⁷ University of Crete School of Medicine, Heraklion, Crete, 71003, Greece.
⁸ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
⁹ Viral Vector Core, University of Massachusetts Medical, School, Worcester, MA, 01605, USA.
¹⁰ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, 01605, USA. erik.sontheimer@umassmed.edu.
¹¹ Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, 01605, USA. erik.sontheimer@umassmed.edu.
¹² Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, 01605, USA. erik.sontheimer@umassmed.edu.

Abstract

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

Publication types

Evaluation Study
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Associated Protein 9 / genetics
CRISPR-Associated Protein 9 / metabolism*
Dependovirus / genetics*
Dependovirus / metabolism
Female
Gene Editing / methods*
Genetic Therapy
Genetic Vectors / genetics*
Genetic Vectors / metabolism
Humans
Male
Mice
Mucopolysaccharidosis II / genetics*
Mucopolysaccharidosis II / therapy
Recombinational DNA Repair*
Tyrosinemias / genetics*
Tyrosinemias / therapy

Substances

CRISPR-Associated Protein 9

Abstract

Publication types

MeSH terms

Substances

Grants and funding