Increasing the Specificity of AAV-Based Gene Editing through Self-Targeting and Short-Promoter Strategies.
Mol Ther
; 29(3): 1047-1056, 2021 03 03.
Article
en En
| MEDLINE
| ID: mdl-33359790
ABSTRACT
Our group previously used adeno-associated viral vectors (AAVs) to express an engineered meganuclease specific for a sequence in the PCSK9 gene (M2PCSK9), a clinical target for treating coronary heart disease. Upon testing this nuclease in non-human primates, we observed specific editing characterized by several insertions and deletions (indels) in the target sequence as well as indels in similar genomic sequences. We hypothesized that high nuclease expression increases off-target editing. Here, we reduced nuclease expression using two strategies. The first was a self-targeting strategy that involved inserting the M2PCSK9 target sequence into the AAV genome that expresses the nuclease and/or fusing the nuclease to a specific peptide to promote its degradation. The second strategy used a shortened version of the parental promoter to reduce nuclease expression. Mice administered with these second-generation AAV vectors showed reduced PCSK9 expression due to the nuclease on-target activity and reduced off-target activity. All vectors induced a stable reduction of PCSK9 in primates treated with self-targeting and short-promoter AAVs. Compared to the meganuclease-expressing parental AAV vector, we observed a significant reduction in off-target activity. In conclusion, we increased the in vivo nuclease specificity using a clinically relevant strategy that can be applied to other genome-editing nucleases.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Regiones Promotoras Genéticas
/
Dependovirus
/
Endonucleasas
/
Edición Génica
/
Vectores Genéticos
/
Inhibidores de PCSK9
/
Lipoproteínas LDL
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Mol Ther
Asunto de la revista:
BIOLOGIA MOLECULAR
/
TERAPEUTICA
Año:
2021
Tipo del documento:
Article
País de afiliación:
Estados Unidos