The therapeutic implications of all-in-one AAV-delivered epigenome-editing platform in neurodegenerative disorders
Boris Kantor, Bernadette O’Donovan, Joseph E. Rittiner, Dellila Hodgson, Nicholas Lindner, Sophia Guerrero, Wendy Dong, Austin Zhang, Ornit Chiba‐Falek
Abstract
Safely and efficiently controlling gene expression is a long-standing goal of biomedical research, and CRISPR/Cas system can be harnessed to create powerful tools for epigenetic editing. Adeno-associated-viruses (AAVs) represent the delivery vehicle of choice for therapeutic platform. However, their small packaging capacity isn’t suitable for large constructs including most CRISPR/dCas9-effector vectors. Thus, AAV-based CRISPR/Cas systems have been delivered via two separate viral vectors. Here we develop a compact CRISPR/dCas9-based repressor system packaged in AAV as a single optimized vector. The system comprises the small Staphylococcus aureus (Sa)dCas9 and an engineered repressor molecule, a fusion of MeCP2’s transcription repression domain (TRD) and KRAB. The dSaCas9-KRAB-MeCP2(TRD) vector platform repressed robustly and sustainably the expression of multiple genes-of-interest, in vitro and in vivo, including ApoE, the strongest genetic risk factor for late onset Alzheimer’s disease (LOAD). Our platform broadens the CRISPR/dCas9 toolset available for transcriptional manipulation of gene expression in research and therapeutic settings. Adeno-associated vectors (AAVs) are frequently used for the delivery of genetic cargo but their small packaging capacity is not suitable for delivery of large constructs, including most CRISPR/dCas9-effector systems. Here the authors report a compact dCas9-based repressor system packaged within a single, optimized AAV vector.