High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells By AAV6 Vectors: strategies for Overcoming Donor-Variation and Implications in Genome Editing

Abstract

We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs). However, the transduction efficiency of the wild-type (WT) AAV6 vector varies greatly in HSPCs from different donors. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced less efficiently with the WT AAV6 vectors. The first strategy involved modifications of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705 + Y731F + T492V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density. The combined use of these strategies resulted in transduction efficiency exceeding ~90% in HSPCs at significantly reduced vector doses. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs.Genetically-modified autologous hematopoietic stem/progenitor cells (HSPCs) transplantation is the most promising therapeutic strategy to treat inherited genetic diseases, such as β -globin disorders 1 , leukodystrophies 2,3 , severe combined immunodeficiency 4 , Wiskott-Aldrich syndrome 5 , as well as acquired diseases such as AIDS 6 . Recent remarkable progress in genome editing tools include zinc-finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and the RNA-guided clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases. These targeted nucleases further expand the application of ex vivo editing of therapeutic genes into patient HSPCs 7 . Most of these methods require the delivery of extracellular DNA into HSPCs, the efficiency of which is frequently sub-optimal. In the present study, we describe a novel, adeno-associated virus (AAV) vector-based DNA delivery strategy to overcome the current drawbacks that limit the clinical use of HSPCs gene editing methods.The current drawbacks of HSPCs gene editing using viral methods such as chimeric adenoviral vectors, retroviral vectors, or integrase-defective lentiviral vectors (IDLV) include inefficient gene delivery, cytotoxicity, or DNA insertion. For example in a phase I/II clinical trial, a self-inactivating lentiviral vector achieved genetic