Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

Abstract

Gene therapy represents an attractive alternative to treat hemophilia B. Here we established three hepatocyte-derived cell lines based on Huh7, PLC/PRF/5, and Hep3B cells stably carrying a mutated canine FIX (cFIXmut) transgene containing a single point mutation in the catalytic domain. Based on these in vitro models resembling a commonly used canine large animal model, the tetracycline-controlled transcriptional activator (Tet-on)-inducible CRISPR/Cas9 system and an optimized donor were used to correct mutated cFIX gene through homology-directed repair (HDR). For efficient delivery of designer nuclease and donor DNA, we produced a high-capacity adenovirus vector type 5 (HCAdV5) containing the Tet-on-inducible cFIX-specific CRISPR/Cas9 system and a single-stranded adeno-associated virus type 2 vector (ssAAV2) containing the modified donor. Moreover, we designed a single HCAdV5 delivering all components for HDR. Our amplification-refractory mutation system based on qPCR analysis (ARMS-qPCR) revealed that the single vector application in Huh7-cFIXmut cells resulted in up to 5.52% HDR efficiencies, which was superior to the two-vector strategy. Furthermore the single vector also resulted in increased phenotypic correction efficiencies assayed by ELISA. We conclude that HDR in combination with viral vector delivery holds great promise for the correction of mutated FIX in disease models.

Adenovirus vectors in hematopoietic stem cell genome editing

“…It can be programmed to target any genomic locus followed by a 5 0 -protospacer adjacent motif sequence of NGG, with the specificity determined by the sgRNA containing a 20-nt guide sequence complementary to the genome locus of interest [92,93]. In addition to our work with ZFNs, TALENs, and CRISPR/Cas9 expressed from HDAd5/35 vectors [52,53,55,103], many other research groups have demonstrated successful genomic editing using Ad vectors, including HDAd, expressing CRISPR/Cas9 [104][105][106][107][108][109]. Moreover, a variety of Cas9 derivatives and orthologs have been developed, for instance, CRISPRa [96], Cpf1 [97], saCas9 [98], and Cas13 [99] to name a few.…”

“…Three of these trials sponsored by different companies have reached Phase III [two for hemophilia B (NCT03861273 and NCT03569891); one for hemophilia A (NCT03370913)] [16]. Efficient targeted integration of a F9 gene was also demonstrated by using a single HDAd vector simultaneously carrying CRISPR/Cas9 and a donor cassette [105]. However, the widespread application of existing rAAV-based approach could face several obstacles: (a) mostly episomal nature of rAAV genomes affecting durability of efficacy, specifically in children with actively dividing hepatocytes (patients under 18 years old are excluded by most of current gene therapy trials); (b) the high cost of rAAV vector production, estimated to be > $1 M per patient; (c) the limited packaging capacity of rAAV which cannot accommodate large transcriptionally regulatory elements (viral titer drops precipitously with transgenes > 5 kb); and (d) the pre-existing immunity to AAV capsid leads to loss of efficacy and circumvents repeated administration (subjects with pre-existing antibodies to the used rAAV serotype are not eligible for current clinical trials) [228].…”

“…A CCR5-ZFN expressed by an Ad5/35 vector from Sangamo Therapeutics is currently tested clinically in HIV/AIDS patients [102]. In addition to our work with ZFNs, TALENs, and CRISPR/Cas9 expressed from HDAd5/35 vectors [52,53,55,103], many other research groups have demonstrated successful genomic editing using Ad vectors, including HDAd, expressing CRISPR/Cas9 [104][105][106][107][108][109]. Tsukamoto et al reported the production of a panel of first generation of Ad vectors containing CRISPR/Cpf1 system [110,111].…”

“…Targeted integration of a corrective F9 gene into the ROSA26 safe harbor locus by AdV-delivered CRISPR/Cas9, and a donor template has led to phenotypic correction in a murine model with juvenile hemophilia B [106]. Efficient targeted integration of a F9 gene was also demonstrated by using a single HDAd vector simultaneously carrying CRISPR/Cas9 and a donor cassette [105]. Instead of liver-directed gene transfer, HSC-directed F8 gene transfer has been investigated and showed advantages in tackling pre-existing immunity [237].…”

Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of genetic payloads for rapid cell engineering

“…Cas9) function in mammalian cells, many strategies have been devised that exploit their reprogrammable capacity for inducing double-strand breaks (DSBs) for performing gene editing. This includes delivering donor sequences via AdV/AAV, highyield PCR product, plasmid, or single stranded DNA oligonucleotides, and Cas/CRISPR components via plasmid, RNA, RNP, or replication-incompetent viruses (Jin et al 2019;Ehrke-Schulz et al 2017;Mangeot et al 2019;Liu et al 2018;Gao et al 2018;Moore et al 2015;Tálas et al 2017). In addition to targeted gene editing for fixing germ-line mutation diseases, the current "killer app" for inserting synthetic transgenes is CAR-T, whereby T cells are programmed to target cancer cells using an engineered chimeric antigen receptor (CAR) (Bloemberg et al 2019).…”