Nitric oxide modulates bone anabolism through regulation of osteoblast glycolysis and differentiation.

Previous studies have shown that nitric oxide (NO) supplements may prevent bone loss and fractures in preclinical models of estrogen deficiency. However, the mechanisms by which NO modulates bone anabolism remain largely unclear. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing arginine, the sole precursor for nitric oxide synthase-dependent (NOS-dependent) NO synthesis. Moreover, ASL is also required for channeling extracellular arginine to NOS for NO production. ASL deficiency (ASLD) is thus a model to study cell-autonomous, NOS-dependent NO deficiency. Here, we report that loss of ASL led to decreased NO production and impairment of osteoblast differentiation. Mechanistically, the bone phenotype was at least in part driven by the loss of NO-mediated activation of the glycolysis pathway in osteoblasts that led to decreased osteoblast differentiation and function. Heterozygous deletion of caveolin 1, a negative regulator of NO synthesis, restored NO production, osteoblast differentiation, glycolysis, and bone mass in a hypomorphic mouse model of ASLD. The translational significance of these preclinical studies was further reiterated by studies conducted in induced pluripotent stem cells from an individual with ASLD. Taken together, our findings suggest that ASLD is a unique genetic model for studying NO-dependent osteoblast function and that the NO/glycolysis pathway may be a new target to modulate bone anabolism.

A Single "All-in-One" Helper-Dependent Adenovirus to Deliver Donor DNA and CRISPR/Cas9 for Efficient Homology-Directed Repair.

In this study, we developed a single helper-dependent adenovirus (HDAd) to deliver all of the components (donor DNA, CRISPR-associated protein 9 [Cas9], and guide RNA [gRNA]) needed to achieve high-efficiency gene targeting and homology-directed repair in transduced cells. We show that these "all-in-one" HDAds are up to 117-fold more efficient at gene targeting than donor HDAds that do not express CRISPR/Cas9 in human induced pluripotent stem cells (iPSCs). The vast majority (>90%) of targeted recombinants had only one allele targeted, and this was accompanied by high-frequency indel formation in the non-targeted allele at the site of Cas9 cleavage. These indels varied in size and nature, and included large deletions of ∼8 kb. The remaining minority of recombinants had both alleles targeted (so-called bi-allelic targeting). These all-in-one HDAds represent an important platform for accomplishing and expanding the utility of homology-directed repair, especially for difficult-to-transfect cells and for in vivo applications.

Production of CRISPR/Cas9-Mediated Self-Cleaving Helper-Dependent Adenoviruses.

Prolonged expression of CRISPR/Cas9 raises concerns about off-target cleavage, cytotoxicity, and immune responses. To address these issues, we have developed a system to produce helper-dependent adenoviruses that express CRISPR/Cas9 to direct cleavage of the vectors' own genome after transduction of target cells. To prevent self-cleavage during vector production, it was necessary to downregulate Cas9 mRNA as well as inhibit Cas9 protein activity. Cas9 mRNA downregulation was achieved by inserting the target sequences for the helper-virus-encoded miRNA, mivaRNAI, and producer-cell-encoded miRNAs, hsa-miR183-5p, and hsa-miR218-5p, into the 3' UTR of the HDAd-encoded Cas9 expression cassette. Cas9 protein activity was inhibited by expressing anti-CRISPR proteins AcrIIA2 and AcrAII4 from both the producer cells and the helper virus. After purification, these helper-dependent adenoviruses will perform CRISPR/Cas9-mediated self-cleavage in the transduced target cells, thereby limiting the duration of Cas9 expression and thus represent an important platform for improving the safety of gene editing by CRISPR/Cas9.

Bi-allelic Homology-Directed Repair with Helper-Dependent Adenoviruses.

We describe a strategy to achieve footprintless bi-allelic homology-directed repair (HDR) using helper-dependent adenoviruses (HDAds). This approach utilizes two HDAds to deliver the donor DNA. These two HDAds are identical except for their selectable marker. One expresses the puromycin N-acetyltransferase-herpes simplex virus I thymidine kinase fusion gene (PACTk), while the other expresses the hygromycin phosphotransferase-herpes simplex virus I thymidine kinase fusion gene (HyTk). Therefore, puromycin and hygromycin double resistance can be used to select for targeted HDAd integration into both alleles. Subsequently, piggyBac-mediated excision of both PACTk and HyTk will confer resistance to gancyclovir, resulting in footprintless HDR at both alleles. However, gene-targeting frequency was not high enough to achieve simultaneous targeting at both alleles. Instead, sequential targeting, whereby the two alleles were targeted one at a time, was required in order to achieve bi-allelic HDR with HDAd.

Gene Editing with Helper-Dependent Adenovirus Can Efficiently Introduce Multiple Changes Simultaneously over a Large Genomic Region.

Helper-dependent adenoviral vectors (HDAds) possess long homology arms that mediate high-efficiency gene editing. These long homology arms may permit simultaneous introduction of multiple modifications into a large genomic region or may permit a single HDAd to correct many different individual mutations spread widely across a gene. We investigated this important potential using an HDAd bearing 13 genetic markers in the region of homology to the target CFTR locus in human iPSCs and found that all markers can be simultaneously introduced into the target locus, with the two farthest markers being 22.2 kb apart. We found that genetic markers closer to the HDAd's selectable marker are more efficiency introduced into the target locus; a marker located 208 bp from the selectable marker was introduced with 100% efficiency. However, even markers 11 kb from the selectable marker were introduced at a relatively high frequency of 21.7%. Our study also revealed extensive heteroduplex DNA formation of up to 10 kb with no bias toward vector or chromosomal repair. However, mismatches escape repair at a frequency of up to 15%, leading to a genetically mixed colony and emphasizing the need for caution, especially if the donor and target sequences are not 100% homologous.