In Vivo Silencing of Regulatory Elements Using a Single AAV-CRISPRi Vector.

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

Regulated expression of a gene of interest is crucial for transgenic research, as well as for safe and efficacious gene therapy. The most commonly used conditional expression system requires the generation of two transgenic strains, one carrying an inducible promoter and the other a transactivator. The generation of conditional transgenic models using this method is costly and time consuming. In this study, we report the design and construction of novel simplified gene delivery vectors that integrate both the regulatory and responsive elements in a single vector. The Tet-On system was used and integrated the inducible promoter and transactivator in a single plasmid, between which a copy of an insulator was inserted to minimize interference between the two units. Another copy of an insulator was inserted upstream of the transgene cassette to eliminate transgene silencing and to lower basal expression. The two insulators were in the same orientation. To further decrease basal expression, the most powerful repressor domain containing a 'kruppel-associated box' of the zinc finger protein NK10 was used and fused to TetR in one of the two vectors. The function of this system was confirmed after in?vitro transient transfection. The two conditional plasmids were successfully constructed, and the expression of the gene of interest was regulated tightly in vitro. In conclusion, the vectors described here may be useful for gene therapy applications, as well as for the establishment of conditional animal models.

BackgroundOur lab has developed a CRISPR‐based, gene‐editing strategy that targets the extreme C‐terminus (C‐term) of APP (amyloid precursor protein) – a gene with a central and indisputable role in AD. We have reported previously that APP C‐terminus CRISPRs effectively attenuate APP β‐cleavage and Alzheimer’s pathology in vivo. Here, we present new data demonstrating the feasibility and efficacy of a clinically‐viable, “all‐in‐one” therapeutic vector that has all the components needed for APP C‐terminus editing (Cas enzyme / gRNAs / regulatory elements) packaged into a single AAV.MethodAPP C‐terminus targeting gRNAs along with a SaCas9 expression cassette were cloned into AAV expression backbones and packaged into AAV‐PHP.eB capsids. Resulting AAVs were injected into APPNL‐G‐F mice and APP C‐terminus editing along with amyloid beta (Aβ) plaque pathology were evaluated.ResultAAV treated brain tissue showed wide‐spread expression of APP CRISPRs and robust APP C‐terminus editing. Both amyloid plaques and insoluble Aβ were decreased in APP CRISPR treated tissue.ConclusionThese data demonstrate in vivo feasibility of our approach and advance our long‐term goal of creating a “one‐and‐done”, single injection CRISPR therapeutic for permanent treatment of AD in human patients.

Single HSV-amplicon vector mediates drug-induced gene expression via dimerizer system

We have engineered a novel herpes simplex virus type 1 (HSV-1)-based amplicon viral vector, whereby gene expression is controlled by cell cycle events. In nondividing cells, trans-activation of the cyclin A promoter via interaction of the Gal4/NF-YA fusion protein with the Gal4-binding sites is prevented by the presence of a repressor protein, cell cycle-dependent factor 1 (CDF-1). CDF-1 is specifically expressed during the G(0)/G(1) phase of the cell cycle and its binding site is located within the cyclin A promoter. In actively proliferating cells, trans-activation could take place because of the absence of CDF-1. Our results showed that when all these cell cycle-specific regulatory elements are incorporated in cis into a single HSV-1 amplicon plasmid vector backbone (pC8-36), reporter luciferase activity is greatly enhanced. Transgene expression mediated by this series of HSV-1 amplicon plasmid vectors and amplicon viral vectors could be regulated in a cell cycle-dependent manner in a variety of cell lines. In a further attempt to target transgene expression to a selected group of actively proliferating cells such as glial cells, we have replaced the cytomegalovirus promoter of the pC8-36 amplicon plasmid with the glial cell-specific GFAP enhancer element. With this latter viral construct, cell type-specific and cell cycle-dependent transgene expression could subsequently be demonstrated specifically in glioma-bearing animals. Taken together, our results suggest that this series of cell cycle-regulatable HSV-1 amplicon viral vectors could potentially be adapted as useful tools for the treatment of human cancers.