Design Solutions

Overall Design Solution

Many viral vectors have limited gene delivery capabilities due to safety concerns with regards to being cytotoxic, and limited capacity for carrying and delivering larger DNA constructs. We chose to make the human simplex virus (HSV) a viral vector since it has the potential in carrying large gene constructs (more than 30 kbs) and an ability to infect a wide range of cell types. In addition, this virus naturally infects humans without integration into the host's genome, making it a potential candidate for gene therapy. However, we need to minimize, or eliminate, the cytotoxicity of the virus for gene therapy purposes. In doing so, we decided to knock out the TK gene, which controls toxicity of the virus, from the viral genome and replace it with the Tn7 site in which our transgene of interest can be inserted into. In addition, we will also knock out certain genes, specifically LoxP sites, that we wish to not have and replace them with FRT sites and a gentamicin resistance gene.

Modification #1: Knock out tk gene and insert Tn7 insertion site.

Modification #1 Design

The TK gene is known to be a major contributor for cytotoxicity HSV vectors. By knocking out this gene and replacing it with a Tn7 insertion site, it will give us a location where we can easily insert large DNA constructs into the HSV KOS-37 BAC. A plasmid with the cassette that includes homology arms (HAs), the LacZ gene, Tn7 insertion site, and Kanamycin resistance will be created then linearized for recombineering into the HSV KOS-37 BAC. Once the cassette is created; it would then be transformed into the original BAC (bPK-024) to create a TK Knockout (KO) BAC (bPK-036)

Modification #2: Replace LoxP sites and remove β-gal gene

The presence of the LoxP recombination sites in the genome could cause complications when used within in vivo models, where the Cre-Lox system is widely used. Therefore, we are replacing the LoxP sites with FRT recombination sites. Through uLoop DNA Assembly, we created a cassette, HA-FRT-F1 replicon-GentR-FRT-HA, that can be linearized and transformed into the TK KO BAC to create the new BAC (bPK-037). In addition, during the recombineering of this cassette into the BAC, the beta-gal gene will be replaced; allowing us to insert genes of interest via blue-white selection cloning.

Modification #2 Design

Expected Outcome

Expected Edited BAC

Main Features:

Customizable Insertion Site via Tn7 insertion site
Blue/White Selection possible via LacZ gene and beta-gal removal
Compatible in Cre Mouse Model due to replacement of LoxP site with FRT sites
Lower cytotoxicity with tk gene knockout
Large gene carrying cassette due to use of HSV genome

Leader: Natalie Maloney

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