Figure 1.

KMMP9 Mediates Expression of Enzymatically Active MMP9 Compared to the KGW Control oHSV

(A) The oHSV vectors were created from the KOS-37 BAC full-length genomic clone of the HSV-1 KOS strain.⁴⁷ They are also deleted for the internal repeat (joint) region⁶ and carry two gain-of-function mutations in the external domain of glycoprotein B (gB) that improve virus entry (D285N/A549T, gB:N/T).⁴⁸ The GFP (EGFP) gene was linked downstream of the glycoprotein C (gC) gene-coding sequence using a T2A skipping sequence in order to track virus replication and spread using fluorescence.⁵³ The vectors were retargeted to tumor cells by modifying glycoprotein D (gD) using a single-chain variable fragment (scFv) antibody that recognizes both EGFR and its constitutively active mutant form EGFRvIII.⁵ Recognition of the natural gD receptors was ablated by deletion of the N-terminal amino acids 2–24 of gD and deletion of the residue at position 38 (Δ38). A double Red recombination technique was used to insert the Gateway cassette (GW) and the bovine growth hormone polyadenylation sequence (bGHpA) between UL3 and UL4 in order to produce KGW^BAC as an MMP9-deficient control vector.⁵⁴ The oncolytic KMMP9^BAC was created by Gateway recombination, replacing the Gateway cassette with the MMP9 gene driven by the highly active CAG promoter. (B) Cell supernatants from U2OS mock-infected cells, U2OS cells transfected with a plasmid expressing MMP9 (+ Control), and U2OS cells infected with KGW or KMMP9 (MOI of 100 gc/cell) were collected 48 hpi and analyzed via a gelatin zymography assay followed by Coomassie blue staining.⁵³ White bands are indicative of gelatinase activity (top panel), signifying enzymatically active MMP9. MMP9 expression in the media was verified by western blot analysis using a monoclonal anti-MMP9 antibody (middle panel). Monoclonal anti-tubulin antibody (bottom panel) was used to detect cellular tubulin gene expression as a loading control.