Adenovirus with Hexon Tat-Protein Transduction Domain Modification Exhibits Increased Therapeutic Effect in Experimental Neuroblastoma and Neuroendocrine Tumors

Abstract

Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surfaces of tumor cells. Furthermore, infected cells overproduce adenovirus fiber proteins, which are released prior to cell lysis. The released fibers block CAR on noninfected neighboring cells, thereby preventing progeny virus entry. Our aim was to add a CAR-independent infection route to Ad5 to increase the infectivity of tumor cells with low CAR expression and prevent the fiber-masking problem. We constructed Ad5 viruses that encode the protein transduction domain (PTD) of the HIV-1 Tat protein (Tat-PTD) in hypervariable region 5 (HVR5) of the hexon protein. Tat-PTD functions as a cell-penetrating peptide, and Tat-PTD-modified Ad5 showed a dramatic increased transduction of CAR-negative cell lines compared to unmodified vector. Moreover, while tumor cell infectivity was severely reduced for Ad5 in the presence of fiber proteins, it was only marginally reduced for Tat-PTD-modified Ad5. Furthermore, because of the sequence alteration in the hexon HVR, coagulation factor X-mediated virus uptake was significantly reduced. Mice harboring human neuroblastoma and neuroendocrine tumors show suppressed tumor growths and prolonged survival when treated with Tat-PTD-modified oncolytic viruses. Our data suggest that modification of Ad5 with Tat-PTD in HVR5 expands its utility as an oncolytic agent.

Tat-PTD-Modified Oncolytic Adenovirus Driven by the SCG3 Promoter and ASH1 Enhancer for Neuroblastoma Therapy

“…Briefly, the als cassette, an expression cassette containing the ampicillin resistance gene (Amp R ), the lacZa gene, and the sucrose-sensitive gene sacB gene, was amplified by PCR using primers pF.E1Ap-als and pR.E1Ap-als (Table 1). The PCR product replaced the E1A promoter in Ad5PTD(WT) (Yu et al, 2011) through a selection step based on Amp and LacZ as selection markers. The ASH1 enhancer-SCG3 promoter cassette was amplified with primers pF.E1Ap-SCG3 and pR.E1Ap-SCG3 (Table 1).…”

“…The PCR product replaced the als cassette to generate Ad5-PTD(ASH1-SCG3-E1A) through a counter-selection step based on sucrose-containing medium. Ad5PTD(GFP) (Yu et al, 2011), herein called Ad5PTD(MOCK), was used as a replication-deficient control virus for animal experiments. …”

“…Because the viral surface protein was modified in this study, the infectious viral titer depended on the cell line used for titration. Therefore, viral titers were determined by quantitative real-time PCR and defined as encapsidated viral genomes (EVG) per microliter (Yu et al, 2011).…”

“…After 8 hr, the 911 cells were washed and harvested; viral DNA was isolated with a High Pure viral nucleic acid kit (Roche). The viral genome copy number in 911 cells was detected by quantitative real-time PCR as described previously (Yu et al, 2011). A time course experiment was performed for one neuroblastoma cell line (SK-N-FI), one nonneuroblastoma cancer cell line (HeLa), and normal fibroblasts (1064Sk), using 10 EVG/cell.…”

“…To overcome this we have developed a capsid-modified Ad5 with the protein transduction domain (PTD) of the Tat protein from human immunodeficiency virus type 1 in the hexon hypervariable region 5 (HVR5). The transduction capacity and oncolytic efficacy of neuroblastoma cells were enhanced for Tat-PTD-modified Ad5 both in vitro and in vivo (Yu et al, 2011).…”