Silk-elastinlike protein polymer hydrogels for localized adenoviral gene therapy of head and neck tumors

Abstract

Vector dissemination, transient gene expression, and rapid clearance are major obstacles to successful human gene therapy. In this study, we investigated the effect of silk-elastinlike protein polymer (SELP) hydrogels on biodistribution and anticancer efficacy of adenoviral gene therapy in a head and neck cancer model. Transcriptional activities of adenovirus carrying beta-galactosidase (Ad-LacZ) and luciferase (Ad-Luc) reporter genes were evaluated in (nu/nu) mice with head and neck cancer as a function of polymer concentration. Antitumor efficacy of thymidine kinase encoding adenovirus (Ad-Tk) and ganciclovir (GSV) combination was also evaluated. SELP (4 wt %) matrices localized viral release, minimized dissemination to liver, and enhanced reporter gene expression levels by 4-8-fold compared to virus alone. SELP- Ad-Tk with GSV reduced tumor volume significantly compared to the virus alone. SELPs provide a means for temporal and spatial control of viral gene delivery to head and neck tumors.

Figure 1

The amino acid sequence of SELP-47K (molecular weight: 69,814 Da). The protein polymer is composed of head and tail portions and a series of silk (GAGAGS) and elastin-like (GVGVP) repeats with the primary repetitive sequences shown in bold.

Figure 2

β-galactosidase expression levels (ng/100mg tissue weight) in tumor (A), and liver (B) from 5 × 10⁸ PFU of Ad-LacZ in10.8, 8, and 4wt% SELP-47K compared to free Ad-LacZ over 3 weeks, * p <0.05.

Figure 3

Typical time course tracking of athymic nude mice with JHU-012 xenografts intratumorally injected with Ad-Luc by bioluminescence (Luciferin/Luciferase assay system). Group on left received an intratumoral injection of Ad-Luc in saline. Mice on right received injection of Ad-Luc in 4 wt% SELP-47K. Luciferase expression one week (A), two weeks (B) and three weeks (C) post injection. Graphs show photon counts from each group. * p <0.05.

Figure 4

Patterns of Ad-LacZ expression in 4 wt% SELP-47K (A, B, and C) and Ad-LacZ in saline (D, E, and F). β-gal expression is highlighted with white arrows. Note the localizing effect of SELP at week 1 (A) and the spread of β-gal expression throughout the tumor tissue in weeks 2 (B) and 3 (C). Virus in saline shows wide distribution of expression in week one (D), reduction in week two (E) and negligible expression in week three (F).

Figure 5

Antitumor efficacy of 4 wt% SELP-47K. A. Antitumor efficacy of SELP-mediated controlled delivery of therapeutic adenoviruses (Ad-Tk) in a Xenograft model of head and neck cancer (JHU-012). Tumor volumes of groups receiving Ad-Tk injections showed significant reduction in volume when compared to PBS and Ad-DL312 treated groups. The SELP-47K + Ad-Tk group prolonged the antitumor effect longer than the Ad-Tk group and at day 14 tumor volume was significantly less with SELP-47K than without. (P<0.05 in all cases); B. Efficacy of SELP-mediated controlled delivery of Ad-TK in nude mice having JHU-012 as measured by RT-PCR of mRNA isolated from the tumors. At day 14, the SELP-47K + Ad-TK group had significantly higher levels of mRNA expression than the Ad-Tk group. The Ad-TK group showed a continuous decrease over the time course of the experiment while SELP-47K sustained the expression of Ad-Tk with a maximum around day 7.