EGFR-Targeted Adenovirus Dendrimer Coating for Improved Systemic Delivery of the Theranostic NIS Gene

Abstract

We recently demonstrated tumor-selective iodide uptake and therapeutic efficacy of combined radiovirotherapy after systemic delivery of the theranostic sodium iodide symporter (NIS) gene using a dendrimer-coated adenovirus. To further improve shielding and targeting we physically coated replication-selective adenoviruses carrying the hNIS gene with a conjugate consisting of cationic poly(amidoamine) (PAMAM) dendrimer linked to the peptidic, epidermal growth factor receptor (EGFR)-specific ligand GE11. In vitro experiments demonstrated coxsackie-adenovirus receptor-independent but EGFR-specific transduction efficiency. Systemic injection of the uncoated adenovirus in a liver cancer xenograft mouse model led to high levels of NIS expression in the liver due to hepatic sequestration, which were significantly reduced after coating as demonstrated by (123)I-scintigraphy. Reduction of adenovirus liver pooling resulted in decreased hepatotoxicity and increased transduction efficiency in peripheral xenograft tumors. (124)I-PET-imaging confirmed EGFR-specificity by significantly lower tumoral radioiodine accumulation after pretreatment with the EGFR-specific antibody cetuximab. A significantly enhanced oncolytic effect was observed following systemic application of dendrimer-coated adenovirus that was further increased by additional treatment with a therapeutic dose of (131)I. These results demonstrate restricted virus tropism and tumor-selective retargeting after systemic application of coated, EGFR-targeted adenoviruses therefore representing a promising strategy for improved systemic adenoviral NIS gene therapy.Molecular Therapy-Nucleic Acids (2013) 2, e131; doi:10.1038/mtna.2013.58; published online 5 November 2013.

Figure 1

In vitro iodide uptake studies of EGFR-targeted adenovirus. In vitro transduction experiments with uncoated Ad5-CMV/NIS showed dose-dependent transduction efficiency in CAR-positive cells (HuH7, HepG2), which was fully retained after EGFR-targeted coating of the adenovirus with increasing amounts of dendrimer (a, c). The CAR-negative cell line SKOV-3 showed no iodide accumulation above background level, even when incubated with high MOI of the uncoated Ad5-CMV/NIS but adenoviral coating with increasing amounts of EGFR-targeted dendrimer caused a significant increase in perchlorate-sensitive iodide uptake activity (b; **P < 0.01). Replacement of the targeting ligand GE11 by a cysteine residue (Cys) lowered transduction efficiency in EGFR-positive HuH7 and SKOV-3 cells (a: *P < 0.05, **P < 0.01; b: **P < 0.01) whereas transfection efficiency in the low EGFR expressing HepG2 cells remained unchanged (c). CAR, coxsackie-adenovirus receptor; EGFR, epidermal growth factor receptor; MOI, multiplicity of infection; NIS, sodium iodide symporter.

Figure 2

In vivo iodide uptake studies of EGFR-targeted adenovirus. ¹²³I-scintigraphy of mice bearing high EGFR-expressing HuH7 xenografts demonstrated high hepatic and low tumoral NIS-mediated radionuclide accumulation after systemic injection of uncoated Ad5-CMV/NIS (a). Coating of Ad5-CMV/NIS with EGFR-targeted PAMAM-G2-PEG-GE11 (dc_300/GE11Ad5-CMV/NIS) before systemic administration strongly reduced liver transduction resulting in significantly increased transduction efficiency of xenograft tumors (b). Replacement of the dendrimer-coupled targeting ligand by a cysteine residue (dc_300/CysAd5-CMV/NIS) still prevented liver pooling of the vector but significantly reduced tumor-specific radionuclide accumulation (c). EGFR, epidermal growth factor receptor; NIS, sodium iodide symporter.

Figure 3

Biodistribution of NIS transgene expression. Quantification of hepatic transgene expression revealed over 80% reduction after intravenous injection of dendrimer-coated dc_300/GE11Ad5-CMV/NIS as compared with injection of uncoated Ad5-CMV/NIS (a: ***P < 0.001). Detargeting of hepatic transgene expression resulted in significantly increased transduction efficiency of xenograft tumors (b). Replacement of the dendrimer-coupled targeting ligand by a cysteine residue (dc_300/CysAd5-CMV/NIS) still prevented liver pooling of the vector but reduced tumor-specific radionuclide accumulation nearly by half (a: ***P < 0.001, b). Ex vivo analysis of NIS mRNA expression in livers and tumors correlated well with the observed radionuclide biodistribution and confirmed the findings of ¹²³I scintigraphy (c: ***P < 0.001). NIS, sodium iodide symporter.

Figure 4

Analysis of liver toxicity. H/E staining of liver sections of mice injected intravenously with Ad5-E1/AFP-RSV/NIS showed fatty degeneration of liver tissue (a), which was not observed in livers of mice treated with dc_300/GE11Ad5-E1/AFP-RSV/NIS (b). Injection of Ad5-E1/AFP-RSV/NIS without surface modification led to a minor increase in ALT level and a strong increase in AST level as compared with mice treated with saline only, which was mostly avoided by coating of the adenovirus before systemic administration (c: ***P < 0.01). AFP, α-fetoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; NIS, sodium iodide symporter.

Figure 5

In vivo analysis of EFGR-specificity. ¹²⁴I-PET-imaging demonstrated strong hepatic transduction after i.v. injection of the uncoated vector (Ad5-E1/AFP-RSV/NIS) (a) and quantification of radioiodine accumulation revealed only poor tumoral transduction (a, d). In contrast, coating of the adenovirus with PAMAM-G2-PEG-GE11 (dc_300/GE11Ad5-E1/AFP-RSV/NIS) before systemic injection resulted in prevention of hepatic radioiodine accumulation and distinct transduction of tumor xenografts (b, d). By pretreatment of mice with the monoclonal anti-EGFR antibody cetuximab before systemic dc_300/GE11Ad5-E1/AFP-RSV/NIS administration tumoral radioiodine accumulation was significantly reduced while liver detargeting of NIS expression was still effective (c, d; **P < 0.01). AFP, α-fetoprotein; EGFR, epidermal growth factor receptor; NIS, sodium iodide symporter.

Figure 6

In vivo therapeutic efficacy. A single i.v. injection of the replication-deficient Ad5-AFP/NIS coated with PAMAM-G2-PEG-GE11 followed by a therapeutic dose of ¹³¹I (dc_300/GE11Ad5-AFP/NIS + ¹³¹I, radiotherapy) showed a significant delay in tumor growth and improved survival (a, b; ***P < 0.01) as compared with mice treated with saline only (NaCl-control, a, b). I.v. application of the oncolytic replication-selective Ad5-E1/AFP-E3/NIS with EGFR-targeted surface modification (dc_300/GE11Ad5-E1/AFP-E3/NIS, virotherapy) revealed a comparable delay in tumor growth and enhancement of survival (a, b). Combined radiovirotherapy treatment (dc_300/GE11Ad5-E1/AFP-E3/NIS + ¹³¹I, radiovirotherapy) resulted in a strongly enhanced therapeutic effect, as seen by significantly delayed tumor growth and further improved survival (a, b; ***P < 0.01). AFP, α-fetoprotein; EGFR, epidermal growth factor receptor; NIS, sodium iodide symporter.