Adenoviral Vectors Coated with PAMAM Dendrimer Conjugates Allow CAR Independent Virus Uptake and Targeting to the EGF Receptor

Abstract

Adenovirus type 5 (Ad) is an efficient gene vector with high gene transduction potential, but its efficiency depends on its native cell receptors coxsackie- and adenovirus receptor (CAR) for cell attachment and α(v)β(3/5) integrins for internalization. To enable transduction of CAR negative cancer cell lines, we have coated the negatively charged Ad by noncovalent charge interaction with cationic PAMAM (polyamidoamine) dendrimers. The specificity for tumor cell infection was increased by targeting the coated Ad to the epidermal growth factor receptor using the peptide ligand GE11, which was coupled to the PAMAM dendrimer via a 2 kDa PEG spacer. Particles were examined by measuring surface charge and size, the degree of coating was determined by transmission electron microscopy. The net positive charge of PAMAM coated Ad enhanced cellular binding and uptake leading to increased transduction efficiency, especially in low to medium CAR expressing cancer cell lines using enhanced green fluorescent protein or luciferase as transgene. While PAMAM coated Ad allowed for efficient internalization, coating with linear polyethylenimine induced excessive particle aggregation, elevated cellular toxicity and lowered transduction efficiency. PAMAM coating of Ad enabled successful transduction of cells in vitro even in the presence of neutralizing antibodies. Taken together, this study clearly proves noncovalent, charge-based coating of Ad vectors with ligand-equipped dendrimers as a viable strategy for efficient transduction of cells otherwise refractory to Ad infection.

Systemic Image-Guided Liver Cancer Radiovirotherapy Using Dendrimer-Coated Adenovirus Encoding the Sodium Iodide Symporter as Theranostic Gene

“…The human cell lines HuH7 (HCC, JCRB 0403 [Japanese Collection of Research Bioresources Cell Bank, National Institute of Health Sciences]), SKOV-3 (ovarian carcinoma, HTB-77 [American Type Culture Collection]), and U87 MG (glioblastoma, HTB-14 [American Type Culture Collection]) were cultured as described previously (7). Cellular CAR levels were analyzed by flow cytometry as described previously (7).…”

“…Amine-terminated generation 5 PAMAM dendrimer was used as described previously (7). For surface modification, the adenovirus was dispensed with serum-free OPTI-MEM (Invitrogen) to a volume of 50 mL (in vitro experiments) or 125 mL (in vivo experiments) and gently mixed with an equal volume of PAMAM-G5 (10 or 300 ng) diluted in HEPES (N-(2-hydroxyethyl) piperazine-N9-(2-ethanesulfonic acid))-buffered glucose (20 mmol/1 HEPES, 5% glucose [w/v], ph 7.4).…”

“…We have recently used chemically well-defined dendritic PAMAM (poly(amidoamine)) dendrimers bearing positively charged terminal amines to coat the negatively charged adenoviral capsid by virtue of electrostatic interaction (7). This modification allowed efficient internalization and transduction of tumor cells in vitro otherwise refractory toward adenoviral transduction.…”

Synthetic virology: engineering viruses for gene delivery

“…Adenoviruses have also been coated with polyamidoamine (PAMAM) dendrimers, leading to increased levels of viral uptake and transduction in tumour cells. 69, 70 …”

Beyond Gene Delivery: Strategies to Engineer the Surfaces of Viral Vectors

“…In vitro assays showed increased transduction of cells with low CAR levels and also a slight increase of transduction into hepatic cells (high CAR levels). Nevertheless, excessive coating led to a decrease in transduction levels into cells [55]. In addition, the PAMAM coating also showed increased resistance to NAbs.…”