Design and construction of a recombinant lentiviral vector with specific tropism to human epidermal growth factor‐overexpressed cancer cells: Developing a new retargeting system for lentivirus vectors

Abstract

Background Targeting of specific tissues and cells by viruses is one of the challenges faced by researchers. Lentiviral vectors (LVs) are one of the most promising gene delivery systems in cancer gene therapy. Therefore, we aimed to design a novel lentiviral delivery system that expresses anti‐ human epidermal growth factor 2 (HER2) designed anykrin repeat protein (DARPin) on the vector envelope to create a pseudotyped lentivirus for targeting HER2‐positive cancer cells. Methods A helper plasmid producing the viral vector envelope containing anti‐HER2 DARPin‐G3 was constructed. LV was produced by transfer vector containing green fluorescent protein (GFP) gene and helper plasmids in human embryonic kidney 293 cells. The human breast cancer cell lines SKBR3 (normal and with inhibited endocytosis) (HER2‐positive) and MDA‐MB‐231 (HER2‐negative) were transduced by the recombinant viral vector. The GFP‐based transduction rate was determined by flow cytometry and fluorescence microscopy. Results The anti‐HER2 DARPin concentration in DARPin‐LVs was significantly higher than the envelope G glycoprotein of the vesicular stomatitis virus‐LVs (non‐anti‐HER2 control) (p < 0.0001). In flow cytometry assays, the percentage of transduction by recombinant LV was significantly higher in SKBR3 cells than in SKBR3 cells with inhibited endocytosis (p = 0.0074) and MDA‐MB‐231 cells (p = 0.0037). In fluorescence microscopy assays, the percentage of transduction by new LV was significantly higher in SKBR3 cells than in SKBR3 cells with inhibited endocytosis (p = 0.0026) and MDA‐MB‐231 cells (p = 0.0014). Conclusions We constructed a new recombinant LV with a defect in cell entry directly, containing an anti‐HER2 DARPin on the vector envelope with specific tropism to HER2 receptor on HER2‐positive cancer cells. We assumed that this viral vector transduces cells via an endocytosis‐dependent process.

ABO blood group antigen therapy: a potential new strategy against solid tumors

“…There are a variety of viral vectors based on adenovirus, alpha virus, herpes simplex virus, coxsackie virus and vaccinia virus for the immunization studies in the experimental model. Lentiviral vectors are one of the most powerful types of gene transfer vectors and are widely used for therapeutic purposes and basic biological research 24 . The drug Glybera delivers a functional gene copy to the patient's muscle cells via a viral vector to treat lipoprotein lipase deficienc 25 .…”

Immunogenicity of CRISPR therapeutics—Critical considerations for clinical translation

“…A non-integrating lentivirus delivering CRISPR/Cas9 has been successfully used for one-time correction of the sickle cell disease mutation in the β-globin gene ( Uchida et al, 2021 ). Lentiviruses have been pseudotyped to alter their host range ( Cronin et al, 2005 ; Humbert et al, 2012 ; Ebrahimabadi et al, 2019 ) and this can help increase their target cell specificity, reduce the dose, and minimize systemic immune responses. Transduction of antigen presenting cells by lentiviruses could be a limitation to the use of these vectors in the delivery of CRISPR therapeutics.…”