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Evaluation of AAV-mediated delivery of shRNA to target basal-like breast cancer genetic vulnerabilities.

Journal of biotechnology (2019-06-01)
Catarina Pinto, Gabriela Silva, Ana S Ribeiro, Mónica Oliveira, Manuel Garrido, Vanessa S Bandeira, André Nascimento, Ana Sofia Coroadinha, Cristina Peixoto, Ana Barbas, Joana Paredes, Catarina Brito, Paula M Alves
ABSTRACT

Adeno-associated viral vectors (AAV) for gene therapy applications are gaining momentum, with more therapies moving into later stages of clinical development and towards market approval, namely for cancer therapy. The development of cytotoxic vectors is often hampered by side effects arising when non-target cells are infected, and their production can be hindered by toxic effects of the transgene on the producing cell lines. In this study, we evaluated the potential of rAAV-mediated delivery of short hairpin RNAs (shRNA) to target basal-like breast cancer genetic vulnerabilities. Our results show that by optimizing the stoichiometry of the plasmids upon transfection and time of harvest, it is possible to increase the viral titers and quality. All rAAV-shRNA vectors obtained efficiently transduced the BLBC cell lines MDA-MB-468 and HCC1954. In MDA-MB-468, transduction with rAAV-shRNA vector targeting PSMA2 was associated with significant decrease in cell viability and apoptosis induction. Importantly, rAAV2-PSMA2 also slowed tumor growth in a BLBC mouse xenograft model, thus potentially representing a therapeutic strategy against this type of cancer.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Calcium Phosphate Transfection Kit, Most cost effective transfection reagent kit for transient and stable transfection of DNA into mammalian cells