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Evaluation of AAV-Mediated Delivery of Shrna to Target Basal-Like Breast T Cancer Genetic Vulnerabilities Catarina Pintoa,B, Gabriela Silvaa, Ana S

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Evaluation of AAV-Mediated Delivery of Shrna to Target Basal-Like Breast T Cancer Genetic Vulnerabilities Catarina Pintoa,B, Gabriela Silvaa, Ana S Journal of Biotechnology 300 (2019) 70–77 Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Evaluation of AAV-mediated delivery of shRNA to target basal-like breast T cancer genetic vulnerabilities Catarina Pintoa,b, Gabriela Silvaa, Ana S. Ribeiroc,d, Mónica Oliveirac,d, Manuel Garridoa, Vanessa S. Bandeiraa, André Nascimentoa, Ana Sofia Coroadinhaa,b, Cristina Peixotoa,b, ⁎ Ana Barbasa,c, Joana Paredesd,e, Catarina Britoa,b, Paula M. Alvesa,b, a iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal b Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal c Bayer Portugal, Carnaxide, Portugal d i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal e IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr Roberto Frias s/n, Porto, Portugal ARTICLE INFO ABSTRACT Keywords: Adeno-associated viral vectors (AAV) for gene therapy applications are gaining momentum, with more therapies Gene therapy moving into later stages of clinical development and towards market approval, namely for cancer therapy. The Adeno-associated viral vectors development of cytotoxic vectors is often hampered by side effects arising when non-target cells are infected, and Basal-like breast cancer their production can be hindered by toxic effects of the transgene on the producing cell lines. In this study,we AAV production 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 apop- tosis 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. 1. Introduction as the only treatment option(Caldas-lopes et al., 2009; Schneider et al., 2008). Standard treatment for these patients, namely surgery, radiation Gene therapy has the opportunity to expand beyond conventional and chemotherapy, often fails to completely eradicate the disease, is drug based therapeutics and deliver a targeted treatment through reg- incapable of maintaining sustained protection, and is accompanied by ulation of endogenous genes(Naldini, 2015). As our knowledge on serious side effects, such as cytotoxicity to non-cancerous cells(Luo cancer´s onset and progression evolves, tumor-specific phenotypic et al., 2015; Santiago-Ortiz and Schaffer, 2016). Therefore, there is a characteristics are uncovered that can be explored in this context, to strong unmet medical need for targeted therapies for BLBC with im- achieve higher specificity and potency of the developed therapies(Miest proved clinical efficacy, targeted effect with lower side effects, and and Cattaneo, 2013). Basal-like breast cancer (BLBC) patients can longer patient survival times(Carey et al., 2010; Santiago-Ortiz and greatly benefit from the development of such therapies. Although this Schaffer, 2016). A lot of effort has been conducted towards the dis- subtype represents up to 20% of worldwide breast cancer (BC) in- covery of actionable molecular targets for this disease(Bianchini et al., cidence, it is responsible for a disproportionate number of deaths as it 2016). Recently, Petrocca et al conducted a genome-wide small inter- often leads to relapse with distant metastasis(Schneider et al., 2008). fering RNA (siRNA) screen to identify selective genetic vulnerabilities The lack of molecular markers and deficient characterization, impaired causing lethality in this breast cancer subtype(Petrocca et al., 2013). by an heterogeneous molecular presentation, leads to lack of targeted Although promising, these leads still face considerable challenges therapies and leaves clinicians with cytotoxic chemotherapeutic drugs reaching the clinic due to ineffective delivery platforms(Kacsinta and ⁎ Corresponding author at: iBET, Apartado 12, 2780-901 Oeiras, Portugal. E-mail addresses: [email protected] (C. Pinto), [email protected] (G. Silva), [email protected] (A.S. Ribeiro), [email protected] (M. Oliveira), [email protected] (M. Garrido), [email protected] (V.S. Bandeira), [email protected] (A. Nascimento), [email protected] (A.S. Coroadinha), [email protected] (C. Peixoto), [email protected] (A. Barbas), [email protected] (J. Paredes), [email protected] (C. Brito), [email protected] (P.M. Alves). https://doi.org/10.1016/j.jbiotec.2019.05.016 Received 1 August 2018; Received in revised form 24 May 2019; Accepted 27 May 2019 Available online 28 May 2019 0168-1656/ © 2019 Published by Elsevier B.V. C. Pinto, et al. Journal of Biotechnology 300 (2019) 70–77 Dowdy, 2016). and orientation were confirmed by restriction analysis and sequencing. Recombinant adeno-associated viral (rAAV) vectors have been in- Gene specific shRNA sequences were obtained from the RNAi con- creasingly successful as gene therapy delivery platforms and have sortium database (http://www.broadinstitute.org/rnai/public/). The proven to be a promising approach to treat a variety of human diseases scramble control shRNA sequences, not targeting any known human or (Brimble et al., 2016; Kaplitt et al., 2007; Testa et al., 2013). The in- mouse genes, were from Sigma. The shRNA catalog numbers are: creasing number of preclinical studies and human clinical trials has PLK1shRNA: TRCN0000006247; MCL1shRNA: TRCN0000196390; demonstrated their favorable safety profile, efficacious gene delivery, PSMA2shRNA: TRCN0000003879; PSMB4shRNA: TRCN0000003914; and robust and persistent transgene expression in vivo, with manageable Ctrl1shRNA: SHC202; and Ctrl2shRNA: SHC016. immune response and minor adverse effects upon injection(Samulski and Muzyczka, 2014; Santiago-Ortiz and Schaffer, 2016; Snyder and 2.3. Production, purification and titer determination of rAAV-shRNA Moullier, 2011). In fact, an rAAV1-based vector for the treatment of a vectors lipoprotein lipase deficiency became the first commercially-approved gene therapy product for clinical applications by the European Com- rAAV-shRNA vectors were produced in HEK293 T cells by 2- or 3- mission(Bryant et al., 2013). plasmid transfection system using 5 μg DNA/1 × 106 cells, with linear rAAV vectors’ potential for cancer applications has also been de- polyethylenimine (PEI; Polysciences) or CaPO4 as transfection reagents. monstrated in several in vitro cancer studies, in vivo pre-clinical cancer In the 2-plasmid system HEK293 T cells (seeded at 4–5 × 104 cell/cm2 models, and clinical trials, with a wide variety of approaches, such as 20–24 h before) were co-transfected at 70–80% confluency with in- the delivery of anti-angiogenesis factors (PEDF, endostatin 34, Kringle dividual pAAV-shRNA plasmids, generated above, plus the pDG 5, Kallistatin), suicide genes (HSV-TK system and polyomavirus middle plasmid(Grimm et al., 2003), encoding the rep and cap genes and the T antigen), immunostimulatory molecules (TRAIL, Interferons, adenovirus encoding products essential for AAV vector production, Interleukins), DNA-encoded small RNA molecules for post-transcrip- using different plasmid ratios as indicated below. 3-plasmid transfec- tional regulation of oncogenes, immunogenic cell surface molecules and tion was performed with plasmids: pAAV-shRNA, pAAV-Helper - en- tumor antigens(Ledford, 2015; Luo et al., 2015; Santiago-Ortiz and coding adenovirus gene products required for the production of in- Schaffer, 2016). fective AAV particles, and pAAV-RC - encoding the rep and cap genes Herein we describe a rAAV-based therapeutic approach to target (necessary trans-acting elements for replication and packaging of the BLBC. Signature genetic dependencies previously identified for this viral genome) (all from Stratagene/Agilent), at a plasmid molar ratio of breast cancer subtype (i.e. MCL1, PSMA2 and PSMB4)(Petrocca et al., 1:1:1. All plasmids, except pAAV-shRNA, were amplified in E. coli 2013) were targeted using rAAV-mediated delivery of shRNA. Con- DH5α cells (Invitrogen). Plasmid DNA was purified with the EndoFree sequent downregulation of the target genes led to a decrease in cell plasmid kit from Qiagen, as per supplier´s instructions. Transfections viability and induced apoptosis in BLBC cell lines. Furthermore, in- with the CaPO4 method were performed using the Calcium Phosphate tratumoral injections of rAAV vectors targeting PSMA2 resulted in Transfection Kit from Sigma (CAPHOS) as per kit´s instructions. slowed tumor growth in a BLBC xenograft model. Moreover, our results Transfections of HEK293 T cells with PEI were performed as per stan- indicate that optimization of plasmid stoichiometry upon transfection dard protocols using optimized DNA:PEI ratio of 1:1.8 (μg), as pre- and time of harvest should be done in a transgene dependent manner, viously described(Merten and Al-Rubeai, 2011). 48–72 h post trans- with the goal of circumventing potential cytotoxic effects of the trans- fection, cells were lysed with 0.1% Triton-X100 (Sigma), releasing the gene in the producer
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