Development of a recombinant Newcastle disease virus capable of expressing the SaCas9 protein

• Grant number: 25/05205-7
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: August 01, 2025
• End date: July 31, 2026
• Field of knowledge: Biological Sciences - Biology
• Principal Investigator: Pedro Luiz Porfirio Xavier
• Grantee: Manoela Brito Lopes
• Host Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil
• Associated research grant: 22/06305-7 - Identifying new therapeutic targets in Pancreatic Ductal Adenocarcinoma by combining relevant models and approaches, AP.GR

Abstract

Newcastle disease virus (NDV) has significant potential as a therapeutic approach for the treatment of neoplasms, due to its natural ability to selectively infect and kill cancer cells, as these often have inefficient antiviral systems. With regard to Pancreatic Ductal Adenocarcinoma (PDA), some studies have demonstrated the oncolytic potential of the NDV virus in this type of cancer. In fact, in the FAPESP Generation project, process 2022/06305-7, we are testing the oncolytic potential of the NDV virus in different ADP models, including three-dimensional (3D) models such as spheroids and tumorspheres.The gene editing complex consisting of guide RNA and the Cas protein, known as CRISPR/Cas9, is a powerful tool for the development of gene therapy approaches against cancer, being able to "fix" genes that are mutated. Interestingly, oncolytic viruses are recognized as promising alternatives for delivering cancer gene therapies, including CRISPR/Cas9 systems. In this context, a genetically modified NDV virus capable of expressing the Cas9 enzyme and selectively transporting it to cancer cells could be a powerful therapeutic tool against cancer, including ADP. In the present scientific initiation project, we will develop a genetically modified NDV virus, expressing the Cas9 protein. The main objective of the project is to determine which is the best Cas protein to be expressed by the NDV virus, without compromising the feasibility of generating and rescuing the virus. To do this, an anti-genome plasmid of the NDV virus will be cloned with the Cas9-encoding gene sequences, along with a strong promoter region. The rescue of the NDV-Cas9 virus will be carried out by reverse genetics, in BHK-T7/9 cells. For this, a transfection cocktail will be prepared using Lipofectamine 3000, the main anti-genome plasmid and the helper plasmids, containing the NP, P and L genes of the NDV virus. After 48 hours, the viral particles will be collected and propagated in embryonated eggs of 10 days of development, for 96 hours. The allantoic liquid from the eggs will be collected and the red blood cell hemagglutination test will be used to determine the presence or absence of the virus. The titration and determination of the virus's PFU (plaque forming units) will be performed in Vero cells. The fidelity and stability of the virus will be realized by sequencing the F gene of the NDV virus. The expression of Cas9 proteins will be verified by RT-qPCR and Western blot. Ten passages of the virus will be performed in embryonated eggs, in order to observe the stability of the expression of Cas proteins. Thus, this scientific initiation has, as its main goal, to generate the NDV virus expressing Cas proteins, determining the first steps towards the development of an oncolytic virus capable of exhibiting anti-tumor potential through oncolysis effects and gene editing of relevant therapeutic targets, through the NDV-CRISPR/Cas9 system. (AU)