Optimization of lentiviral transduction by lentiviral vectors

• Grant number: 25/24990-7
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: December 01, 2025
• End date: November 30, 2026
• Field of knowledge: Biological Sciences - Immunology
• Principal Investigator: Bruna Cunha Gondim de Alencar
• Grantee: Jhonnatan Olivetti Klarosk
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 25/13226-4 - Optimization of lentiviral transduction by lentiviral vectors, AP.R

Abstract

Although transduction of human macrophages by lentiviral vectors offers a powerful resource for gene knockdown, it faces several challenges that directly impact laboratory execution. The lack of methodological details in scientific studies leads to difficulties in reproducing existing protocols. The laboratory hosting the current project has encountered difficulties in genetically manipulating macrophages, a factor that prevents the use of this cell type in studies that require manipulations such as gene silencing.The overall objective of this work is to determine an optimized protocol with high success rates for transduction using an HIV-1-derived lentiviral vector in macrophages derived from human primary monocytes. The specific objectives are: to test cell differentiation methods with different culture media (RPMI medium with FBS or human AB serum, with or without M-CSF); to evaluate transduction techniques during differentiation (with fresh or frozen virus); to test different lentiviral transduction optimization techniques (modulation of serum percentage (2), CD47 overexpression in the packaging cell and lentiviral vector (3), centrifuge inoculation (spinoculation) (4), use of retronectin (5) or protamine (6), pseudotyping with baboon endogenous virus glycoprotein - BaEV (7)); and finally, to silence the MYO9B gene to validate the use of the protocol for gene silencing. To achieve the proposed objectives, analytical techniques will be used. These include: cell morphology by optical microscopy; cell provision by trypan blue staining and/or live/dead; differentiation success by flow cytometry for macrophage markers; percentage of transduction with and without antibiotics; and silencing success by qPCR and Western Blot.