Work

Protocol for generating human CAR-engineered macrophages by Vpx-containing lentivirus

Year: 2024

Type: article

Abstract: Human-derived macrophages are notoriously difficult to infect with HIV-1-based lentiviruses, posing a limitation to the advancement of chimeric antigen receptor macrophage (CAR-M) therapy. Here, we present a protocol for generating human chimeric antigen receptor (CAR)-engineered macrophages using the viral protein Vpx (encoded by the Sooty Mangabey simian immunodeficiency virus [SIV] and HIV-2 lineages) incorporated into the lentivirus vector, which enhances infection efficiency. We describe steps for cell cultivation, lentivirus production, concentration, infection procedures, and efficiency assessments. This protocol provides a foundation to study macrophage manipulation, especially with CAR or other immune engagers. For complete details on the use and execution of this protocol, please refer to Gao et al. 1 • A high-efficiency approach for generating human-derived CAR macrophages by lentivirus • A protocol for engineering lentiviruses to incorporate the Vpx protein • Steps for infecting human monocyte-derived macrophages by lentivirus Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Human-derived macrophages are notoriously difficult to infect with HIV-1-based lentiviruses, posing a limitation to the advancement of chimeric antigen receptor macrophage (CAR-M) therapy. Here, we present a protocol for generating human chimeric antigen receptor (CAR)-engineered macrophages using the viral protein Vpx (encoded by the Sooty Mangabey simian immunodeficiency virus [SIV] and HIV-2 lineages) incorporated into the lentivirus vector, which enhances infection efficiency. We describe steps for cell cultivation, lentivirus production, concentration, infection procedures, and efficiency assessments. This protocol provides a foundation to study macrophage manipulation, especially with CAR or other immune engagers.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2024.103350

Authors: Yun Gao, Xiaobin Fang, Luo Zhang, Xiushan Yin

Institutions: Chinese PLA General Hospital, Soochow University

Language: English

Topic: Virus-based gene therapy research

Subfield: Genetics

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,500