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Use of a lentivirus/VSV pseudotype virus for highly efficient genetic redirection of human peripheral blood lymphocytes

Abstract

Genetic redirection of lymphocytes that have been engineered to recognize antigens other than those originally programmed by their rearranged germlines is a potentially powerful immunotherapeutic tool. The rationale for the protocol described here is that many cancers and persistent or latent viruses have developed strikingly similar mechanisms of evading attack by host immunity that can often be overcome by redirection of host lymphocytes using chimeric T-cell receptor (chTCR) genes. However, for human peripheral blood lymphocytes (PBLs), this is generally regarded as a technically demanding procedure with unacceptably low efficiency using either contemporary transfection methods or retroviral transduction. One of the main difficulties with retroviruses is their reliance on rapidly dividing cells for integration of their genomes carrying the desired chTCR. Here we describe a highly efficient protocol that uses a lentivirus/vesicular stomatitis virus pseudotyped virus to engineer CD3/CD28-stimulated human peripheral blood cells (i.e., primarily T cells), with near 100% efficiency.

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Figure 1: A summary of the creation and testing of a lentivirus/VSV pseudotype for redirection of human PBLs with an anti-cancer chimeric receptor.
Figure 2: Shown here is the nucleotide sequence of p-Jantz-2, which encodes the scFv for human CEA (a) and the nucleotide plus amino acid sequences of p-Jantz-1, containing modified human CD28 (b) indicating desirable and undesirable domains for inclusion into chTCR cassettes.
Figure 3: FACS analysis of transduced PBL, visualized directly in Figure 1.
Figure 4: Schematic of a PBL transduced with an scFv-based chTCR of the structure described.

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Acknowledgements

The authors wish to thank Robert Davey and Andrey Kolokoltsov of UTMB for invaluable advice and assistance with this procedure and the Gillson Longenbaugh Foundation of Houston for funding the project.

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Correspondence to Anthony Simmons.

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Simmons, A., Jantz, K. Use of a lentivirus/VSV pseudotype virus for highly efficient genetic redirection of human peripheral blood lymphocytes. Nat Protoc 1, 2688–2700 (2006). https://doi.org/10.1038/nprot.2006.409

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