1. ¹Department of Hematology and Oncology, UCLA AIDS Institute, David Geffen School of Medicine, University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA
2. ²Department of Immunology, M. D. Anderson Cancer Center, University of Texas, Unit 901, 7455 Fannin Street, Houston, TX 77030, USA
3. ³Division of Biology, California Institute of Technology, 1200 E. California Boulevard, Pasadena, CA 91125, USA
4. ⁴Department of Microbiology, Immunology, and Molecular Genetics and Medicine, UCLA AIDS Institute, David Geffen School of Medicine, University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA
5. ⁵Department of Immunology, University of Manitoba, 608 Basic Medical Sciences Building, 730 William Avenue, Winnipeg, MB, Canada, R3E 0W3

Correspondence: Irvin S. Y. Chen, Fax: +1 310 267 1875. E-mail: SYUChen@mednet.ucla.edu

^*These authors contributed equally to this work.

Received 18 October 2005; Revised 17 May 2006; Accepted 17 May 2006.

Abstract

Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. We examined the effects of shRNA expression in primary human lymphocytes (PBLs) using lentiviral vectors bearing different RNA polymerase III promoters. We found that the U6 promoter is more efficient than the H1 promoter for shRNA expression and for reducing expression of CCR5 in PBLs. However, shRNA expression from the U6 promoter resulted in a gradual decline of the transduced cell populations. With one CCR5 shRNA this decline could be attributed to elevated apoptosis but another CCR5 shRNA that caused cytotoxicity did not show evidence of apoptosis, suggesting sequence-specific mechanisms for cytotoxicity. In contrast to the U6 promoter, PBLs transduced by vectors expressing shRNAs from the H1 promoter could be maintained without major cytotoxic effects. Since a lower level of shRNA expression appears to be advantageous to maintaining the shRNA-transduced population, lentiviral vectors bearing the H1 promoter are more suitable for stable transduction and expression of shRNA in primary human T lymphocytes. Our results suggest that functional shRNA screens should include tests for both potency and adverse metabolic effects upon primary cells.