1. ¹Division of Molecular Medicine, Beckman Research Institute and City of Hope National Medical Center, Duarte, CA 91010, USA
2. ²Division of Molecular Biology, Beckman Research Institute and City of Hope National Medical Center, Duarte, CA 91010, USA
3. ³Division of Pediatric Hematology–Oncology, Beckman Research Institute and City of Hope National Medical Center, Duarte, CA 91010, USA
4. ⁴Division of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute and City of Hope National Medical Center, Duarte, CA 91010, USA

Correspondence: John J. Rossi, Division of Molecular Biology, Beckman Research Institute of the City of Hope National Medical Center, 1450 East Duarte Road, Duarte, CA 91010-3000, USA. Fax: (626) 301-8271. E-mail: jrossi@bricoh.edu

Received 29 November 2004; Accepted 27 December 2004.

Abstract

Posttranscriptional suppression of gene expression can be achieved by introduction of sequence-specific small interfering (si) RNA duplexes and by de novo intracellular synthesis of short sequence-specific double-stranded RNAs. However, achieving desired levels of knockdown is a barrier to successful analytic and therapeutic application. We demonstrate that increasing expression of introduced short hairpin RNA (shRNA) can markedly enhance RNA interference (RNAi) and that this approach can be used to achieve maximal target down-regulation, when the choice of optimal siRNA-binding sites is restricted or when multiple genes are simultaneously targeted and the amount of siRNA is limiting. A dose-dependent RNAi effect was accomplished by placing copies of shRNA under control of the Pol III U6 small nuclear RNA promoter in tandem in a DNA vector. Using this system, we achieved simultaneous down-regulation of expression of classical human leukocyte antigen (HLA) class I genes in cultured and primary human T cells, which might be applied to help circumvent T-cell-mediated rejection of immunogenic and/or HLA-disparate allografts.