Inhibition of HIV-1 infection by a unique short hairpin RNA to chemokine receptor 5 delivered into macrophages through hematopoietic progenitor cell transduction.
BACKGROUND
We recently expressed a potent and noncytotoxic short hairpin (sh)RNA directed against chemokine (c-c motif) receptor 5 (CCR5) using lentiviral mediated transduction of CD34+ hematopoietic progenitor cells (HPCs) and demonstrated the stable reduction of CCR5 expression in T-lymphocytes.


METHODS
In the present study, we further assessed the activity of the shRNA through HPC transduction and differentiation into macrophages derived from fetal liver CD34+ (FL-CD34+) HPCs. Transduced lentiviral vector encoding the human CCR5 shRNA was stably maintained in FL-CD34+ cells and in the terminally differentiated macrophages using macrophage colony-stimulating factor, granulocyte macrophage colony-stimulating factor, interleukin-3 and stem cell factor.


RESULTS
Quantitative real-time polymerase chain reaction for CCR5 mRNA indicated over 90% reduction of CCR5 mRNA levels in CCR5 shRNA-transduced population. The cells with knockdown of CCR5 expression acquired resistance to R5 tropic HIV-1 NFN-SX strain. We also developed a novel approach utilizing a mCherry-CCR5 chimeric reporter to assess the effectiveness of CCR5 target down-regulation in macrophages directly. Both the shRNA and the reporter were maintained throughout HPC differentiation to macrophages without apparent cytotoxicity.


CONCLUSIONS
The present study demonstrates a novel method to simply and directly assess the function of small interfering RNA and the effective inhibition of HIV-1 infection by a potential potent shRNA to CCR5 delivered into macrophages derived from HPCs.
Liang
Min
M
Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, USA.
Kamata
Masakazu
M
Chen
Kevin N
KN
Pariente
Nonia
N
An
Dong Sung
DS
Chen
Irvin S Y
IS
D065100:CCR5 Receptor Antagonists; D002460:Cell Line; D055785:Gene Knockdown Techniques; D015316:Genetic Therapy / Q000379:methods*; D015658:HIV Infections / Q000517:prevention & control*; D015497:HIV-1; D006412:Hematopoietic Stem Cells / Q000502:physiology; D006801:Humans; D008264:Macrophages / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics*; D019713:Receptors, CCR5 / Q000235:genetics; D014161:Transduction, Genetic
D065100:CCR5 Receptor Antagonists; D034741:RNA, Small Interfering; D019713:Receptors, CCR5