ANTI-HIV TYPE-1 ACTIVITY OF WILD-TYPE AND FUNCTIONAL DEFECTIVE RANTESINTRAKINE IN PRIMARY HUMAN-LYMPHOCYTES

We have developed a genetic ''intrakine'' strategy to inactivate the C C-chemokine receptor 5 (CCR-5), the principal coreceptor for macrophag e (M)-tropic HIV-1 viruses (Yang et al., 1997). The inactivation of CC R-5 was achieved by targeting a modified CC-chemokine (RANTES) to the lumen of the endoplasmic reticulum (ER) to block the transport of the newly synthesized CCR-5. The transduced lymphocytes with the phenotypi c CCR-5 knockout were shown to be resistant to M-tropic HIV-1 infectio n. This study illustrated the feasibility of the intrakine strategy to block HIV-1 infection. In our current study, the potential clinical a pplication of the intrakine approach was further evaluated in human pe ripheral blood lymphocytes (PBLs), PBLs were transduced with the RANTE S intrakine gene by using retroviral vectors with the truncated low-af finity human nerve growth factor receptor (Delta NGFR) marker, and the n isolated by an anti-NGFR antibody/magnetic bead method. The surface expression of CCR-5 in the transduced lymphocytes was dramatically inh ibited, as demonstrated by flow cytometric assays. The transduced PBLs were shown to resist various types of M-tropic HIV-1 virus infection. The cell viability, cell proliferation rates, and cell surface marker s of the intrakine-transduced PBLs were shown to be comparable to thos e of control PBLs. The transduced PBLs were also found to respond to t he stimulation of various CXC- and CC-chemokines, other than RANTES. T he transduced PBLs responded to tetanus antigen stimulation by increas ing IL-2 production and cell proliferation. In addition, a functionall y defective mutant of RANTES that retains its binding activity to CCR- 5, but loses its signaling ability, was used to generate a mutant RANT ES intrakine. The primary lymphocytes transduced with the mutant RANTE S intrakine were found to be resistant to M-tropic HIV-1 infection. Fr om these results, we conclude that the primary human lymphocytes trans duced with either the wild-type or functionally defective RANTES intra kine are resistant to M-tropic HIV-1 infection, and maintain their bas ic biological functions. This study, therefore, indicates the potentia l clinical application of the intrakine approach for HIV-1 gene therap y.