Lentiviral transduction of Tar Decoy and CCR5 ribozyme into CD34+ progenitor cells and derivation of HIV-1 resistant T cells and macrophages

Akhil Banerjea,Ming-Jie Li,Ramesh Akkina,John Rossi,Leila Remling

doi:10.1186/1742-6405-1-2

Abstract

BackgroundRNA based antiviral approaches against HIV-1 are among the most promising for long-term gene therapy. These include ribozymes, aptamers (decoys), and small interfering RNAs (siRNAs). Lentiviral vectors are ideal for transduction of such inhibitory RNAs into hematopoietic stem cells due to their ability to transduce non-dividing cells and their relative refractiveness to gene silencing. The objective of this study is to introduce an HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme into CD34+ hematopoietic progenitor cells via an HIV-based lentiviral vector to derive viral resistant progeny T cells and macrophages.ResultsHigh efficiency and sustained gene transfer into CD34+ cells were achieved with lentiviral vector constructs harboring either Tar decoy or Tar decoy in combination with CCR5 ribozyme. Cells transduced with these constructs differentiated normally into T-lymphocytes in vivo in thy/liv grafts of SCID-hu mice, and into macrophages in vitro in the presence of appropriate growth factors. When challenged in vitro, the differentiated T lymphocytes and macrophages showed marked resistance against HIV-1 infection.ConclusionsViral resistant transgenic T cells and macrophages that express HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme could be obtained by lentiviral gene transduction of CD34+ progenitor cells. These results showed for the first time that expression of these anti-HIV-1 transgenes in combination do not interfere with normal thymopoiesis and thus have set the stage for their application in stem cell based gene therapy for HIV/AIDS.

Highlights

RNA based antiviral approaches against HIV-1 are among the most promising for long-term gene therapy
Using an in vitro cell differentiation system and in vivo SCID-hu mouse model, we show that expression of transactivation response element (Tar) decoy alone or in combination with an anti-CCR5 ribozyme has no adverse effect on lineage specific differentiation of CD34+ cells into macrophages and T lymphocytes
Tar and Tar-CCR5Rz vector transduced CD34+ cells differentiate normally into mature macrophages It is not known if lentivirus transduced Tar decoy and TarCCR5Rz, will have any adverse effects on the lineage specific differentiation of CD34+ cells into different end stage cells

Summary

Introduction

RNA based antiviral approaches against HIV-1 are among the most promising for long-term gene therapy. These include ribozymes, aptamers (decoys), and small interfering RNAs (siRNAs). A 32-base pair deletion in the CCR5 gene found in a segment of the normal European and North-American population rendered their macrophages resistant to infection by R5-tropic HIV-1 [3]. Since these individuals lacking a functional CCR5 are apparently normal, this gene has been targeted by many investigators to confer HIV-1 resistance. Efficacy of siRNAs in down regulating the CCR5 coreceptor and thereby preventing HIV-1 entry was described recently [7,8]

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