Inducible Expression of Small Interfering RNAs to Inhibit HIV Replication

Jing-Kuan Yee, Beckman Research Institute of the City of Hope
Biomedical and Clinical Sciences

Background: Gene therapy strategies using genetically modified T cells or hematopoietic stem cells (HSCs) have been proposed as an adjuvant to chemotherapy for treating HIV infection. The discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells raises the possibility of harnessing RNA interference (RNAi) technology as a therapeutic tool against HIV infection. This is confirmed by stable expression of short hairpin RNAs (shRNAs) via HIV vector-mediated gene transfer in T cells, rendering cells resistant to HIV replication. However, HIV resistant strains with mutations in the siRNA target site emerge quickly. Thus, expression of siRNAs against multiple targets in the HIV genome may be necessary to effectively suppress the emergence of resistant strains. Using multiple pol II promoters in a HIV vector to drive siRNA expression is technically difficult. Using multiple pol III promoters is feasible but constitutive siRNA expression from strong pol III promoters will most likely generate undesirable "off-target" effect in the target cell. In contrast to siRNA, multiple microRNAs (miRNAs) can be expressed as a single transcript from a pol II promoter and can be processed efficiently by Drosha based on their unique stem-loop structures.

Methods: We propose to evaluate whether multiple siR-NAs with each siRNA flanked by the stem-loop structure of pri-miR-30a can be expressed and processed properly from a pol II promoter-derived polycistronic transcript. We will determine whether inducible pol II promoters can be used to drive the expression of multiple siRNAs against HIV in the target cells.

Results or expected results: We have so far inserted an shRNA gene against a common exon for the tat and rev genes into the stem-loop structure of pri-miR-30a. A constitutive pol II promoter and the GFP gene were placed upstream from the shRNA gene and the whole cassette was then inserted into a HIV vector. H9 cells were transduced with this vector and challenged with HIV-1. We will determine whether the siRNA is properly processed and whether it can render the cell resistance to HIV-1 replication. If this design allows efficient expression and proper processing of this siRNA, we will evaluate whether inserting two shRNA genes targeted to different regions of the HIV genome can lead to a similar efficiency of expression and processing. We will then determine whether the well-characterized human interleukin 2 (IL-2) promoter can drive T cell-specific and inducible expression of the siRNA genes. We will also evaluate whether HIV long terminal repeat (LTR) which responses strongly to the HIV Tat protein can also serve as an inducible promoter for siRNA expression.