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Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model
Published on: July 2, 2016
Targeting CCR5 with siRNAs: using recombinant SV40-derived vectors to protect macrophages and microglia from
Pierre Cordelier1, Barry Morse, David S Strayer
1Department of Pathology and Cell Biology, Jefferson Medical College, Philadelphia, PA 19107, USA.
Abstract:
Transducing macrophages and other phagocytic cells has been problematic because these cells are largely nondividing and can phagocytose and degrade viral gene delivery vectors. Because of their carriage of the CCR5 chemokine receptor that functions as a coreceptor for most clinical strains of HIV, these cells are also key targets in early HIV infection and dissemination. We describe here a strategy to transduce these phagocytes, reduce cell membrane CCR5, and protect from infection with R5-tropic HIV. Recombinant Tag-deleted SV40 vectors were used to transduce unselected CCR5-bearing cell lines and primary cells with >98% efficiency. rSV40s were designed to express two different anti-CCR5 small interfering RNAs (siRNAs), driven by the adenoviral VA1 polymerase III (pol III) promoter, which localizes the transcripts in the cytoplasm. Transduction with both siRNAs substantially reduced CCR5 mRNA, which in turn decreased detectable cell membrane CCR5. Both CCR5+ cell lines and primary cells were used: SupT1/CCR5 cells, monocyte-derived macrophages (MDM), and primary human brain microglia. In addition, one siRNA, siRNA R5 #5, was designed to recognize conserved sequences in both murine and human CCR5 mRNA and effectively reduced CCR5 transcript in cells of both species. These siRNAs largely protected CCR5+ cell lines and primary human macrophages and brain microglia from challenge with R5-tropic HIV. Therefore, strategies to target CCR5 using rSV40-delivered, VA promoter-driven siRNAs may be useful therapeutic options for treating HIV infection.
Insights
This study introduces a novel method using modified SV40 vectors to deliver small interfering RNAs (siRNAs) targeting CCR5. This approach effectively reduces CCR5 on phagocytic cells, offering protection against R5-tropic HIV infection.
Area of Science:
- Gene therapy and virology
- Immunology and infectious diseases
- Molecular and cellular biology
Background:
- Macrophages and phagocytic cells are difficult to transduce due to their non-dividing nature and vector degradation.
- These cells express CCR5, a coreceptor crucial for early HIV infection and spread.
- Targeting these cells is vital for developing effective HIV therapies.
Purpose of the Study:
- To develop a strategy for transducing phagocytic cells.
- To reduce CCR5 expression on cell membranes.
- To protect these cells from R5-tropic HIV infection.
Main Methods:
- Utilized recombinant Tag-deleted SV40 vectors (rSV40s) for transduction.
- Engineered rSV40s to express anti-CCR5 small interfering RNAs (siRNAs) via the adenoviral VA1 polymerase III promoter.
- Tested efficacy in CCR5+ cell lines, monocyte-derived macrophages (MDM), and primary human brain microglia.
Main Results:
- Achieved >98% transduction efficiency in unselected CCR5-bearing cells.
- Demonstrated substantial reduction in CCR5 mRNA and cell membrane CCR5 expression.
- Showed significant protection of various cell types against R5-tropic HIV challenge.
- One siRNA (siRNA R5 #5) effectively reduced CCR5 in both murine and human cells.
Conclusions:
- rSV40-delivered, VA promoter-driven siRNAs targeting CCR5 represent a promising therapeutic strategy.
- This approach can be used to transduce phagocytic cells and confer resistance to HIV.
- Further development may lead to novel treatments for HIV infection.
