RNA interference (RNAi) is an evolutionarily conserved process by which both plants and animals protect their genome, using small, double-stranded RNA to degrade targeted RNAs in a sequence-specific manner. Post-transcriptional gene silencing by these moieties can lead to degradation of both cellular and viral RNAs. As such, this approach has been utilized by our group and others to target HIV-1 in human cells. As the CNS of HIV-1-infected individuals is an important site for viral pathogenesis, the use of this potent new approach should now be targeted to inhibit HIV-1 in the CNS. We hypothesize that both HIV-1 neuroinvasion and neuropathogenesis can be selectively targeted by RNAi against viral replication in certain human CNS-based cells, plus attacking apoptosis pathways induced by HIV-1. In this proposal, we will use our previous data on small interfering RNAs (siRNAs) to:
Specific Aim I - Design and construct RNAi moieties to inhibit specific viral and cellular targets towards inhibiting HIV-1 in the CNS. These targets will include cellular factors such as chemokine co-receptors, CCR5, CXCR4 and the CNS-Iocalized co-receptor APJ, as well as intracellular targets such as the DEAD box helicase DDX1, recently shown by our laboratory to be a co-factor for HIV-1 Rev. In addition, targets against viral open reading frames will be utilized, including targeting multiply-spliced plus unspliced HIV-1-specific RNAs. The most potent and specific sites, by evaluating possible homology with cellular genes by Blast searches will be utilized. Targets to conserved areas of the viral genome between different strains, as well as chemokine receptor-dependent, strain-specific siRNAs will be utilized via consensus sequences of CNS-isolated viral strains. Selected sites in the molecular pathways of apoptosis induced by HIV-1 virions and proteins in human neurons will also be targeted with siRNAs. Transfer of siRNAs will include both direct transfection with chemical-synthesized siRNAs and expression of siRNAs using hairpin motifs expressed from polymerase III promoters. HIV-1-based and spleen necrosis virus (SNV) vector systems will be utilized.
Specific Aim II; Modeling of anti-HIV-1 and anti-apoptosis RNAi approaches in CNS-relevant systems. After initial proof of concept studies using T-cell lines and primary CD4+ T-lymphocytes on various strains of virus, with controls including siRNAs to other cellular proteins as well as scrambled siRNAs, we will then target relevant human CNS-based cells individually and then in mixed cell cultures. We will utilize the lead siRNAs constructs to target human macrophages and microglia, plus differentiated human neurons. Viral infection will be analyzed in macrophages, microglia and human brain microvascular endothelial cells. Importantly, in neurons, blockade of CXCR4 will be utilized to inhibit the apoptosis induced by gp120 and free HTV-1 virions. The mixed cultures will include a morphologically intact human blood:brain barrier with microglia/macrophages, microvascular endothelium, neurons and astrocytes. In addition to HIV-1 replication, apoptosis assays using TUNEL and Annexin V will be analyzed. These initial approaches will target the human CNS utilizing RNAi to both inhibit HIV-1 replication in this potential sanctuary site, as well as evaluate targeted effects on neuronal drop put from programmed cell death. ? ? ? ?
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