Friday, November 22, 2024
11/22/2024
DESCRIPTION (provided by applicant): The immune response integrates interactions between diverse cell types, signaling molecules and pathways that can most effectively be studied in vivo. The immune response to infection or vaccination is the outcome of such a complex network of positive and negative regulatory pathways that it is often difficult to predict from in vitro experiments what will happen in vivo. Because immune genes are amongst the most highly evolving genes and the cytokine and chemokine responses in mice and humans often differ, mouse immune responses often differ qualitatively from human responses. Understanding and manipulating human immune responses is challenging, given the genetic diversity and unique history of environmental/infectious exposure of each individual. Experimental manipulations (i.e. gene knockout, blocking antibodies, and cytokines) that have been so powerful for dissecting the molecular basis of mouse immunology are rarely possible in humans. Two recent technologies, RNA interference (RNAi) and humanized mice with robust functional human immune responses, now provide an opportunity for genetic manipulation of the in vivo human immune response. The goal of this proposal is to join these two technologies to establish a method to knock down individual genes in CD4+ and CD8+ cells in humanized mice transplanted with human bone marrow, fetal liver and thymus (BLT mice) and use it to manipulate human immunity in vivo. BLT mice reconstitute an immune system that mimics human immune cell distribution in uninfected mice, as well as viral dynamics and B and T cell responses to HIV infection. RNAi is a ubiquitous mechanism for suppressing gene expression, which can be harnessed to knockdown individual genes by introducing exogenous small double-stranded RNAs with sequences complementary to a target gene (siRNAs) into a cell. The main obstacle to harnessing the power of RNAi for in vivo studies is delivering small RNAs across the plasma membrane, which is especially challenging for lymphocytes, which are both a moving target and resistant to transduction. We engineered chimeric small RNAs, composed of an aptamer that recognizes human CD4, linked at its 3'-end to a siRNA. CD4 aptamer- siRNA chimeras (CD4-AsiC) knockdown genes specifically in human CD4+ cells in peripheral blood, human tissue explants, and in BLT mice after intravaginal administration. Our first aim is to test and optimize CD4- AsiCs for systemic knockdown of gene expression in CD4 T cells, monocytes and macrophages. We will also engineer similar CD8 aptamer-siRNA chimeras for gene knockdown in CD8 T cells and NK cells. To test the utility of the AsiC method for studying human immunity in vivo, we will design CD4-AsiCs to knockdown FOXP3 and BCL6, transcription factors that direct CD4 T cell differentiation into suppressive Treg cells and antibody-promoting TFH cells, respectively. We will use them to test the hypothesis that enhancing TFH cells and suppressing Treg cells augments the humoral response to two HIV candidate vaccines, a gp120 subunit vaccine and whole killed virus. PUBLIC HEALTH RELEVANCE: Attempts to design effective vaccines for many of the world's serious chronic infectious diseases, including HIV, malaria and hepatitis C, have not been successful; vaccines that look promising in other species often fail in human clinical trials. This proposal will develop a powerful tool that will be useful for dissecting the contribution of individual immune cell proteins to human immune responses by knocking down individual genes one at a time in human CD4+ or CD8+ cells in vivo in humanized mice. These tools should not only provide a more scientific basis for vaccine design and understanding human immune protection and immunopathology, but the methods developed in this proposal could also be applied to knockdown gene expression in other cell types to study other complex human disease processes in vivo in human cells; this strategy potentially could also be adapted to design RNAi-based drugs suitable for systemic targeted delivery of small RNAs directed against disease-causing genes.
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