Background. Sickle cell disease (SCD) is the most common form of inherited blood disorder affecting approximately 100,000 Americans. Patients with SCD suffer from repeated red cell sickling and vaso-occlusion episodes, which, since early age, lead to acute and chronic pain, stroke, anemia, infections, organ failure and premature death. Vaso-occlusion episodes cause ischemia-reperfusion injury, activation of endothelial cells, immune cells and the coagulation cascade. These processes lead to a state of chronic inflammation in SCD, associated with increased pro-inflammatory cytokines and induction of TLR4/NF-kB activation and oxidative stress in all organs, including the bone marrow (BM). To date, little is known about how inflammation impacts the BM microenvironment and HSC functions in SCD. Notably, few and limited studies have been conducted on human samples. To address this gap, we will examine the impact of SCD in the molecular profile and functional status of bone marrow-resident cells. Preliminary results. We observed that SCD patients enrolled for transplant could be divided in two groups based on the frequency of CD34+ cells. Group 1 displayed reduced frequency of CD34+ population whereas group 2 exhibited higher or similar frequency when compared to controls. Single cell RNA-sequencing (scRNA-seq) of CD34+ enriched cells from one patient assigned in group 1 showed changes in the number of distinct cell populations and their transcriptomic profiles, with enrichment in pathway related to cellular stress. We also found that SCD BM-derived mesenchymal cells exhibit an inflammatory profile, compared to controls, characterized by increased expression of the proinflammatory related genes miR-155, miR-146a, IL-8, and IL-6 by qPCR. Furthermore, we observed that BM tissue biopsies of patients at pre-transplant show evidence of dysfunctional stroma. We hypothesize that a pro-inflammatory state induced by SCD alters the composition of the BM microenvironment and damages bone-marrow resident HSPC cells.
Aims and Strategy. This study will be conducted on BM specimens of SCD patients (minimum n=10) prior HSCT and at different times after HSCT. To better understand the heterogeneity of SCD BM samples and correlate it with disease severity and clinical outcome after transplant, we propose an in-depth molecular analysis,which will be integrated with data from the parent grant. We will examine: i) the transcriptomic landscape of the HSCs in the BM of SCD patients by single cell RNA-sequencing (scRNA-seq); ii) determine the molecular pathways contributing to a pro-inflammatory phenotype in BM-derived mesenchymal cells by bulk RNA-seq, and iii) characterize the morphological and histological features in the BM biopsies of SCD patients. Relevance. This study represents a unique opportunity to better understand the biology of SCD in human specimens. It will also provide critical insights into the function of SCD CD34+ cells and the BM niche to explore new therapeutic approaches for improving current HSCT and gene therapy strategies.

Public Health Relevance

Approximately 100,000 Americans have sickle cell disease, the most common form of inherited blood disorder in which abnormal hemoglobin induces the red blood cells to block blood flow, causing pain and infections and numerous complications, including stroke, acute chest syndrome, and organ damage and in some cases premature death. Current treatments for SCD aim at pain management and prevention of complications; up to now the only curative treatment is bone marrow transplant, which presents risks and it is limited by the presence of available matched donors. In this application we propose to study the bone marrow microenvironment of SCD patients prior and after bone marrow transplant to identify determinants of bone marrow engraftment and to identify novel targets of intervention to improve transplant strategies and other therapeutic approaches, including gene therapy.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Bai, C Brian
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Beckman Research Institute/City of Hope
United States
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