Kurre Lab Current Projects
Hematopoietic Stem Cell Biology
EXOSOMAL RNA IN THE LEUKEMIA MICROENVIRONMENT
Acute Myeloid Leukemia (AML) is a disease that owes much of its tenacity to its ability to communicate with and subvert the normal bone marrow niche. The conversion of the bone marrow into the leukemic niche is believed to mediate effects including spread of disease, immune response, and resistance to treatment. One means by which this is accomplished is by the transfer of RNA in small extracellular vesicles called exosomes. Our research focuses on the transfer of exosomal RNA (and particularly microRNA) between AML and the cells that make up the marrow microenvironment (Image). In order to understand the relevant facets of this process, we have developed a number of models, both in vitro (using an oxygen-controlled environment and culture materials designed to allow for cells to grow in connected but separate environments) and in vivo (using an immune-deficient mouse strain engrafted with exosome-treated hematopoietic cells or with human leukemia).
Anchoring' Lentiviral Vectors
Vector integration events can cause transcriptional aberration and proto-oncogene activation in the host genome. Non-integrating retroviral vectors are safer in that context but without a cell dependent replication mechanism with their ‘floating’ genomes are rapidly lost during cell division. We are developing mitotically stable non-integrating Lentiviral vectors for safe and stable gene delivery into hematopoietic stem cells. In our first generation vectors the beta interferon Scaffold/Matrix Associated Region (S/MAR), which provides potential cell cycle-dependent replication and nuclear retention, was incorporated into the vector backbone to meet this elusive goal. This circular virus enters rapidly dividing cells and is retained over many cell generations in tissue culture and after transfer to murine hematopoietic stem cells. On going studies are currently towards using this vector in preclinical models to deliver therapeutically relevant transgenes and further improve the vector efficacy.
Replication deficient particles access the cell through a series of steps involving no specific (receptor independent) receptor dependent mechanisms. Previous work demonstrated the limitations posed by cell surface receptor expression and, by implication, heterologous pseudotyping with the cognate envelope. In the context of more recent studies using vesicular stomatitis virus G envelope protein we have identified additional aspects of the initial cell-vector interaction that reveal cell specific, cell entry kinetics associated with the complexity of the fibronectin extracellular matrix (Figure). Experiments are ongoing to understand how transduction of stem cell target population can be further optimized in light of these findings.
Ex vivo culture of HIV-derived lentivirus particles is generally thought to result in non specific attachment, receptor mediated binding, followed by uptake of the particle by the cell and proviral integration, or degradation via lysosomal / proteasomal mechanisms. Studies in the laboratory over the past years suggest that additional non-degradative cytoplasmic fates may exist. Based on extensive experiments a model (Figure) is now emerging whereby a minority of particles are capable of cytoplasmic persistence in association with key marker proteins of an exosomal pathway. And while DC-SIGN mediated uptake of native HIV particles can lead to exosomal sequestration by dendritic cells and ' trans-infection' of T-cells, our observation offers no apparent cell type restriction, suggesting a generally conserved cellular mechanism. The underlying biology and potential therapeutic implications of these studies are subject of ongoing investigation.
Fanconi Anemia | Genetics and Biology
Bone marrow failure is the most common cause of morbidity and mortality from Fanconi anemia (FA), a recessively inherited disorder resulting from mutations in one of at least 15 genes that cooperate in a DNA repair pathway. The underlying etiology is thought to reflect an accelerated postnatal exhaustion of the hematopoietic stem and progenitor cell (HSPC) pool. However, laboratory evidence of compromised hematopoietic function in patients generally precedes symptoms of cytopenia, and several other mesodermal-derived organ systems show defects with prenatal onset, including the skeletal system, heart, kidneys, and others.
Although spontaneous bone marrow failure does not occur in most murine models of FA, animals recapitulate impaired repopulating ability, characteristic cell cycle abnormalities, and impaired cytokine responses. The fetal liver provides a unique microenvironment for development of definitive hematopoietic function and serves as a site of massive HSPC expansion. Our ongoing studies suggest that bone marrow failure begins early during development with a progressive deficiency in progenitor number and function. Results contrast with a conventional model of postnatal stem cell attrition and may impact the development of preemptive therapies for FA patients.