Gene transfer technologies and new gene therapy strategies

SR-Tiget Unit - ERC Advanced Grant
Luigi Naldini, Head of Unit

Research activities

 2. Investigating microRNA biology in the hematopoietic system

Bernhard Gentner (Project Leader), Alice Giustacchini (PhD student), Francesco Boccalatte (PhD student), Cristiana Fanciullo (undergraduate student)

microRNA are a large class of small non-coding RNA molecules which shape gene expression by negatively regulating mRNA stability and translation when binding to a specific 7bp+ motif contained within the 3’UTR of mRNA transcripts. We have developed vectors containing artificial miRNA target sequences whose expression is negatively regulated by a given miRNA. The optimization of these vectors has led to the development of several invaluable tools to study microRNA function. These include miRNA reporter vectors (both miRNA-OFF and miRNA-ON versions built within bidirectional LV) allowing to monitor miRNA activity in real time, at the single cell level and in a multitude of cell populations simultaneously, as well as positive or negative selection of a desired cell population based on miRNA expression. Moroever, we have developed miRNA sponge vectors which can stably antagonize miRNA function and achieve a near-complete miRNA knockdown in primary cells and in vivo. We have applied these vector tools to study the function of miR-223 and miR-126 in both mouse and human hematopoiesis, and have identified a key role of these miRNAs in the regulation of myeloid differentiation and HSC cell cycle progression, respectively. We showed that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function (see figure taken from Lechman, Gentner et al., Cell Stem Cell 2012, 11:799-811).

Upon establishing miRNA gain and loss of function, we are employing unbiased proteomic and transcriptomic approaches to identify relevant miRNA targets and the molecular networks they act on. These data are providing important insights into how HSC quiescence, commitment and differentiation are regulated. Ongoing work is focused on deciphering the pathways these miRNAs act on and investigating additional miRNAs which have a role in HSC and early progenitor functions. We will exploit the knowledge obtained in these studies to further improve HSC manipulation for gene and cell therapy (see Project 1). There is also growing evidence that deregulation of miRNAs (including miR-126) can contribute to disease such as leukemia/lymphoma. In collaboration with the hematology and pathology department, we are studying patient samples to shed light on the clinical relevance of these miRNAs in health and disease.


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