Keynote Conference - Interevent

Symp#3 Gene Therapy
Chair Martin Bonamino
Gene Therapy of Limb Ischemia with GM-CSF
Sang Won Han
Department of Biophysics, Federal University of São Paulo, São Paulo, Brazil
Peripheral arterial diseases (PAD) affect about 1000 per million every year and about 200 of them suffer limb
amputations. The PAD incidence increases in the case of diabetic patients and people with more than 60 years. Therefore,
PAD has a high socio-economic and medical impact today. Approximately half of critical limb ischemic patients can be
treated by vascular surgery, but the remainder depends mainly on the velocity of adaptation of the existing collateral
vessels, a process known as arteriogenesis.
Several growth factors, cytokines and proteases are required for arteriogenesis, which is the process of remodeling preexistent
vessels during the ischemia. The monocytes, attracted by the presence of tumor necrosis
factor-α and monocyte chemoattractant protein-1 at the inflammatory sites, are the main producers of factors for
arteriogenesis. The time and concentration of the factors required to complete arteriogenesis varies in each case of PAD,
consequently the action of the activated monocytes may not be enough to resolve the ischemic problem. One way to
prolong the life of monocytes is by inhibiting apoptosis by granulocyte-colony-stimulating factor (GM-CSF), which is a
hematopoietic-stimulator that enhances the survival, proliferation and rate of differentiation of hematopoietic cells.
In my presentation, therapeutic effects of GM-CSF in limb ischemia by gene therapy will be presented and discussed.
A Hematopoietic Stem Cell (HSC) Specific microRNA Gives Novel Insights into the Regulation of HSC Homeostasis and
Allows Safer HSC-based Gene Therapy
Bernhard Gentner 1,2 , Alice Giustacchini 1,2 , Ilaria Visigalli 1 , Eric Lechman 3 , Peter van Galen 3 , Hidefumi Hiramatsu 3 , Francesco
Boccalatte 1,2 , Massimo Saini 1 , Silvia Ungari 1,2 , John E Dick 3 , Alessandra Biffi 1 and Luigi Naldini 1,2 .
1 San Raffaele Telethon Institute for Gene Therapy; 2 Vita-Salute San Raffaele University, San Raffaele Scientific Institute,
Milan, Italy; 3 Division of Stem Cell and Developmental Biology, University of Toronto, Toronto, Ontario
We report that miR-126, a microRNA preferentially expressed in Hematopoietic Stem Cells (HSC) among the
hematopoietic lineage, plays a pivotal role in restraining cell cycle progression of HSC in vitro and in vivo. miR-126
knockdown expanded functional mouse and human HSC in vivo, without HSC exhaustion. Conversely, enforced miR-126
expression increased the fraction of phenotypic HSC in G 0. miR-126 control of proliferation is attributable, in part, to
attenuation of signal transduction by the PI3K/AKT/GSK3β axis. We propose that miR-126 establishes a threshold for HSC
activation and provides negative feedback aiding the return of stimulated HSC to quiescence. Our results establish that
the HSC quiescence/activation equilibrium is regulated by miRNAs in a mechanism conserved between mouse and human.
The discovery of HSC-specific microRNAs also prompted us to design novel vectors for HSC-mediated gene therapy with
enhanced efficacy and safety. By adding miR-126 target sequences (miRT) to a vector cassette, miR-126 activity was
harnessed to negatively regulate transgene expression in HSC and target expression to differentiated hematopoietic cells.
These HSC-off vectors allow delivering a transgene into HSC without affecting its proteome, while achieving sustained
multi-lineage expression in the progeny. The utility of this new vector was demonstrated for the gene therapy of Krabbe
disease in the mouse model. miR-126 regulated vectors overcame hematopoietic toxicity associated to unregulated
galactocerebrosidase (GALC) expression in HSC, while delivering substantial amounts of GALC enzyme in the nervous
system with improved survival of the affected mice.
Conditional models for Chimeric Antigen Receptor (CAR) based activation of T lymphocytes
Martin Bonamino
Instituto Nacional de Cancer – Rio de Janeiro – Brazil
The modification of T lymphocytes with Chimeric Antigen Receptors (CARs) represents a promising strategy for combined
adoptive T cell based immune-gene therapy of cancer. Current CAR molecules are constructed fusing an immunoglobulinderived
Fab-based antigen recognition domain in the scFv configuration, a transmembrane domain and signaling domains
promoting T cell activation. Clinical trials based on this strategy have reported impressive hematological responses for
leukemia and lymphomas for CARs designed against CD19, CD20 or CD30 antigens.
One potential limitation of this strategy is the off-target effects observed when target antigens are expressed in healthy
tissues. To circumvent this limitation we are considering conditional activation based on two CARs in a way that only the
correct antigen combination induces complete T cell activation. This strategy has the potential to expand the panel of
CAR-targeted antigens, narrowing T cell responses based on CAR mediated antigen recognition and increasing the safety
of CAR-based immune-gene therapies.
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