EURON and THEME joint PhD meeting

64
EURON and THEME joint meeting 2011
Local gene targeting and cell positioning using
magnetic nanoparticles for the generation of
biological cardiac pacemakers
Carsten Kilgus, Tobias Bruegmann, Bernd K. Fleischmann and Philipp Sasse
Institute of Physiology I, Life&Brain Center, University Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany.
Implantation of electronic implantable pacemakers is the primary therapy for
patients with sinus node dysfunction or high degree of atrio-ventricular block.
Although complications are rare, this treatment has some limitations which is
why the development of biological pacemakers is studied. A biological pacemaker
should be confined to an area within the heart and we have therefore developed
new technologies for localized cell therapy and gene transfer to cardiomyocytes
using magnetic nanoparticles (MNP) and magnetic fields.
First initial proof of principle studies, a monolayer cardiomyocytes from the HL-1
line transfected with a lentivirus that expressed the green fluorescent protein
(GFP). Localization was achieved by generation of complexes with MNP and
lentiviruses (200 fg iron per virus particle) and transduction of cells in a confined
magnetic field that was generated by neodymium-iron-boron permanent
magnets with specially designed soft iron tips. This approach allowed reliably
and stable transduction of cells within an area of < 1mm diameter indicated by
localized GFP expression.
To provide a functional pacemaker we plated HL-1 cells onto multi-electrode
arrays (MEA, 60 electrodes, 200 µm spacing) to read out the local field potentials
and calculated the site from where pacemaking was initiated. We performed
localized transduction with MNP-lentivirus complexes that express the light-gated
ion channel channelrhodopsin2 (ChR2). This lead to localized ChR2 expression
and light flashes paced the whole monolayer. Importantly latency analysis of the
MEA recordings proved that pacing was always initiated from the transduced sites
despite global illumination. To be able to generate an autonomeous pacemaker,
we have successfully generated lentiviruses to express the classical pacemaker
channels HCN2 and HCN4 as well as a dominant-negative potassium channel
(Kir2.1-AAA). Preliminary long-term MEA recordings and localized transduction
with the Kir2.1-AAA virus indicated that pacemaking is initiated from the
transduced site 2-3 days after localized gene transfer.
Besides gene transfer we have also tested localized cell positioning for pacemaker
generation. Therefore we have differentiated and purified ChR2-expressing