Artificial reporter gene providing MRI contrast based on proton exchange

Existing magnetic resonance reporter genes all rely on the
presence of (super)paramagnetic substances and employ water
relaxation to gain contrast. We designed a nonmetallic,
biodegradable, lysine rich–protein (LRP) reporter, the prototype
of a potential family of genetically engineered reporters
expressing artificial proteins with frequency-selective contrast.
This endogenous contrast, based on transfer of radiofrequency
labeling from the reporter’s amide protons to water protons, can
be switched on and off.

Imaging the Beta-Cell Mass: Why and How

Diabetes is a disorder characterized by beta-cell loss or exhaustion
and insulin deficiency. At present, knowledge is
lacking on the underlying causes and for the therapeutic recovery
of the beta-cell mass. A better understanding of diabetes
pathogenesis could be obtained through exact monitoring
of the fate of beta-cells under disease and therapy
conditions. This could pave the way for a new era of intervention
by islet replacement and regeneration regimens.
Monitoring the beta-cell mass requires a reliable method for
noninvasive in vivo imaging. Such a method is not available at
present due to the lack of a beta-cell-specific contrast agent.
The only existing method to monitor islet cells in vivo consists
of labeling islet transplants with iron nanoparticles prior
to transplantation and visualization of the transplanted islets
by magnetic resonance imaging (MRI). Therefore, accurate
assessment of the native beta-cell mass is still limited to autopsy
studies. Endeavors to find a biological structure specific
for beta-cells led to the discovery of potential candidates
that have been tested for noninvasive imaging. Among
them are the ligand to the vesicular monoamine transporter
type 2 (VMAT-2), which is called dihydrotetrabenazine
(DTBZ), antibodies to zinc transporter (ZnT-8) and the
monoclonal antibody IC2. While DTBZ and antibodies to
ZnT-8 showed binding activities to more than beta-cells, the
anti-IC2 monoclonal antibody showed binding properties
exclusively to insulin-producing beta-cells. This effect was
demonstrated in many previous investigations, and has been
further substantiated more recently. Thus, at present, IC2
seems to be the only useful marker for noninvasive functional
imaging of native beta-cells. Experiments with a radioisotope-
chelated IC2 structure on pancreas ex vivo
showed that the tracer specifically bound to the beta-cell
surface and could be detected by nuclear imaging. In the
near future, these promising findings may offer a new way
to monitor the beta-cell mass in vivo under disease and therapy
conditions so that we can learn more about diabetes
pathogenesis and options for disease prevention.
Keywords: diabetes · beta-cell · noninvasive imaging ·
IC2 · islet transplantation · MRI · PET · photoacoustic

Deconvolution of a Complex Target Using DNA Aptamers

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 40, pp. 34193–34201

In vitro selection of single-stranded nucleic acid aptamers from large random sequence libraries is now a straightforward process particularly when screening with a single target molecule. These libraries contain considerable shape diversity as evident by the successful isolation of aptamers that bind with high affinity and specificity to chemically diverse targets.Wepropose that aptamer libraries contain sufficient shape diversity to allow deconvolution of a complex mixture of targets. Using unfractionated human plasma as our experimental model, we aim to develop methods to obtain aptamers against as many proteins as possible. To begin, it is critical that we understand how aptamer populations change with increasing rounds of in vitro selection when using complex mixtures. Our results show that sequence representation in the selected population changes dramatically with increasing rounds of selection. Certain aptamer families were apparent after only three selection rounds. Two additional cycles saw a decline in the relative abundance of these families and the emergence of yet another family that accounted for more than 60% of sequences in the pool. To overcome this population convergence, an aptamer-based target depletion method was developed, and the library screen was repeated. The previous dominant family effectively disappeared from the selected
populations but was replaced by other aptamer families. Insights gained from these initial experiments are now being applied in the creation of second generation plasma protein screens and also to
the analysis of other complex biological targets.

Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery

Gene Therapy (2007) 14, 682–689

This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirusmediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies.

Noninvasive in vivo imaging of pancreatic islet cell biology

Dimitri presented this paper on 1/22/09

Nature Medicine, 14, 2008, 574

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions.