Andreas M. Loening

Self-illuminating quantum dot conjugates for in vivo imaging.

Fluorescent semiconductor quantum dots hold great potential for molecular imaging in vivo. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and a paucity of excitation light at nonsuperficial locations. Here we present quantum dot conjugates that luminesce by bioluminescence resonance energy transfer in the absence of external excitation. The conjugates are prepared by coupling carboxylate-presenting quantum dots to a mutant of the bioluminescent protein Renilla reniformis luciferase. We show that the conjugates emit long-wavelength (from red to near-infrared) bioluminescent light in cells and in animals, even in deep tissues, and are suitable for multiplexed in vivo imaging. Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 103 for 5 pmol of conjugate.

AMIDE: a free software tool for multimodality medical image analysis.

Amides a Medical Image Data Examiner (AMIDE) has been developed as a user-friendly, open-source software tool for displaying and analyzing multimodality volumetric medical images. Central to the packages abilities to simultaneously display multiple data sets (e.g., PET, CT, MRI) and regions of interest is the on-demand data reslicing implemented within the program. Data sets can be freely shifted, rotated, viewed, and analyzed with the program automatically handling interpolation as needed from the original data. Validation has been performed by comparing the output of AMIDE with that of several existing software packages. AMIDE runs on UNIX, Macintosh OS X, and Microsoft Windows platforms, and it is freely available with source code under the terms of the GNU General Public License.

Red-shifted Renilla reniformis luciferase variants for imaging in living subjects

The use of R. reniformis luciferase (RLuc) as a reporter gene in small-animal imaging has been hampered by its 481 nm peaked emission spectrum, as blue wavelengths are strongly attenuated in biological tissues. To overcome this, we generated variants of RLuc with bathochromic (red) shifts of up to 66 nm (547 nm peak) that also had greater stability and higher light emission than native RLuc.

A versatile shear and compression apparatus for mechanical stimulation of tissue culture explants

We have developed an incubator housed, biaxial-tissue-loading device capable of applying axial deformations as small as 1 microm and sinusoidal rotations as small as 0.01 degrees. Axial resolution is 50 nm for applying sinewaves as low as 10 microm (or 1% based on a 1 mm thickness) or as large as 100 microm. Rotational resolution is 0.0005 degrees. The machine is small enough (30 cm high x 25 cm x 20 cm) to be placed in a standard incubator for long-term tissue culture loading studies. In metabolic studies described here, application of sinusoidal macroscopic shear deformation to articular cartilage explants resulted in a significant increase in the synthesis of proteoglycan and proteins (uptake of (35)S-sulfate and (3)H-proline) over controls held at the same static offset compression.

An Improved Bioluminescence Resonance Energy Transfer Strategy for Imaging Intracellular Events in Single Cells and Living Subjects

Bioluminescence resonance energy transfer (BRET) is currently used for monitoring various intracellular events, including protein-protein interactions, in normal and aberrant signal transduction pathways. However, the BRET vectors currently used lack adequate sensitivity for imaging events of interest from both single living cells and small living subjects. Taking advantage of the critical relationship of BRET efficiency and donor quantum efficiency, we report generation of a novel BRET vector by fusing a GFP(2) acceptor protein with a novel mutant Renilla luciferase donor selected for higher quantum yield. This new BRET vector shows an overall 5.5-fold improvement in the BRET ratio, thereby greatly enhancing the dynamic range of the BRET signal. This new BRET strategy provides a unique platform to assay protein functions from both single live cells and cells located deep within small living subjects. The imaging utility of the new BRET vector is shown by constructing a sensor using two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. This new BRET vector should facilitate high-throughput sensitive BRET assays, including studies in single live cells and small living subjects. Applications will include anticancer therapy screening in cell culture and in small living animals.

BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals

Taking advantage of the bioluminescence resonance energy transfer (BRET) phenomenon, we report the development of a highly photon‐efficient, self‐illuminating fusion protein combining a mutant red fluorescent protein (mOrange) and a mutant Renilla reniformis luciferase (RLuc8). This new BRET fusion protein (BRET3) exhibits severalfold improvement in light intensity in comparison with existing BRET fusion proteins. BRET3 also exhibits the most red‐shifted light output (564‐nm peak wavelength) of any reported bioluminescent protein that utilizes its natural substrate coelenterazine, a benefit of which is demonstrated at various tissue depths in small animals. The imaging utility of BRET3 at the single‐cell level is demonstrated using an intramolecular sensor incorporating two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. With its increased photon intensity, red‐shifted light output, and good spectral resolution (~85 nm), BRET3 shows improved spatial and temporal resolution for measuring intracellular events in single cells and in living small animal models. The development of further BRET3‐based assays will allow imaging of protein‐protein interactions using a single assay directly scalable from intact living cells to small living subjects, allowing accelerated drug discovery.— De, A.,Ray, P., Loening, A. M., Gambhir, S. S. BRET3: a red‐shifted bioluminescence resonance energy transfer (BRET) based integrated platform for imaging protein‐protein interactions from single live cells and living animals. FASEBJ. 23, 2702–2709 (2009)

Creating self-illuminating quantum dot conjugates

Semiconductor quantum dots are inorganic fluorescent nanocrystals that, because of their unique optical properties compared with those of organic fluorophores, have become popular as fluorescent imaging probes. Although external light excitation is typically required for imaging with quantum dots, a new type of quantum dot conjugate has been reported that can luminesce with no need for external excitation. These self-illuminating quantum dot conjugates can be prepared by coupling of commercially available carboxylate-presenting quantum dots to the light-emitting protein Renilla luciferase. When the conjugates are exposed to the luciferases substrate coelenterazine, the energy released by substrate catabolism is transferred to the quantum dots through bioluminescence resonance energy transfer, leading to quantum dot light emission. This protocol describes step-by-step procedures for the preparation and characterization of these self-illuminating quantum dot conjugates. The preparation process is relatively simple and can be done in less than 2 hours. The availability of self-illuminating quantum dot conjugates will provide many new possibilities for in vivo imaging and detection, such as monitoring of in vivo cell trafficking, multiplex bioluminescence imaging and new quantum dot-based biosensors.

A red-shifted Renilla luciferase for transient reporter-gene expression

To the Editor: The principal limitation when using Renilla reniformis luciferase (RLuc) as a reporter for bioluminescence imaging in small-animal models has been that, because of its blue-peaked (481-nm) emission spectrum and the preferential absorption of short-wavelength photons by biological tissues, RLuc has diminished sensitivity at non-superficial locations1. To overcome this, we previously developed the red-shifted variant RLuc8.6-535 (ref. 2), which, in addition to having enhanced enzymatic activity compared to the native luciferase, shows an additional threefold increase in sensitivity at non-superficial tissue depths because of its green-peaked (535nm) emission spectrum. RLuc8.6-535 was derived from the variant RLuc8 (ref. 3) and retains RLuc8’s stabilized phenotype (intracellular half-life >50 h). Though advantageous for following constitutive gene expression, this may obscure transient changes in gene expression. We have therefore now developed a red-shifted RLuc variant with intracellular stability comparable to that of the native enzyme. We carried out site-specific random mutagenesis and selection as previously described2 (Supplementary Methods). A full description of the mutagenesis screens is in Supplementary Results. Briefly, we used the destabilized variant RLuc/M185V/Q235A3 as starting template and performed ten rounds of mutagenesis at the residue pairs Asp162/Ile163, Val185/Leu186, Asp154/Glu155, Ile159/Leu163, Glu160/Glu161, Trp156/Pro157, Ala164/Leu165, Lys136/Ile137, Phe286/Ser287 and Pro220/Arg221. Asp162 and residues in close proximity were mutated because Asp162 substitutions have previously yielded large shifts in the emission spectrum2, as was the case here. We also identified Asp162 mutations leading to blue-shifts (Supplementary Fig. 1), but did not pursue these further. Mutations at residue 185 were probed because this residue lies at the top of the active site4 and prior substitutions have increased quantum yield and

Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase.

Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc production using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biology provides the freedom to control both the catalyst and chemical compositions in biological reactions, and we capitalized on this to produce large amounts of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell extract source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metabolism, and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412mug/mL of purified GLuc, relative to 5mug/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2x10(24)photons/s/mol, the highest reported activity for any characterized luciferase.

Pilot Comparison of ⁶⁸Ga-RM2 PET and ⁶⁸Ga-PSMA-11 PET in Patients with Biochemically Recurrent Prostate Cancer.

Glu-NH-CO-NH-Lys-(Ahx)-[68Ga(HBED-CC)] (68Ga-PSMA-11) is a PET tracer that can detect prostate cancer relapses and metastases by binding to the extracellular domain of PSMA. 68Ga-labeled DOTA-4-amino-1-carboxymethyl-piperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (68Ga-RM2) is a synthetic bombesin receptor antagonist that targets gastrin-releasing peptide receptors. We present pilot data on the biodistribution of these PET tracers in a small cohort of patients with biochemically recurrent prostate cancer. Methods: Seven men (mean age ± SD, 74.3 ± 5.9 y) with biochemically recurrent prostate cancer underwent both 68Ga-PSMA-11 PET/CT and 68Ga-RM2 PET/MRI scans. SUVmax and SUVmean were recorded for normal tissues and areas of uptake outside the expected physiologic biodistribution. Results: All patients had a rising level of prostate-specific antigen (mean ± SD, 13.5 ± 11.5) and noncontributory results on conventional imaging. 68Ga-PSMA-11 had the highest physiologic uptake in the salivary glands and small bowel, with hepatobiliary and renal clearance noted, whereas 68Ga-RM2 had the highest physiologic uptake in the pancreas, with renal clearance noted. Uptake outside the expected physiologic biodistribution did not significantly differ between 68Ga-PSMA-11 and 68Ga-RM2; however, 68Ga-PSMA-11 localized in a lymph node and seminal vesicle in a patient with no abnormal 68Ga-RM2 uptake. Abdominal periaortic lymph nodes were more easily visualized by 68Ga-RM2 in two patients because of lack of interference by radioactivity in the small intestine. Conclusion: 68Ga-PSMA-11 and 68Ga-RM2 had distinct biodistributions in this small cohort of patients with biochemically recurrent prostate cancer. Additional work is needed to understand the expression of PSMA and gastrin-releasing peptide receptors in different types of prostate cancer.