Introduction: Recent advances in molecular biology, genetics and live imaging allow direct comparison between invertebrates (nematodes, fruit flies) or vertebrates (zebra fish) and human development. Optogenetics and fluorescent gene probes have also made some advances in our ability to see connectivity and functional aspects of the central nervous systems in higher species, but these modalities may not easily achieve functional imaging because current imaging modalities suffer insufficient depth of penetration. Recent technological developments in magnetic resonance (MR) hardware and contrast agents have enabled high-resolution imaging of the soft brain tissue in living subjects at the anatomical, functional and molecular levels. MRI is a relatively safe technology that has excellent depth of penetration. Nanoparticle-enhanced MR imaging (MRI) is highly sensitive and can track neural progenitor cells in vivo after stem cell therapy in preclinical and perhaps clinical disease models. The mechanism of cell labeling with contrast agent alone is not well understood (1-4).
Rationale: We have developed nucleic acid-conjugated MR contrast agent from investigations in the literature including our own delivery of antisense DNA to living brains (5-7). Our rationale for imaging functional transcription is based on: (1) gene transcripts have short half-lives, and therefore represent real-time gene action in life forms; (2) Watson and Crick's hybridization by sequence homology can serve as the basis of mRNA tagging; (3) short DNA or RNA can be made more easily than antibodies against cellular antigens; and (4) antisense DNA transfects human cells.
Methods: Our MR contrast agent has three components (please see the Research page): Superparamagnetic iron oxide nanoparticles (SPION), NeutrAvidin (NA) and biotinylated phosphorothioate-modified oligoDNA (sODN), individually they are IND except NA. The MR contrast agent tags transcripts of various genes, i.e., transcriptome tagging, in living brains by direct injection via intracerebral (icv) route (40 mg Fe or 120 pmol per kg) to normal mice, or intraperitoneal (ip) injection (4 mg Fe or 12 nmol per kg) after cerebral ischemia.
The specificity of this contrast agent may have a therapeutic application (8,9). MR contrast agent tags intracellular mRNA in vivo for ex vivo primer-free reverse transcription to cDNA from mRNA (10); MR contrast agents with sequence not complementary to mRNA do not served as primer for reverse transcription and they are excluded in vivo (8,11).
The advantages of transcriptome targeted MRI:
- Real-time gene expression for longitudinal imaging/assays during the lifetime of the host with and without combination of neurological or behavioral tests (8,9).
- Delivery using a delivery route that is the same as that of Ommaya reservoir biopsy of brain tumors in humans.
- The U.S. FDA has approved the sODN (single-stranded DNA with phosphorothioate modification) as an IND.
- Molecular assays go green: MR-contrast agent-based assays eliminate many organic solvents that need special handling (12).
- The sODN we used is single-stranded DNA; therefore, it has a very minimal off-target effect by sense sequence if a double-stranded RNA (siRNA) were used.
- This nanotechnology can have application in transcriptome targeting by MR contrast agent for in vivo MRI with theranostic application.
Research supported by NIH (1R01EB013768 & 1R01DA029889 [EUREKA award]) and Am Heart Association GIA.
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