Imaging Genetic Information - Nanotechnology for Biomedical Imaging and Diagnostics - page 370

from transcription of retroviral DNA (such as HIV), viral DNA (such as adenovirus),
or RNA viruses resident in the cytoplasm (such as hepatitis c, influenza) [52]. The
most highly expressed mRNAs belong to the ribosomal proteins, in which mutations
have been associated with disease [53]. mRNA is usually in much lower abundance
than protein targets, with mRNAs ranging from 1 to 100,000 copies/cell corresponding
to 1 pm to 100 nm. mRNAs also fold, though most of the highly structured and pro-
tein binding sections of the mRNA are in the noncoding regions [37], thereby offering
the opportunity for antisense agents to binding in the coding sections.

13.3 antIsense rna ImagIng

The ability to noninvasively image RNA in a patient would enable one to monitor
disease states in which the disease state is associated with the expression of a unique
gene, such as an oncogene [54], or a viral gene [55, 56], or an overexpressed gene
[57]. Antisense imaging of RNA could also be used to monitor the level and distribu-
tion of therapeutic genes in gene therapy or to monitor the effectiveness of therapeutic
treatments that are based on suppressing gene expression. Once a disease-specific
mRNA sequence for a patient is identified, it would be straightforward, at least in
principle, to design and synthesize a patient-specific antisense nucleic acid probe
within days that can recognize and bind the RNA with high affinity by using only
Watson-crick base-pairing rules. The problem at the moment is that currently avail-
able antisense oligonucleotides and analogs are membrane impermeable, and
attempts to attach cell-targeting and cell-penetrating ligands to enable antisense
imaging by PeT have not been very successful [54, 58]. Thus, much of the
development of antisense imaging agents has shifted to the design of the antisense
delivery system and the use of optical turn-on probes that circumvent problems with
membrane permeability. The universality and patient specificity of an antisense
imaging approach, however, make it well worth the initial investment in developing
generic, FDA-approved delivery systems for delivering RNA imaging probes.
To design an antisense imaging probe for a specific patient requires (i) identification
of a suitable RNA for imaging and (ii) identification of sites on the RNA accessible
to an antisense oligomer. To make any given antisense probe functional will require
(i) a method for the efficient delivery of the antisense agent to the candidate cells,
(ii) efficient intracellular delivery of the antisense agent to the cytoplasm or target
organelle, (iii) efficient efflux of unbound antisense probe, or (iv) RNA target-
specific probe activation. We will now briefly discuss these technical requirements
individually in the following sections.

13.3.1 selection of an rna target

The RNA target will depend on the particular disease state. In the case of cancer,
there may be a specific mutation or rearrangement in the DNA that becomes tran-
scribed into a mutated or rearranged mRNA sequence [59-61]. In principle, genetic
changes could be detected by sequencing genomic DNA from a biopsy and compared