Gene therapy, selectively correcting the genetic code of diseased cells, offers the prospect of treating many ailments at the molecular level.1
However, introducing new genetic code into cells is not a trivial exercise. The polynucleic acids in which genes are contained consist of long, negatively charged polymers and strong Coulombic repulsions with highly negatively charged cell membranes provides a natural barrier for gene delivery. To overcome this barrier transfection agents are required to deliver genetic code across these membranes.1
Successful gene therapy also requires the subsequent delivery of the new genetic code into the cell nucleus where genetic expression takes place. Viruses, which possess an innate ability to introduce foreign genetic code into cells, have been adapted for gene therapy with some success.1–3
Viral vectors have limitations however, particularly with respect to their propensity to stimulate an immune response.4
An alternative approach frequently considered for gene therapy is to bundle the anionic nucleic acids with cationic polymers, thereby neutralizing the charge and allowing passage of the polynucleic acid into cells.1
Many cationic polymers have been investigated for their potential utility to reliably transfect cells with non-native DNA.1
Although the development of gene therapy in clinical practice has stalled at the clinical trail stage, recent developments have shown extremely promising results: for example curing adenosine deaminase (ADA) deficiency,2,3
or restoring the sight of those with Leber's congenital amaurosis.5
However, one limitation of current gene therapy technology, whether viral or polymer based, is that it is difficult to track its progress in vivo
. There is considerable interest in being able to track gene therapy on two levels: first, by monitoring the distribution of the new genetic code throughout all tissue and second, assessing the outcome of gene transfection. This has stimulated modification of gene therapy agents to include reporters for detection by imaging modalities such as MRI.6–8
For practical applications it is often only possible to measure the success of gene therapy by waiting to see if the new genetic code is expressed and to measure the biological effects of that expression. Clearly, a method by which the success of gene therapy can be measured immediately and non-invasively would be a valuable addition to the gene therapy tool kit.
MRI is a technique by which images of soft tissue can be generated in a safe and non-invasive manner. As such, it is the ideal technique for consideration as a method by which gene therapy might be monitored. Recent reports have suggested that the cationic polymers used to bundle nucleic acids,9,10
and even the nucleic acids11
themselves could function as markers for gene therapy in MRI scans. These reports were based upon the idea that it is possible to apply a frequency selective, low energy pulse to protons of the polymer or nucleic acid that are in exchange with those of the bulk water of tissue. It is these bulk water protons that are monitored by MRI and by applying a frequency selective pulse we ‘saturate’, or wipe out the signal, from these exchangeable protons. When these protons move into the bulk water, the signal intensity of bulk water is reduced, generating image contrast in an MRI through a chemical exchange saturation transfer (CEST) mechanism.12,13
Although this approach allows image contrast to be generated specifically from the nucleic acid or polymer with which it was bundled, it cannot provide a means of tracking the effectiveness of gene therapy. The exchangeable protons used to probe the nucleic acid and polymer are ubiquitous in vivo
and no means exist to discriminate contrast arising from the gene therapy agent and that arising from the background biological medium. Furthermore, this approach would not afford any change in contrast intensity when transfection has occurred successfully.
These reports suggesting that gene therapy could be imaged by MRI did, however, prompt us to consider a polymeric MRI contrast agents () developed recently in our lab.14
Although these polymeric contrast agents were originally conceived as a method of increasing agent delivery in targeted imaging applications, one of these polymeric agents is highly positively charged—with an average of 51 positive charges spread over an average of 17 monomer units per polymer—and thus, to our mind, potentially had the necessary attributes to facilitate gene therapy tracking by MRI. We had previously demonstrated14
that these polymeric agents could be used to generate image contrast by MRI using the same CEST mechanism used by van Zijl and coworkers.9–11
However, in this case the active component of the agent, from an MRI perspective, was a chelate of the paramagnetic ion Eu3+
. Among other advantages,12
the paramagnetic CEST (PARACEST) approach allows the specific detection of the cationic polymer in biological media because activation of the agent requires that the frequency selective pre-saturation pulse be applied at ~50 ppm, far away from the resonance frequency of any exchangeable endogenous protons.