Noninvasive measurement of gene expression in skeletal muscle

Abstract

We have developed a noninvasive detection method for expression of viral-mediated gene transfer. A recombinant adenovirus was constructed by using the gene for arginine kinase (AK), which is the invertebrate correlate to the vertebrate ATP-buffering enzyme, creatine kinase. Gene expression was noninvasively monitored using 31 P-magnetic resonance spectroscopy ( 31 P-MRS). The product of the AK enzyme, phosphoarginine (PArg), served as an MRS-visible reporter of AK expression. The recombinant adenovirus coding for arginine kinase (rAdCMVAK) was injected into the right hindlimbs of neonatal mice. Two weeks after injection of rAdCMVAK, a unique 31 P-MRS resonance was observed. It was observable in all rAdCMVAK injected hindlimbs and was not present in the contralateral control or the vehicle injected limb. PArg and phosphocreatine (PCr) concentrations were calculated to be 11.6 ؎ 0.90 and 13.6 ؎ 1.1 mM respectively in rAdCMVAK injected limbs. AK activity was demonstrated in vivo by monitoring the decreases in PArg and ATP resonances during prolonged ischemia. After 1 h of ischemia intracellular pH was 6.73 ؎ 0.06, PCr͞ATP was decreased by 77 ؎ 8%, whereas PArg͞ATP was decreased by 50 ؎ 15% of basal levels. PArg and PCr returned to basal levels within 5 min of the restoration of blood flow. AK activity persisted for at least 8 mo after injection, indicating that adenoviral-mediated gene transfer can produce stable expression for long periods of time. Therefore, the cDNA encoding AK provides a useful reporter gene that allows noninvasive and repeated monitoring of gene expression after viral mediated gene transfer to muscle. nuclear magnetic resonance ͉ gene therapy ͉ adenovirus ͉ creatine kinase ͉ arginine kinase

Non-invasive gamma camera imaging of gene transfer using an adenoviral vector encoding an epitope-tagged receptor as a reporter

“…8,9 Magnetic resonance spectroscopy has been used to detect gene transfer using cytosine deaminase, arginine kinase, and the murine creatine kinase as reporter genes. [10][11][12] These studies observed the changes in chemical shift of the 19 F or the 31 P isotopes. However, these magnetic strategies have limited temporal resolution and sensitivity.…”

Molecular imaging perspectives

“…Magnetic resonance spectroscopy (MRS) has also been used to follow the expression of marker genes. Viral-mediated gene transfer using recombinant adenovirus construct with the gene for the invertebrate enzyme arginine kinase was introduced into the hind limb of neonatal mice and the appearance of the enzyme in muscle was detected by 31 P MRS following the formation of phosphoarginine (Walter et al 2000).…”

Dynamic MRS and MRI of skeletal muscle function and biomechanics

“…31 P MRS may also serve as a noninvasive tool to report on the level of gene expression following gene therapy, as demonstrated by the pioneering example of the virally-mediated transfer of the arginine kinase gene in mouse skeletal muscle (98). Arginine kinase was active, as evidenced by the fact that a phosphoarginine resonance appeared, which could be simply distinguished from the PCr peak and, alike PCr, was reduced during muscle ischemia.…”