We report a new platform technology for visualizing transgene expression in living subjects using magnetic resonance imaging (MRI). Using a vector, we introduced an MRI reporter, a metalloprotein from the ferritin family, into specific host tissues. The reporter is made superparamagnetic as the cell sequesters endogenous iron from the organism. In this new approach, the cells construct the MRI contrast agent in situ using genetic instructions introduced by the vector. No exogenous metal-complexed contrast agent is required, thereby simplifying intracellular delivery. We used a replication-defective adenovirus vector to deliver the ferritin transgenes. Following focal inoculation of the vector into the mouse brain, we monitored the reporter activity using in vivo time-lapse MRI. We observed robust contrast in virus-transduced neurons and glia for several weeks. This technology is adaptable to monitor transgene expression in vivo in many tissue types and has numerous biomedical applications, such as visualizing preclinical therapeutic gene delivery.

To test the utility of gene therapeutic approaches for the treatment of pain, a recombinant herpes simplex virus, type 1, has been engineered to contain the cDNA for an opioid peptide precursor, human preproenkephalin, under control of the human cytomegalovirus promoter. This virus and a similar recombinant containing the Escherichia coli lacZ gene were applied to the abraded skin of the dorsal hindpaw of mice. After infection, the presence of beta-galactosidase in neuronal cell bodies of the relevant spinal ganglia (lacZ-containing virus) and of human proenkephalin (preproenkephalin-encoding virus) in the central terminals of these neurons indicated appropriate gene delivery and expression. Baseline foot withdrawal responses to noxious radiant heat mediated by Adelta and C fibers were similar in animals infected with proenkephalin-encoding and beta-galactosidase-encoding viruses. Sensitization of the foot withdrawal response after application of capsaicin (C fibers) or dimethyl sulfoxide (Adelta fibers) observed in control animals was reduced or eliminated in animals infected with the proenkephalin-encoding virus for at least 7 weeks postinfection. Hence, preproenkephalin cDNA delivery selectively blocked hyperalgesia without disrupting baseline sensory neurotransmission. This blockade of sensitization was reversed by administration of the opioid antagonist naloxone, apparently acting in the spinal cord. The results demonstrate that the function of sensory neurons can be selectively altered by viral delivery of a transgene. Because hyperalgesic mechanisms may be important in establishing and maintaining neuropathic and other chronic pain states, this approach may be useful for treatment of chronic pain and hyperalgesia in humans.

Diseases of the nervous system have devastating effects and are widely distributed among the population, being especially prevalent in the elderly. These diseases are often caused by inherited genetic mutations that result in abnormal nervous system development, neurodegeneration, or impaired neuronal function. Other causes of neurological diseases include genetic and epigenetic changes induced by environmental insults, injury, disease-related events or inflammatory processes. Standard medical and surgical practice has not proved effective in curing or treating these diseases, and appropriate pharmaceuticals do not exist or are insufficient to slow disease progression. Gene therapy is emerging as a powerful approach with potential to treat and even cure some of the most common diseases of the nervous system. Gene therapy for neurological diseases has been made possible through progress in understanding the underlying disease mechanisms, particularly those involving sensory neurons, and also by improvement of gene vector design, therapeutic gene selection, and methods of delivery. Progress in the field has renewed our optimism for gene therapy as a treatment modality that can be used by neurologists, ophthalmologists and neurosurgeons. In this Review, we describe the promising gene therapy strategies that have the potential to treat patients with neurological diseases and discuss prospects for future development of gene therapy.

The DNA templates containing immediate early (IE) genes of human cytomegalovirus (CMV) were transcribed in vitro by using a HeLa cell extract. When IE region 1, 2, and 3 were used, transcription was detected qualitatively only from IE region 1. Transcription was detected with DNA representing IE region 2 when the IE region 1 promoter was not present. DNA sequence analysis of the upstream regulatory region of IE region 1 detected two distinct repeats of 19 and 18 nucleotides, both being repeated four times. A putative cruciform structure could form through the surrounding sequences with each 18-nucleotide repeat being located in the unpaired region. The potential secondary structure and the repeat sequences in the regulatory region of IE region 1 are presumably related to the high level of transcription of this IE gene.Human cytomegalovirus (CMV), a member of the' herpesvirus classification group, has a large double-stranded DNA genome of 240 kilobases (kb). The viral genome consists of a long-and short unique region flanked by differept repeat sequences 'that are inverted relative to each other. Four genome arrangements, resulting from the possible combination of inversions of the two sections of the genome, are present in DNA preparations in approximately equal amounts (1-7).At immediate early (IE) times after infection-i.e., in the absence of de novo viral protein synthesis, 88% or more of the viral RNA originates from a region in the long unique component of the viral genome (6,8,9) In Vitro Transcription and RNA Fractionation. In vitro transcription was as described by Manley et al. (14). Recombinant plasmids cut with various restriction enzymes to generate linear templates were at a concentration of 100 ig per ml. Some reactions contained a-amanitin (1 pg/ml; Sigma) to inhibit RNA polymerase II activity. The 32P-labeled RNA was subjected to electrophoresis in 1.5% agarose gels containing 10 mM methylmercury (II) hydroxide as described by Bailey and Davidson (15). Molecular weight standards were 23S (3.3 kb) and 16S (1.7 kb) Escherichia coli rRNA (16), 28S (5.3 kb) and 18S (2.0 kb) human' cell rRNA (17), and approximately 0.160 kb tRNA. To visualize the RNA, the slab gels were stained in a solution containing 0.5 M ammonium acetate, 0.005 M 2-mercaptoethanol, and 1 ,ug of ethidium bromide per ml. The gels were dried and exposed to Kodak XOmat AR film. RNA sizes were interpolated from a standard 659The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.- §1734 solely to indicate this fact.