The Human Norepinephrine Transporter and [11C]-mHED, a Novel Reporter Gene-Tracer Combination for PET Imaging of Gene Therapy

PRESIDENTIAL SYMPOSIUM within a narrow inducer concentration range of a few nanograms/ml. We have developed a gene regulatory network tailored to lock transgene expression at desired levels in response to clinical doses of different inducers rather than different concentrations of a single inducer. The regulatory cascade was designed by interconnecting streptogramin-, macrolide- and tetracycline- repressible gene regulation systems. Four different expression levels could be achieved by clinical dosing of a single antibiotic: high expression level in the absence of any antibiotic (+++), medium level expression following addition of tetracycline (++), low level expression in response to the macrolide erythromycin (+), and complete repression by streptogramins (-). We have furthermore developed a mammalian epigenetic transcription control system, which can be switched between two stable expression states following transient integration of pharmacological signals, thus eliminating the need for sustained inducer administration. We will present the latest progress in the development of gene regulation systems, gene regulatory networks and their building blocks, as well as advances in in vivo applications and tissue engineering.

The tracer accumulated in transduced cells, and not in untransfected cells. The accumulation was reduced to control level in the presence of DMI. To further assess the feasibility of this approach we constructed a first-generation adenoviral vector containing two transgenes, GFP and hNET, which are expressed from identical but separate expression cassettes. Cell lines were transformed with increasing amounts of this virus, and exposed to [11C]-mHED. The tracer accumulated in transformed cells to approximately 30 times control levels. Good correlation (R²=0.95), was found between [11C]mHED uptake, GFP fluorescence and the amount of virus used. These results confirm that hNET may be used reporter in gene therapy studies. The linear relationship between gene expression and indicating a good correspondence between gene expression and tracer accumulation should allow for quantification of gene expression in vivo. Experiments are ongoing to confirm the power of this imaging system in an in vivo model.

716. The Human Norepinephrine Transporter and [11C]-mHED, a Novel Reporter Gene - Tracer Combination for PET Imaging of Gene Therapy

PRESIDENTIAL SYMPOSIUM

Antoine M. J. Beerens,1 Anne Rixt Buursma,2 Marianne G. Rots,1 Aren van Waarde,2 Hidde J. Haisma,1 Erik F. J. de Vries.2 1 Departement of Therapeutic Gene Modulation, University of Groningen, Groningen, Netherlands; 2PET Center, Groningen University Hospital, Groningen, Netherlands. Currently many clinical protocols for gene therapy are being evaluated for use in the treatment of human disease. In the majority of these protocols however, it is difficult to determine the exact fate of the vector, or to determine the location and extend of expression of the introduced gene. Data about the expression of transgenes is not only invaluable in research, but is also of major importance in clinical gene therapy. If the therapy is devised for destroying cells, information about the transgene expression can minimize the risk of side effects. Numerous techniques are in use to measure gene expression, of which the use of non-invasive imaging using bioluminescence, SPECT (single photon emission tomography) and PET (positron emission tomography) are the most promising. For human applications, PET is the most promising technique because of its sensitivity. Because PET utilizes positron-emitting tracers that generate two gamma photons, a three-dimensional model can be created of the subject. Several combinations of tracer and reporter gene have been investigated. However most tracers require complicated synthesis or have rate limiting steps like membrane transport that influence tracer accumulation. We report upon a novel, generally applicable PET imaging method to monitor gene therapy in human subjects. We selected the tracer [11C]-meta-hydroxyephedrine (mHED), which is used in clinical PET studies for the diagnosis of heart neuropathy. mHED is actively transported into the cell by hNET (human norepinephrine transporter). In humans the expression of hNET is restricted to the sympatic nervous system. Therefore, hNET can be used as a reporter in all other tissues. hNET is a human gene, so no immune response is to be expected and the small size (< 2 kb) allows it to be easily incorporated as a reporter gene. To confirm the specificity of mHED uptake, we transformed cell lines with a plasmid vector containing the hNET gene.Transformed cells were exposed to [11C]-mHED with or without desipramine (DMI), a specific inhibitor of hNET. After washing, accumulation of the tracer in cells was measured by gamma counting of cell lysates. Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy

717. RNAi Therapy for Dominant Neurodegenerative Diseases Haibin Xia,1,2 Qinwen Mao,1,2 Steven L. Eliason,1,2 Nathan Kiewiet,1,2 Jaime Critchfield,1,2 Ines H. Martins,1,2 Scott Q. Harper,1,2 Xiaohua He,1,2 Robert M. Kotin,5 Huda Y. Zoghbi,6,7,8,9 Harry T. Orr,10 Henry L. Paulson,1,3 Beverly L. Davidson.1,2,3,4 1 Program in Gene Therapy; 2Departments of Internal Medicine; 3 Neurology; 4Physiology & Biophysics, University of Iowa, Iowa City, IA; 5NHLBI/NIH, Bethesda, MD; 6Howard Hughes Medical Institute; 7Departments of Molecular and Human Genetics; 8 Pediatrics; 9Program in Developmental Biology, Baylor College of Medicine, Houston, TX; 10Institute of Human Genetics, University of Minnesota, Minneapolis, MN. In this study we investigated gene silencing by RNA interference (RNAi) as a potential therapy for Spinocerebellar ataxia type 1 (SCA1), a dominant neurodegenerative disease caused by expansion of a polyglutamine tract in ataxin-1. Therapies for SCA1 do not exist, and studies in mouse models of other polyglutamine repeat diseases support that silencing of the disease allele can improve disease manifestations. Short hairpin RNAs (shRNAs) specific to SCA1 were screened for effective silencing against a mutant human ataxin (Q = 82) in vitro prior to testing in a transgenic mouse model of SCA1. SCA1 trangenic mice express a mutant human ataxin-1 (Q = 82) from the Purkinje cell specific promoter, and recapitulate many aspects of human SCA1, including progressive ataxia, shrinkage of the molecular layer, Purkinje cell degeneration and nuclear inclusion formation. AAV1 vectors expressing the most effective shRNA and an hrGFP reporter (AAVshSCA1.hrGFP) were injected into the cerebellar lobules of 7-week old SCA1 or wildtype mice. Viruses expressing an irrelevant hairpin (AAVshlacZ.hrGFP), or saline, were included as hairpin and injection controls. Rotarod tests of motor coordination were done 2-week prior and every two weeks following injections, until sacrifice at 16 weeks of age. Beginning at 9 weeks of age and continuing until sacrifice, SCA1 mice treated with shSCA1expressing viruses showed significantly improved performance on the rotarod relative to saline- or shlacZ-treated SCA1 mice. There was no effect of the therapy on the performance of wildtype mice. The efficiency of transduction, quantified by hrGFP expression, ranged from ∼ 5–10% of all Purkinje cells. We took advantage of hrGFP reporter expression to directly compare transduced and untransduced lobules for ataxin-1 nuclear inclusions, Purkinje cell morphology, and the width of the cerebellar molecular layer. In shSCA-treated SCA1 mice, Purkinje cell dendritic arbors remained S273