Prolonged Bioactive Transgene Expression Driven by the HSV Latency Active Promoter 2 (LAP2) in the Peripheral Nervous System

CENTRAL NERVOUS SYSTEM I 219. Prolonged Bioactive Transgene Expression Driven by the HSV Latency Active Promoter 2 (LAP2) in the Peripheral Nervous System 1

2

220. Prolonged Biologically Active Transgene Expression Driven by HSV Latency Active Promoter 2 (LAP2) in Brain In Vivo 2

Munmun Chattopadhyay, Darren P. Wolfe, William C. Goins, Shaohua Huang,2 Joseph C. Glorioso,2 Marina Mata,1,3 David J. FINK. 1,2,3 1 Neurology, University of Pittsburgh, Pittsburgh, PA; 2Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA; 3GRECC, VA Pittsburgh Healthcare System, Pittsburgh, PA.

We have previously demonstrated in subacute experiments lasting up to 2 months that HSV-mediated transfer of neurotrophin genes to dorsal root ganglion by peripheral subcutaneous inoculation can prevent the progression of neuropathy caused by pyridoxine (PDX), cisplatin or diabetes in rodents. In order to further examine the duration of biologically active transgene expression we compared non-replicating HSV vectors expressing nerve growth factor (NGF) under the control of the HCMV IEp (SHN) or HSV LAP2 (SLN), and neurotrophin 3 (NT-3) under the control of LAP2 (QL2NT3) or a LAP2-HCMV fusion promoter (QL2HNT3), and a control lacZ expressing vector (SHZ) in a delayed model of PDX neuropathy. Each animal received 25µl containing 25x106pfu of SHN, SLN, QL2HNT3, QL2NT3, or the control vector SHZ in a single subcutaneous injection under the skin of the plantar surface of each hind paw. Six months after vector inoculation neuropathy was induced by injection of PDX twice a day for 8 days, and the animals assessed for neuropathy by electrophysiologic, behavioral and histologic parameters 7 days later. Rats intoxicated with PDX, or intoxicated and treated with QOZHG had no detectable H reflex. Animals inoculated with SHN had a dramatic reduction in H-wave amplitude. Animals inoculated with QL2HNT3, QL2NT3, or SLN 6 months prior to intoxication showed substantial preservation of the H-wave (SLN: 0.71 ± 0.2 mV; QL2HNT3: 1.35 ± 0.09 mV; QL2NT3: 1.28 ± 0.18 mV). The evoked sensory nerve action potential (SNAP) was markedly decreased in amplitude in rats intoxicated with PDX compared to control (control 22.03 ± 0.7 µV, PDX 8.3 ± 0.6 µV). Animals transduced with QL2HNT3, QL2NT3 and SLN but not SHN 6 months prior to intoxication showed substantial preservation of SNAP (SLN: 13.9 ± 2.7 µV; QL2HNT3 16.8 ± 1.2 µV; QL2NT3: 17.7 ± 1.5 µV). Control animals with normal proprioceptive function had no difficulty traversing a narrow beam. Animals intoxicated with PDX experienced substantial difficulty, recording an average of 15 slips from the beam during the test period, as did animals inoculated with SHN (13.25 ± 0.9 slips). Rats transduced with QL2HNT3, QL2NT3 and SLN performed substantially better than PDX-only animals both qualitatively and quantitatively (SLN: 5.8 ± 0.8 slips; QL2HNT3: 5 ± 0.5 slips; QL2NT3: 5 ± 0.7 slips). The sciatic nerve from animals transduced with QOZHG was indistinguishable from the nerve of PDX treated animals, while the sciatic nerve of vector treated animals except SHN showed preservation of normal nerve morphology. These results demonstrate that the HSV LAP2 element produces prolonged expression biologically active transgenes from latent HSV vector genomes in the DRG at levels sufficient to prevent the development of neuropathy up to 6 months after inoculation.

Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

Veljko Puskovic,1,3 Darren Wolfe,2 James R. Goss,1,3 Shaohua Hang,2 Joseph C. Glorioso,2 Marina Mata,1,3 David J. Finnk.1,3 1 Neurology, University of Pittsburgh-School of Medicine, Pittsburgh, PA; 2Molecular Genetics and Biochemistry, University of Pittsburgh-School of Medicine, Pittsburgh, PA; 3GRECC, VA Pittsburgh Healthcare System, Pittsburgh, PA. The herpes simplex virus (HSV) latency associated promoter (LAP2) is a transposable element that drives life-long expression of latency associated transcripts (LATs) from latent HSV genomes. We examined the ability of this promoter to drive prolonged expression of a biologically active transgene from latent HSV vector genomes in brain in vivo using the 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models of Parkinson disease. Animals injected with an HSV vector with GDNF under the control of LAP2 five and a half months before 6OHDA injection showed preservation dopaminergic neuron function in the face of 6-OHDA toxicity measured by amphetamine-induced rotation, number of tyrosine hydroxylase (TH) neurons in the substantia nigra (SN), and immunoreactivity for TH and the dopamine transporter (DAT) in striatum determined 6 months after vector inoculation. The amount of GDNF produced in injected SN determined by ELISA remained stable at 1-2 pg/mg over 6 months. Mice inoculated with the LAP2-GDNF HSV vector followed by 3 months of daily low dose MPTP injection were substantially protected against the consequences of that treatment measured by weekly behavioral testing (contralateral pellet retrieval in a staircase apparatus) and by histologic measures including number of TH-IR neurons in SN, and TH-IR and DAT-IR in striatum determined at the conclusion of the experiment 13 weeks after vector inoculation. These studies demonstrate that the HSV LAP2 promoter element provides prolonged expression of relevant amounts of a transgene to produce significant biological effects in brain in vivo over the course of many months.

221. Developing HSV-Mediated Gene Therapy β Vectors To Adjust the Metabolic Imbalance of Aβ in Alzheimer’s Disease Chang-Sook Hong,1 James R. Goss,2 William F. Goins,1 David J. Fink,1,2 Joseph C. Glorioso.1 1 Molecular Genetics & Biochemistry; 2Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA. Alzheimer’s disease (AD) is the most common type of dementia occurring in mid- to late-life, affecting more than 4 million individuals in United States. The amyloid β-protein (Aβ), which is generated from the amyloid precursor protein (APP) by β- and γ-secretase cleavages, is a major component of pathological plaques that accumulate in AD brains. The majority of AD patients develop the disease due to over-secretion or faulty clearance of Aβ peptide. Thus down-regulation of the expression of Aβ or stimulation of the Aβ clearance process may ameliorate or cure the devastating disease. We are developing gene therapy vectors to favorably alter the metabolic imbalance of Aβ in AD brain. Replication-defective HSV vectors have proven to be attractive vehicles for central nervous system gene therapy because of the natural tropism of the virus for neurons. Recently, siRNA has been shown to be a powerful tool for suppressing gene expression in cells from many organisms. However, the methods of delivering siRNA have limited mammalian application, especially in vivo. To investigate whether siRNA technologies can efficiently knock down the APP gene, we have designed a siRNA expression cassette (APP-siRNA) targeting APP S85