ORIGINAL ARTICLE Herpes simplex virus RNAi and neprilysin gene transfer vectors reduce accumulation of Alzheimer’s disease-related amyloid-b peptide in vivo C-S Hong 1 , WF Goins 1 , JR Goss 1 , EA Burton 1,2 and JC Glorioso 1 1 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA and 2 Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Accumulation of insoluble aggregates of amyloid-b peptide (Ab), a cleavage product of amyloid precursor protein (APP), is thought to be central to the pathogenesis of Alzheimer’s disease (AD). Consequently, downregulation of APP, or enhanced clearance of Ab, represent possible therapeutic strategies for AD. We generated replication-defective herpes simplex virus (HSV) vectors that inhibit Ab accumulation, both in vitro and in vivo. In cell culture, HSV vectors expressing either (i) short hairpin RNA directed to the APP transcript (HSV-APP/shRNA), or (ii) neprilysin, an endopep- tidase that degrades Ab (HSV-neprilysin), substantially inhibited accumulation of Ab. To determine whether these vectors showed similar activity in vivo, we developed a novel mouse model, in which overexpression of a mutant form of APP in the hippocampus, using a lentiviral vector (LV-APP Sw ), resulted in rapid Ab accumulation. Co-inocula- tion of LV-APP Sw with each of the HSV vectors showed that either HSV-APP/shRNA or HSV-neprilysin inhibited Ab accumulation in this model, whereas an HSV control vector did not. These studies demonstrate the utility of HSV vectors for reducing Ab accumulation in the brain, thus providing useful tools to clarify the role of Ab in AD that may facilitate the development of novel therapies for this important disease. Gene Therapy (2006) 13, 1068–1079. doi:10.1038/ sj.gt.3302719; published online 16 March 2006 Keywords: herpes simplex virus; Alzheimer’s disease; amyloid-b protein; siRNA; neprilysin; lentivirus Introduction Alzheimer’s disease (AD) is the most common type of dementia, and the most prevalent neurodegenerative disease, affecting more than 4 million individuals in the United States alone. Although significant improvement has been made in clinical diagnosis and care for AD patients, treatments that prevent clinical progression have not yet been identified. Amyloid-b peptide (Ab) is generated from the amyloid precursor protein (APP) by the combined cleavage activity of b- and g-secretases. Amyloid-b peptide is a major component of neuritic plaques that accumulate in the brains of AD patients and together with neurofibrillary tangles represent the hall- mark pathological features of the disease. Studies performed to investigate Ab accumulation suggest that overproduction and/or ineffective clearance of the peptide contributes to the disease development. 1,2 Thus, downregulation of APP gene expression or upregulation of Ab clearance may prevent or slow the disease process. Recently, inhibitors of b- and g-secretases have been developed. 3 However, these enzymes have additional functions, other than APP processing, and the long-term effects of inhibiting these processes are unknown. For example the g-secretase complex is involved in post- translational processing of the Notch receptor, 4 which has crucial physiological roles in the brain, raising the concern that long-term inhibition of g-secretase may adversely affect normal brain function. Amyloid-b peptide might also be eliminated by antibody-directed scavenging of plaque-forming proteins through opsoni- zation. Immunotherapy against Ab has been shown to reduce the establishment of plaques in an AD transgenic mouse model, 5 suggesting a promising approach for clinical evaluation. However, clinical studies demon- strated serious adverse events related to the develop- ment of meningoencephalitis in the vaccination groups, which forced termination of phase II trials. 6 Another approach to treatment might involve gene therapy strategies to reduce the synthesis of Ab or prevent its accumulation. The recent development of short interfer- ing RNA (siRNA) technology, and its successful applica- tion in a number of experimental systems, suggests that targeting APP mRNA could represent a viable strategy for downregulating APP synthesis. Alternatively, meth- ods to increase expression of natural membrane-bound cellular proteases, such as neprilysin, could be used to degrade extracellular Ab. RNA interference (RNAi; reviewed in Dykxhoorn et al. 7 ) is a natural mechanism found in plants and animals that is believed to provide a host defense Received 9 September 2005; revised 30 November 2005; accepted 1 December 2005; published online 16 March 2006 Correspondence: Dr JC Glorioso, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, E1240 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261, USA. E-mail: glorioso@pitt.edu, usgt@mgb.pitt.edu Gene Therapy (2006) 13, 1068–1079 & 2006 Nature Publishing Group All rights reserved 0969-7128/06 $30.00 www.nature.com/gt