For scFv9, we used mAb40

For scFv9, we used mAb40.1 as capture, and for scFv40.1 and scFv42.2, mAb9 was used as capture (Fig. mouse models. Keywords: single-chain variable fragments, immunotherapy, Alzheimer’s disease, adeno-associated virus, amyloid, A Introduction Numerous studies RIPK1-IN-3 of Alzheimer’s disease (AD) support the hypothesis that cerebral accumulation of amyloid protein (A) triggers pathological changes leading to cognitive dysfunction. Strategies to prevent A aggregation by reducing A levels or targeting pathogenic A aggregates are being developed (Golde, 2003). A immunotherapies are potential AD therapeutics that could work by reducing A levels, enhancing clearance of A aggregates, neutralizing toxic aggregates, or combining some of these mechanisms. In amyloid precursor protein (APP) transgenic mice, active A immunization and passive immunization with anti-A monoclonal antibodies (mAbs) reduces cerebral A deposition, neuritic dystrophy, and gliosis and improves cognitive deficits (Schenk et al., 1999; Bard et al., 2000; Bennett and Holtzman, 2005). A clinical trial using aggregated A1C42 in combination with QS-21 adjuvant was halted when 6% of the vaccinated subjects developed meningoencephalitis (Orgogozo et al., 2003; Gilman et al., 2005). However, patients who generated anti-A antibodies had reduced cerebrospinal levels of tau and showed a slower cognitive decline (Gilman et al., 2005; Masliah et al., 2005). Because passive immunization with anti-A antibodies is theoretically safer than active vaccination with A peptides, several humanized anti-A mAbs are currently being evaluated in early-phase clinical trials. Studies have suggested that to reduce A deposition, it is not necessary to use intact anti-A antibodies (Bacskai et al., 2002; Matsuoka et al., 2003; Wilcock et al., 2003; Tamura et al., 2005). It may be possible to avoid potential side effects simply by targeting A with high-affinity binding agents that lack immune effector functions. We have identified multiple anti-A mAbs that are effective as passive immunogens in APP mice (Levites et al., 2006a) and generated recombinant single-chain variable fragments (scFvs) from the hybridomas expressing these anti-A mAbs. We have injected adeno-associated virus serotype 1 (AAV1) vectors encoding anti-A40-specific, anti-A42-specific, and anti-pan-A scFvs into neonatal CRND8 mice brains and examined their effects on A levels and plaque deposition. Such studies establish a novel rapid, flexible, and cost-effective paradigm for evaluating potential gene- or protein-based modifiers of pathology in AD mouse models. Materials and Methods AAV construction and preparation AAV was prepared by standard methods. Briefly, AAV vectors expressing the scFv under the control of the cytomegalovirus enhancer/chicken -actin promoter, a woodchuck post-transcriptional regulatory element, and the bovine growth hormone, poly(A), were generated by plasmid transfection with helper plasmids in HEK293T cells. Forty-eight hours after transfection, the cells were harvested and lysed in the presence of 0.5% sodium deoxycholate and 50 U/ml Benzonase (Sigma, St. Louis, MO) by freeze thawing, and the virus RIPK1-IN-3 was isolated using a discontinuous iodixanol gradient and affinity purified on a HiTrap HQ column (Amersham Biosciences, Arlington Heights, IL). The genomic titer of each virus was determined by quantitative PCR. Mice All animal husbandry procedures performed were approved by the Mayo Clinic Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines under protocol A29803. To generate CRND8 mice, male CRND8 mice containing a double mutation in the human APP gene (KM670/671NL and V717F) (Chishti et al., 2001) were mated with female B6C3F1/Tac mice that were obtained from Taconic (Germantown, NY). Genotyping of Tg2576 and CRND8 mice was performed by PCR as described previously (Levites et al., 2006a). All animals were housed three to five to a cage and maintained on food and water with a 12 h light/dark cycle. mRNA isolation, cDNA synthesis, amplification of cDNAs encoding variable heavy and variable light regions, and construction of scFvs mRNA was isolated from hybridoma cell lines using an mRNA isolation kit (Qiagen, Chatsworth, CA). cDNA was synthesized using Moloney murine leukemia RIPK1-IN-3 virus reverse transcriptase (Promega, Madison, WI) and random hexamers. The cDNA was then poly(G)-tailed Mouse monoclonal to HA Tag with terminal transferase (New England Biolabs, Beverly, MA). cDNAs encoding the variable heavy (VH) and variable light (VL) chains were amplified using anchor PCR with a forward poly(C) anchor primer and a reverse primer specific for a constant region sequence of IgG2a (for pan Ab) and IgG1 for Ab40.1 and Ab42.2, as described by Gilliland et al. (1996). PCR products were then sequenced using the same primers, and the consensus VH and VL subunits were determined. cDNAs encoding scFvs of three anti-A antibodies were constructed by ligating the VH and VL cDNAs in VH-linker-VL orientation separated by a Gly4Ser3 linker. Nonspecific scFv (scFv ns).