A unifying characteristic of all transmissible spongiform encephalopathies (TSE) is the conversion of the normal cellular prion protein (PrPc) to an abnormal conformation, designated PrPsc. The conversion confers an increase in beta- sheet content and a decrease in sensitivity to protease K. Recent data in PrPsc challenged transgenic mice that constitutively express PrPc-specific antibodies and PrPc-specific antibody treated cell cultures chronically infected with Prpsc showed a failure to accumulate PrPsc and demonstrated an ability to clear chronic infection of PrPsc. This application proposes to positively affect the conversion of PrPc in C57BL/6 mice challenged with PrPsc by utilizing a herpes amplicon-based gene therapy strategy to express novel single-chain variable fragment (scFv) antibody coding sequences.
Three aims are proposed. Isolation, subcloning, and in vitro expression of PrPc-specific scFv antibody coding sequences: A previously characterized combinatorial phage display library expressing human immunoglobin heavy and light chains variable regions will be used to identify phage clones capable of binding to recombinant mouse prion protein (rec MoPrP). Immunoglobulin coding sequences from identified clones and a murine Ig kappa secretion signal will be subcloned into HSV amplicon vectors. Efficiency of in vitro expression and secretion of functional PrPc-specific scFv antibodies will be assessed by real-time quantitative RT-PCR, ELISA, and Western blot analysis. Characterization of in vivo expression of PrPc-specific scFv antibodies in C57BL/6 mice: C57B1/6 mice will be administered HSV amplicons via intracerebral (IC) route. In vivo secreted expression levels, kinetics, and migration from IC administration site of PrPc-specific scFvs in vaccinated mice will be assessed via immunohistochemical (IHC) at 3, 7, 14, 30, 60, and 90 days post-injection time-points via detection of an scFv epitope tag. Deleterious effects of expressing PrPc-specific scFvs on behavior of C57BL/6 mice and signs of autoimmune disease will be examined. Assessment of vaccine efficacy in PrPsc-challenged mice: Efficacy of approach will be tested in C57BL/6 mice challenged with mouse-adapted PrPsc. Mice will be IC-challenged with PrPsc at the apex of PrPc-specific scFv expression. Efficacy will be assessed by end-point PrPsc levels in brains and spleens of treated, challenged mice. Gross motor coordination behavioral will also be evaluated in treated mice by rota-rod performance.
| Wuertzer, Charles A; Sullivan, Mark A; Qiu, Xing et al. (2008) CNS delivery of vectored prion-specific single-chain antibodies delays disease onset. Mol Ther 16:481-6 |
