ABSTRACT
The 37/67 kDa laminin receptor (LRP/LR) acts as a receptor for prions providing a promising target for the treatment of prion diseases. Recently, we selected anti-LRP/LR single-chain antibodies (scFvs) and proved a reduction of the peripheral PrP(Sc) propagation by passive immunotransfer into scrapie-infected mice. Here, we report the development of an in vivo gene delivery system based on adeno-associated virus (AAV) vectors expressing scFvs-S18 and -N3 directed against LRP/LR. Transduction of neuronal and non-neuronal cells with recombinant (r)AAV serotype 2 vectors encoding scFv-S18, -N3 and -C9 verified the efficient secretion of the antibodies. These vectors were administered via stereotactic intracerebral microinjection into the hippocampus of C57BL/6 mice, followed by intracerebral inoculation with 10 % RML at the same site 2 weeks post-injection of rAAV. After 90 days post-infection, scFv-S18 and -N3 expression resulted in the reduction of peripheral PrP(Sc) propagation by approximately 60 and 32 %, respectively, without a significant prolongation of incubation times and survival. Proof of rAAV vector DNA in spleen samples by real-time PCR strongly suggests a transport or trafficking of rAAV from the brain to the spleen, resulting in rAAV-mediated expression of scFv followed by reduced PrP(Sc) levels in the spleen most likely due to the blockage of the prion receptor LRP/LR by scFv.
Subject(s)
Dependovirus/genetics , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Immunoglobulin Fc Fragments/administration & dosage , Prion Diseases/therapy , Receptors, Laminin/immunology , Animals , Antibodies/genetics , Antibodies/immunology , Antibodies/metabolism , Antibody Specificity , Gene Transfer Techniques , Hippocampus/virology , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fc Fragments/metabolism , Mice , Mice, Inbred C57BL , PrPSc Proteins/metabolism , Recombination, Genetic , Spleen/metabolism , Spleen/virology , Transduction, GeneticABSTRACT
Transmissible spongiform encephalopathies are a group of neurological disorders associated with the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(c). The 37 kDa/67 kDa laminin receptor (LRP/LR) has been identified as a prion receptor and several lines of evidence strongly suggest that this protein plays a role during prion pathogenesis. Here we report the selection of recombinant single chain antibodies (scFvs) directed against LRP from naïve and synthetic phage scFv libraries for therapeutic application. Western blotting and FACS analysis confirmed a specific LRP/LR recognition pattern of the two selected scFvs S18 and N3. Both scFvs specifically interfered with the PrP/LRP interaction in vitro. High yield production of the scFvs of approx. 1mg/l of culture medium was achieved in E. coli. Passive immunotransfer of the scFv S18 antibody reduced PrP(Sc) levels by approx. 40% in the spleen of scrapie infected C57BL/6 mice 90 days post scFv injection, suggesting that scFv S18 interferes with peripheral PrP(Sc) propagation, without a significant prolongation of incubation and survival times.
Subject(s)
Antibodies/pharmacology , Immunoglobulin Variable Region/pharmacology , Prion Diseases/therapy , Receptors, Laminin/antagonists & inhibitors , Amino Acid Sequence , Animals , Antibodies/chemistry , Antibody Specificity/drug effects , Antibody Specificity/immunology , Disease Models, Animal , Epitope Mapping , Female , Humans , Immunization, Passive , Kinetics , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Molecular Weight , PrPC Proteins/metabolism , PrPSc Proteins/metabolism , Protein Binding/drug effects , Scrapie/metabolism , Spleen/drug effects , Spleen/metabolism , Spleen/pathologyABSTRACT
BACKGROUND: Recently, we showed that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor of the cellular prion protein. METHODS: For the present study, we investigated the binding of the murine scrapie prion protein (moPrP27-30) to baby hamster kidney (BHK) cells, using the Semliki Forest virus system. RESULTS: The enhanced binding of moPrP27-30 to BHK cells expressing moLRP::FLAG was inhibited by the LRP/LR-specific antibody W3, which suggests that LRP/LR acts as a receptor for the scrapie form of the prion protein, PrP(Sc). This finding was confirmed by a parallel study that showed that bovine prions are internalized by human enterocytes via LRP/LR. The heparan sulfate mimetics HM5004 and HM2602 reduced PrP27-30 binding to moLRP-expressing cells to approximately 30% and approximately 20%, respectively, at a concentration of 10 microg/mL, whereas pentosan polysulfate (SP54) and phycarin sulfate (PS3) both reduced the binding to approximately 40% at a concentration of 100 microg/mL. CONCLUSIONS: We suggest that the inhibition reported elsewhere of PrP(Sc) synthesis and the incubation times prolonged in rodent models by these sulfated glycans are due to the inhibition of the LRP/LR-dependent binding of prions to the target cells.
Subject(s)
Heparitin Sulfate/pharmacology , PrPSc Proteins/metabolism , Prions/physiology , Receptors, Laminin/physiology , Animals , Cell Line , Cricetinae , Kidney , Mice , Plasmids , Prions/drug effects , RNA, Viral/genetics , Semliki forest virus/drug effects , Semliki forest virus/genetics , Semliki forest virus/physiologyABSTRACT
Many interferon (IFN)-stimulated genes are also induced by double-stranded RNA (dsRNA), a component closely associated with the IFN system in the context of virus-host interactions. Recently, we demonstrated that the IFN-induced 3' --> 5' exonuclease ISG20 possesses antiviral activities against RNA viruses. Here we show that ISG20 induction by synthetic dsRNA (pIpC) is stronger and faster than its induction by IFN. Two families of transcription factors are implicated in the transcriptional activation of ISG20 by dsRNA. Initially, the NF-kappaB factors p50 and p65 bind and activate the kappaB element of the Isg20 promoter. This is followed by IRF1 binding to the ISRE. As pIpC often induces protein movements in the cells, we questioned whether it could influence ISG20 localization. Interestingly and contrary to IFN, dsRNA induces a nuclear matrix enrichment of the ISG20 protein. dsRNA induction of ISG20 via NF-kappaB and its antiviral activity led us to suggest that ISG20 could participate in the cellular response to virus infection.
Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Exonucleases/metabolism , NF-kappa B/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , RNA, Double-Stranded/pharmacology , Binding Sites , Carrier Proteins/drug effects , Carrier Proteins/genetics , Cytoplasm/metabolism , DNA-Binding Proteins/genetics , Exonucleases/genetics , Exoribonucleases , HeLa Cells , Humans , Interferon Regulatory Factor-1 , Interferon Type I/pharmacology , Mutagenesis, Site-Directed , NF-kappa B/genetics , Nuclear Proteins/drug effects , Nuclear Proteins/genetics , Phosphoproteins/genetics , Promoter Regions, Genetic , RNA, Double-Stranded/chemical synthesis , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The accumulation of PrP(Sc) in scrapie-infected neuronal cells has been prevented by three approaches: (i) transfection of ScMNB cells with an antisense laminin receptor precursor (LRP) RNA-expression plasmid, (ii) transfection of ScN2a cells and ScGT1 cells with small interfering RNAs (siRNAs) specific for the LRP mRNA, and (iii) incubation of ScN2a cells with an anti-LRP/LR antibody. LRP antisense RNA and LRP siRNAs reduced LRP/LR expression and inhibited the accumulation of PrP(Sc) in these cells. The treatments also reduced PrP(c) levels. The anti-LRP/LR antibody, W3, abolished PrP(Sc) accumulation and reduced PrP(c) levels after seven days of incubation. Cells remained free of PrP(Sc) after being cultured for 14 additional days without the antibody, whereas the PrP(c) level was restored. Our results demonstrate the necessity of the laminin receptor (LRP/LR) for PrP(Sc) propagation in cultured cells and suggest that LRP/LR-specific antibodies could be used as powerful therapeutic tools in the treatment of transmissible spongiform encephalopathies.
