Treatment of Prion Disease with Heterologous Prion Proteins.

Prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are fatal neurodegenerative diseases for which there is no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPsc or PrPres). Both in vitro (cell culture and cell free conversion assays) and in vivo (animal) studies have demonstrated the strong dependence of this conversion process on protein sequence homology between the initial prion inoculum and the host's own cellular prion protein. The presence of non-homologous (heterologous) proteins is often inhibitory to this conversion process. We hypothesize that the presence of heterologous prion proteins from one species might therefore constitute an effective treatment for prion disease in another species. To test this hypothesis, we infected mice intracerebrally with murine adapted RML-Chandler scrapie and treated them with heterologous prion protein (purified bacterially expressed recombinant hamster prion protein) or vehicle alone. Treated animals demonstrated reduced disease associated pathology, decreased accumulation of protease-resistant disease-associated prion protein, with delayed onset of clinical symptoms and motor deficits. This was concomitant with significantly increased survival times relative to mock-treated animals. These results provide proof of principle that recombinant hamster prion proteins can effectively and safely inhibit prion disease in mice, and suggest that hamster or other non-human prion proteins may be a viable treatment for prion diseases in humans.

Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice.

Prion diseases are caused by a conformational modification of the cellular prion protein (PrP (C)) into disease-specific forms, termed PrP (Sc), that have the ability to interact with PrP (C) promoting its conversion to PrP (Sc). In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP (C) region involved in the interaction with PrP (Sc) thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP (Sc) in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.

Effects of a brain-engraftable microglial cell line expressing anti-prion scFv antibodies on survival times of mice infected with scrapie prions.

We first verified that a single chain Fv fragment against prion protein (anti-PrP scFv) was secreted by HEK293T cells and prevented prion replication in infected cells. We then stably expressed anti-PrP scFv in brain-engraftable murine microglial cells and intracerebrally injected these cells into mice before or after infection with prions. Interestingly, the injection before or at an early time point after infection attenuated the infection marginally but significantly prolonged survival times of the mice. These suggest that the ex vivo gene transfer of anti-PrP scFvs using brain-engraftable cells could be a possible immunotherapeutic approach against prion diseases.

Adoptive transfer of T lymphocytes sensitized against the prion protein attenuates prion invasion in scrapie-infected mice.

There is to date no effective way of preventing or curing neurodegenerative diseases such as Alzheimer disease or transmissible spongiform encephalopathies. The idea of treating those conditions by immunological approaches has progressively emerged over the last ten years. Encouraging results have been reported in Alzheimer disease and in peripheral forms of mouse prion diseases following passive injection of Abs or active immunization against the peptides or proteins presumably at the origin of those disorders. Still, major difficulties persist due to some characteristics of those conditions such as slow evolution, brain location, uncertainties regarding precise pathogenic pathways, and, above all, the fact that the target Ag is self, meaning that it is poorly immunogenic and potentially harmful if tolerance was transgressed. To analyze some of those difficulties, we are developing adoptive cell transfer approaches. In this study, lymphocytes sensitized against the prion protein in nontolerant Prnp(-/-) mice were transferred into histocompatible wild-type recipients which were partly or totally devoid of their own lymphocytes. Under such conditions, we found that the engrafted T lymphocytes resisted peripheral tolerance, remained reactive for several months against epitopes of the prion protein, and significantly attenuated the progression of prions in secondary lymphoid organs with subsequent delay in the evolution of the neurological disease. Interestingly, those protective T lymphocytes secreted lymphokines and migrated more readily into the host CNS but did not appear to be engaged in cooperation with host B cells for Ab production.

Development of antibody fragments for immunotherapy of prion diseases.

Prions are infectious proteins responsible for a group of fatal neurodegenerative diseases called TSEs (transmissible spongiform encephalopathies) or prion diseases. In mammals, prions reproduce themselves by recruiting the normal cellular protein PrP(C) and inducing its conversion into the disease-causing isoform denominated PrP(Sc). Recently, anti-prion antibodies have been shown to permanently cure prion-infected cells. However, the inability of full-length antibodies and proteins to cross the BBB (blood-brain barrier) hampers their use in the therapy of TSEs in vivo. Alternatively, brain delivery of prion-specific scFv (single-chain variable fragment) by AAV (adeno-associated virus) transfer delays the onset of the disease in infected mice, although protection is not complete. We investigated the anti-prion effects of a recombinant anti-PrP (D18) scFv by direct addition to scrapie-infected cell cultures or by infection with both lentivirus and AAV-transducing vectors. We show that recombinant anti-PrP scFv is able to reduce proteinase K-resistant PrP content in infected cells. In addition, we demonstrate that lentiviruses are more efficient than AAV in gene transfer of the anti-PrP scFv gene and in reducing PrP(Sc) content in infected neuronal cell lines. Finally, we have used a bioinformatic approach to construct a structural model of the D18scFv-PrP(C) complex. Interestingly, according to the docking results, Arg(PrP)(151) (Arg(151) from prion protein) is the key residue for the interactions with D18scFv, anchoring the PrP(C) to the cavity of the antibody. Taken together, these results indicate that combined passive and active immunotherapy targeting PrP might be promising strategies for therapeutic intervention in prion diseases.

Effect of intraventricular infusion of anti-prion protein monoclonal antibodies on disease progression in prion-infected mice.

It is well known that anti-prion protein (PrP) monoclonal antibodies (mAbs) inhibit abnormal isoform PrP (PrPSc) formation in cell culture. Additionally, passive immunization of anti-PrP mAbs protects the animals from prion infection via peripheral challenge when mAbs are administered simultaneously or soon after prion inoculation. Thus, anti-PrP mAbs are candidates for the treatment of prion diseases. However, the effects of mAbs on disease progression in the middle and late stages of the disease remain unclear. This study carried out intraventricular infusion of mAbs into prion-infected mice before and after clinical onset to assess their ability to delay disease progression. A 4-week infusion of anti-PrP mAbs initiated at 120 days post-inoculation (p.i.), which is just after clinical onset, reduced PrPSc levels to 70-80 % of those found in mice treated with a negative-control mAb. Spongiform changes, microglial activation and astrogliosis in the hippocampus and thalamus appeared milder in mice treated with anti-PrP mAbs than in those treated with a negative-control mAb. Treatment with anti-PrP mAb prolonged the survival of mice infected with Chandler or Obihiro strain when infusion was initiated at 60 days p.i., at which point PrPSc is detectable in the brain. In contrast, infusion initiated after clinical onset prolonged the survival time by about 8 % only in mice infected with the Chandler strain. Although the effects on survival varied for different prion strains, the anti-PrP mAb could partly prevent disease progression, even after clinical onset, suggesting immunotherapy as a candidate for treatment of prion diseases.

Antiprion prophylaxis by gene transfer of a soluble prion antagonist.

Prion diseases are untreatable neurodegenerative disorders characterized by accumulation of PrP(Sc), an aggregated isoform of the normal prion protein PrP(C). Here, we delivered the soluble prion antagonist PrP-Fc(2) to the brains of mice by lentiviral gene transfer. Although naïve mice developed scrapie at 175 +/- 5 days postintracerebral prion inoculation (dpi), gene transfer before inoculation delayed disease onset by 72 +/- 4 days. At 170 days postintracerebral prion inoculation, PrP(Sc) accumulation and prion infectivity in PrPFc-treated brains were reduced by 3.6 and 4.2 logs, respectively. When PrP-Fc(2) was delivered 30 days after prion inoculation, survival of the treated animals was extended by 25 days. We then used tissue-specific recombination to express PrP-Fc(2) in the entire central nervous system, in only astrocytes, or in only oligodendrocytes. Oligodendrocyte-restricted PrP-Fc(2) expression impaired PrP(Sc) deposition and delayed disease even though oligodendrocytes are completely resistant to prion infection, suggesting that PrP-Fc(2) affords protection via noncell autonomous mechanisms. These results suggest that somatic gene transfer of prion antagonists may be effective for postexposure prophylaxis of prion diseases.

RNAi: a novel strategy for the treatment of prion diseases.

Prion disease refers to a group of fatal transmissible neurodegenerative diseases for which no pharmacological treatment is available. The cellular prion protein (PrP(C)) is required for both prion replication and pathogenesis, and reducing PrP(C) levels has been shown to extend survival time after prion infection. RNA interference (RNAi) is a sequence-specific posttranscriptional gene silencing mechanism. In this issue of the JCI, Pfeifer et al. report that lentivector-mediated RNAi significantly reduced neuronal PrP(C) expression; effectively suppressed accumulation of the infectious protease-resistant form of PrP (PrP(Sc)) in a persistently infected neuroblastoma cell line; and markedly slowed the progression of prion disease in a unique chimeric mouse model (see the related article beginning on page 3204). These findings indicate that lentivector-mediated RNAi could, in principle, be developed for the therapy of prion disease.

Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice.

Prion diseases are fatal neurodegenerative diseases characterized by the accumulation of PrP(Sc), the infectious and protease-resistant form of the cellular prion protein (PrP(C)). We generated lentivectors expressing PrP(C)-specific short hairpin RNAs (shRNAs) that efficiently silenced expression of the prion protein gene (Prnp) in primary neuronal cells. Treatment of scrapie-infected neuronal cells with these lentivectors resulted in an efficient and stable suppression of PrP(Sc) accumulation. After intracranial injection, lentiviral shRNA reduced PrP(C) expression in transgenic mice carrying multiple copies of Prnp. To test the therapeutic potential of lentiviral shRNA, we used what we believe to be a novel approach in which the clinical situation was mimicked. We generated chimeric mice derived from lentivector-transduced embryonic stem cells. Depending on the degree of chimerism, these animals carried the lentiviral shRNAs in a certain percentage of brain cells and expressed reduced levels of PrP(C). Importantly, in highly chimeric mice, survival after scrapie infection was significantly extended. Taken together, these data suggest that lentivector-mediated RNA interference could be an approach for the treatment of prion disease.

Investigation of prion removal/inactivation from chromatographic gel.

BACKGROUND AND OBJECTIVES: Concerns about the potential for prions to be retained on chromatography gels during the manufacture of plasma products prompted development of an investigational strategy for detecting infectious prions bound to gels. The objective was to firstly examine methods of implanting gels intracerebrally (IC) in mice, then to examine prion cleaning from a scaled-down version of the DEAE Sepharose column used in a production process to fractionate immunoglobulins and albumin from human plasma. MATERIALS AND METHODS: The study consisted of two parts: (i) the pathophysiological impact by IC inoculation of ground gel beads was compared to whole gel beads; (ii) the feedstreams to two DEAE Sepharose columns were spiked with scrapie ME7. One column was subjected to the protein loading and elution portions of the chromatography cycle. The other column was subjected to the full cycle of protein loading and elution, followed by regeneration with 0.5 m NaCl, 1 m NaOH and solvent/detergent washes. The gels were unpacked and bioassayed by IC implantation in mice to quantify infectivity. RESULTS: IC inoculation of ground gel beads resulted in unacceptably high pathological impact in the mice whereas whole gel bead inoculation resulted in a reduced affect. Accordingly, the whole bead model system was used to assess prion removal/inactivation from chromatography gels at the pre- and postcleaning stage of the chromatography cycle. Infectious prions were detected on the DEAE Sepharose prior to the cleaning step; however, the gel cleaning cycle reduced infectivity by a log reduction factor (LRF) of > or = 2.75, thus reducing infectivity by bioassay to below detectable limits. CONCLUSIONS: A model system for assessment of prion inactivation/removal from chromatography gels has been established. Spiked prion infectivity does bind to DEAE Sepharose gel; however, the cleaning cycle removed infectivity to levels below that detectable by bioassay.

Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis.

The mechanisms involved in prion neurotoxicity are unclear, and therapies preventing accumulation of PrPSc, the disease-associated form of prion protein (PrP), do not significantly prolong survival in mice with central nervous system prion infection. We found that depleting endogenous neuronal PrPc in mice with established neuroinvasive prion infection reversed early spongiform change and prevented neuronal loss and progression to clinical disease. This occurred despite the accumulation of extraneuronal PrPSc to levels seen in terminally ill wild-type animals. Thus, the propagation of nonneuronal PrPSc is not pathogenic, but arresting the continued conversion of PrPc to PrPSc within neurons during scrapie infection prevents prion neurotoxicity.

Specific inhibition of pathological prion protein accumulation by small interfering RNAs.

Development of transmissible spongiform encephalopathies (TSEs) pathogenesis requires the presence of both the normal host prion protein (PrP-sen) and the abnormal pathological proteinase-K resistant isoform (PrP-res). PrP-res forms highly insoluble aggregates, with self-perpetuating properties, by binding and converting PrP-sen molecules into a likeness of themselves. In the present report, we show that small interfering RNA (siRNA) duplexes trigger specific Prnp gene silencing in scrapie-infected neuroblastoma cells. A non-passaged, scrapie-infected culture transfected with siRNA duplexes is depleted of PrP-sen and rapidly loses its PrP-res content. The use of different murine-adapted scrapie strains and host cells did not influence the siRNA-induced gene silencing efficiency. More than 80% of transfected cells were positive for the presence of fluorescein-labeled siRNA duplexes. No cytotoxicity associated with the use of siRNA was observed during the time course of these experiments. Despite a transient abrogation of PrP-res accumulation, our results suggest that the use of siRNA may provide a new and promising therapeutic approach against prion diseases.

Anti-prion antibodies for prophylaxis following prion exposure in mice.

Prion disease is characterized by a conformational change of the normal form of the prion protein (PrP(C)) to the scrapie-associated form (PrP(Sc)). Since the emergence of new variant Creutzfeldt-Jakob disease a potentially large human population is at risk for developing prion disease. Currently, no effective treatment or form of post-exposure prophylaxis is available for prion disease. We recently showed that active immunization with recombinant PrP prolongs the incubation period of scrapie. Here we show that anti-PrP antibodies following prion exposure are effective at increasing the incubation period of the infection. Stimulation of the immune system is an important therapeutic target for the prion diseases, as well as for other neurodegenerative illnesses characterized by abnormal protein conformation.

Modulation of proteinase-K resistant prion protein by prion peptide immunization.

Prion diseases are caused by conformational alterations in the prion protein (PrP). The immune system has been assumed to be non-responsive to the self-prion protein, therefore, PrP autoimmunity has not been investigated. Here, we immunized various strains of mice with PrP peptides, some selected to fit the MHC class II-peptide binding motif. We found that specific PrP peptides elicited strong immune responses in NOD, C57BL/6 and A/J mice. To test the functional effect of this immunization, we examined the expression of proteinase-K-resistant PrP by a scrapie-infected tumor transplanted to immunized syngeneic A/J mice. PrP peptide vaccination did not affect the growth of the infected tumor transplant, but significantly reduced the level of protease-resistant PrP. Our results demonstrate that self-PrP peptides are immunogenic in mice and suggest that this immune response might affect PrP-scrapie levels in certain conditions.

The prion diseases.

The human prion diseases are fatal neurodegenerative maladies that may present as sporadic, genetic, or infectious illnesses. The sporadic form is called Creutzfeldt-Jakob disease (CJD) while the inherited disorders are called familial (f) CJD, Gerstmann-Straussler-Scheinker (GSS) disease and fatal familial insomnia (FFI). Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. In fCJD, GSS, and FFI, mutations in the PrP gene located on the short arm of chromosome 20 are the cause of disease. Considerable evidence argues that the prion diseases are disorders of protein conformation.

Effect of hyperbaric oxygenation on experimental scrapie in mice.

The effect of hyperbaric oxygenation upon experimental infection of BALB/c mice with the scrapie agent was investigated by virological methods, histology and electron microscopy. A multiple exposure of scrapie-infected mice to hyberbaric oxygenation during the incubational period led to a certain aggravation of infection as evidenced by a greater accumulation of the agent in the central nervous system (CNS) and spleen, as well as by more pronounced ultramicroscopic changes in CNS. A single exposure to hypoxia failed to alter any manifestations of infection.

Failure to modify scrapie in mice by administration of interferon or anti-interferon globulin.

Administration of potent mouse interferon preparations or anti-mouse interferon globulin did not influence the evolution of scrapie in mice after intraperitoneal injection of the agent. We conclude that the interferon system is probably not involved in scrapie.