Treatment of prion diseases using gene therapy

Abstract

Provided herein is a nucleic acid encoding a prion protein, wherein the prion protein (a) comprises an amino acid sequence comprising a protective sequence variant such as a G127V mutation and (b) lacks a fully functional 3' glycosylphosphatidylinositol (GPI)-anchor attachment sequence. Also provided are viral vectors and pharmaceutical compositions comprising the nucleic acids. Further provided are methods and uses of treating prion diseases or other neurodegenerative diseases that confer toxicity through cell surface- anchored prion proteins in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition or viral vector described herein.

Description

TITLE: TREATMENT OF PRION DISEASES USING GENE THERAPYRELATED APPLICATION

[0001] This application claims benefit of United States Provisional Patent Application serial no. 63 / 661 ,707, filed June 19, 2024, incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “P96495985PCT00_SequenceListing.xml” (18530 bytes), created on June 17, 2025 is herein incorporated by reference.FIELD

[0003] The present disclosure relates to a nucleic acid or recombinant protein comprising an anchorless prion protein harboring a protective sequence variant and methods and uses of these nucleic acids and proteins for treating CNS disorders whose etiologies center on the cellular prion protein.INTRODUCTION

[0004] Prion diseases are neurodegenerative diseases that afflict humans and a subset of mammals1. To date, human manifestations of these diseases have remained incurable and are the cause of approximately 1 in 5000 deaths2. In these diseases, the cellular prion protein (PrPc), a small glycosylphosphatidylinositol (GPI)-anchored protein3that is expressed in most vertebrate cells, converts through templated polymerization to p-sheet-rich alternative conformers, referred to as PrP Scrapie (PrPSc). Several strands of evidence suggest that the main function of PrPc, the substrate of this diseasecausing process, relates to its ability to control the polysialylation of the neural cell adhesion molecule 1 (NCAM1)4, thereby fulfilling a non-essential role in adult mammals5. Consequently, PrPclowering strategies may not only be effective for the treatment of these diseases but may also be safe. This expectation is corroborated by good health reported in elderly individuals that were found to carry only a single functional allele of the human prion gene(PRNP)2. Moreover, mice, cattle, and goats can live without PrPc, exhibiting only a mild subclinical peripheral neuropathy, with no overt phenotypes6-8.

[0005] Previous prion therapy strategies include recombinant AAV (rAAV) vectors coding for zinc fingers proteins (ZFPs) tailored to block prion gene transcription. Although this alternative PrPclowering strategy has been presented to achieve robust survival extension in prion-infected mice9, ongoing challenges with the delivery of rAAVs to human brain cells may limit its efficacy until rAAV capsids with broad and efficient human brain tropism or immunosuppressive regimes become available that allow repeat rAAV vector administrations. Consequently, there is a need to explore other treatment modalities that require a lower percentage of brain cells to be transduced.

[0006] Although recent rAAV capsids represent an improvement to earlier delivery vehicles, the difficulty to target most human brain cells remains the chief obstacle to the broad and effective use of gene therapies for the treatment of human neurodegenerative diseases that afflict the entire brain32'34. Accordingly, strategies are needed that can boost the potency of gene therapies delivered to a minority of brain cells.SUMMARY

[0007] The present disclosure describes the use of an anchorless prion protein harboring a G127V mutation (PrPG127V-AGPI) as a gene therapy for conferring protection against prion diseases. Secretion of PrPG127V-AGPI by the subset of transduced brain cells leads to enhanced cross-correction. Indeed, the present inventor has shown that delivering PrP without its glycosylphosphatidylinositol (GPI)-anchor attachment sequence and containing the V127 protective mutation through a recombinant adeno- associated virus (rAAV) extends prion disease survival. This approach was shown to preempt notorious delivery limitations of rAAVs through enhanced cross-correction.

[0008] Accordingly, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a prion protein, wherein the prionprotein (a) comprises an amino acid sequence comprising a protective sequence variant and (b) lacks a fully functional 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence.

[0009] In some embodiments, the protective sequence variant is a M129V polymorphism or a G127V, E219K, or E168R mutation. In some embodiments, the protective sequence variant is a G127V mutation.

[0010] In some embodiments, the prion protein lacks a 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence.

[0011] In some embodiments, the nucleotide sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 2, and wherein the protein encoded by said nucleotide sequence confers protection against prion disease.

[0012] In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 2.

[0013] In some embodiments, the amino acid sequence further comprises a prion disease-associated mutation. Optionally, the prion disease- associated mutation is E200K.

[0014] In some embodiments, the open reading frame coding for the prion protein is operably linked to a promoter.

[0015] In some embodiments, the promoter is selected from the group consisting of: chicken P-actin hybrid (CBh), chicken beta actin (CBA), CMV early enhancer (CAG), synapsin I (Syn1), Thy1 (Thy1), neuron-specific enolase (NSE), CamKII (CamKII), neural cell adhesion molecule 1 (NCAM1 ), glial fibrillary acidic protein (GFAP), nestin (Nes), tyrosine hydroxylase (TH), dopamine transporter (DAT), microtubule-associated protein 2 (MAP2), a- calcium / calmodulin-dependent protein kinase II (a-CaMKII), Elongation Factor 1a (eF-1a), phosphoglycerate kinase 1 (PGK), cytomegalovirus immediate- early promoter (CMV) and human P-actin (hACTB).

[0016] The disclosure further provides a recombinant prion protein, wherein the protein (a) comprises an amino acid sequence comprising a protective sequence variant and (b) lacks a fully functional 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence.

[0017] In some embodiments, the protective sequence variant is a M129V polymorphism or a G127V, E219K, or E168R mutation.

[0018] In some embodiments, the protective sequence variant is a G127V mutation.

[0019] In some embodiments, the prion protein lacks a 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence.

[0020] In some embodiments, the amino acid sequence is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino sequence of SEQ ID NO: 1 , and the protein confers protection against prion disease.

[0021] In some embodiments, the amino sequence is set forth in SEQ ID NO: 1.

[0022] In some embodiments, the amino acid sequence further comprises a prion disease-associated mutation.

[0023] In some embodiments, the prion disease-associated mutation is E200K.

[0024] In some embodiments, the prion protein is fused to one or more proteins, optionally wherein the one or more proteins are selected from the group consisting of viral capsid proteins, transferrin, semaphorin, Tat peptide, angiopep peptides, ligands targeting the insulin receptor, fragments of capsids from AAVs, fragments of capsids from Zika viruses, bacteriophage capsids and plant virus capsid.

[0025] The disclosure further provides a viral vector comprising a nucleic acid or a nucleic acid encoding a recombinant protein as described herein.

[0026] In some embodiments, the viral vector is an Adeno-Associated Virus (AAV) vector or a derivative thereof.

[0027] In some embodiments, the AAV vector is selected from the group consisting of: AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.32, AAVrh.33 AAVrh.43 and AAVrh.64R1.

[0028] In some embodiments, the viral vector is a chimeric, shuffled or capsid modified derivative of the AAV.

[0029] Further provided is a pharmaceutical composition comprising a nucleic acid, recombinant protein, or viral vector as described herein, and a pharmaceutically acceptable carrier or diluent.

[0030] In some embodiments, the pharmaceutically acceptable carrier or diluent is a lipid nanoparticle (LNP) or an exosome.

[0031] In some embodiments, the pharmaceutical composition is formulated for parenteral administration or intracerebral administration.

[0032] In some embodiments, the pharmaceutical composition is permeable to the blood-brain barrier.

[0033] Also provided is a method of increasing the therapeutic efficiency of a protective prion protein variant, comprising administering a viral vector or a pharmaceutical composition as described herein.

[0034] Also provided is a method of increasing secretion of a protective prion protein variant from transduced cells in a subject, comprising administering a viral vector or a pharmaceutical composition as described herein.

[0035] Also provided is a method of treating or preventing a prion disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a viral vector or a pharmaceutical composition as described herein.

[0036] Also provided is a method of treating or preventing a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a viral vector or a pharmaceutical composition as described herein, wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity.

[0037] In some embodiments, the neurodegenerative disease is selected from the group consisting of: Alzheimer’s disease (AD), frontotemporal dementia, tauopathies, Parkinson’s disease (PD), multiple system atrophy (MSA) and synucleinopathies,

[0038] In some embodiments, the viral vector or the pharmaceutical composition is administered parenterally or administered intracerebrally.

[0039] Further provided is a use of a viral vector or a pharmaceutical composition as described herein, for treating or preventing a prion disease and / or a neurodegenerative disease in a subject in need thereof, wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity.

[0040] Further provided is a viral vector or a pharmaceutical composition as described herein, in the manufacture of a medicament for treating or preventing a prion disease and / or a neurodegenerative disease in a subject in need thereof, wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity.

[0041] Further provided is a viral vector or a pharmaceutical composition as described herein, for use in treating or preventing a prion disease and / or a neurodegenerative disease in a subject in need thereof, wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity.

[0042] Also provided is a method of increasing the therapeutic efficiency of a prion protein composing a protective sequence variant, comprising the stepof deleting or mutating the 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence of the prion protein or a nucleic acid encoding the prion protein.

[0043] In some embodiments, the protective sequence variant is a M129V polymorphism or a G127V, E219K, or E168R mutation.

[0044] In some embodiments, the protective sequence variant is a G127V mutation.

[0045] In some embodiments, the 3’ glycosylphosphatidylinositol (GPI)- anchor attachment sequence is deleted from the prion protein.

[0046] In some embodiments, the prion protein further comprises a prion disease-associated mutation.

[0047] In some embodiments, the method further comprises administering the prion protein or the nucleic acid encoding the prion protein to a subject.

[0048] These and other features and advantages of the present disclosure will become apparent from the following detailed description taken together with the accompanying drawings. It should be understood, however, that the detailed description and specific examples, while indicating preferred implementations of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those of skill in the art from this detailed description.DRAWINGS

[0049] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0050] Figures 1A-F shows design of a therapeutic all-in-one rAAV vector that exhibits low immunogenicity and facilitates enhanced crosscorrection. Figure 1A depicts the genome organization of human PRNP gene. Zoom-in depicts the sequence organization of the ORF. From left to right the coding segments as follows: signal peptide for co-translational ER targeting; octarepeats; hydrophobic region; P-sheets; a-helices; signal sequence for attachment of GPI-anchor. The protein structure model shown on the right identifies the approximate position of the G127V residue within the tertiary fold of PrPcwith a circle. For ease of orientation, the image depicts a PrPcmolecule that is inserted into a membrane bilayer with a GPI-anchor. Figure 1B depicts the nucleotide sequence of a codon-optimized, synthetic bank vole PrnpG127Vcoding sequence (SEQ ID NO: 11 ). The G127V codon and the GPI-SS are shown in bold font and underlined, respectively. Figure 1C depicts the design of self-complementary rAAV vector for the expression of synthetic bank vole PrPG127V-AGPL Figure 1D shows a schematic summarizing key steps of alternative rAAV purifications schemes based on AAVX affinity capture or iodixanol gradient centrifugation. Figure 1E shows assessment of purity and yield of representative rAAV vector preparations coding for wild-type BvPrnpv127AGPI, or BvPrnpV127. Figure 1F depicts a representative image showing pronounced expression and brain-wide distribution of spEGFP signal in sagittal cut of mouse brain following retro-orbital administration of 9P31- spEGFP vector.

[0051] Figures 2A-F show heterologous expression of rAAV vector- delivered BvPrPv127AGPI causes survival extension in RML-infected BvPrnp ki mice. Figure 2A shows a timeline of pilot in vivo prion infection and treatment study in BvPrnp ki mice. Note that all endogenous and heterologous bank vole sequences employed in this work carried the 1109 polymorphism. Figure 2B shows a Kaplan-Meier chart comparing the survival of negative control mice and treated mice. Figure 2C shows a weight analysis chart depicts the relative weights of mice from the time of 100 dpi to the time of sacrifice when the animals met humane prion disease endpoints. Note that weights of individualmice were normalized to their weight at 100 dpi to account for individual weight differences and sex-specific weight differences. Figure 2D shows a nesting analysis chart showing the nesting score of mice from the time of 100 dpi to the end of the study. Figure 2E shows a chart documenting that the alternative rAAV vector preparation methods based on AAVX affinity capture or iodixanol gradient centrifugation for purifying the therapeutic 9P31-BvPrnpv127AGPI had an insignificant influence on survival extension. Figure 2F shows no significant differences in the survival times afforded by the expression of BvPrn pv127AGPI was observed whether the therapeutic payload was encapsulated in PHP.eB or 9P31 capsid. In all graphs, 95% confidence intervals are indicated by shading around the curves.

[0052] Figures 3A,B show expression of heterologous BvPrnpv127AGPI in RML-inoculated BvPrnp ki mice mitigates accumulation of PrPScand increases total PrP signal intensities up to threefold. Figure 3A (top) shows western blot analysis of extracts from sagittal half brains of BvPrnp ki mice that were either from a cohort of naive mice, or RML-inoculated mice that were mock-treated with a 9P31-spEGFP vector or treated with the 9P31- BvPrnpv127AGPI vector. Note that all animals were sacrificed by 152 days, i.e., when the RML-inoculated, mock-treated cohort reached its humane prion disease endpoint. In other words, neither the naive BvPrnp ki cohort nor the mice treated with 9P31-BvPrnpv127AGPI showed overt signs of prion disease at the time of their sacrifice. A western blot depicting steady-state beta actin (Actb) levels served as a loading control in this analysis. The bar graph on the right revealed significant differences in total PrP levels in the three cohorts. Figure 3A (bottom) shows analysis of proteinase K-resistant PrPScin protein extracts analyzed in the top panel. The bar graph on the right quantified band intensities of proteinase K-resistant PrPSc. It was normalized to PrPSclevels in the mock-treated cohort transduced with the 9P31-spEGFP vector. Figure 3B (top) shows therapeutic vectors encapsulated in 9P31 caused robust expression of BvPrnpv127AGPI that exceeded endogenous total PrP levels threefold. Western blot analysis of extracts from sagittal half brains of BvPrnpki mice that were RML-inoculated but then were either mock-treated through transduction with the spEGFP vector or transduced with therapeutic vectors coding for BvPrnpv127AGPI. For this analysis, brains from all mice were harvested after the animals reached their individual humane prion disease endpoint as shown by their unique dpi. To facilitate the recognition of the therapeutic BvPrnpv127AGPI signal amongst a complex mixture of N- glycosylated endogenous BvPrP expression products, N-glycans were removed by PNGase F digestion prior to the western blot analysis. Note the pronounced signal of the BvPrnpv127AGPI band, which migrated as expected a bit faster than the endogenous wild-type full-length BvPrP band on account of it lacking the GPI-anchor. The detection of Actb served again as loading control in this analysis. The bar graph on the right quantifies the intensities of BvPrnpv127AGPI signals in the treatment cohorts. Note that the weak respective signal in the mock-treated BvPrnp ki mice can be interpreted to represent shed, deglycosylated BvPrP. Figure 3B (bottom) shows a side-by-side analysis of one brain extract from each of the four treatment cohorts analyzed in the top panel with or without digestion with Proteinase K followed by digestion with PNGase F. Note that the bands visible in lanes 6, 9, and 12 do not appear to constitute doublet signals, which would be expected if the anchorless BvPrPv127AGPI had a propensity to contribute to the Proteinase K-resistant PrPSc. This is an important observation as it showed that the presence of the V127 mutation caused the BvPrPv127AGPI to behave different from wild-type anchorless prion proteins, which had been shown to form proteinase K-resistant aggregates.

[0053] Figures 4A-D depicts that global proteome analyses reveal that BvPrnpG127V- GP\ delays prion disease by slowing perturbations to the proteome observed in prion-inoculated mice. Figure 4A is a showing sample workup scheme for global proteome analysis. Figure 4B shows a global proteome summary diagram documenting benchmarks of the analysis, including the number of proteins whose steady-state levels in RML-inoculated and mock-treated brains were > 33% altered relative to their average proteinlevels in naive mouse brains. Figure 4C (left) shows results from hierarchical clustering analysis of global proteome dataset. Note that the unbiased clustering analysis grouped the cohorts in accordance with the study design, i.e., it identified three groups of highly similar brain extracts. The global proteomic signature of the mice, which were RML-inoculated and transduced with 9P31 -delivered BvP / T?pv127AGPI vectors, places them between the naive and the RML-inoculated but mock-treated mouse cohort. Figure 4C (right) shows a zoom-in into the hierarchical clustering results focused on proteins whose steady-state levels are upregulated in RML-inoculated mice. The graph indicates highly consistent changes in the direction of perturbation for a given protein and corroborates that many proteins are to a lesser degree altered in their steady-state levels in the 9P31-BvPrnpv127AGPI treatment cohort than the 9P31-spEGFP mock-treated cohort. Figure 4D shows the short-listed results from KEGG pathway analyses undertaken with proteins whose levels were > 33% up- or down-regulated.

[0054] Figures 5A-I show 9P31 -delivered BvPrnpG127V- GP\ gave rise to similar gene levels as endogenous BvPrnp yet increased protein levels approximately threefold and amplified unglycosylated PrP >20-fold. Figure 5A shows a schematic of PCR and restriction enzyme (RE) digest analysis of genomic brain DNA preparations of mice for determining the relative abundance of endogenous and heterologous BvPrnp ORFs. Figure 5B shows transduction of mice with 9P31-BvPrnpv127AGPI gave rise to the detection of RE cleavage products specific for heterologous BvPrnpv127AGPL Note that the signal intensities of RE digested PCR products in lanes 10 and 11 are approximately matched, indicating similar levels of BvPrnp and BvPrnpv127AGPI ORFs in the genome. Figure 5C shows steady-state total BvPrP levels, comprising both endogenous and heterologous BvPrP expression products, revealed in global proteomic analysis based on 52% sequence coverage and the assignment of 151 PSMs to 12 unique BvPrP- derived peptides. Figure 5D shows MS2 spectrum of the tryptic peptide ESQAYYEGR contributed by endogenous and heterologous pools of BvPrP.Figure 5E shows MS2 spectrum of the tryptic peptide GENFTETDVK comprising the second ‘NxT’ N-glycan acceptor consensus motif within BvPrP. Figures 5F-I show quantitation of tryptic BvPrP peptides that are either present in all BvPrP sequences analyzed (F), only observed in the subpool of BvPrP that is non-glycosylated at GENFTETDVK (G), only present in endogenous BvPrnp expression products (H), or only present in the heterologous BvPrnpv127AGPI expression product (I).

[0055] Figures 6A,B show lower steady-state levels of cell surface proteins residing in proximity to PrPccannot be accounted for by the loss of neurons that occurs late in the disease. Figure 6A shows proteins reported to reside in proximity to PrPcat the cell surface are downregulated in end-stage RML prion-inoculated mice. The list depicts all but one protein, which was not captured in this global proteome analysis, in the order in which they were previously reported, i.e., based on their relative enrichment in an affinity-capture PrP interactome from an in vivo crosslinked mouse brains86The intensity of the shading reflects the level of reduction of a given protein entry relative to its average steady-state levels in age-matched naive control mice. Note the consistent shading in proteins whose dominant subcellular compartment is the cell surface. Figure 6B shows that although neurons are understood to die in late-stage prion disease, the steady-state levels of several neuronal markers (NeuN, nestin, or Tuj1 ) are not noticeably lowered in a sagittal half brain extracts generated at the humane prion disease end-stage, indicating that for a majority of brain neurons the disease merely causes the retraction of synapses but not their full disappearance. The table also shows well-known brain cell marker proteins that are preferentially expressed in astrocytes, microglia, or oligodendrocytes, indicating the activation of astrocytosis and microgliosis, as well as consistent loss oligodendrocytes.

[0056] Figure 7 shows a model interpreting results and highlighting facets of ECO that may translate into advantages of expressing PrPv127AGPI over anchored PrPV127. The column on the left depicts outcomes of a treatment based on the expression of GPI-anchored PrPG127V. The middle column depictsconsequences of the expression of PrPG127V-AGPL The column on the right indicates features of the therapy that lead to enhanced cross-correction. Panel A shows that whereas in germ line-expressing heterozyogous kuru survivors carrying the V127 mutation, PrPV127is expressed in all cells, only a subset of cells will be transduced when the protective mutant is delivered through rAAV vectors. This deficiency may be partially compensated through an increase in the half-life of PrPv127AGPI, relative to PrPV127. Panel B shows the secretion of PrPv127AGPI promotes local spread, thereby promoting access to extracellular PrPScseeds. Panel C shows that whereas the germline expressed PrPV127is present in all brain areas due to its ubiquitous expression, PrPv127AGPI can be highly expressed only in a subset of brain cells. However, a strong promoter may allow it to achieve a similar brain-wide protection through its diffusion within extracellular spaces and the CSF. Panel D shows the presence of a GPI-anchor and N-glycans has the capacity to slow access to nascent PrPSc. This may not be a hindrance if PrPV127is germline expressed alongside wild-type PrP as the immediacy of contact between the conversion susceptible and refractory isoforms may still provide the earliest possible protection (here depicted as a capping mechanism, one of several possible scenarios through which the V127 may protect in a dominant negative fashion). Note that in this and the final subpanel, the size of PrPScseeds is meant to indicate the readiness with which the V127 mutant can block aggregation, i.e., a capped aggregate comprising just one PrPScand one V127 mutant molecule is meant to indicate immediate inhibition, whereas four, six, and eight building blocks in a PrPScaggregate symbolizes moderate, slow, and impaired inhibition of PrPScaccumulation. To simplify the cartoon, only one N-glycan is shown when a population of mostly N-glycosylated PrP molecules is expected, and steric hindrances are indicated with red-crossed arrows. Panel E shows that the absence of a GPI-anchor or N-glycans generate reduced steric hindrances for PrPv127AGPI, which may broaden its protective properties to a wider range of strains, here depicted as hypothetical strains ‘a’, ‘b’, and ‘c’.DESCRIPTION OF VARIOUS EMBODIMENTS

[0057] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0058] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature described herein may be combined with any other feature or features described herein.I. Definitions

[0059] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0060] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components,groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[0061] The term “consisting” and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0062] All numerical values herein are modified by “about” or “approximately” the indicated value, and take into account experimental error and variations that would be expected by a person having ordinary skill in the art.

[0063] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art.

[0064] As used herein, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0065] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1 .5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0066] As used herein, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0067] The term “prion protein” or “PrP” as used herein refers to the prion protein, which is coded by the prion gene, designated PRNP in humans and Prnp in mice and many other species. Prion proteins can be classified as cellular prion proteins (PrPc) or Scrapie prion proteins (PrPSc). PrPcexist in most vertebrate cells. They can turn into transmissible agents, referred to as PrPSc, which cause neurodegenerative diseases when they acquire distinct physicochemical characteristics through a mechanism of templated protein misfolding. The amino acid sequence of the human prion protein can be found, for example, at UniProt ID: P04156. The amino acid sequence of the mouse prion protein can be found, for example, at UniProt ID: P04925.

[0068] The term “prion disease” as used herein refers to neurodegenerative diseases associated with templated protein misfolding of the prion protein. Prion diseases include without limitation, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Fatal Familial Insomnia (FFI), and the now extinct disease known as Kuru. Other neurodegenerative diseases whose toxicity is mediated by soluble aggregates, including without limitation, Alzheimer’s disease (AD), frontotemporal dementia, and other tauopathies, as well as Parkinson’s disease (PD), multiple system atrophy (MSA) and other synucleinopathies, are not considered prion diseases, but benefit from the prion treatments described herein due to their aggregates forming interactions with cell surface prion proteins.

[0069] The term “anchorless” refers to a prion protein lacking the glycosylphosphatidylinositol (GPI) anchor attachment sequence (also referred to herein as a GPI signal sequence (GPI-SS), which directs the cell to attach a GPI anchor to the prion protein. The GPI anchor serves to insert the prion protein into a membrane bilayer. Accordingly, an anchorless prion protein is released from the cell into the extracellular space. For example, the PrPcamino acid and PRNP nucleotide sequences of the human GPI-SS are shown in SEQ ID NOs: 3 and 4, respectively.

[0070] The term “cross-correction” or “CO” as used herein, refers to the ability of a biologic therapeutic agent, such as a protein, to treat both the cell producing the therapeutic agent as well as neighbouring cells. In some embodiments of the present disclosure, cross-correction of a prion protein is accomplished by deleting the GPI Anchor Signal Sequence (GPI-SS) of the prion protein. Secretion of the anchorless prion protein variant by the subset of transduced brain cells leads to cross-correction.

[0071] The term “enhanced cross-correction” or “ECO” as used herein, refers to the method of facilitating CO not only through increasing the extracellular spread of a biologic therapeutic agent but also through altering the biochemical properties of a biologic therapeutic agent in a manner that naturally enhances its therapeutic effectiveness.

[0072] The term “nucleic acid molecule” and its derivatives, as used herein, are intended to include unmodified DNA or RNA or modified DNA or RNA. For example, the nucleic acid molecules or polynucleotides of the disclosure can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically double-stranded or a mixture of single- and double-stranded regions. In addition, the nucleic acid molecules can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acidmolecules of the disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus “nucleic acid molecule” embraces chemically, enzymatically, or metabolically modified forms. The term “polynucleotide” shall have a corresponding meaning.

[0073] The term “nucleic acid construct” as used herein refers to a nucleic acid molecule comprising an expression cassette, the expression cassette comprising a DNA sequence encoding a prion protein operably linked to a promoter and a transcription termination site.

[0074] The term “operably linked” as used herein refers to a relationship between two components that allows them to function in an intended manner. For example, where a coding sequence is operably linked to a promoter, the promoter actuates expression of the coding sequence.

[0075] The term “promoter” or “promoter sequence” generally refers to a regulatory DNA sequence capable of being bound by an RNA polymerase to initiate transcription of a downstream (i.e. 3’) sequence to generate an RNA. Suitable promoters may be derived from any organism and may be bound or recognized by any RNA polymerase. Suitable promoters for the expression cassette will be known to the skilled person. In some embodiments, the promoter is an inducible promoter. Examples of inducible promoters include, without limitation, a tetracycline response element (TRE) (e.g. Tet-ON or Tet- OFF systems), ponA-inducible expression systems (Agilent Technologies), or cumate-inducible promoters such as CuO (System Biosciences). In some embodiments, the promoter is a constitutive promoter. Examples of constitutive promoters include human Ubiquitin C (UBC), human Elongation Factor 1a (EF1A), phosphoglycerate kinase 1 (PGK), cytomegalovirus immediate-early promoter (CMV), chicken b-Actin promoter coupled with CMV early enhancer (CAG), chicken P-actin hybrid (CBh) and EF1-HTLV. In some embodiments, the promoter is a brain-specific promoter. Examples of brain specific promotersinclude, without limitation synapsin I (Syn1), Thy1 (Thy1), neuron-specific enolase (NSE), CamKII (CamKII), neural cell adhesion molecule 1 (NCAM1), glial fibrillary acidic protein (GFAP), nestin (Nes), tyrosine hydroxylase (TH), dopamine transporter (DAT), microtubule-associated protein 2 (MAP2), a- calcium / calmodulin-dependent protein kinase II (a-CaMKII),

[0076] The term “expression cassette” refers to a DNA molecule encoding an RNA or protein operably linked to a promoter and a polyadenylation signal, such that certain portions of the expression cassette are capable of being transcribed into RNA (such as antisense RNA, Long noncoding RNA or for the genome of a virus such as a lentivirus) and / or as a messenger RNA that is subsequently translated into protein by cellular machinery. The term “expression cassette” is also used to refer to a nucleic acid molecule comprising a promoter, a polyadenylation signal, and a cloning site for insertion of a nucleic acid molecule encoding an RNA or protein of interest in operable linkage with the promoter and the polyadenylation signal.

[0077] The term “transcription termination site” as used herein refers generally to a polyadenylation signal (pA) that terminates transcription of messenger RNA (mRNA). As used herein, the phrase “polyadenylation signal” refers to sequences from various genes that can be added to mammalian vectors to ensure proper mRNA processing and stability. For example, a 100- 200 nucleotide polyadenylate tail can be added to the 3’ end of a coding sequence to protect mRNA from degradatory action of phosphatases and nucleases. Suitable pAs may be derived from any organism and are known to the skilled person. Examples of pA signals include, without limitation, rabbit beta-globin pA (GenBank accession number K03256), SV40 late polyA, and hGH polyA and strong bovine growth hormone pA (BGHpA).

[0078] The term “functional variant” as used herein includes modifications of the nucleic acid or polypeptide sequences disclosed herein that perform substantially the same function as the nucleic acid molecules or polypeptides disclosed herein in substantially the same way. For example, thefunctional variant may comprise sequences having at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity to the sequences disclosed herein. In the case of nucleic acids, functional variants include nucleotide sequences that hybridize to the nucleic acid sequences set out above, under at least moderately stringent hybridization conditions, optionally stringent hybridization conditions, or the functional variant nucleic acid sequences may comprise degenerate codon substitutions or codon-optimized nucleic acid sequences. In the case of polypeptides, the functional variant may also comprise conservatively substituted amino acid sequences of the sequences disclosed herein. In one embodiment, a functional variant of a prion protein with a G127V mutation is a prion protein that confers protection against prion disease.

[0079] The term “degenerate codon substitution” as used herein refers to variant nucleic acid sequences in which the second and / or third base of a codon is substituted with a different base that does not result in a change in the amino acid sequence encoded therein.

[0080] The term “sequence identity” or “identity” as used herein refers to the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = [number of identical overlapping positions] I [total number of positions] X 100%). In one embodiment, the two sequences are the same length. The determination of percent identity betweentwo sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g. for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g. to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. of XBLAST and NBLAST) can be used (see, e.g. the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0081] With reference to nucleic acids, the terms “anneal” and “hybridize” as used herein refer to the ability of a nucleic acid to non-covalently interact with another nucleic acid through base-pairing. The terms “complementary” or “complementary nucleic acid” refer to a nucleic acid or a portion of a nucleicacid that is able to anneal with a nucleic acid of a given sequence. In some cases, this is referred to as the “reverse complement” of a given sequence.

[0082] By “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. The term “at least moderately stringent hybridization conditions” encompasses stringent hybridization conditions and moderately stringent hybridization conditions. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm = 81 ,5°C - 16.6 (Log10 [Na+]) + 0.41 (%(G+C) - 600 / l), orsimilarequation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1 % mismatch may be assumed to result in about a 1 °C decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In some embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt’s solution / 1.0% SDS at Tm - 5°C based on the above equation, followed by a wash of 0.2x SSC / 0.1 % SDS at 60°C. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al.,Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0083] The term "vector" as used herein comprises any intermediary vehicle for a nucleic acid molecule which enables said nucleic acid molecule, for example, to be introduced into prokaryotic and / or eukaryotic cells and / or integrated into a genome, and include plasmids, phagemids, bacteriophages or viral vectors such as retroviral based vectors, including lentiviral vectors, Adeno Associated viral (AAV) vectors and the like. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0084] The term “viral vector” as used herein is intended to include viral particles or virus-like particles capable of transduction of a target cell. Common viral vectors include, but are not limited to, HIV-derived lentiviral vectors, retroviral vectors, adenoviral vectors, and recombinant adeno-associated virus (AAV) vectors. Other viral vectors may be derived from rhabdovirus (such as vesicular stomatitis virus (VSV)), or herpes virus (such CMV and HSV-1 ). Typical components of the viral vector are the structural components of the viral particle, such as the proteins making the capsid and the envelope of the vector. Other components are the enzymes involved in the replication of the vector RNA or DNA. Such enzymes can be also involved in the synthesis, maturation or transport of the virus RNA. These enzymes can also be involved in the processing and maturation of viral components, as well as in the integration of the genome of the virus into the cell chromosomes. Enzymes that are components of the viral vectors can also be involved in the reverse transcription of the virus genomic RNA into DNA. Other components of the vector can be protein or peptide that regulate the replication, transcription, transport or translation of the genes or gene products of the viral vector. Such factors can also activate or decrease the expression of cellular genes and they can modulate the defense mechanism of the cells against viruses.

[0085] The term "subject" as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans. Optionally, the term “subject” includes mammals that have been diagnosed with a prion disease. In one embodiment, the term “subject” refers to a human having, or suspected of having, a prion disease.

[0086] The term "subject in need thereof" refers to a subject that could benefit from the method(s) or treatment(s) described herein, and optionally refers to a subject with a prion disease, or optionally a subject with increased risk of a prion disease, or a subject with a genetic disposition.

[0087] The term "pharmaceutically acceptable carrier" refers to any carrier, agent, excipient, or vehicle that is not biologically or otherwise undesirable. Except where the carrier, agent, excipient or vehicle is incompatible with the active ingredient, its use in the therapeutic formulations is contemplated. The use of such pharmaceutically acceptable carrier is well known in the art.

[0088] The term "treating" or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0089] “Palliating” a disease, disorder or condition means that the extent and / or undesirable clinical manifestations of a disease, disorder or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to not treating the disorder.

[0090] The term “prevention” or “prophylaxis”, or synonym thereto, as used herein refers to a reduction in the risk or probability of a subject becoming afflicted with a disease, disorder or condition or manifesting a symptom associated with a disease, disorder or condition.

[0091] The term “administered” or “administering” as used herein means administration of a therapeutically effective amount of a compound or composition of the disclosure to a cell either in cell culture or in a subject.

[0092] The term “parenteral” as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.

[0093] The term “intracerebral” as used herein refers to direct delivery, such as injection or infusion techniques, into the brain.

[0094] As used herein, the phrase "effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, in the context of treating a prion disease, an effective amount is an amount that for example increases expression of the desired gene, reduces disease symptoms, and / or reduces disease severity compared to the response obtained without administration of the compound. Effective amounts may vary according to factors such as the disease state, age, sex and weight of the animal. The amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

[0095] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0096] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.II. Nucleic Acids, Constructs and Viral Vectors

[0097] Described herein is a nucleic acid comprising a nucleotide sequence encoding a prion protein, wherein the prion protein (a) comprises an amino acid sequence comprising a protective sequence variant and (b) lacks a fully functional 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence.

[0098] As used herein, the term “protective sequence variant” refers to a mutation in a prion protein which confers protection against prion disease. A protective sequence variant, may, for example, be found to exist in humans or may originally be identified in a non-human species and subsequently observed to confer protection to prion diseases in humans as well.

[0099] The G127V mutation, first identified amongst the Fore people of Papua New Guinea, is an example of a protective sequence variant. The G127V mutation is a dominant positive mutation conferring protection against prion disease. A single G127V allele is sufficient to confer protection when exposed to PrPSc. Thus, as used herein, the term “G127V mutation” refers to the substitution of a glycine with a valine in position 127 (G127V) within the human prion protein (PrP). The G127V mutation is well known in the art23’64 66’67. A person of skill in the art will also appreciate that the corresponding mutation to the G127V mutation in other species may be at a slightly different position. For example, in mouse prion protein, the corresponding mutation is a G126V mutation.

[0100] In one embodiment, the G127V mutation is shown in SEQ ID NO: 1 , or a functional variant thereof.

[0101] Other human protective sequence variants include, but are not limited, to a M129V polymorphism or a G127V, E219K, or E168R mutation. In other species, protective sequence variants include, without limitation, V136A, R154H and Q171 R / H in sheep and Q95H and G96S in white-tailed deer.

[0102] In one embodiment, the protective sequence variant is an amino acid from a non-human PrP sequence that has been shown to confer resistance to conversion but is not a mutation or polymorphism in the respective species. For example, dogs and rabbits are naturally resistant to prion diseases and certain amino acids, including S174 in rabbit and D159 in dogs, contribute to this resistance. By converting the orthologous amino acids in the human PrP sequence, a protective sequence variant may be generated.

[0103] In one embodiment, the prion protein lacks a fully functional, or a functional 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence (also referred to herein as a GPI signal sequence (GPI-SS)). Examples of the human PrPcamino acid and PRNP nucleotide sequences of the human GPI- SS are shown in SEQ ID NOs: 3 and 4, respectively.

[0104] The expression “a fully functional 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence” refers to a prior protein that has a mutation or deletion in the GPI-SS such that ability of a cell to attach a GPI anchor to the prion protein is diminished or eliminated. In one embodiment, the entire GPI- SS of the prion protein is deleted. In another embodiment, a fragment of the GPI-SS of the prion protein is deleted or the GPI-SS of the prion protein is altered such that the GPI-SS is not fully functional or is not functional.

[0105] In an embodiment, the amino acid sequence of the prion protein is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1 , and the prion protein confers protection against prion disease.

[0106] In an embodiment, the nucleotide sequence of the nucleic acid encoding the prion protein is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 2, and whereinthe protein encoded by said nucleotide sequence confers protection against prion disease.

[0107] In an embodiment, the nucleotide sequence is set forth in SEQ ID NO: 2.

[0108] In one embodiment, the nucleotide sequence hybridizes to SEQ ID NO: 2, under at least moderately stringent hybridization conditions, optionally stringent hybridization conditions.

[0109] In another embodiment, the nucleic acid sequence comprises SEQ ID NO: 2 with one or more degenerate codon substitutions.

[0110] In another embodiment, the amino acid sequence of the prion protein further comprises a familial prion disease-associated mutation. This allows delivery of protection within a bespoke therapeutic construct that also comprises a specific prion disease-associated mutation, for example.

[0111] As used herein, the term “prion disease-associated mutation” refers to a mutation in the amino acid sequence which is associated with a prion disease. For example, a prion disease-associated mutation may cause or increase the propensity for an individual to develop prion disease. In some embodiments, the prion disease-associated mutation is a familial prion disease- associated mutation. Numerous prion disease-associated mutations are known, including, but not limited to, those shown in the table below, adapted from Bernardi and Bruni, 201996. In one embodiment, the prion disease- associated mutation is a E200K mutation.Table 1 : Prion disease-associated mutations

[0112] In one embodiment, the nucleic acid is comprised in a nucleicacid construct, the nucleic acid construct comprising a promoter and a transcriptiontermination site, wherein the open reading frame coding for the prion protein is operably linked to the promoter.

[0113] In an embodiment, the promoter is a constitutive promoter, or a tissue- or cell-specific promoter.

[0114] In an embodiment, the promoter is selected from the group consisting of: Synapsin I (Syn1), Thy1 (Thy1), neuron-specific enolase (NSE), CamKII (CamKII), neural cell adhesion molecule 1 (NCAM1 ), glial fibrillary acidic protein (GFAP), nestin (Nes), tyrosine hydroxylase (TH), dopamine transporter (DAT), microtubule-associated protein 2 (MAP2), a- calcium / calmodulin-dependent protein kinase II (a-CaMKII), chicken beta actin (CBA), chicken P-actin hybrid (CBh), CMV early enhancer (CAG), Elongation Factor 1a (eF-1a), Phosphoglycerate kinase 1 (PGK), cytomegalovirus immediate-early promoter (CMV) and human P-actin (hACTB). In one embodiment, the promoter is chicken beta actin (CBA), chicken P-actin hybrid (CBh) or CMV early enhancer (CAG), optionally chicken P-actin hybrid (CBh).

[0115] In an embodiment, the nucleic acid further comprises, an enhancer, a post-transcription regulatory sequence, one or more sequences that facilitate incorporation of the nucleic acid into a viral particle and / or integration into the host genome, or any combination thereof, operably linked to the nucleic acid encoding the PrP protein. Post transcriptional regulatory sequences include, for example, without limitation, sequences of nucleotides that when placed in an AAV transfer plasmid results in the increased or decreased expression of the transgene. As used herein, the phrase “enhancer” refers to a sequence of nucleotides that argument the activity of a promoter in an orientation, position, and distance-dependent manner. Enhancers play a significant role in the regulation of tissue-specific gene expression in high eukaryotes but have been repurposed for use in recombinant DNA technologies to impact the transcriptional activity of an associated promoter. Typically, a trans-acting gene regulatory protein binds the enhancer in order to affect transcriptional activity of the associated promoter.

[0116] Also described herein is a recombinant prion protein wherein the prion protein (a) comprises an amino acid sequence comprising a protective sequence variant and (b) lacks a fully functional 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence.

[0117] In some embodiments, the recombinant prion protein is fused to one or more proteins, for example to one or more proteins that confer uptake by brain cells. Examples of such proteins include, but are not limited to, viral capsid proteins, transferrin, semaphorin, Tat peptide, angiopep peptides, ligands targeting the insulin receptor, fragments of capsids from AAVs, fragments of capsids from Zika viruses, bacteriophage capsids and plant virus capsids. The recombinant prion protein can be fused directly or indirectly to the at least one more protein

[0118] In some embodiments, the recombinant protein is a fusion protein engineered to cross the blood-brain barrier, as described in Chen et al71.

[0119] Also described herein is a viral vector comprising a nucleic acid or recombinant protein described herein. Viral vectors are made of DNA or RNA and they contain some of the genetic material of the viruses they are derived from (such as lentivirus, retrovirus, AAV and adenoviruses). For example, viral vectors may include sequences that facilitate incorporation of the nucleic acid into a viral particle and / or integration into the host genome. In some embodiments, the viral vectors may include inverted terminal repeats (ITRs) for example from an AAV such as AAV9, or other viral sequences. Viral vectors have been modified to carry and to deliver a gene of interest that will produce a protein or an RNA of interest and can be used for example for the treatment of diseases by gene therapy. Suitable viral vectors are known in the art.

[0120] One aspect of the disclosure is a viral vector comprising a nucleic acid, nucleic acid construct or nucleic acid encoding a recombinant prion protein. Replication incompetent viral vectors are particularly useful in gene therapy applications as they allow for efficient transduction of delivery of a transgene to target tissues. Differences between viral vectors includeavailability of tropisms, packaging capacity, safety, and transduction efficiencies in different tissues.

[0121] Several viral vectors are well known in the art including adenovirus, adenoviral associated virus (AAV), lentivirus, retrovirus, and herpes simplex virus 1. Accordingly, in an embodiment, the viral vector is a lentivirus, adenovirus, adenoviral associated virus (AAV), retrovirus, or herpes simplex virus 1 vector. Optionally, the viral vector is an AAV vector.

[0122] AAV is particularly useful for gene therapy applications as it elicits a limited immune response, exhibits a wide variety of serotypes, and has a stable expression profile. Accordingly, in an embodiment, the viral vector is an AAV vector or a derivative thereof. Optionally, the AAV vector is selected from the group consisting of AAV1 , AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh.8, AAV9, AAVr10, AAVrh.32, AAVrh.33 AAVrh.43, AAVrh.64R1 or a derivative thereof.

[0123] In an embodiment, the viral vector is a chimeric, shuffled, or capsid modified derivative of AAV.

[0124] In one embodiment there is provided a pharmaceutical composition comprising a nucleic acid construct, recombinant protein construct or viral vector described herein or a derivative of it, and a pharmaceutically acceptable carrier or diluent. The composition may be formulated for use or prepared for administration to a subject using pharmaceutically acceptable formulations known in the art. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington’s Pharmaceutical Sciences (2003 - 20thedition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. The term “pharmaceutically acceptable” means compatible with the treatment of animals, in particular, humans.

[0125] On this basis, the pharmaceutical compositions could include an active compound or substance, such as a nucleic acid construct, a recombinant protein construct or viral vector described herein, in association with one ormore pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and isosmoticwith the physiological fluids. The methods of combining viral vectors the vehicles or combining them with diluents is well known to those skilled in the art. The composition could include a targeting agent for the delivery or transport of the active compound to specified sites within the body, organ, tissue, or cell.

[0126] The pharmaceutical compositions, formulations, dosages, etc. described herein can be administered for example, by parenteral, intracerebral, intravenous, intrathecal, subcutaneous, or intramuscular administration in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

[0127] In an embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle (LNP) or an exosome.

[0128] The nucleic acid, recombinant proteins or viral vectors described herein are suitably formulated in a conventional manner into compositions using one or more carriers or diluents. Accordingly, the present description also includes a composition comprising one or more nucleic acid, recombinant proteins or viral vectors described herein and a carrier or diluent. The nucleic acids, recombinant proteins or viral vectors described herein are suitably formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. Accordingly, the present description further includes a pharmaceutical composition comprising the nucleic acid, recombinant proteins or viral vectors described herein, and a pharmaceutically acceptable carrier. In some embodiments the pharmaceutical compositions are used in the treatment of any of the diseases, disorders or conditions described herein. In an embodiment, the disease, disorder, or condition is a prion disease. In an embodiment, the disease, disorder or condition is a neurodegenerative disease, wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity.

[0129] In some embodiments, the nucleic acid, recombinant proteins or viral vectors described herein are formulated for parenteral administration or intracerebral administration by injection, including using conventional catheterization techniques or infusion. Formulations for injection are, for example, presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. In some embodiments, the compositions take such forms as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulating agents such as suspending, stabilizing and / or dispersing agents. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. Alternatively, nucleic acids, recombinant proteins or viral vectors described herein are suitably in a sterile powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0130] In an embodiment, the pharmaceutical composition is formulated for parenteral administration or intracerebral injection.

[0131] In some embodiments, the pharmaceutical composition is permeable to the blood-brain barrier.

[0132] In some embodiments, the pharmaceutical composition is impermeable to the blood-brain barrier. In some embodiments, the nucleic acid or viral vector is fused to a vehicle that facilitates crossing of the blood-brain barrier. In some embodiments, the pharmaceutical composition comprises the nucleic acid or viral vector fused to the vehicle.

[0133] In some embodiments, the vehicle that facilitates crossing of the blood-brain barrier is transferrin, semaphorin, Tat peptide, angiopep peptides, ligands targeting the insulin receptor, fragments of capsids from AAVs, fragments of capsids from Zika viruses, bacteriophage capsids, or plant virus capsid.

[0134] Also provided are kits comprising a nucleic acid, viral vector, or pharmaceutical composition as described herein, along with suitable containeror packaging and / or instructions for the use thereof, such as for the treatment of a prion disease in a subject.II. Methods and Uses

[0135] Another aspect of the disclosure is a method of treating or preventing a prion disease in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid, the viral vector or the pharmaceutical composition described herein. A further aspect includes use of the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein for treating or preventing a prion disease in a subject in need thereof. An aspect also includes use of the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein in the manufacture of a medicament for treating or preventing a prion disease in a subject in need thereof. Another aspect includes the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein for use in treating or preventing a prion disease in a subject in need thereof.

[0136] Another aspect of the disclosure is a method of treating or preventing a neurodegenerative disease in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid, the viral vector or the pharmaceutical composition described herein, wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity. A further aspect includes use of the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein for treating or preventing a neurodegenerative disease in a subject in need thereof, wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity. An aspect also includes use of the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein in the manufacture of a medicament for treating or preventing a neurodegenerative disease in a subject in need thereof, wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity. Another aspect includes the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein for use intreating or preventing a neurodegenerative disease in a subject in need thereof, wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity.

[0137] Another aspect of the disclosure is a method of increasing the therapeutic efficiency of a protective prion protein variant, comprising administering the nucleic acid, the viral vector or the pharmaceutical composition described herein to a subject in need thereof. A further aspect includes use of the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein for increasing the therapeutic efficiency of a protective prion protein variant. An aspect also includes use of the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein in the manufacture of a medicament for increasing the therapeutic efficiency of a protective prion protein variant. Another aspect includes the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein for increasing the therapeutic efficiency of a protective prion protein variant.

[0138] Another aspect of the disclosure is a method of increasing secretion of a protective prion protein variant from transduced cells in a subject, comprising administering the nucleic acid, the viral vector orthe pharmaceutical composition described herein to a subject in need thereof. A further aspect includes use of the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein for increasing secretion of a protective prion protein variant from transduced cells in a subject. An aspect also includes use of the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein in the manufacture of a medicament for increasing secretion of a protective prion protein variant from transduced cells in a subject. Another aspect includes the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein for increasing secretion of a protective prion protein variant from transduced cells in a subject.

[0139] In some embodiments, the neurodegenerative disease is selected from the group consisting of: Alzheimer’s disease (AD), frontotemporaldementia, tauopathies, Parkinson’s disease (PD), multiple system atrophy (MSA) and synucleinopathies

[0140] In some embodiments, the methods or uses disclosed herein comprise administering the therapeutically effective amount of the nucleic acid, the viral vector or the pharmaceutical composition disclosed herein by parenteral injection or intracerebral injection.

[0141] The nucleic acids, recombinant proteins or viral vectors described herein may be administered to or used in a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. For example, the nucleic acids, recombinant proteins or viral vectors described herein may be administered by parenteral administration or intracerebral administration and the pharmaceutical compositions formulated accordingly. In some embodiments, administration is by means of a pump for periodic or continuous delivery. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington’s Pharmaceutical Sciences (2000 - 20thedition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0142] Suitable administration schedules may include, without limitation, at least once a week, from about once in lifetime, one time per two weeks, three weeks or one month, about one time per week to about once daily. The length of the treatment period may depend on a variety of factors, such as the severity of the disease, disorder or condition, the age of the subject, the concentration and / or the activity of the nucleic acids or viral vectors described herein. It will also be appreciated that the effective dosage of the nucleic acids or viral vectors described herein used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration is required. For example, the nucleic acid orviral vector described herein is administered to the subject in an amount and for duration sufficient to treat the subject.

[0143] The disclosure also provides a method of increasing the therapeutic efficiency of a prion protein comprising a protective sequence variant (for example, a M129V polymorphism or a G127V, E219K, or E168R mutation) comprising the step of deleting or mutating the 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence of the prion protein or a nucleic acid encoding the prion protein. The method optionally further comprises administering the prion protein or the nucleic acid encoding the prion protein to a subject in need thereof. Methods of deleting or mutating the 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence of the prion protein or a nucleic acid encoding the prion protein are well known in the art and are also described herein.

[0144] The following non-limiting examples are illustrative of the present disclosure:ExamplesExample 1 : Study Design

[0145] To develop a pre-clinical in vivo model, mice that express bank vole (Bv) prion proteins were used. Recent work has established that BvPrPG127Vexpressed in cells confers resistance to infection with several prion strains25. BvPrPcexpressing mice were generated by a knock-in that replaced the endogenous mouse Prnp ORF with the respective BvPrnp ORF69. For proof-of-concept experiments BvPrnp expressing mice offer the advantage that they are expected to be susceptible to infection with a broad range of prion strains, including human prions, thereby facilitating the downstream crosscomparison of data generated with various prion inocula.

[0146] To document the improvement in therapeutic potency that can be attained with PrPG127V-AGPI, cohorts of BvPrnp mice were inoculated with prions through intracerebral injection of prion-infected brain homogenates. Atsuitable intervals, i.e., days post inoculation (dpi), separate cohorts of mice were transduced with highly purified rAAV vectors whose payloads code for either BvPrPG127V-AGPI or GPI-anchored BvPrPG127V

[0147] To promote the expression of rAAV vector-delivered singlestranded DNA constructs that code for heterologous BvPrPG127V-AGPI or GPI- anchored BvPrPG127V, the rAAV vectors were assembled as self- complementary vectors, thereby bypassing the rate-limiting second strand DNA synthesis70. To minimize the immunogenicity of the rAAV vector payload and make it distinguishable from the endogenously expressed wild-type BvPrP by RT-qPCR, the ORFs coding for BvPrPG127V-AGPI and GPI-anchored BvPrPG127Vwere based on synthetic sequences that feature a minimal number of CpG dinucleotides, which was achieved by capitalizing on the redundancy of the genetic code through the selection of appropriate codons.

[0148] Transduction with rAAV vectors was performed 60 or 90 days after prion-infection to mimic real-world scenario of individuals learning about the risk of developing the disease, either due to genetic predisposition or dietary exposure to prions. In a third treatment group, treatment was initiated 120 days after prion infection, when first clinical symptoms may occur, to model treatment onset of symptomatic human cases in which the disease manifests sporadically. Animals were euthanized when they met predefined endpoint criteria that include inability to reach food or water, 20% body weight loss, severe respiratory stress, or severe prion disease-characteristic neurological deficits. Subsequently, brain and blood were collected for pharmacological studies and biomarker analyses. A subset of animals were fixated by transcardiac perfusion for neuropathological analyses using (immuno-) histochemistry. Disease onset was tracked, and median survival times were computed, using two-sided statistical tests, stratifying results also by sex for all cohorts.Example 2: Prion Proteins in Neurodegenerative Diseases

[0149] The expression of PrPG127V-AGPI not only confers protection against prion diseases but also against other neurodegenerative diseases, which require for neurotoxicity to manifest that the prion protein is accessible at the cell surface of brain cells. This is in stark contrast to gene therapy approaches that might be based on the heterologous expression of a GPI- anchored PrPG127Vmutant, which would heighten the risk associated with such a neurodegenerative disease by adding PrP molecules to the cell surface (prpd27v jogsnotjjffgp initsability to serve as a cell surface receptor). The release of PrPG127V-AGPI ameliorates the risk of toxicity in such neurodegenerative diseases by competing with cell surface PrPcfor binding of toxicity-inducing entities, thereby sequestering them away from the cells.

[0150] PrPcis thought to play a central role as a mediator of cellular toxicity in Alzheimer’s disease (AD). Specifically, PrPchas been shown to act as the cell surface receptor necessary for small oligomeric assemblies of the Abeta peptide (oAP) that are endoproteolytically released from the larger amyloid precursor protein (APP) in AD45. An imbalance in Ap production and clearance in the brain is an early step in the development of the disease46-49that eventually gives rise to the deposition of large Ap amyloid plaques50. Its small size allows oAp to diffuse within the extracellular space, spreading the disease51. Once bound to neurons, oAp elicits a toxic cascade of events that is only partially understood52-56. Consistent with this scenario, reduced levels of PrPcprevent toxicity in AD models45’57-63.

[0151] Although the precise mechanism by which G127V confers protection is debated, there is agreement that the G127V mutant of PrPcacquires the same fold as native PrPc, barring minor structural differences surrounding the mutated residue64-67. This is important for immunological considerations, as so long as the synthetic coding sequence for the transgenic expression of PrPG127Vavoids immunogenic regions of DNA, such as cytosine- phosphate-guanine dinucleotides68, this therapeutic agent exhibits low immunogenicity that is largely restricted to the rAAV capsid itself.Example 3: Materials and MethodsAntibodies

[0152] Primary antibodies: Human monoclonal anti-prion F(ab’)2 antibody, clone D13, 1 :5000. Rabbit monoclonal IgG anti-prion (epitope: 221SQA223 and Y225) antibody, clone EP1802Y, 1 :10,000 (catalog number ab52604, Abeam, Cambridge, United Kingdom). Mouse monoclonal lgG2b anti-beta-actin antibody, clone BA3R, 1 :40,000 (catalog number MA5-15739- HRP, Thermo Fisher Scientific, Waltham, MA, USA). Secondary antibodies: Goat polyclonal IgG F(ab')2 anti-human HRP secondary antibody, 1 :5000 (catalog number 31414, Thermo Fisher Scientific). Goat polyclonal IgG antirabbit HRP secondary antibody, 1 :5000 (catalog number 31460, Thermo Fisher Scientific).Cloning

[0153] The self-complementary therapeutic BvPrnp vectors were built using Gibson assembly. Briefly, the synthetic BvPrnpV127sequence avoiding CpG motifs was purchased from a local gene synthesis service (Bio Basic Inc, Markham, ON, Canada). The backbone of the recombinant AAV transfer plasmid with its regulatory elements, designated as pscAAV-CBh-Null-WPRE3- enSV40pA, were a gift from Michael J Fox Foundation MJFF (catalog number 194245, http: / / n2t.net / addgene: 194245, Addgene, Watertown, MA, USA). The transfer plasmid was opened with Stul and Agel restriction enzymes. A parallel Gibson assembly, which removed the GPI signal sequence as needed and inserted a translation termination nonsense codon, was undertaken in parallel with a PCR reaction mix composed of Q5 High-Fidelity 2x Master Mix (catalog number M0492S, New England Biolabs), template DNA and the following primers:Forward: 5’- tcaggttggaccggctagcaccggtgccaccATGGCCAACCTcagctactg - 3’, (SEQ ID NO: 7)Reverse: 5’ - gtaatccagaggttgattaggTCATCTGCCCTCATAGTAGGCCTGGG - 3’. (SEQ ID NO: 8)

[0154] The reaction ran for 10 cycles with an annealing temperature of 55 °C, followed by 20 cycles at the elevated annealing temperature of 65 °C. Finally, the gel-purified open vector backbone and BvPrnpV127or BvPrnpv127AGPI sequences were assembled with the help of the HiFi DNA Assembly Master Mix (catalog number E2621 L, New England Biolabs) during a 1 hour incubation at 50 °C, then transformed into NEB Stable Competent E. coli (catalog number C3040H, New England Biolabs).Cell culture

[0155] HEK293T cells were maintained in DMEM (catalog number 119650-92, Thermo Fisher Scientific) supplemented with 50 U / mL Pen / Strep (catalog number 15140122, Thermo Fisher Scientific), NEAA (catalog number 11140-050, Thermo Fisher Scientific) and 10% FBS (catalog number 12483020, Thermo Fisher Scientific). Cultures were grown at 37°C, in an atmosphere of 5% CO2 and 95% relative humidity. The media were replaced every 48 to 72 hours and the cells were passaged at 90% confluency in using 0.25% Trypsin-EDTA (catalog number 15050065, Thermo Fisher Scientific). rAAV vector production rAAV vector purification methodsAAVX-based affinity capture-based purification of rAAV vectors:

[0156] The affinity capture of rAAV vectors made use of 50 pm POROS CaptureSelect AAVX resins (catalog number A36652, Thermo Fisher Scientific). The resin was washed with 20 column volumes (CVs) of Low Salt Wash Buffer (50mM Tris / HCI, pH 7.4, 150mM NaCI, 0.0051 % Pluronic™F-68 surfactant, 1 % Triton-X100). The affinity capture step was initiated by the slow (0.3 mL / min) loading of rAAV vectors harvested from HEK293 supernatants onto the AAVX resins. Once the rAAV vectors were loaded, the resin was washed with 20 CVs of Low Salt Wash Buffer, 20 CVs of High Salt Wash Buffer(300 mM NaCI, 50mM Tris, pH 7.4, 0.01 %, 0.01 % Pluronic™F-68 surfactant), and 20 CVs of Low Salt Wash Buffer without T riton X100. lodixanol density gradient centrifugation-based purification of rAAV vectors:

[0157] The iodixanol purification was based on a widely used protocol18. Briefly, 29.9 mL OptiSeal tubes (catalog number 361625, Beckman Coulter, Brea, CA, USA) were pre-loaded with step gradients of 15%, 25%, 40%, 60% (w / v) iodixanol (catalog number D1556, MilliporeSigma). Next, the HEK293 supernatant containing virus particles was precipitated with polyethylenglycol (PEG) as detailed by the authors of the protocol, then loaded directly on top of the 15% gradient layer with an 18-gauge needle. The carefully balanced ultracentrifugation tubes were then spun in a Type 50.2 Ti rotor (Beckman Coulter) at 47,500 rpm for 2 h 57 min at 18°C. During the density gradient centrifugation, the rAAV vectors were concentrated at the 40% to 60% iodixanol transition zone. They were collected by puncturing the tube with a 16-gauge needle. The rAAV vectors recovered were loaded into a 100 kDa Amicon centrifugation filter (catalog number UFC910024, Thermo Fisher Scientific) that had been pre-coated with PBS containing 0.001 % (vol / vol) Pluronic F-68 (catalog number 24040032, Thermo Fisher Scientific). Four consecutive buffer exchanges were performed during 5 min centrifugations at 3,000 g and room temperature.Animals

[0158] All animal procedures were based on guidelines by the Canadian Council on Animal Care and were authorized by the University Health Network (UHN) Animal Care Committee (Animal Use Protocol 6840). C57BL / 6J mice were purchased from the Princess Margaret Cancer Centre at UHN. Homozygous BvPrnp 1109 ki mice (Background: C57BL / 6J) were provided to us by the Joel Watts group77. A maximum of five mice per cage were kept at artificial 12-hour day and night cycles, drinking water ad libitum, and given 18% protein chow as solid food source. The mice received daily checks to assess their health and appearance. Their cages were changed weekly. Prion-inoculated mice were closely monitored for signs of distress or pain and humane endpoints were predefined (>20% weight loss, abnormal posture, lethargy, and impaired ambulation). Mice meeting these criteria were euthanized.Intracerebral prion inoculations

[0159] Intracerebral prion inoculations were performed on mice that were 4-6 weeks old. The inoculant was generated by infecting a C57BL / 6J mouse with RML-prions, then sacrificing the animal when terminally ill with prion diseases and homogenizing the brain in PBS to a final concentration of 3% brain extract in PBS (v / v). To immobilize the mice, which were to be inoculated, anesthesia was induced with inhaled 5% isoflurane and maintained at 2%. The anesthetic depth was assessed by performing a toe pinch. Then, a free-hand injection of 20 pL of inoculant into the right parietal lobe at a depth of 3 mm was performed using a 29-gauge Safetyglide insulin syringe (catalog number 305930, Becton Dickinson Canada, Mississauga, ON, Canada).Retro-orbital injections

[0160] Ahead of this procedure, the mice were anesthetized as described for the intracerebral prion inoculations. Additionally, all mice received for this procedure one eye drop of 0.5% proparacaine hydrochloride ophthalmic solution as local anesthetic before injection. The injections were performed using a 28-gauge insulin syringe (catalog #329420, Becton Dickinson Canada) into the right orbital sinus. All animals received a single retro-orbital injection of 1 xio12viral genomes (vg), diluted in PBS to a final volume of 100 pL. A six- week old female C57BL / 6J mouse was injected with 9P31-spEGFP to test the CNS spread (Fig 1) after retroorbital injection.Mouse tissue collection

[0161] Mice were deeply anesthetized using 5% isoflurane and euthanized by six-minute transcardiac perfusion with PBS. Next, the brains were carefully extracted, their meninges removed, and the two hemispheressplit sagittally. The right hemisphere was designated for biochemical analyses and kept at -80°C until homogenization. The left hemisphere was post-fixed in 10 mL of neutral-buffered formalin (catalog number #HT501128-4L, Sigma- Aldrich) for subsequent pathological analyses.Cryo-sectioning of mouse brains for direct fluorescence detection

[0162] Three weeks post-injection, mice were deeply anesthetized using 5% isoflurane and euthanized by six-minute transcardiac perfusion with PBS. Next, brains were promptly extracted, the meninges removed, and tissues postfixed in 10 mL of neutral-buffered formalin (catalog number #HT501128-4L, Sigma-Aldrich) for two hours at 4°C. After fixation, brains were cryoprotected by immersion in 30% sucrose in PBS at 4°C for up to 36 hours causing the brain tissue to sink. Brains were then incubated in a 1 :1 mixture of 30% sucrose and Tissue-Tek O.C.T. Compound (catalog 25608-930, VWR, Radnor, PA, USA). Next, brains were positioned in a cryomold (catalog number 70182, Electron Microscopy Sciences, Hatfield, PA, USA) and embedded in O.C.T for another one hour at 4°C degrees. Finally, the brains were frozen by partially immersing the cryomolds in a liquid nitrogen-chilled 2-methylbutane bath for 2-3 mins until the O.C.T completely froze. The frozen blocks were briefly air-dried on dry ice to remove residual 2-methylbutane and stored at -80°C.

[0163] Before cryo-sectioning, brains were allowed to warm up in the Cryostat chamber (model HM525 NX, Thermo Fisher Scientific) to a temperature of -21 °C during a two-hour acclimatization period. Finally, 16 pm sagittal sections were cut and collected on a piece of adhesive Cryofilm (catalog number C-FUF303, Section-Lab Co. Ltd., Yokohama, Kanagawa Prefecture, Japan) using Kawamoto’s film method78. Sections were then washed for five minutes in PBS and mounted with mounting media containing DAPI (catalog number ab104139, Abeam) before imaging.Microscopy

[0164] Brain samples were imaged under #1 .5 glass coverslips (catalog number 48393-060, VWR) on Fisherbrand Superfrost Plus microscope slides(catalog number 22-037-246, Thermo Fisher Scientific) using a Zeiss AXIO Observer 7 inverted LED fluorescence microscope (Carl Zeiss Canada Ltd., North York, ON, Canada). To reconstruct full sagittal brain views, individual fields of view were acquired sequentially across each section and digitally stitched into a composite image using ZEN Blue microscopy software (Carl Zeiss Canada Ltd.).Homogenization and protein extraction

[0165] Mouse brain tissue was homogenized in 100 mM Tris-HCI (pH 8.3) using a Minilys homogenizer (catalog number P000673-MLYS0-A, Bertin Technologies, Rockville, MD, USA) and Zirconia beads (cat. No. 11079110zx, Biospec, Bartlesville, OK, USA) with three sets of 30 seconds bead-beating pulses. The resulting 20% homogenates were aliquoted, and a protease inhibitor cocktail (catalog number 78425, Thermo Fisher Scientific) was added to samples not designated for Proteinase K digest. A solution of 0.5% deoxycholic acid (DOC) (catalog number DCA333.50, BioShop, Burlington, ON, Canada) and 0.5% NP-40 (catalog number NON505.100, BioShop) was added to the homogenate to extract proteins, followed by gentle vortexing and 30 minute incubation on ice. Next, insoluble debris were removed during a five- minute spin at 500 g, followed by a ten-minute spin at 5,000 g. Protein concentrations of the samples were determined using the Pierce BCA Protein Assay Kit (catalog number 23225, Thermo Fisher Scientific) and subsequently adjusted with lysis buffer to 4 pg / pL.PNGase F digest

[0166] To remove N-linked glycans by digestion with PNGase F (catalog number P0704S, New England Biolabs, Ipswich. MA, USA), we made use of supplier provided buffers. Specifically, equal amounts of proteins were denatured using 10* Denaturing Buffer at 95 °C for 10 minutes. Next, NP-40, GlycoBuffer, and PNGase F enzyme were added to the denatured samples. To inhibit undesired proteolytic activity, 3 mM phenylmethanesulfonyl fluoride (PMSF) (catalog number PMS123.5, BioShop) was added to the reaction tubes.Next, samples were incubated for 2 h at 37°C with gentle shaking. Finally, an equal volume of 2x LDS sample buffer containing 5% P-mercaptoethanol (BME) was added directly to the samples to stop the reaction and prepare the samples for western blot analysis.Proteinase K digest

[0167] To assess the relative PrPSccontent of brain extracts, 200 pg each of BCA-adjusted total protein were treated with Proteinase K (catalog number 25530049, Thermo Fisher Scientific) at a final concentration of 50 pg / mL and a ratio of total protein to Proteinase K of 1 :20. Next, samples were incubated for 45 min at 37°C under gentle shaking. The digestion was terminated by adding PMSF to a final concentration of 2 mM, followed by the addition of 2% Sarkosyl (catalog number SLS002.100, BioShop). Next, the digests were ultracentrifuged for 1 hour at 48,000 rpm and 4°C using an Optima TLX Ultracentrifuge (Beckman Coulter, Brea, CA, USA). Finally, the pelleted insoluble PrPScwas resuspended in 1 x Bolt LDS sample buffer (catalog number B0007, Thermo Fisher Scientific), heat-denatured at 95°C for 10 minutes, and analyzed by western blotting.Western blotting

[0168] To detect total PrP within brain extract samples, proteins were denatured in 1x Bolt LDS sample buffer at a final concentration of 2 pg / pL. This was followed by heating the samples at 95 °C for 10 min, followed by briefly cooling on ice, immediately before gel loading. Proteins were separated by SDS-PAGE on 10% Bolt Bis-Tris gels (catalog number NW00105BCX, Thermo Fisher Scientific). For PNGase F digested samples, 12% NuPage Bis-T ris gels (NP0342BOX, Thermo Fisher Scientific) were used. After the gel electrophoresis, proteins were transferred to 0.45 pm PVDF membranes (catalog number IPVH00010, Sigma-Aldrich, St. Louis, MO, USA). Following the blocking of the membrane in 5% skimmed milk (catalog number SKI400, BioShop Canada Inc) for 1 h at room temperature, membranes were incubated in primary antibodies overnight at 4 °C with gentle rocking. Next, membraneswere washed three times in 1x Tris-buffered saline containing 0.1 % Tween-20 (TBST) (catalog number TWN508, BioShop) and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. After washing the membranes thrice again in 1x TBST, they were incubated with Western lightning pro enhanced chemiluminescent (ECL) reagent (catalog number NEL120001 EA, Rewity Health Sciences Inc., Mississauga, ON, Canada) for 1 min. Finally, membranes were exposed to autoradiography film (catalog number CLMS810, MedStore, Toronto, ON, Canada) and developed using a film developer.Sample preparation for mass spectrometry

[0169] The protein concentration of brain extracts was adjusted to 4 pg / uL. To denature all proteins in the sample, including the PrPSc, 20 pg (in 5 uL) of total proteins were transferred to Protein LoBind (PLB, catalog number: PRE050LR-N, Diamed, Mississauga, ON, Canada) tubes and diluted with 9M urea (catalog number UR001.1 , Bioshop) in a 1 :2 ratio (v / v) to achieve a 6 M urea concentration in the sample, followed by gentle vortexing and 30-minute incubation at room temperature to ensure complete denaturation. The pH of the samples was checked and, if below pH 8, brought up to this pH using 1 M Triethylammonium Bicarbonate (TEAB) (catalog number 1861436, Thermo Fisher Scientific). Next the denatured proteins were reduced with 6.5 mM TCEP (catalog number TCE101 , Bioshop) during a 30-minute incubation at60°C, then alkylated for another 30 minutes in the presence of 15 mM iodoacetamide (catalog number 1861445, Thermo Fisher Scientific) in the dark at room temperature.

[0170] Next, the proteins were subjected to solvent precipitation to maximize recovery for proteomic analyses79. To this end, samples were threefold diluted in preparation of precipitation. In parallel, silica beads of 9-13 pm mean particle diameter (catalog number 440345) which had been precleaned and resuspended in ACN at a concentration of 70 pg / pL, were added at a 10:1 bead-to-protein ratio and the tubes were gently vortexed.Subsequently, 100% acetonitrile (ACN) was added to each sample at a 1 :5 (v / v) ratio to achieve an 80% ACN concentration without pipette mixing, followed by gentle vortexing for 10 seconds. Next, the precipitates were centrifuged for 5 minutes at 16,000g and room temperature, before supernatants were carefully removed, using the tube hinge as a guide to avoid disturbing the pellet. To further remove non-protein contaminants, the pellets were washed three times with 80% ethanol, using at least twice the total precipitation volume for each wash, followed by 2-minute centrifugation at 16,000g and room temperature.

[0171] After the final wash, the remaining supernatant was carefully removed, and the protein pellet resuspended by gently vortexing in 100 mM ABC with a 1 :50 trypsin-to-protein ratio. To fully disrupt the pellet, the tubes were placed in a sonication bath for 2 minutes. To ensure complete digestion, the samples were then incubated at 37°C for 18 hours at 800 rpm in a thermomixer (Thermomixer Comfort). Following digestion, 1 pL of 10% formic acid (FA) (catalog number A117-50, Thermo Fisher Scientific) was added to each sample to adjust the pH to approximately pH 3. The beads and any insoluble debris were precipitated by 10-minute centrifugation at 16,000 g and room temperature, before transferring the peptide-enriched supernatants to a new tube. To further recover additional peptides from the silica beads, the centrifugation pellets were washed once with 2% ACN in 0.1 % formic acid, and the resultant wash supernatants combined with the initial peptide-containing supernatants. The final peptide concentration was adjusted to 0.25 to 0.5 pg / pL in 1 % acetonitrile with 0.1 % formic acid.Mass spectrometry data acquisition

[0172] Samples injected were three biological replicates from three treatment cohorts, with each sample composed of tryptic digests of 200 ng of total brain extract proteins. All data were acquired using a Vanquish Neo UHPLC system (Thermo Fisher Scientific) coupled to an Orbitrap Astral mass spectrometer (Thermo Fisher Scientific) through an EASY-Spray source (Thermo Fisher Scientific). During peptide separation, a 25 cm EASY-Spray™HPLC Column (ES900) (Thermo Fisher Scientific) was maintained at 50°C. The mobile phase A consisted of 0.1 % formic acid in water, while mobile phase B consisted of 0.1 % formic acid and 80% acetonitrile in water. The gradient was as follows: the mobile phase was initially held at 2% B, increased from 2% to 4% in 0.5 min at a flow rate of 0.7 pL / min, then further increased to 5% B from 0.5 to 0.6 min, increased to 7% B from 0.6 to 1 min, ramped to 22.5% B from 1 to 18 min, then increased to 35% B from 18 to 25.5 min, and further increased to 50% B from 25.5 to 27 min. The flow rate was set to 0.5 pL / min. The column was then washed with 99% B from 27 to 30 min at a flow rate of 0.7 pL / min. Data were acquired in data independent acquisition (DIA) mode with a normalized collision energy of 25% and a default charge state of +2. MS1 spectra were acquired in the embedded orbitrap mass analyzer at a resolving power of 240,000 every 0.6 s. MS2 spectra were acquired in the embedded astral analyzer, with precursor isolation windows of 2 Th spanning the range of 380-980 Th. The custom normalized AGC target was set as 500% for both MS1 and DIA. The MS1 mass range was the same as the MS / MS precursor range.Processing of global proteomic data set

[0173] Protein identification and peptide peak integration were performed using Proteome Discoverer (PD) software (Version 3.2, Thermo Fisher Scientific) with CHIMERYS, using the Mus musculus database (Taxi D= 10090, release=407, supplemented with the entry for the major prion protein from bank vole (Accession number Q8VHV5). The database search was restricted to tryptic peptides, allowing up to two missed cleavages per peptide. The maximum fragment mass tolerance was set to 10 ppm. Carbamidomethylation at cysteine residues was specified as a fixed modification, while oxidation at methionine residues was included as a variable modification. The sample-to-sample normalization in PD was based on the ‘Total Peptide Amount’ computed for each sample. Prion peptide comparisons were conducted using Skyline (Version 24.1)80, with tubulin alpha and tubulin beta used for sample normalization. The assignment of peptide transitions toBvPrP were manually reviewed to ensure accurate peak integration. Both the Proteome Discoverer and Skyline data were exported to Excel for further conditional formatting.Cluster and KEGG analyses

[0174] Hierarchical clustering was undertaken with Cluster (Version 3.0, using C Clustering Library Version 1.59)81 82. Prior to clustering, data columns were generated that inform about steady-state protein level ratios, computed by dividing cumulative MS1 ion intensities assigned to a given protein in a specific brain extract to the average MS1 ion intensities for the same protein in brain extracts from naive mice. Subsequently, these steady-state protein level ratio data for all proteins, i.e. , the rows within the Excel dataset, were clustered using centroid linkage clustering methods based on a ‘City-block distance’ metric and the relationship across the brain samples, i.e., the columns within the Excel dataset, were inferred by hierarchical clustering using a Spearman Rank Correlation metric. Finally, hierarchical cluster analysis results were visualized in Java TreeView (Version 1.21 ) open source software23.

[0175] KEGG pathway analyses were undertaken with the DAVID Bioinformatics suite (release DAVID 2021 , Version 2023q4) of functional annotation tools made available by the National Institutes of Health83’84. Briefly, to initiate the analyses, lists of official gene symbols of genes whose expression gave rise to top- or bottom-ranked proteins in the global proteomic dataset were uploaded into the DAVID Analysis Wizard along with the selection of Mus musculus as the biological sample source. Next, a KEGG_PATHWAY analysis was undertaken from within the Annotation Summary Results page. KEGG_PATHWAYS, whose charts were returned with the lowest p-values and as such emerged top-ranked were shortlisted and their charts downloaded to capture the specific proteins identified based on the ‘red asterisk’ markings assigned to them.Assignment of proteins increased in their steady-state levels in prion disease to mouse brain cell types

[0176] The cell type assignment made use of previously reported mouse brain cell type annotations, which were originally obtained through single-cell transcriptomic analyses85. To interrogate these data with genes coding for proteins whose steady-state levels were most increased in RML-infected mice, which had reached the humane prion disease end-stage, the shortlist of genes were submitted to the online Transcriptom ics Explorer algorithm (https: / / portal.brain-map.org / atlases-and-data / rnaseq). Separately, the ggplot package within RStudio was used to depict the non-neuronal portion of the brain cell type dendogram, which this analysis revealed to comprise the genes whose levels were most profoundly increased in RML prion disease. qPCR

[0177] Quantitative PCRs were undertaken to determine virus titers using a well-developed protocol76. Briefly, serial dilutions of DNA standards were generated using the respective transgene plasmids which were linearized with Seal (catalog number R3122S, New England Biolabs). Viral preparations were digested with 50 U / mL of DNase I (catalog number EN0521 , Thermo Fisher Scientific) in 2 mM CaCI2, 10 mM Tris-HCI, 10 mM MgCI2. Next, viral capsids were digested using Proteinase K (catalog number EO0491 , Thermo Fisher Scientific) in 1 M NaCI, 34 mM N-lauroylsarcosine. The final qPCR measurement was conducted using a LightCylcler 480 II (catalog number 05015278001 , Roche Diagnostics, Indianapolis, IN, USA) with the help of the SYBR green master mix (catalog number 4367659, Thermo Fisher Scientific) as well as the following primers which target the CBh promoter: Forward: GTTACTCCCACAGGTGAGC (SEQ ID NO: 9) and reverse: CCAACCAACCATCCCTTAAAC (SEQ ID NO: 10). gDNA extraction and restriction digest

[0178] The gDNAs of study mice were extracted with the Monarch Spin gDNA Extraction Kit (catalog number T301 OS, New England Biolabs). For the subsequent PCR reaction, 50 ng of gDNA was amplified with Q5 Hot Start High-Fidelity 2X Master Mix (catalog number M0494S, New England Biolabs) and forward and reverse primers (final concentration 500 nM) that map to gDNA sequences shared by the endogenous and heterologous BvPrnp ORFs. Equal volumes of PCR product were then subject to a restriction enzyme (RE) digest with 1 pL of Eagl (catalog number R3505S, New England Biolabs), Csil (catalog number FD2114, Thermo Fisher Scientific), or both REs for cutting the endogenous or the heterologous amplicons or both, respectively. The RE digestion products were finally separated on a 1 % agarose gel containing SYBR safe DNA gel stain (catalog number S33102, Thermo Fisher Scientific). The fluorescent image of the gel was captured with the ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA). Forward: AAGAAGCGGCCAAAG (SEQ ID NO: 12) and reverse: TAGTAGGCCTGGGACTC (SEQ ID NO: 13).Statistical analyses

[0179] Western blot signal intensities from three biological replicates were quantified by densitometric analysis using Imaged software and corresponding statistics were performed using Microsoft Excel. First, cohorts were subject to an F-test to assess the variance of the two compared groups. A Welch’s t-test or pooled t-test was subsequently chosen to assess the statistical significance of the differences between the averages of cohorts that were compared. Results were considered significant if p < 0.05 or denoted as non-significant with “ns” if p >0.05. Asterisks were used to signify varying levels of significance: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

[0180] Survival, weight, and nesting score charts were assembled in R using RStudio (Version 4.4.3). For all analyses, Excel raw data were uploaded into RStudio using the ‘readxl’ package. For the Kaplan-Meier analyses, the Greenwood formula computed a variance estimate, which was used by the Kaplan-Meier estimator (kmfit) to compute the 95% confidence interval. Plots were then generated with basic plotting functions embedded in ‘ggplot2’ and ‘ggfortify’ packages.

[0181] For generating the weight and nesting score charts, the geom_smooth() function within ggplot2 was used to depict the linear trend and the confidence interval using the Locally Estimated Scatterplot Smoothing ‘loess’ method.

[0182] A hypergeometric distribution was assumed when determining if it is statistically significant that 27 out of 30 cell surface proteins, which had been shown to reside in proximity to PrP in mouse brains28, were downregulated > 33% (out of 455 other proteins sharing this characteristic) in a global proteome dataset of 4874 proteins.Example 4: Design of watermarked all-in-one rAAV vectors to achieve low immunogenicity and enhanced cross-correction

[0183] Although the precise mechanism by which V127 confers protection is still being debated, there is broad agreement that the V127 mutant of PrPcacquires the same fold as native PrPc, barring minor structural differences surrounding the mutated residue64'67(Fig 1A). This insight was important for immunological considerations, as it predicted that so long as the design of the synthetic coding sequence for the transgenic expression of PrPV127avoids highly immunogenic CpG islets68, this therapeutic agent can be expected to exhibit low immunogenicity that is largely restricted to the rAAV capsid and its DNA payload. The immunogenicity of the latter is increasingly well understood, suggesting that upon first exposure, the rAAV capsids will produce a moderate immune response that would only pose a hindrance if repeat administrations were planned, which was not the case. The immunogenicity of DNA is largely governed by CpG islet. Consequently, redundancies in the genetic code were capitalized on to eliminate occurrences of CpG (Fig 1B).

[0184] Rather than working with wild-type mice, the recently introduced bank vole knock-in (BvPrnp ki) model in which the endogenous mouse Prnp ORF was replaced with the respective BvPrnp ORF was selected for this study. Two considerations guided this choice: 1) BvPrnp-expressing mice had been shown to develop disease faster than wild-type mice87. 2) In anticipation offuture inoculation work with human prions, bank vole PrP’s universal prion acceptor characteristics were capitalized88’89.

[0185] To maximize the expression of the heterologous BvPrnpV127, the payload was designed as a self-complementary DNA, thereby avoiding the replication step that single-stranded AAV vectors must undertake upon transduction90, a biology that is limited by host cell factors (Fig 1C). The expression cassette of the construct was flanked by inverted terminal repeats (ITRs) derived from the AAV2 serotype, with one of the ITRs carrying a small deletion within the terminal repeat sequence (TRS) motif to facilitate selfcomplementarity91. The expression of the prion gene was driven by the chimeric chicken P-actin promoter with intron (CBh) that had been shown to promote the steady expression of transgenes in mouse brains for extended periods92.

[0186] While the study was ongoing several innovations emerged in the rAAV vector field, including alternative ways to purify rAAV vectors as well as capsids with optimized blood brain barrier (BBB) penetrance. Comparisons of two of these advances were incorporated into the study design: 1 ) an affinity capture-based method for rAAV vector purification using AAVX matrices (Fig 1 D)93, and 2) a capsid, designated 9P31 (Voyager Therapeutics Inc.), which was reported to feature several-fold higher CNS penetrance than PHP.eB, often the choice capsid for targeting the CNS in mice following intravenous administration94. Purity assessments of rAAV vectors that we prepared by AAVX affinity chromatography showed predominantly the expected three bands derived from the three capsid isoforms VP1 , VP2, and VP3 in the expected ratio of 1 :1 :10 after SDS-PAGE separation (Fig 1 E). To test the 9P31 vector, a self-complementary rAAV vector identical to the one described for the expression of BvPrPV127was assembled, except that the BvPrnpV127ORF was replaced with a coding sequence for a variant of the enhanced green fluorescent protein, which was directed to the endoplasmic reticulum (ER) by the addition of a signal peptide (spEGFP, with the signal peptide-encoding sequence borrowed from the calreticulin gene) (Fig 1F)95.Example 5 - Retro-orbital injection of BvPrnpG727' / AGPI into prion- inoculated mice caused similar survival extension regardless of rAAV vector preparation method or capsid choice

[0187] Prion inoculations were performed as staggered inoculations. The in vivo studies began by comparing side-by-side mice that had been prion- inoculated on the same day, then were either left untransduced or were retro- orbitally transduced 60 days later with 9P31 -spEGFP (negative controls) or 9P31-BvP / 7?pGy27VAGPI vectors (Fig 2A). The latter cohort consisted of three sub-cohorts. Two of these were transduced with 9P31 vectors that had been purified either by iodixanol density centrifugation or AAVX affinity chromatography (Fig 1 D). The third sub-cohort was transduced with rAAV vectors encapsulated in the PHP.eB capsid that had also been purified by AAVX affinity chromatography. Deferred were the analyses of mice transduced with 9P31 encapsulated vectors coding for anchored BvPrP1,727.

[0188] RML-prion inoculated BvPrnp ki mice began to show symptoms 130-150 dpi and had to be sacrificed shortly thereafter when they reached the humane prion disease endpoint. Whether the prion-inoculated mice were left untransduced or transduced with the negative control 9P31 -spEGFP vector made no significant difference to their survival. In contrast, the cohort of mice that had been made to express the therapeutic BvPrnpv127GP\ construct, survived approximately 50 days longer. One characteristic of their Kaplan- Meier curve is a broader spread of survival times from 150 to a maximum of 232 days, possibly reflecting variances associated with the administration of the therapeutic capsids (Fig 2B). Plotted results from monitoring body weights (Fig 2C) and nesting scores (Fig 2D) of the mice reflected both the survival extension as well as the more gradual decline in the BvPrnpG127V- GP\ cohort. Interestingly, when the latter results were deconvoluted based on how the 9P31-BvP / T?p' / 727AGPI capsids were prepared (Fig 2E) or whether the payload was encapsulated in 9P31 or PHP.eB, no significant differences emerged (Fig 2F)Example 6: Survival extension afforded by Bv Prnpv121AGPI was accompanied by reduced PrPScaccumulation but did not correlate linearly with steady-state expression levels of therapeutic payload

[0189] To begin to understand how the expression of B PrnpV127AGPI had delayed the disease, at 152 dpi, i.e. , when the prion-inoculated control mice reached the humane prion disease endpoint, a small number of BvPrnp ki mice, which had been treated with the 9P31 encapsulated vector coding for the protective anchorless V127 mutant were also sacrificed, along with age- matched control BvPrnp ki mice that had neither been prion-inoculated nor transduced. Sagittal half brains of these mice were formalin-fixed, and the remaining half brains were homogenized and extracted, then processed for western blot analyses with or without prior digestion with proteinase K. This analysis revealed the expected increase in total PrP levels in RML prion- inoculated mice (relative to uninoculated age-matched control mice) that had not been transduced with the therapeutic vector (Fig 3A, top). Whether or not these prion-inoculated mice had been transduced with 9P31 -spEGFP made no difference to this outcome, consistent with the interpretation that the mere transduction itself has little, if any, influence on disease progression. In contrast, RML prion-inoculated mice that had been transduced with the therapeutic 9P31 -BvP / T?p' / 727AGPI vector — although exhibiting an increase in total steadystate PrP levels relative to naive age-matched BvPrnp ki mice — could be distinguished by the lower intensity of their PK-resistant PrPScsignals from the non-treated prion-inoculated mice (Fig 3A, bottom).

[0190] Once the remaining cohort of the 9P31-BvPrnp' / 727AGPI-treated mice had reached the humane prion disease endpoint, additional western blot analyses were conducted, after processing their half brains as above. These experiments were guided by an interest in understanding why the sub-cohort, which had been treated with the PHP.eB-encapsulated payload coding for BvPrnp' / 727AGPI had shown a similar survival extension as the mice whose identical therapeutic construct had been encapsulated in the 9P31 capsid, a counterintuitive result based on the prior observation that 9P31 vectorsmediated an approximately seven-fold higher CNS transduction than PHP.eB vectors when payloads, virus preparation and administration steps were identical. It was of particular interest to determine the steady-state BvPrnp' / 727AGPI expression levels that had been reached in the treated cohort, for this western blot analysis the total brain extracts were first digested with PNGase F so that PrP isoforms differing solely in their N-linked glycans would be reduced to a single band (Fig 3B, top). Intriguingly, this analysis corroborated the relative CNS transduction potencies of 9P31 and PHP.eB by showing that the BvPrnp' / y27AGPI-derived western blot signals were considerably stronger when the payload had been encapsulated in 9P31 than the respective signal in brain extracts of mice that had been transduced with the corresponding PHP.eB vector. In fact, the western blot signal that was interpreted to represent BvPrnpv127GP\ by its absence in non-transduced RML-inoculated mice or in mice that had been transduced with 9P31 -spEGFP emerged as the strongest PrP antibody-reactive signal. The intensity of this band in 9P31-BvPrnp' / 727AGPI mice exceeded approximately threefold the respective signals for endogenous wild-type BvPrP. This result indicated that the transduction worked better than anticipated but it also indicated that the therapeutic potency of Bv Prnpv127AGPI, when measured based on the survival extension it conferred, did not correlate linearly to its expression level in BvPrnp ki mice.

[0191] Finally, it was sought to determine if the highly expressed BvPrP ' / 727AGPI contributed to the formation of Proteinase K resistant material in the treatment cohort after these mice had succumbed to prion disease. To reduce the complexity of signals expected if isoforms with varying N-glycan occupancy were present, representative brain extract from each of the four cohorts were first digested with Proteinase K, then removed N-glycans from the resolubilized PrPScmaterial by an additional digestion with PNGase F. The side-by-side analysis of the resultant PrP products next to undigested or only PNGase F digested samples revealed that mice, which expressed both the endogenous BvPrnp gene and the synthetic Bv Prnpv127AGPI expressionconstruct, still only gave rise to a single PrPScsignal which ran at the same level as the respective band in those mice, which were not transduced (Fig 3B, bottom). Previous work with prion-inoculated transgenic mice that expressed both wild-type PrP and anchorless PrPAGPI documented that PK-resistant unglycosylated bands derived from anchorless PrPAGPI ran considerably faster than the corresponding wild-type-derived bands and could be easily distinguished in a western blot analysis39. These results are consistent with the interpretation that in this work the BvPrP' / 727AGPI did not contribute in the treated mice to the formation of PK-resistant PrPSc, even though the mice expressed relatively high level of the protective variant and had eventually succumbed to prion disease.Example 7: Global proteome analyses reveal that Bv Prnpv121AGPI delays prion disease by slowing perturbations to the proteome observed in prion-inoculated mice.

[0192] Although a fair bit is known about how prion diseases affect specific proteins, systematic in-depth global proteome comparisons of age- matched naive and prion-inoculated end-state prion disease brains have not been reported. Moreover, the understanding of how V127 confers its protection against prion diseases is limited. Although it is apparent that the expression of V127 slows the accumulation of PrPSc, it is not known if its protective effect manifests in a mere slowing of prion-disease-associated perturbations or involves more specific protective changes to the proteome. To fill these knowledge gaps, the proteome of brain extracts from age-matched naive BvPrnp ki mice, and the corresponding prion-inoculated and mock 9P31- spEGFP- versus 9P31-BvPrnp' / 727AGPI- treated mice, i.e., the same samples we had subjected to western blot analysis (Fig 3A, top) were interrogated.

[0193] To prepare the samples for the mass spectrometry analyses, the total brain extracts were fully denatured in the presence of urea, then reduced and alkylated, and finally trypsinized. Using 30-minute gradients, tryptic mixtures were separated on a reversed phase column. The effluents from thisseparation were online coupled by nanospray ionization to an Orbitrap Astral mass spectrometer, which was operated in data-independent acquisition (DIA) mode (Fig 4A). A high degree of run-to-run proteomic sequence coverage afforded by the DIA acquisition mode obviated the need for isobaric labeling, thereby enabling MS2-based relative quantitation of consecutively analyzed samples. Taken together, this configuration allowed the deep unsupervised characterization of global proteomes, leading to the relative quantitation of 4,874 proteins in all cohorts, comprised of three biological replicates for each of the three cohorts (Fig 4B). More than 800 of these proteins were identified based on peptide-to-spectrum matches (PSMs) that accounted for more than 50% coverage of their protein sequence, with approximately half of the protein identifications supported by >100 PSMs, and more than 4,800 protein identifications based on >10 PSMs. Even at cursory inspection, it was apparent that the three biological replicates for each cohort were more like one another than samples from other cohorts, an impression supported by results from an unsupervised hierarchical cluster analysis (see below). When the relative abundances of individual proteins in all nine samples were computed by forming ratios of their combined ion intensities in each sample and their respective average ion intensities in the naive BvPrnp ki cohort, it became apparent that most proteins were not altered by more than 33% in their steadystate levels in the disease. Yet, looking at it from the opposite direction, 780 genes were >33% upregulated in their expression and 455 proteins were >33% down-regulated at end-stage prion disease, relative to age-matched naive BvPrnp ki mice. BvPrnp ' / 727AGPI-treated mice fell between these two extremes and, importantly, exhibited no proteomic drifts relative to naive mice that were not also observed in the RML-inoculated mice that had been sacrificed at the humane prion disease endpoint. However, the abundance changes were less pronounced for these treated mice than what was observed for the respective protein abundances in the untreated or 9P31-spEGFP-treated mice (Fig 4C, left). Taken together, these observations were consistent with the interpretation that the expression of BvPrnpv127AGPI does not prolong survival by having aspecific effect on a subproteome that compensates for the prion disease- induced perturbations to the proteome.Example 8: End-state prion disease was accompanied by profound loss of synapses, calcium homeostasis, and circadian entrainment, as well as upregulation of spliceosome genes, ribosome assembly, and ER protein processing.

[0194] Pathway analyses was undertaken to investigate more systematically the changes to the proteome that manifested in RML-inoculated BvPrnp ki mice. To this end, the KEGG pathway repository was interrogated with identifiers of proteins that were either >33% upregulated or downregulated in the disease (Fig 4C, right). Consistent with expectations, these analyses revealed a highly significant reduction in the steady-state levels of proteins associated with synaptic biology. Interestingly, within the various types of synapses, glutamatergic synapses were the most significantly impacted (p = 8.0 E-37), with the GABAergic (2.3 E-19), adrenergic (1.5 E-13), dopaminergic (5.9 E-13), and cholinergic synapses (1.2 E-12), showing lesser impacts but still returning highly significant p-values. Consistent with known perturbations of prion diseases to sleep-wake patterns, the KEGG pathway defining components of circadian entrainment was also one of the most downregulated (p = 2.6 E-18).

[0195] The analyses also revealed that brain cells in RML-inoculated BvPrnp ki mice impacted by PrPScaccumulation do not succumb without a fight, as several pathways can be seen to be >33% upregulated relative to age- matched naive BvPrnp ki mice. Chief among them are the KEGG pathway defining components that facilitate ‘Protein processing in the endoplasmic reticulum’ (p = 4.3 E-12). Along with ER proteins, spliceosome proteins were next in this category in the order of relative significance (p = 3.5 E-11).

[0196] Taken together, these analyses added compelling granularity to preceding reports, which had pointed toward synaptic decay and sleep perturbations in prion diseases. Perhaps less appreciated had been theconcerted efforts that brain cells undertake to fight their demise by turning on the expression of spliceosome and ER processing biology.Example 9: 9P31 -delivered BvPrnpG727' / -AGPI gave rise to similar gene levels as endogenous BvPrnp yet increased protein levels approximately threefold and amplified unglycosylated PrP >20-fold.

[0197] Also assessed was the extent to which the brain-wide delivery of the genetic payload of the gene therapy had occurred. To this end, nucleotide sequence differences in the endogenous BvPrnp ORF and the synthetic BvPrnpv127AGPI-ORF were capitalized on. Specifically, a stretch of the respective sequence ORFs was identified that could be amplified with the same PCR primers because it was flanked by identical sequences yet could only be digested by one or another restriction enzyme, namely Eagl for endogenous BvPrnp gene sequences and Csil for BvPrnpv127AGPI inserted into the genome following transduction (Fig 5A). By comparing the signal intensity of the fragments released by the respective restriction enzymes, this assay was designed to inform about the relative amount of genomic DNA (gDNA) coding for BvPrP that was contributed by the endogenous and heterologous synthetic ORF. When applied, this analysis revealed that the relative amounts of genomic endogenous BvPrnp ORFs were approximately matched to those of the heterologous synthetic BvPrnpv127AGPI ORFs when the mice had been transduced with the 9P31- BvPrnpv127AGPI vectors (Fig 5B).

[0198] The depth of the proteome dataset enabled additional questions to be answered by comparing the total levels of BvPrP observed in the respective brain extracts to the levels at which specific BvPrP-derived peptides were detected. Consistent with the western blot data and the genomic DNA data, the mass spectrometry-based quantitation indicated that the total steadystate BvPrP in mice which had been transduced with the 9P31- BvPrnpv127AGPI-ORF vector was approximately three-fold higher than the respective quantity in the naive BvPrnp ki mice (Fig 5C). The levels of tryptic peptides that were observed in the nine samples were investigated for 1 ) aBvPrP-derived peptide (ESQAYYEGR; SEQ ID NO: 14) present in both endogenous and heterologous PrP (Fig 5D), 2) a BvPrP-derived peptide (GENFTETDVK; SEQ ID NO: 15) harboring one of the two N-linked glycan acceptor sites (NxT) present in the protein (Fig 5E), as well as BvPrP peptides marked by the presence of G127 or V127 residues in them, which can be unequivocally assigned to endogenous BvPrP or the heterologous BvPrPv127AGPI, respectively. The quantitation of the mass spectrometry results across the nine samples revealed the generic BvPrP to be observed at lowest levels in the naive mice, at slightly increased levels in RML-inoculated mice, and to be present at approximately three-fold higher levels in the 9P31- BvPrnpv127AGPI-ORF-transduced mice (Fig 5F). The peptide harboring the N- glycan acceptor site was present at approximately 20-fold higher levels in the BvPrnpv127AGPI-ORF-transduced mice. When considering the three-fold higher total BvPrP levels in these mice, this result suggests that the anchorless protein is approximately six-fold less likely to carry the N-glycan at the respective acceptor site than the endogenous protein — note that the attachment of the N-glycan precluded detection of the respective peptide in this analysis (Fig 5G). The BvPrP peptide comprising the wild-type BvPrP G127 residue was observed at about twice the level in the RML-inoculated samples (Fig 5H), consistent with the stabilization and overall increase in steady-state PrP levels that are commonly observed in prion-infected mice. Finally, as anticipated, the V127-comprising peptide was not detected in naive or RML inoculated mice transduced with the control 9P31-spEGFP vector but was robustly detected in mice transduced with 9P31-BvPrnpv127AGPI (Fig 5I).

[0199] These results corroborated the increase in total BvPrP levels observed by western blot analyses (Fig 3). However, these data also indicated that this increase at the protein level was not reflected at the gDNA level, where endogenous and heterologous BvPrnp ORFs were approximately matched in 9P31-BvP / T?pv127AGPI transduced brains. This divergence of steady-state protein levels versus gene copy number may reflect differences in the relative strengths of the endogenous mouse Prnp promoter versus the CBh promoterused to drive the heterologous expression. Alternatively, these data may indicate differences in the half-lives of anchored versus secreted 9P31- BvPrnpv127AGPI. The results further validated the anticipation that the expression of anchorless BvPrnpv127AGPI is accompanied by a profound reduction in the N-glycan occupancy of the heterologous protein relative to endogenous anchored BvPrP.Example 10: Lower steady-state levels of cell surface proteins residing in proximity to PrPccannot be accounted for by the loss of neurons that occurs late in the disease

[0200] The molecular environment of PrPcin a mouse brain was previously reported using an in vivo crosslinking affinity capture paradigm and had published the top-ranked 40 proteins86. With the deep global proteome analyses at hand, this shortlist was revisited to determine if proteins in proximity to PrPcawait a specific fate late in the disease. All but one of the proteins in the original paper were identified in this global proteome analysis with at least 10 unique peptides and a range of 185 to 1325 PSMs, thereby generating highly robust relative quantitation data. Interestingly, at late-stage prion disease, none of these proteins were upregulated, many were approximately two-fold down regulated, and only a few were not affected in their steady-state levels. A common feature of the latter subgroup seems to be that they are understood to encounter the prion protein within its passage along the secretory pathway, whereas the downregulated subcohort of proteins are almost invariably residing next to PrP at the cell surface. Many of these proteins belong to the subset of proteins that were most strongly and consistently downregulated in the entire dataset (Fig 6A). In fact, if proteins which were reported to reside in proximity to PrP at the cell surface were captured in a KEGG pathway, its downregulation would emerge among all known KEGG pathways annotations as a highly significantly downregulated pathway (p = 2.37E-25), with only three proteins (contactin-1 , the amyloid precursor protein, and the myelin associated glycoprotein) not showing this trend.

[0201] It was considered if neurons dying in late-stage prion diseases might account for this selective downregulation of these proteins, since most of them are associated with neurons. To address this question, the relative quantities of proteins were extracted from the global proteome dataset, which are commonly associated with specific brain cell types. Specifically, this incomplete list was comprised of ten neuronal, seven astrocytic, four oligodendrocytic, and two microglial markers (Fig 6B). Within the ten neuronal markers, it was observed that steady-state level of NeuN and Tuj 1 , proteins that are frequently used to quantify homeostatic and mature neurons, did not change in late-stage-disease. Neither did other neuronal markers which identify neuronal stem cells (Nestin) or specific subtypes of neurons (choline-O- acetyltransferase for cholinergic neurons and tyrosine 3-monooxygenase for dopaminergic or noradrenergic neurons). Only neuronal markers associated with synapses, including Synaptophysin or Synapsin, were observed to be down regulated. These results indicate that the neuronal death that occurs late in the disease cannot account for the reduction in the steady-state levels of the previously reported interactors and candidate interactors of the prion protein.Example 11 : Discussion

[0202] This study evaluated the therapeutic potential of a virus-delivered gene therapy based on a secreted bank vole PrPv127AGPI expression product. The study documented an approximately 50-day survival extension in RML prion-inoculated mice using this approach. The subsequent analyses of brain samples indicated that this survival extension was obtained irrespective of whether the heterologous therapeutic protein was expressed at low or high levels, suggesting a ceiling of therapeutic potency in this paradigm. A deep proteomic analysis of brain samples, collected at the time when negative control mice succumbed to prion disease yet the treated mice were still free of overt symptoms, showed that the heterologous expression of BvPrnpv127AGPI mitigated proteomic perturbations observed in non-treated RML-inoculated mice and was not in itself causing notable changes to the proteome. Further investigation of the global proteome dataset strongly corroborated that priondisease was associated with astrocytosis and microgliosis, as well as a deterioration of synapses and proteins underpinning the sleep-wake cycle. These data also revealed that prion-inoculated mice invest in a rescue biology centered on cellular Ca2+influx and a replenishment of cell surface proteins, prominently manifest in increases in steady state levels of an endoplasmic reticulum protein processing subproteome and components of the spliceosome.

[0203] This report is the first to test the protective effect of the V127 mutation in vivo using a paradigm that introduced the therapeutic protein through viral delivery. Key differences of the experimental design to the natural protective V127 heterozygosity that evolved in the Kuru endemic region in Papua New Guinea are: 1 ) The heterologous germ-line expression of this protective mutant in all cells in the few dozen humans who were reported to carry this mutation naturally versus the heterologous expression following the transduction of a subset of brain cells weeks after the brain had been exposed to the prion agent. 2) The human sequence context of the naturally evolved V127 mutation versus the decision to work with the BvPrP sequence. It was previously shown that the V127 mutations retains at least some of its protective capacity when it is embedded in the BvPrP sequence by showing that mouse CAD5 cells, in which the expression of the endogenous mouse Prnp gene was functionally depleted and instead expressed BvPrnpV127as a transgene became resistant to prion infection25. Due to biosafety restrictions, it was not possible to study mice expressing human PRNP as a knock-in ORF, inoculated with human prion inocula, and treated with a virus-delivered gene therapy based on the expression of human PrPv127-AGPL 3) The expression of the naturally evolved protective PrPV127mutant as a GPI-anchored protein versus the choice to secrete BvPrPv127AGPI to achieve enhanced cross-correction (ECC) (Fig 7). Some level of cross-correction (CC) may occur naturally, because a subset of PrPcand its PrPv12Vmutant are shed by a-disintegrin-and- metalloproteinase 10 (ADAM 10) from the cell surface through endoproteolytic cleavage at a site adjacent to the G Pl-attachment site45, thereby giving rise toendoproteolytic products referred to as shed PrPc(sPrPcand sPrPV127)37. However, the natural proportion of sPrPcto total PrPcis low, estimated to be 7-10% in rodents38. Therefore, the natural ADAMIO-mediated release of PrPcwould provide limited protection to the brain if only a small subset of cells can be made to express the GPI-anchored PrPv127mutant.

[0204] PrPcis also thought to play a central role as a mediator of cellular toxicity in Alzheimer’s disease (AD), where it has been shown to act as the cell surface receptor necessary for small oligomeric assemblies of the Abeta peptide (oAP) that are endoproteolytically released from the larger amyloid precursor protein (APP) in AD45. Consistent with this scenario, reduced levels of cell surface PrPchave been reported to prevent toxicity in AD models45 57'63. The small structural differences reported for PrPV127cannot be expected to interfere with its ability to serve as a cell surface receptor for oAP, since the necessary binding site map to the N-terminus of PrP that are shared between wild-type PrP and PrPV127. Consequently, the secretion of PrPv127AGPI could be expected to ameliorate the risk of toxicity in such neurodegenerative diseases by competing with cell surface PrPcfor binding of toxicity-inducing entities, thereby sequestering them away from the cells.

[0205] Since prion diseases require templated polymerization for their propagation, any mismatch in the prion sequence has the potential to hinder disease progression. The idea to harness the power of sequence variants for therapy is not new. In the prion field, this idea has had traction ever since sequence variants that manifest in animal and human populations as polymorphisms or mutations were shown to confer partial or full protection against prion diseases. Initial work in this area was mostly based on the transient or stable transfection of protective PrP mutants in vitro based on the ScN2a cell model and the measurement of PK resistance as a surrogate for disease burden17’18. To date, in vivo work in this area has remained limited. For instance, it was shown that transgenic mice expressing the protective sequence variants Q167R or Q218K at the same level as wt PrP could slow prion disease in mice after prion inoculation but did not prevent it completelyunless the protective transgene was exclusively expressed19. A follow-on paper documented that the Q218K variant could reduce the burden of PK- resistant PrPScin ScN2a cells when the protein was added to the cell culture medium in recombinant form, i.e., lacking N-glycans and GPI anchor20. The subsequent infusion of the same recombinant protein into mouse brains through an intracerebroventricular catheter was reported to have prolonged the prion disease incubation period from 1 17 days to 131 days21. A separate report based on the same therapeutic concept but a different means of delivery documented that the lentiviral transduction of a gene therapy coding for the protective Q167R variant achieved a 30-day survival extension in prion- inoculated mice22. Several experimental differences to the work reported here stand in the way of interpreting differences in outcomes, including that the protective Q167R mutant was expressed in the context of a mouse sequence that coded for the attachment of a GPI anchor, the use of the Me7 prion inoculum, the administration of the treatment through an intracerebral cannula implant, and the choice to administer repeat treatments at 80 and 95 dpi.Example 12: Summary and Conclusions

[0206] Since prion diseases require templated polymerization for their propagation, any mismatch in the prion sequence has the potential to hinder disease progression. This hindrance manifests as species barriers when the species from which the infectious prion inoculum was sourced differs from the prion sequence of the inoculated host10Since there are natural polymorphisms in the prion gene in most mammals, this hindrance can also be observed if the disease is transmitted within a species and the sequence of the prion inoculum differs from the host prion sequence. In fact, for a transmission barrier to manifest, it can be sufficient for the host to be heterozygous in such a manner that one prion allele codes for a prion protein that is a perfect sequence match to the prion inoculum and the other differs at a single amino acid11’13. The most common polymorphisms in humans are the presence of a methionine or valine amino acid at residue 12914, which occurs in around 30% of individuals worldwide, and a glutamic acid or lysine amino acid at residue 219 that isrelatively prevalent in East Asia15. Finally, although several mutations in prion genes are the cause of inherited prion diseases, others are understood to confer protection based on the sequence mismatch they contribute. In light of this reality, one therapeutic approach for the treatment of prion diseases that has enjoyed lasting traction is the idea to prevent disease by introducing a protective sequence variant into the brain.

[0207] This strategy has been shown to be effective in proof-of-concept studies undertaken with mouse neuroblastoma cells16'18and mice19that were made to express protective Prnp variants through transfection or transgene technology, respectively. A first, more realistic attempt at evaluating the merits of this therapeutic strategy made use of recombinantly expressed dominant negative mouse prion protein (rPrP-Q218K; 218 in mice represents the orthologous residue to human 219). Specifically, the purified recombinant protein was introduced into the medium of cells grown in culture, thereby blocking the conversion to the protease resistant PrPSc 20. The subsequent infusion of the same recombinant protein into mouse brains through an intracerebroventricular catheter was reported to have prolonged the prion disease incubation period from 117 days to 131 days21. A separate report based on the same therapeutic concept but a different means of delivery documented that the lentiviral expression of PrPQ167R— another human PrP sequence variant that had been shown to interfere with the conversion of PrPcto PrPSc— led to a 30 day survival extension in prion-inoculated mice22.

[0208] The substitution of a glycine with a valine in position 127 (G127V) within the prion protein (PrP) constitutes a remarkable case of recent human evolution that has been credited with the survival of dozens of individuals among the Fore people of Papua New Guinea who had been exposed to kuru disease23. Subsequent work in transgenic mice established that this G127V mutation conferred prion disease protection even when present at sub- stoichiometric levels24.

[0209] Since its discovery, several ideas have been proposed for how the protective properties of this mutant may be harnessed for the development of a treatment for prion diseases. One approach is to borrow the therapeutic strategies used before, namely, to introduce the gene exogenously as a recombinant protein or drive its heterologous expression of G127V following its delivery to the brain through some vehicle, such as rAAVs or lipid nanoparticles (LNPs).

[0210] Another therapeutic avenue is to gene-edit the G127V mutation into the endogenous prion gene (PRNP) using CRISPR technology25. Specific implementations could be based on homology-directed repair (HDR)26, base editing (BE)2728, or prime editing (PE)29,

[0211] All the above approaches suffer from a major flaw, namely the reality that technologies, which can deliver recombinant proteins or genes to most human brain cells, are not available at this time30. Even rAAV vectors with potent brain tropism and cross-species activity achieve no more than the transduction of 20-30% of neurons (S M 1311 -positive) and 60-77% of astrocytes (Sox9-positive), respectively, in mice and non-human primates31, which realistically can be expected to translate into still considerably lower transduction levels in human brains.

[0212] In prior transgenic mouse work that validated the protective capacity of PrPG127V, the expression of the mutant was driven by the germ-line integration of the transgene, thereby causing the co-expression of endogenous wild-type PrP and PrPG127Vat consistent ratios in all cells24. In contrast, upon delivery of gene therapies into the brain using existing vehicles, including rAAV vectors, only a subset of cells will be positive for the heterologous PrPG127Vtransgene and others will express none. This is a major shortcoming because it can be expected to translate into prion disease continuing to spread to and manifest in most cells unhindered.

[0213] Cross-correction (CC) is a concept and mechanism that has had traction in the context of lysosomal storage disorders35. When applied, CC canturn a small number of cells into factories for the release of a therapeutic agent, for instance, a lysosomal enzyme whose deficiency causes disease. Thereby, the therapeutic agent can act not only on the cells that produce it but also on neighboring cells. In the context of prion proteins, a low level of CC may occur naturally, because a subset of PrPcand its PrPG127Vmutant are shed by a- disintegrin-and-metalloproteinase 10 (ADAM 10) from the cell surface through endoproteolytic cleavage at a site adjacent to the GPI-attachment site36, thereby giving rise to shed endoproteolytic products of PrPc(sPrPc) andSprpGi27v 37 However, the natural proportion of sPrPcto total PrPcis low, estimated to be 7-10% in rodents38. Therefore, the natural ADAM10-mediated release of PrPcwould provide limited protection to the brain if only a small subset of cells can be made to express the GPI-anchored PrPG127Vmutant.

[0214] In the context of prion disease therapy, the present inventors have shown herein that effective cross-correction is implemented by expressing the protective PrPG127Vmutant without its GPI-anchor. In contrast to reliance on ADAM 10-based endoproteolytic cleavage, this is achieved by removing the 3’ portion that encodes the GPI-anchor attachment signal sequence (GPI-SS) from the rAAV vector-encoded open reading frame (ORF) of the prion gene, thereby causing the biogenesis of anchorless prion protein (PrPc-AGPI)39. In contrast to the CC example of the expression of lysosomal enzymes for the treatment of lysosomal storage diseases, when applied to the prion protein, the method of facilitating CC not only merely increases the extracellular spread of the protective PrPG127Vvariant. Rather, it also alters the biochemical properties of PrPG127V-AGPI in a manner that naturally enhances its therapeutic effectiveness, hence the term enhanced cross-correction (ECC). Moreover, this enhancement makes the treatment safer and increases its usefulness for the treatment of other neurodegenerative diseases that cause brain cells to die upon docking of specific disease-associated conformers to PrPcexposed at the cell surface.

[0215] In the 15 years since the discovery of the G127V mutation during which several ideas for exploiting the G127V mutation for gene therapy, the useof an anchorless PRP harboring the G127V mutation has not been formulated or implemented. In fact, several prior observations can be interpreted as discouragement for pursuing a therapeutic avenue that relies on anchorless PrPcas follows.

[0216] First, genetic manipulations, which increase levels of PrPc-AGPI on a background of wild-type PrPc, have been reported to accelerate clinical scrapie39.

[0217] Second, it has been observed repeatedly that the lack of the GPI- SS causes nascent PrPc-AGPI to become less N-glycosylated during its passage through the secretory pathway39 40. When combined with data, which established that the N-glycans can pose steric hindrances to the templated conversion41, the central event underlying prion diseases, these insights can lead to the conclusion that PrPc-AGPI promotes conversion to PrPScand disease. Consistent with this interpretation, PrPc-AGPI was shown to undergo templated in vitro conversion reactions more readily42and was observed to form denser deposits than wild-type PrPC 41. Similarly, the recruitment of PrPcinto the templated polymerization was shown to be hindered by N-glycans — a phenomenon that has been attributed to the negative charges contributed by the natural sialylation of N-glycans attached to PrPC 43.

[0218] Third, PrPc-AGPI has been shown to serve as a malleable substrate that exhibits a low seeding barrier in the templated conversion of a wide range of prion strains43. This enhanced conversion susceptibility of PrPc- AGPI can raise the concern that sub-stoichiometric strains that may exist in individuals afflicted with prion disease, whose templated proliferation is constrained by the presence of N-glycans, may come to the fore if the proportion of under-glycosylated PrPcsubstrate is increased by the additional expression of PrPc-AGPL

[0219] Finally, the half-life of PrPc-AGPI has been reported to exceed considerably the half-life of normal PrPc, with an approximately sixfold longer half-life reported in a specific cell model44. If this observation was to translateto brain-expressed prion proteins, then PrPc-AGPI could be expected to accelerate disease progression in an outsized manner that is not governed by its relative expression level but its disproportionate contribution to steady-state levels of total PrPc.

[0220] These characteristics of PrPc-AGPI are undesirable when applied to the wild-type PrPcsequence as they increase the risk of it acting as a substrate for the templated polymerization. However, by the methods disclosed herein, in the context of PrPG127Vthese characteristics turn into a therapeutic advantage as follows.

[0221] First, without being bound by theory, it is believed that the same quality that causes PrPc-AGPI to accelerate prion disease when expressed in the presence of normal PrPc, makes anchorless PrPG127V-AGPI an even more potent dominant negative inhibitor of the disease.

[0222] The lower steric hindrance to conversion that is afforded to PrPG127V-AGPI by its|owerlevels of post-translational N-glycans increases its access to PrPSctemplates relative to the endogenous GPI-anchored PrPcthat carries these post-translational modifications, thereby allowing it to block further templated polymerization more effectively. This is a key distinction also to the low levels of ADAM10-shed full-length sPrPG127Vthat might be observed if prpd27vwasexpressed with its GPI-anchor present, as this molecule would have undergone the normal passage through the secretary pathway with its GPI-anchor present and therefore would carry the normal level of N- glycosylation observed in PrPc.

[0223] Further, without being bound by theory, because anchorless PrPG127V-AGPI is not constrained by a GPI-anchor or extensive N-glycosylation, it is expected to have a broader compatibility with a wide range of prion strains. This feature not only improves its ability to slow disease progression in a given prion-infected host, where more than one strain may compete at any time for PrPcsubstrate, but the malleability conferred by this biochemical differenceallows the treatment to be effective against a larger range of prion disease manifestations in humans and mammals.

[0224] Finally, without being bound by theory, it is expected that the increased half-life of PrPc-AGPI will translate to an increased half-life of PrPG127V-AGPI,anc| this biochemical feature enables the treatment to be more effective, as it shifts the relative contributions of endogenous PrPcand heterologous PrPG127V-AGPI to total steady-state levels of PrP in favor of anchorless PrPG127V-AGPL

[0225] T aken together, without being bound by theory, it is proposed that if brain cells are instructed to produce PrPG127V-AGPI, the percentage of brain cells that need to take up the heterologous transgene to achieve complete protection from prion disease is lower than with any of the alternative currently pursued gene therapy approaches for targeting prion diseases.

[0226] It has been well established that the survival extension that can be achieved with PrPclowering approaches is approximately proportional to the level of suppression in steady-state PrPclevels that they can achieve, and no complete protection from disease has been shown in vivo with even the most effective approach. Currently, none of the PrPclowering strategies incorporate cross-correction, thereby limiting their effectiveness.

[0227] The expression of PrPG127V-AGPI is more effective for the treatment of prion diseases than a therapy based on the expression of a GPI- anchored PrPG127Vmutant, whose dominant negative influence is largely restricted to cells that can be made to express the heterologous protein.

[0228] The key conceptual benefit of the G127V mutation, relative to PrP lowering approaches, namely the observation that sub-stoichiometric transgene expression of PrPG127Vcan provide complete protection against various strains of prion disease, is unlikely to manifest if GPI-anchored PrPG127Vcan only be expressed in a subset of brain cells. This potential is unleashed through enhanced cross-correction when PrPG127V-AGPI is secreted by the subset of brain cells that can be transduced.

[0229] Indeed, the data presented herein suggest that brain-wide protection may be possible through ECC even when only a subset of brain cells can be reached. This conclusion is based on steady-state PrPv127AGPI levels having exceeded endogenous expression levels for this highly expressed protein at a 3:1 ratio. Considering the 1 :3 gene dosage ratio of V127 mutant to wild-type human PRNP that had been shown to be sufficient to provide complete protection in humanized mice24, this excess in protective PrPv127AGPI may buffer efficiency reductions that must be anticipated when translating this treatment modality to humans. The survival extension ceiling encountered in the BvPrnp sequence context is expected to be lifted by moving this approach to human prion gene sequences and human prion inocula.

[0230] While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.SequencesSEQ ID NO: 1 : Human PrPG127V-AGPIMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQG GGTHSQWN KPS KPKTN M KH M AGAAAAGAVVGG LGVYM LGS AMS R P 11 H FG SDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSEQ ID NO: 2: Human PRN P^^-AGPI-SSATGGCGAACCTTGGCTGCTGGATGCTGGTTCTCTTTGTGGCCACATGGAGTGACCTGGGCCTCTGCAAGAAGCGCCCGAAGCCTGGAGGATGGAACACTGGGGGCAGCCGATACCCGGGGCAGGGCAGCCCTGGAGGCAACCGC TACCCACCTCAGGGCGGTGGTGGCTGGGGGCAGCCTCATGGTGGTGGC TGGGGGCAGCCTCATGGTGGTGGCTGGGGGCAGCCCCATGGTGGTGGCTGGGGACAGCCTCATGGTGGTGGCTGGGGTCAAGGAGGTGGCACCCA CAGTCAGTGGAACAAGCCGAGTAAGCCAAAAACCAACATGAAGCACATG GCTGGTGCTGCAGCAGCTGGGGCAGTGGTGGGGGGCCTTGGCGTCTACATGCTGGGAAGTGCCATGAGCAGGCCCATCATACATTTCGGCAGTGACTATGAGGACCGTTACTATCGTGAAAACATGCACCGTTACCCCAACCAAGT GTACTACAGGCCCATGGATGAGTACAGCAACCAGAACAACTTTGTGCAC GACTGCGTCAATATCACAATCAAGCAGCACACGGTCACCACAACCACCAAGGGGGAGAACTTCACCGAGACCGACGTTAAGATGATGGAGCGCGTGG TTGAGCAGATGTGTATCACCCAGTACGAGAGGGAATCTCAGGCCTATTA CCAGAGAGGATCGSEQ ID NO: 3: Human GPI Anchor Signal Sequence (GPI-SS)SMVLFSSPPVILLISFLIFLIVGSEQ ID NO: 4: Nucleotide sequence coding for Human GPI Anchor Signal SequenceAGCATGGTCCTCTTCTCCTCTCCACCTGTGATCCTCCTGATCTCTTTCCTCATCTTCCTGATAGTGGGATGASEQ ID NO: 5: Human PrPG127VMANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQG GGTHSQWN KPS KPKTN M KH M AGAAAAGAVVGG LGVYM LGS AMS R P 11 H FG SDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTT KGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVGSEQ ID NO: 6: Human PRA / P0127VATGGCGAACCTTGGCTGCTGGATGCTGGTTCTCTTTGTGGCCACATGGA GTGACCTGGGCCTCTGCAAGAAGCGCCCGAAGCCTGGAGGATGGAACA CTGGGGGCAGCCGATACCCGGGGCAGGGCAGCCCTGGAGGCAACCGC TACCCACCTCAGGGCGGTGGTGGCTGGGGGCAGCCTCATGGTGGTGGCTGGGGGCAGCCTCATGGTGGTGGCTGGGGGCAGCCCCATGGTGGTGG CTGGGGACAGCCTCATGGTGGTGGCTGGGGTCAAGGAGGTGGCACCCA CAGTCAGTGGAACAAGCCGAGTAAGCCAAAAACCAACATGAAGCACATG GCTGGTGCTGCAGCAGCTGGGGCAGTGGTGGGGGGCCTTGGCGTCTACATGCTGGGAAGTGCCATGAGCAGGCCCATCATACATTTCGGCAGTGACT ATGAGGACCGTTACTATCGTGAAAACATGCACCGTTACCCCAACCAAGT GTACTACAGGCCCATGGATGAGTACAGCAACCAGAACAACTTTGTGCAC GACTGCGTCAATATCACAATCAAGCAGCACACGGTCACCACAACCACCAAGGGGGAGAACTTCACCGAGACCGACGTTAAGATGATGGAGCGCGTGG TTGAGCAGATGTGTATCACCCAGTACGAGAGGGAATCTCAGGCCTATTA CCAGAGAGGATCGAGCATGGTCCTCTTCTCCTCTCCACCTGTGATCCTC CTGATCTCTTTCCTCATCTTCCTGATAGTGGGATGAReferences1. 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Claims

CLAIMS:

1. A nucleic acid comprising a nucleotide sequence encoding a prion protein, wherein the prion protein (a) comprises an amino acid sequence comprising a protective sequence variant and (b) lacks a fully functional 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence.

2. The nucleic acid of claim 1 , wherein the protective sequence variant is a M129V polymorphism or a G127V, E219K, or E168R mutation.

3. The nucleic acid of claim 1 , wherein the protective sequence variant is a G127V mutation.

4. The nucleic acid of any one of claims 1 -3, wherein the prion protein lacks a 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence.

5. The nucleic acid of any one of claims 1-4, wherein the nucleotide sequence is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 2, and wherein the protein encoded by said nucleotide sequence confers protection against prion disease.

6. The nucleic acid of any one of claims 1-5, wherein the nucleotide sequence is set forth in SEQ ID NO: 2.

7. The nucleic acid of any one of claims 1-4, wherein the amino acid sequence further comprises a prion disease-associated mutation.

8. The nucleic acid of claim 7, wherein the prion disease-associated mutation is selected from E200K, D178N, A117V, P102L, V210I, M232R, and V180I.

9. The nucleic acid of any one of claims 1-8, wherein the open reading frame coding for the prion protein is operably linked to a promoter.

10. The nucleic acid of claim 9, wherein the promoter is selected from the group consisting of: chicken P-actin hybrid (CBh), chicken beta actin (CBA), CMV early enhancer (CAG), synapsin I (Syn1), Thy1 (Thy1), neuron-specific enolase (NSE), CamKII (CamKII), neural cell adhesion molecule 1 (NCAM1), glial fibrillary acidic protein (GFAP), nestin (Nes), tyrosine hydroxylase (TH), dopamine transporter (DAT), microtubule-associated protein 2 (MAP2), a- calcium / calmodulin-dependent protein kinase II (a-CaMKII), Elongation Factor 1a (eF-1a), phosphoglycerate kinase 1 (PGK), cytomegalovirus immediate- early promoter (CMV) and human P-actin (hACTB).

11. A recombinant prion protein, wherein the protein (a) comprises an amino acid sequence comprising a protective sequence variant and (b) lacks a fully functional 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence.

12. The recombinant protein of claim 11 , the protective sequence variant is a M129V polymorphism or a G127V, E219K, or E168R mutation.

13. The recombinant protein of claim 11 , wherein the protective sequence variant is a G127V mutation.

14. The recombinant protein of any one of claims 11-13, wherein the prion protein lacks a 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence.

15. The recombinant protein of any one of claims 11-14, wherein the amino acid sequence is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino sequence of SEQ ID NO: 1 , and the protein confers protection against prion disease.

16. The recombinant protein of any one of claims 11-15, wherein the amino sequence is set forth in SEQ ID NO: 1 .

17. The recombinant protein of any one of claims 11-15, wherein the amino acid sequence further comprises a prion disease-associated mutation.

18. The recombinant protein of claim 17, wherein the prion disease- associated mutation is selected from E200K, D178N, A117V, P102L, V210I, M232R, and V180I.

19. The recombinant protein of any one of claims 11-18, wherein the prion protein is fused to one or more proteins that confers uptake by brain cells, optionally wherein the one or more proteins are selected from the group consisting of viral capsid proteins, transferrin, semaphorin, Tat peptide, angiopep peptides, ligands targeting the insulin receptor, fragments of capsids from AAVs, fragments of capsids from Zika viruses, bacteriophage capsids and plant virus capsid.

20. A viral vector comprising the nucleic acid of any one of claims 1 -10 or a nucleic acid encoding the recombinant protein of any one of claims 11-19.

21. The viral vector of claim 20, wherein the viral vector is an Adeno- Associated Virus (AAV) vector or a derivative thereof.

22. The viral vector of claim 21 , wherein the AAV vector is selected from the group consisting of: AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.32, AAVrh.33 AAVrh.43, AAVrh.64R1 .

23. The viral vector of claim 21 or 22, wherein the viral vector is a chimeric, shuffled or capsid modified derivative of the AAV.

24. A pharmaceutical composition comprising the nucleic acid of any one of claims 1-10, the recombinant protein of any one of claims 11-19, or the viral vector of any one of claims 20-23, and a pharmaceutically acceptable carrier or diluent.

25. The pharmaceutical composition of claim 24, wherein the pharmaceutically acceptable carrier or diluent is a lipid nanoparticle (LNP) or an exosome.

26. The pharmaceutical composition of claim 24 or 25, wherein the pharmaceutical composition is formulated for parenteral administration or intracerebral injection.

27. The pharmaceutical composition of any one of claims 24-26, wherein the pharmaceutical composition is permeable to the blood-brain barrier.

28. A use of viral vector of any one of claims 20-23 or the pharmaceutical composition of any one of claims 24-27 for increasing secretion of a protective prion protein variant from transduced cells in a subject.

29. A use of the viral vector of any one of claims 20-23 or the pharmaceutical composition of any one of claims 24-27 for treating or preventing a prion disease and / or a neurodegenerative disease in a subject in need thereof, wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity.

30. A use of the viral vector of any one of claims 20-23 or the pharmaceutical composition of any one of claims 24-27 in the manufacture of a medicament for treating or preventing a prion disease and / or a neurodegenerative disease in a subject in need thereof, wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity.

31. The use of claim 29 or 30, wherein the neurodegenerative disease is selected from the group consisting of: Alzheimer’s disease (AD), frontotemporal dementia, tauopathies, Parkinson’s disease (PD), multiple system atrophy (MSA) and synucleinopathies,32. The use of any one of claims 29-31 , wherein the viral vector or the pharmaceutical composition is for administration parenterally or intracerebrally.

33. The viral vector of any one of claims 20-23 or the pharmaceutical composition of any one of claims 24-27 for use in treating or preventing a prion disease and / or a neurodegenerative disease in a subject in need thereof,wherein the neurodegenerative disease is mediated by soluble aggregates requiring PrPcfor toxicity.

34. The viral vector or the pharmaceutical composition for use of claim 33, wherein the neurodegenerative disease is selected from the group consisting of: Alzheimer’s disease (AD), frontotemporal dementia, tauopathies, Parkinson’s disease (PD), multiple system atrophy (MSA) and synucleinopathies,35. The viral vector or the pharmaceutical composition for use of claim 33 or 34, wherein the viral vector or the pharmaceutical composition is for administration parenterally or intracerebrally.

36. A method of increasing the therapeutic efficiency of a prion protein composing a protective sequence variant, comprising the step of deleting or mutating the 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence of the prion protein or a nucleic acid encoding the prion protein.

37. The method of claim 36, wherein the protective sequence variant is a M129V polymorphism or a G127V, E219K, or E168R mutation.

38. The method of claim 36, wherein the protective sequence variant is a G127V mutation.

39. The method of any one of claims 36-38, wherein the 3’ glycosylphosphatidylinositol (GPI)-anchor attachment sequence is deleted from the prion protein.

40. The method of any one of claims 37-40, wherein the prion protein further comprises a prion disease-associated mutation.41 . The method of any one of claims 37-40, wherein the prion protein or the nucleic acid encoding the prion protein is for administration to a subject.

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