WO2014102267A1 - Means and methods for the treatment of synucleinopathies - Google Patents

Abstract

The present invention relates to the field of neurological diseases, in particular to the field of synucleinopathies, more particularly to the field of Parkinson's disease. In particular, the invention provides polynucleotides and polypeptides which can be used for treating Parkinson's disease.

Description

Means and methods for the treatment of synucleinopathies

Field of the invention

The present invention relates to the field of neurological diseases, in particular to the field of synucleinopathies, more particularly to the field of Parkinson's disease. In particular, the invention provides polynucleotides and polypeptides which can be used for treating Parkinson's disease.

Introduction to the invention

Parkinson's disease is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra leading to motor dysfunction^1,10. Most forms of Parkinson's disease are sporadic; however, -5% cases are attributed to mutations in several genes leading to early onset or familial forms of the disease¹⁰. One of these genes, SNCA1, codes for the alpha-synuclein protein, a natively unfolded protein¹¹ with a poorly characterized post-synaptic function. Point mutations¹ and overexpression^12,13 of SNCA1 lead to the accumulation and aberrant interaction of alpha- synuclein in the cell, including the formation of insoluble fibrils¹⁴. These alpha-synuclein aggregates constitute the primary structural component of Lewy bodies, abnormal cytoplasmic inclusions found in neural cells of patients with most forms of Parkinson's disease, including sporadic forms^10,15. The close association of alpha-synuclein with Parkinson's disease suggests that its aberrant accumulation and aggregation may have a direct role in dopaminergic neuronal cell loss. Thus, both neuronal death and the accumulation of alpha- synuclein are hallmark cellular phenotypes of Parkinson's disease. There is currently no satisfactory cure for Parkinson's disease. Dopaminergic replacement is believed to be the most effective therapeutic strategy currently in use for Parkinson's disease. Symptomatic treatment of the disease-associated motor impairments involves oral administration of the dopamine precursor dihydroxyphenylalanine, also known as levodopa (L-Dopa). In early stage Parkinson's disease, oral L-Dopa is efficacious, but patients progressively lose the ability to convert L-Dopa to dopamine as more and more dopaminergic neurons degenerate. One alternative strategy for the treatment of Parkinson's disease is gene therapy. Viral vector- based approaches are being evaluated for the treatment of various neurological diseases, through the introduction of therapeutic genes by transduction of the viral vector into neuronal and/or support cells.

A widely studied mutation that causes early-onset Parkinson's disease is the alanine to threonine substitution at site 53 (A53T) in human alpha-synuclein. The pathogenic allele 53T is present in many other mammalian species without pathogenic manifestations^1,2,5,6. Specifically, the SNCA1 Parkinson's disease allele 53T is a recent ancestral state present in the common ancestor of Catarrhini and non-Catarrhini primates. Non-Catarrhini mammals are not known to suffer from Parkinson's disease despite carrying the 53T allele that causes the disease in humans. The difference in the phenotypic manifestation of the 53T allele between humans and non-Catarrhini mammals is likely to be due to a functional intra- or inter-genic interaction that modulates the impact of amino acids present at site 53. Thus, in the course of evolution of the Catarrhini lineage at least one substitution must have occurred that changed the previously benign manifestation of alpha-synuclein 53T into the currently observed pathogenic one². Evolution at sites involved in a functional interaction often proceeds in a co-dependent manner^2,16,17. Therefore, we hypothesized that the substitution that altered the deleterious effect of 53T likely occurred nearly simultaneously as the T->A substitution at site 53, close to the last common ancestor of the Catarrhini clade.

Summary of the invention The present invention has identified two variants of the human LAMP2A polypeptide (LAMP2A stands for Lysosomal-Associated Membrane Protein 2, a lysosomal membrane protein involved in the degradation of alpha-synuclein) comprising (i) a serine, or (ii) a phenylalanine residue at amino acid position 94 (instead of the wild-type residue proline), that can be successfully used to obliterate the deleterious effect of the alpha-synuclein A53T mutation in human neurons.

Figures

Figure 1 : Mammalian phylogeny with allele states in amino acid sites 53 of alpha-synuclein and 94 of LAMP2A.

Figure 2: Rates of alpha-synuclein degradation observed in differentiated SH-SY5Y cells expressing combinations of alpha-synuclein and LAMP2A alleles. The effect of the combination of allele states in sites 53 of alpha-synuclein and 94 of LAMP2A present in mammals relative to the disease combination 53T/94P is shown in (a), while other combinations, including two control combinations: 53T/94G and 53A 94G, are shown in (b). The values are averages of three independent experiments with standard errors. A representative blot of each experimental condition is shown in (c). Levels of endogenous LAMP2A protein are shown in (d). The values indicate mean ratio standard errors of three independent experiments, with instances of significant difference from 53A 94P marked with an asterisk (t-test, p<0.05). Figure 3: Effect of the co-expression of combinations of allele states in alpha-synuclein and LAMP2A on cell viability of differentiated dopaminergic SH-SY5Y cells. The effect of combinations present in mammals relative to the disease 53T/94P combination is shown in (a) and the effect of all other combinations is shown in (b). The values represent averages of six independent replicates, each of them performed in quintuplicate, with standard errors. Instances of a significant difference from the effect of 53A 94P combination (Fisher's t-test, p<0.05) are marked with an asterisk.

Detailed description of the invention

As used herein, each of the following terms has the meaning associated with it in this section. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein.

Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4^th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

Mutations that cause or contribute to human diseases are present in genomes of other species without apparent deleterious consequences^1"4. In the present invention we have investigated the in vitro phenotype manifestation of the Parkinson's disease-causing A53T (G209A) mutation in the SNCA 1 gene, which is present in wild-type non-Catarrhini mammals^1,2,5,6. Using a phylogenetic approach we identified a clear pattern of co-evolution between site 53 in alpha- synuclein and site 94 in LAMP2A, a lysosomal membrane protein involved in the degradation of alpha-synuclein^7"9. Specifically, alanine at site 53 (53A) was always present with proline at site 94 (94P), while threonine at site 53 (53T) was present with serine at site 94 (94S) in all species but one (Mus musculus, with phenylalanine (F) in orthologous position 90 of LAMP2A). We recreated the genotype linked to Parkinson's disease by co-expressing vectors coding for 53T and 94P alleles in a human dopaminergic neuronal cell model, which manifested in a decrease in the rate of degradation of alpha-synuclein and increased neuronal toxicity, two hallmark phenotypes of the disease¹⁰. Conversely, when the pathogenic 53T allele was co- expressed with either the 94S or 94F alleles, recreating the combinations present in non- Catarrhini species, the rates of alpha-synuclein degradation and cell death were indistinguishable from those observed when the 53A and 94P alleles—those present in wild- type Catarrhini— were co-expressed. The capacity of the P94S/P94F mutations to compensate for the manifestation of the A53T mutation shows that the effect of pathogenic mutations can be modulated by changes in other genes, suggesting a novel strategy for treatment of human genetic pathologies. The present findings can be used as an approach to develop therapeutic strategies for Parkinson's disease.

In one embodiment, the invention provides a human variant LAMP2A nucleic acid which encodes a variant LAMP2A protein, wherein said variant LAMP2A comprises a serine residue at position 94. Said variant LAMP2A protein with a serine residue at position 94 is depicted in SEQ ID NO: 2.

In a particular embodiment, the invention provides a human variant polynucleotide sequence depicted in SEQ ID NO: 1 . SEQ ID NO: 1 encodes a variant LAMP2A protein with a serine at position 94.

In another embodiment, the invention provides a human variant LAMP2A nucleic acid which encodes a variant LAMP2A protein, wherein said variant LAMP2A comprises a phenylalanine residue at position 94. Said variant LAMP2A protein with a phenylalanine residue at position 94 is depicted in SEQ ID NO: 4. In another particular embodiment, the invention provides a human variant polynucleotide sequence depicted in SEQ ID NO: 3. SEQ ID NO: 3 encodes a variant LAMP2A protein with a phenylalanine at position 94

In the present invention the terms 'protein' and 'polypeptide' can be used interchangeably. As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the specific variant LAMP2A proteins of the present invention, depicted in SEQ ID NO: 2 and SEQ ID NO: 4. Thus, having the particular amino acid sequence of the variants of the invention, those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the variant LAMP2A protein.

In a particular embodiment, the variant LAMP2A proteins and related nucleic acids of the present invention are recombinant. As used herein, "nucleic acid" may refer to either DNA or RNA, or molecules that contain both desoxy- and ribonucleotides. The nucleic acids include genomic DNA, cDNA and oligonucleotides, including sense and anti-sense nucleic acids. Such nucleic acids may also contain modifications in the ribose-phosphate backbone to increase stability and half-life of such molecules in physiological environments. The nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence. As will be appreciated by those in the art, the depiction of a single strand ("Watson") also defines the sequence of the other strand ("Crick"). By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature. Thus, an isolated variant LAMP2A nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell. However, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention. A "recombinant LAMP2A protein" is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid. A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild-type host, and thus may be substantially pure. In a less preferred embodiment, variant LAMP2A proteins may be prepared by in vitro synthesis using established techniques (e.g. chemical synthesis, see for example Wilken et al, Curr. Opin. Biotechnol. 9:412-26 (1998)).

In yet another particular embodiment, one of either SEQ ID NO: 1 or SEQ ID NO:3 is used for the manufacture of a medicament to treat synucleinophathies such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), pure autonomic failure (PAF), and multiple system atrophy (MSA).

In yet another particular embodiment, the polynucleotide used for the manufacture of a medicament to treat synucleinophathies is preferably SEQ ID NO: 1 .

The term alpha-synucleinopathies comprises progressive, neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) with the major pathological hallmark being alpha-synuclein (AS)-positive inclusions in neuronal and glial cells. Neuronal inclusions, Lewy bodies (LBs) and Lewy neurites (LNs) are characteristic for PD and DLB, while AS-positive glial cytoplasmic inclusions (GCIs) are distinctive of MSA, and occur predominantly in oligodendroglial cells. Astroglial AS- positive inclusions may also occur in PD. PD pathology has been partly related to point mutations or duplications and triplications of the gene coding for alpha-synuclein (SNCA). Moreover, SNCA variants can increase the risk of developing PD and MSA. AS inclusion formation may be related to posttranslational modifications of AS (nitration, ubiquitination and phosphorylation), which can lead to pathological accumulation of AS, and enhance the progression of alpha-synucleinopathies.

In yet another embodiment, one of either SEQ ID NO: 2 or SEQ ID NO:4 is used for the manufacture of a medicament to treat synucleinophathies such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), pure autonomic failure (PAF), and multiple system atrophy (MSA). In yet another particular embodiment, the polypeptide used for the manufacture of a medicament to treat synucleinophathies is preferably SEQ ID NO: 2.

In what follows, nucleic acids encoding SEQ ID NO: 2 and SEQ ID NO: 4, SEQ I D NO: 1 and SEQ ID NO: 3, and the polypeptides SEQ ID NO: 2 and SEQ ID NO: 4 are collectively designated as agents. Thus an agent of the invention can be a polynucleotide, or a polypeptide, or both.

In a particular embodiment, the agents are deliverable to the brain, by itself or via an agent delivery vehicle or method. Example agent delivery vehicles and methods include nanoparticles, micelles, dendrimers, liposomes, mists, microdroplets, aerosols, atomizations, gels, artifical DNA nanostructures and biological vectors, to name a few. Some of these will be described further.

In another particular embodiment, the agents are delivered by a cyclodextrin-based nanoparticle. Polycation formulated nanoparticles have been used for agent delivery into the brain and are useful for delivery of agents of the invention.

In some other embodiments, the agent delivery particles for use to deliver the agents of the present invention can be a micro-lipid particle or nano-lipid particle e.g., spheres, micelles, or dendrimers. In some embodiments, the agent delivery particles are unilaminar, (meaning the agent delivery particles comprise more than one layer or are multi-layered). In some other embodiments, an agent delivery particle is a liposome which is used to capture and deliver agents of the invention to the brain using the methods herein. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration. Liposomes are non-toxic, non-hemolytic and non-immunogenic even upon repeated injections; they are biocompatible and biodegradable. Lipid based, ligand coated nanocarriers can store their payload in the hydrophobic shell or the hydrophilic interior depending on the nature of the drug/contrast agent being carried.

In some other embodiments, agents to be delivered are encapsulated in a virosome. A virosomes is an agent delivery particle comprising lipid bilayers containing viral glycoproteins derived from enveloped viruses. Virosomes (or virosome-like-particles, considering that the exact size and shape of the particles) are generally produced by extraction of membrane proteins and lipids from enveloped viruses with a detergent, followed by removal of this detergent from the extracted lipids and viral membrane proteins, in fact reconstituting or reforming the characteristic lipid bilayers (envelopes) that surround the viral core or nucleocapsid. The term "virosome" defines a specific form of virus-like particles (YLPs). Virosomes are semi-synthetic complexes derived from viral particles and produced by an in vitro procedure. They are essentially reconstituted viral coats, while the viral nucleocapsid is replaced by a compound of choice. Virosomes retain their fusogenic activity and thus deliver the incorporated compound (antigens, agents, genes) inside the target cell. They can be used for agent delivery or gene transfer. Virus-like particles (VLPs) are particle structures that are in size and shape reminiscent of or even indistinguishable from their parental virus but are lacking the capability to infect and replicate in host cells. VLPs are multimeric structures composed of viral proteins (authentic or modified variants of it). In addition, VLPs may or may not contain nucleic acids, lipids, and include lipid membrane structures or not. Two typical but very distinct examples for VLPs derived from a single Virus (HBV) are HBs and HBc particles. Virosomes are unilamellar phospholipid bilayer vesicles incorporating virus derived proteins to allow the virosomes to fuse with target cells. Virosomes are not able to replicate but are pure fusion-active vesicles. In contrast to liposomes, virosomes contain functional viral envelope glycoproteins, for example, influenza virus hemagglutinin (HA) and neuraminidase (NA) intercalated in the phospholipid bilayer membrane. Virosomes typically have a mean diameter of 150 nm, and without being limited to theory, virosomes represent reconstituted empty influenza virus envelopes, devoid of the nucleocapsid including the genetic material of the source virus.

In some embodiments, agents delivered to the brain can be administered in the form of an aerosol or by nebulization, e.g. in the form of a mist, microdroplet, aerosols and atomizations. For use as aerosols, an agent can be present in a solution or suspension and can be connected to a pressurized aerosol present in the device, and can be delivered with a suitable propellant, for example, air, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. Agents can also be administered in a non-pressurized form such as in a nebulizer or atomizer. In some embodiments, agents are comprised in a gel. A gel is a substantially dilute cross- linked system which resembles a solid in steady state. By weight, gels are mostly liquid, yet they behave like solids due to a three-dimensional cross-linked network within the liquid. This internal network structure may result from physical bonds (physical gels) or chemical bonds (chemical gels), as well as crystallites or other junctions that remain intact within the extending fluid. Virtually any fluid can be used as an extender including water (hydrogels), oil, and air (aerogel).

In some embodiments, agents are embedded in a nanofiber hydrogel scaffold. Such a gel is comprised of small, woven protein fragments which can successfully carry and release proteins of different sizes. The rate of release of the agents of the invention can be controlled by changing the density of the gel, allowing for continuous agent delivery over a specific period of time. The proteins are released from the gel over hours, days or even months and the gel itself is eventually broken down into harmless amino acids. Such peptide hydrogels are ideally suited for agent delivery as they are pure, easy to design and use, non-toxic, non- immunogenic, bio-absorbable, and can be locally applied to a particular tissue. In addition, proteins carried by the gel emerge unscathed after delivery, with no adverse effect on their function.

In some embodiments, agents comprise an artificial DNA nanostructure. An artificial DNA nanostructure is DNA that is used as a structural material rather than as a carrier of genetic information. DNA nanotechnology makes use of the fact that, due to the specificity of Watson- Crick base-pairing, only portions of the strands which are complementary to each other will bind to each other to form duplex DNA. DNA nanotechnology attempts to rationally design sets of DNA strands so that desired portions of each strand will assemble in the correct positions for some desired target structure, a process called nucleic acid design.

It may be appreciated that the principles of DNA nanotechnology apply equally well to other nucleic acids such as RNA and PNA and may be used in a similar fashion as agents as described herein.

In specific embodiments, the polynucleotides of the invention are incorporated into a viral vector such as a gene therapy vector. A wide variety of gene transfer/gene therapy vectors and constructs are known in the art. These vectors are readily adapted for use to deliver the polynucleotide agents of the present invention. By the appropriate manipulation using recombinant DNA/molecular biology techniques to insert an operatively linked nucleic acid encoding a protein agent into the selected expression/delivery vector, many equivalent vectors for the practice of the methods described herein can be generated. A vector comprising a nucleic acid molecule of the invention linked to expression control elements and capable of replicating inside the cells is prepared. Alternatively the vector can be replication deficient and can require helper cells for replication and use in gene therapy. Vectors, recombinant viruses, and other expression systems can comprise any nucleic acid which can infect, transfect, transiently or permanently transduce a neuronal cell or neuronal support cell, e.g. dopaminergic neurons, neurons, glia, astrocytes and the like. In one aspect, a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. In one aspect, a vector can comprise viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). In one aspect, expression systems can be replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. In one aspect, expression systems also include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and include both the expression and non-expression plasmids. In a particular embodiment, a vector can be an expression vector including both (or either) extra-chromosomal circular and/or linear nucleic acid (DNA or RNA) that has been incorporated into the host chromosome(s). In one aspect, where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.

A number of viral vector systems are available including retroviral systems, adenoviral vectors, adeno-associated virus vectors, viral vectors from the pox family including vaccinia virus and the avian pox viruses, viral vectors from the alpha virus genus, such as those derived from Sindbis and Semliki Forest Viruses, and rhabdoviruses, such as vesicular stomatitis virus and papillomaviruses. In an alternative embodiment, a polynucleotide of the invention, or a vector containing the same, can be packaged into liposomes. Suitable lipids and related analogs are described in the art.

Gene therapy vectors or naked DNA can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, nasal, gastric, intradermal, intrathecal, intracerebroventricular, intramuscular, subdermal, or intracranial infusion) or topical application. Such vectors can further include facilitating agents. DNA can also be administered using a gene gun (Xiao & Brandsma, Nucleic Acids. Res. 24:2630-2622 (1996), which is hereby incorporated by reference in its entirety). The DNA encoding a polynucleotide of the invention is precipitated onto the surface of microscopic metal beads. The microprojectiles are accelerated with a shock wave or expanding helium gas, and penetrate tissues to a depth of several cell layers. In a further variation, vectors encoding polynucleotides can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, and tissue biopsies) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.

In a specific embodiment, the agents of the invention are used for the treatment of Parkinson's disease, wherein the patients suffering from Parkinson's disease are selected from patients carrying a mutation in the alpha-synuclein gene leading to the functional expression of a mutant alpha-synuclein protein harboring the 53T mutation. Thus, in a specific embodiment, suspected patients of Parkinson's disease are first diagnosed for the 53T alpha-synuclein mutation to be eligible for treatment with the agents of the present invention.

The agents of the invention, preferably incorporated in an agent delivery, are preferably formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical formulations" include formulations for human use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example, as described in Remington: The Science and practice of Pharmacy, 21 ^st ed., ISBN 0-7817-4673-6 (2006), the entire disclosure of which is herein incorporated by reference. The present pharmaceutical formulations comprise at least one agent of the invention (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a physiologically acceptable carrier medium. Preferred physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. Pharmaceutical compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality-adjusting agents, buffers, and pH-adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as, for example, calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form or can be lyophilized. For solid compositions, conventional nontoxic solid carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For example, a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10 - 95%, preferably 25% - 75%, of one or more agents of the invention. A pharmaceutical composition for aerosol (for inhalation) administration can comprise 0.01 - 20% by weight, preferably 1 % - 10% by weight, of one or more agents of the invention encapsulated in a liposome as described above. A carrier can also be included as desired; e.g., lecithin for intranasal delivery.

The term "medicament to treat" relates to a composition comprising agents as described above and a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to prevent and/or to treat synucleinopathies such as, for example, Parkinson's disease. Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers.

It is clear to the person skilled in the art that the use of a therapeutic composition comprising an agent of the invention for the manufacture of a medicament to prevent and/or to treat Parkinson's disease can be administered by any suitable means, including, but not limited to parenteral, subcutaneous, intraperitoneal, intrapulmonary, intracerebroventricular, intrathecal and intranasal administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.

Still another aspect of the invention is the use of the compensatory variants/mutations of the invention for the construction of a transgenic murine model for Parkinson's disease. It is known in the art that mice and other non-human mammals harbor the 53T variant in the alpha- synuclein gene without causing any symptom of Parkinson pathogenicity. Wild type mice also harbor a phenylalanine amino acid at position 90 in the LAMP2A protein, instead of the wild- type serine usual in the orthologous position in all other non-human species. Additionally, our results show that the combination of the A53T mutation in human alpha-synuclein with the proline residue at position 94 in the human LAMP2A polypeptide leads to an aggregation of alpha-synuclein and the subsequent neuronal cell death, as a reflection of what causes the disease in patients affected with such A53T mutation. However, replacement in human LAMP2A of the 94P wild-type residue by the 94S of 94F variants rescues the apoptotic effect. Please note that position 90 in the mice LAMP2A polypeptide corresponds with the orthologous position 94 in the human LAMP2A polypeptide, and vice versa. Therefore, mice harboring the alpha-synuclein 53T/LAMP2A 90P combination have been designed as putative models for Parkinson's disease, while those harboring the 53T/90S combination have been designed as controls to distinguish them from the wild-type 53T/90F background. Methods to make variant SEQ ID NO: 5-based transgenic animals are well known to a person skilled in the art. Such murine model organism can be used for screening compounds and testing medication useful for the treatment of Parkinson's disease. SEQ ID NO: 5 is the murine polynucleotide sequence encoding a LAMP2A variant polypeptide harboring a proline at position 90.

Aspects of the present invention are described in the following examples, which are not intended to limit the scope of the invention described in the claims. 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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

Examples 1 . Mammalian phylogenetic analysis of alpha-synuclein and interacting proteins

We screened alpha-synuclein and proteins known to interact with alpha-synuclein, Hsp70 [ref. 9], Hsp90 [ref. 18], UCH-L1 [Ref. 19], Parkin²⁰ and LAMP2A⁷, for sites in which a substitution has occurred in the same place of the phylogeny as the T->A substitution at site 53 of alpha- synuclein. We found only a single amino acid site, 94 in lysosome-associated membrane protein type 2, LAMP2A, at which a substitution has occurred in the last common ancestor of Catarrhini that showed a near-perfect match between the amino acid states at site 53 of alpha- synuclein (Figure 1 ). Specifically, 53A was always present with 94P while 53T was always present with 94S, except for Mus musculus, which contained a phenylalanine at site 90, the site orthologous to site 94 in the human sequence (94F from here on). The evolutionary history in these two sites suggests that 53T is not pathogenic to species that carry it because 94S modulates the phenotypic manifestation of 53T. Thus, we should expect that dopaminergic human cells that carry either of the two combinations found in nature, 53A/94P in Catarrhini and 53T/94S in most other mammals, to show normal rates of alpha-synuclein degradation and no effect on cell viability. In contrast, the 53T/94P combination, which corresponds to the Parkinson's disease-causing genotype, should lead to a decrease in the degradation of alpha- synuclein and increased neuronal toxicity.

2. Effect of the expression in neuronal cells of recombinant constructs comprising alpha- synuclein mutants and LAMP2A variants

We generated vectors coding for 53A and 53T alleles in the human alpha-synuclein cDNA and 94P, 94S, 94F and 94G alleles in the human LAMP2A sequence. The 94G allele was chosen as a negative control variant because of the simplicity and neutrality of the glycine side chain. Moreover, in the specific context of the LAMP2A sequence this variant creates 93-GGG-95 pattern that may dramatically increase protein chain flexibility²¹ and could, therefore, lead to loss of function. We made pairwise co-transfections of the two SNCA1 variants with each of the four variants of LAMP2A in differentiated SH-SY5Y cells, an in vitro model of the dopaminergic neuronal cells that are primarily affected in the course of Parkinson's disease^22,23. The rate of alpha-synuclein degradation in these eight combinations was then measured over the course of 24 hour time-series. As expected the co-expression of 53T and 94P led to a substantial decrease in the rate of alpha-synuclein degradation compared to the rate of degradation found when 53A and 94P are co-expressed (Figure 2). However, as predicted by our evolutionary analysis, the co-expression of the 53T/94S genotype that is present in the common ancestor of Catarrhini, led to the same rate of alpha-synuclein degradation as the co-expression of 53A 94P. When the 53T/94F combination was expressed, which is present only in M. musculus, we observed rates of alpha-synuclein degradation similar to the human wild type combination of 53A 94P. All other combinations, including the two control combinations with 94G revealed impaired alpha-synuclein degradation rates.

Dopaminergic neuronal cell death is thought to be the cause of Parkinson's disease motor symptoms and, therefore, we evaluated the effect of the co-expression of eight combinations of SNCA 1 and LAMP2A vectors on neurotoxicity assayed by cell viability. We found that co- expression of 53T/94S or 53A 94P had no significant effect on cell viability (Figure 3). In contrast, other combinations induced neuronal cell death with 53T/94P, 53A 94S, 53T/94G, 53A 94G and 53A 94F having the strongest impact. The 53T/94F combination that is present in the mouse genome had a weaker effect on cell death, such that the corresponding cell viability was not significantly different either from the normal 53A 94P or the pathogenic 53T/94T combination (t-test, p = 0.22 and p = 0.20, respectively). This intermediate effect was observed in a human cell line and in human SNCA 1 and LAMP2A cDNA background suggesting that a third substitution could be affecting the interaction between sites 53 and 94 in mice.

The A53T mutation causes an autosomal dominant early-onset form of Parkinson's disease^1,13 and is one of the most studied pathogenic forms of alpha-synuclein^10,14. The present invention shows that the pathogenic effect of this mutation can be compensated by a single mutation at an interacting protein, LAMP2A. By restoring the amino acid in site 94 of LAMP2A to the allele present in the common ancestor of Catarrhini and non-Catarrhini primates we show to revert two hallmark phenotypes of the disease. LAMP2A acts as a lysosomal receptor in chaperone- mediated autophagy²⁴, CMA, a selective pathway of degradation of cytosolic proteins, and one of the pathways for alpha-synuclein degradation. In a human background, alpha-synuclein bearing the 53A allele binds to LAMP2A with the help of Hsp70 and crosses the lysosomal membrane into the lysosome, where it is degraded. However, in the human background the alpha-synuclein with the 53T allele binds to LAMP2A with high affinity resulting in CMA blockage that increases alpha-synuclein levels and leads to neurotoxicity⁷. The modulation of alpha-synuclein degradation by substitutions in LAMP2A is consistent with the function of LAMP2A as an initiator of the CMA pathway^7"9,25. Furthermore, the present data linking the modulation of alpha-synuclein levels by substitutions in LAMP2A with toxicity in dopaminergic neurons suggests that alpha-synuclein accumulation contributes to neuronal dysfunction and the development of at least some forms of Parkinson's disease. 3. Conclusions

The invention shows that 53A 94S and 53T/94P, the allele combinations intermediate between the 53A 94P and 53T/94S combinations observed in nature do not correspond to wild-type phenotypes. Therefore, the T->A and S->P substitutions at sites 53 and 94, respectively, were probably fixed simultaneously in the course of evolution, as it is unlikely that a pathogenic mutation can reach fixation. The simultaneous fixation of two individually deleterious but jointly neutral mutations has been predicted theoretically²⁶ and observed in mitochondrial tRNAs¹⁷, although such events have not been previously reported in protein coding genes. Whether or not inter-genic and intra-genic interactions between sites play a role in evolution and in the connection between phenotype and genotype is one of the main questions in contemporary genetics. The observed interaction between site 53 in alpha-synuclein and site 94 in LAMP2A is in agreement with data suggesting that such interactions were common in the course of evolution^2,17,27,28. The suspected involvement of a third, unidentified site in the evolution of S->F substitution in the murine lineage is also consistent with such interactions being common. It is likely that two sites that co-evolved in a short timeframe, such as the 53/94 interaction observed in primates, are also involved in interactions with other sites in the course of longer term evolution. The findings that the pathogenic effect of the A53T mutation can be completely alleviated in vitro by a compensatory substitution P94S (or P94F) in LAMP2A that restores the interaction of these two sites to the ancestral state has several implications for Parkinson's disease therapy and for the application of evolutionary methods in genetics. First, the consequences of the A53T mutation in patients with Parkinson's disease can be reversed through exogenous expression of LAMP2A carrying the P94S (or P94F) mutation.

Materials and Methods

1 . Evolutionary analysis Substitutions along the mammalian phylogeny were analyzed in multiple alignments of orthologous sequences of alpha-synuclein, Hsp70, Hsp90, UCH-L1 , Parkin and LAMP2A. Criteria for the identification of sites potentially interacting with site 53 of alpha-synuclein included having a substitution in the common ancestor of Catarrhini and few or no other substitutions elsewhere along the mammalian phylogeny. LAMP2A was cloned from human RNA into pCMV-Tag2 mammalian expression vectors and SNCA 1 cDNA was obtained from pT7.7 vector and subcloned into pCDNA3-Myc mammalian expression vector. Both cDNAs were modified by site-directed mutagenesis to code for the studied mutations. Transfections were done into the dopaminergic differentiated SH-SY5Y cells leading to near-endogenous level of exogenous expression. Protein degradation rates and cell viability were measured according to standard protocols described in the online methods section. Statistical analyses were performed using the two-tailed unpaired t-student's test for single comparisons (p<0.05). We obtained sequences of alpha-synuclein, Hsp70, Hsp90, UCH-L1 , Parkin and LAMP2A from GenBank using the Entrez retrieval system. Orthologs were identified using the two directional best BLAST hit approach²⁹ and aligned using MUSCLE³⁰. If a substitution at a site was present in the common ancestor of Catarrhini and with few, if any, other substitutions in other clades of the phylogeny, then such sites were considered to be potential candidates for an interaction with alpha-synuclein site 53.

2. Cell culture

Human neuroblastoma cells (SH-SY5Y) were grown on Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin and 100 μg ml Streptomycin (Invitrogen). FBS was heat inactivated for 45 min at 56°C before use. SH-SY5Y cells were differentiated toward a post-mitotic dopaminergic phenotype by the administration of 10μΜ retinoic acid (RA, Sigma) in the cell culture medium during four days and 80 nM of 12-0-tetradecanoylphorbol-13-acetate (TPA, Sigma) during four additional

3. Generation of alpha-synuclein and LAMP2A expressing vectors

Human LAMP2A cDNA were cloned from total human RNA isolated previously from the human brain using the following primers: LAMP2A forward: 5'- ATGGTGTGCTTCCGCCTCTTCCCGG-3'; LAMP2A reverse: 5 -

CTAAAATTGCTCATATCCAGCATGA-3'. The PCR products were purified and ligated into pGEMT-easy vector and subcloned into pCMV-Tag2 vector (Clonetech). Human SNCA 1 cDNA was subcloned from pT7.7 vector (provided by CW Bertoncini, IRB, Barcelona) into pCDNA3- MYC. Site-directed mutagenesis (QuickChange, Stratagene) was performed to generate SNCA 1 mutant constructs changing codon number 53 from GCA to ACA in SNCA1 leading to A->T residue change, and LAMP2A variant vectors at codon 94 from CCT to TCT, TTT or GGT leading to P->S, P->F and P->G residue changes, respectively. The orientation and sequence of the inserts were verified by DNA sequencing. 4. Cell Transfections

Transfection assays were performed using Lipofectamine 2000™ (Invitrogen), according to the manufacturer's instruction. Experiments were carried out using a double transfection protocol. The first step consisted in the delivery of a siRNA against LAMP2A 3'UTR (75nM) in order to knock down endogenous LAMP2A levels. The second transfection was performed 48 hours later, with the co-administration of LAMP2A siRNA (75nM) and the different combinations of alpha-synuclein/LAMP2A expressing vectors (750ng for MW6 plates). Each transfection was performed with a modest amount of vector to maintain a near-wild type level of expression of the exogenous genes. For each co-transfection we knocked down the levels of expression of endogenous LAMP2A. The endogenous alpha-synuclein was not knocked down and we differentiated between the endogenous and exogenous alpha-synucleins by the presence of a 5' Myc tag in the exogenous alpha-synuclein. LAMP2A siRNA (5'- UUUCCCUACUCUCUUUCUC-3') and control, scrambled siRNA used as a negative control (5'-GCGACGUUCCUGAAACCAC-3') were purchased from Dharmacon.

5. Western blotting Total protein extraction was performed at different time points by rinsing the cells with ice-cold PBS. Cells were then solubilized in ice-cold lysis buffer [50 mM Tris-HCI pH 7.4, 10mM Sodium Pyrophosphate, 50mM NaF, 0.5% Triton X-100, 0.5% deoxycholate and 1 X Protease Inhibitor Cocktail (Roche)] and cell lysates were centrifuged at 13000 rpm for 15 min. Samples were resolved in 10% SDS-PAGE gels and transferred to nitrocellulose membranes using the iBlot™Dry Blotting System (Invitrogen). Membranes were blocked for 1 h with 3% BSA in TBS- T (Tris-HCI, pH 7.5, 10 mm; NaCI, 100 mm containing 0.1 % Tween-20), and incubated at 4°C and overnight with primary antibodies (diluted in TBS-T). After washing with TBS-T, membranes were incubated for 45 min at room temperature with the appropriate secondary antibodies (1 :2000 in TBS-T. Detection was performed by ECL™ (Amersham Bioscience) using a LAS-3000 image analyzer (Fuji PhotoFilm Co., Carrollton, TX, USA). Primary antibodies were anti-alpha-synuclein (1 :2000, in 3%BSA-TBS-T; BD Bioscience, mouse), anti- LAMP2A (1 :1000, Santa Cruz Biotechnology, mouse). Anti-alpha-Tubulin antibody (1 :20000, Sigma, mouse) was used as loading controls. Secondary antibodies were HRP-conjugated anti-mouse from DAKO (1 :2000).

6. Determination of alpha-synuclein degradation rate

Protein synthesis was blocked in SH-SY5Y cells expressing the different alpha- synuclein/LAMP2A combinations 21 hours after transfection by replacing cell medium with fresh medium containing 5Dg/ml cycloheximide (CHX). Cells were collected 0, 4, 8, 12 and 24 hours after CHX exposure. Cell lysates at each specific time point were resolved by SDS- PAGE and relative alpha-synuclein levels were determined by western blotting using Tubulin levels as loading control.

7. Cell toxicity assays

Cell death was assayed 24 hours after CHX treatment by using the CytoTox 96 non- radioactive cytotoxicity assay (Promega) according to the manufacturer's protocol. Absorbance was recorded at 490 nm and total release was measured by lysing the cells with 1 % Triton X- 100. In each experiment, determinations were performed in quintuplicate.

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Claims

Claims

1 . A polynucleotide coding for a LAMP2A polypeptide wherein said polypeptide comprises a serine residue at position 94 in SEQ ID NO: 2.

2. A polynucleotide according to claim 1 which is depicted in SEQ ID NO: 1 .

3. A polynucleotide coding for a LAMP2A polypeptide wherein said polypeptide comprises a phenylalanine residue at position 94 in SEQ ID NO: 4.

4. A polynucleotide according to claim 3 which is depicted in SEQ ID NO: 3.

5. A polynucleotide according to claims 1 to 4 for the treatment of synucleinopathies such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), pure autonomic failure (PAF), and multiple system atrophy (MSA).

6. A polynucleotide according to claims 1 to 4 for the treatment of Parkinson's disease wherein the subject has a mutation in the alpha-synuclein gene leading to a threonine at position 53 in the alpha-synuclein protein.

7. A recombinant vector comprising a polynucleotide according to claims 1 , 2, 3 or 4.

8. A recombinant vector according to claim 7 which is a viral vector.

9. A recombinant vector according to claim 7 which is a gene therapy vector.

10. A recombinant vector according to claims 7, 8 or 9 for the treatment of Parkinson's disease.

1 1 . A recombinant vector according to claims 7, 8 or 9 for the treatment of Parkinson's disease wherein the subject has a mutation in the alpha-synuclein gene leading to a threonine at position 53 in the alpha-synuclein protein.

12. A transgenic murine model comprising a variant polynucleotide of LAMP2A in the genome, wherein said variant polynucleotide expresses a LAMP2A polypeptide comprising a proline residue at position 90 of the mice gene.

13. A murine model according to claim 12, wherein said transgenic murine model comprises SEQ ID NO: 5 in the genome.