VECTORS

FIELD OF THE INVENTION

The present invention relates to compounds for use in the gene therapy of neurodegenerative diseases. More specifically, the invention relates to adeno-associated viral (AAV) vectors, for use in the treatment or prevention of neurodegenerative diseases, wherein the vectors enable systemic delivery of β-glucocerebrosidase (GBA1) or fragments or derivatives thereof to a subject. BACKGROUND TO THE INVENTION

Neurodegeneration refers to the progressive loss of structure or function of nerve cells (neurons) including neuronal cell death. Many diseases occur as a result of neurodegeneration and are known collectively as neurodegenerative diseases. These diseases result in progressive degeneration and/or death of neuronal cells and are incurable. There are many similarities between different neurodegenerative diseases including atypical protein assemblies and induced cell death. It is hoped that the similarities between neurodegenerative diseases will allow therapeutic approaches which could ameliorate several diseases simultaneously.

Widespread accumulation of alpha-synuclein (alpha-syn) protein aggregates in Lewy bodies is a key neuropathological hallmark of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), leading to complex and heterogeneous symptomatic manifestations.

GBA1 encodes the enzyme lysosomal glucocerebrosidase (GCase), and heterozygous mutations of the gene are the most common genetic risk factor for PD and DLB. The glucocerebrosidase deficiency in Gaucher disease patients promotes widespread accumulation of substrate glycosphingolipids in various organs, including the brain. Evidence suggests that GCase activity impairment affects lysosomal activity and overall autophagic flux negatively affecting aggregate degradation and catabolism.

It is known in the art that the metabolism and function of different subgroups of neurons is different. For example, lysosomal activity has specific functions in each neuronal cell type. Therefore the treatment of one subgroup of neurons with a particular agent may have a different outcome to the treatment of a different neuron subgroup with the same agent. For example, lysosomal activity has been shown to regulate synaptic plasticity in neurons of the hippocampus (Padamsey et al., Neuron, 2017, Jan 4;93(1):132-146), but this has not been demonstrated for other neurons, such as the dopaminergic neurons in the nigra. Brefeldin A- inhibited guanine nucleotide exchange protein 3 (BIG3) has been shown to regulate GABA neurotransmitters in inhibitory neurons of the cerebral cortex. This is a specific function of the lysosomes for this particular class of neurons. In another example, glutamate (the principal neurotransmitter in the central nervous system) produced by glutamatergic neurons in the cerebral cortex has been shown to induce permeabilisation of lysosomal membranes. Taken together, these studies demonstrate how lysosomes perform different activities in the different classes of neurons. Accordingly, it will be appreciated by one skilled in the art that mechanisms of lysosomal degradation will also depend on the neuronal cell type.

Virus-mediated gene-transfer has been considered as a strategy for gene modification in the nervous system. However, intraparenchymal injection of adeno-associated viruses supports robust but relatively localized transduction in the brain tissue. Thus, while this approach is meaningful for assessing the function of small neuronal clusters, its application to wider neuronal circuitries or large neural areas up to the entire brain remains unfeasible. For the treatment of neurodegenerative diseases with pathology widespread throughout the central nervous system, alternative approaches are needed.

There is a need in the art for new approaches to treat neurodegenerative diseases which globally affect the brain or central nervous system.

SUMMARY OF THE INVENTION

The inventors have surprisingly provided methods for treating and/or preventing neurodegenerative diseases which globally affect the brain or central nervous system without impairing the blood-brain barrier. Herein, the inventors showed that AAV-PHP.B-mediated GBA1 overexpression enabled a robust and long-lasting reduction of alpha-synuclein inclusions in the whole forebrain accompanied by a significant recovery in lifespan and cognitive performance. The inventors also showed that AAV2-BR1 -mediated GBA1 overexpression enabled a robust and long- lasting reduction of alpha-synuclein inclusions in the cerebral cortex and hippocampus.

This present inventors have developed therapeutic methods for the global expression of GBA1. While not wishing to be bound by theory, the inventors' findings suggest that the invention may have a particularly beneficial effect because it is able to provide GBA1 to several different neuron cell types simultaneously and enhance the degradation of protein aggregates and/or the prevention of protein aggregate formation in those different neuron cell types by promoting lysosome activity.

The present method of treatment is able to treat neurons which are otherwise extremely difficult to access.

Surprisingly, the present inventors have demonstrated that the systemic administration of GBA1 is able to target neurons in multiple regions including the hippocampus, the cerebral cortex, and the peripheral nervous system (e.g. dorsal root ganglia), thereby reducing pathology (e.g. a-synuclein aggregates) in these regions and improving learning and/or cognitive performance. A disadvantage of the previous local administration-based approaches is that neurodegenerative diseases (e.g. disorders associated with α-synuclein aggregates) do not typically affect small, localised regions. The pathology of neurodegenerative diseases is usually widespread throughout the CNS. Advantageously, the present invention provides a method to treat multiple regions of the CNS and the PNS afflicted with a neurodegenerative disorder simultaneously.

Without wishing to be bound by theory, this may be particularly beneficial in the treatment of disorders which demonstrate pathology in the hippocampus, cerebral cortex and peripheral nervous system. The hippocampus is critical for controlling cognitive and memory function. The cerebral cortex and the hippocampus are main areas of α-synuclein accumulation which lead to dementia in patients with Parkinson's disease. Therefore in one advantageous embodiment, the present invention provides a method for treating Lewy body associated disorders such as dementia (e.g. Lewy body dementia associated with Parkinson's disease). The inventors surprisingly found that not only was the present invention able to express GBA1 in different types of cells in different parts of the central nervous system and peripheral nervous system, furthermore the present invention provides GBA1 to the correct part of the cell (i.e. the lysosome) and said GBA1 is functional within the lysosome (i.e. is able to reduce α-synuclein pathology). These findings were particularly surprising because it is well known in the art that neurons are extremely heterogeneous in their function and metabolism. It was completely unexpected that the use of an AAV would be able to target and have a functional effect in different types of neurons. The global expression of GBA1 provided by the present invention is also able to target dopaminergic neurons which make up less than 1 % of the total number of brain neurons. These neurons are vital to diverse brain functions including: voluntary movement, reward, addiction and stress. The progressive loss of dopaminergic neurons is responsible for Parkinson's disease amongst other neurodegenerative diseases. Dopaminergic neurons are located in the diencephalon, mesencephalon, medulla oblangata, hypothalamus, retina and the olfactory bulb. More than 70% of all dopaminergic neurons are located in the substantia nigra.

The present inventors now provide an approach for providing GBA1 to the central nervous system (e.g. the brain) or the brain endothelium which is useful, for example, in the treatment of neurodegenerative diseases. The inventors provide GBA1 delivered by gene therapy with the purpose of reducing alpha-synuclein pathology and/or glucocerebroside pathology. The result is an improvement in learning and/or cognitive performance.

In one aspect, the invention provides an adeno-associated viral (AAV) vector particle adapted for targeting the brain endothelium (e.g. for transducing brain endothelial cells), wherein the AAV vector particle comprises a nucleotide sequence encoding β- glucocerebrosidase (GBA1 ) or a fragment or derivative thereof. In another aspect, the invention provides an adeno-associated viral (AAV) vector particle adapted for crossing the blood-brain barrier, wherein the AAV vector particle comprises a nucleotide sequence encoding β-glucocerebrosidase (GBA1 ) or a fragment or derivative thereof.

In one embodiment, the nucleotide sequence encoding GBA1 or fragment or derivative thereof comprises a sequence selected from the group consisting of:

(a) a nucleotide sequence encoding an amino acid sequence that has at least 70% identity to SEQ ID NO: 1 (GBA1 );

(b) a nucleotide sequence that has at least 70% identity to SEQ ID NO: 2 (GBA1 ); and

(c) the nucleotide sequence of SEQ ID NO: 2 (GBA1). Preferably the nucleotide sequence encodes an amino acid sequence that has at least 75%, 80%, 85% or 90% identity to SEQ ID NO: 1. More preferably the nucleotide sequence encodes an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1. Preferably the nucleotide sequence has at least 75%, 80%, 85% or 90% identity to SEQ ID NO: 2. More preferably the nucleotide sequence has at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2.

In one embodiment, the AAV vector particle comprises an artificial capsid amino acid sequence.

In a preferred embodiment, the AAV vector particle comprises an artificial capsid amino acid sequence which targets the viral particle to brain microvascular endothelial cells.

Suitably, the AAV vector particle comprises a capsid protein comprising the amino sequence NRGTEWD (SEQ ID NO: 5) or DWETGRN (SEQ ID NO: 6). In a preferred embodiment, the AAV vector particle comprises a BR1 AAV2 capsid.

In one embodiment, the artificial capsid amino acid sequence enables the vector particle to cross the blood-brain barrier.

Suitably, the AAV vector particle comprises a VP1 capsid protein comprising an amino acid sequence comprising at least four contiguous amino acids from the sequence TLAVPFK (SEQ ID NO: 3) or KFPVALT (SEQ ID NO: 4).

In a preferred embodiment, the AAV vector particle comprises a PHP.B AAV9 capsid.

In another aspect, the invention provides a cell transduced with the AAV vector particle of the invention.

Preferably the invention provides a blood-brain barrier associated endothelial cell transduced with the AAV vector particle of the invention. Preferably the invention provides a neuronal cell transduced with the AAV vector particle of the invention. Preferably the invention provides a dopaminergic neuronal cell transduced with the AAV vector particle of the invention. Preferably the invention provides a glial cell transduced with the AAV vector particle of the invention. In yet another aspect, the invention provides a pharmaceutical composition comprising the AAV vector particle of the invention, or the cell of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In one embodiment, the pharmaceutical composition of the invention is formulated for systemic delivery. In a preferred embodiment, the pharmaceutical composition of the invention is formulated for intravascular or intra-arterial delivery.

In one aspect the invention provides a method for expressing GBA1 in a subject, the method comprising: providing the AAV vector particle of the invention, or the cell of the invention, or the pharmaceutical composition of the invention; and administering the AAV vector particle, cell, or pharmaceutical composition to the subject.

In a preferred embodiment, GBA1 is expressed in or provided to the central nervous system and/or the peripheral nervous system of the subject. In a preferred embodiment, GBA1 is expressed in or provided to the brain of the subject. In a preferred embodiment, GBA1 is expressed in or provided to blood-brain barrier associated endothelial cells. In a preferred embodiment, GBA1 is expressed in or provided to the lysosome. In a preferred embodiment GBA1 is expressed in or provided to a neuron. Suitably, GBA1 is expressed in or provided to a dopaminergic neuron.

In one embodiment, the AAV vector particle, cell or pharmaceutical composition is administered systemically.

In one embodiment, the AAV vector particle, cell or pharmaceutical composition is administered intravenously or intra-arterially.

In a preferred embodiment, the AAV vector particle, cell or pharmaceutical composition of the invention is administered to the internal carotid artery. Advantageously, in a preferred embodiment, the administration of the AAV vector particle does not impair blood-brain barrier integrity or affect permeability.

In a preferred embodiment, following administration the peripheral organs do not comprise the AAV vector particle and/or the exogenous nucleotide sequence encoding the GBA1 gene or fragment or derivative thereof. In another aspect, the invention provides a method of treating or preventing a neurodegenerative disease comprising delivering to a subject having or at risk of having the neurodegenerative disease an AAV vector particle of the invention, or the cell of the invention, or the pharmaceutical composition of the invention.

In a preferred embodiment, the method of treating or preventing a neurodegenerative disease comprises transducing one or more cells in the subject with the AAV vector particle of the invention. Suitably, the method of treatment comprises transducing blood-brain barrier associated endothelial cells. Suitably the method of treatment comprises transducing neuronal cells. Suitably the method of treatment comprises transducing dopaminergic neuronal cells. Suitably the method of treatment comprises transducing glial cells. Suitably, GBA1 is expressed by the transduced cells at a therapeutically effective level, thereby treating or preventing the neurodegenerative disease. In one embodiment, the subject is mammalian, preferably a human.

In one embodiment, the AAV vector particle of the invention, or the cell of the invention, or the pharmaceutical composition of the invention is delivered to the subject by systemic administration.

In one embodiment, the AAV vector particle of the invention, or the cell of the invention, or the pharmaceutical composition of the invention is delivered to the subject by intravenous or inter-arterial administration.

In one embodiment, GBA1 is provided to the central nervous system and/or the peripheral nervous system of said subject.

In one embodiment, GBA1 is provided to the forebrain, midbrain, cortex, hippocampus and/or cerebellum.

In another embodiment, GBA1 is provided to the dorsal root ganglia and/or sympathetic ganglia.

In one embodiment, GBA1 is provided to glial and/or neuronal cells, preferably to dopaminergic neurons. In a preferred embodiment GBA1 is provided to the lysosome. Preferably the treatment reduces protein aggregates in the lysosome.

In another preferred embodiment, a-synuclein pathology is reduced in the subject.

In one embodiment, a-synuclein pathology is reduced in at least one region selected from the group consisting of the visual cortex, the somatosensory cortex and the hippocampus. In one embodiment, the neurodegenerative disease is Parkinson's disease. In a preferred embodiment, the neurodegenerative disease is Parkinson's dementia.

In another embodiment, the neurodegenerative disease is Lewy Body dementia. Preferably the neurodegenerative disease is diffuse Lewy body dementia. Preferably the neurodegenerative disease is Lewy body variant of Alzheimer's disease. In another embodiment the neurodegenerative disease is multiple system atrophy. In another preferred embodiment, glucocerebroside pathology is reduced in the subject.

In one embodiment, glucocerebroside pathology is reduced in at least one region selected from the group consisting of the visual cortex, the somatosensory cortex and the hippocampus. In a further embodiment, the neurodegenerative disease is Gaucher disease.

In a preferred embodiment, the subject has or is at risk of developing one or more neurodegenerative diseases. Preferably the subject has Parkinson's disease and dementia, preferably Parkinson's disease and Parkinson's dementia or Parkinson's disease and Lewy body dementia. Preferably the subject has Parkinson's disease and Gaucher disease. Preferably the subject has Gaucher disease and dementia, preferably Lewy body dementia.

In one aspect, the invention provides a method for reducing alpha-synuclein pathology e.g. for reducing alpha-synuclein aggregates, the method comprising, administering the AAV vector particle of the invention to a cell. Suitably the cell is transduced by the AAV vector particle and expresses GBA1 which is encoded by the AAV vector particle. Suitably GBA1 expressed by the transduced cell reduces alpha-synuclein pathology in the transduced cell and/or the GBA1 is secreted by the transduced cell, wherein it is taken up by a second, for example neighbouring, cell and alpha-synuclein pathology is reduced in the second cell. Suitably the method for reducing alpha-synuclein pathology is an in vivo method. Suitably the method for reducing alpha-synuclein pathology is an in vitro method.

In another aspect, the invention provides a method for reducing glucocerebroside pathology, e.g. for reducing glucocerebroside aggregates, the method comprising, administering the AAV vector particle of the invention to a cell. Suitably the cell is transduced by the AAV vector particle and expresses GBA1 which is encoded by the AAV vector particle. Suitably GBA1 expressed by the transduced cell reduces glucocerebroside pathology in the transduced cell and/or the GBA1 is secreted by the transduced cell, wherein it is taken up by a second, for example neighbouring, cell and glucocerebroside pathology is reduced in the second cell. Suitably the method for reducing glucocerebroside pathology is an in vivo method. Suitably the method for reducing glucocerebroside pathology is an in vitro method.

In one aspect, the invention provides a method for improving learning and/or cognitive function in a subject with a neurodegenerative disease, the method comprising, administering the AAV vector particle or agent of the invention to the subject. Suitably the AAV vector particle transduces at least one cell in the subject and preferably, the transduced cell expresses GBA1 which is encoded by the AAV vector particle. Suitably GBA1 expressed by the transduced cell reduces alpha-synuclein pathology in the transduced cell and/or the GBA1 is secreted by the transduced cell, wherein it is taken up by a second, for example neighbouring, cell and alpha-synuclein pathology is reduced in the second cell, thereby improving learning and/or cognitive function.

In one aspect, the invention provides a method for improving learning and/or cognitive function in a subject with a neurodegenerative disease, the method comprising, administering the AAV vector particle or agent of the invention to the subject. Suitably the AAV vector particle transduces at least one cell in the subject and preferably, the transduced cell expresses GBA1 which is encoded by the AAV vector particle. Suitably GBA1 expressed by the transduced cell reduces glucocerebroside pathology in the transduced cell and/or the GBA1 is secreted by the transduced cell, wherein it is taken up by a second, for example neighbouring, cell and glucocerebroside pathology is reduced in the second cell, thereby improving learning and/or cognitive function.

DESCRIPTION OF THE DRAWINGS

Figure 1. Global GFP expression in the central and peripheral nervous system with a single AAV-PHP.B intravenous injection.

A. Schematic view depicting the transgenic cassette integrated in the AAV-PHP.B vector and injection of the viral particles into the tail vein of an adult mouse. B. GFP immunofluorescence in brain transduced with an empty AAV-PHP.B (negative control). C-E. GFP localization on coronal hemi-sections at different rostro-caudal levels of the AAV- PHP.B-GFP transduced brain. F-H High-magnification images of cerebral cortex (F), substantia nigra (G) and cerebellum (H) showing the double staining for GFP and the neuronal marker NeuN, TH and Calbl , respectively. I. Bar graph showing the fraction of cells positive for a specific neuronal marker (NeuN, TH and Calbl ) and expressing the viral GFP transgene. J. GFP staining on coronal section of the AAV-PHP.B-GFP transduced spinal cord. K, L. Section of thoracic dorsal root (DRG) or sympathetic ganglion (SG) co-stained for GFP and beta-lll-Tubulin (K) or TH (L), respectively. M. Bar graph showing the fraction of cells positive for a specific neuronal marker (Hb9, beta-lll-Tubulin and TH) and expressing the viral GFP transgene. (n = 12 mice for B-J; n = 4 mice for K-M). Scale bars: 500 pm (B- E,J); 100 pm (G,H); 50 pm (K,L). Figure 2. AAV-PHP.B transduction associated to Cre-loxP technology enables the labeling of a specific neural subpopulation throughout the brain.

A. Schematic view depicting the GFP FLEX cassette integrated in the AAV-PHP.B vector and injection of the viral particles into the tail vein of Cre-expressing transgenic mouse strains. B. tdTomato staining on a forebrain coronal section of AAV-PHP.B-Cre transduced Ai9 tdTomato reporter strain. The highly diffuse activation of the reporter demonstrates the highly efficient Cre-mediated recombination occurred after viral transduction (positive control). C,D. GFP localization in the transduced NeuroD6-Cre forebrain (C) and cortical tissue (D). E. Co-labeling of GFP and NeuN in NeuroD6-Cre transduced cortical tissue. F. Bar graph showing the percentage of cortical GFP positive on total NeuN positive cells. G-l. Co-staining between GFP and Parvalbumin (PV) in the PV-Cre transduced cortical tissue. J. Bar graph depicting the fraction of GFP positive on total PV expressing neurons in the cortex. K-O. Double staining for GFP and TH on infected DAT-Cre ventral midbrain tissue and quantification of the percentage of GFP expressing cells within the TH cellular fraction. P. GFP immunofluorescence on infected Olig2-Cre cortical tissue. Q,R. GFP transduced cells co-express the oligoglial CC1 (Q), but not astrocytic GFAP (R) marker, identifying them as oligodendrocytes. S. Bar graph quantifying the percentage of transduced GFP cells within the CC1 expressing cellular fraction, (n = 3 mice for each Cre-transgenic line). Scale bars: 200 pm (A,B); 200 pm (K-M); 100 pm (D); 50 pm (G-I,N,P); 30 pm (E); 20 pm (Q,R).

Figure 3. AAV-PHP.B-mediated targeting of the DREADD M4 chemogenetic inhibitory receptor in PV ⁺ cortical interneurons sensitizes the mice to pro-epileptic insults.

A. Schematic view depicting the chemogenetic DREADD M4 inhibitory receptor fused to mCherry cloned in a FLEX cassette and integrated in the AAV-PHP.B vector and injection of the viral particles into the tail vein of a PV-Cre adult mouse. B, C. Parvalbumin (PV) (B) and mCherry (C) immunofluorescence on transduced forebrain coronal sections. D. High- magnification images of cortical tissue co-stained for PV and mCherry. E. Bar graph depicting the relative fractions of PV and mCherry double-positive cells, (n = 4 PV-Cre mice). F. Electrophysiological recordings on transduced PV-Cre brain slices showing that CNO perfusion strongly inhibits the membrane excitability of mCherry positive neurons (n = 6). G. Representative EEG traces of 12hr recordings after CNO injection into transduced PV-Cre mice. (3 recordings in 4 mice). H. Bar graph showing the number of mice succumbed after treatment with kainic acid (KA) between the two animal groups treated either with the GFP or the DM4C expressing viruses. Scale bars: 500 pm (B,C); 00 pm (D).

Figure 4. Complete or partial loss of Tsc1 in the adult brain mediated by the Cre-expressing AAV-PHP.B leads to severe epileptic seizures. A. Experimental set up for the tail vein injection of the virus AAV-PHP.B-Cre in Tsc1 ^{flox flox} mice. B. Representative traces of EEG recordings in baseline state (above) and during seizure (bottom) in treated Tsc1 ^flox/flox mice injected with a viral dose of 2x10 ¹² vg (LE: left emisphere; RE: Right emisphere). C. Quantification of epileptic events in 12 hrs (3 recordings in 3 mice). D,E. tdTomato direct signal and pS6 immunofluorescence on cortical tissue of transduced Ai9 tdTomato reporter mice. F,G. Images at different magnification of transduced TSC1 ^{fl0X fl0X} cortical tissue stained for pS6. H. Quantification of tdTomato and pS6 positive cells on the NeuN neuronal fraction in transduced Ai9 or TSC1 ^{flox flox} mice, respectively (n = 3 mice). I. Representative traces of EEG recordings in baseline state (above) and during seizure (bottom) in treated Tsc1 ^floxfflox mice injected with a viral dose of 5x10 ¹⁰ vg (LE: left emisphere; RE: Right emisphere). J. Quantification of epileptic events in 12 hrs (3 recordings in 2 mice). K,L. tdTomato direct signal and pS6 immunofluorescence on cortical tissue of transduced Ai9 tdTomato reporter mice. M,N. Images at different magnification of transduced TSC1 ^fl0X/fl0X cortical tissue stained for pS6. O. Quantification of tdTomato and pS6 positive cells on the NeuN neuronal fraction in transduced Ai9 or _{TSC 1} flox/flox _micei respectively, (n = 3 mice). Scale bars: 100 pm (D-F.K-M); 50 pm (G,N).

Figure 5. AAV-PHP.B intravenous delivery enables a global stimulation of GCase activity in adult A53T-SCNA mice.

A-C. Immunostaining for phospho-S129-a-syn show the diffuse accumulation of PK resistant a-syn deposits in the somato-sensory cortex (SCx) of 8 month olds A53T-SCNA transgenic mice mainly localized in the neuronal soma (arrowheads in B and arrows in C). D-G. Representative pictures showing the amount and distribution of GFP transduced brain cells in cortex (D), dentate gyrus (E), striatum (F) and thalamus (G) in 10 month old A53T-SCNA mice infused with the AAV-PHP.B-GBA1-P2A-GFP virus. H. Immunoblotting analysis showing the total amount of GCase protein in cortical and hippocampal tissues of wild-type (WT) and A53T-SCNA transgenic mice treated with the GFP- or GBA1 -expressing AAV- PHP.B. i. Bar graph illustrating the quantification of the GCase the immunoblotting signal (n = 3 A53T-SCNA + GFP; n = 3 A53T-SCNA + GBA1 ; n = 3 WT tested at 3 months after infection; p < 0.05). J. Direct quantification of total GCase catalytic activity showing a significant recovery of the enzymatic activity in spinal cord (Sc), cerebral cortex (Sc), Hippocampus (Hp), striatum (Str), midbrain (Mb) and cerebellum (Cb) of the treated A53T- SCNA transgenic mice (yellow bars). The GCase selective inhibitor conduritol-B- epoxide (CBE) was included to evaluate the specificity of the reaction, (n = 3 WT; n = 3 A53T-SCNA + GFP; n = 3 A53T-SCNA + GBA1 tested at 3 months after infection). Data are expressed as mean + SEM and analyzed by unpaired Student's f-test (* < 0.05, ** < 0.01 , *** < 0.001 ). Scale bars: 100 pm (B,C, D-G); 10 pm (C). Figure 6. Global brain GCase gene transfer ensures a diffuse protection from a-syn deposits throughout all the forebrain regions in adult A53T-SCNA mice.

A-D. Immunohistochemistry (A,B) and immunofluorescence (C,D) analysis for pS129-a-syn on PK-treated visual cortical tissue from 10 month old A53T-SCNA transgenic mice treat with GFP (control) or GCase expressing AAV-PHP.B. Insets in A and B are high power enlargements showing the a-syn deposits concentrated in the cytoplasm of the cortical neurons, (n = 6 A53T-SCNA + GFP; n = 5 A53T-SCNA + GBA1 ). E. Total number of insoluble a-syn inclusions in different forebrain regions as quantified by semi-automatic stereology counting in selected forebrain areas (vCx, ventral cortex; sCx, somatosensory cortex; mCx, motor cortex; cCx, cingulate cortex). Counting was automatically performed in a selected patterning within the brain tissue as highlighted in the drawing. F. Immunoblotting with TBS and SDS soluble tissue lysates from GFP and GBA1 transduced brains detecting the monomeric (m) and high- (HMW) and low-molecular weight (LMW) a-syn aggregates. * Unspecific band. Quantitative analysis showed a significant reduction of a-syn monomeric and aggregated species protein after GCase treatment, (n = 3 A53T-SCNA + GFP; n = 3 A53T-SCNA). Data are expressed as mean + SEM and analyzed by unpaired Student's t- test (* < 0.05, ** < 0.01 , *** < 0.001). Scale bars: 50 μηη (A-D); 10 Mm (insets in A, B). Figure 7. AAV-PHP.B carotid artery delivery efficiently targets the neuraxis while sparing peripheral organ viral transduction.

A. Schematic view depicting the transgenic cassette integrated in the AAV-PHP.B vector and injection of the viral particles into the carotid artery of an adult mouse. B. GFP immunofluorescence in brain transduced with an empty AAV-PHP.B (negative control). C-E. GFP localization on coronal hemi-sections at different rostro-caudal coordinates of a brain transduced with the AAV-PHP.B-GFP virus. F-H. High-magnification images of cerebral cortex (F), mesencephalic nigral tissue (G) and cerebellum (H) showing the double staining for GFP and the neuronal marker NeuN, TH and Calbl , respectively. I. Bar graph showing the fraction of cells positive for a specific neuronal marker (NeuN, TH and Calbl ) and expressing the viral GFP transgene. J-Q. GFP localization in the peripheral organs liver (J,N), heart (Κ,Ο) and muscles (L,P) after injection into either the tail vein (J-M) or the carotid artery (N-Q). ,Q. Bar graphs showing the percentage of GFP positive on total cells. Note that the artery route substantially reduces viral targeting in peripheral organs, (n = 3 mice). Scale bars: 500 μηι (B-E); 200 μιη (J-L, N-P); 100 μηι (G); 50 μηη (H); 20 μηη (F).

Figure 8. The AAV-PHP.B brain transduction does not affect BBB permeability in vivo and endothelial integrity in vitro. A. Experimental set up for the tail vein injection of the AAV-PHP.B-GFP in wild-type adult mice. B. Viral capsid staining using the B1 anti-AAV VP3 antibody reveals a robust targeting of the AAV-PHP.B-GFP in the brain endothelium 4 hrs after infection. C. Alexa Fluor-555 conjugated with cadaverine is undetectable in brain parenchyma 24 hrs after AAV-PHP.B injection. D. No evident sign of astrogliosis (GFAP staining) is present 2 days after AAV- PHP.B injection. E. Cadaverine staining in the brain parenchyma in kainic acid (KA) treated animals F. High magnification of cadaverine staining in the cortical tissue. G. Strong GFAP positive astrogliosis in KA injected animals. H. Schematic view depicting the infection of brain microvascular endothelial cells (BMVECs) with the AAV-PHP.B-GFP. i-l. Immunofluoresce for the cell-cell junction markers Z01 and Claudin-5 (Cld5) in confluent BMVECs either untreated (l,J) or infected with the AAV-PHP.B-GFP (K,L). Transduced cells are visible for GFP expression in K and L. M. Transendothelial electrical resistance (TEER) analysis of confluent BMVEC cultures infected with AAV-PHP.B-GFP at time 0 (purple line). Cultures never exposed to virus were used as stable baseline controls (blue line). EDTA in the culture medium leads to a strong loss of the TEER signal (yellow line). All treatments were performed in triplicate. Scale bars: 50 pm (B-G); 200 pm (K-M); 10 pm (l-L).

Figure 9. Intravenous delivery of AAV-PHP.B exerts a diffuse and efficient transduction of the adult central and peripheral nervous system. Related to Figure 1.

A. Schematic view depicting the transgenic cassette integrated in the AAV-PHP.B vector and injection of the viral particles into the tail vein of an adult mouse. B-G. Representative images of GFP immunofluoresce on coronal sections of the whole forebrain (B,E), cortical tissue (C), thoracic spinal cord (D), whole cerebellum (F) and dorsal root ganglia (DRG) (G) 3 weeks after in vivo transduction, (n = 12 mice). Scale bars: 500 pm (B,E,F); 100 pm (D,G); 50 pm (C).

Figure 10. AAV-PHP.B transduced cells in peripheral ganglia exhibit different levels of transgene GFP expression. Related to Figure 1.

Single channel photographs and relative merge of the immunofluorescence stainings for dorsal root (DRG) (A-C) and sympathetic ganglia (SG) (D-F). In B and E is evident the presence of AAV-PHP.B-GFP transduced cells with different levels of GFP expression and some with very dim GFP signal. Arrows in inset in B indicate viral transduced cells with low expression levels, while arrowheads point to GFP negative cells. Scale bar: 100 pm. Figure 11. Injection of the AAV-PHP.B into the facial vein of neonatal mice promotes an efficient and global transduction of the neuraxis. Related to Figure 1. A. Schematic view depicting the transgenic cassette integrated in the AAV-PHP.B vector and injection of the viral particles into the facial vein in a neonatal mouse. B-H. GFP localization on coronal sections of forebrain (B), hippocampus (C), cortical tissue (D), substantia nigra (E), cerebellum (F,G) and spinal cord (H). D,E,G. Co-staining between GFP and the neuronal markers NeuN (D), TH (E) and Calbl (G). I. Bar graph showing the fraction of cells positive for a specific neuronal marker (NeuN, TH and Calbl ) and expressing the viral GFP transgene. J,K. GFP staining in liver and heart peripheral organs. L. Bar graph showing the fraction GFP transduced cells in liver and heart, (n = 6 neonatal pups). Scale bars: 500 μπι (B); 200 Mm (F-H.J); 100 pm (C-Ε,Κ).

Figure 12. Low-dosed AAV-PHP.B virus enables spare and single neuronal labeling throughout the brain. Related to Figure 2.

A. Schematic view depicting the transgenic cassette constitutively expressing the Cre recombinase and integrated in the AAV-PHP.B vector employed for injection of the viral particles into the tail vein of an adult Ai9 tdTomato reporter mouse. B-K. tdTomato immunofluorescence on coronal sections of cortex (B-E), hippocampus (F-H) and cerebellum (l-K) of Ai9 mice transduced with the Cre-expressing AAV-PHP.B at three (dentate gyrus and cerebellum) or four (cortex) different doses. Note that at lowest dose (10 ⁹ vg) of AAV-PHP.B-Cre, the viral transduction targeted few sparse neurons in these tissues enabling a close morphological inspection of the infected neurons, (n = 3 mice for each viral dose). Scale bars: 100 pm.

Figure 13. Exogenous GCase is targeted to the lysosomes and acquires functional activity. Related to Figure 5.

A. Confocal image of HeLa cells transfected with a GBA1-mCherry expression vector show partial co-labelling between mCherry and LAMP2 (arrows) indicating correct targeting of the exogenous GCase into the lysosomes. B. GCase enzymatic activity assay shows a substantial increase in total catalytic activity in the GBA1-mCherry transfected as compared to wild-type (WT) cells. The GCase selective inhibitor conduritol-B-epoxide (CBE) was included to evaluate the specificity of the reaction. C. Immunoblotting for GCase protein levels in wild-type and GBA1 transfected HeLa cells. Arrowheads point to the faint, but specific, GCase protein signal detectable in wild-type cells. Scale bar: 10 pm (A).

Figure 14. GBA1 treated A53T-SCNA mice showed a significant extended survival and behavioral improvement. Related to Figure 6.

A. Kaplan-Meier survival curves of GFP and GBA1 treated A53T-SCNA mice showing a significant increase in lifespan and rescue from mortality after global GBA1 gene transduction. B. In the novel object recognition test, GBA1 treated A53T-SCNA mice show a significant recovery in learning and memory as revealed by an increase discrimination index (Dl) indicating a significantly higher exploration time for the novel object as compared to control GFP transduced A53T-SCNA mice (n = 6 wild-type; n = 8 GBA1 treated A53T-SCNA; n = 8 GFP treated A53T-SCNA; tests at 3 and 5 months after gene transduction; * < 0.05, *** < 0.001 )

Figure 15. AAV2-BR1 infects the blood brain barrier. Intravascular administration of the AAV2-BR1-GFP virus is transducing the entire brain endothelium (blood-brain barrier) as shown by double immunohistochemistry for GFP and the endothelium specific marker CD31 in the cerebral cortex and hippocampus.

Figure 16. AAV2-BR1 is not transducing the endothelium of peripheral organs such as liver. A. Intravascular delivery of the AAV2-BR1-GFP virus did not enable the transduction of the endothelium in peripheral organs such as liver. B. Bar graph showing the fraction of CD31 + endothelial cells transduced with the AAV2-BR1-GFP virus. STR, striatum; CRB, Cerebellum; CRT, cerebral cortex; HP, Hippocampus; SP, spinal cord.

Figure 17. GCase enzyme is secreted and re-uptaked by surrounding cells. A. Overview of the experimental setting. B. Bar graph showing the GCase levels in C EM endothelial cells transduced with either AAV-BR1-GFP or AAV-BR1-GBA. C. Bar graph showing GCase activity in GBA knock-out HeLA cells exposed to conditioned media of AAV-BR1 -GFP or AAV-BR1 -GBA transduced cells. Data are expressed as means ± SEM and were analyzed with the unpaired Student's t test (**p < 0.01 ).

Figure 18. GCase is released by the transduced endothelium and re-uptaked by GBA knock-out cells. A. Scheme showing the experimental procedure. B. Bar graph showing the GCase enzymatic activity in the immuno-isolated CD31 + primary brain endothelial cells from animals transduced with AAV-BR1-GFP (green) or AAV-BR1 -GBA (blue) viruses. C. Bar graph showing the GCase enzymatic activity in the immuno-isolated CD31- cellular fraction, which includes neural and glial cells, from animals transduced with AAV-BR1-GFP (green) or AAV-BR1-GBA (blue) viruses. Data are expressed as means + SEM and were analyzed with the unpaired Student's t test (*p < 0.05, **p < 0.01). Figure 19. AAV2-BR1 -GBA transduced A53T-SCNA mice show a significant reduction of alpha-synuclein aggregates. A. Immunostaining for pS129-alpha-synuclein in PK-treated brain sections of A53T-SCNA transgenic mice transduced with AAV-BR1 -GFP or AAV-BR1 - GBA viruses. Arrows point to alpha-synuclein aggregates in the cerebral cortical tissues and are strongly reduced in the AAV-BR1-GBA transduced tissue. B. Bar graph showing the quantification of the average number of alpha-synuclein aggregates in visual (vCx) and somatosensory (sCx) cortex and hippocampus (hip) in AAV-BR1-GFP (blue) or AAV-BR1- GBA (violet) treat mice. Data are expressed as means ± SEM and were analyzed with the unpaired Student's t test (**p < 0.01 , ***p < 0.001).

DETAILED DESCRIPTION OF THE INVENTION

Neurodegenerative disease Neurodegenerative diseases are diseases which primarily affect neurons. These diseases result in the progressive loss of structure or function of nerve cells (neurons) including neuronal cell death. This causes problems with movement (called ataxias), or mental functioning (called dementias).

The invention encompasses agents, compositions and methods for treating and/or delaying progression of a neurodegenerative disease in a subject. The subject may have a neurodegenerative disease, i.e. has been diagnosed as having the neurodegenerative disease or is suspected as having the neurodegenerative disease, or may be asymptomatic. The subject can have a genetic predisposition to the neurodegenerative disease. The subject may have one or more family members with a neurodegenerative disease. In one embodiment, the neurodegenerative disease is a synucleinopathy. Synucleinopathies are neurodegenerative diseases characterised by the abnormal accumulation of aggregates of alpha-synuclein in neurons, nerve fibres or glial cells. Three types of synucleinopathies are Parkinson's disease, dementia with Lewy bodies and multiple system atrophy.

In one embodiment, the neurodegenerative disease is Parkinson's disease. In one embodiment, the neurodegenerative disease is a Parkinson's disease-related disorder. Preferably the Parkinson's disease-related disorder is Parkinson's dementia. Preferably, the Parkinson's disease-related disorder is dementia with Lewy bodies. In one embodiment, the neurodegenerative disease is lower body Parkinson's syndrome.

In one embodiment, the neurodegenerative disorder is associated with Lewy bodies. Lewy bodies are abnormal aggregates of protein that develop inside nerve cells and may be found within the substantia nigra or within the cortex. Lewy bodies comprise alpha-synuclein associated with other proteins which may include ubiquitin, neurofilament protein and alpha B crystallin. Lewy neurites may be associated with Lewy bodies. In one embodiment, the neurodegenerative disease is dementia with Lewy bodies.

In one embodiment, the neurodegenerative disease is multiple system atrophy. In one embodiment, the neurodegenerative disease is multisystemic atrophy cerebellar type (MSA- C). In one embodiment, the neurodegenerative disease is fronto-temporal dementia. In one embodiment, the neurodegenerative disease is fronto-temporal dementia with Parkinson's disease.

In one embodiment, the neurodegenerative disease is Gaucher disease.

The neurodegenerative disease may be associated with defects in lysosomal storage. The invention encompasses agents, compositions and methods for treating or delaying progression of a lysosomal storage disease in a subject. The subject may have a lysosomal storage disease, i.e. has been diagnosed as having the lysosomal storage disease or is suspected as having the lysosomal storage disease, or may be asymptomatic. The subject can have a genetic predisposition to the lysosomal storage disease. The subject may have one or more family members with a lysosomal storage disease.

"Lysosome storage disease", as used herein, refers to any condition that involves defects in or disruption of lysosomal function. These defects are usually a consequence of a deficiency of an enzyme required for the metabolism of lipids, glycoproteins or mucopolysaccharides.

Gaucher disease is one of the most common lysosomal storage diseases. It is a genetic disorder in which glucocerebroside accumulates in cells and certain organs. The disease is caused by a hereditary deficiency in the enzyme glucocerebrosidase which is located in the lysosomes. When the enzyme is defective, glucocerebroside accumulates and can collect in the spleen, live, kidneys, lungs, brain and bone marrow. Macrophages are unable to eliminate the accumulated enzyme, which accumulates in fibrils, and turn into 'Gaucher cells'. The glucocerebrosidase deficiency in Gaucher patients promotes widespread accumulation of substrate glycosphingolipids in various organs, including the brain.

Neurological symptoms occur in some types of Gaucher disease. Neurological symptoms in Type I Gaucher disease include impaired olfaction and cognition. Neurological symptoms in Type II Gaucher disease include serious convulsions, hypertonia, mental retardation, and apnea. Neurological symptoms in Type III Gaucher disease include muscle twitches known as myoclonus, convulsions, dementia, and ocular muscle apraxia. Parkinson's disease is more common in Gaucher disease patients and their heterozygous carrier relatives than in the general population. Gaucher disease types II and III exhibit neurological defects which cannot be treated with protein replacement since the protein injected into the blood stream cannot penetrate the blood-brain barrier. Gene therapy (such as with an intravascular injection) may be advantageous when capable of transferring GBA1 to wide areas of the brain to treat the widespread pathology of the neurological disorder.

In one embodiment the neurodegenerative disease associated with defects in lysosomal storage is Gaucher disease.

In a preferred embodiment the neurodegenerative disease associated with defects in lysosomal storage is Gaucher disease type I (non-neuropathic). In a preferred embodiment the neurodegenerative disease associated with defects in lysosomal storage is Gaucher disease type II (acute infantile neuropathic).

In a preferred embodiment, the neurodegenerative disease associated with defects in lysosomal storage is Gaucher disease type III (chronic neuropathic).

In one embodiment the subject has one or more neurodegenerative diseases or lysosome storage diseases. Preferably the subject has one or more neurodegenerative disease or lysosome storage diseases described herein.

Suitably, the subject may have Parkinson's disease and dementia. Preferably the subject may have Parkinson's disease and Parkinson's dementia. Preferably the subject may have Parkinson's disease and dementia with Lewy bodies. Suitably, the subject may have Gaucher disease and Parkinson's disease. Suitably, the subject may have Gaucher disease and dementia. β-Glucocerebrosidase (GBA1)

In one aspect the invention relates to the treatment or prevention of a glucocerebrosidase mediated neurodegenerative disease or lysosomal storage disease. For example, in pathological conditions, glucocerebroside aggregates and causes dysfunction. β-Glucocerebrosidase (also called acid β-glucosidase, D-glucosyl-N-acylsphingosine glucohydrolase, or GCase) is an enzyme with glucosylceramidase activity (enzyme accession EC 3.2.1.45) that is needed to cleave, by hydrolysis, the beta-glucosidic linkage of the chemical glucocerebroside, an intermediate in glycolipid metabolism. It is localized in the lysosome. Mutations in this gene cause Gaucher disease, a lysosomal storage disease characterised by accumulation of glucocerebrosidase substrates. An example of β-glucocerebrosidase is the human β-glucocerebrosidase protein having the UniProtKB accession number P04062.

In one embodiment, the amino acid sequence of β-glucocerebrosidase is the canonical (i.e. wild-type) amino acid sequence of β-glucocerebrosidase which is not associated with glucocerebrosidase pathology.

In one embodiment β-glucocerebrosidase has the NCBI Reference Sequence Number: NP_000148.2.

In one embodiment β-glucocerebrosidase has the UniProtKB accession number P04062-1. An example β-glucocerebrosidase amino acid sequence is that of SEQ ID NO: 1 : MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSWCVCNATY CDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFG GAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDF QLH NFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIY HQ TWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPT L ANSTHHNVRLLMLDDQRLLLPHWAKWLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHR LFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHWGWTDWNLALNPEG GPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVAL M HPDGSAWWLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ (SEQ ID NO:1)

In one embodiment, the nucleotide sequence of β-glucocerebrosidase has the NCBI Reference Sequence Number: NM_0001573.

An example β-glucocerebrosidase nucleotide sequence is that of SEQ ID NO: 2: atcacatgac ccatccacat cgggaagccg gaattacttg cagggctaac ctagtgccta tagctaaggc aggtacctgc atccttgttt ttgtttagtg gatcctctat ccttcagaga ctctggaacc cctgtggtct tctcttcatc taatgaccct gaggggatgg agttttcaag tccttccaga gaggaatgtc ccaagccttt gagtagggta agcatcatgg ctggcagcct cacaggattg cttctacttc aggcagtgtc gtgggcatca ggtgcccgcc cctgcatccc taaaagcttc ggctacagct cggtggtgtg tgtctgcaat gccacatact gtgactcctt tgaccccccg acctttcctg cccttggtac cttcagccgc tatgagagta cacgcagtgg gcgacggatg gagctgagta tggggcccat ccaggctaat cacacgggca caggcctgct actgaccctg cagccagaac agaagttcca gaaagtgaag ggatttggag gggccatgac agatgctgct gctctcaaca tccttgccct gtcaccccct gcccaaaatt tgctacttaa atcgtacttc tctgaagaag gaatcggata taacatcatc cgggtaccca tggccagctg tgacttctcc atccgcacct acacctatgc agacacccct gatgatttcc agttgcacaa cttcagcctc ccagaggaag ataccaagct caagataccc ctgattcacc gagccctgca gttggcccag cgtcccgttt cactccttgc cagcccctgg acatcaccca cttggctcaa gaccaatgga gcggtgaatg ggaaggggtc actcaaggga cagcccggag acatctacca ccagacctgg gccagatact ttgtgaagtt cctggatgcc tatgctgagc acaagttaca gttctgggca gtgacagctg aaaatgagcc ttctgctggg ctgttgagtg gatacccctt ccagtgcctg ggcttcaccc ctgaacatca gcgagacttc attgcccgtg acctaggtcc taccctcgcc aacagtactc accacaatgt ccgcctactc atgctggatg accaacgctt gctgctgccc cactgggcaa aggtggtact gacagaccca gaagcagcta aatatgttca tggcattgct gtacattggt acctggactt tctggctcca gccaaagcca ccctagggga gacacaccgc ctgttcccca acaccatgct ctttgcctca gaggcctgtg tgggctccaa gttctgggag cagagtgtgc ggctaggctc ctgggatcga gggatgcagt acagccacag catcatcacg aacctcctgt accatgtggt cggctggacc gactggaacc ttgccctgaa ccccgaagga ggacccaatt gggtgcgtaa ctttgtcgac agtcccatca ttgtagacat caccaaggac acgttttaca aacagcccat gttctaccac cttggccact tcagcaagtt cattcctgag ggctcccaga gagtggggct ggttgccagt cagaagaacg acctggacgc agtggcactg atgcatcccg atggctctgc tgttgtggtc gtgctaaacc gctcctctaa ggatgtgcct cttaccatca aggatcctgc tgtgggcttc ctggagacaa tctcacctgg ctactccatt cacacctacc tgtggcgtcg ccagtgatgg agcagatact caaggaggca ctgggctcag cctgggcatt aaagggacag agtcagctca cacgctgtct gtgactaaag agggcacagc agggccagtg tgagcttaca gcgacgtaag cccaggggca atggtttggg tgactcactt tcccctctag gtggtgccag gggctggagg cccctagaaa aagatcagta agccccagtg tccccccagc ccccatgctt atgtgaacat gcgctgtgtg ctgcttgctt tggaaactgg gcctgggtcc aggcctaggg tgagctcact gtccgtacaa acacaagatc agggctgagg gtaaggaaaa gaagagacta ggaaagctgg gcccaaaact ggagactgtt tgtctttcct ggagatgcag aactgggccc gtggagcagc agtgtcagca tcagggcgga agccttaaag cagcagcggg tgtgcccagg cacccagatg attcctatgg caccagccag gaaaaatggc agctcttaaa ggagaaaatg tttgagccca gtca (SEQ ID NO: 2)

As used herein, the term "glucocerebroside pathology" includes the dysfunction of glucocerebrosidase and/or the widespread accumulation and/or deposition and/or aggregation of substrate glycosphingolipids. This accumulation is characteristic of "Gaucher cells". Accumulation may be particularly evident in the lysosome. Suitably a decrease in glucocerebroside pathology is a reduction the aggregation, concentration, number or distribution of aggregates in a subject. Suitably treated patients have a decrease in the size and number of Gaucher cells.

Gaucher disease is caused by mutations in the canonical amino acid sequence of GBA1.

Mutations in Type II Gaucher disease include E80K, R170C, L483P and/or D513Y, wherein the amino acid numbering is with reference to the canonical sequence set forth above.

Mutations in Type III Gaucher disease include V437L, D448H and/or T530I, wherein the amino acid numbering is with reference to the canonical sequence set forth above.

Alpha-synuclein (a-synuclein) In one aspect the invention relates to the treatment or prevention of an alpha-synuclein- mediated neurodegenerative disease or synucleinopathies. For example, in pathological conditions, alpha-synuclein aggregates to form insoluble fibrils.

Alpha-synuclein is essential for normal development of cognitive functions. Knock-out mice with the targeted inactivation of the expression of alpha-synuclein show impaired spatial learning and working memory. In one embodiment, the invention provides a method for improving learning and/or cognitive function of a subject.

Alpha synuclein is primarily found in neural tissue and is predominantly expressed in the neocortex, hippocampus, substantia nigra, thalamus and cerebellum. Although it is predominantly a neuronal protein, alpha-synuclein can also be found in neuroglial cells.

Alpha-synuclein lacks a single stable 3D structure and is considered to be an intrinsically disordered protein. Aggregates of alpha-synuclein form insoluble fibrils in pathological conditions characterized by Lewy bodies, such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy Among the strategies for treating synucleinopathies are compounds that inhibit aggregation of alpha-synuclein. In rare cases of familial forms of Parkinson's disease, there is a mutation in the gene coding for alpha-synuclein. Five point mutations have been identified thus far: A53T, A30P, E46K, H50Q, and G51 D. It has been reported that some mutations influence the initiation and amplification steps of the aggregation process. Genomic duplication and triplication of the gene appear to be another rare cause of Parkinson's disease in other lineages, although more common than point mutations. Hence certain mutations of alpha- synuclein may cause it to form amyloid-like fibrils that contribute to Parkinson's disease and other neurodegenerative diseases.

In one embodiment, the amino acid sequence of α-synuclein is the canonical amino acid sequence of a-synuclein:

MDVFMKGLSKAKEGWAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGWHGVATVAEKT K EQVTNVGGAWTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDP DNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 7).

An example of α-synuclein is the human α-synuclein protein having the UniProtKB accession number P37840-1.

In one embodiment, the amino acid sequence of α-synuclein is an isoform which differs from the canonical sequence. In one embodiment, the amino acid sequence of a-synuclein is an isoform in which amino acid residues 103-130 are missing from the canonical sequence: DVFMKGLSKAKEGWAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGWHGVATVAEKTK EQVTNVGGAWTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKEGYQDYEPEA (SEQ ID NO: 8)

An example of α-synuclein is the human α-synuclein protein having the UniProtKB accession number P37840-2.

In another embodiment, the amino acid sequence of α-synuclein is an isoform in which amino acid residues 41-54 are missing from the canonical sequence: MDVFMKGLSKAKEGWAAAEKTKQGVAEAAGKTKEGVLYWAEKTKEQVTNVGGAWTGV TAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQ DYEPEA (SEQ ID NO: 9)

An example of α-synuclein is the human α-synuclein protein having the UniProtKB accession number P37840-3. The α-synuclein may comprise a natural variant. One skilled in the art is able to identify natural variants of α-synuclein for example variants A53T, A30P, E46K, H50Q, and/or G51 D, wherein the amino acid numbering is relative to the canonical sequence set forth above.

As used herein, the term "alpha-synuclein pathology" includes the aggregation, deposition and dysfunction of alpha synuclein. Alpha synuclein deposits include aberrant soluble oligomeric conformations, or protofibrils or fibrils comprising alpha-synuclein. Protofibrils are soluble oligomeric forms of alpha-synuclein. The protofibrils gradually become insoluble and coalesce into fibrils. Dopamine and its metabolites act as inhibitors to the conversion of protofibrils to mature fibrils. Protofibrils, are the toxic species which mediate disruption of cellular homeostasis and neuronal death, through effects on various intracellular targets, including synaptic function.

The term alpha-synuclein pathology may also refer to secreted alpha-synuclein which contributes to alpha-synuclein pathology by exerting effects on neighboring cells, including seeding of aggregation, thought to contribute to disease progression.

Neuronal Lewy bodies and Lewy neurites, oligodendroglial cytoplasmic inclusions and large aconal spheroids are examples of alpha-synuclein pathology as a result of insoluble aggregates. Suitably a decrease in alpha-synuclein pathology is a reduction the concentration, number or distribution of fibrils, or of protofibrils. Alternatively, a decrease in alpha-synuclein pathology may be a reduction in levels of secreted alpha-synuclein. The distribution and progression pattern of alpha-synuclein pathology is considered to be linked to clinical dysfunction. Blood-brain barrier (BBB)

The term "blood brain barrier" (BBB) as used herein means the highly selective semipermeable membrane barrier which separates the circulating blood from the brain and extracellular fluid in the central nervous system. It is formed by the selectivity of tight junctions between endothelial cells. The blood-brain barrier occurs along all capillaries of the brain and consists of tight junctions.

Overcoming the difficulty of delivering therapeutic agents to specific regions of the brain presents a major challenge to treatment of most brain disorders.

In one aspect of the invention there is provided an AAV vector particle adapted for crossing the blood-brain barrier. Preferably, the AAV vector particle is adapted for crossing an intact blood brain barrier. Preferably the AAV vector particle does not impair blood-brain barrier integrity and/or selectivity and/or affect permeability.

In other words, the vector particle is adapted to cross a blood-brain barrier which has not been compromised or weakened, i.e. which maintains tight junctions between endothelial cells. Methods are known in the art which can determine whether or not a blood brain barrier is intact. For example, the permeability of the blood-brain barrier can be detected by perfusion of Evan's blue dye. Alternatively a fluorescent-conjugated cadaverine dye can be used as a blood-brain barrier permeability marker, together with the AAV particle carrying a fluorescent marker. (See Example 1.) In a preferred embodiment, the AAV vector particle of the invention does not cause microgliosis. Suitably, the AAV vector particle does not cause sustained inflammation in the central nervous system.

The "central nervous system" as used herein means the nervous system consisting of the brain and spinal cord. The "peripheral nervous system" as used herein means the components of the nervous system outside of the central nervous system. The peripheral nervous system consists of the nerves and ganglia outside of the brain and spinal cord. Brain endothelium or brain microvascular endothelial cells

As used herein "brain endothelial cells", or brain endothelium or brain microvascular endothelial cells are the cerebral endothelial cells which present the interface between the blood and the central nervous system i.e. blood-brain barrier endothelial cells. Suitably the brain endothelial cells may form the blood-brain barrier.

In one embodiment, the AAV vector particle of the invention targets the brain endothelium. In a preferred embodiment, the AAV vector particle targets the endothelium in at least one of the cerebellum, olfactory bulb, striatum and cerebral cortex. Preferably the AAV vector particle targets the endothelium in the cerebellum, olfactory bulb, striatum and cerebral cortex.

In one embodiment the AAV vector particle targets the blood-brain barrier-associated endothelial cells. Preferably the vector particle targets the blood-brain barrier associated endothelium of the entire central nervous system, such as including the spinal cord.

As used herein, the term "targets" or "targeting" means that the concentration of the AAV vector particle is increased in a specific cell type relative to other cell types. For example, after administration the AAV vector particle may be found in higher concentrations in endothelial cells compared to pericytes or astrocytic endfeet.

Methods for assessing the targeting of the viral vector particle are known to those skilled in the art. For example to confirm endothelial targeting, one may use immunohistochemistry. A viral vector particle comprising a fluorescent tag could be used along with antibodies against an endothelial cell marker (such as CD31 ) and a cell marker for another cell type, such as aquaporin 4 which is a marker for astrocytic endfeet or CD13 which is a marker for pericytes. The viral vector particle will demonstrate increased co-localisation with the fluorescence marker of the targeted cell type. As used herein "peripheral organs" include but are not limited to the liver, heart and muscles.

In one embodiment, the AAV vector particle does not substantially target the peripheral organs. Preferably the AAV vector particle crosses the blood-brain barrier and does not substantially target the peripheral organs. Preferably the AAV vector particle targets blood- brain endothelial cells and does not substantially target the peripheral organs. Suitably the peripheral organs do not comprise the AAV vector particle. Suitably the peripheral organs do not comprise the exogenous nucleotide sequence encoding GBA1 or a fragment or derivative thereof. Vectors

A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the invention, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. The vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid and/or facilitating the expression of the protein encoded by a segment of nucleic acid. The vectors used in the invention may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter.

Vectors comprising polynucleotides used in the invention may be introduced into cells using a variety of techniques known in the art, such as transfection, transduction and transformation. Transfection may refer to a general process of incorporating a nucleic acid into a cell and includes a process using a non-viral vector to deliver a polynucleotide to a cell. Transduction may refer to a process of incorporating a nucleic acid into a cell using a viral vector.

Preferably, the vectors used to transduce cells in the invention are viral vectors. The vectors of the invention are preferably adeno-associated viral (AAV) vectors, although it is contemplated that other viral vectors may be used.

Preferably, the viral vector for use according to the present invention is in the form of a viral vector particle.

In one aspect the invention provides a viral vector particle adapted for targeting the brain endothelium or for crossing the blood-brain barrier, wherein the viral vector particle comprises a nucleotide sequence encoding β-glucocerebrosidase (GBA1) or a fragment or derivative thereof.

In one embodiment the viral vector particle adapted for targeting the brain endothelium or for crossing the blood-brain barrier for use according to the invention is a retroviral, lentiviral, adeno-associated viral (AAV) or adenoviral vector particle. Preferably, the viral vector particle is a lentiviral or AAV vector particle, more preferably an AAV vector particle. Although, some embodiments of the invention have been described with respect to AAV vector particles, it will be appreciated that some embodiments may apply mutatis mutandis to other viral vectors disclosed herein.

Adeno-associated viral (AAV) vectors In one aspect, the invention provides an AAV vector particle adapted for targeting the brain endothelium or for crossing the blood-brain barrier, wherein the AAV vector particle comprises a nucleotide sequence encoding β-glucocerebrosidase (GBA1) or a fragment or derivative thereof.

Methods of preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art.

The AAV vector may comprise an AAV genome or a fragment or derivative thereof.

An AAV genome is a polynucleotide sequence, which may encode functions needed for production of an AAV particle. These functions include those operating in the replication and packaging cycle of AAV in a host cell, including encapsidation of the AAV genome into an AAV particle. Naturally occurring AAVs are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, the AAV genome of the AAV vector of the invention is typically replication-deficient.

The AAV genome may be in single-stranded form, either positive or negative-sense, or alternatively in double-stranded form. The use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression.

The AAV genome may be from any naturally derived serotype, isolate or clade of AAV. Thus, the AAV genome may be the full genome of a naturally occurring AAV. As is known to the skilled person, AAVs occurring in nature may be classified according to various biological systems. Commonly, AAVs are referred to in terms of their serotype. A serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, a virus having a particular AAV serotype does not efficiently cross- react with neutralising antibodies specific for any other AAV serotype. AAV serotypes include AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV1 1 , and also recombinant serotypes, such as Rec2 and Rec3, recently identified from primate brain.

Several rAAV vectors have been reported to efficiently cross the blood-brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system (Zhang et al., Molecular therapy vol. 19, no 8, 1440-1448).

In one embodiment the AAV is an AAV1 , AAV6, AAV6.2, AAV7, AAV9, rh10, rh39 or rh43 serotype. In one embodiment the AAV vector particle comprises an AAV1 , AAV6, AAV6.2, AAV7, AAV9, rh10, rh39 or rh43 serotype capsid protein. In one embodiment of the invention the AAV vector particle is an AAV1 , AAV6, AAV6.2, AAV7, AAV9, rh10, rh39 or rh43 vector particle.

In one embodiment the AAV is an AAV2 or AAV9 serotype. In one embodiment the AAV vector particle comprises an AAV2 or AAV9 serotype capsid protein.

The capsid protein may be an artificial or mutant capsid protein. The term "artificial capsid" as used herein means that the capsid particle comprises an amino acid sequence which does not occur in nature or which comprises an amino acid sequence which has been engineered (e.g. modified) from a naturally occurring capsid amino acid sequence.

In other words the artificial capsid protein comprises a mutation or a variation in the amino acid sequence compared to the sequence of the parent capsid from which it is derived where the artificial capsid amino acid sequence and the parent capsid amino acid sequences are aligned. Methods of sequence alignment are well known in the art and referenced herein.

As used herein the term "adapted for crossing the blood brain barrier" means that the vector particle has the ability to cross the blood brain barrier, for example the vector particle may comprise a mutation or modification relative to the wild type vector particle which improves the ability to cross the blood brain barrier relative to an unmodified or wild type viral particle. Improved ability to cross the blood brain barrier may be measured for example by measuring the expression of a transgene, e.g. GFP, carried by the vector particle, wherein expression of the transgene in the brain correlates with the ability of the viral particle to cross the blood brain barrier. In one embodiment, the AAV vector particle comprises an artificial capsid amino acid sequence which enables the viral particle to cross the blood-brain barrier.

In one embodiment, the AAV vector particle capable of crossing the blood-brain barrier comprises a VP1 capsid protein comprising an amino acid sequence comprising at least four contiguous amino acids from the sequence TLAVPFK (SEQ ID NO: 3) or KFPVALT (SEQ ID NO: 4).

In one embodiment, the AAV vector particle capable of crossing the blood-brain barrier comprises a VP1 capsid protein comprising an amino acid sequence comprising at least five contiguous amino acids from the sequence TLAVPFK (SEQ ID NO: 3) or KFPVALT (SEQ ID NO: 4).

In one embodiment, the AAV vector particle capable of crossing the blood-brain barrier comprises a VP1 capsid protein comprising an amino acid sequence comprising at least six contiguous amino acids from the sequence TLAVPFK (SEQ ID NO: 3) or KFPVALT (SEQ ID NO: 4). In one embodiment, the AAV vector particle capable of crossing the blood-brain barrier comprises a VP1 capsid protein comprising an amino acid sequence comprising the sequence TLAVPFK (SEQ ID NO: 3) or KFPVALT (SEQ ID NO: 4).

In a preferred embodiment, the nucleic acid sequence encoding the at least four, at least five, at least six or all seven contiguous amino acids from the sequence TLAVPFK (SEQ ID NO: 3) or KFPVALT (SEQ ID NO: 4) is inserted at a position corresponding to the position between a sequence encoding for amino acids 588 and 589 of AAV9 (SEQ ID NO: 10).

The amino acid sequence of the (wild-type) AAV9 capsid (SEQ ID NO: 10) is:

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGL DKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVF QAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDT ESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGD RVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRD WQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLG S AHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFE NVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNY I PGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPL SGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQ NQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPAD P PTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTE G VYSEPRPIGTRYLTRNL In a preferred embodiment, the AAV vector particle comprises a PHP.B AAV9 capsid, preferably the AAV-PHP.B VP1 capsid protein.

In a preferred embodiment, the AAV vector particle capable of crossing the blood-brain barrier is PHP.B AAV9. In one embodiment, the sequence of the AAV-PHP.B capsid VP1 protein is (SEQ ID NO: 1 1 ):

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGN GL DKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVF QAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDT ESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGD RVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRD WQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLG S AHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFE NVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNY I PGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPA ASHKEGEDRFFPL SGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQTLAVPFKAQ A QTGVWQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKN TPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNV E FAVNTEGVYSEPRPIGTRYLTRNL

In one embodiment, the AAV vector particle comprises a capsid comprising an amino acid sequence that has at least 75%, 80%, 85% or 90% identity to SEQ ID NO: 11 , more preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 1 , wherein the AAV vector particle is capable of crossing the blood-brain barrier.

The AAV-PHP.B vector is described in Deverman et al., Nat Biotechnol. 2016 February; 34(2):204-209 and WO2015/038958 which are incorporated herein by reference.

In one embodiment, the AAV vector particle comprises an artificial capsid amino acid sequence which targets the viral particle to brain microvascular endothelial cells.

In one embodiment the AAV vector particle comprises an artificial capsid amino acid sequence which comprises the amino acid sequence NRGTEWD (SEQ ID NO: 5) or DWETGRN (SEQ ID NO: 6).

In a preferred embodiment, the amino acid sequence NRGTEWD (SEQ ID NO: 5) or DWETGRN (SEQ ID NO: 6) is inserted after (preferably immediately after) an amino acid corresponding to the position of any one of amino acids 550-600 of the AAV2 capsid (preferably the AAV2 VP1 capsid protein), wherein the AAV2 capsid amino acid sequence is numbered with reference to SEQ ID NO: 12. Suitably, SEQ ID NO: 5 is inserted in the region of amino acids 560-600, 570-600, 560-590 or 570-590 of the VP1 amino acid sequence.

Suitably SEQ ID NO: 5 or SEQ ID NO: 6 is inserted after (preferably immediately after) one of the following amino acids of the VP1 protein, wherein the numbering is with reference to SEQ ID NO:12, amino acid position 550, 551 , 552, 553, 554, 555, 556, 557, 558, 559, 560, 561 , 562, 563, 564, 565, 566, 567, 568, 569, 570, 571 , 572, 573, 574, 575, 576, 577, 578, 579, 580, 581 , 582, 583, 584, 585, 586, 587, 588, 589, 590, 591 , 592, 593, 594, 595, 596, 597, 598, 599 or 600.

In a preferred embodiment, the amino acid sequence NRGTEWD (SEQ ID NO: 5) or DWETGRN (SEQ ID NO: 6) is inserted after (preferably immediately after) a position corresponding to the position at amino acid R588 of the AAV2 capsid, wherein the AAV2 capsid amino acid sequence is numbered with reference to SEQ ID NO: 12.

An example sequence of the wild type VP1 capsid protein of AAV2 is set forth in SEQ ID NO: 12. MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGV LPGMV QDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGL HPPPQILIKNTPVPANPSTT FSAAKFASFITQYSTGQVSVEIEWELQKENS RWNPEIQYTSNYNKSVNVDFTVDTNGVY SEPRPIGTRYLTRNL (SEQ ID NO: 12)

In one embodiment, the AAV vector particle comprises an artificial capsid amino acid sequence. In a preferred embodiment the AAV vector particle comprises a VP1 amino acid sequence set forth in SEQ ID NO: 13.

MAADGYLPDWLEDTLSEGIRQWWKL PGPPPPKPAERH DDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYL YNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPAR RLNFGQTGDAD SVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQV EVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPG PCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVL IFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGQRGNRGTEWDAQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNT PVPANPSTTFSAAKFASFITQYSTGQVSVEIE ELQ ENSKRWNPEIQYTSNYNKSVNVD FTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 13) SEQ ID NO: 13 is the amino acid sequence of the VP1 protein of AAV2 after introduction of SEQ ID NO: 5, flanked at its N terminus by a glycine in position 589 and at its C-terminus by an alanine in position 597. In addition, the asparagine at position 584 of the wild-type AAV2 sequence is replaced with a glutamine.

In one embodiment, the AAV vector particle comprises a BR1 AAV2 capsid, preferably the BR1 AAV2 VP1 capsid protein.

In one embodiment, the sequence of the BR1 AAV capsid VP1 protein is (SEQ ID NO: 13):

In one embodiment, the AAV vector particle comprises a capsid comprising an amino acid sequence that has at least 75%, 80%, 85% or 90% identity to SEQ ID NO: 13, more preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13, wherein the AAV vector particle targets the brain microvascular endothelial cells.

The AAV-BR1 vector is described in Korbelin et al., EMBO Molecular medicine Vol 8, No.6, 2016 which is incorporated herein by reference. In one embodiment, the AAV vector particle comprises a BR1 AAV2 capsid which targets the viral particle to brain microvascular endothelial cells.

Reviews of AAV serotypes may be found in Choi et al. (2005) Curr. Gene Ther. 5: 299-310 and Wu et al. (2006) Molecular Therapy 14: 316-27. The sequences of AAV genomes or of elements of AAV genomes including ITR sequences, rep or cap genes for use in the invention may be derived from the following accession numbers for AAV whole genome sequences: Adeno-associated virus 1 NC_002077, AF063497; Adeno-associated virus 2 NCJD01401 ; Adeno-associated virus 3 NC_001729; Adeno-associated virus 3B NC_001863; Adeno-associated virus 4 NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associated virus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC_005889, AY388617.

AAV may also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally derived AAVs, and typically to a phylogenetic group of AAVs which can be traced back to a common ancestor, and includes all descendants thereof. Additionally, AAVs may be referred to in terms of a specific isolate, i.e. a genetic isolate of a specific AAV found in nature. The term genetic isolate describes a population of AAVs which has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining a recognisably distinct population at a genetic level.

The skilled person can select an appropriate serotype, clade, clone or isolate of AAV for use in the invention on the basis of their common general knowledge. The AAV serotype determines the tissue specificity of infection (or tropism) of an AAV virus.

Typically, the AAV genome of a naturally derived serotype, isolate or clade of AAV comprises at least one inverted terminal repeat sequence (ITR). An ITR sequence acts in cis to provide a functional origin of replication and allows for integration and excision of the vector from the genome of a cell. In preferred embodiments, one or more ITR sequences flank the nucleotide sequences encoding the GBA1 nucleotide sequence (or fragments or derivatives thereof). The AAV genome may also comprise packaging genes, such as rep and/or cap genes which encode packaging functions for an AAV particle. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins such as VP1 , VP2 and VP3 or variants thereof. These proteins make up the capsid of an AAV particle.

A promoter will be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19 and p40 promoters (Laughlin et al. (1979) Proc. Natl. Acad. Sci. USA 76: 5567-5571 ). For example, the p5 and p19 promoters are generally used to express the rep gene, while the p40 promoter is generally used to express the cap gene. As discussed above, the AAV genome used in the AAV vector of the invention may therefore be the full genome of a naturally occurring AAV. For example, a vector comprising a full AAV genome may be used to prepare an AAV vector or vector particle in vitro. However, while such a vector may in principle be administered to patients, this will rarely be done in practice. Preferably the AAV genome will be derivatised for the purpose of administration to patients. Such derivatisation is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art. Derivatisation of the AAV genome and of the AAV capsid are reviewed in Coura and Nardi (2007) Virology Journal 4: 99, and in Choi et al. and Wu et al., referenced above. Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV vector of the invention in vivo. Typically, it is possible to truncate the AAV genome significantly to include minimal viral sequence yet retain the above function. This is preferred for safety reasons to reduce the risk of recombination of the vector with wild-type virus, and also to avoid triggering a cellular immune response by the presence of viral gene proteins in the target cell.

Typically, a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR. A preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences, i.e. a self- complementary AAV genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression.

In one embodiment, the AAV vector comprises at least one, such as two, AAV serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 ITRs. Preferably, the AAV vector comprises at least one, such as two, AAV serotype 2 ITRs.

The one or more ITRs will preferably flank the nucleotide sequence encoding GBA1 (or fragment or derivatives thereof) at either end. The inclusion of one or more ITRs is preferred to aid concatamer formation of the vector of the invention in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases. The formation of such episomal concatamers protects the vector construct during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.

In preferred embodiments, ITR elements will be the only sequences retained from the native AAV genome in the derivative. Thus, a derivative will preferably not include the rep and/or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene.

The following portions could therefore be removed in a derivative of the invention: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes. However, in some embodiments, derivatives may additionally include one or more rep and/or cap genes or other viral sequences of an AAV genome. Naturally occurring AAV integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the vector may be tolerated in a therapeutic setting. Where a derivative comprises capsid proteins i.e. VP1 , VP2 and/or VP3, the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e. a pseudotyped vector). Thus, in one embodiment the AAV vector is in the form of a pseudotyped AAV vector particle.

Chimeric, shuffled or capsid-modified derivatives will be typically selected to provide one or more desired functionalities for the AAV vector. Thus, these derivatives may display increased efficiency of gene delivery, decreased immunogenicity (humoral or cellular), an altered tropism range and/or improved targeting of a particular cell type compared to an AAV vector comprising a naturally occurring AAV genome, such as that of AAV2. Increased efficiency of gene delivery may be effected by improved receptor or co-receptor binding at the cell surface, improved internalisation, improved trafficking within the cell and into the nucleus, improved uncoating of the viral particle and improved conversion of a single- stranded genome to double-stranded form. Increased efficiency may also relate to an altered tropism range or targeting of a specific cell population, such that the vector dose is not diluted by administration to tissues where it is not needed.

Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This may be performed for example by a marker rescue approach in which non-infectious capsid sequences of one serotype are co-transfected with capsid sequences of a different serotype, and directed selection is used to select for capsid sequences having desired properties. The capsid sequences of the different serotypes can be altered by homologous recombination within the cell to produce novel chimeric capsid proteins. Chimeric capsid proteins also include those generated by engineering of capsid protein sequences to transfer specific capsid protein domains, surface loops or specific amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes.

Shuffled or chimeric capsid proteins may also be generated by DNA shuffling or by error- prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes e.g. those encoding capsid proteins of multiple different serotypes and then subsequently reassembling the fragments in a self-priming polymerase reaction, which may also cause crossovers in regions of sequence homology. A library of hybrid AAV genes created in this way by shuffling the capsid genes of several serotypes can be screened to identify viral clones having a desired functionality. Similarly, error prone PCR may be used to randomly mutate AAV capsid genes to create a diverse library of variants which may then be selected for a desired property.

The sequences of the capsid genes may also be genetically modified to introduce specific deletions, substitutions or insertions with respect to the native wild-type sequence. In particular, capsid genes may be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of a capsid coding sequence, or at the N- and/or C-terminus of a capsid coding sequence.

The unrelated protein or peptide may advantageously be one which acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or improving the specificity of targeting of the vector to a particular cell population (e.g. to brain microvascular endothelial cells). The unrelated protein may also be one which assists purification of the viral particle as part of the production process, i.e. an epitope or affinity tag. The site of insertion will typically be selected so as not to interfere with other functions of the viral particle e.g. internalisation, trafficking of the viral particle. The skilled person can identify suitable sites for insertion based on their common general knowledge.

The invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome. The invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.

The AAV vector of the invention may take the form of a nucleotide sequence comprising an AAV genome or derivative thereof and a sequence encoding the GBA1 transgene or derivatives thereof.

The AAV particles of the invention include transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype. The AAV particles of the invention also include mosaic forms wherein a mixture of unmodified capsid proteins from two or more different serotypes makes up the viral capsid. The AAV particle also includes chemically modified forms bearing ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting a particular cell surface receptor. Thus, for example, the AAV particles of the invention include those with an AAV2 genome and AAV9 capsid proteins (AAV2/9).

The AAV vector may comprise multiple copies (e.g., 2, 3 etc.) of the nucleotide sequence referred to herein. Promoters and regulatory sequences

The AAV vector of the invention may also include elements allowing for the expression of GBA1 (or fragment or derivatives thereof) in vitro or in vivo. These may be referred to as expression control sequences. Thus, the AAV vector typically comprises expression control sequences (e.g. comprising a promoter sequence) operably linked to the nucleotide sequence encoding the transgene.

Any suitable promoter may be used, the selection of which may be readily made by the skilled person. The promoter sequence may be constitutively active (i.e. operational in any host cell background), or alternatively may be active only in a specific host cell environment, thus allowing for targeted expression of the transgene in a particular cell type (e.g. a tissue- specific promoter such as an endothelial specific promoter). The promoter may show inducible expression in response to presence of another factor, for example a factor present in a host cell. In any event, where the vector is administered for therapy, it is preferred that the promoter should be functional in the target cell background.

In some embodiments, it is preferred that the promoter shows neural cell expression in order to allow for the transgene to only be expressed in neural cell populations. Thus, expression from the promoter may be neural-cell specific.

In other embodiments, it is preferred that the promoter shows endothelial cell expression in order to allow for the transgene to only be expressed in endothelial cell populations. Thus, expression from the promoter may be endothelial-cell specific. Example promoters, which are not cell specific, include the chicken beta-actin (CBA) promoter, optionally in combination with a cytomegalovirus (CMV) enhancer element. An example promoter for use in the invention is a CAG promoter.

The AAV vector of the invention may also comprise one or more additional regulatory sequences which may act pre- or post-transcriptionaliy. The regulatory sequence may be part of the native transgene locus or may be a heterologous regulatory sequence. The AAV vector of the invention may comprise portions of the 5'-UTR or 3'-UTR from the native transgene transcript. Regulatory sequences are any sequences which facilitate expression of the transgene, i.e. act to increase expression of a transcript, improve nuclear export of mRNA or enhance its stability. Such regulatory sequences include for example enhancer elements, post- transcriptional regulatory elements and polyadenylation sites. An example of a polyadenylation site is the Bovine Growth Hormone poly-A signal.

In the context of the AAV vector of the invention, such regulatory sequences will be cis- acting. However, the invention also encompasses the use of trans-acting regulatory sequences located on additional genetic constructs.

An example of a post-transcriptional regulatory element for use in a AAV vector of the invention is the woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof.

Another regulatory sequence which may be used in a AAV vector of the invention is a scaffold-attachment region (SAR). Additional regulatory sequences may be readily selected by the skilled person. Retroviral and lentiviral vectors

A retroviral vector may be derived from or may be derivable from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include murine leukaemia virus (MLV), human T-cell leukaemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV). A detailed list of retroviruses may be found in Coffin, J.M. et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. Retroviruses may be broadly divided into two categories, "simple" and "complex". Retroviruses may be even further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses.

The basic structure of retrovirus and lentivirus genomes share many common features such as a 5' LTR and a 3' LTR. Between or within these are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome, and gag, pol and env genes encoding the packaging components - these are polypeptides required for the assembly of viral particles. Lentiviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.

In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes.

The LTRs themselves are identical sequences that can be divided into three elements: U3, R and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA. U5 is derived from the sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.

In a defective retroviral vector genome gag, pol and env may be absent or not functional.

In a typical retroviral vector, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective. Portions of the viral genome may also be replaced by a library encoding candidate modulating moieties operably linked to a regulatory control region and a reporter moiety in the vector genome in order to generate a vector comprising candidate modulating moieties which is capable of transducing a target host cell and/or integrating its genome into a host genome. Lentivirus vectors are part of the larger group of retroviral vectors. A detailed list of lentiviruses may be found in Coffin, J.M. et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. In brief, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype "slow virus" visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV). The lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis, P et al. (1992) EMBO J. 1 1 : 3053-8; Lewis, P.F. et al. (1994) J. Virol. 68: 510-6). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.

A lentiviral vector, as used herein, is a vector which comprises at least one component part derivable from a lentivirus. Preferably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated.

The lentiviral vector may be a "primate" vector. The lentiviral vector may be a "non-primate" vector (i.e. derived from a virus which does not primarily infect primates, especially humans). Examples of non-primate lentiviruses may be any member of the family of lentiviridae which does not naturally infect a primate. As examples of lentivirus-based vectors, HIV-1 - and HIV-2-based vectors are described below.

The HIV-1 vector contains cis-acting elements that are also found in simple retroviruses. It has been shown that sequences that extend into the gag open reading frame are important for packaging of HIV-1. Therefore, HIV-1 vectors often contain the relevant portion of gag in which the translational initiation codon has been mutated. In addition, most HIV-1 vectors also contain a portion of the env gene that includes the RRE. Rev binds to RRE, which permits the transport of full-length or singly spliced mRNAs from the nucleus to the cytoplasm. In the absence of Rev and/or RRE, full-length HIV-1 RNAs accumulate in the nucleus. Alternatively, a constitutive transport element from certain simple retroviruses such as Mason-Pfizer monkey virus can be used to relieve the requirement for Rev and RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Similar to HIV-1 - based vectors, HIV-2 vectors also require RRE for efficient transport of the full-length or singly spliced viral RNAs. In one system, the vector and helper constructs are from two different viruses, and the reduced nucleotide homology may decrease the probability of recombination. In addition to vectors based on the primate lentiviruses, vectors based on FIV have also been developed as an alternative to vectors derived from the pathogenic HIV-1 genome. The structures of these vectors are also similar to the HIV-1 based vectors. Preferably, the viral vector used in the present invention has a minimal viral genome.

By "minimal viral genome" it is to be understood that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998/017815.

Preferably, the plasmid vector used to produce the viral genome within a host cell/packaging cell will have sufficient lentiviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle which is capable of infecting a target cell, but is incapable of independent replication to produce infectious viral particles within the final target cell. Preferably, the vector lacks a functional gag-pol and/or env gene and/or other genes essential for replication. However, the plasmid vector used to produce the viral genome within a host cell/packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell/packaging cell. These regulatory sequences may be the natural sequences associated with the transcribed viral sequence (i.e. the 5' U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g. the CMV promoter).

The vectors may be self-inactivating (SIN) vectors in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be generated and transduce non-dividing cells in vivo with an efficacy similar to that of wild-type vectors. The transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilisation by replication-competent virus. This should also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR.

The vectors may be integration-defective. Integration defective lentiviral vectors (IDLVs) can be produced, for example, either by packaging the vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site; Naldini, L. et al. (1996) Science 272: 263-7; Naldini, L. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 11382-8; Leavitt, A.D. et al. (1996) J. Virol. 70: 721 -8) or by modifying or deleting essential att sequences from the vector LTR (Nightingale, S.J. et al. (2006) Mol. Ther. 13: 1 121 -32), or by a combination of the above.

Adenoviral vectors The adenovirus is a double-stranded, linear DNA virus that does not go through an RNA intermediate. There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on the genetic sequence homology. The natural targets of adenovirus are the respiratory and gastrointestinal epithelia, generally giving rise to only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenoviral vector systems and are normally associated with upper respiratory tract infections in the young.

Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes. The large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and is able to replicate efficiently in complementing cell lines to produce very high titres of up to 1012. Adenovirus is thus one of the best systems to study the expression of genes in primary non-replicative cells.

The expression of viral or foreign genes from the adenovirus genome does not require a replicating cell. Adenoviral vectors enter cells by receptor mediated endocytosis. Once inside the cell, adenovirus vectors rarely integrate into the host chromosome. Instead, they function episomally (independently from the host genome) as a linear genome in the host nucleus. Hence the use of recombinant adenovirus alleviates the problems associated with random integration into the host genome. Variants, derivatives, analogues, homologues and fragments

In addition to the specific proteins and nucleotides mentioned herein, the invention also encompasses variants, derivatives, analogues, homologues and fragments thereof.

In the context of the invention, a "variant" of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one residue present in the naturally occurring polypeptide or polynucleotide.

The term "derivative" as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence, providing that the resultant protein or polypeptide retains at least one of its endogenous functions.

The term "analogue" as used herein in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics. Typically, amino acid substitutions may be made, for example from 1 , 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

Proteins used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

The term "homologue" as used herein means an entity having a certain homology with the wild type amino acid sequence or the wild type nucleotide sequence. The term "homology" can be equated with "identity".

In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence. Although homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.

Preferably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.

Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent homology or identity between two or more sequences.

Percent homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum percent homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1 ): 187-8).

Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

"Fragments" are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. "Fragment" thus refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

Such variants may be prepared using standard recombinant DNA techniques such as site- directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

Codon optimisation

The polynucleotides used in the invention may be codon-optimised. Codon optimisation has previously been described in WO 1999/41397 and WO 2001/79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular ceil type. Thus, an additional degree of translational control is available. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms.

Method of treatment All references herein to treatment include curative, palliative and prophylactic treatment. The treatment of mammals, particularly humans, is preferred. Both human and veterinary treatments are within the scope of the invention.

In one embodiment, the method of treatment provides GBA1 to the central nervous system and/or the peripheral nervous system of said subject. In one embodiment, the method of treatment provides GBA1 to the forebrain, midbrain, cortex, hippocampus and/or cerebellum of the subject.

In another embodiment, the method of treatment provides GBA1 to the dorsal root ganglia and/or sympathetic ganglia.

In one embodiment, the method of treatment provides GBA1 to glial and/or neuronal cells, preferably dopaminergic neurons.

In a preferred embodiment, the method of treatment provides GBA1 to the lysosome.

Preferably the treatment reduces protein aggregates in the lysosome.

In another preferred embodiment, a-synuclein pathology is reduced in the subject.

In one embodiment, a-synuclein pathology is reduced in at least one region selected from the group consisting of the visual cortex, the somatosensory cortex and the hippocampus. In one preferred embodiment, glucocerebroside pathology is reduced in the subject.

In one embodiment, glucocerebroside pathology is reduced in at least one region selected from the group consisting of the visual cortex, the somatosensory cortex and the hippocampus. In one embodiment the method of treatment reduces protein aggregates in neuronal cells. In a preferred embodiment the method of treatment reduces protein aggregates in dopaminergic neurons.

In one embodiment, the method of treatment provides an improvement in learning and/or cognitive function in the subject. Methods for measuring learning and/or cognitive function are known to those skilled in the art. For example, in humans the General Practicioner Assessment of Cognition (GPCOG) test may be used. Alternative cognitive tests include but are not limited to the Mini Mental State Examination (MMSE), The Six-item Cognitive Impairment Test (6CIT), Abbreviated Mental Test (AMT) and Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE). Advantageously, the present invention provides a method for treating dopaminergic neurons by systemically administering the vector particle of the invention. These neurons are vital to diverse brain functions, including: voluntary movement, reward, addiction and stress. Progressive loss of dopaminergic neurons is responsible for neurodegenerative diseases such as Parkinson's Disease. Pharmaceutical compositions and injected solutions

Although the agents for use in the invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy.

A "pharmaceutical composition" is a preparation which is stable and in a form which is acceptable to the patient.

The medicaments, for example AAV vector particles, of the invention may be formulated into pharmaceutical compositions. These compositions may comprise, in addition to the medicament, a pharmaceutically acceptable carrier, diluent, excipient, buffer, stabiliser or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration, e.g. intravenous or intra-arterial. The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001 % may be used. In some cases, serum albumin may be used in the composition.

For injection, the active ingredient may be in the form of an aqueous solution which is pyrogen-free, and has suitable pH, isotonicity and stability. The skilled person is well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included as required.

For delayed release, the medicament may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

Handling of the cell therapy products is preferably performed in compliance with FACT- JACIE International Standards for cellular therapy.

Administration

In one embodiment the agent of the invention is systemically administered to the subject. The term "systemic delivery" or "systemic administration" as used herein, means that the agent of the invention is administered into the circulatory system e.g. to achieve broad distribution of the agent. In contrast, topical or local administration restricts the delivery of the agent to a localised area e.g. intracerebral administration entails direct injection into the brain. In a preferred embodiment, the agent of the invention is administered intravenously.

In a preferred embodiment, the agent of the invention is administered intra-arterially.

Suitably, in one embodiment the agent of the invention is administered to the internal carotid artery.

As used herein, the term "agent" may refer to the vector particle, cell or pharmaceutical composition of the invention. Dosage

The skilled person can readily determine an appropriate dose of an agent of the invention to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of the invention.

Subject

The term "subject" as used herein refers to either a human or non-human animal.

Examples of non-human animals include vertebrates, for example mammals, such as non- human primates (particularly higher primates), dogs, rodents (e.g. mice, rats or guinea pigs), pigs and cats. The non-human animal may be a companion animal.

Preferably, the subject is a human.

In one embodiment the subject is a mouse model of a neurodegenerative disease. Suitably the mouse is a model of Parkinson's disease. Suitably the mouse is a A53T-SCNA transgenic mouse. A A53T-SCNA transgenic mouse overexpresses human a-synuclein A53T specifically in dopamine (DA) neurons

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference. Various preferred features and embodiments of the present invention will now be described by way of non limiting examples.

EXAMPLES

Example 1

Materials and Methods Generation of the AA V transfer vectors

The pAAV-CBA-eGFP construct was kindly donated by Dr. G. Gonzalez (CIMA, Pamplona, Spain). This plasmid was further modified to express under the control of the CBA promoter: the CreNLS cassette, the multicistronic sequence including GBA1 coding region and GFP and the FLEX eGFP cassette. In FLEX cassettes the transgene is antisense respect to promoter driven transcription and flanked by two consecutive but different pairs of flox sequences. In presence of Cre recombination the transgene gets reverted and its gene expression activated. The GBA1 coding region was purchased from Origene (RG216061). Subsequently, GBA1 was cloned in frame with a P2A sequence followed by the eGFP coding region. Finally, for the GBA1-mCherry expression vector, the GBA1 and mCherry coding regions were cloned in frame but separated by a Gly-Ser-Gly linker and inserted downstream to the EF1 alpha promoter. The pAAV-hSyn-DIO-hM4D(Gi)-MCherry plasmid was obtained by Prof. B.L. Roth (Chapel Hill, NC, USA) through Addgene (#44362).

Cell cultures

293T cells were cultured in Iscove's Modified Eagle Medium (Sigma-Aldrich) containing 10% fetal bovine serum (Sigma-Aldrich), 1 % non-essential amino acids (Gibco), 1 % sodium pyruvate (Sigma-Aldrich), 1 % glutamine (Sigma-Aldrich) and 1 % penicillin/streptomycin (Sigma-Aldrich). Cells were split every 3-4 days using Trypsin 0.25% (Sigma-Aldrich). Isolation of mouse brain microvascular endothelial cells was performed as previously reported by Liebner et al. ⁴⁶ . BMVECs were grown in EBM-2 plus bullet kit (Lonza). For immunostaining 2 x 10 ⁵ cells were seeded on 24 well-plate, coated with rat tail collagen type-1 (150 Mg/ml; Sigma). The day after, fresh culture medium was added supplemented with LiCI (10mM, Sigma) and the cells were infected 48 hours after seeding in 300 μΙ of total volume. The medium was changed every 2-3 days. Hela cells were cultured in Dulbecco Modified Eagle Medium - high glucose (Sigma-Aldrich) containing 10% fetal bovine serum (Sigma-Aldrich), 1 % non-essential amino acids (Gibco), 1 % sodium pyruvate (Sigma- Aldrich), 1 % glutamine (Sigma-Aldrich) and 1 % penicillin/streptomycin (Sigma-Aldrich). Cells were split every 2-3 days using Trypsin 0.25% (Sigma-Aldrich). For transfection Lipofectamine LTX® (Thermo Fisher Scientific) was used, according to manufacturer's protocol.

Virus production and purification

Replication-incompetent, recombinant viral particles were produced in 293T cells by polyethylenimine (PEI) (Polyscience) co-transfection of three different plasmids: transgene- containing plasmid, packaging plasmid for rep and cap genes and pAdDeltaF6 for the three adenoviral helper genes, The cells and supernatant were harvested at 120 hrs. Cells were lysed in Tris buffer (50mM Tris pH=8,5, 150mM NaCI, Sigma-Aldrich) by repetitive freeze- thawing cycles (3 times) whereas the viral particles present in the supernatant were concentrated by precipitation with 8% PEG8000 (Polyethylene glycol 8000, Sigma-Aldrich), lysed in Tris buffer and combined with correspondent cell lysates. In order to clarify the lysate, Benzonase treatment was performed (250U/ml_, 37°C for 30min, Sigma-Aldrich) in presence of 1 mM MglCI2 (Sigma-Aldrich) and cellular debris separated by centrifugation (2000g, 30min). The viral phase was isolated by iodixanol step gradient (15%, 25%, 40%, 60% Optiprep, Sigma-Aldrich) in the 40% fraction and concentrated in PBS (Phosphate Buffer Saline) with 100K cut-off concentrator (Vivaspin20, Sartorius Stedim). Virus titers were determined by measuring the number of DNase l-resistant viral particles, using qPCR with linearized genome plasmid as a standard.

Animals

Mice were maintained at the San Raffaele Scientific Institute Institutional mouse facility (Milan, Italy) in micro-isolators under sterile conditions and supplied with autoclaved food and water. The following transgenic mouse strains were used: NeuroD6-Cre ⁴⁷ , DAT-Cre ⁴⁸ , Ai9 ¹⁸ , PV-Cre ⁴⁹ , Olig2-Cre ⁵⁰ and Tsc1 conditional mutants ⁵¹ . All procedures were performed according to protocols approved by the internal IACUC and reported to the Italian Ministry of Health according to the European Communities Council Directive 2010/63/EU.

Viral injections

For tail vein injection, 2-6 month old mice were previously warmed under an heat lamp for 10 minutes and, then, placed into a restrainer for further manipulation. Mice were injected with variable viral concentrations depending on the experimental set-up in a total volume 200 μΙ of PBS (from 1x10 ⁹ to 2x10 ¹² vg/each mouse). For injections into mouse neonates, 1-old day pups were rested on a bed of ice for anesthetization. 50 μΙ of AAV viral suspension (1.5x10 ¹¹ vg) was manually injected into the facial vein using a 29 gauge insulin syringe. After injection, pups were rubbed with bedding to prevent rejection before reintroducing the mother into the cage. For carotid artery injections, 8-10 weeks old C57BI6/J mice were anesthetized with a mixture of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively) and temperature maintained during the procedure between 36 and 36.5 °C using a feedback-controlled heating system. Under a stereomicroscope a midline neck incision was performed, the common carotid artery exposed and the external carotid artery ligated. A micro-catheter was placed inside the common carotid artery and advanced up to the internal carotid artery. Under a sterile hood, the 50 μΙ of viral suspension (2x10 ¹² vg) in PBS was infused for 5-6 minutes with an infusion pump (World Precision Instruments) ⁵² . At the end of the procedure the microcatheter was withdrawn, the incisions sutured and animals allowed recovering.

Immunohistochemistry

Immunohistochemical analysis of the tissue sections was conducted essentially as previously described ⁵³ . Briefly, mice were anesthetized with Ketamine/Xylazine and transcardially perfused with 0.1 M phosphate buffer (PB) at room temperature (RT) at pH 7.4 with freshly prepared, ice-cold 4% paraformaldehyde (PFA) in PB. Tissues were post-fixed in 4% PFA overnight and then soaked in cryoprotective solution (30% Sucrose in PBS). After OCT embedding in dry ice, tissues were sectioned using cryostat. For immunofluorescence, free-floating 30 pm thickness coronal sections were rinsed in PBS, incubated for 10 min with 3% H ₂ 0 ₂ and 10% methanol, then for 20 min with Triton X 100 2%. 3% BSA for 1 hour was used to saturate the unspecific binding site before the overnight incubation with primary antibody (diluted in a solution containing 1 % BSA and Triton X 100 at room temperature). Following incubation, sections were rinsed three times in PBS and incubated for 1 hour with the secondary antibody. For immunohistochemistry, free-floating slices were rinsed in PBS and treated for one hour with a blocking solution containing 3% BSA and 0.3% Triton X 100 in PBS. After blocking, samples were incubated with the primary antibody diluted with a solution containing 1 % BSA, 0.3% Triton X 100 over night at room temperature. The slices were then incubated with the secondary antibody, followed by Vectastain ABC enhancing reaction, and finally the staining was revealed in DAB solution. After mounting the slices were dehydrated in xylene and the coverslip sealed with Eukitt™ mounting medium. The slices treated with Proteinase K (PK) were incubated for 10 minutes in a solution with 1 pg/mL of PK prior any step and the tissue processed for the immunostaining.

Primary antibodies for the following epitopes were used: GFP (1 :500, Molecular Probes), TH (1 :1000, Immunological Sciences), Calbindin (1 :200, Swant), Parvalbumin (1 :500, Sigma), beta-lll-Tubulin (1 :1000, Covance), NeuN (1 :1000, Immunogical Sciences), human alpha- Syn (1 :200, Syn211 , BDbiosciences), phosphoS129-alpha-syn (1 :100, Abeam). Slices were mounted with fluorescent mounting medium (Dako). For blood-brain barrier integrity evaluation, cadaverine (0.2 mg/animal) conjugated to Alexa Fluor-555 (Life Technologies) was injected intravenously into the tail vein 2 hours before sacrifice. Images were captured with a Nikon Eclipse 600 fluorescent microscope. Images were then imported and processed with the Photoshop Suite applications.

Stereological counting

Unbiased semi-automatic stereological sampling and counting was performed with a Leica DM4000B microscope equipped with MAC 6000 system and Stereo Investigator 9 software (MFB Bioscience, Williston, Vermont, USA). After structure boundaries delimitation, cortical phosphoS129-a-syn positive cells were automatically counted at 40X magnification. Slices were collected every 180 μηι, encompassing about 1.5 mm of cortex (antero-posterior +1 to -0.5 from bregma). The optical fractionator stereological probe (40 X 40 sized, 240 X 240 spaced) was then used to estimate the total number of phosphoS129-a-syn positive neurons.

GCase enzymatic assay

Dissected brain parts were lysates and mechanical homogenized in GCase assay buffer PH 5.4 (Citrate buffer 1 x, Triton X-100 0.25% w/w, Taurocholic acid 0.25% w/w, H20) supplemented with 1 % protease inhibitor mixture (Roche Diagnostics). After 30 min of lysis on ice, samples were then centrifuged at 13,000g for 15 minutes at 4°C. Supernatant was collected, and used for activity assays and Western blotting analysis. GCase activity in tissues was measured using 10 [ig of protein/well, with protein quantification assessed by the Pierce BCA protein Assay Kit (Thermo Fisher Scientific). The substrate 4- methylumbelliferyl β-D-glucopyranoside (4MUG, Sigma M3633) was then assembled with the tissue lysates to a final concentration of 5 mM for 1 hr at 37°C. After blocking the reaction with 1 M glycine solution, the signal was detected at the Victor plate reader (Perkin Elmer) with excitation and emission wavelengths at 360 nm and 440 nm, respectively. The standard used for this assay was the fluorescent product 4-Methylumbelliferone (4MU) (Sigma, M1381 ). The specific activity was calculated with 4MU standard curve by converting the relative fluorescence units (RFUs) to the concentration of the fluorescent cleaved product (GraphPad Prism 5.1). This interpolated value was then used to calculate the GCase enzymatic activity in the lysed tissue, which was expressed as nmol/hr/mg. Specificity of the enzymatic activity was assessed by adding the specific GCase inhibitor Conduritol-B-Epoxide (CBE) at 16 mM (Sigma). Immunoblotting

Brain lysate samples (-30 of protein lysates) were separated using 10% or 15% polyacrylamide gel and, then, transferred to PVDF membranes. Membranes were incubated overnight at 4°C with the following primary antibodies: C-terminal GCase antibody (Sigma, G4171 , 1 : 1 ,000), anti-actin (Sigma, A3853, 1 : 10,000), anticalnexin (1 :5000; Sigma, Cat. C4731). Subsequently, membranes were incubated with the corresponding horseradish- peroxidase-conjugated secondary antibodies (1 :5000, Dako). The signal was, then, revealed with a chemiluminescence solution (ECL reagent, RPN2232, GE Healthcare) and detected with the ChemiDoc imaging system (Bio-Rad). For alpha-syn immunoblotting, brain homogenates were processed in order to collect the TBS and SDS-soluble fraction of a-syn as described ⁵⁴ . Briefly, brains were homogenated with TBS (pH 7.4), clarified from non- homogenated residue and submitted to 100.000 g centrifugation at 4 °C for 1 hr. The resulting supernatant represents the TBS-soluble fraction. Then, pellets were solubilized in TBS-SDS (SDS 5% w/v) by sonication and centrifuged at 100.000 g for 30 min at 25°C. Supernatants were collected and referred to as the SDS-soluble fractions. Sampled (15 ug/uL of total proteins) were loaded in a gradient gel (Bis-tris gel 4-12%, NP0322BOX, Invitrogen) with MOPS as running buffer (NuPAGE MOPS SDS, NP0001 , Invitrogen) at 200V. The transfer was performed for 2hr at 40V on nitrocellulose membrane (nitrocellulose membrane 0.45 Mm, 1060003, GE Helthcare). Membranes were, then, blocked in 5% BSA for 1 hr and the primary antibody (Syn21 1) incubated overnight at 4 °C. After the incubation with the appropriate HRP-conjugated secondary antibody for 30 min the signal was then revealed and processed as previously described.

EEG recordings

At least three days before recording, epidural stainless steel screw electrodes (0.9 mm diam./3 mm long) were surgically implanted under ketamine/xylazine anesthesia and secured using dental cement (Ketac Cem, ESPE Dental AG, Seefeld, Germany). Two active electrodes were placed on right and left parietal areas (2mm lateral to midline, 1 mm posterior to bregma) and one over the occipital area (1 mm posterior to lambda) as a common reference. Freely moving 12 hour sessions of digital EEG monitoring were performed via a flexible cable connected to the amplifier (Micromed Mogliano Veneto, Italy) in a Faraday cage, with food and water available ad libitum. EEG traces were filtered between 0.53 and 60 Hz and sampled at 256 Hz (16 bits). EEG recordings were visually inspected to detect epileptiform discharges and/or seizures, defined as high-amplitude (at least 2 times the baseline) rhythmic discharges lasting at least 5 seconds.

Ex vivo electrophysiological recordings Mice (60-90 days of age) were anesthetized with an intraperitoneal injection of a mixture of ketamine/xylazine and transcardially perfused with ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCI, 2.5 KCI, 1.25 NaH ₂ P0 ₄ , 2 CaCI ₂ , 25 NaHC0 ₃ , 1 MgCI ₂ and 1 1 D-glucose saturated with 95% 0 ₂ , 5% C0 ₂ (pH 7.3). After decapitation, brains were removed from the skull and mounted in a VT1000S vibratome chamber (Leica Microsystems, Wetzlar, Germany) filled with ACSF at 4°C. Sagittal brain slices were cut at a 300-μιη thickness. Individual slices were submerged in a recording chamber mounted on the stage of an upright BX51WI microscope (Olympus) equipped with differential interference contrast optics (DIC) and an optical filter set for the detection of mCherry red fluorescence (Semrock, Rochester, NY). The slices were continuously perfused with ACSF (3-5 ml/min) at room temperature. Fast-spiking interneurons expressing DREADD were visually identified by tdTomato fluorescence. Whole-cell patch clamp recordings were performed using pipettes filled with a solution containing the following (in mM): 124 KH ₂ P0 ₄ , 2 MgCI ₂ , 10 NaCI, 10 HEPES, 0.5 EGTA, 2 Na ₂ -ATP, 0.02 Na-GTP (pH 7.2, adjusted with KOH; tip resistance: 6-8 ΜΩ). CNO (10 μΜ) was added through extracellular perfusion. All recordings were performed using a MultiClamp 700B amplifier interfaced with a PC through a Digidata 1440A (Molecular Devices, Sunnyvale, CA, USA). Data were acquired using pClampI O software (Molecular Devices) and analyzed with Prism 5 (GraphPad Software, Inc., La Jolla, CA). Current-clamp traces were sampled at a frequency of 10 kHz and low-pass filtered at 2 kHz.

Behavioral studies

The novel object recognition test was performed in a square arena of 40 ^χ 40 cm. On day 1 , mice were habituated to the open-field apparatus in a 5 min session. On day 2, animals underwent the training phase (10 min), in which two identical objects were introduced into the arena before allowing the mouse to explore. The amount of time that the rodents spent exploring each object was scored. Finally, on day 3, mice were tested for their memory (10 min). The discrimination index (Dl) is defined as the difference between the exploration time for the novel object and the one for the familiar object, divided by total exploration time, was calculated.

Statistics

The results were analyzed with GraphPad Prism version 6.0c for Macintosh. Unpaired Student's i-test or two-way ANOVA followed by Bonferroni's post-tests, was used in the datasets to be analyzed. Results Global-scale neural transduction and single neuronal cell labeling by AAV-PHP.B intravenous delivery in neonatal and adult mice.

An AAV2 transfer plasmid was used to clone the GFP cDNA downstream of a constitutive CBA promoter and combined with the PHP.B rep/cap and helper plasmids for productive viral infection. Viral particles were then harvested from both cells and supernatants, separately concentrated and finally mixed together in order to obtain high-titer viral preparations. A dose of 2 x 10 ¹² vg of AAV-PHP.B-GFP was administered by tail vein injection into 8-week-old mice (Figure 1A). Transduction efficiency was evaluated between 3 and 5 weeks post-injection by assessing GFP expression in various organs. As previously reported ¹⁰ , GFP signal was widely detected in all CNS regions, with diffuse and robust staining in the forebrain, midbrain, and cerebellum and along the entire spinal cord axis. (Figures 1 B-E and 9). Co-labeling for regional neuronal markers and GFP expression revealed that a very significant fraction of neurons, generally higher than 65%, was targeted by AAV-PHP.B in these regions (Figures 1 F-J). Interestingly, beyond the CNS, robust GFP expression was detected in the dorsal root (DRG) and sympathetic (SG) ganglia as well (Figures 1 K-M). In fact, the majority of blll-tubulin ⁺ DRG and tyrosine hydroxylase (TH) ⁺ thoracic SG neurons were effectively transduced by the virus (Figures 1 K-WI and 10). Therefore, a single i.v. administration of AAV-PHP.B-GFP is sufficient for global and robust transduction of the adult mouse CNS and provides new evidence of efficient tropism for PNS structures as well. Furthermore, we assessed the AAV-PHP.B viral distribution after i.v. injection in neonatal mice. Interestingly, 3 weeks after viral administration, global targeting of the nervous system was confirmed, with a pattern similar but not identical to that obtained in adult mice (Figure 11). In fact, the GFP signal was particularly strong in selected glial populations in the cerebral cortex, hippocampus and striatum (arrows in Figure 11). Although neuronal transduction was generally very efficient, especially in the cortex, hippocampus, cerebellum and spinal cord, it was very limited in the substantia nigra, revealing notable differences with respect to the transduction pattern obtained through i.v. delivery in adult animals (Figure 11). Thus, at the perinatal stage, although administered by a comparable systemic delivery route, the AAV-PHP.B had a close but distinct tropism for glial and neuronal cells, probably as a result of some phenotypic differences between neonatal and adult cells likely associated with their maturation state.

Sparse and selective labeling of distinct neuronal populations is a prerequisite for accurate tracing of nerve projections. To facilitate morphological analysis, we infused the AAV-PHP.B- Cre at different doses in adult Ai9 reporter mice ¹⁸ and evaluated the extent of transduction in the brain parenchyma (Figure 12A). Interestingly, a low viral dose enabled sparse to single- cell labeling in the brain tissue as detected by tdTomato immunofluorescence imaging (Figures 12B-K). Given the whole-brain targeting profile of this virus, single-cell labeling was simultaneously obtained in different brain areas including the cortex, hippocampus and cerebellum (Figures 12E, H, K). These results demonstrate that a single administration of this virus at a low titer enables single-neuron visualization in multiple brain regions simultaneously.

Facile AAV-PHP.B-based Cre-loxP conditional gene activation and control of neuronal activity in selected neuronal subtypes throughout the brain

Global targeting of the mouse nervous system by AAV-PHP.B has considerable implications, but it might represent a drawback when the aim is to study more specific neuronal targets or circuits. Thus, we conceived the idea to combine the widespread targeting of AAV-PHP.B with Cre-loxP technology in order to target specific neuronal subtypes in large brain areas (Figure 2A). Initially, we confirmed that systemic transduction of Cre-expressing AAV-PHP.B into Ai9 mice carrying a fluorescent tdTomato protein downstream of a loxP-flanked STOP cassette triggered very efficient Cre excision of the cassette and subsequent expression of the reporter throughout the brain (Figure 2B). Next, we generated an AAV-PHP.B carrying GFP in a FLEX switch cassette whose expression is activated by Cre recombinase. Thus, AAV-PHP.B-FLEX-GFP was infused in different transgenic mouse strains expressing Cre in specific neural subtypes (Figure 2A). Interestingly, this configuration enabled very selective expression of the GFP transgene according to the Cre expression pattern for each transgenic line. In infused NeuroD6-Cre mice, GFP expression was specifically confined to neurons in the cortex and hippocampus (Figures 2C, D and data not shown). Co-staining with NeuN and GFP revealed that more than 80% of all the neurons in the aforementioned territories expressed GFP (Figures 2E, F). Conversely, neither S100 ⁺ nor GFAP ⁺ glial cells were found to co-express GFP (data not shown). Likewise, systemic viral transduction of parvalbumin (PV)-Cre and dopamine transporter (DAT)-Cre transgenic mice led to a very specific pattern of GFP expression restricted to the forebrain GABAergic interneurons or the midbrain dopaminergic neurons, respectively (Figures 2G-0). In both cases, GFP expression was detected in the majority of these neuronal cell populations and not in other neuronal or glial cell types (Figures 2J, O). However, a similar strategy could be equally employed to selectively target glial cells. In fact, i.v. viral transduction of Olig2-Cre mice enabled specific GFP labeling of CC1 ⁺ oligodendrocytes but not GFAP ⁺ astrocytes (Figures 2P-R). Although the overall viral targeting was not as efficient as for neurons, approximately 60% of the oligodendrocyte lineage was targeted (Figure 2S).

Thus, combining Cre-loxP cell lineage specificity with spatially broad AAV-PHP.B transduction enabled the targeting of a specific neural subtype in wide regions up to the whole brain. This is a favorable setting for evaluating the function of a specific neuronal cell type within the brain and its resulting effects in live animals. Current optogenetic methods evaluate the effects of altering a specific neuronal circuit, but only in a confined brain territory limited by the intraparenchymal spreading of conventional viruses ¹⁹ . To move beyond this, we conceived the idea to combine the AAV-PHP.B/Cre-loxP system with the chemogenetic DREADD technology to obtain global modulation of the neuronal activity ²⁰ . Moreover, the DREADD receptors are activated by the brain-penetrant small molecule clozapine N-oxide (CNO), which additionally provides fine temporal control of this system. Thus, as a proof of concept, we sought to test the effects of altering PV ⁺ neuronal function in the whole brain. Although PV has some expression in subcortical regions, especially in cerebellar Purkinje neurons, its major expression selectively localizes to the forebrain fast-spiking inhibitory interneurons. Therefore, we transduced adult PV-Cre transgenic mice with an AAV-PHP.B- FLEX-DREADDM4-mCherry (PHP.B-FLEX-M4C) to inhibit activity exclusively in the PV- expressing neurons (Figure 3A). Double immunofluorescence for the viral mCherry reporter and PV showed a common pattern of staining in the somatosensory cortex (Figures 3B-C). Quantitative analysis confirmed that the mCherry reporter was restricted exclusively to the PV ⁺ interneurons (Figure 3E). Conversely, a small fraction of PV ⁺ cells did not express mCherry, probably because they were not transduced by the virus (Figure 3E). To determine whether the system was functional, we acutely sliced brains from transduced mice and the recorded the electrical activity of mCherry ⁺ neurons during patch-clamp experiments. Soon after CNO was added to the slice extracellular medium, recorded neurons silenced their activity with an abrupt loss of action potentials and membrane potential hyperpolarization (Figure 3F). Next, PHP.B-FLEX-M4C-transduced PV-Cre mice were implanted with epidural electrodes and EEG recordings performed before and after CNO injection. Interestingly, after CNO, the EEG showed slowed background activity and mild epileptic abnormalities (sharp waves), with no clear epileptic behavior in mice recorded for 12 hrs (n = 3) (Figure 3G). We then hypothesized that loss of PV forebrain interneuron activity could lead to increased seizure susceptibility. Accordingly, kainic acid-induced seizure activity was strongly enhanced in PHP.B-FLEX-M4C, leading to animal death immediately after treatment (5/5), while the majority of control mice treated with PHP.B-GFP survived the same treatment (4/5) (Figure 3H). These findings exemplify a strategy, sophisticated yet extremely easy to implement, by which to control neuronal activity in the whole brain and determine its underlying behavioral consequences in live animals.

Rapid analysis of Tsc1 gene function in adulthood by systemic injection of Cre- expressing AAV-PHP.B

Global nervous system targeting by AAV-PHP.B might also be convenient to regulate gene activity in transgenic mice carrying floxed gene alleles. In fact, deleting genes with Cre-loxP technology to study their effects exclusively in adulthood requires a rather extended time to obtain the mutant mice for phenotypic analysis. Thus, we generated a constitutively Cre- expressing AAV-PHP.B and systemically injected it in adult mice carrying a floxed allele for Tsc1 (Figure 4A). Mutations in TSC1 in humans are responsible for tuberous sclerosis complex (TSC), a disorder characterized by severe intellectual disability and intractable seizures ^{21 ,22} . Inactivation of TSC1 or its homolog TSC2, with which it forms a multimeric complex, causes hyperactivation of mTOR complex 1 (mTORCI) and hyperphosphorylation of its downstream effectors including ribosomal protein S6 ^23,24 . TSC patients present with focal brain lesions, known as cortical tubers and subependymal nodules, characterized by general cellular disorganization and giant cells. It is believed that these structural brain alterations are the primary cause of the chronic epileptogenic state ²⁵ . Homozygous Tsc1 or Tsc2 mutant mice recapitulate the pathological milestones described in patients ²⁶ . In fact, Tsc1/2 gene deletion causes overt brain pathology associated with severe epileptic crises and consequent death soon after birth ²⁶ . Whether epilepsy is a result of the cortical tissue disorganization occurring during development or, conversely, is caused by a cell- autonomous dysfunction in the mutated neurons has remained controversial. Recently, full- body acute Tsc1 inactivation in adulthood by classical mouse transgenic breeding was found to lead to profound epileptic seizures in the absence of neurodevelopmental brain lesions ²⁷ . To extend this analysis, we infused AAV-PHP.B-Cre at a high dose (2 x 10 ¹² vg) in adult Tsc1 ^f/f and Ai9 reporter mice. Starting 1 week after systemic viral injection, mice developed severe epileptic seizures, with a minimum of 6 crises detected in a 12-hr continuous EEG recording (Figures 4B, C). About half of these animals died in the following 4 weeks (4 out of 9). A similar dose of AAV-PHP.B-Cre robustly activated tdTomato in the brains of the Ai9 mice (Figure 4D). To assess mTOR activation at the cellular level, we performed immunohistochemistry for phospho-S6 (pS6). As expected, pS6 staining was strongly increased in transduced floxed Tsc1 brains but not in Ai9 control brains, with most of the neurons in the cerebral cortex and hippocampus presenting a strong positive cytoplasmatic signal (Figures 4E-H). We then asked whether loss of Tsc1 in only a fraction of neurons would be sufficient to cause a disease state. Thus, we injected AAV-PHP.B-Cre at a low dose (10 ¹¹ vg) in Tsc1 ^f/f mice. This dose of virus transfused in the brain of Ai9 conditional mice activated the tdTomato reporter in approximately 35% of cells in the cerebral cortex (Figure 4K). Nonetheless, even with this dose of virus, all the animals developed severe seizures starting 3 weeks after treatment, although the number of crises was reduced to an average of 1-2 events in 12 hrs (Figures 4I, J). Remarkably, strong pS6 staining was detectable only in a mosaic fashion in the cortex and hippocampus, accounting for only a 25% of neurons, in treated Tsc1 mice but not in Ai9 control mice (Figures 4K-0). These results demonstrate that the Tsc1 gene has an indispensable cell-autonomous role in adult neurons and that its loss triggers severe epileptogenesis in mice, even when only a fraction of neurons carry mutant alleles for this gene.

Whole brain GBA1 gene transfer significantly prevents alpha-syn inclusion formation in A53T-SCNA transgenic mice

The brain-penetrating AAV-PHP.B is an unprecedented platform to exploit gene therapy protocols to treat neurodegenerative disorders affecting the whole nervous system. In particular, this system can be explored to sustain diffuse expression of GBA1 in the brain to potentially counteract the gradual widespread accumulation of alpha-syn inclusions in the nervous system. To test this hypothesis, we employed A53T-SCNA transgenic mice that overexpress the SCNA mutation responsible for a genetic form of PD in humans. Starting from 6 months of age, these mice gradually accumulate insoluble alpha-syn deposits throughout the brain, with particular enrichment within the cerebral cortex, the midbrain and the pons, and at 10-12 months of age most of them die after developing a severe and rapid loss of voluntary movements and fatal paresis ²⁸ . We focused particularly on the somatosensory (sCx) and visual (vCx) cortical areas, where alpha-syn aggregates were particular evident and diffuse (Figures 5A,B, arrowheads), resembling the alpha-syn toxicity in the cerebral cortex of PD patients, which leads to cognitive disabilities and dementia. Initially, we asked whether the overexpressed GCase enzyme encoded by the GBA1 transgene could be properly targeted to the lysosome and acquire functionality. Thus, GBA1 was tagged with mCherry, a fluorescence tag, which maintains its activity in the acidic lysosomal environment, and transfected into HeLa cells. Co-staining for mCherry and Lamp2 revealed that the exogenous GCase protein was at least in part correctly localized in the lysosomes (Figure 13A). To determine whether the expressed GCase was functional, we assessed the overall enzymatic activity using a quantitative assay with a specific synthetic substrate. GBA1-overexpressing cells exhibited a significant increase in GCase catalytic activity compared with untransfected cells, demonstrating the complete functional maturation of the exogenous GCase (Figure 13B, C). Hence, we cloned the GBA1 cDNA upstream of a P2A-GFP cassette driven by the EF1 alpha promoter in a shuttle vector and used it to generate AAV-PHP.B viral particles. Then, 5-month-old A53T-SCNA transgenic mice were infused with either the GFP- (control) or the GBA1 -P2A-GFP-expressing virus. A group of animals was subsequently euthanized at 10 months of age, when control mice started to perish, and brain tissue was isolated for molecular and neuropathological inspection. Since GBA1 antibodies failed to give reliable immunohistochemical staining, we investigated the global pattern of brain transduction in A53T-SCNA mice by GFP reporter analysis. As shown in Figures 5D-G, GBA1-P2A-GFP (hereinafter referred to as GBA1 only) gene transfer was efficient and diffuse in all the forebrain regions, infecting both neurons and glia. Accordingly, the immunoblotting profiling of cortical and hippocampal tissues from GBA1 -transduced animals confirmed a robust increase in the overall amount of GCase protein (Figures 5H, I). We then evaluated the levels of GCase activity in control and treated animals. Interestingly, GCase enzymatic activity was strongly reduced in control A53T-SCNA transgenic mice in most of the neural regions tested, in line with previous data suggesting that alpha-syn pathology affects GCase protein processing and targeting to lysosomes ¹⁴ . Conversely, GBA1 -transduced animals exhibited a strong rescuing of GCase enzymatic levels, which were at least comparable to those detected in wild-type animals in all the CNS regions (Figure 5J). Then, alpha-syn pathology was specifically assessed by immunostaining for pS129-alpha-syn in PK-treated brain sections to enable the accurate identification of insoluble intracellular alpha-syn deposits. In the visual cortex, alpha-syn aggregates were mainly detected within the somata of neurons, as revealed by both immunohistochemistry and immunofluorescence imaging (Figures 6A-D). Remarkably, GBA1 gene transfer elicited a strong reduction of alpha-syn pathology in the visual cortical areas (Figure 6A-E). To extend this analysis, we performed stereological semi-automatic counting of alpha-syn inclusions within the visual (vCx, anteroposterior position from -3 mm to -4 mm, centered on bregma), cingulate (cCx), motor (mCx) and somatosensory (sCx) cortical areas as well as the striatum (anteroposterior position from +1 to -0.5 mm, centered on bregma). Accordingly, the overall quantity of PK-resistant deposits was significantly diminished in all these brain domains to a comparable extent, confirming efficacious and widespread protection from alpha-syn pathology (Figure 6E). To confirm the effects of the exogenous GCase activity on a-syn protein processing, the various forms of a-syn were resolved and analyzed by Western blotting of TBS-soluble and TBS-insoluble fractions of forebrain lysates (Figure 6F). Indeed, a significant decrease in both monomeric and oligomeric forms of a-syn (including low- (LMW) and high-molecular-weight (HMW) aggregates) was observed in GBA1 compared with GFP transduced tissues (Figure 6F).

Next, we wondered whether the acute reduction of alpha-syn pathology in adulthood correlated with any behavioral amelioration. GBA1 -transduced animals showed a consistent increase in median survival compared with control treated mice, with a consistent fraction of animals surviving when all control mice had expired (Figure 14A). In addition, GBA1-treated, but not control mice exhibited a strong recovery in learning and cognitive performance as revealed by a significant improvement in the novel object recognition test both at 3 and 5 months after treatment (Figure 14B). Overall, GBA1 -transduced mice showed a robust reduction of alpha-syn pathology in the whole forebrain, suggesting that the exogenous GCase provided sufficient supplemental activity to limit and counteract the widespread development and accumulation of alpha-syn deposits. Hence, these data strongly indicate that AAV-PHP.B-mediated gene transfer in the adult brain is an outstanding system to express a therapeutic gene throughout the brain tissue in order to curb pervasive pathological manifestations often associated with the progression of neurodegenerative diseases.

AAV-PHP.B viral brain transduction through the carotid artery route limited viral diffusion in peripheral organs

Systemic i.v. delivery enables the effective spreading of the virus throughout the brain vasculature and subsequently in the neural parenchyma. However, the peripheral venous route diffuses the virus to the whole body and its peripheral organs. Thus, a single i.v. injection of AAV-PHP.B is sufficient to transduce, beyond the nervous system, a non- marginal fraction of cells in all the peripheral organs ¹⁰ . This undesired viral spreading might result in a serious drawback for many potential applications of this system. To restrict the viral delivery to the nervous system, we sought to inject the virus directly into the brain circulation. For this, 2 x 10 ¹² vg of AAV-PHP.B-GFP was directly infused into the internal carotid artery via a microcatheter (Figure 7A). Brains and peripheral organs were retrieved 3 weeks after injection for immunofluorescence analysis. Notably, GFP transgene expression was detected diffusely throughout the brain with high transduction efficiency (Figures 7B-D). Co-labeling between GFP and either NeuN, TH or calbindin-1 showed that a high fraction of cortical and mesencephalic nigral neurons as well as cerebellar Purkinje cells were effectively transduced (Figures 7F-I). We then compared the viral GFP gene transfer in peripheral organs after tail vein and carotid artery injections. Analysis of GFP expression in the liver, heart and muscles showed that carotid infusion substantially reduced the viral distribution in all these peripheral organs (Figures 7J-Q). In particular, the viral transduction in the liver and heart was decreased by more than 5- and 10-fold, respectively (Figure 7Q). These data indicate that the carotid artery route is advantageous since the nervous system targeting is coupled to a reduction in peripheral spread.

AAV-PHP.B brain targeting does not impair blood-brain barrier integrity or selectivity

Considering the extremely efficient brain diffusion of the AAV-PHP.B after i.v. injection, we wondered whether it could alter blood-brain barrier (BBB) properties. We therefore analyzed BBB permeability and inflammation after AAV-PHP.B transduction in vivo. For this aim, mice were intravenously injected with fluorescent-conjugated cadaverine dye, a small (640 Da) BBB permeability marker, together with the virus AAV-PHP.B-GFP (Figure 8A). Staining for viral capsids with the AAV-VP3-specific antibody (B1) confirmed the localization of the viral particles within the brain endothelium 24 hrs after viral delivery (Figure 8B). However, the transduced brain tissue did not show any evident diffusion of the cadaverine dye (Figure 8C). In addition, no signs of astrocytosis were revealed by GFAP staining in the targeted tissue 2 days after viral transduction (Figure 8C). As a positive control, diffuse cadaverine staining and astrocyte activation were detected in the brain parenchyma of kainic acid- treated mice that developed seizure-induced BBB permeability and severe inflammation (Figure 8E-G). To further assess BBB integrity upon AAV-PHP.B transduction, we employed a simplified in vitro BBB model obtained by isolating and culturing primary mouse brain microvascular endothelial cells (BMVECs). Acutely dissociated BMVECs were cultured to confluence to form an organized epithelial layer and then either infected with AAV-PHP.B- GFP or left untreated for 5 days (Figure 8H). In these conditions, untreated cells maintained cell-cell contacts positive for the tight junction markers ZO-1 and claudin-5 (Figure 8I, J). Similarly, virally transduced cells, identified by GFP expression, displayed comparable ZO-1 and claudin-5 protein localization at cell junctions (Figures 8K, L). Finally, we asked whether the viral infection could perturb the transendothelial electrical resistance (TEER), a key measurement of tight junction resistance in endothelial cells. Notably, there was no significant difference in TEER values between untreated and infected cells as measured up to 5 days from viral loading (Figure 8M). Conversely, TEER signal was strongly abolished when EDTA was added to the culture, causing a loss of calcium-dependent cell junctions (Figure 8M) ²⁹ . Altogether, these data indicate that AAV-PHP.B targeting to the BBB does not alter the basic properties of the brain endothelium, maintaining unaltered its barrier selectivity in vivo and morphological integrity in vitro.

Discussion

AAV9 is the only AAV serotype able to cross the BBB when delivered through the vascular system. However, this ability is considerably diminished in adulthood, raising significant hurdles for pervasive targeting of the adult brain through a peripheral route. Remarkably, the AAV-PHP.B variant maintains efficient penetration even in the mature BBB and widely diffuses in the brain parenchyma in adult mice ¹⁰ . Herein, we confirmed and extended these data, showing that a single i.v. injection of AAV-PHP.B can globally transduce both the central and the peripheral nervous system. Interestingly, we showed that DRGs and SGs are both efficiently targeted by AAV-PHP.B, which mostly infects the sensory neurons. Given that satellite glial cells or and interneurons were poorly transduced by the virus, it is likely that the infection mainly followed a retrograde route, with initial uptake at the periphery followed by retrograde transport to the neuronal soma. Our results and those of previous studies have shown that the systemic i.v. route is intrinsically associated with widespread transduction of peripheral organs ^30,31 . In this work, we employed single-stranded AAVs (ssAAV) exclusively. Recent studies have shown that self-complementary AAV9 (scAAV9) can transduce the adult brain parenchyma ⁹ . However, in all cases, the efficiency of transduction was far from the level necessary for supporting the technical approaches herein presented. In addition, scAAV9 facilitates viral transduction by circumventing the limiting step of the synthesis of the viral DNA complementary strand, but it also causes a loss of half of the coding packaging, reducing it to only 2.2 kb for the entire expression cassette including the promoter, coding gene and polyA sequences ³² . Thus, AAV-PHP.B supports the global spread of the virus in the nervous system while maintaining the full packaging space for AAV, providing convenient flexibility in designing the transgene cassette.

Herein, we established straightforward approaches to control gene expression and neuronal activity in the adult mouse brain with a single-step protocol. These procedures will have a strong impact by accelerating functional studies of genes and molecular labeling of neuronal cell types for anatomical tracing. Furthermore, the AAV-PHP.B whole-brain delivery of the chemogenetic DREADD system opens the opportunity to manipulate the activity of selected neurons in large brain areas and eventually in the entire brain and subsequently evaluate the resulting behavioral response.

We provided a strong proof of concept of this strategy by showing that whole-forebrain inactivation of PV* GABAergic interneurons, while not sufficient per se to elicit spontaneous epileptic seizures, creates a strong predisposition to them after a proepileptic insult. However, PV is also expressed in some caudal brain areas and in Purkinje cerebellar neurons. Overall, we could not exclude the possibility that other cell populations might have influenced this phenotype. Thus, the choice of selective genetic tracing, when available, is a crucial prerequisite to subsequently retrieve conclusive functional data. Wide-scale access to the adult mouse nervous system makes feasible to misexpress genes and evaluate their direct impact on brain functions and consequent behavior.

Taken together, these results provide solid evidence that the brain-penetrant AAV-PHP.B is an ideal platform for transducing therapeutic genes to treat neurodegenerative disorders that globally affect the brain tissue. Herein, we focused on alpha-syn inclusions, that spread over time throughout large brain areas in PD, LBD and MSA and are responsible for cortical functional decline leading to severe dementia ^33,34 . Approximately 5-8% of PD patients are carriers of a heterozygous GBA1 mutation, causing a detectable reduction in GCase global activity ^11,12 . PD patients carrying GBA1 mutations often develop more severe symptoms then GBA1 non-carrier patients, including an accelerated cognitive decline associated with increased a-syn accumulation ^35,36 . Thereby, stimulating GCase activity in these patients represents a direct and valuable therapeutic approach. Furthermore, GCase activity gradually declines with aging in healthy individuals, and in addition, sporadic patients showed a further reduction in GCase functionality ^37,38 . Given this, increasing GCase levels can also be an effective therapeutic strategy for age-related and sporadic forms of PD. Herein, we showed that AAV-PHP.B-mediated global expression of GCase is sufficient to provide robust and long-lasting protection from alpha-syn deposits in a mouse model of synucleinopathy. Exogenous virally delivered GCase is targeted to the lysosome and acquires functionality, which resulted in significantly diminished accumulation of insoluble alpha-syn species in all the forebrain regions. Previous studies have shown that alpha-syn accumulation is promoted by diminished GCase activity in vitro and in vivo, which leads to an abnormal accumulation of its glycolipid substrates in the lysosomes ^39,40 . Conversely, alpha-syn inhibits the lysosomal activity of GCase, thereby causing a loss of its catalytic function upon progressive accumulation of alpha-syn ¹⁴ . Hence, pathological conditions establish a vicious cycle between alpha-syn and GCase that can sustain and progressively worsen the disease ^14,15 . Along these lines, stimulating GCase activity has been shown to counteract alpha-syn pathology in mouse and human neurons in Wire ^16,17,41 . Our results support this view, showing that GCase is a strong determinant of alpha-syn accumulation and that increasing its enzymatic levels significantly protects against alpha-syn pathology and toxicity. Intriguingly, although GBA1 viral transduction did not target the entire neuronal population, we nonetheless observed a general and even reduction of alpha-syn inclusions throughout the neural parenchyma. These results are plausible considering that the GCase enzyme has non-cell-autonomous action, reducing alpha-syn deposits even in cells in which it is not directly stimulated as long as they are placed close enough to virally transduced cells. In support of this hypothesis, classical cell biology experiments have shown that lysosomal enzymes can be released from producing cells, endocytosed by their neighbors, and correctly trafficked to their lysosomes both in vitro and in vivo ⁴² . Therefore, our findings imply that it is not necessary to transduce the entire brain neuronal population; GCase overproduction in a partial subset of neural cells (either neurons or glia, or both) is sufficient to achieve widespread protection from alpha-syn inclusions. Strategies to reduce alpha-syn toxicity by active immunization or by stimulating GCase activity through small-molecule noninhibitory chaperones are currently being explored to establish therapeutic approaches for these diseases ^43"45 . However, limited crossing of the BBB, toxic side effects and restricted efficacy to only some disease forms are some of the important hurdles that remain to be fully cleared to translate these treatments to patients. The present results strongly imply that gene therapy should be considered as a further therapeutic opportunity for synucleinopathies with the potential advantage of providing a long-lasting beneficial effect with a single treatment. The introduction of the AAV-PHP.B viral platform, which sustains effective BBB crossing and global spreading of the therapeutic GBA1 gene in the adult brain tissue, fulfills the necessary conditions for the development of an effective and non-invasive gene therapy approach for synucleinopathies. Further studies on wild-type animals will be necessary to address whether the chronic stimulation of the GCase activity might cause any long-term adverse effect on neuronal homeostasis and function, regardless of whether this stimulation will be achieved by a small-molecule or gene- based approach. However, this might not be a serious hurdle in our approach since the GBA1 viral transduction elicited a strong rescuing of GCase activity in most brain regions of A53T-SCNA transgenic mice, but without causing evident supraphysiological activity compared with wild-type conditions.

The massive penetration of this virus into the brain upon acute delivery in the circulation raises some concerns about altering normal BBB physiology. Thus, we searched for any sign of acute derangement of BBB integrity after virus administration. Importantly, no evidence of loss of functional selectivity was found; our work showed that a small (640 Da) fluorescent dye was continuously excluded from entering the brain space immediately or soon after viral infection. This is in line with the absence in transduced brain tissues of any overt sign of inflammation, which is closely associated with abnormal BBB permeability. Thus, the acute targeting of AAV-PHP.B to the brain appears to be substantially harmless, at least in regards to major BBB functionality.

To concentrate the viral transduction to the brain, we administered the virus through the carotid artery, thereby maintaining high transduction efficiency within the neural parenchyma while strongly diminishing the spread of the virus in the peripheral organs. Although viral targeting in the liver and heart was strongly restrained, some remaining infected cells were still detectable, indicating that even direct arterial brain delivery can diffuse some viral particles into the systemic circulation. However, this delivery route has important advantages, especially from a therapeutic prospective. In fact, this viral administration is feasible in large apes and human clinical practice, limiting unnecessary viral spreading to peripheral organs while concentrating the viral particles to the therapeutic target, namely, the adult nervous system.

The diffuse penetration of the AAV-PHP.B in the adult mouse brain parenchyma is a unique property among all the recombinant viral strains in current use. Future studies will address whether this capacity will remain intact and equally efficient when tested in large animals. If so, this viral strain might become the system of choice to deliver therapeutic genes such as GBA1 to the brain, enabling the development of effective and non-invasive gene therapy approaches for synucleinopathies.

Example 2

Materials and Methods

The materials and methods for the A53T-SCNA mice and the procedure for viral production in Example 2 are the same as described in Example 1.

Results

AAV2-BR1 was generated to specifically target the brain endothelium (Korbelin et al., 2016). To properly evaluate this, we produced a GFP-expressing AAV2-BR1 and injected intravenously in adult mice and 3 weeks later evaluated its transduction ability. As expected, we detected a strong GFP signal in the endothelium of different brain regions including the cerebral cortex and hippocampus (Figure 15). No other cells beyond endothelium were found expressing GFP in the brain suggesting a tight cell type specificity of this virus. Importantly, GFP expression was completely lacking in vessels of the liver or other peripheral organs demonstrating that the virus was unable to successfully transduce endothelial cells outside the brain tissue (Figure 16).

Next, we asked whether the GBA encoded lysosomal enzyme GCase could be released from cells and re-uptaked by the neighbor cells. To this goal, the CMEM endothelial cells were transduced with the AAV-BR1-GBA virus. 4 days after the transduction and two changes of culture medium, the supernatant was harvested and transferred to GBA knockout HeLa cells (Figure 17). Remarkably, cells exposed to the AAV-BR1-GBA cell supernatant showed a significant increase in intracellular GCase activity. These data provide strong evidence that GCase can be released by endothelial cells overexpressing GBA and can be uptaken from the culture medium.

The extracellular release of GCase can be exploited as a therapeutic process in Parkinson's disease. In fact, the brain endothelium can be specifically targeted by the AAV-BR1 virus to express the GBA1 gene, and therefore becoming a long-lasting source of GCase throughout the brain tissue. To prove its relevance, wild-type adult mice were intravenously injected with the AAV-BR1-GBA or the AAV-BR1 -GFP viruses and sacrificed 2 months later. To assess the release of the enzyme, the endothelial cells were purified by CD31 immunopanning, and the negative fraction was containing the remaining cell types including neurons and glia (Figure 18). Importantly, in both cellular fractions GCase activity was significantly increased only using the AAV-BR1 -GBA, strongly indicating that GCase released by the endothelial cells was incorporated and functional active in other brain cells (Figure 18). We next moved to assess whether this approach could reach a therapeutic significance in a model of Parkinson's disease and synucleinopathy. 4 months old A53T-SCNA transgenic mice were intravenously transduced with the AAV-BR1 -GBA (n = 6) or the AAV-BR1 -GFP (n = 6) viruses and euthanized at 8 months of age. Immunohistochemistry for pS129-alpha- synuclein on brain sections treated with proteinase K showed that alpha-synuclein aggregates were evidently reduced in the cerebral cortex and hippocampus of animals treated with the GBA expressing virus (Figure 19). Altogether, these data provide evidence that GCase release from the brain endothelium can be up-taken by brain cells and strongly protect them by the progressive accumulation of alpha-synuclein aggregates in A53T-SCNA transgenic mice.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described agents, compositions, uses and methods of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to those skilled in biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims.

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