Adeno-associated virus compositions for treating parkinson's disease (PD)

Abstract

Aspects of the invention provide an AAV product that delivers an GBA1 gene supplementation strategy throughout the human CNS at levels of DNA Biodistribution, RNA expression, and GBA1 protein levels expected to restore function in patients with Parkinson's disease.

Description

[0001] ADENO-ASSOCIATED VIRUS COMPOSITIONS FOR TREATING PARKINSON’S DISEASE (PD)

[0002] I. FIELD OF THE INVENTION

[0003] The invention is related to the field of treatment of Parkinson’s disease (PD) by adeno- associated virus (AAV) compositions comprising therapeutic cargo.

[0004] II. SEQUENCE LISTING

[0005] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named CAPS-036-01WO-Seq-Listing.xml, created on April 4, 2025, which is 15KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0006] III. BACKGROUND

[0007] Parkinson's disease is a progressive disorder that affects the nervous system and the parts of the body controlled by the nerves. Nerve cell damage in the brain causes dopamine levels to drop, leading to the development of Parkinson’s disease in patients. Parkinson's disease often starts with a resting tremor in one hand. Other symptoms are slow movement, stiffness, and loss of balance. Parkinson’s disease may be caused by defect or absence of the GBA1 gene in the central nervous system. There is no known cure for Parkinson’s disease.

[0008] Recombinant adeno-associated viruses (rAAVs) are widely used as vectors for gene delivery in therapeutic applications because of their ability to transduce both dividing and nondividing cells, their long-term persistence as episomal DNA in infected cells, and their low immunogenicity. These characteristics make them appealing for therapeutic applications, such as gene therapy. However, there is a need to significantly improve the performance of existing AAV serotypes to selectively and efficiently express in distinct cell-types upon systemic delivery to a subject. This need is especially acute when the AAV must be expressed in the brain.

[0009] IV. SUMMARY OF THE INVENTION

[0010] Compositions and methods of the invention use recombinant adeno-associated viruses (rAAV) to deliver a viral vector comprising a human glucosylceramidase P-1 (GBA1') gene encoding a functional glucosylceramidase enzyme (“GBA protein” or “GCase”). By allowing cells to produce functional GCase, compositions and methods of the invention can be used to treat neurological conditions caused by reduction in GCase function, including Parkinson’s disease.

[0011] The present invention is directed to a recombinant adeno-associated virus (rAAV) engineered to enrich delivery of cargo to the central nervous system (CNS). The present invention provides rAAVs with widespread transduction to the brain. Following IV injection, unmodified rAAVs such as those derived from AAV9 (SEQ ID NO: 1) may not have sufficient tissue enrichment to treat certain human diseases by delivery of an AAV cargo. Accordingly, engineered rAAVs described herein are particularly useful in delivering DNA cargo to the brain. The rAAVs of the invention are beneficial for the treatment of certain neurological conditions.

[0012] In certain embodiments, the rAAVs of the present invention enhance the delivery of cargo to the brain. In certain aspects, the invention is directed to a rAAV comprising the human glucosylceramidase pi (GBA / ) gene. The GBA I gene may be the cargo in the rAAVs of the invention.

[0013] Specifically, the present invention provides an adeno-associated virus (AAV) vector comprising an engineered capsid protein that comprises an amino acid sequence having at least 80% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 4). In certain embodiments, the AAV may comprise an engineered capsid protein that comprises an amino acid sequence having at least 90% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 4). The vector further comprises (e.g. encapsidates) a nucleic acid sequence encoding a promoter and an GBA1 transgene. In preferred aspects of the invention the AAV capsid protein comprises the amino acid sequence ATRNGEVFIAQ (SEQ ID NO: 4).

[0014] In certain aspects, the invention provides an rAAV comprising a capsid protein comprising an amino acid sequence having at least 80% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 4), a promoter, and a therapeutic gene, wherein the therapeutic gene is the human GBAJ transgene. In certain embodiments, the invention provides an rAAV comprising a capsid protein comprising an amino acid sequence having at least 90% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 4), a promoter, and a therapeutic gene, wherein the therapeutic gene is human GBA1 transgene.

[0015] The capsid protein may be engineered from an AAV9 capsid protein. For example, the capsid protein is engineered relative to an AAV9 capsid protein by substituting amino acid position 588 with the amino acid T and by peptide insertion of amino acids RNGEVFI between amino acid positions 588 and 589. Parental AAV9 amino acids in positions 589 and 590, AQ, are unchanged.

[0016] As a result, the amino acid positions 587-597 of the AAV capsid comprises the sequence ATRNGEVFIAQ. In certain embodiments, the engineered capsid protein comprises amino acid sequence that is at least 90% identical to the amino acid sequence provided in SEQ ID NO: 7. In certain embodiments, the engineered capsid protein comprises amino acid sequence that is at least 95% identical to the amino acid sequence provided in SEQ ID NO: 7. In certain embodiments, the engineered capsid protein comprises amino acid sequence that is at least 98% identical to the amino acid sequence provided in SEQ ID NO: 7. In certain embodiments, the engineered capsid protein comprises amino acid sequence provided in SEQ ID NO: 7.

[0017] A person of skill in the art will understand the equivalent positions of AAV vectors other than AAV9 in which the sequence ATRNGEVFIAQ (SEQ ID NO: 4) may be adapted.

[0018] Accordingly, in certain embodiments, the AAV vector may comprise an AAV9 backbone. For example, the AAV vector may comprise an AAV capsid protein comprising an amino acid sequence that is at least 98% identical to amino acid 217 through amino acid 736 of AAV9 (SEQ ID NO: 1).

[0019] In certain embodiments, the therapeutic gene is a human GBA1 transgene. In certain embodiments, the therapeutic gene is a wild-type human GBA1 transgene. In certain embodiments, the therapeutic gene is a modified or recombinant human GBA1 transgene. In certain embodiments, the GBA1 gene is codon optimized. In certain embodiments, the GBA1 transgene is at least 95% identical to the human transgene provided in NCBI reference NM_000157.4 (SEQ ID NO: 2). In certain embodiments, the GBA1 transgene is the transgene provided in NCBI reference NM 000157.4 (SEQ ID NO: 2) or a substantially similar gene.

[0020] In certain embodiments, the rAAV of the invention further comprises a CAG promoter. In certain embodiments, the CAG promoter comprises three (3) regulatory elements. In certain embodiments, the regulatory elements are: human cytomegalovirus immediate early enhancer (CMV enhancer), a chicken beta-actin promoter, and a chimeric rabbit P-globin (rBG) intron.

[0021] In certain embodiments, the rAAV of the invention further comprises a post-transcriptional regulatory element. The regulatory element increases the efficiency of transcription of the therapeutic drug in the complex. In certain embodiments, the post-translational regulatory element (WPRE) is a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In certain embodiments, the rAAV of the invention further comprises a poly(A) signal. The poly(A) signal enhances stability of the cargo in the rAAV. In certain embodiments, the poly(A) signal is a bovine growth hormone polyadenylation signal (bGH-polyA).

[0022] In certain embodiments, the rAAV of the invention may further comprise an HA epitope tag. In certain embodiments, the HA epitope tag is at the C-terminus of the hGBA protein. In certain embodiments, the HA epitope sequence is YPYDVPDYA (SEQ ID NO: 3).

[0023] Accordingly, in certain embodiments, the invention provides a rAAV comprising a capsid protein comprising an amino acid sequence having at least 80% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 4) and a payload comprising a human GBA1 gene. In certain embodiments, the invention provides a rAAV comprising a capsid protein comprising an amino acid sequence having at least 90% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 4), and a payload comprising a human GBA1 gene. In certain embodiments, the rAAV of the invention comprises capsid protein comprising an amino acid sequence having at least 90% identical to ATRNGEVFIAQ (SEQ ID NO: 4), a payload comprising a human GBA1 gene, a CAG promoter, a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a bovine growth hormone polyadenylation signal (bGH-polyA). In certain embodiments, the invention further provides that the rAAV of the invention may optionally include a HA epitope tag at the C-terminus of the hGBAl protein.

[0024] In some instances, the 5' ITR and the 3' ITR are derived from an AAV2 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV5 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV9 serotype. In some instances, the 5’ ITR and the 3’ ITR each originate from different serotypes, e.g. 5’ ITR from serotype AAV2 and 3’ ITR from AAV5. In some instances, the 5’ ITR and / or the 3’ ITR originate from another natural serotype or have been engineered for improved transduction or transgene expression efficiency.

[0025] For example, the 5’ ITR (referred to as L-ITR) may comprise a nucleic acid having at least 90% sequence identity with the sequence of SEQ ID NO: 8. The 3’ ITR (referred to as R-ITR) may comprise a nucleic acid having at least 90% sequence identity with the sequence of SEQ ID NO: 9.

[0026] In certain aspects, the invention provides methods of treatment of diseases by administration of a composition comprising rAAVs of the invention. In certain embodiments, the disease is a neurological disease. Parkinson’s disease (PD) is associated with motor and non-motor symptoms, including bradykinesia, rigidity and resting tremor, cognitive decline, sleep disturbances, hyposmia and psychiatric symptoms. A key pathological hallmark of PD is the loss of nigrostriatal dopaminergic neurons and development and spread of aggregated protein inclusions known as Lewy bodies, with the most abundant protein being alpha-synuclein. The invention recognizes that Parkinson’s disease may be caused by mutations in the GBA1 gene. The mutations in the GBA1 gene are a significant risk factor in development and progression of Parkinson’s disease in patients. PD resulting from mutations in the GBA1 gene is clinically indistinguishable from sporadic PD, but it does tend to present with an earlier age of onset and greater cognitive dysfunction. Thus, there is a significant, but unmet need to develop interventions to assist PD patients with GBA1 mutations.

[0027] In certain aspects, the invention provides methods for the treatment of Parkinson’s disease. The invention provides methods for the treatment of Parkinson’s disease by administration of compositions comprising rAAVs of the invention. In certain embodiments, the administration of compositions comprising rAAVs of the invention results in delivery of rAAVs to the central nervous system, including the brain. Because the rAAVs of the invention are designed to have a high level of expression in the central nervous system, the payload in the rAAVs is delivered to the central nervous system, including the brain, of the patient. Advantageously, the rAAVs of the invention deliver functional human GBA1 gene to neurons in the CNS, thereby providing a permanent source of the GBA1 protein in the brain, and allowing for alleviation of symptoms of PD.

[0028] In certain embodiments, the composition comprising rAAVs of the invention are administered as an injection. In certain embodiments, the composition comprising rAAVs of the invention are administered as an intravenous injection. In certain embodiments, the composition comprising rAAVs of the invention are administered as a subcutaneous injection. In certain embodiments, the composition comprising rAAVs of the invention is administered as a single dose. In certain embodiments, the composition comprising rAAVs of the invention is administered as a single dose of an intravenous injection. In certain embodiments, the compositions of the invention are administered once daily.

[0029] Advantageously, the exemplary therapies and products provided in the invention result in a high degree of transduction across the CNS. The methods of the invention provide an GCase increase of greater than 200% on average across key regions of the brain. The invention beneficially relies on non-invasive intravenous delivery which limits the risks and allows for broad transduction of the therapeutic cargo and / or expression of GCase across the CNS. Moreover, the methods of the invention result in no adverse histopathology findings in non-human primate (NHP) studies.

[0030] V. BRIEF DESCRIPTION OF DRAWINGS

[0031] FIG. 1 provides the study overview.

[0032] FIG. 2A and FIG. 2B provide data showing DNA level changes in CNS and liver following administration of the rAAV of the invention to NHPs.

[0033] FIG. 3A and FIG. 3B provide data pertaining to the expression of GBA1 transgene in CNS and liver respectively following administration of the rAAV of the invention.

[0034] FIG. 4 provides data pertaining to neuronal transduction after administration of the rAAV of the invention.

[0035] FIGS. 5A, 5B, 5C, 5D, and 5E demonstrate the images for neuronal transduction in putamen, frontal cortex, substantia nigra, thalamus, and caudate respectively, as images, with a representative section zoomed in at the dose of 1.4E13 vg / kg.

[0036] FIG. 6A and FIG. 6B provide data pertaining to GCase activity in the brain, using two analytical methods (fluorescence and mass spectrometry, respectively) following administration of the rAAV of the invention.

[0037] FIG. 7A provides GBA1 protein expression data in treated and untreated mice.

[0038] FIG. 7B provides GCase activity data in treated and untreated mice.

[0039] FIG. 7C provides data pertaining to the lipid concentrations in treated and untreated mice.

[0040] FIG. 8A is a graph of RNA expression in NHP brain regions following administration of capsids of the invention.

[0041] FIG. 8B is a graph of vector genomes in NHP livers following administration of capsids of the invention.

[0042] FIG. 9A-B are graphs of DNA biodistribution and RNA expression in mice following administration of delivering therapeutic cargo.

[0043] FIG. 10A-C are graphs of GCase protein level and activity in mice following administration of delivering therapeutic cargo. FIG. 11A-B are graphs of brain GluSph and GalSph ratios in mice following administration of delivering therapeutic cargo.

[0044] FIG. 12A-C are graphs of DNA biodistribution and RNA expression in non-human primate brain regions following administration of an AAV product of the invention.

[0045] FIG. 13A-B are graphs of DNA biodistribution in NHP liver and RNA expression in non- human primate dorsal root ganglia (DRG) following administration of an AAV product of the invention.

[0046] FIG. 13C-D are graphs providing data for biodistribution of NHP liver DNA and DRG RNA expression 3 months after the administration of an AAV product of the invention compared to the control AAV9 capsid.

[0047] FIG. 14A-B are fluorescent images of NHP substantia nigra showing tissue level expression of the development candidate following administration of an AAV product of the invention.

[0048] FIG. 15A-C are graphs of GCase protein level and activity in NHP brain regions following administration of an AAV product of the invention.

[0049] FIG. 15D-E provide data related to the GCase levels and GCase activity 3 months after the administration in NHPs.

[0050] FIG. 16A-C are graphs of GCase protein and activity levels in the CSF of NHPs and average brain GCase activity following administration of an AAV product of the invention.

[0051] FIG. 16D-E are graphs of GCase protein levels in the CSF of NHPs and correlation with average brain GCase activity 3 months after administration of an AAV product of the invention.

[0052] FIG. 17A is a graph of plasma GluSph in NHPs following administration of an AAV product of the invention.

[0053] FIG. 17B provides data related to target engagement via decreases in plasma GluSph levels 3 months after administration of an AAV product of the invention, when normalized to the vehicle- administered control.

[0054] FIG. 18 provides a schematic of the key elements of an exemplary plasmid of the invention.

[0055] FIG. 19 provides a schematic representation of the exemplary capsid of the invention.

[0056] FIG. 20A provides a schematic design of the study for mouse pharmacology study.

[0057] FIG. 20B provides group details related to the mouse pharmacology study. FIG. 21A provides data pertaining to Brain GCase protein expression, Brain GCase activity, and Brain GluSph levels 6 months after administration of the cargo of the rAAVs of the invention after systemic injection via a surrogate capsid to the mice.

[0058] FIG. 21B provides the data pertaining to WPRE DNA and RNA copies 2 months and 6 months after the administration of cargo of the rAAVs of the invention after systemic injection via a surrogate capsid to mice.

[0059] FIG. 22 provides the in vitro data for DNA, RNA and GCAse activity after the administration of rAAVs of the invention.

[0060] VI. DETAILED DESCRIPTION

[0061] The present invention provides an AAV product that delivers a human GBA1 supplementation / replacement strategy throughout the human CNS at levels of DNA Biodistribution, RNA expression, and hGBAl protein levels expected to restore function in GBA1 mutation carriers suffering from Parkinson’s disease.

[0062] Parkinson’s disease:

[0063] Parkinson's disease is a progressive disorder that affects the nervous system and the parts of the body controlled by the nerves. Parkinson’s disease is very common overall, ranking second among degenerative brain diseases. The predictive prevalence of PD is 1.2 million people in the United States by 2030. It’s also the most common motor (movement-related) brain disease.

[0064] PD is associated with motor and non-motor symptoms, including bradykinesia, rigidity and resting tremor, cognitive decline, sleep disturbances, hyposmia and psychiatric symptoms. PD leads to slowed movements, tremors, balance problems and more. It causes deterioration of cognitive function and motor skills.

[0065] A key pathological hallmark of PD is the loss of nigrostriatal dopaminergic neurons and development and spread of aggregated protein inclusions known as Lewy bodies, with the most abundant protein being alpha-synuclein.

[0066] GBA 1 gene and Parkinson’s disease:

[0067] Over the past decade, several large cohort studies investigated the link between mutations in the GBA1 gene and the risk of developing Parkinson’s disease (PD). Current evidence suggests approximately 5-15% of PD patients have GBA mutations making this the most significant genetic risk factor for PD. PD resulting from mutations in the GBA1 gene is clinically indistinguishable from sporadic PD, but it does tend to present with an earlier age of onset and greater cognitive dysfunction.

[0068] GBA1 encodes a lysosomal hydrolase enzyme, glucocerebrosidase (GCase). GCase cleaves the beta-glucosidic linkage of glucosylceramide, an intermediate in glycolipid metabolism. Specifically, GCase hydrolyzes glucosylceramide and glucosyl sphingosine to ceramide, glucose, and sphingosine. GCase is a 497-amino-acid membrane-associated protein with a 39-amino-acid leader sequence and five glycosylation sites. The protein is synthesized in the endoplasmic reticulum (ER) and glycosylated, but the enzyme only becomes active when transferred to the acidic lumen of the lysosome.

[0069] Treatment of Parkinson’s disease:

[0070] Currently, there are no approved disease modifying treatments for PD, including PD associated with GBA mutations. Levodopa is the standard of care for motor symptoms; however, motor fluctuations and dyskinesias develop in many patients, which can be debilitating in and of themselves. Adjunctive medications, or deep brain stimulation in advanced refractory cases, may be used to manage or mitigate motor fluctuations and dyskinesias. Nonmotor symptoms are also treated symptomatically, most typically with antidepressants, pain medications, acetylcholinesterase inhibitors, NMDA antagonists, antipsychotic agents, and various medications for autonomic dysfunction.

[0071] Compositions and methods of the invention use recombinant adeno-associated viruses (rAAV) to deliver a viral vector comprising a human glucosylceramidase P-1 (GBA1) gene encoding a functional glucosylceramidase enzyme (“GBA protein” or “GCase”). By allowing cells to produce functional GCase, compositions and methods of the invention can be used to treat neurological conditions caused by lack of GCase function, including Parkinson’s disease.

[0072] The invention provides methods of treatment of Parkinson’s disease. In certain embodiments, the rAAVs of the invention are designed to replace or supplement the human GBA1 gene in the cells in the brain, including neurons. Because the rAAVs of the invention are designed to be enriched in the brain, the rAAVs, along with their cargo, are enriched in the brain as compared to other parts of the body. In certain embodiments, the cargo included with rAAVs comprises GBA1 gene. Accordingly, in certain embodiments, the rAAVs of the invention deliver the functional hGBAl gene to neurons in the CNS, including the brain, thereby providing a permanent source of GBA1 gene, which can encode functional GCase protein.

[0073] The administration of compositions comprising rAAVs of the invention, including the GBA1 gene as payload, offers the potential to stably replace wild type GCase protein with a single administration, enabling long-term disease modification and substantially slowing disease progression with limited treatment burden.

[0074] In some embodiments, the viral particle comprises a nucleic acid sequence that encodes for an amino acid sequence having, having at least, or having at most 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 2, or any range or value derivable therein. In certain embodiments, the viral particle comprises a nucleic acid sequence that encodes for the GCase amino acid sequence of SEQ ID NO:2. In certain embodiments, the viral particle comprises a nucleic acid that encodes for an amino acid sequence having, having at least, or having at most 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO 2, or a homolog thereof.

[0075] SEQ ID NO: 2:

[0076] MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCV CNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQK VKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYAD TPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSL KGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEH QRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHW YLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNL L YHVVGWTDWNLALNPEGGPNWVRNF VD SPIIVDITKDTF YKQPMF YHLGHF SKFIPEG SQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQ

[0077] In certain embodiments, the nucleic acid sequence encodes for part or all of SEQ ID NO: 2. In some embodiments, the viral particle comprises a sequence that encodes for an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2. In some embodiments, the viral particle comprises a nucleic acid sequence encoding SEQ ID NO: 2, wherein 1, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 40 or fewer, or 50 or fewer of the codons within SEQ ID NO: 2 is substituted with another codon, optionally comprising a conservative amino acid substitution or silent mutation, and / or are deleted and / or an insertion (including 5 ’ and / or 3 ’ extensions) of 1 , 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 40 or fewer, or 50 or fewer codons or any combination of substitutions, deletions and / or insertions, wherein the substitutions, deletions and / or insertions do not unduly impair the structure and / or function of GBA protein.

[0078] Conservative amino acid substitutions are known in the art. In particular embodiments, a conservative amino acid substitution includes substitutions within one or more of the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and / or phenylalanine, tyrosine.

[0079] Accordingly, provided herein are methods of treating PD, or a symptom of PD, in a subject, comprising: (a) diagnosing a subject with PD affecting a target in vivo environment; and (b) treating PD by administering to the subject a therapeutically effective amount of a composition disclosed herein (e.g., rAAV particle, AAV vector, pharmaceutical composition), wherein the composition is engineered with an increased enrichment or specificity for the target in vivo environment.

[0080] Transgene delivery

[0081] In some embodiments, an individual is treated by a method comprising administering a therapeutically effective amount of one or more compositions encompassed herein, including any viral particle herein, to the individual. The composition may increase the level of a heterologous transgene in the individual, including in cells of the individual. In some embodiments, the composition administered to the individual restores the level of the transgene to a level found in a control individual. In some embodiments, the compositions restore cognitive abilities in the individual. In some embodiments, the compositions restore motor functions in the individual. In some embodiments, the compositions restore psychiatric functions in the individual. The transgene may be in cis with two inverted terminal repeats (ITRs) flanking the transgene. Due to the limited packaging capacity of the rAAV (~5kB), in some cases, the transgene may be split between two AAV vectors, the first with 3’ splice donor and the second with a 5’ splice acceptor. Upon co-infection of a cell, concatemers form, which are spliced together to express a full-length transgene.

[0082] Effective dosages of the viral particles to be administered to a subject will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, the particular virus vector, and the nucleic acid sequence to be delivered, and can be determined in a routine manner. Examples of effective doses for achieving therapeutic effects include virus titers of at least about 105, 106, 107, 108, 109, IO10, 1011, 1012, 1013, 1014, 1015transducing units or more.

[0083] In some embodiments, the viral particle is administered directly to the CNS, e.g., the brain or the spinal cord. Direct administration can result in high specificity of transduction of CNS cells, e.g., wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the transduced cells are CNS cells. Any method known in the art to administer vectors directly to the CNS can be used. The vector may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (corpus striatum, cerebrum including the occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and amygdala), limbic system, neocortex, corpus striatum, cerebrum, and inferior colliculus. The vector may also be administered to different regions of the eye such as the retina, cornea or optic nerve. The vector may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more disperse administration of the vector.

[0084] The delivery vector may be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intracerebral, intraventricular, intraci sternal, intranasal, intra-aural, intra-ocular (e.g., intra- vitreous, sub-retinal, anterior chamber) and peri ocular (e.g., sub-Tenon's region) delivery or any combination thereof.

[0085] Typically, the viral vector will be administered in a liquid formulation by direct injection to the desired region or compartment in the CNS. In some embodiments, the vector can be delivered via a reservoir and / or pump. In other embodiments, the vector may be provided by topical application to the desired region or by intra-nasal administration of an aerosol formulation. Administration to the eye or into the ear, may be by topical application of liquid droplets. As a further alternative, the vector may be administered as a solid, slow-release formulation.

[0086] In some embodiments, one can inject the AAV particles directly into the brain tissues. In some embodiments, one can deliver the particles into cerebrospinal fluid (CSF), such as by injection into the ventricle or lumbar intrathecal space. In some embodiments, one can deliver the particles systemically, such as by injection into a blood vessel, and then let the AAV particles cross the blood brain barrier (BBB). In particular embodiments, one or more AAV particles of the disclosure has the ability to cross the blood brain barrier (BBB). In embodiments wherein any AAV particle is considered to have very weak or no ability to cross BBB, one can inject the AAV particles directly into the brain tissues, such as by intraparenchymal injection.

[0087] In general, methods disclosed herein comprise administering a therapeutic rAAV composition by systemic administration. In some instances, methods comprise administering a therapeutic rAAV composition by intraperitoneal injection. In some instances, methods comprise administering a therapeutic rAAV composition by intravenous (“i.v ”) administration. It is conceivable that one may also administer therapeutic rAAV compositions disclosed herein by other routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection percutaneous administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration, intraocular administration, intracerebroventricular administration, intraci sternal administration, intrathecally, or any other suitable parenteral administration. Routes, dosage, time points, and duration of administrating therapeutics may be adjusted. In some embodiments, administration of therapeutics is prior to, or after, onset of either, or both, acute and chronic symptoms of the disease or condition.

[0088] The term “CNS” or “central nervous system” means a tissue selected from brain, thalamus, cortex, putamen, lateral ventricles, medulla, pons, amygdala, motor cortex, caudate, hypothalamus, striatum, ventral midbrain, neocortex, basal ganglia, hippocampus, cerebrum, cerebellum, brainstem, and / or spinal cord. The brain includes a variety of cortical and subcortical areas, including the frontal, temporal, occipital, and parietal lobes.

[0089] The term “systemic delivery” is defined as a route of administration of medication or other substance into a circulatory system so that the entire body is affected. Administration can take place via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally injection, infusion, or implantation). “Circulatory system” includes blood and / or cerebrospinal fluid circulatory systems. Examples of systemic administration for the CNS include intraarterial, intravenous or intrathecal injection. Other examples include administration to the cerebrospinal fluid at any location, in the spine (i.e., but not limited to lumbar) or brain (i.e., but not limited to cistema magna). The terms “systemic administration” and “systemic delivery” are used interchangeably.

[0090] In some embodiments, routes for administration includes administration into the CSF, for example via an intracerebroventricular (ICV), intrathecal cisternal, or intrathecal lumbar route. Particular embodiments result in delivery to neurons and glial cells of the brain. Other routes of delivery to the CNS / brain include, but are not limited to intracranial administration, lateral cerebroventricular administration, intranasal administration, endovascular administration, and intraparenchymal administration.

[0091] An effective dose and dosage of pharmaceutical compositions to prevent or treat the disease or condition disclosed herein is defined by an observed beneficial response related to the disease or condition, or symptom of the disease or condition. Beneficial response comprises preventing, alleviating, arresting, or curing the disease or condition, or symptom of the disease or condition. In some embodiments, the beneficial response may be measured by detecting a measurable improvement in the presence, level, or activity, of biomarkers, transcriptomic risk profile, or intestinal microbiome in the subject. An “improvement,” as used herein refers to shift in the presence, level, or activity towards a presence, level, or activity, observed in normal individuals (e g., individuals who do not suffer from the disease or condition). In certain instances, the dosage amount and / or route of administration may be changed, or an additional agent may be administered to the subject, along with the therapeutic rAAV composition. In some embodiments, as a patient is started on a regimen of a therapeutic rAAV composition, the patient is also weaned off (e.g., step-wise decrease in dose) a second treatment regimen.

[0092] In some cases, a dose of the pharmaceutical composition may comprise a concentration of infectious particles of at least or about 107, 108, 109, IO10, 1011, 1012, 1013, 1014, 1015, 1016, or 1017. In some cases, the concentration of infectious particles is 2xl07, 2xl08, 2xl09, 2xlO10, 2xlOn, 2xl012, 2xl013, 2xl014, 2xl015, 2xl016, or 2xl017. In some cases, the concentration of the infectious particles is 3xl07, 3xl08, 3xl09, 3xlO10, 3xlOn, 3xl012, 3xl013, 3xl014, 3xl015, 3xl016, or 3xlO17In some cases, the concentration of the infectious particles is 4xl07, 4xl08, 4xl09, 4xlO10, 4xlOn, 4xl012, 4xl013, 4xl014, 4xl013, 4xl016, or 4xl017. In some cases, the concentration of the infectious particles is 5xl07, 5xl08, 5xl09, 5xlO10, 5xlOn, 5xl012, 5xlO13, 5xl014, 5xlO15, 5xl016, or 5xl017. In some cases, the concentration of the infectious particles is 6xl07, 6xl08, 6xl09, 6xlO10, 6xlOn, 6xl012, 6xl013, 6xl014, 6xl015, 6xl016, or 6xl017. In some cases, the concentration of the infectious particles is 7xl07, 7xl08, 7xl09, 7xlO10, 7xlOn, 7xl012, 7xl013, 7xl014, 7xl015, 7xl016, or 7xlO17In some cases, the concentration of the infectious particles is 8xl07, 8xl08, 8xl09, 8xlO10, SxlO11, 8xl012, 8xl013, 8xl014, 8xl015, 8xl016, or 8xl017. In some cases, the concentration of the infectious particles is 9xl07, 9xl08, 9xl09, 9xlO10, 9xlOn, 9xl012, 9xl013, 9xl014, 9xl015, 9xl016, or 9xl017.

[0093] Disclosed herein, in some embodiments are formulations of pharmaceutically-acceptable excipients and carrier solutions suitable for delivery of the rAAV compositions described herein, as well as suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. In some embodiments, the amount of therapeutic gene expression product in each therapeutically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. In some instances, the rAAV compositions are suitably formulated pharmaceutical compositions disclosed herein, to be delivered either intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricularly, intramuscularly, intrathecally, intraperitoneally, by nasal inhalation, or by direct injection to one or more cells, tissues, or organs.

[0094] In some embodiments, the pharmaceutical forms of the AAV-based viral compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0095] In some cases, for administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.

[0096] Disclosed herein are sterile injectable solutions comprising the rAAV compositions disclosed herein, which are prepared by incorporating the rAAV compositions disclosed herein in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Injectable solutions may be advantageous for systemic administration, for example by intravenous administration.

[0097] Also provided herein are formulations in a neutral or salt form. Pharmaceutically- acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like. Suitable dose and dosage administrated to a subject is determined by factors including, but not limited to, the particular therapeutic rAAV composition, disease condition and its severity, the identity (e.g., weight, sex, age) of the subject in need of treatment, and can be determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0098] The amount of AAV compositions and time of administration of such compositions will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically-effective amounts of the disclosed compositions may be achieved by a single administration, for example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. This is made possible, at least in part, by the fact that certain target cells (e.g., neurons) do not divide, obviating the need for multiple or chronic dosing.

[0099] For example, the number of infectious particles administered to a mammal may be on the order of about 107, 108, 109, IO10, 1011, 1012, 1013, 1014, or even higher, infectious particles / ml given either as a single dose or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated. In fact, in certain embodiments, it may be desirable to administer two or more different AAV vector compositions, either alone, or in combination with one or more other therapeutic drugs to achieve the desired effects of a particular therapy regimen. In various embodiments, the daily and unit dosages are altered depending on a number of variables including, but not limited to, the activity of the therapeutic rAAV composition used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0100] The effective dosage ranges may be adjusted based on subject’s response to the treatment. Some routes of administration will require higher concentrations of effective amount of therapeutics than other routes.

[0101] In certain embodiments, the daily dosage range and / or the unit dosage amount varies within this range depending upon the dosage form employed and the route of administration utilized. Viral Vectors

[0102] Certain embodiments of the disclosure concern methods of producing viral particles. In some embodiments, the method comprises providing to a cell in vitro, (a) a template comprising (i) a nucleic acid encoding for a gene product, and (ii) packaging signal sequences sufficient for the encapsidation of an AAV template into virus particles (e.g., one or more (e.g., two) terminal repeats, such as AAV terminal repeats), and (b) AAV sequences sufficient for replication and encapsidation of the template into viral particles (e.g., the AAV rep and AAV cap sequences encoding an AAV capsid). The template and AAV replication and capsid sequences are provided under conditions such that recombinant virus particles comprising the template packaged within the capsid are produced in the cell. The method can further comprise the step of collecting the virus particles from the cell. Virus particles may be collected from the medium and / or by lysing the cells.

[0103] Recombinant adeno-associated virus (rAAV) mediated gene delivery leverages the AAV mechanism of viral transduction for nuclear expression of an episomal heterologous nucleic acid sequence (e.g., a transgene, therapeutic nucleic acid sequence). Upon delivery to a host in vivo environment, a rAAV will (1) bind or attach to cellular surface receptors on the target cell, (2) endocytose, (3) traffic to the nucleus, (4) uncoat the virus to release the encapsidated heterologous nucleic acid , (5) convert of the heterologous nucleic acid from single-stranded to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribe the episomal heterologous nucleic acid sequence in the nucleus of the host cell. rAAVs engineered to have an increased specificity (binding to cellular surface receptors on the target cell), transduction efficiency (the effectiveness of a virus , engineered or naturally occurring, at delivering its DNA component to a host cell), and / or transgene expression (transcription of the episomal heterologous nucleic acid in the host cell) are desirable for gene therapy applications.

[0104] An rAAV comprises an AAV capsid that can be engineered to encapsidate a heterologous nucleic acid sequence (e.g., therapeutic nucleic acid, gene editing machinery). The AAV capsid is made up of three AAV capsid protein monomers, VP1, VP2, and VP3. Sixty copies of these three VP proteins interact in an approximately a 1 :1 :10 ratio to form the viral capsid. VP1 covers the whole of VP2 protein in addition to a -137 amino acid N-terminal region (VPlu), VP2 covers the whole of VP3 in addition to -65 amino acid N-terminal region (VP1 / 2 common region). The three capsid proteins share a conserved amino acid sequence of VP3, which in some cases is the region beginning at amino acid position 138 (e.g., AA139-736).

[0105] While not wishing to be bound by theory, it is understood that a parent AAV capsid sequence comprises a VP1 region. In certain embodiments, a parent AAV capsid sequence comprises a VP1, VP2 and / or VP3 region, or any combination thereof. A parent VP1 sequence may be considered synonymous with a parent AAV capsid sequence.

[0106] The AAV VP3 structure contains highly conserved regions that are common to all serotypes, a core eight-stranded P-barrel motif (PB-pi) and a small a-helix (aA). The loop regions inserted between the P-strands consist of the distinctive HI loop between P-strands H and I, the DE loop between P-strands D and E, and nine variable regions (VRs), which are typically surface exposed on the capsid structure. These VRs, such as VR-VIII containing the engineered amino acids 587-590, can be associated with specific functional roles in the AAV life cycle including receptor binding, transduction, and antigenic specificity.

[0107] Disclosed herein are AAV capsids comprising AAV capsid proteins with a substitution at AA588 and peptide insertion between AA588 and 589 which confers a desired tropism characterized by a higher efficiency and specificity for transduction in specific cell-types, including, for example, cells within the CNS or brain cell types (e.g., brain endothelial cells, neurons, astrocytes, glia). In particular, the AAV capsid proteins disclosed herein enable rAAV- mediated transduction of a heterologous nucleic acid sequence (e.g., transgene) in the CNS of a subject. The AAV capsids of the present disclosure, or the AAV capsid proteins, may be formulated as a pharmaceutical composition. In addition, the AAV capsids or the AAV capsid proteins can be isolated and purified to be used for a variety of applications. Disclosed herein are recombinant AAV (rAAV) capsids which comprise AAV capsid proteins that are engineered with a modified capsid protein (e.g., VP1, VP2, VP3). In some embodiments, the rAAV capsid proteins of the present disclosure are generated using the methods disclosed herein. In some embodiments, the AAV capsids are used in the methods of delivering a therapeutic nucleic acid sequence (e.g., a transgene) to a subject. In some instances, the rAAV capsids have desired AAV tropisms rendering them particularly suitable for certain therapeutic applications, e.g., the treatment of a disease or disorder in a subject such as those disclosed herein.

[0108] The rAAV capsid proteins are engineered for optimized transduction and transgene expression in the CNS, for example the brain, of a subject upon systemic administration of the rAAV to the subject. The rAAV capsid proteins are engineered to have tropisms that eliminate the need for intracranial injection, while also achieving widespread and efficient transduction of an encapsidated transgene. In particular, the tropisms comprise at least one of an increased specificity and efficiency (e.g., of viral transduction) in the CNS of a subject, as compared to a reference AAV.

[0109] The engineered AAV capsid proteins described herein have, in some cases, a peptide insertion and amino acid substitution that is heterologous to the parental AAV capsid between positions 587 and 590. In some embodiments, the amino acids flanking the peptide insertion do not originate from the parental AAV capsid protein sequence. The amino acids flanking the insertion may share sequence identity with the amino acid at the same positions in the parental serotype or equivalent amino acid position as the substitutions and peptide insertion and in alternative AAV serotypes or engineered variant capsid proteins.

[0110] Also disclosed herein are rAAVs with engineered capsid proteins that are optimized for targeting specific organ or tissue within a subject. In a non-limiting example, the rAAVs of the present embodiment, have increased specificity, transduction, and transgene expression in the CNS.

[0111] Seven amino acids comprise the peptide insertion sequence (7-mer, respectively) that is inserted within VR-VIII in the parental AAV capsid protein. Aspects provided herein provide amino acid insertions comprising seven amino acid polymer (7-mer) inserted between AA588- 589, and may additionally include a substitution of one or two amino acids at amino acid positions flanking the 7-mer sequence (e.g., AA587-588 and / or AA589-590) to produce an eleven amino acid polymer (11-mer) at the 588 loop of a parental AAV capsid protein.

[0112] The capsid protein is engineered, relative to an AAV9 capsid protein, by substituting amino acid positions 588 with amino acid T, inserting between amino acid positions 588 and 589 the amino acids RNGEVFI, and with the amino acids A and Q from parental AAV9 present at positions 589 and 590.

[0113] As a result, the amino acid positions 587-597 of the AAV capsid comprises the sequence ATRNGEVFIAQ.

[0114] Peptide insertion sequences of the disclosure include sequences that have been modified in any way and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) alter binding affinities, and (3) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., equivalent, conservative or nonconservative substitutions, deletions or additions) may be made in a sequence.

[0115] An AAV vector can comprise a viral genome comprising a nucleic acid encoding the recombinant AAV (rAAV) capsid protein described herein. The viral genome can comprise a Replication (Rep) gene encoding a Rep protein, and Capsid (Cap) gene encoding an AAP protein in the first open reading frame (ORF1) or a Cap protein in the second open reading frame (ORF2). The Rep protein is selected from Rep78, Rep68, Rep52, and Rep40. In some instances, the Cap gene is modified encoding a modified AAV capsid protein described herein. A wild-type Cap gene encodes three proteins, VP1, VP2, and VP3. In some cases, VP1 is modified. In some cases, VP2 is modified. In some cases, VP3 is modified. In some cases, all three VP1-VP3 are modified. The AAV vector can comprise nucleic acids encoding wild-type Rep78, Rep68, Rep52, Rep40 and AAP proteins.

[0116] In some instances, the 5' ITR and the 3' ITR are derived from an AAV2 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV5 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV9 serotype. In some instances, the 5’ ITR and the 3’ ITR each originate from different serotypes, e.g. 5’ ITR from serotype AAV2 and 3’ ITR from AAV5. In some instances, the 5’ ITR and / or the 3’ ITR originate from another natural serotype or have been engineered for improved transduction or transgene expression efficiency. In some instances, the 5' ITR and the 3' ITR are provided in Earley et al., Adeno-Associated Virus Serotype- Specific Inverted Terminal Repeat Sequence Role in Vector Transgene Expression, Hum Gene Ther., February 2020; 31(3-4): 151-162, which is incorporated by reference in its entirety.

[0117] A conservative amino acid substitution refers to the substitution of an amino acid in an insertion sequence with a functionally similar amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity.

[0118] In some embodiments, methods of increasing transduction of an encoded gene in a target in vivo environment comprise delivering a rAAV particle described herein, with the rAAV engineered to have an increased transduction enrichment in a target in vivo environment (e.g., tissue or cell type). In some instances, the increased transduction enrichment comprises a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40- fold, 50-fold or 100-fold increase, or more, relative to a reference AAV. In some instances, the increased transduction enrichment is at least 2-fold. In some instances, the increased transduction enrichment is at least 10-fold. In some instances, the increased transduction enrichment is at least 20-fold.

[0119] Methods of delivering a heterologous nucleic acid sequence to a target in vivo environment are also provided comprising delivering the rAAV particle described herein that has been engineered to have an increased expression or specificity in an in vivo environment (e.g., tissue or cell type), as compared to a reference AAV. Methods, in some cases, comprise detecting whether a rAAV possesses more specificity for an in vivo environment, includes measuring a level of gene expression product expressed from the vector encapsidated by the rAAV in a tissue sample obtained from the in vivo environment in a subject.

[0120] In some instances, the reference AAV has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or variants thereof.

[0121] Provided herein are methods of delivering a heterologous nucleic acid sequence to a target in vivo environment comprising delivering a composition to the target in vivo environment selected from the CNS in a subject, the composition comprising a rAAV particle with a rAAV capsid protein, the rAAV capsid proteins encapsidating a viral vector encoding a heterologous nucleic acid sequence (e.g., therapeutic nucleic acid sequence). In some embodiments, the rAAV particle encapsidating the heterologous nucleic acid sequence comprises a rAAV capsid protein engineered with an increased transduction enrichment when measured in the CNS of the subject, even when administered to the subject systemically.

[0122] Methods may comprise delivering a rAAV particle comprising an rAAV capsid protein with increased transduction and transgene expression when measured in the CNS in the subject. In some embodiments, delivery is systemic. Alternatively, delivery is direct (e.g., into the affected area of the CNS).

[0123] Promoter / Enhancers

[0124] A variety of promoter / enhancer elements may be used depending on the level and tissuespecific expression desired. The promoter / enhancer may be constitutive or inducible, depending on the pattern of expression desired. The promoter / enhancer may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. Promoter / enhancer elements can be native to the target cell or subject to be treated and / or native to the heterologous nucleic acid sequence. The promoter / enhancer element is generally chosen so that it will function in the target cell(s) of interest. In representative embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhance element may be constitutive or inducible.

[0125] Promoters are DNA regions that initiate gene transcription by controlling the binding of RNA polymerase to the vector DNA to begin the process toward expression of the encoded protein. Promoters control the binding of RNA polymerase to DNA. RNA polymerase transcribes DNA to mRNA which is ultimately translated into a functional protein. Thus, the promoter region controls when and where in the organism the gene of interest is expressed. Exemplary promoters include CMV, CBh, human synapsin I, EFla, SV40, PGK1, Ubc, human beta actin, and CAG. In preferred embodiments, the vector comprises a promoter selected from a CAG synthetic promoter, a CBh synthetic promoter, and a human synapsin I promoter. See Miyazaki, J; Takaki, S; Araki, K; Tashiro, F; Tominaga, A; Takatsu, K; Yamamura, K (Jul 15, 1989). "Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5". Gene. 79 (2): 269-77; Grey et al., Optimizing Promoters for Recombinant Adeno-Associated Virus- Mediated Gene Expression in the Peripheral and Central Nervous System Using Self- Complementary Vectors, Hum Gene Ther. 2011 Sep; 22(9): 1143-1153; Glover et al., Adenoviral- mediated, High-Level, Cell-Specific Transgene Expression: A SYN1-WPRE Cassette Mediates Increased Transgene Expression With No Loss of Neuron Specificity, Mol Ther. 2002 May; 5(5 Pt 1 ): 509- 16; the content of each of which is incorporated herein by reference.

[0126] In some instances, the vector may comprise a promoter and / or enhancer, for example, a constitutive promoter or an inducible or tissue / cell specific promoter. As a non-limiting example, the promoter may be CMV promoter, a CMV-P-Actin-intron-P-Globin hybrid promoter (CAG), CBA promoter, FRDA or FXN promoter, UBC promoter, GUSB promoter, NSE promoter, Synapsin promoter, MeCP2 promoter, GFAP promoter, Hl promoter, U6 promoter, NFL promoter, NFH promoter, SCN8A promoter, or PGK promoter. As a non-limiting example, promoters can be tissue-specific expression elements include, but are not limited to, human elongation factor la-subunit (EFla), immediate-early cytomegalovirus (CMV), chicken P-actin (CBA) and its derivative CAG, the P glucuronidase (GUSB), and ubiquitin C (UBC). The vector may include a tissue-specific expression elements for neurons such as, but not limited to, neuronspecific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor Bchain (PDGF-0), the synapsin (Syn), the methyl-CpG binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor (mGluR2), NFL, NFH, np32, PPE, Enk and EAAT2 promoters. The vector may comprise a tissuespecific expression element for astrocytes such as, but not limited to, the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. The vector may comprise tissue-specific expression elements for oligodendrocytes such as, but not limited to, the myelin basic protein (MBP) promoter.

[0127] Various regulatory elements may be included in vectors of the invention including posttranscriptional regulatory elements (PREs), such as those derived from hepatitis B virus (HPRE), woodchuck hepatitis virus (WPRE), human heat shock protein 70 mRNA (Hsp70), the vascular endothelial growth factor (SP163), the tripartite leader sequence of human adenovirus mRNA linked with a major late promoter enhancer (TM), or the first intron of human cytomegalovirus immediate early gene (Intron A). Posttranscriptional regulatory elements can help enhance gene expression when included in expression vectors such as those described herein. Particular PREs may exhibit cell-specific and / or gene-specific regulatory enhancement and those factors are considered when selecting a PRE.

[0128] Methods and compositions of the invention:

[0129] The present invention is directed to a recombinant adeno-associated virus (rAAV) engineered to enrich delivery of cargo to the central nervous system (CNS). The present invention provides rAAVs with widespread transduction to cells in the brain. Following IV injection, unmodified rAAVs such as those derived from AAV9 (SEQ ID NO: 1) may not have sufficient tissue enrichment to treat many human diseases by delivery of an AAV cargo. Accordingly, engineered rAAVs described herein are particularly useful in delivering DNA cargo to the brain. The rAAVs of the invention are beneficial for the treatment of certain neurological conditions.

[0130] In certain embodiments, the rAAVs of the present invention enhance the delivery of cargo to the brain. In certain aspects, the invention is directed to a rAAV comprising the human glucosylceramidase pi GBA1) gene. The GBA1 gene may be the cargo in the rAAVs of the invention.

[0131] In certain aspects, the invention provides an rAAV comprising an amino acid sequence having at least 80% shared sequence identity with ATRNGEVFIAQ (SEQ ID NO: 4) for delivery of a GBA1 transgene to the CNS. In certain embodiments, the invention provides an rAAV comprising an amino acid sequence having at least 90% shared sequence identity with ATRNGEVFIAQ (SEQ ID NO: 4) for delivery of a GBA1 transgene to the CNS. In certain aspects, the invention provides an rAAV comprising an amino acid sequence having at least 80% identical to ATRNGEVFIAQ (SEQ ID NO: 4). In certain embodiments, the AAV capsid comprises an amino acid sequence ATRNGEVFIAQ (SEQ ID NO: 4). In certain embodiments, the capsid protein in the rAAV of the invention is engineered from an AAV capsid protein. In certain embodiments, the capsid protein in the rAAV of the invention is engineered from an AAV9 capsid protein. In certain embodiments, the AAV capsid protein is engineered from AAV9 capsid protein provided in SEQ ID NO: 1.

[0132] In certain aspects, the invention provides an rAAV comprising a capsid protein comprising an amino acid sequence having at least 80% identical to ATRNGEVFIAQ (SEQ ID NO: 4), a promoter, and a therapeutic gene, wherein the therapeutic gene is human GBA1 transgene. In certain embodiments, the invention provides an rAAV comprising a capsid protein comprising an amino acid sequence having at least 90% identical to ATRNGEVFIAQ (SEQ ID NO: 4), a promoter, and a therapeutic gene, wherein the therapeutic gene is human GBA1 transgene.

[0133] In certain embodiments, the rAAV of the invention is engineered by peptide insertion between amino acid position 588 and 589 of AAV9 capsid protein or an equivalent location in another AAV serotype or engineered variant. Advantageously, the rAAV of the invention is characterized by at least one of an increased specificity and / or increased transduction efficiency in the central nervous system (CNS). In certain embodiments, the rAAV capsid is characterized by at least one of an increased specificity and / or increased transduction efficiency in the brain.

[0134] In certain embodiments, the therapeutic gene is a human GBA1 transgene. In certain embodiments, the therapeutic gene is a wild-type human GBA1 transgene. In certain embodiments, the therapeutic gene is a modified or recombinant human GBA1 transgene. In certain embodiments, the GBA1 transgene is at least 95% identical to the human transgene provided in NCBI reference NM_000157.4 (SEQ ID NO: 2). In certain embodiments, the GBA1 transgene is the transgene provided in NCBI reference NM_000157.4 (SEQ ID NO: 2) or a substantially similar gene.

[0135] In certain embodiments, the rAAV of the invention further comprises a CAG promoter. In certain embodiments, the CAG promoter comprises three (3) regulatory elements. In certain embodiments, the regulatory elements are: human cytomegalovirus immediate early enhancer (CMV enhancer), a chicken beta-actin promoter, and a chimeric rabbit P-globin (rBG) intron.

[0136] In certain embodiments, the rAAV of the invention further comprises a post-transcriptional regulatory element. The regulatory element increases the efficiency of transcription of the therapeutic drug in the complex. In certain embodiments, the post-translational regulatory element (WPRE) is a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0137] In certain embodiments, the rAAV of the invention further comprises a poly(A) signal. The poly(A) signal enhances stability of the cargo in the rAAV. In certain embodiments, the poly(A) signal is a bovine growth hormone polyadenylation signal (bGH-polyA).

[0138] In certain embodiments, the rAAV of the invention may further comprise an HA epitope tag. In certain embodiments, the HA epitope tag is at the C-terminus of the hGBAl protein. In certain embodiments, the HA epitope sequence is YPYDVPDYA (SEQ ID NO: 3).

[0139] Accordingly, in certain embodiments, the invention provides a rAAV comprising a capsid protein comprising an amino acid sequence having at least 80% shared sequence identity with ATRNGEVFIAQ (SEQ ID NO: 4), and a payload comprising a human GBA1 gene. In certain embodiments, the rAAV of the invention comprises capsid protein comprising an amino acid sequence having at least 80% shared sequence identity with ATRNGEVFIAQ (SEQ ID NO: 4), a payload comprising a human GBA1 gene, a CAG promoter, a Woodchuck hepatitis virus post- transcriptional regulatory element (WPRE), and a bovine growth hormone polyadenylation signal (bGH-polyA). In certain embodiments, the invention further provides that the rAAV of the invention may optionally include a HA epitope tag at the C-terminus of the hGBAl protein.

[0140] In certain aspects, the invention provides methods of treatment of diseases by administration of a composition comprising rAAVs of the invention. In certain embodiments, the disease is a neurological disease.

[0141] Parkinson’s disease is associated with motor and non-motor symptoms, including bradykinesia, rigidity and resting tremor, cognitive decline, sleep disturbances, hyposmia, and psychiatric symptoms. A key pathological hallmark of PD is the loss of nigrostriatal dopaminergic neurons and development and spread of aggregated protein inclusions known as Lewy bodies, with the most abundant protein being alpha-synuclein. The invention recognizes that Parkinson’s disease may be caused by mutations in the GBA1 gene. Mutations in GBA1 gene are a significant risk factor in development and progression of Parkinson’s disease in patients. PD resulting from mutations in GBA1 gene is clinically indistinguishable from sporadic PD, but it does tend to present with an earlier age of onset and greater cognitive dysfunction. Thus, there is a significant, but unmet need to develop interventions to assist PD patients with GBA1 mutations.

[0142] In certain aspects, the invention provides methods for the treatment of Parkinson’s disease. The invention provides methods for the treatment of Parkinson’s disease by administration of compositions comprising rAAV of the invention. In certain embodiments, the administration of compositions comprising rAAVs of the invention results in delivery of rAAVs to the central nervous system, including the brain. Because the rAAVs of the inventions are designed to have a high level of expression in the central nervous system, the payload in the rAAVs is delivered to the central nervous system, including the brain of the patient. Advantageously, the rAAVs of the invention deliver functional human GBA1 gene to neurons in the CNS, thereby providing a permanent source of the GBA1 protein in the brain, and allowing for alleviation of symptoms of PD

[0143] In certain embodiments, the composition comprising rAAVs of the invention are administered as an injection. In certain embodiments, the composition comprising rAAVs of the invention are administered as an intravenous injection. In certain embodiments, the composition comprising rAAVs of the invention are administered as a subcutaneous injection. In certain embodiments, the composition comprising rAAVs of the invention is administered as a single dose. In certain embodiments, the composition comprising rAAVs of the invention is administered as a single dose of an intravenous injection. In certain embodiments, the compositions of the invention are administered once daily.

[0144] VII. EXAMPLES

[0145] Example 1 : Exemplary rAAV nucleic acid sequence of the invention

[0146] An exemplary sequence included in the plasmids of the invention are provided in SEQ ID NO: 5.

[0147] Example 2: Exemplary hGBAl nucleic acid sequence of the invention

[0148] An exemplary sequence included in the plasmids of the invention are provided in SEQ ID NO: 6.

[0149] 3’ ITR (L-ITR) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCC CGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAG AGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 8)

[0150] 5’ ITR (R-ITR)

[0151] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTC AGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG (SEQ ID NO: 9)

[0152] Example 3: Performance of rAAV after systemic administration in cynomolgus macaques

[0153] A study was conducted to assess the performance of an rAAV variant of the present invention, encapsidating viral genomes produced from an hGBAl cargo (SEQ ID NO: 6), after systemic administration in male and female cynomolgus macaques (Macaca fascicularis) aged 37- 46 months of age. The rAAV packaged an HA-tagged human GBA1 under control of the ubiquitous CAG promoter. FIG. 1 provides the study overview. Animals were administered the rAAV at a dose of either 1.4E13 vg / kg or 2.8E13 vg / kg (doses based on previous titer method using WPRE primers are 1.25E13 and 2.5E13 vg / kg, respectively) as a single intravenous (IV) infusion with necropsy following a 6-week in-life duration. Downstream analyses included DNA and RNA rAAV biodistribution in the CNS and peripheral organs, quantification of neuronal transduction in key brain regions using histological immunostaining of the HA tag, and analysis of GCase activity in the brain.

[0154] FIG. 2A and FIG. 2B provide data pertaining to the DNA biodistribution of rAAV in the CNS and liver, respectively. The data provided in FIG. 2A and FIG. 2B demonstrates targeted delivery of the rAAVs of the invention to the CNS, with dose-dependent detection in the brain and reduced delivery to liver compared to AAV9. Thus, the data demonstrates that that the rAAVs of the invention are delivered to the brain in a dose dependent manner with de-targeting from the liver. Advantageously, at a dose of 2.8E13 vg / kg, the data demonstrates 16-fold de-targeting of the liver with the rAAV of the invention compared to historical data from TV-delivered AAV9.

[0155] FIG. 3A and FIG. 3B provide data pertaining to the expression of GBA1 transgene in the CNS and liver, respectively. The data provided in FIG. 3A demonstrates that the rAAV efficiently delivers GBA1 transgene to the CNS, resulting in widespread expression in the brain at both doses. This data demonstrates improved expression observed at both tested doses for the rAAV, suggesting improved transduction in the brain. Historical comparison to IV-delivered AAV9 at a dose of 2.8E13 vg / kg, shows greater than 100-fold average improvement in vector mRNA detected in brain with the rAAV.

[0156] FIG. 4A provides the data pertaining to neuronal transduction after the administration of the rAAV of the invention. As the data provided in FIG. 4 demonstrates, rAAV administration results in widespread neuronal transduction at both doses, including greater than 50% neuronal transduction in substantia nigra. An antibody against TH was used to identify dopaminergic neurons in the substantia nigra pars compacta; all other regions used an antibody against NeuN as a neuronal marker. The administration of rAAVs of the invention resulted in high levels of neuronal transduction in the brain with an average of 28% in the frontal cortex, 26% in caudate, 30% in putamen, 65% in thalamus, and 59% in substantia nigra at both doses. Moreover, greater than 99% of transduced cells were identified as neurons in these quantified brain regions. The data provided in FIG. 4 confirms substantial GBA1 protein expression with both doses of the rAAVs of the invention.

[0157] Images in FIGS. 5A, 5B, 5C, 5D, and 5E demonstrate neuronal transduction and transgene expression in putamen, frontal cortex, substantia nigra, thalamus, and caudate, respectively, with a representative section zoomed in at the dose of 1.4E13 vg / kg. Similar data was observed for the dose of 2.8E13 vg / kg.

[0158] FIGS. 6A and 6B provide data pertaining to GCase activity in the brain following administration of the rAAVs of the invention. The data demonstrates that the administration of rAAVs of the invention enhances delivery to the CNS, resulting in significant increase in GCase activity. Specifically, administration of rAAV of the invention resulted in greater than 30% increase in both bulk and per transduced neuron GCase activity at 2.8E13 vg / kg dose. Administration of rAAVs of the invention at a dose of 1.4E13 vg / kg dose achieves a 26% increase in bulk brain GCase activity and a 69% average increase in transduced cells, suggesting meaningful clinical efficacy may also be achieved at this dose. The shaded gray box in FIG. 6A represents normalization range of GCase activity from published post-mortem patient tissue data. FIG. 6B provides data using mass spectrometry to measure GCase activity, and this highly sensitive analytical method confirms increases in GCase activity in the brain of rAAV treated NHPs that are expected to be clinically meaningful. Thus, the data provided above shows widespread expression in the brain at both a nucleic acid and protein level. Furthermore, the GBA1 protein expressed results in a significant increase in GCase activity in the brain of treated primates.

[0159] Example 4: Target engagement study

[0160] A study was conducted to assess target engagement of the cargo of the rAAVs of the invention after systemic injection via a surrogate capsid in both wild-type (WT) and mice harboring the homozygous point mutation D409V in the mGbal gene. Mice were administered either 2E12 vg / kg or 1E13 vg / kg of AAV.CAP-B10:CAG-hGBAl-HA-WPRE-bGH via a single retroorbital IV injection at 2-months of age with a necropsy following a 4-week in-life duration. A group of untreated animals of both genotype types were euthanized at 3 months of age as controls. Downstream analyses included neuronal transduction using histological immunostaining of the HA tag and measurements of GBA1 protein, GCase activity, and glycosphingolipid levels in the brain. FIG. 7A and FIG. 7B provide the data for the GBA1 protein expression and GCase activity respectively in treated and untreated mice. FIG. 7C provides the data for the levels of glucosylsphingosine (GluSph) 18: 1 in the treated and untreated mice. The results demonstrate increased GBA1 protein levels that correlate with an increase in GCase activity and reduction of lipid substrates.

[0161] Another study was conducted to assess target engagement of the cargo of the rAAVs of the invention after systemic injection via a surrogate capsid in mice harboring the homozygous point mutation D409V in the mGbal gene and transgene overexpression of alpha-synuclein. Mice were administered 1E12, 3E12, or 8E12 vg / kg of AAV.CAP-B10:CAG-hGBAl-WPRE-bGH via a single retroorbital IV injection at 2-months of age with a necropsy following 2 and 6 month in-life duration.

[0162] FIG. 20A provides a schematic design of the study, and FIG. 20B provides an overview of the number of mice and administered doses in this study. FIG. 21A provides data pertaining to Brain GCase protein expression, Brain GCase activity, and Brain GluSph levels 6 months after expression of the cargo of the rAAVs of the invention to the mice. IV delivery of hGBAl via a surrogate capsid resulted in dose-dependent increases in both GCase protein and activity in the brain which coincided with significant decreases in brain GluSph levels. Expression was durable (6-mo in life) and well -tolerated with no safety signals.

[0163] FIG. 21B provides the data pertaining to WPRE DNA and RNA copies 2 months and 6 months after expression of the cargo of the rAAVs of the invention.

[0164] In vitro studies:

[0165] A study was conducted to assess transduction and cargo expression efficiency in human iPSC-derived neurons with GBA1 mutations. GBA1 N370S dopaminergic neurons were characterized to exhibit reduced GCase activity in comparison to its isogenic control. N370S neurons were administered 5E3, 5E4, 1E5, or 5E5 vg / cell of the rAAV and cultured for 13-14 days along with a control (no vector administration). Cells were harvested and assessed for vector DNA, RNA, and GCase activity.

[0166] The table below provides an overview of the study: The neurons were cultured for 8 days for DNA and RNA measurement prior to the dose administration, and 9 days for GCase activity measurement prior to the dose administration. Moreover, the duration of culture (including the time after the dose administration) was 13 days and 14 days for DNA / RNA measurement and GCase activity measurement respectively. FIG. 22 provides the in vitro data for DNA, RNA and GCAse activity after the administration of rAAVs of the invention. The data demonstrates a dose-dependent increase in transduction and GCase activity in the neurons.

[0167] Example 5: Gene supplementation therapy administered as a single intravenous (IV) infusion to non-human primates and mice

[0168] Parkinson’s disease (PD) is the second most common neurodegenerative disorder with an estimated prevalence of over 1.2 million people in the United States by 2030 and no approved disease modifying treatments today. While there are several genetic risk factors associated with PD, mutations within the GBA1 gene have been shown to be the most significant, and current evidence suggests up to 15% of PD patients have GBA1 mutations.

[0169] A gene supplementation therapy candidate of the invention is administered as a single intravenous (IV) infusion to non-human primates. The candidate consists of an engineered AAV capsid that is designed to deliver the functional human GBA1 gene broadly across the CNS while de-targeting the liver, thereby providing a permanent source of the GBA1 protein to the brain and allowing for long term correction of GCase activity. Substantial clinical and preclinical evidence suggests that interventions targeted to normalize GCase activity in the brain would enable longterm disease modification and substantially slow or stop the disease progression of PD-GBA. Using a GCase loss-of-function mouse model, proof of concept pharmacology was provided demonstrating that administration of the intended clinical cargo results in dose-dependent increases in GCase activity that coincide with dose dependent decreases in glycolipid accumulation. Importantly, normalization of GCase activity to wild-type levels resulted in significant reductions in glycolipid species that are thought to contribute either directly or indirectly to a-synuclein pathology in PD-GBA patients.

[0170] In non-human primates, it was shown that at low to moderate doses administration of the candidate results in robust neuronal transduction across the entire CNS, with particularly high levels in disease relevant brain regions such as the substantia nigra and putamen. These levels of transduction resulted in increases in bulk GCase activity that exceeded levels expected to normalize GCase activity in the patient population. Importantly, these levels of GCase supplementation are achieved at doses that are well tolerated and without any clinical pathology or immunogenicity findings, including a lack of histopathology in the liver and dorsal root ganglia, that are associated with higher-dose systemic gene therapies. Taken together, the candidate is advanced with the ultimate goal of achieving disease modifying clinical benefit for patients with PD-GBA through a convenient single dose IV administration.

[0171] In summary, systemic AAV gene therapy with CNS-targeted engineered capsids achieved significant GCase activity increases in the primate brain to support the potential treatment of PD- GBA.

[0172] Example 6: Systemic AAV gene therapy with CNS-targeted engineered capsids achieves significant GCase activity increases in the primate brain to support potential treatment of PD- GBA

[0173] Biallelic mutations in the GBA1 gene cause autosomal recessive Gaucher’s Disease, the most common lysosomal storage disorder. More recently, epidemiological studies have revealed a role for mutations in this gene in the development of Parkinson’s disease (PD), and current evidence suggests that heterozygous mutations within the GBA1 gene are present in up to 15% of PD patients, making it the most significant genetic risk factor. The GBA1 gene encodes a lysosomal hydrolase enzyme, glucocerebrosidase (GCase), which metabolizes glycolipids within the lysosomal compartment. Mutations in this gene result in decreased GCase activity, leading to accumulation of glycolipid species which are thought to contribute either directly or indirectly to a-synuclein pathology, the pathological hallmark of PD. While certain aspects of the pathogenesis underlying PD-GBA remain unclear, substantial preclinical evidence suggests that interventions targeted to restore GCase activity in the brain could slow or stop the progression of PD-GBA.

[0174] The present invention provides a gene supplementation therapy candidate that is administered as a single intravenous (IV) infusion to non-human primates. The candidate consists of an engineered AAV capsid that is designed to deliver the functional human GBA1 gene broadly across the CNS while de-targeting the liver and DRGs, which have been shown to be associated with safety issues in previous gene therapy programs. Using a loss-of-function mouse model it was demonstrating that administration of the therapeutic cargo using a surrogate capsid results in dosedependent increases in GCase activity that coincide with dose-dependent decreases in glycolipid accumulation. In non-human primates using the clinical candidate, it was shown that administration of the candidate at low to moderate doses results in robust expression across the entire CNS along with increases in levels of GCase protein and activity which show strong correlations with similar measurements in biofluids. Given that a -30% decrease in brain GCase activity is expected in the patient population, the observed increases are expected to normalize activity levels, reduce glycolipids, and potentially slow or stop the progression of PD-GBA.

[0175] Methods and Materials

[0176] GBA1 LOF mouse model: GBA D409V KI (Jax Strain# 019106) and WT mice (Jax Strain# 005304) received a retroorbital IV injection at 8-weeks of age. After 4-weeks cargo DNA and RNA were assessed using PCR. GCase protein, activity, and glycosphingolipid content were assessed in CNS tissue using mass spectrometry. A surrogate capsid was used to deliver the therapeutic cargo enabling assessment of target engagement in CNS tissue achieved by a capsid that crosses the BBB in mice.

[0177] Non-human primates (NHPs): The development candidate was administered IV to WT male and female cynomolgus macaques at -33 months of age. After 6-weeks, DNA biodistribution and mRNA expression were assessed using ddPCR. GCase protein, GCase activity, and glycosphingolipid content were measured via mass spectrometry in both tissue as well as plasma and CSF to allow for early assessment of planned clinical target engagement biomarkers.

[0178] IV Administration to NHPs of candidate capsids

[0179] FIG. 8A is a graph of RNA expression in NHP brain regions following administration of lead capsids of the invention. For each candidate capsid, RNA expression of a Gene of Interest (GO I) was increased across the CNS, with up to -300% increased CBS expression.

[0180] FIG. 8B is a graph of vector genomes in NHP liver following administration of lead capsids of the invention. For each candidate capsid, capsid MOI was decreased in the liver up to ~16x.

[0181] Capsids selected for the development candidate demonstrated high expression across the CNS and significant liver de-targeting with IV-dosing.

[0182] IV administration to mice of capsids delivering therapeutic cargo

[0183] As described above, GBA D409V KI (Jax Strain# 019106) and WT mice (Jax Strain# 005304) received a retroorbital IV injection at 8-weeks of age. After 4-weeks cargo DNA and RNA were assessed using PCR. GCase protein, activity, and glycosphingolipid content were assessed in CNS tissue using mass spectrometry. A surrogate capsid was used to deliver the therapeutic cargo enabling assessment of target engagement in CNS tissue achieved by a capsid that crosses the BBB in mice.

[0184] FIG. 9A-B are graphs of DNA biodistribution and RNA expression in mice following administration of capsids delivering therapeutic cargo.

[0185] FIG. 10A-C are graphs of GCase protein level and activity in mice following administration of capsids delivering therapeutic cargo.

[0186] FIG. 11A-B are graphs of brain GluSph and GalSph levels in mice following administration of capsids delivering therapeutic cargo. GluSph 18: 1 = Glucosyl sphingosine, GalSph 18: 1 = Galactosylsphingosine.

[0187] AAV treatment resulted in dose-dependent increase in GBA1 protein and GCase activity. These increases coincided with significantly reduced GluSph levels in the CNS and validate target engagement of the hGBAl cargo.

[0188] Overall, in vivo target engagement in a GBA1 LOF mouse model following administration of capsids delivering therapeutic cargo hGBAl showed robust target engagement and significant reductions in GluSph.

[0189] IV Administration to NHPs of development candidate

[0190] As described above, the development candidate was administered IV at two doses (2.2E13 vg / kg and 5.5E13 vg / kg) to WT male and female cynomolgus macaques at ~33 months of age. After 6-weeks, DNA biodistribution, mRNA expression, GCase protein levels, GCase activity, and GluSph levels were assessed.

[0191] In a second study, the development candidate was administered IV at three doses (9.8E12, 3.1E13 and 5E13 vg / kg) to WT male and female cynomolgus macaques. After 3 months, DNA biodistribution, mRNA expression, GCase protein levels, GCase activity, and GluSph levels were assessed.

[0192] FIG. 12A-C are graphs of DNA biodistribution and RNA expression in NHP brain regions from the first study after 6 weeks following administration of the development candidate. DNA biodistribution and RNA expression of hGBAl was increased across the CNS. The development candidate achieved superior expression compared to ICM-delivered AAV9 in NHPs, achieving >150-fold higher expression in cortical regions and >6000-fold higher expression in sub-cortical regions compared to ICM-delivered AAV9 (2E11 vg / g ICM).

[0193] FIG. 13A-B are graphs of DNA biodistribution in NHP liver and RNA expression in nonhuman primate dorsal root ganglia (DRG) from the first study after 6 weeks following administration of the development candidate. DNA biodistribution in NHP liver and RNA expression of hGBAl in DRG was decreased at both doses, with reductions up to 19x in the liver and 17x in DRGs. The administration resulted in no adverse histopathology findings. FIG. 13C- D are graphs providing data for biodistribution of NHP liver DNA and DRG RNA expression from the second study, 3 months after the administration of an AAV product of the invention, compared to the control AAV9 capsid.

[0194] IV administration of the development candidate in primates resulted in robust DNA biodistribution and mRNA expression across key areas of interest in the CNS while de-targeting peripheral organs including the liver and DRGs reducing safety risks associated with WT AAV9. AAV9 was IV delivered at a similar low dose.

[0195] FIG. 14A-B are fluorescent images of NHP substantia nigra showing tissue level expression of the development candidate after 6 weeks following administration of the development candidate.

[0196] Representative histology in the substantia nigra was derived from an epitope tagged version of the development candidate to enable histological quantification of transduced cells.

[0197] Overall, IV administration of the development candidate in NHPs showed expression >200-fold higher in the brain and shows significant de-targeting of the liver and dorsal root ganglion (~20-fold) relative to systemically administered AAV9.

[0198] FIG. 15A-C are graphs of GCase protein level and activity in NHP brain regions from the first study, after 6 weeks following administration of the development candidate. All doses of the development candidate exceeded the 30% efficacy threshold for normalizing GCase activity in patients. Across doses and brain regions, including the substantia nigra, GCase activity was 2-8- fold higher than the threshold needed to overcome the expected deficit in patients and normalize GCase activity. Average GCase protein level increases of the development candidate in NHPs was 8-24x greater than ICM AAV9 (8E10 vg / g brain). FIG. 15D-E provide data related to the GCase levels and GCase activity from the second study, 3 months after the administration in NHPs. The administration of the rAAVs of the invention results in sustained increase in GCase levels and GCase activity in the brain.

[0199] FIG. 16A-C are graphs of GCase protein and activity levels in the CSF of NHPs and average brain GCase activity from the first study following administration of the development candidate. Marked increases in GCase protein and activity in NHP CSF support use as early target engagement biomarkers. FIG. 16D-E are graphs of GCase protein and activity levels in the CSF of NHPs and average brain GCase activity from the second study following administration of an AAV product of the invention. Average GCase activity in the brain showed significant positive correlation with GCase protein levels in the CSF and a trend of positive correlation with GCase activity in the CSF. This data raises confidence in the use of CSF GCase biomarkers in the clinic.

[0200] FIG. 17A is a graph of Plasma GluSph in NHPs from the first study, 6 weeks following administration of the development candidate. GluSph shows decreased levels in the terminal plasma of development candidate treated NHPs, providing evidence of lysosomal activity and target engagement. CSF GluSph levels in healthy animals are below limit of quantification and decrease is not detectable. The data shows strong target engagement in the key glycolipid substrates by the development candidate. FIG. 17B provides data related to target engagement via decreases in plasma GluSph levels 3 months after administration of an AAV product of the invention, when normalized to the vehicle-administered control.

[0201] Administration of the development candidate in primates resulted in up to 6-fold increases in GCase protein level and up to 3-fold increases in GCase activity compared to untreated animals in therapeutically relevant brain regions, exceeding levels that are expected to be clinically meaningful. Measurement of key fluid biomarkers show strong relationship to GCase activity in the brain across the doses tested. The increases in GCase protein and activity in the NHP brain significantly surpass expected therapeutic threshold and correlate with key biomarkers.

[0202] Conclusion

[0203] Previous investigation therapies showed low neuronal transduction (especially in the substantia nigra) and limited GCase elevation. Direct injection to the brain or CSF is invasive and results in inconsistent expression across brain regions, whereas non-invasive IV delivery limits risks and allows for broad coverage across the CNS. . In contrast, the development candidate showed up to 70% neuronal transduction, with 57% in the substantia nigra. GCase increases greater than levels needed to treat PD-GBA were shown, reaching 172% in the cortex and 249% in the putamen.

[0204] Non-invasive IV delivery limited risks and allowed for broad coverage across the CNS while showing no adverse histopathology finding in surveyed NHP organs, including liver and DRGs.

[0205] Overall, the lead capsids of the invention showed significant improvements in CNS penetrance and peripheral de-targeting relative to AAV9. The development candidate was well tolerated and shows a favorable safety profile.

[0206] Proof-of-concept pharmacology in a GBA1 LOF mouse demonstrated dose-dependent increases in GCase protein levels and activity following administration of the hGBAl cargo. These increases coincided with dose-dependent decreases in GluSph and indicate successful target engagement.

[0207] The development candidate achieved broad CNS distribution in non-human primates, including high expression in regions impacted by PD-GBA pathology and increases in GCase activity expected to normalize levels in the PD-GBA patient population. Importantly, exploratory biomarkers show strong relationships to GCase activity in the brain which sets expectations for clinical outcomes in similar measures.

[0208] Example 7: Methods for treatment of Parkinson’s disease:

[0209] The invention provides novel recombinant adeno-associated virus (rAAV) gene replacement therapy designed utilizing a novel AAV packaging the human glucosylceramidase beta 1 (GBA 1) transgene. This transgene encodes the protein that comprises the human P- Glucocerebrosidase (GCase) enzyme. The schematic representation of key elements of exemplary plasmids used to produce the capsid for the novel therapy of the invention is provided in FIG. 18, and the schematic representation of key elements of the produced plasmid used to prepare the therapeutic cargo for the methods of the invention is provided in FIG. 19. The structure provided in FIG. 19 is L-ITR-CAG-hGBAl-WPRE-bGHpA-R-ITR .

[0210] Specifically, in certain embodiments, the product is a novel rAAV gene replacement therapy product that will used for patients with Parkinson’s disease who carry mutations in the GBA1 gene (PD-GBA). The product is formulated as a suspension for infusion and dosed in vector genomes per kilogram (vg / kg). The formulation will be administered intravenously as a single infusion.

[0211] Parkinson’s disease:

[0212] Parkinson’s disease (PD) is the second most common neurodegenerative disorder, with a predicted prevalence of over 1.2 million people in the United States (US) by 2030. The incidence has increased over time, likely due to factors such as the aging of the population, industrialization and pollution. PD is more prevalent in nations with high levels of social and economic development (high Socio-demographic Index; “SDI”) and has a rising prevalence in nations with low and middle SDIs. Upward trends in the age standard rate of PD were observed in most settings over the past 30 years. A new study reveals that the actual annual incidence of Parkinson’s disease among older adults in US is 50% higher than the current estimates of 90,000 diagnoses annually (Parkinson’s Foundation). Worldwide prevalence in 2019, based on the Global Burden of Disease Study was estimated at 8.4 million, up from 2.4 million in 1990 and 6.1 million in 2016.

[0213] PD is associated with both motor and non-motor symptoms. The classic triad of motor symptoms includes bradykinesia, rigidity and resting tremor with balance and gait disturbances often developing with disease progression. Non-motor symptoms frequently include cognitive decline, sleep disturbances, hyposmia, psychiatric symptoms, and autonomic dysfunction. Some non-motor symptoms, such as REM sleep disorder, constipation, depression and anosmia often precede the development of motor symptoms.

[0214] While the majority of PD cases are idiopathic, current evidence suggests approximately 5- 15% of PD patients have GBA1 mutations, making this the most significant genetic risk factor for PD. Not all GBA1 mutations in the general population will result in PD. However, among patients diagnosed with PD, the prevalence of GBA1 mutations is well documented and varies among different ethnic groups. For example, the frequency of GBA1 mutations in the European Ashkenazi Jewish population is 10-31%, while it is lower in the European non-Ashkenazi population (2.9- 12%), Asian population (1.8-8.7%), and North and South America overall (2.9-8%). In clinical practice, the sending of genetic panels for younger patients with PD has become commonplace; however, testing rate is not uniform across all age groups and geographies, so it is possible that some portion of ethnic and racial differences is due to ascertainment bias.

[0215] PD-GBA manifests similarly to sporadic PD but tends to present with an earlier age of onset and greater cognitive dysfunction. Patients with PD and a GBA1 mutation are more likely to have an earlier age of onset, with an average of 53 years old for a severe GBA1 mutation and 58 years old for mild mutations, versus 61 to 65 years old for average age of onset of PD. On average, PD-GBA is associated with a more rapid progression than sporadic PD. A pathological hallmark of PD is the presence of intraneuronal inclusions called Lewy bodies, which are made up primarily of abnormally aggregated alpha-synuclein. Mutations in the GBA1 gene may lead to loss of GCase activity and lysosomal dysfunction, which may impair alpha-synuclein metabolism and contribute to its aggregation.

[0216] Patients with PD-GBA exhibit the same cardinal motor symptoms as patients with idiopathic PD, but with more rapid progression. Non-motor symptoms are reported to be more common and severe in PD-GBA, with patients often showing advanced clinical decline and a higher risk for cognitive impairment.

[0217] GBA1 Mutations in Parkinson’s disease (PD-GBA)

[0218] GBA1 encodes the lysosomal hydrolase enzyme “GCase”, comprises 10 introns and 11 exons, and is located on chromosome 1 q21. GCase is a 497- amino-acid (AA) active protein with 5 glycosylation regions. The protein is synthesized in the ER, transported by the lysosomal integral membrane protein-2 (LIMP2), and becomes active upon reaching the acidic lumen of the lysosome by interacting with its activator protein saposin C.

[0219] GBA1 gene mutations are much more prevalent in most PD populations than other genetic subtypes, such as a-synuclein SNCA, PARK2, and LRRK2. Mutations of GBA1 are a significant risk factor for PD. The variants of GBA1 can raise the PD risk by up to 10-fold and present in 2 to 30% of all age groups of PD patients. Over 300 mutations have been identified in GBA1. The most common mutations are point mutations, including N370S and L444P. Point mutations contribute to 70%-80% of PD-GBA. There is a 10%- 30% probability of developing PD among the heterozygous mutation carriers at the age of 80, a 20-fold rise as compared to the non-carriers.

[0220] Patients with PD-GBA, when compared with patients who had PD but lacked a GBA1 pathogenic variant, are more likely to have the features of PD with younger age onset; less prominent tremor, bradykinesia, and rigidity; lower frequency of asymmetric onset; higher frequency of a family history of PD; greater likelihood of cognitive impairment; and faster and worse motor progression.

[0221] Evidence from in vitro and in vivo models suggests that reduced GCase function is associated with alpha synuclein accumulation, which is a pathological hallmark of PD. Analysis of post-mortem tissue also reveals decreased GCase activity in the brain of PD-GBA subjects. While certain aspects of the pathogenesis underlying PD-GBA remain incomplete, growing clinical and preclinical data suggest that developing an effective treatment that can restore GCase levels in the brain could slow or stop the progression of PD-GBA. Various approaches to the restoration of GCase function in the central nervous system are under investigation, including chaperone molecules, substrate reduction therapy, and gene therapy.

[0222] Available Therapy and Unmet Need:

[0223] Currently, there are no approved disease-modifying treatments for any form of PD, including PD-GBA. Levodopa is the standard of care for motor symptoms; however, motor fluctuations and dyskinesias develop in many patients as a result, which can be debilitating in and of themselves. Adjunctive medications, or deep brain stimulation, may be used to manage or mitigate motor fluctuations and dyskinesias. Nonmotor symptoms are also treated symptomatically, most typically with antidepressants, pain medications, acetylcholinesterase inhibitors, N-Methyl-D-aspartate receptor (NMD A) antagonists, antipsychotic agents, and various medications for autonomic dysfunction.

[0224] Gene therapy specifically offers the potential to stably replace wild-type GCase protein with a single administration, enabling long-term disease modification and substantially slowing disease progression with a limited treatment burden. Given the absence of targeted, diseasemodifying therapies and only modest efficacy of currently available therapies, Capsida is pursuing the development of products for the treatment of products targeting GCase replacement.

[0225] The product has the potential to be a practical and feasible therapy that addresses significant unmet needs in patients with a serious disease that has a substantial impact on patients, caregivers, and society as a whole. Specific targeting of GBA offers the potential for disease modification in PD patients with GBA1 mutations, addressing both motor and non-motor symptoms.

[0226] Table 1 : Select Sequences

[0227] Incorporation by Reference

[0228] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0229] Equivalents

[0230] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Claims1. An adeno-associated virus (AAV) vector comprising: an engineered capsid protein comprising the amino acid sequence having at least 80% shared sequence identity with ATRNGEVFIAQ (SEQ ID NO: 4); and a nucleic acid encoding: a promoter; and a GBA1 transgene.

2. The AAV of claim 1, wherein the AAV capsid protein comprises an amino acid sequence ATRNGEVFIAQ (SEQ ID NO: 4).

3. The AAV of claim 1, wherein the capsid protein is engineered from AAV9 capsid protein.

4. The AAV of claim 3, wherein the capsid protein is engineered, relative to an AAV9 capsid protein, by substituting amino acid position 588 with the amino acid T, peptide insertion between amino acid positions 588 and 589 the amino acids RNGEVFI, and parental AAV9 substituting amino acids AQ in positions 589 and 590.

5. The AAV of claim 4, wherein the AAV is characterized by at least one of an increased specificity and / or increased transduction efficiency in the central nervous system (CNS).

6. The AAV of claim 5, wherein the AAV capsid is characterized by at least one of an increased specificity and / or increased transduction efficiency in the brain.

7. The AAV of claim 1, wherein the GBA1 transgene is codon optimized.

8. The AAV of claim 1, wherein the promoter is a CAG promoter.

9. The AAV of claim 8, wherein the CAG promoter comprises human cytomegalovirus immediate early enhancer (CMV enhancer), a chicken P-actin promoter, and a chimeric rabbit -globin (rBG) intron.

10. The AAV of claim 8, wherein the AAV further comprises at least one of regulatory element, poly(A) signal for stability, and a HA epitope tag.

11. The AAV of claim 10, wherein the regulatory element is Woodchuck hepatitis virus Post- transcriptional Regulatory Element (WPRE).

12. The AAV of claim 10, wherein the poly(A) signal is bGH polyA.

13. The AAV of claim 10, wherein the HA epitope tag is on the C-terminus of hGBAl protein.

14. The AAV of claim 13, wherein the HA epitope tag comprises an amino acid sequence YPYDVPDYA (SEQ ID NO: 3).

15. A method of treatment of a disease, the method comprising administering a composition comprising adeno-associated virus (AAV) comprising: an engineered capsid protein comprising the amino acid sequence having at least 80% shared sequence identity with ATRNGEVFIAQ (SEQ ID NO: 4); and a nucleic acid encoding: a promoter; and a GBA1 transgene.

16. The method of claim 15, wherein the AAV capsid protein comprises an amino acid sequence ATRNGEVFIAQ (SEQ ID NO: 4).

17. The method of claim 16, wherein the capsid protein is engineered from AAV9 capsid protein.

18. The method of claim 17, wherein the capsid protein is engineered, relative to an AAV9 capsid protein, by substituting amino acid positions 588 with the amino acid T, peptide insertion between amino acid positions 588 and 589 the amino acids RNGEVFI, and parental AAV9 amino acids AQ in positions 589 and 590.

19. The method of claim 18, wherein the AAV is characterized by at least one of an increased specificity and / or increased transduction efficiency in the central nervous system (CNS).

20. The method of claim 19, wherein the AAV capsid is characterized by at least one of an increased specificity and / or increased transduction efficiency in the brain.

21. The method of claim 15, wherein the GBA1 transgene is codon optimized.

22. The method of claim 15, wherein the promoter is a CAG promoter.

23. The method of claim 22, wherein the CAG promoter comprises human cytomegalovirus immediate early enhancer (CMV enhancer), a chicken P-actin promoter, and a chimeric rabbit P-globin (rBG) intron.

24. The method of claim 17, wherein the AAV further comprises at least one of regulatory element known to increase expression of the therapeutic gene, poly(A) signal for stability, and a HA epitope tag.

25. The method of claim 24, wherein the regulatory element is Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE).

26. The method of claim 24, wherein the poly(A) signal is bGH polyA.

27. The method of claim 24, wherein the HA epitope tag is on the C-terminus of hGBAl protein.

28. The method of claim 27, wherein the HA epitope tag an amino acid sequence YPYDVPDYA (SEQ ID NO: 3).

29. The method of claim 15, wherein the disease is Parkinson’s disease (PD).

30. The method of claim 29, wherein the Parkinson’s disease is associated with a mutation of GBA in the patient.

31. The method of claim 15, wherein the composition is administered as an injection.

32. The method of claim 31, wherein the injection is an intravenous injection.

33. The method of claim 32, wherein the administration of the composition results in the delivery of hGBAl to the central nervous system.

34. The method of claim 33, wherein the method results in administration of the composition results in preferential delivery of hGBAl to brain cells as compared to other cells.

35. The AAV vector of claim 1, wherein the transgene is flanked by inverted terminal repeats (ITRs), wherein: the 3’ ITR comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 8, and / or the 5’ ITR comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 9.

36. The method of claim 15, wherein the transgene is flanked by inverted terminal repeats (ITRs), wherein: the 3’ ITR comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 8, and / or the 5’ ITR comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 9.

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