[go: up one dir, main page]

WO2025160452A1 - Procédés d'administration basée sur fus de particules virales au cerveau - Google Patents

Procédés d'administration basée sur fus de particules virales au cerveau

Info

Publication number
WO2025160452A1
WO2025160452A1 PCT/US2025/013023 US2025013023W WO2025160452A1 WO 2025160452 A1 WO2025160452 A1 WO 2025160452A1 US 2025013023 W US2025013023 W US 2025013023W WO 2025160452 A1 WO2025160452 A1 WO 2025160452A1
Authority
WO
WIPO (PCT)
Prior art keywords
aav
fus
viral particle
administering
brain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/013023
Other languages
English (en)
Inventor
Isabelle AUBERT
Bradford Miller ELMER
Kullervo Hynynen
Rikke H. KOFOED
Christian Mueller
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sunnybrook Research Institute
Genzyme Corp
Original Assignee
Sunnybrook Research Institute
Genzyme Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sunnybrook Research Institute, Genzyme Corp filed Critical Sunnybrook Research Institute
Publication of WO2025160452A1 publication Critical patent/WO2025160452A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0085—Brain, e.g. brain implants; Spinal cord
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5184—Virus capsids or envelopes enclosing drugs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates to methods for treating neurodegenerative disorders in a patient in need thereof, the methods comprising administering to the patient a viral vector via focused ultrasonic delivery.
  • BACKGROUND Gene therapy has demonstrated long-lasting therapeutic benefit for the treatment of neurological disorders affecting large parts of the central nervous system, such as spinal muscular atrophy(1).
  • Recombinant adeno-associated virus (AAV) is the most advanced vector for gene delivery in vivo, and some AAV serotypes, such as AAV9, can cross the blood-brain barrier (BBB) after intravenous administration(2).
  • BBB crossing of AAV9 requires high intravenous dosages of up to 2x10 14 genome copies per kilogram (GC/kg) in children and more in adults where the ability of AAV9 to cross the BBB is limited(2, 3).
  • the recent deaths of patients with X-linked myotubular myopathy receiving 3x10 14 GC/kg AAV8 highlight the risks associated with intravenous AAV administration and the need to develop new strategies for AAV delivery to the central nervous system(4).
  • the permeability of the BBB can be increased transiently by the application of focused ultrasound combined with intravenous microbubbles (FUS-MB)(5).
  • FUS induces an oscillation of the microbubbles, which decreases tight-junction proteins and increases sf-6501845 Attorney Docket No.:15979-20190.40 transcytosis across the endothelial cells(6, 7).
  • the temporary increase in BBB permeability facilitates non-invasive delivery of intravenous AAV to FUS-targeted brain areas at dosages 50- 100 times lower than needed for BBB crossing by AAV9 alone(8). Still, the delivery is limited to FUS-targeted brain areas, and while FUS can be targeted simultaneously to multiple brain regions it remains unsuitable for treating whole-brain diseases(9).
  • a challenge and unmet need of gene therapy is to deliver gene vectors to deep brain structures using a minimally invasive strategy.
  • the disclosure provides methods of delivering viral particles to the brain of a mammal (e.g., a human patient), comprising administering the viral particles to the cerebral spinal fluid (CSF) of the patient and administering a plurality of microbubbles intravenously to the mammal, wherein one or more regions of the brain of the mammal are subjected to focused ultrasound (FUS).
  • the viral particles are delivered to the CSF of the mammal by injection of the viral particles into the cisterna magna of the patient.
  • the combination of CSF administration and focused ultrasound with microbubbles facilitates gene delivery of the viral particles to the superficial and deep brain regions of the mammal that are difficult to reach.
  • the combination of ICM administration of viral particles along with focused ultrasound-microbubble (FUS-MB) techniques enable viral particle delivery to both superficial brain areas and FUS-targeted deep brain structures.
  • the viral particles are adeno-associated virus (AAV) particles.
  • the AAV particles is a recombinant adeno-associated virus (rAAV) particles.
  • the administered viral particles comprise a dose level of from about 8 x 10 11 to about 5 x 10 13 genome copies per kilogram (GC/kg). In some embodiments, the administered viral particles comprise a dose level of from about 8 x 10 11 to about 2 x 10 13 genome copies per kilogram (GC/kg). sf-6501845 Attorney Docket No.:15979-20190.40 [0010] In some embodiments, methods comprising FUS-MB increases delivery of viral particles (e.g., AAV) administered via ICM to the striatum. In some embodiments, methods comprising FUS-MB increases delivery of viral particles administered via ICM to the striatum about 5-fold compared to delivery of viral particles administered via ICM alone.
  • viral particles e.g., AAV
  • methods comprising FUS-MB increases delivery of viral particles administered via ICM to the striatum about 2-fold, 3-fold, 4-fold, 5-fold, or more, compared to delivery of viral particles administered via ICM alone.
  • methods comprising FUS-MB increases delivery of viral particles (e.g., AAV) administered via ICM to the cerebral cortex.
  • methods comprising FUS-MB increases delivery of viral particles (e.g., AAV) administered via ICM to the thalamus.
  • Application of the FUS, administration of the microbubbles and administration of the viral particles can occur in any order. In some embodiments, the application of FUS occurs prior to the administering of the viral particles and/or the plurality of microbubbles.
  • the applying of the FUS occurs simultaneously with the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs less than one minute after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs at about 1 minute after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs at about 1, 2, 3, 4, 5, 10, 60, 120, 360 minutes, or more after the administering of the viral particles and/or the plurality of microbubbles.
  • the applying of the FUS occurs at about 1, 2, 3, 4, 5, 10, 24 hours, or more after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs at about 1 or 2 hours after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs between about 1 minute to about 180 minutes after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs between about 10 minutes to about 180 minutes after the administering of the viral particles and/or the plurality of microbubbles.
  • the applying of the FUS occurs between about 30 minutes to about 180 minutes after the administering of the viral particles and/or the plurality of microbubbles.
  • sf-6501845 Attorney Docket No.:15979-20190.40
  • provided herein are methods for treating neurological disorders requiring delivery of viral particles (e.g., AAV) to the brain of patients.
  • methods comprise CSF administration (e.g., intracisternal magna (ICM) administration) of a viral particle (e.g., adeno-associated virus (AAV)) for treating neurological disorders comprising beta-galactosidase-1 deficiency, Huntington’s Disease, Batten disease, Alzheimer’s disease, Parkinson’s disease, multiple system atrophy, progressive supranuclear palsy, frontotemporal dementia, amyotrophic lateral sclerosis, or spinal muscular atrophy.
  • a viral particle e.g., adeno-associated virus (AAV)
  • AAV adeno-associated virus
  • FIG. 1A shows intracisternal magna (ICM) administration of viral particles (e.g., AAV particles) may lead to widespread transduction, especially of superficial brain areas, but the transduction of deeper brain structures (e.g. striatum) is negligible.
  • FIG.1B showa focused ultrasound (FUS) combined with intravenous (IV) microbubbles and intravenous viral particles delivers viral particles to any FUS-targeted brain region, including deep structures such as the striatum.
  • FIG.1C shows that the combination of FUS and IV microbubbles with viral particles administered ICM results in viral particle delivery widespread to superficial brain areas as well as to deep brain structures targeted with FUS.
  • ICM intracisternal magna
  • FIG.1D shows a first possible mechanisms of action describing how FUS-MB may increase brain delivery of ICM-administered viral particles.
  • FIG. 1E shows a second possible mechanisms of action describing how FUS-MB may increase brain delivery of ICM-administered viral particles.
  • FIGS.2A-K show experimental design schematics and results for a pilot trial of adeno associated vector (AAV) dosing timepoints relative to focused ultrasound (FUS) applications, in accordance with some embodiments.
  • AAV adeno associated vector
  • FIGS.2F-H Five groups were tested for FUS-MB-mediated delivery of AAV and results are depicted in FIGS.2F-H.
  • AAV was injected intravenously during FUS application as a positive control as depicted in FIG.2F.
  • AAV was injected ICM either 60 minutes or 120 minutes prior to FUS-MB application or 10 minutes post FUS-MB sf-6501845 Attorney Docket No.:15979-20190.40 application as depicted in FIG.
  • FIG. 2G The injection of AAV after FUS application was used to determine whether distribution route #2 was possible.
  • One group of animals received ICM injection without FUS-MB application as depicted in FIG. 2H. After 4 weeks, the animals were sacrificed, and one hemisphere was used for GFP immunohistochemistry (IHC) and the other for analysis of GFP mRNA expression as depicted in FIG. 2I.
  • IHC GFP immunohistochemistry
  • GFP protein expression was seen in the FUS-targeted spots in all animals except those not treated with FUS-MB where GFP expression was visible only in one area in the striatum close to the cortex (k10) as depicted in FIG. 2J.
  • FIGS.3A-D show results demonstrating AAV is significantly cleared from the cerebrospinal fluid into the blood, in accordance with some embodiments.
  • AAV was found to be significantly cleared from the cerebrospinal fluid into the blood as depicted in FIGS. 3A-D.
  • the distribution of AAV to peripheral organs following intravenous (IV) and ICM administration was measured by quantifying the GFP genome copies in the peripheral organs 4 weeks after AAV administration (FIG.3A). There were no significant differences between the GFP genome copies in the peripheral organs with IV injection of the AAV compared to ICM injection (FIG. 3B).
  • GFP genome copies were quantified in blood samples taken 60 and 153 minutes post-ICM injection and 21 minutes post IV injection (FIG.3C).
  • GFP genome copies in the blood 60 minutes after ICM injection was not significantly higher than negative control samples (FIG. 3D).
  • GFP genome copies in the blood 153 minutes after ICM injection was significantly higher than the 60 minutes time point and the negative control.
  • the level of GFP genome copies was the highest of all groups 21 minutes after IV injection of the AAV. Bars represent mean +/- standard deviation.
  • FIGS.4A-L show results demonstrating FUS-mediated delivery of intracisternal magna (ICM) administered AAV to the striatum, in accordance with some embodiments.
  • AAV was sf-6501845 Attorney Docket No.:15979-20190.40 injected intravenously (IV) during FUS-MB, ICM 120 minutes pre-FUS-MB or ICM without FUS-MB application.
  • IV intravenously
  • FUS was targeted bilaterally to the striatum. After 4 weeks the animals were sacrificed, one hemisphere was used for IHC and the other hemisphere which was dissected into brain regions for RNA extraction and qPCR analysis (FIG.4A).
  • AAV ICM injection did not result in higher GFP mRNA expression in the FUS-targeted cortical structures compared to animals injected with AAV IV and treated with FUS-MB.
  • cortical structures where FUS was not targeted there was significantly higher GFP mRNA expression in animals injected ICM with AAV, both with and without FUS-MB application in the striatum, than in animals injected IV with AAV and FUS-MB (FIG. 4K).
  • FIG.5 shows accurate, large volume BBB opening achieved in both caudate nucleus and putamen using methods of the disclosure. Blood brain barrier opening was achieved throughout the left caudate (Cd) and putamen (Put) of all four animals.
  • FIG.5A shows axial MR images taken using a modified T2* relaxation map “T2*map” to detect minor perturbation of the BBB immediately following sonication. Animals 1001, 1002, 1003 and 1004 received between 1-3 sonication rounds per target depending on the extent of hypointense signal change observed.
  • FIG. 5B shows axial MR images taken following intravenous administration of gadolinium (Gd) contrast agent to indicate opened brain regions by showing as hyperintense contrast as compared to the same regions in the opposite, nontargeted hemisphere. Accurate opening throughout the targeted regions was demonstrated for all animals immediately after FUS.
  • FIG.5C shows that opening largely resolved in 1001 and 1003 by 2 days post-sonication, suggestive of excessive BBB opening in the other two animals.
  • Gd gadolinium
  • FIGS.5D and 5E show axial, T2 weighted imaging hyperintensities two days after sonication, indicative of inflammation/edema that did not resolve, as this signal remained for the duration of the study, out to 20 days post-sonication.
  • FIG. 6 shows enhancement of AAV vector genome biodistribution to the NHP striatum using MRIgFUS in combination with intra-CSF delivery of AAV.
  • Vector genome biodistribution of AAV2HBKO and AAV.SAN006 was quantified 3 weeks following intra-CSF AAV administration and BBB opening. DNA was extracted from 49 gray matter punches encompassing 20 brain regions.
  • FIGS. 6C and 6D show VG data plotted to highlight spatial differences in BBB opening and resulting transduction within the targeted brain regions, VG data show transduction at the tissue punch level for both AAV2HBKO (FIG. 6C) and AAV.SAN006 (FIG.
  • FIG. 6E shows quantification of AAV VG levels in the blood following intra-CSF administration of AAV. Genomic DNA was extracted from 200uL of whole blood drawn at the indicated timepoints to track the amount of AAV2HBKO-GFP and AAV.SAN006-mCherry in the blood before, during and 1 week after FUS. Values are VG per uL of blood from each animal with the average, predose background subtracted. Vector genome levels in spinal cord (SC) (FIG.
  • SC spinal cord
  • FIG. 7 shows enhancement of AAV-mediated transgene mRNA expression in the NHP striatum using MRIgFUS in combination with intra-CSF delivery of AAV.
  • FIGS.7A and 7B show graphs of mRNA expression (GFP for AAV2HBKO, mCherry for AAV.SAN006) for the indicated capsids in the untreated (“- FUS”) hemisphere vs sonicated (“+FUS”) hemisphere.
  • FIGS. 7C and 7D highlight potential spatial differences in BBB opening and resulting transduction within the targeted brain regions VG data are plotted to show transduction at the tissue punch level for both AAV2HBKO (FIG.7C) and AAV.SAN006 (FIG. 7D) in the caudate and putamen of each animal. 1003 and 1004 are denoted by blue symbols. Data are mean +/-SEM. [0019] FIG.8 shows Histology and imaging assessments.
  • FIGS. 8A and 8B show representative images showing hematoxylin and eosin (H&E) staining of 5 ⁇ m sections from animal 1003 indicative of cellular infiltrates and necrosis in the dorsal caudate of the FUS-treated, left hemisphere. These findings were consistently found in all animals but were restricted to dorsal caudate in each case. Putamen was spared in this animal and in all other animals. A1 and A2 insets are shown to detail findings in the caudate but not putamen. Scale bars are indicated in each panel.
  • FIG. 8C and 8D shows the evaluation of the pattern and extent of transduction in the anterior striatum in situ hybridization was performed on adjacent sections with probes specific to GFP or mCherry to visualize cells transduced by AAV2HBKO or AAV.SAN006 respectively. Tissue was counterstained with hematoxylin. High magnification images of the same region as shown in A2 and B2 are shown in C (GFP ISH) and D (mCherry ISH). Scale bars are indicated in each panel. [0020] FIG.9 shows a table of Data comprising the individual and summed actual cavitation doses for caudate and putamen.
  • Acoustic dose is provided in arbitrary units and is the dose sf-6501845 Attorney Docket No.:15979-20190.40 measured following the completion of each sonication round using ExAblate Neuro software. Due to the size of the region, ventral putamen was divided into two separate posterior (A) and anterior (B) regions within the same axial slice as indicated. Animals 1003 and 1004 were treated on 2 axial planes per structure to attempt more comprehensive tissue coverage. Total dose is shown as the sum of sonication rounds for each targeted region. DETAILED DESCRIPTION I. Introduction [0021] Gene delivery via adeno-associated viral particles can provide lasting clinical benefits following a one-time treatment.
  • focused ultrasound combined with intravenous microbubbles increases the delivery of the clinically relevant virus (e.g., gene vector, or adeno-associated virus) to deep brain structures as compared to methods without FUS-MB.
  • virus e.g., gene vector, or adeno-associated virus
  • administration of viral vector e.g., adeno-associated virus
  • the cerebrospinal fluid e.g., through the cisterna magna
  • focused ultrasound e.g., FUS
  • intravenous microbubbles e.g., MB
  • CSF intracisternal magna
  • focused ultrasound with microbubbles facilitate gene delivery, to superficial and deep brain structures.
  • intracisternal magna administration is less invasive than intracerebroventricular (IVC) administration.
  • methods may provide increased therapeutic efficacy of gene therapies.
  • methods may provide increased therapeutic efficacy of gene therapies particularly for disorders with brain regions that have remained difficult to reach.
  • methods may provide increased therapeutic efficacy of gene therapies particularly for disorders with brain regions that have remained difficult to reach.
  • neurodegenerative disorders e.g., disease
  • the combination of intracisternal magna administration and focused ultrasound enables viral vector delivery to both superficial brain areas and focused ultrasound-targeted deep brain structures.
  • the use of the proper viral vector (e.g., the proper AAV serotype) in conjunction with focused ultrasound microbubble-enhanced administration of the viral vector may also allow for delivery to the whole brain at dose levels low enough to allow for safe delivery to both children and adult human patients. II.
  • a “vector,” as used herein, refers to a recombinant plasmid or virus that comprises a nucleic acid to be delivered into a host cell, either in vitro or in vivo.
  • the terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full- length proteins and fragments thereof are encompassed by the definition.
  • polypeptide refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • a “recombinant viral vector” refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of viral origin).
  • the recombinant nucleic acid is flanked by at least one and in some embodiments two, inverted terminal repeat sequences (ITRs).
  • a “recombinant AAV vector” refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin) that are flanked by at least one, and in some embodiments two, AAV inverted terminal repeat sequences (ITRs).
  • rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins).
  • a rAAV vector When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the rAAV vector may be referred to as a “pro-vector” which can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions.
  • a rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, particularly an AAV particle.
  • a rAAV vector can be packaged into an AAV virus capsid to generate a “recombinant adeno-associated viral particle (rAAV particle)”.
  • rAAV particle recombinant adeno-associated viral particle
  • Heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).
  • a cellular sequence (e.g., a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector.
  • transgene refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and/or expressed under appropriate conditions. In aspects, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome.
  • CBA Chicken ⁇ -actin
  • CBA cytomegalovirus
  • chicken ⁇ -actin promoter may refer to a promoter containing a cytomegalovirus (CMV) early enhancer element, the promoter and first exon and intron of the chicken ⁇ -actin gene, and the splice acceptor of the rabbit beta-globin gene, such as the sequences described in Miyazaki, J. et al. (1989) Gene 79(2):269-77.
  • CMV cytomegalovirus
  • CAG promoter may be used interchangeably.
  • CAG early enhancer/chicken beta actin (CAG) promoter may be used interchangeably.
  • vector genome (vg) may refer to one or more polynucleotides comprising a set of the polynucleotide sequences of a vector, e.g., a viral vector.
  • a vector genome may be encapsidated in a viral particle.
  • a vector genome may comprise single-stranded DNA, double-stranded DNA, or single- stranded RNA, or double-stranded RNA.
  • a vector genome may include endogenous sequences associated with a particular viral vector and/or any heterologous sequences inserted into a particular viral vector through recombinant techniques.
  • a recombinant AAV vector genome may include at least one ITR sequence flanking a promoter, a stuffer, a sequence of interest (e.g., an RNAi), and a polyadenylation sequence.
  • a complete vector genome may include a complete set of the polynucleotide sequences of a vector.
  • the sf-6501845 Attorney Docket No.:15979-20190.40 nucleic acid titer of a viral vector may be measured in terms of vg/mL. Methods suitable for measuring this titer are known in the art (e.g., quantitative PCR).
  • infection unit (iu), “infectious particle,” or “replication unit,” as used in reference to a viral titer, refer to the number of infectious and replication-competent recombinant AAV vector particles as measured by the infectious center assay, also known as replication center assay, as described, for example, in McLaughlin et al. (1988) J. Virol., 62:1963-1973.
  • transducing unit (tu) refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product as measured in functional assays such as described in Examples herein, or for example, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or in Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).
  • An “inverted terminal repeat” or “ITR” sequence is a term well understood in the art and refers to relatively short sequences found at the termini of viral genomes which are in opposite orientation.
  • An “AAV inverted terminal repeat (ITR)” sequence is an approximately 145-nucleotide sequence that is present at both termini of the native single-stranded AAV genome.
  • the outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome.
  • the outermost 125 nucleotides also contains several shorter regions of self-complementarity (designated A, A', B, B', C, C' and D regions), allowing intrastrand base-pairing to occur within this portion of the ITR.
  • a “terminal resolution sequence” or “trs” is a sequence in the D region of the AAV ITR that is cleaved by AAV rep proteins during viral DNA replication. A mutant terminal resolution sequence is refractory to cleavage by AAV rep proteins.
  • AAV helper functions refer to functions that allow AAV to be replicated and packaged by a host cell. AAV helper functions can be provided in any of a number of forms, including, but not limited to, helper virus or helper virus genes which aid in AAV replication and packaging. Other AAV helper functions are known in the art such as genotoxic agents.
  • a “helper virus” for AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by a host cell.
  • a helper virus provides “helper functions” which allow for the replication of AAV.
  • helper viruses have been identified, including adenoviruses, herpesviruses and, poxviruses such as vaccinia and baculovirus.
  • the adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used.
  • adenoviruses of human, non- human mammalian and avian origin are known and are available from depositories such as the ATCC.
  • Viruses of the herpes family which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV).
  • HSV herpes simplex viruses
  • EBV Epstein-Barr viruses
  • CMV cytomegaloviruses
  • PRV pseudorabies viruses
  • Examples of adenovirus helper functions for the replication of AAV include E1A functions, E1B functions, E2A functions, VA functions and E4orf6 functions.
  • Baculoviruses available from depositories include Autographa californica nuclear polyhedrosis virus.
  • a preparation of rAAV is said to be “substantially free” of helper virus if the ratio of infectious AAV particles to infectious helper virus particles is at least about 10 2 :l; at least about 10 4 :l, at least about 10 6 :l; or at least about 10 8 :l or more.
  • preparations are also free of equivalent amounts of helper virus proteins (i.e., proteins as would be present as a result of such a level of helper virus if the helper virus particle impurities noted above were present in disrupted form).
  • an “effective amount” is an amount sufficient to effect beneficial or desired results, including clinical results (e.g., amelioration of symptoms, achievement of clinical endpoints, and the like).
  • An effective amount can be administered in one or more administrations. In terms of a disease state, an effective amount is an amount sufficient to ameliorate, stabilize, or delay development of a disease.
  • An “individual” or “subject” is a mammal.
  • Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non- human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • the individual or subject is a human.
  • sf-6501845 Attorney Docket No.:15979-20190.40
  • treatment is an approach for obtaining beneficial or desired clinical results.
  • g-FUS comprises ultrasound imaging-guided FUS (US-g-FUS).
  • g-FUS comprises magnetic resonance-guided focused ultrasound (MRg-FUS).
  • the guided FUS comprises imaging-guided FUS.
  • the imaging-guided FUS comprises US-g-FUS.
  • imaging-guided FUS comprises magnetic resonance imaging-guided FUS (MRI-g-FUS). Specific types of viral particles are discussed in Section IV, below.
  • an ultrasound transducer is noninvasively used to focus the ultrasound (FUS) within the brain to specific regions of interest.
  • FUS is sf-6501845 Attorney Docket No.:15979-20190.40 used in combination with gas-filled, microscopic bubbles that have been infused into the bloodstream in conjunction with the treatment molecule to be delivered to the brain.
  • a FUS signal excites the bubbles, temporarily permeabilizing the blood brain barrier in that location and allowing localized entry of viral particles to regions not normally accessible surgically but critical for treatment of neurologic diseases.
  • viral particles are introduced via the CSF, allowing for much lower vector dose required and potential avoidance of peripheral immune responses, making clinical translation more realistic.
  • Administration of viral particles to the CSF e.g., via intracisternal magna (ICM) administration, mitigates problems for clinical translation of gene therapies via systemic administration, which generally require high doses of viral particles.
  • ICM intracisternal magna
  • administration of viral particles (e.g., AAV particles) 101 may lead to widespread transduction, especially of superficial brain areas, but the transduction of deeper brain structures (e.g. striatum) is negligible.
  • FIG. 1A intracisternal magna
  • FUS focused ultrasound
  • IV intravenous
  • ICM intravenous
  • FIG. 1B focused ultrasound (FUS) 104 combined with intravenous (IV) microbubbles 105 and intravenous viral particles 101 delivers viral particles to any FUS-targeted brain region, including deep structures such as the striatum.
  • the transduction is only seen in the FUS spots 104, as depicted in FIG 1B.
  • FIG. 1C the combination of FUS and IV microbubbles with viral particles administered ICM results in viral particle delivery widespread to superficial brain areas as well as to deep brain structures targeted with FUS.
  • two possible mechanisms of action describing how FUS-MB may increase brain delivery of ICM-administered viral particles have been identified.
  • the viral particle 101 may travel in the cerebrospinal fluid (CSF) to the perivascular space and from there the interaction between FUS and intravenous MBs 105 (e.g., IV administered MBs) may create a pumping effect, which may increase the distribution of the viral particle from the CSF into the brain parenchyma.
  • CSF cerebrospinal fluid
  • intravenous MBs 105 e.g., IV administered MBs
  • FIG.1E viral particle (e.g. AAV) 101 injected via ICM may eventually be cleared from the CSF into the blood, where the viral particle can then enter the brain from the blood at FUS-targeted sites in a similar manner as when the viral particle is injected (e.g., administered) intravenously.
  • the first mechanism is active, and the second mechanism may not be.
  • the second mechanism is active, sf-6501845 Attorney Docket No.:15979-20190.40 and the first mechanism may not be.
  • the mechanism of action is related to distribution route #1, distribution route #2, or a combination of both.
  • methods may comprise injection directly into the cerebrospinal fluid through the cisterna magna as the route of administration for AAV for treatments of whole-brain diseases.
  • methods comprising FUS-MB may increase the delivery of intracisternal magna (ICM)-injected viral particles (e.g., AAV) to deep brain regions (e.g., thalamus) compared to methods without FUS-MB.
  • ICM intracisternal magna
  • the combination of ICM administration and FUS-MB may mediate (e.g., enhance) viral particle delivery to both superficial brain areas and FUS-targeted deep brain structures (FIG. 1C).
  • methods comprising FUS-MB may increase delivery of viral particles (e.g., AAV) administered via ICM to the striatum.
  • the striatum is a deep brain structure where the viral particles (e.g., AAV) may not be efficiently transduced following ICM injection alone.
  • the striatum controls both motor movements and emotional control/motivation and has been implicated in many neurological diseases, such as Huntington’s disease.
  • Several cell types of interest are located in the striatum, including without limitation spiny projection neurons (also known as medium spiny neurons), GABAergic interneurons, and cholinergic interneurons.
  • medium spiny neurons make up most of the striatal neurons. These neurons are GABAergic and express dopamine receptors.
  • Each hemisphere of the brain contains a striatum.
  • methods comprising FUS-MB may increase delivery of viral particles (e.g., AAV) administered via ICM to the cerebral cortex.
  • the cerebral cortex is a deep brain structure where the viral particles (e.g., AAV) may not be efficiently transduced following ICM injection alone.
  • methods comprising FUS-MB may increase delivery of viral particles (e.g., AAV) administered via ICM to the striatum and the cerebral cortex of the brain of a human.
  • a heterologous nucleic acid carried by the viral particle is expressed in one or more regions of interest in the CNS.
  • the heterologous nucleic acid is expressed in at least the cerebral cortex and striatum.
  • the heterologous nucleic acid is expressed in the frontal cortex, occipital cortex, and/or layer IV of the mammal.
  • the cerebral cortex is known as the outer layer of the mammalian brain important for language, consciousness, memory, attention, sf-6501845 Attorney Docket No.:15979-20190.40 and awareness.
  • the cerebral cortex is subdivided into a number of different components and regions due to its extensive anatomy and complex functions.
  • the cerebral cortex is divided into left and right hemispheres. In addition, it contains four gross lobes: frontal, parietal, temporal, and occipital.
  • Frontal cortex may refer to the frontal lobe of the cortex and is known to provide a wide range of neurological functions related to non-task-based memory, social interactions, decision making, and other complex cognitive functions.
  • Occipital cortex may refer to the occipital lobe of the cortex and is known to be involved in visual processing.
  • Parietal cortex may refer to the parietal lobe of the cortex and is known to be involved in language processing, proprioception, and sensory inputs related to touch.
  • Temporal cortex may refer to the temporal lobe of the cortex and is known to be involved in language, memory, and emotional association. [0057] In addition, three general types of areas of the cortex are described: sensory, motor, and association.
  • primary motor cortex involved in muscle control
  • premotor cortex higher order motor areas that command primary motor areas
  • association areas e.g., parietal-temporal-occipital or prefrontal; these areas are involved in planning, memory, attention, and other higher cognitive tasks and assume the majority of the human cortex
  • higher order areas sensor processing
  • primary sensory areas e.g., auditory, visual, and somatosensory
  • the heterologous nucleic acid is expressed in the prefrontal association cortical areas, the premotor cortex, the primary somatosensory cortical areas, sensory motor cortex, parietal cortex, occipital cortex, and/or primary motor cortex.
  • the cerebral cortex is divided into different cortical layers (moving from superficial to deep), each containing a characteristic pattern of neuronal connectivities and cell types. These layers are divided into supragranular layers (layers I-III), internal granular (IV), and infragranular (V and VI). Supragranular layers typically project to other cortical layers, whereas infragranular layers receive input from supragranular layers and send output to structures outside the cortex (e.g., motor, sensory, and thalamic regions).
  • Layer V contains pyramidal neurons with axons that connect to subcortical structures like the basal ganglia.
  • Layer V neurons in the primary motor cortex also form the corticospinal tract that is critical for voluntary motor control.
  • Layer IV receives inputs from the thalamus and connects to the rest of the column, thereby providing critical functions related to integration of the thalamus and cortex.
  • Characteristic cells of layer IV include stellate cells (e.g., spiny stellate cells) and pyramidal neurons.
  • the heterologous nucleic acid is further expressed in the thalamus, substantia nigra and/or hippocampus. In some embodiments, the heterologous nucleic acid is expressed in the thalamus, midbrain, cerebellum, hippocampus, and brainstem.
  • the thalamus is between the cortex and midbrain, sends signals (e.g., sensory and motor) to the cortex from subcortical areas, and plays a role in alertness and sleep.
  • the thalamus also connects to the hippocampus, part of the limbic system and a critical mediator of long-term memory consolidation.
  • the substantia nigra contains many dopaminergic neurons and is important for movement and reward.
  • CNS disorders like Parkinson’s disease are associated with loss of dopaminergic neurons in the substantia nigra. It further provides dopamine to the striatum that is critical for proper striatal function.
  • the heterologous nucleic acid is further expressed in the thalamus, subthalamic nucleus, globus pallidus, substantia nigra, putamen and/or hippocampus.
  • a viral particle e.g., AAV particle
  • administering i) a viral particle; and ii) a plurality of microbubbles 105; and b) applying focused ultrasound (FUS) to at least one region of interest (e.g., deep brain structure or striatum), thereby causing entry of the viral particle to the region of interest of the brain.
  • FUS focused ultrasound
  • the administering the viral particle 101 comprises a dose level of less than 1 x 10 14 genome copies per kilogram (GC/kg) for a patient (e.g., human patient). [0062] In some embodiments, the administering the viral particle 101 comprises a dose level of at most 5 x 10 13 genome copies per kilogram (GC/kg) for a patient (e.g., human patient).
  • the administering the viral particle comprises a dose level of at most 1 x 10 12 , 2 x 10 12 , 3 x 10 12 , 5 x 10 12 , 8 x 10 12 , 1 x 10 13 , 2 x 10 13 , 3 x 10 13 , 4 x 10 13 , 5 x 10 13 , or 8 x 10 13 genome copies per kilogram (GC/kg).
  • the administering the viral particle comprises a dose level of from about 1 x 10 11 to about 8 x 10 13 genome copies per kilogram (GC/kg).
  • the administering the viral particle comprises a dose level of from about 8 x 10 11 to about 5 x 10 13 genome copies per kilogram (GC/kg).
  • the administering the viral particle comprises a dose level of from about 8 x 10 11 sf-6501845 Attorney Docket No.:15979-20190.40 to about 2 x 10 13 genome copies per kilogram (GC/kg). In some embodiments, the administering the viral particle comprises a dose level of from about 1 x 10 12 to about 1 x 10 13 genome copies per kilogram (GC/kg). In some embodiments, the administering the viral particle comprises a dose level of from about 1 x 10 12 to about 8 x 10 12 genome copies per kilogram (GC/kg). In some embodiments, the administering the viral particle comprises a dose level of from about 2 x 10 12 to about 6 x 10 12 genome copies per kilogram (GC/kg).
  • methods comprising applying focused ultrasound in combination with administration of microbubbles (FUS-MB).
  • applying the FUS may occur after the administering of the viral particle and/or the plurality of microbubbles.
  • the applying the FUS may occur prior to the administering of the viral particles and/or the plurality of microbubbles.
  • the applying of the FUS occurs simultaneously with the administering of the viral particles and/or the plurality of microbubbles.
  • the applying of the FUS occurs after the administering of the viral particles and/or the plurality of microbubbles.
  • the applying of the FUS occurs less than one minute after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs at about 1 minute after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs at about 1, 2, 3, 4, 5, 10, 60, 120, 360 minutes, or more after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs at about 1, 2, 3, 4, 5, 10, 24 hours, or more after the administering of the viral particles and/or the plurality of microbubbles.
  • the applying of the FUS occurs at about 1 or 2 hours after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs between about 1 minute to about 180 minutes after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs between about 10 minutes to about 180 minutes after the administering of the viral particles and/or the plurality of microbubbles. In some embodiments, the applying of the FUS occurs between about 30 minutes to about 180 minutes after the administering of the viral particles and/or the plurality of microbubbles.
  • the placement of the FUS spot is determined by imaging. In some embodiments, the placement of the FUS spot is determined by magnetic resonance imaging (MRI).
  • the administering further comprises administering a tracer.
  • the tracer may comprise an image contrast agent.
  • the image contrast agent may comprise an MRI contrast agent.
  • the MRI contrast agent may comprise Gadolinium. In some embodiments, the tracer may comprise Gadolinium.
  • the applying the FUS comprises applying the FUS at a FUS frequency. In some embodiments, the FUS frequency may comprise about 0.58 MHz.
  • the FUS frequency may comprise 0.58 MHz. In some embodiments, the FUS frequency may comprise about 0.58 MHz or more. In some embodiments, the FUS frequency may comprise about 0.58 MHz or less. In some embodiments, the applying the FUS may comprise applying FUS via a 0.58 MHz spherically focused transducer. In some embodiments, the spherically focused transducer may comprise a 75 mm outer diameter, 26 mm inner diameter, and/or 60 mm radius of curvature. [0067] In some embodiments, the plurality of microbubbles 105 is administered at about 0.2 mL/kg. In some embodiments, the plurality of microbubbles is administered at about 0.2 mL/kg or more.
  • the plurality of microbubbles is administered at about 0.2 mL/kg or less. In some embodiments, the plurality of microbubbles is administered at about 0.2 mL/kg. to about 0.4 mL/kg. In some embodiments, the plurality of microbubbles is administered at about 0.1 mL/kg. to about 0.2 mL/kg. [0068] In some embodiments, the plurality of microbubbles 105 is administered at a fixed pressure of about 0.32 MPa. In some embodiments, the plurality of microbubbles is administered at a fixed pressure of about 0.32 MPa or more. In some embodiments, the plurality of microbubbles is administered at a fixed pressure of about 0.32 MPa or less.
  • the plurality of microbubbles comprises a gas.
  • the gas may comprise octafluoropropane gas.
  • each of the microbubbles of the plurality of microbubbles 105 may comprise a shell.
  • the shell may comprise a synthetic phospholipid shell. sf-6501845 Attorney Docket No.:15979-20190.40
  • the plurality of microbubbles may comprise octafluoropropane gas encapsulated by a synthetic phospholipid shell.
  • the plurality of microbubbles may comprise DEFINITY® microbubbles.
  • methods for treating neurological disorders requiring delivery of viral particles (e.g., AAV) to the brain of patients may comprise intracisternal magna administration (e.g., ICM administration) of adeno-associated virus (e.g., AAV) for treating neurological disorders comprising beta-galactosidase-1 deficiency, Batten disease, Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, aromatic L ⁇ amino acid decarboxylase (AADC) deficiency, MSA, or spinal muscular atrophy.
  • ICM administration e.g., ICM administration
  • adeno-associated virus e.g., AAV
  • AADC aromatic L ⁇ amino acid decarboxylase
  • provided herein are methods for gene delivery to the entire brain for the treatment of neurological disorders causing widespread pathology such as monogenic disorders.
  • methods for gene delivery via adeno- associated viral particles configured to provide lasting clinical benefits following a one-time treatment.
  • a method may comprise gene delivery to the entire brain for the treatment of neurological disorders causing widespread pathology such as monogenic disorders or advanced disease stages of Alzheimer, Parkinson disease and amyotrophic lateral sclerosis.
  • FUS-MB may increase the delivery of the clinically relevant viral particles to deep brain structures.
  • FUS-MB may increase the delivery of the clinically relevant viral particles to deep brain structures following injection in the cisterna magna.
  • FUS-MB increases the delivery of the clinically relevant viral particle (e.g., gene vector, or adeno-associated virus) by 2-fold, 3-fold, 4-fold, 5-fold, or more to deep brain structures following injection in the cisterna magna.
  • the permeability of the BBB can be increased transiently by the application of FUS combined with intravenous microbubbles (FUS-MB).
  • FUS induces an oscillation of the microbubbles, thereby decreasing tight-junction proteins and increasing transcytosis across the endothelial cells.
  • Such embodiments allow non- invasive delivery AAV via intravenous (IV) or ICM administration to FUS-targeted brain areas at dosages 50-100 times lower than needed for BBB crossing by AAV alone (e.g., without FUS sf-6501845 Attorney Docket No.:15979-20190.40 or without FUS-MB).
  • FUS-MB may decrease the intravenous AAV dose needed for brain delivery.
  • Current methods of using FUS for AAV delivery to the brain involve intravenous (IV) infusion of high doses of AAV.
  • methods may comprise delivery comprising AAV in CSF while keeping microbubbles in blood to avoid the high material requirements needed for IV administration of AAV.
  • Viral particles and methods of producing viral particles [0077] The disclosure provides, inter alia, administering viral particles (e.g., recombinant viral particles) to the CSF in the brain of a patient.
  • viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus).
  • AAV particles [0079]
  • the viral particle to be administered to the CSF is a recombinant AAV particle comprising a nucleic acid comprising a transgene flanked by one or two ITRs.
  • the nucleic acid is encapsidated in the AAV particle.
  • the AAV particle also comprises capsid proteins.
  • the nucleic acid comprises the coding sequence(s) of interest, operatively linked components in the direction of sf-6501845 Attorney Docket No.:15979-20190.40 transcription, control sequences including transcription initiation and termination sequences, thereby forming an expression construct.
  • the expression construct is flanked on the 5' and 3' end by at least one functional AAV ITR sequences.
  • functional AAV ITR sequences it is meant that the ITR sequences function as intended for the rescue, replication and packaging of the AAV virion. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J.
  • the recombinant vectors comprise at least all of the sequences of AAV essential for encapsidation and the physical structures for infection by the rAAV.
  • AAV ITRs for use in the vectors of the disclosure need not have a wild-type nucleotide sequence (e.g., as described in Kotin, Hum.
  • AAV serotypes may be derived from any of several AAV serotypes. More than 40 serotypes of AAV are currently known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18): 11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810. Use of any AAV serotype is considered within the scope of the present disclosure.
  • a rAAV vector is a vector derived from an AAV serotype, including without limitation, AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV capsid serotype or the like.
  • AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV capsid serotype or the like.
  • the nucleic acid in the AAV comprises an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV capsid serotype or the like.
  • the nucleic acid in the AAV further encodes a miRNA as described herein.
  • the rAAV particle comprise an AAV1, an AAV2HBKO capsid (e.g., as described in WO2015168666), an AAV9 capsid, a PHP.B capsid, a PHP.eB capsid, or an Olig001 capsid.
  • a rAAV particle can comprise viral proteins and viral nucleic acids of the same serotype or a mixed serotype.
  • a rAAV particle can comprise AAV1 capsid proteins and at least one AAV2 ITR or it can comprise AAV2 capsid proteins and at least one sf-6501845 Attorney Docket No.:15979-20190.40 AAV1 ITR. Any combination of AAV serotypes for production of a rAAV particle is provided herein as if each combination had been expressly stated herein.
  • the invention provides rAAV particles comprising an AAV1 capsid and a rAAV vector of the present disclosure (e.g., an expression construct comprising nucleic acid encoding a miRNA of the present disclosure), flanked by at least one AAV2 ITR.
  • the invention provides rAAV particles comprising an AAV2 capsid.
  • the rAAV particle comprise an AAV1, an AAV2HBKO capsid (e.g., as described in WO2015168666), an AAV9 capsid, a PHP.B capsid, a PHP.eB capsid, or an Olig001.
  • the capsid of an rAAV viral particle is known to include three capsid proteins: VP1, VP2, and VP3. These proteins contain significant amounts of overlapping amino acid sequence and unique N-terminal sequences.
  • an AAV9 capsid includes 60 subunits arranged by icosahedral symmetry.
  • AAV9 includes VP1 (SEQ ID NO: 6), VP2 (SEQ ID NO: 10), and VP3 (SEQ ID NO: 11), capsid proteins in a ratio of about 5:5:50.
  • the VP proteins of AAV9 are products of the structural protein-encoding open reading frame of the genome, designated cap, VP1 ( ⁇ 82 kDa) and VP2 ( ⁇ 73 kDa), which are the minor capsid proteins, and VP3 ( ⁇ 61 kDa), the major capsid protein. Due to the utilization of both alternative splicing and leaky scanning, when expressed, the individual VPs share a C terminus that encompasses the entire VP3, while VP1 and VP2 are N-terminal VP3 extensions.
  • the rAAV particle can comprise a modified AAV9 capsid.
  • a targeting peptide e.g., SEQ ID NO: 7
  • a targeting peptide e.g., SEQ ID NO: 7
  • the targeting peptide (e.g., SEQ ID NO: 7) is incorporated into VP3. In some embodiments of the modified AAV9 capsid proteins, the targeting peptide (e.g., SEQ ID NO: 7) is incorporated into VP1, VP2 and VP3. [0083] In particular embodiments, the targeting peptide of the modified AAV9 capsids are inserted after residue 588 of the AAV9 structural protein. In some embodiments, the targeting peptide has SEQ ID NO: 7.
  • the targeting peptide is flanked by linker sf-6501845 Attorney Docket No.:15979-20190.40 sequences on the N-terminal and the C-terminal end of the targeting peptide.
  • the linker sequence on the N-terminal side has the sequence AAA.
  • the linker sequence on the C-terminal side is AS.
  • the full sequence inserted after residue 588 of the AAV9 capsid structural protein has SEQ ID NO: 8.
  • the full modified AAV9 capsid structural protein has SEQ ID NO: 9.
  • the modified AAV9 capsid structural protein having SEQ ID NO: 9 is also referred to herein as capsid SAN006.
  • the full modified AAV9 capsid structural protein that it at least 90% (e.g., at least 92%, at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.5%, or at least 99.8%) identical to SEQ ID NO: 9, wherein the modified AAV9 structural capsid comprises the targeting peptide of SEQ ID NO: 7.
  • the transgene encoding a disorder-related polypeptide is codon-optimized.
  • the transgene encoding a disorder-related polypeptide is codon optimized for expression in a particular cell, such as a eukaryotic cell.
  • eukaryotic cells are those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • Codon usage tables are readily available, for example, at the “Codon Usage Database”, and these tables can be adapted in a number of ways (see, e.g., Nakamura, Y.
  • a transgene encoding the disorder-related polypeptide is codon optimized using the GA algorithm.
  • the expression cassette further comprises an intron.
  • introns for use in the disclosure are known to those of skill in the art, and include the MVM intron, the F IX truncated intron 1, the ⁇ -globin SD/immunoglobin heavy chain SA, the adenovirus SD/immunoglobin SA, the SV40 late SD/SA (19S/16S), and the hybrid adenovirus SD/IgG SA.
  • MVM intron the F IX truncated intron 1
  • ⁇ -globin SD/immunoglobin heavy chain SA the adenovirus SD/immunoglobin SA
  • the SV40 late SD/SA (19S/16S) the hybrid adenovirus SD/IgG SA.
  • the intron is a chicken ⁇ -actin (CBA)/rabbit ⁇ -globin hybrid intron.
  • intron is a chicken ⁇ -actin (CBA)/rabbit ⁇ -globin hybrid promoter and intron where all the ATG sites are removed to minimize false translation start sites.
  • the intron is an MVM intron, a F IX truncated intron 1, a ⁇ -globin SD/immunoglobin heavy chain SA, an adenovirus SD/immunoglobin SA, a SV40 late SD/SA (19S/16S), or a hybrid adenovirus SD/IgG SA.
  • the intron is a chicken ⁇ -actin (CBA)/rabbit ⁇ -globin hybrid intron.
  • the expression cassette further comprises a polyadenylation signal.
  • the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or a HSV TK pA.
  • the polyadenylation signal is a synthetic polyadenylation signal as described in Levitt, N et al. (1989), Genes Develop.3:1019-1025.
  • the expression cassette comprises a stuffer nucleic acid.
  • the stuffer nucleic acid may comprise a sequence that encodes a reporter polypeptide.
  • the stuffer nucleic acid may be located in a variety of regions within the nucleic and may be comprised of a continuous sequence (e.g., a single stuffer nucleic acid in a single location) or multiple sequences (e.g., more than one stuffer nucleic acid in more than one location (e.g., 2 locations, 3 locations, etc.) within the nucleic acid.
  • the stuffer nucleic acid is located downstream of the transgene encoding the disorder-related polypeptide.
  • the stuffer nucleic acid is located upstream of the transgene encoding the disorder-related polypeptide (e.g., between the promoter and the transgene).
  • a variety of nucleic acids are used as a stuffer nucleic acid.
  • the stuffer nucleic acid comprises all or a portion of a human alpha-1-antitrypsin (AAT) stuffer sequence or a C16 P1 chromosome 16 P1 clone (human C16) stuffer sequence.
  • the stuffer sequence comprises all or a portion of a gene.
  • the stuffer sequence may comprise a portion of the human AAT sequence.
  • a gene e.g., the human AAT sequence
  • the stuffer fragment is from the 5’ end of the gene, the 3’ end of the gene, the middle of a gene, a non-coding portion of the gene (e.g., an intron), a coding region of the gene (e.g., an exon), or a mixture of non-coding and coding portions of a gene.
  • a non-coding portion of the gene e.g., an intron
  • a coding region of the gene e.g., an exon
  • all or a portion of sf-6501845 Attorney Docket No.:15979-20190.40 stuffer sequence is used as a stuffer sequence.
  • the stuffer sequence is modified to remove internal ATG codons.
  • the expression cassette is incorporated into a vector. In some embodiments, the expression cassette is incorporated into a viral vector. In some embodiments, the viral vector is a rAAV vector as described herein. [0089]
  • the expression cassette for expressing a disorder-related polypeptide or RNAi molecule is contained in a vector. In some embodiments, the present disclosure contemplates the use of a recombinant viral genome for introduction of nucleic acid sequences encoding the disorder-related polypeptide or RNAi molecule for packaging into a viral particle, e.g., a viral particle described below.
  • the recombinant viral genome may include any element to establish the expression of the disorder-related polypeptide, for example, a promoter, an ITR, a ribosome binding element, terminator, enhancer, selection marker, intron, polyA signal, and/or origin of replication.
  • the disclosure provides viral particles comprising a single- stranded genome.
  • the disclosure provides viral particles comprising a recombinant self-complementing genome.
  • the vector is a self- complementary vector. AAV viral particles with self-complementing genomes and methods of use of self-complementing AAV genomes are described in US Patent Nos.
  • a rAAV comprising a self-complementing genome will quickly form a double stranded DNA molecule by virtue of its partially complementing sequences (e.g., complementing coding and non-coding strands of a transgene).
  • the disclosure provides an AAV viral particle comprising an AAV genome, wherein the rAAV genome comprises a first heterologous polynucleotide sequence (e.g., the coding strand of the disorder-related polypeptide of the disclosure) and a second heterologous polynucleotide sequence (e.g., the noncoding or antisense strand of the disorder-related polypeptide or RNAi molecule) wherein the first heterologous polynucleotide sequence can form intrastrand base pairs with the second polynucleotide sequence along most or all of its length.
  • a first heterologous polynucleotide sequence e.g., the coding strand of the disorder-related polypeptide of the disclosure
  • a second heterologous polynucleotide sequence e.g., the noncoding or antisense strand of the disorder-related polypeptide or RNAi molecule
  • the first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence are linked by a sequence that facilitates intrastrand basepairing; e.g., a hairpin DNA structure. Hairpin structures are known in the art, for example in siRNA molecules.
  • the first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence are linked by a mutated ITR (e.g., the right ITR).
  • the mutated ITR may comprise a deletion of the D region comprising the terminal resolution sequence.
  • the rep proteins may not cleave the viral genome at the mutated ITR and as such, a recombinant viral genome comprising the following in 5' to 3' order is packaged in a viral capsid: an AAV ITR, the first heterologous polynucleotide sequence including regulatory sequences, the mutated AAV ITR, the second heterologous polynucleotide in reverse orientation to the first heterologous polynucleotide and a third AAV ITR.
  • the first heterologous nucleic acid sequence and a second heterologous nucleic acid sequence are linked by a mutated ITR (e.g., the right ITR).
  • the ITR comprises the polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCT CGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGG GCG - 3' (SEQ ID NO: 12).
  • the mutated ITR may comprise a deletion of the D region comprising the terminal resolution sequence.
  • the rep proteins do not cleave the viral genome at the mutated ITR and as such, a recombinant viral genome comprising the following in 5' to 3' order is packaged in a viral capsid: an AAV ITR, the first heterologous polynucleotide sequence including regulatory sequences, the mutated AAV ITR, the second heterologous polynucleotide in reverse orientation to the first heterologous polynucleotide and a third AAV ITR.
  • the heterologous nucleic acid encodes a therapeutic polypeptide or therapeutic nucleic acid.
  • the heterologous nucleic acid encodes a therapeutic polypeptide.
  • the therapeutic polypeptide is an enzyme, a neurotrophic factor, a polypeptide that is deficient or mutated in an individual with a CNS-related disorder, an antioxidant, an anti- apoptotic factor, an anti-angiogenic factor, and an anti-inflammatory factor, alpha-synuclein, acid beta-glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), sf-6501845 Attorney Docket No.:15979-20190.40 galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase (MAN2B1), beta- mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1- phosphotransferase (GNPT)
  • the heterologous nucleic acid encodes a therapeutic nucleic acid.
  • the therapeutic nucleic acid is an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme.
  • the therapeutic polypeptide or the therapeutic nucleic acid is used to treat a disorder of the CNS.
  • the disorder of the CNS is a lysosomal storage disease (LSD), Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticogasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) or cancer of the brain.
  • LSD lysosomal storage disease
  • CBD corticobasal degeneration
  • CBGD corticogasal ganglionic degeneration
  • FTD frontotemporal dementia
  • MSA multiple system atrophy
  • PSP progressive supranuclear palsy
  • the disorder is a lysosomal storage disease selected from the group consisting of Aspartylglusoaminuria, Fabry, Infantile Batten Disease (CNL1), Classic Late Infantile Batten Disease (CNL2), Juvenile Batten Disease (CNL3), Batten form CNL4, Batten form CNL5, Batten form CNL6, Batten form CNL7, Batten form CNL8, Cystinosis, Farber, Fucosidosis, Galactosidosialidosis , Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, GM1 gangliosidosis, Hunter disease, Krabbe disease, ⁇ mannosidosis disease, ⁇ mannosidosis disease, Maroteaux-Lamy, metachromatic leukodystrophy disease, Morquio A, Morquio B, mucolipidosisII/III disease, Niemann-Pick A disease, Niemann-Pick B disease, Niemann-Pick C disease, Pompe disease, Sandhoff disease, Sanfillipo A
  • the heterologous nucleic acid is operably linked to a promoter.
  • the promoter expresses the heterologous nucleic acid in a cell of the CNS.
  • the promoter expresses the heterologous nucleic acid in a brain cell.
  • the promoter expresses the heterologous nucleic acid in a neuron and/or a glial cell.
  • the neuron is a medium spiny neuron of the caudate nucleus, a medium spiny neuron of the putamen, a neuron of the cortex layer IV and/or a neuron of the cortex layer V.
  • the glial cell is an astrocyte.
  • the promoter is a CBA promoter, a minimum CBA promoter, a CMV promoter or a GUSB promoter. In other embodiments, the promoter is inducible.
  • the rAAV vector comprises one or more of an enhancer, a splice donor/splice acceptor pair, a matrix attachment site, or a polyadenylation signal. In some embodiments, the rAAV vector is a self-complementary rAAV vector.
  • the vector comprises a first nucleic acid sequence encoding the heterologous nucleic acid and a second nucleic acid sequence encoding a complement of the heterologous nucleic acid, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length.
  • the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence.
  • rAAV vectors Numerous methods are known in the art for production of rAAV vectors, including transfection, stable cell line production, and infectious hybrid virus production systems which include adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71(11):8780-8789) and baculovirus-AAV hybrids (Urabe, M. et al., (2002) Human Gene Therapy 13(16):1935-1943; Kotin, R. (2011) Hum Mol Genet.20(R1): R2-R6).
  • rAAV production cultures for the production of rAAV virus particles all require; 1) suitable host cells, 2) suitable helper virus function, 3) AAV rep and cap genes and gene products; 4) a nucleic acid (such as a therapeutic nucleic acid) flanked by at least one AAV ITR sequences (e.g., an AAV sf-6501845 Attorney Docket No.:15979-20190.40 genome encoding a peptide of interest); and 5) suitable media and media components to support rAAV production.
  • the suitable host cell is a primate host cell.
  • the suitable host cell is a human-derived cell lines such as HeLa, A549, 293, or Perc.6 cells.
  • the suitable helper virus function is provided by wild-type or mutant adenovirus (such as temperature sensitive adenovirus), herpes virus (HSV), baculovirus, or a plasmid construct providing helper functions.
  • adenovirus such as temperature sensitive adenovirus
  • HSV herpes virus
  • baculovirus or a plasmid construct providing helper functions.
  • the AAV rep and cap gene products are from any AAV serotype.
  • the AAV rep gene product is of the same serotype as the ITRs of the rAAV vector genome as long as the rep gene products may function to replicated and package the rAAV genome.
  • suitable media known in the art are used for the production of rAAV vectors.
  • These media include, without limitation, media produced by Hyclone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), custom formulations such as those described in U.S. Patent No.6,566,118, and Sf-900 II SFM media as described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for use in production of recombinant AAV vectors.
  • the AAV helper functions are provided by adenovirus or HSV.
  • the AAV helper functions are provided by baculovirus and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cells).
  • the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cells).
  • one method for producing rAAV particles is the triple transfection method.
  • a plasmid containing a rep gene and a capsid gene, along with a helper adenoviral plasmid are transfected (e.g., using the calcium phosphate method) into a cell line (e.g., HEK-293 cells), and virus are collected and optionally purified.
  • the rAAV particle was produced by triple transfection of a nucleic acid encoding the rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions into a host cell, wherein the transfection of the nucleic acids to the host cells generates a host cell capable of producing rAAV particles.
  • rAAV particles are produced by a producer cell line method (see Martin et al., (2013) Human Gene Therapy Methods 24:253-269; U.S. PG Pub. No. US2004/0224411).
  • a cell line (e.g., a HeLa, 293, A549, or Perc.6 cell line) is stably transfected with a plasmid containing a rep gene, a capsid gene, and a vector genome comprising a promoter-heterologous nucleic acid sequence (e.g., a disorder-related sf-6501845 Attorney Docket No.:15979-20190.40 polypeptide).
  • cell lines are screened to select a lead clone for rAAV production, which may then be expanded to a production bioreactor and infected with a helper virus (e.g., an adenovirus or HSV) to initiate rAAV production.
  • a helper virus e.g., an adenovirus or HSV
  • viruses are subsequently be harvested, adenovirus are inactivated (e.g., by heat) and/or removed, and the rAAV particles are purified.
  • the rAAV particle is produced by a producer cell line comprising one or more of nucleic acid encoding the rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions.
  • the producer cell line method is advantageous for the production of rAAV particles with an oversized genome, as compared to the triple transfection method.
  • nucleic acid encoding AAV rep and cap genes and/or the rAAV genome are stably maintained in the producer cell line.
  • nucleic acid encoding AAV rep and cap genes and/or the rAAV genome is introduced on one or more plasmids into a cell line to generate a producer cell line.
  • the AAV rep, AAV cap, and rAAV genome are introduced into a cell on the same plasmid.
  • the AAV rep, AAV cap, and rAAV genome are introduced into a cell on different plasmids.
  • a cell line stably transfected with a plasmid maintains the plasmid for multiple passages of the cell line (e.g., 5, 10, 20, 30, 40, 50 or more than 50 passages of the cell).
  • the plasmid(s) may replicate as the cell replicates, or the plasmid(s) may integrate into the cell genome.
  • a variety of sequences that enable a plasmid to replicate autonomously in a cell have been identified (see, e.g., Krysan, P.J. et al. (1989) Mol. Cell Biol. 9:1026-1033).
  • the plasmid(s) may contain a selectable marker (e.g., an antibiotic resistance marker) that allows for selection of cells maintaining the plasmid.
  • selectable markers commonly used in mammalian cells include without limitation blasticidin, G418, hygromycin B, zeocin, puromycin, and derivatives thereof.
  • Methods for introducing nucleic acids into a cell include without limitation viral transduction, cationic transfection (e.g., using a cationic polymer such as DEAE- dextran or a cationic lipid such as lipofectamine), calcium phosphate transfection, microinjection, particle bombardment, electroporation, and nanoparticle transfection (for more details, see e.g., Kim, T.K. and Eberwine, J.H. (2010) Anal. Bioanal. Chem.397:3173-3178).
  • cationic transfection e.g., using a cationic polymer such as DEAE- dextran or a cationic lipid such as lipofectamine
  • calcium phosphate transfection e.g., using a cationic polymer such as DEAE- dextran or a cationic lipid such as lipofectamine
  • calcium phosphate transfection e.g., using a cationic polymer such as DEAE- dextran or
  • nucleic acid encoding AAV rep and cap genes and/or the rAAV genome are stably integrated into the genome of the producer cell line.
  • nucleic acid encoding AAV rep and cap genes and/or the rAAV genome is introduced on one or more plasmids into a cell line to generate a producer cell line.
  • the AAV rep, AAV cap, and rAAV genome are introduced into a cell on the same plasmid.
  • the AAV rep, AAV cap, and rAAV genome are introduced into a cell on different plasmids.
  • the plasmid(s) may contain a selectable marker (e.g., an antibiotic resistance marker) that allows for selection of cells maintaining the plasmid.
  • a selectable marker e.g., an antibiotic resistance marker
  • Methods for stable integration of nucleic acids into a variety of host cell lines are known in the art. For example, repeated selection (e.g., through use of a selectable marker) is used to select for cells that have integrated a nucleic acid containing a selectable marker (and AAV cap and rep genes and/or a rAAV genome).
  • nucleic acids are integrated in a site-specific manner into a cell line to generate a producer cell line.
  • the producer cell line is derived from a primate cell line (e.g., a non-human primate cell line, such as a Vero or FRhL-2 cell line).
  • the cell line is derived from a human cell line.
  • the producer cell line is derived from HeLa, 293, A549, or PERC.6® (Crucell) cells.
  • the cell line Prior to introduction and/or stable maintenance/integration of nucleic acid encoding AAV rep and cap genes and/or the oversized rAAV genome into a cell line to generate a producer cell line, the cell line is a HeLa, 293, A549, or PERC.6® (Crucell) cell line, or a derivative thereof.
  • the producer cell line is adapted for growth in suspension.
  • anchorage-dependent cells are typically not able to grow in suspension without a substrate, such as microcarrier beads.
  • Adapting a cell line to grow in suspension may include, for example, growing the cell line in a spinner culture with a stirring paddle, using a culture medium that lacks calcium and magnesium ions to prevent clumping (and optionally an antifoaming agent), using a culture vessel coated with a siliconizing compound, and selecting cells in the culture (rather than in large clumps or on the sides of the vessel) at each passage.
  • a method for producing any rAAV particle as disclosed herein comprising (a) culturing a host cell under a condition that rAAV particles are produced, wherein the host cell comprises (i) one or more AAV package genes, wherein each said AAV packaging gene encodes an AAV replication and/or encapsidation protein; (ii) a rAAV pro- vector comprising a nucleic acid encoding a heterologous nucleic acid as described herein flanked by at least one AAV ITR, and (iii) an AAV helper function; and (b) recovering the rAAV particles produced by the host cell.
  • said at least one AAV ITR is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, a goat AAV, bovine AAV, or mouse AAV serotype ITRs or the like.
  • the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10.
  • the nucleic acid in the AAV comprises an AAV2 ITR.
  • said encapsidation protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAVSAN006.
  • the encapsidation protein is an AAV8 capsid protein.
  • the rAAV particles comprise an AAV9 capsid and a recombinant genome comprising AAV2 ITRs, and nucleic acid encoding a therapeutic transgene/nucleic acid (e.g., an expression cassette for expressing a disorder-related polypeptide).
  • the rAAV particles comprise an AAV.SAN006 capsid and a recombinant genome comprising AAV2 ITRs, and nucleic acid encoding a therapeutic transgene/nucleic acid (e.g., an expression cassette for expressing a disorder-related polypeptide).
  • Suitable rAAV production culture media of the present disclosure is supplemented with serum or serum-derived recombinant proteins at a level of 0.5%-20% (v/v or w/v).
  • rAAV vectors are produced in serum-free conditions which may also be referred sf-6501845 Attorney Docket No.:15979-20190.40 to as media with no animal-derived products.
  • commercial or custom media designed to support production of rAAV vectors may also be supplemented with one or more cell culture components know in the art, including without limitation glucose, vitamins, amino acids, and or growth factors, in order to increase the titer of rAAV in production cultures.
  • rAAV production cultures can be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized.
  • rAAV production cultures include attachment- dependent cultures which can be cultured in suitable attachment-dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors.
  • rAAV vector production cultures may also include suspension-adapted host cells such as HeLa, 293, and SF-9 cells which can be cultured in a variety of ways including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system.
  • rAAV vector particles of the disclosure are harvested from rAAV production cultures by lysis of the host cells of the production culture or by harvest of the spent media from the production culture, provided the cells are cultured under conditions known in the art to cause release of rAAV particles into the media from intact cells, as described more fully in U.S. Patent No.6,566,118). Suitable methods of lysing cells are also known in the art and include for example multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and/or proteases. [0107] In a further embodiment, the rAAV particles are purified.
  • purified includes a preparation of rAAV particles devoid of at least some of the other components that may also be present where the rAAV particles naturally occur or are initially prepared from.
  • isolated rAAV particles are prepared using a purification technique to enrich it from a source mixture, such as a culture lysate or production culture supernatant.
  • Enrichment can be measured in a variety of ways, such as, for example, by the proportion of DNase-resistant particles (DRPs) or genome copies (gc) present in a solution, or by infectivity, or it can be measured in relation to a second, potentially interfering substance present in the source mixture, such as contaminants, including production culture contaminants or in- process contaminants, including helper virus, media components, and the like.
  • DNase-resistant particles DNase-resistant particles
  • gc genome copies
  • sf-6501845 Attorney Docket No.:15979-20190.40 [0108]
  • the rAAV production culture harvest is clarified to remove host cell debris.
  • the production culture harvest is clarified by filtration through a series of depth filters including, for example, a grade DOHC Millipore Millistak+ HC Pod Filter, a grade A1HC Millipore Millistak+ HC Pod Filter, and a 0.2 ⁇ m Filter Opticap XL1O Millipore Express SHC Hydrophilic Membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as, centrifugation or filtration through any cellulose acetate filter of 0.2 ⁇ m or greater pore size known in the art.
  • the rAAV production culture harvest is further treated with Benzonase® to digest any high molecular weight DNA present in the production culture.
  • the Benzonase® digestion is performed under standard conditions known in the art including, for example, a final concentration of 1-2.5 units/ml of Benzonase® at a temperature ranging from ambient to 37°C for a period of 30 minutes to several hours.
  • rAAV particles are isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anionic exchange filtration; tangential flow filtration (TFF) for concentrating the rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anionic exchange chromatography, cationic exchange chromatography, or affinity chromatography.
  • steps are used alone, in various combinations, or in different orders.
  • the method comprises all the steps in the order as described below.
  • Certain aspects of the present disclosure relate to methods of treating various neurodegenerative diseases in an individual in need thereof.
  • the disclosure provides methods of treating a neurodegenerative disease by administering an effective amount of a viral particle of the disclosure for expressing a disorder-related polypeptide or RNAi molecule.
  • the viral particles are capable of expressing a sf-6501845 Attorney Docket No.:15979-20190.40 polypeptide associated with a neurodegenerative disorder.
  • the viral particles are capable of expressing an RNAi molecule capable of reducing or eliminating the expression of a particular polypeptide implicated in a neurodegenerative disorder as set forth herein.
  • the RNAi molecule is an artificial miRNA.
  • the RNAi molecule is a small interfering RNA (siRNA).
  • the RNAi molecule is a short hairpin RNA (shRNA).
  • the administration of the viral particle includes direct administration into the cerebrospinal fluid (CSF).
  • the viral particle is administered via direct injection into the spinal cord, via intrathecal injection, or via intracisternal injection. In some embodiments, the viral particle is administered to more than one location of the spinal cord or cisterna magna via ICM injection. In some embodiments, the viral particle is administered to the cisterna magna. In some embodiments, the viral particle is administered by intracerebroventricular injection (also referred to as intraventricular injection). [0113] In some embodiments, an effective amount or therapeutically effective amount of viral particles are administered, depending on the objectives of treatment. For example, where a low percentage of transduction can achieve the desired therapeutic effect, then the objective of treatment is generally to meet or exceed this level of transduction.
  • this level of transduction can be achieved by transduction of only about 1 to 5% of the target cells of the desired tissue type, in some embodiments at least about 20% of the cells of the desired tissue type, in some embodiments at least about 50%, in some embodiments at least about 80%, in some embodiments at least about 95%, in some embodiments at least about 99% of the cells of the desired tissue type.
  • the viral particle composition is administered by one or more administrations, either during the same procedure or spaced apart by days, weeks, months, or years.
  • the disclosure provides a method for treating a human with a neurodegenerative disorder by administering an effective amount of a pharmaceutical sf-6501845 Attorney Docket No.:15979-20190.40 composition comprising a recombinant viral particle encoding a disorder-related polypeptide or RNAi molecule.
  • the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.
  • the methods comprise administering an effective amount of a pharmaceutical composition comprising a recombinant viral vector encoding a disorder-related polypeptide of the present disclosure to treat a neurodegenerative disorder in an individual in need thereof.
  • the viral titer of the viral particles e.g., rAAV particles
  • the viral titer of the viral particles is at least about any of 5 ⁇ 10 12 , 6 ⁇ 10 12 , 7 ⁇ 10 12 , 8 ⁇ 10 12 , 9 ⁇ 10 12 , 10 ⁇ 10 12 , 11 ⁇ 10 12 , 15 ⁇ 10 12 , 20 ⁇ 10 12 , 25 ⁇ 10 12 , 30 ⁇ 10 12 , or 50 ⁇ 10 12 genome copies/mL.
  • the viral titer of the viral particles is about any of 5 ⁇ 10 12 to 6 ⁇ 10 12 , 6 ⁇ 10 12 to 7 ⁇ 10 12 , 7 ⁇ 10 12 to 8 ⁇ 10 12 , 8 ⁇ 10 12 to 9 ⁇ 10 12 , 9 ⁇ 10 12 to 10 ⁇ 10 12 , 10 ⁇ 10 12 to 11 ⁇ 10 12 , 11 ⁇ 10 12 to 15 ⁇ 10 12 , 15 ⁇ 10 12 to 20 ⁇ 10 12 , 20 ⁇ 10 12 to 25 ⁇ 10 12 , 25 ⁇ 10 12 to 30 ⁇ 10 12 , 30 ⁇ 10 12 to 50 ⁇ 10 12 , or 50 ⁇ 10 12 to 100 ⁇ 10 12 genome copies/mL.
  • the viral titer of the viral particles is about any of 5 ⁇ 10 12 to 10 ⁇ 10 12 , 10 ⁇ 10 12 to 25 ⁇ 10 12 , or 25 ⁇ 10 12 to 50 ⁇ 10 12 genome copies/mL.
  • the viral titer of the viral particles is at least about any of 5 ⁇ 10 9 , 6 ⁇ 10 9 , 7 ⁇ 10 9 , 8 ⁇ 10 9 , 9 ⁇ 10 9 , 10 ⁇ 10 9 , 11 ⁇ 10 9 , 15 ⁇ 10 9 , 20 ⁇ 10 9 , 25 ⁇ 10 9 , 30 ⁇ 10 9 , or 50 ⁇ 10 9 transducing units /mL.
  • the viral titer of the viral particles is about any of 5 ⁇ 10 9 to 6 ⁇ 10 9 , 6 ⁇ 10 9 to 7 ⁇ 10 9 , 7 ⁇ 10 9 to 8 ⁇ 10 9 , 8 ⁇ 10 9 to 9 ⁇ 10 9 , 9 ⁇ 10 9 to 10 ⁇ 10 9 , 10 ⁇ 10 9 to 11 ⁇ 10 9 , 11 ⁇ 10 9 to 15 ⁇ 10 9 , 15 ⁇ 10 9 to 20 ⁇ 10 9 , 20 ⁇ 10 9 to 25 ⁇ 10 9 , 25 ⁇ 10 9 to 30 ⁇ 10 9 , 30 ⁇ 10 9 to 50 ⁇ 10 9 or 50 ⁇ 10 9 to 100 ⁇ 10 9 transducing units /mL.
  • the viral titer of the viral particles is about any of 5 ⁇ 10 9 to 10 ⁇ 10 9 , 10 ⁇ 10 9 to 15 ⁇ 10 9 , 15 ⁇ 10 9 to 25 ⁇ 10 9 , or 25 ⁇ 10 9 to 50 ⁇ 10 9 transducing units /mL.
  • the viral titer of the viral particles is at least any of about 5 ⁇ 10 10 , 6 ⁇ 10 10 , 7 ⁇ 10 10 , 8 ⁇ 10 10 , 9 ⁇ 10 10 , 10 ⁇ 10 10 , 11 ⁇ 10 10 , 15 ⁇ 10 10 , 20 ⁇ 10 10 , 25 ⁇ 10 10 , 30 ⁇ 10 10 , 40 ⁇ 10 10 , or 50 ⁇ 10 10 infectious units/mL.
  • the viral titer of the viral particles is at least any of about 5 ⁇ 10 10 to 6 ⁇ 10 10 , 6 ⁇ 10 10 to 7 ⁇ 10 10 , 7 ⁇ 10 10 to 8 ⁇ 10 10 , 8 ⁇ 10 10 to 9 ⁇ 10 10 , 9 ⁇ 10 10 to 10 ⁇ 10 10 , 10 ⁇ 10 10 to 11 ⁇ 10 10 , 11 ⁇ 10 10 to 15 ⁇ 10 10 , 15 ⁇ 10 10 to 20 ⁇ 10 10 , 20 ⁇ 10 10 to 25 ⁇ 10 10 , 25 ⁇ 10 10 to 30 ⁇ 10 10 , 30 ⁇ 10 10 to 40 ⁇ 10 10 , 40 ⁇ 10 10 to 50 ⁇ 10 10 , or 50 ⁇ 10 10 to 100 ⁇ 10 10 infectious units/mL.
  • the viral titer of the viral particles is at sf-6501845 Attorney Docket No.:15979-20190.40 least any of about 5 ⁇ 10 10 to 10 ⁇ 10 10 , 10 ⁇ 10 10 to 15 ⁇ 10 10 , 15 ⁇ 10 10 to 25 ⁇ 10 10 , or 25 ⁇ 10 10 to 50 ⁇ 10 10 infectious units/mL.
  • the viral particles are rAAV particles.
  • the rAAV particles comprise an AAV.SAN006 capsid protein.
  • the dose of viral particles administered to the individual is at least about any of 1 ⁇ 10 8 to about 6 ⁇ 10 13 genome copies/kg of body weight. In some embodiments, the dose of viral particles administered to the individual is about any of 1 ⁇ 10 8 to about 6 ⁇ 10 13 genome copies/kg of body weight.
  • the dose of viral particles administered to the individual is about any of 1 ⁇ 10 10 , 2 ⁇ 10 10 , 3 ⁇ 10 10 , 4 ⁇ 10 10 , 5 ⁇ 10 10 , 6 ⁇ 10 10 , 7 ⁇ 10 10 , 8 ⁇ 10 10 , 9 ⁇ 10 10 , 1 ⁇ 10 11 , 2 ⁇ 10 11 , 3 ⁇ 10 11 , 4 ⁇ 10 11 , 5 ⁇ 10 11 , 6 ⁇ 10 11 , 7 ⁇ 10 11 , 8 ⁇ 10 11 , 9 ⁇ 10 11 , 1 ⁇ 10 12 , 2 ⁇ 10 12 , 13 ⁇ 10 12 , 4 ⁇ 10 12 , 5 ⁇ 10 12 , 6 ⁇ 10 12 , 7 ⁇ 10 12 , 8 ⁇ 10 12 , 9 ⁇ 10 12 , or 1 ⁇ 10 13 genome copies/kg of body weight.
  • the dose of viral particles administered to the individual is about any of 0.5 x 10 10 , 1.5 x 10 10 , 2.5 x 10 10 , 3.5 x 10 10 , 4.5 x 10 10 , 5.5 x 10 10 , 6.5 x 10 10 , 7.5 x 10 10 , 8.5 x 10 10 , 9.5 x 10 10 , 10.5 x 10 10 , 11.5 x 10 10 , or 12.5 x 10 10 genome copies/kg of body weight.
  • the total amount of viral particles administered to the individual is at least about any of 1 ⁇ 10 9 to about 1 ⁇ 10 14 genome copies.
  • the total amount of viral particles administered to the individual is about any of 1 ⁇ 10 9 to about 1 ⁇ 10 14 genome copies. In some embodiments, the total amount of viral particles administered to the individual is about any of 1 ⁇ 10 11 , 2 ⁇ 10 11 , 3 ⁇ 10 11 , 4 ⁇ 10 11 , 5 ⁇ 10 11 , 6 ⁇ 10 11 , 7 ⁇ 10 11 , 8 ⁇ 10 11 , 9 ⁇ 10 11 , 1 ⁇ 10 12 , 2 ⁇ 10 12 , 3 ⁇ 10 12 , 4 ⁇ 10 12 , 5 ⁇ 10 12 , 6 ⁇ 10 12 , 7 ⁇ 10 12 , 8 ⁇ 10 12 , 9 ⁇ 10 12 , 1 ⁇ 10 13 , 2 ⁇ 10 13 , 13 ⁇ 10 13 , 4 ⁇ 10 13 , 5 ⁇ 10 13 , 6 ⁇ 10 13 , 7 ⁇ 10 13 , 8 ⁇ 10 13 , 9 ⁇ 10 13 , or 1 ⁇ 10 14 genome copies.
  • compositions of the disclosure can be used either alone or in combination with one or more additional therapeutic agents for treating a neurodegenerative disorder.
  • the interval between sequential administration can be in terms of at least (or, alternatively, less than) minutes, hours, or days.
  • a viral particle composition of the present disclosure is used for administration to a human. In some embodiments, a viral particle composition of the present disclosure is used for pediatric administration.
  • an effective amount of sf-6501845 Attorney Docket No.:15979-20190.40 viral particles is administered to a patient that is less than one month, less than two months, less than three months, less than four months, less than five months, less than six months, less than seven months, less than eight months, less than nine months, less than ten months, less than eleven months, less than one year, less than 13 months, less than 14 months, less than 15 months, less than 16 months, less than 17 months, less than 18 months, less than 19 months, less than 20 months, less than 21 months, less than 22 months, less than two years, less than three years old, less than five years old or less than seven years old.
  • a rAAV composition of the present disclosure is used for administration to a young adult.
  • an effective amount of viral particles is administered to a patient that is less than 12 years old, less than 13 years old, less than 14 years old, less than 15 years old, less than 16 years old, less than 17 years old, less than 18 years old, less than 19 years old, less than 20 years old, less than 21 years old, less than 22 years old, less than 23 years old, less than 24 years old, or less than 25 years old.
  • the invention provides rAAV vectors for use in methods of preventing or treating one or more gene defects (e.g., heritable gene defects, somatic gene alterations, and the like) in a mammal, such as for example, a gene defect that results in a polypeptide deficiency or polypeptide excess in a subject, or for treating or reducing the severity or extent of deficiency in a subject manifesting a CNS-associated disorder linked to a deficiency in such polypeptides in cells and tissues.
  • gene defects e.g., heritable gene defects, somatic gene alterations, and the like
  • methods involve administration of a rAAV vector that encodes one or more therapeutic peptides, polypeptides, functional RNAs, inhibitory nucleic acids, shRNAs, microRNAs, antisense nucleotides, etc. in a pharmaceutically- acceptable carrier to the subject in an amount and for a period of time sufficient to treat the CNS- associated disorder in the subject having or suspected of having such a disorder.
  • a rAAV vector may comprise as a transgene, a nucleic acid encoding a protein or functional RNA that modulates or treats a CNS-associated disorder.
  • neuronal apoptosis inhibitory protein NAIP
  • nerve growth factor NEF
  • GDNF glial-derived growth factor
  • BDNF brain-derived growth factor
  • CNTF ciliary neurotrophic factor
  • TM tyrosine hydroxlase
  • GTPCH GTP- cyclohydrolase
  • ASPA aspartoacylase
  • SOD1 superoxide dismutase
  • AADC amino acid decarboxylase
  • a transgene encoding GTPCII which generates the TII cofactor tetrahydrobiopterin, may also be used in the treatment of Parkinson's disease.
  • a transgene encoding GDNF or BDNF, or AADC, which facilitates conversion of L-Dopa to DA, may also be used for the treatment of Parkinson's disease.
  • a useful transgene may encode: GDNF, BDNF or CNTF.
  • a useful transgene may encode a functional RNA, e.g., shRNA, miRNA, that inhibits the expression of SOD1.
  • a useful transgene may encode NAIP or NGF.
  • a transgene encoding Beta-glucuronidase is useful for the treatment of certain lysosomal storage diseases (e.g., Mucopolysacharidosis type VII (MPS VII)).
  • a transgene encoding a prodrug activation gene e.g., HSV- Thymidine kinase which converts ganciclovir to a toxic nucleotide which disrupts DNA synthesis and leads to cell death, is useful for treating certain cancers, e.g., when administered in combination with the prodrug.
  • a transgene encoding an endogenous opioid such a ⁇ -endorphin is useful for treating pain.
  • the heterologous nucleic acid may encode a therapeutic nucleic acid.
  • a therapeutic nucleic acid may include without limitation an siRNA, an shRNA, an RNAi, an miRNA, an antisense RNA, a ribozyme or a DNAzyme.
  • a therapeutic nucleic acid may encode an RNA that when transcribed from the nucleic acids of the vector can treat a disorder of the invention (e.g., a disorder of the CNS) by interfering with translation or transcription of an abnormal or excess protein associated with a disorder of the invention.
  • a disorder of the invention e.g., a disorder of the CNS
  • the nucleic acids of the invention may encode for an RNA which treats a disorder by highly specific elimination or reduction of mRNA encoding the abnormal and/or excess proteins.
  • RNA sequences include RNAi, small inhibitory RNA (siRNA), micro RNA (miRNA), and/or ribozymes (such as hammerhead and hairpin ribozymes) that can treat disorders by highly specific elimination or reduction of mRNA encoding the abnormal and/or excess proteins.
  • the heterologous nucleic acid may encode a therapeutic polypeptide.
  • a therapeutic polypeptide may, e.g., supply a polypeptide and/or enzymatic activity that is absent or present at a reduced level in a cell or organism.
  • a sf-6501845 Attorney Docket No.:15979-20190.40 therapeutic polypeptide may supply a polypeptide and/or enzymatic activity that indirectly counteracts an imbalance in a cell or organism.
  • a therapeutic polypeptide for a disorder related to buildup of a metabolite caused by a deficiency in a metabolic enzyme or activity may supply a missing metabolic enzyme or activity, or it may supply an alternate metabolic enzyme or activity that leads to reduction of the metabolite.
  • a therapeutic polypeptide may also be used to reduce the activity of a polypeptide (e.g., one that is overexpressed, activated by a gain-of-function mutation, or whose activity is otherwise misregulated) by acting, e.g., as a dominant-negative polypeptide.
  • the therapeutic polypeptide or therapeutic nucleic acid is used to treat a disorder of the CNS.
  • a therapeutic polypeptide or therapeutic nucleic acid may be used to reduce or eliminate the expression and/or activity of a polypeptide whose gain-of-function has been associated with a disorder, or to enhance the expression and/or activity of a polypeptide to complement a deficiency that has been associated with a disorder (e.g., a mutation in a gene whose expression shows similar or related activity).
  • Non-limiting examples of CNS disorders of the invention that may be treated by a therapeutic polypeptide or therapeutic nucleic acid of the invention (exemplary genes that may be targeted or supplied are provided in parenthesis for each disorder) comprise stroke (e.g., caspase-3, Beclin1, Ask1, PAR1, HIF1 ⁇ , PUMA, and/or any of the genes described in Fukuda, A.M. and Badaut, J. (2013) Genes (Basel) 4:435-456), Huntington’s disease (mutant HTT), epilepsy (e.g., SCN1A, NMDAR, ADK, and/or any of the genes described in Boison, D.
  • stroke e.g., caspase-3, Beclin1, Ask1, PAR1, HIF1 ⁇ , PUMA, and/or any of the genes described in Fukuda, A.M. and Badaut, J. (2013) Genes (Basel) 4:435-456
  • Huntington’s disease mutant HTT
  • epilepsy e
  • Parkinson’s disease alpha-synuclein
  • Lou Gehrig’s disease also known as amyotrophic lateral sclerosis; SOD1
  • Alzheimer’s disease tau, amyloid precursor protein
  • SOD1 amyotrophic lateral sclerosis
  • AD amyotrophic lateral sclerosis
  • Alzheimer’s disease tau, amyloid precursor protein
  • disorders of the invention may include those that involve large areas of the cortex, e.g., more than one functional area of the cortex, more than one lobe of the cortex, and/or the entire cortex.
  • Other non-limiting examples of disorders of the invention that may be treated by a therapeutic polypeptide or therapeutic nucleic acid of the invention comprise traumatic brain injury, enzymatic dysfunction disorders, psychiatric disorders (including post-traumatic stress sf-6501845 Attorney Docket No.:15979-20190.40 syndrome), neurodegenerative diseases, and cognitive disorders (including dementias, autism, and depression).
  • Enzymatic dysfunction disorders include without limitation leukodystrophies (including Canavan’s disease) and any of the lysosomal storage diseases described below.
  • the therapeutic polypeptide or therapeutic nucleic acid is used to treat a lysosomal storage disease.
  • lysosomal storage disease are rare, inherited metabolic disorders characterized by defects in lysosomal function. Such disorders are often caused by a deficiency in an enzyme required for proper mucopolysaccharide, glycoprotein, and/or lipid metabolism, leading to a pathological accumulation of lysosomally stored cellular materials.
  • Non-limiting examples of lysosomal storage diseases of the invention that may be treated by a therapeutic polypeptide or therapeutic nucleic acid of the invention (exemplary genes that may be targeted or supplied are provided in parenthesis for each disorder) include Gaucher disease type 2 or type 3 (acid beta-glucosidase, GBA), GM1 gangliosidosis (beta-galactosidase-1, GLB1), Hunter disease (iduronate 2-sulfatase, IDS), Krabbe disease (galactosylceramidase, GALC), a mannosidosis disease (a mannosidase, such as alpha-D- mannosidase, MAN2B1), ⁇ mannosidosis disease (beta-mannosidase, MANBA), metachromatic leukodystrophy disease (pseudoarylsulfatase A, ARSA), mucolipidosisII/III disease (N- acetylglucosamine-1
  • the therapeutic polypeptide is caspase-3, Beclin1, Ask1, PAR1, HIF1 ⁇ , PUMA,SCN1A, NMDAR, ADK, alpha-synuclein, SOD1, acid beta- glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase (MAN2B1), beta- mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1- phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick C protein sf-6501845 Attorney Docket No.:15979-20190.40 (NPC1), acid alpha-1,4-glucosidase
  • the therapeutic polypeptide may increase or decrease the function of the target polypeptide in the subject (e.g., it may supply the missing function in a lysosomal storage disease, or reduce the level of alpha-synuclein in MSA, such as by blocking its function or dysfunction).
  • the therapeutic nucleic acid is caspase-3, Beclin1, Ask1, PAR1, HIF1 ⁇ , PUMA,SCN1A, NMDAR, ADK, alpha-synuclein, SOD1, acid beta-glucosidase (GBA), beta- galactosidase-1 (GLB1), iduronate 2-sulfatase (IDS), galactosylceramidase (GALC), a mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), Niemann-Pick C protein (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sulfo
  • the therapeutic nucleic acid may increase or decrease the function of the target polypeptide in the subject (e.g., it may supply the missing function in a lysosomal storage disease or reduce the level of alpha-synuclein in MSA, such as by RNAi).
  • an exemplary disease for which AAV expression in the cortex and striatum is useful is Huntington’s disease (HD).
  • Huntington’s disease is caused by a CAG repeat expansion mutation that encodes an elongated polyglutamine (polyQ) repeat in the mutant huntingtin protein (mHTT).
  • HD is a particularly attractive target for DNA- and RNA-based therapies as it is an autosomal dominant disease resulting from mutation on a single allele.
  • AAV vectors provide an ideal delivery system for nucleic acid therapeutics and allow for long lasting and continuous expression of these huntingtin lowering molecules in the brain.
  • delivery to both the striatum and cortex will likely be required.
  • Postmortem analysis of HD patient brains revealed extensive medium spiny neuronal loss in the sf-6501845 Attorney Docket No.:15979-20190.40 striatum, in addition to loss of pyramidal neurons in the cerebral cortex and hippocampus.
  • Parkinson’s Disease PD
  • LBD lewy body dementias
  • MSA multiple system atrophy
  • PD and its associated disorders are related to dysfunction of any of several disease associated genes, including, but not limited to: ATP13A2, PLA2G6, VPS35, DJ1, GBA1, SNCA, PINK1, PARKIN or LRRK2.
  • ATP13A2 gene encodes a member of the P5 subfamily of ATPases which transports inorganic cations as well as other substrates. Mutations in this gene are associated with Kufor-Rakeb syndrome (KRS), also referred to as Parkinson disease 9.
  • KRS is a very rare form of inherited juvenile-onset Parkinson's disease.
  • the SNCA gene expresses Synucleins (e.g., small, soluble proteins) in the brain. Defects in the SNCA gene have been implicated in the pathogenesis of Parkinson disease.
  • Certain aspects of the present disclosure relate to methods of treating Parkinson’s disease (PD) and associated disorders in an individual in need thereof.
  • the disclosure provides methods of treating PD and/or associated disorders by administering an effective amount of viral particles as disclosed herein.
  • the disclosure provides methods of treating PD and/or associated disorders administering an effective amount of viral particles comprising expression cassette for expressing a disorder-related polypeptide of the present disclosure.
  • the disorder-related polypeptide is a wild type disorder-related polypeptide.
  • the method for treating PD and/or associated disorders may comprise delivering a viral particle expressing a transgene for modulating the function or levels of at least one target.
  • the method may comprise delivering an expression cassette expressing a transgene for modulating the function or levels of at least one target intracellularly or extracellularly.
  • the method may comprise delivering a miRNA (e.g., artificial miRNA) targeting SNCA to lower expression.
  • the method may comprise intracellular delivery of a viral particle comprising an artificial miRNA configured to target SNCA and lower expression.
  • the method may comprise extracellular delivery of a viral particle comprising an expression cassette configured for expression of an antibody fragment for secretion and binding to a target of interest either intra- or extracellularly.
  • expression of the disorder-related polypeptide is under control of a promoter.
  • the expression cassette is under the control of a promoter.
  • the transgene is under the control of a promoter.
  • the promoter may comprise a ubiquitous promoter or a neuron-specific promoter.
  • the ubiquitous promoter may comprise CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • the neuron-specific promoter may comprise SYN1, NSE, CAMKII, or TUBB3.
  • the promoter e.g., promoter activity
  • the expression cassette may comprise at least one transgene.
  • at least one transgene e.g., gene
  • transgene expression is regulated by an exogenous small molecule.
  • the expression cassette may comprise at least one additional nucleotide sequence.
  • At least one additional nucleotide sequence is configured to improve RNA stability. In some embodiments, at least one additional nucleotide sequence configured to improve RNA stability may comprise WPRE. In some embodiments, at least one additional nucleotide sequence is configured to detarget expression from liver and/or DRG. sf-6501845 Attorney Docket No.:15979-20190.40 Methods for treating Alzheimer’s Disease and associated disorders [0136] Another common neurodegenerative disorder is Alzheimer’s Disease (AD). In fact, AD and associated disorders comprise the most common form of dementia. Two main neuropathological lesions of AD are amyloid plaques and neurofibrillary tangles (NFTs).
  • AD Alzheimer’s Disease
  • NFTs neurofibrillary tangles
  • AD and CAA are characterized by intracellular aggregation of hyperphosphorylated tau protein with extracellular aggregation of amyloid beta (A ⁇ ) plaques.
  • the associated disorders including PSP, CBD, FTDP-17, PiD, GGT, and AGD are characterized by intracellular aggregation of hyperphosphorylated tau protein without extracellular aggregation of amyloid beta (A ⁇ ) plaques.
  • These pathological protein aggregates can cause synaptic dysfunction, neuroinflammation, and neurodegeneration in cerebral cortex, cerebellum, basal ganglia, midbrain, and brainstem.
  • AD Alzheimer’s Disease
  • the disclosure provides methods of treating AD and/or associated disorders by administering an effective amount of a viral particle of the disclosure.
  • the disclosure provides methods of treating AD and/or associated disorders by administering an effective amount of a viral particle for expressing a disorder-related polypeptide or RNAi molecule.
  • the method comprises delivering a viral particle comprising am expression cassette configured to target tau pathology in AD and/or associated disorders.
  • the method comprises delivering a viral particle encoding an artificial sf-6501845 Attorney Docket No.:15979-20190.40 miRNA.
  • the method comprises delivering a viral particle encoding an antisense oligonucleotide (ASO).
  • the method may comprise delivering a viral particle comprising an expression cassette configured to target MAPT or BIN1 mRNA.
  • the method may comprise delivering a viral particle comprising an expression cassette configured to target a vectorized antibody targeting tau or Bin1 protein.
  • the disorder-related polypeptide comprises the vectorized antibody targeting tau. In some embodiments, the disorder-related polypeptide comprises the vectorized antibody targeting the Bin1 protein.
  • the expression cassette configured to target tau pathology in AD and/or associated disorders is under control of a promoter.
  • the promoter may comprise a ubiquitous or neuron-specific promoter. In some embodiments, the ubiquitous promoter may comprise CBA, CAG, CAGG, CMV, H1, U6 or 7SK. In some embodiments, the neuron-specific promoter may comprise SYN1, NSE, CAMKII, or TUBB3. In some instances, the promoter is regulated by an exogenous small molecule.
  • the method may comprise delivering a viral particle comprising an expression cassette configured to target A ⁇ pathology in AD and/or associated disorders.
  • the method comprises delivering a viral particle comprising an expression cassette (e.g., AAV genome) encoding an artificial miRNA.
  • the method comprises delivering a viral particle comprising an expression cassette (e.g., AAV genome) encoding an antisense oligonucleotide (ASO).
  • the method comprises delivering a viral particle comprising an expression cassette configured to target APP or PS1 gene.
  • the method comprises delivering a viral particle comprising an expression cassette configured to target a vectorized antibody targeting A ⁇ .
  • the expression cassette configured to target A ⁇ pathology in AD and/or associated disorders is under control of a promoter.
  • the promoter may comprise a ubiquitous or neuron-specific promoter.
  • the ubiquitous promoter may comprise CBA, CAG, CAGG, CMV, H1, U6 or 7SK.
  • the neuron-specific promoter may comprise SYN1, NSE, CAMKII, or TUBB3. In some instances, promoter is regulated by an exogenous small molecule.
  • the method for treating AD and/or associated disorders may comprise delivering an expression cassette (e.g., AAV genome) encoding an ASO targeting APOE4 and/or encoding the protective allele APOE2.
  • the method for treating AD and/or associated disorders is configured to target innate immune pathways contributing to neuroinflammation.
  • the method may comprise delivering an expression cassette (e.g., AAV genome) encoding an artificial miRNA or vectorized antibody targeting CD33 or encoding the soluble form of TREM2.
  • the expression cassette is under the control of a promoter.
  • AAV-encoded therapeutics are expressed under control of ubiquitous or neuron-specific promoters.
  • the ubiquitous promoter comprises CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • the neuron- specific promoter comprises SYN1, NSE, CAMKII, or TUBB3.
  • the viral particle encodes at least one nucleotide sequence. In some embodiments, at least one nucleotide sequence is configured to improve RNA stability.
  • At least one of the nucleotide sequences may comprise WPRE. In some embodiments, at least one nucleotide sequence is configured to target expression from liver and/or DRG.
  • the expression cassette may comprise at least one transgene. In some embodiments, at least one transgene may comprise MAPT, BIN1, APP, PS1, APOE, CD33, TREM2, or a combination thereof. In some instances, the transgene expression is regulated by an exogenous small molecule.
  • Metachromatic leukodystrophy is an autosomal recessive neurodegenerative disorder caused by mutations in the enzyme arylsulfatase A (ARSA).
  • ARSA arylsulfatase A
  • Reduced levels of ARSA activity result in toxic accumulation of sulfatide characterized by degeneration of myelin- sf-6501845 Attorney Docket No.:15979-20190.40 forming cells (oligodendrocytes and Schwann cells) in the central and peripheral nervous system. This results in demyelination, dysfunction, degeneration of neurons and neuroinflammation (astrocytosis, microglial activation).
  • Certain aspects of the present disclosure relate to methods of treating MLD and/or increasing levels of an ARSA polypeptide in an individual in need thereof.
  • the disclosure provides methods of treating MLD by administering an effective amount of a viral particle of the disclosure for expressing an ARSA polypeptide.
  • the ARSA polypeptide is a wild type ARSA polypeptide.
  • the method may provide for treating a human with MLD by administering an effective amount of a pharmaceutical composition comprising a recombinant viral vector encoding an ARSA polypeptide of the present disclosure.
  • the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.
  • Methods for treating Adrenoleukodystrophy and Adrenomyeloneuropathy [0150] Certain aspects of the present disclosure relate to methods of treating Adrenoleukodystrophy (ALD) and Adrenomyeloneuropathy (ALM) in an individual in need thereof.
  • the disclosure provides methods of treating ALD and/or ALM by administering an effective amount of a viral particle of the disclosure.
  • the disclosure provides methods of treating a ALD and/or ALM by administering an effective amount of a viral particle comprising an expression cassette for expressing a disorder-related polypeptide or RNAi molecule.
  • the disorder-related polypeptide may comprise human ABCD1 polypeptide.
  • the expression cassette of the viral particle may comprise a transgene for encoding human ABCD1 polypeptide. sf-6501845 Attorney Docket No.:15979-20190.40 [0151]
  • expression of the disorder-related polypeptide is under control of a promoter.
  • the promoter may comprise a ubiquitous, neuron-specific, or astrocyte-specific promoter.
  • the expression cassette of the viral particle may comprise at least one additional nucleotide sequence.
  • the at least one additional nucleotide sequence is for improving RNA stability.
  • the at least one additional nucleotide sequence is for improving RNA stability may comprise WPRE.
  • the at least one additional nucleotide sequence is for detargeting expression from liver and/or DRG.
  • the expression cassette comprising the transgene encoding for the ABCD1 polypeptide is combined with an artificial miRNA.
  • two viral particle can be delivered to the patient, one encoding the ABCD1 polypeptide and the other encoding the artificial miRNA.
  • the artificial miRNA is configured for targeting ELOVL1. In some embodiments, the artificial miRNA is configured for targeting ELOVL1 to reduce expression of ELOVL1.
  • promoter activity and/or transgene expression is/are regulated by an exogenous small molecule.
  • the expression cassette e.g., expression cassette delivered in a rAAV particle
  • for expressing a disorder-related polypeptide is administered through various routes as provided in the present disclosure.
  • ALS and FTD together are a heterogeneous group of disorders characterized by degeneration of the cerebral cortex and/or the spinal cord. This degeneration is related to dysfunction of one of several disease-causing genes, including, but not limited to: SOD1, TARDBP, C9Orf72, FUS, GRN, VCP, TBK1, RIPK1, MAPT, NEK1, SQSTM1, CHCHD10. Studies involving these genes has led to the identification sf-6501845 Attorney Docket No.:15979-20190.40 of neurodegeneration pathways such as autophagy, RNA regulation, and vesicle and inclusion formation.
  • Certain aspects of the present disclosure relate to methods of treating Amyotrophic Lateral Sclerosis (ALS) and/or Frontotemporal Dementia (FTD) in an individual in need thereof.
  • the method of treating ALS and/or FTD comprises administering an effective amount of a viral particle of the disclosure.
  • the expression cassette of the viral particle may comprise a transgene.
  • the expression cassette is configured for encoding an artificial miRNA.
  • the expression cassette is configured for encoding a vectorized antibody.
  • the vectorized antibody is configured to target genes comprising SOD1, TARDBP, C9orf72 repeat expansion, FUS, RIPK1, or MAPT genes.
  • the expression cassette is under control of a promoter.
  • the promoter may comprise a ubiquitous or neuron-specific promoter.
  • the ubiquitous promoter may comprise CBA, CAG, CAGG, CMV, H1, U6 or 7SK.
  • the neuron-specific promoter may comprise SYN1, NSE, CAMKII, or TUBB3.
  • promoter is regulated by an exogenous small molecule.
  • the method may comprise regulating transgene expression by an exogenous small molecule.
  • expression cassettes e.g., AAV genomes
  • the expression cassette (e.g., AAV genome, genome, etc.) may encode artificial miRNA.
  • the artificial miRNA is configured to target endogenous, affected gene, as well as nucleotide sequences to improve RNA stability.
  • the artificial miRNA e.g., at least one additional nucleotide sequence
  • WPRE e.g., at least one additional nucleotide sequence
  • at least one additional nucleotide sequence is configured to detarget expression from liver and/or DRG.
  • NP is characterized by dysfunction of dorsal root ganglia, spinal cord, and/or cerebral cortex. Often, NP is caused by nerve injury or trauma related to dysfunction of at least one of several genes, including, but not limited to: ADORA2A, CACNA1B, CACNA2D1, CACNA2D3, CACNA1H, CALCA, CALCB, GABBR1, GABBR2, GABRA1, GABRA2, GABRG1, GABRB3, GABRG2, GAD1, GAD2, HCN1, HCN2, HCN4, KCNA1, KCNA2, KCNB1, P2RX7, SCN9A, SCN10A, SCN11A, SLC12A2, SLC12A5, TRPA1, TRPV1.
  • Certain aspects of the present disclosure relate to methods of treating Neuropathic Pain (NP) in an individual in need thereof.
  • the method of treating MP may comprise administering an effective amount of a viral particle of the disclosure.
  • the expression cassette may comprise a transgene.
  • the transgene (e.g., gene) may comprise ADORA2A, CACNA1B, CACNA2D1, CACNA2D3, CACNA1H, CALCA, CALCB, GABBR1, GABBR2, GABRA1, GABRA2, GABRG1, GABRB3, GABRG2, GAD1, GAD2, HCN1, HCN2, HCN4, KCNA1, KCNA2, KCNB1, P2RX7, SCN9A, SCN10A, SCN11A, SLC12A2, SLC12A5, TRPA1, or TRPV1.
  • the expression cassette of the viral particle is configured for encoding an artificial miRNA.
  • the rAAV particle may comprise an artificial RNA.
  • the expression cassette of the viral particle is configured for encoding a target polypeptide.
  • the expression cassette of the viral particle is configured for encoding a target polypeptide (e.g., disorder-related polypeptide).
  • the expression cassette of the viral particle is configured for encoding the disorder-related polypeptide, wherein the disorder-related polypeptide may comprise ADORA2A, CACNA1B, CACNA2D1, CACNA2D3, CACNA1H, CALCA, CALCB, GABBR1, GABBR2, GABRA1, GABRA2, GABRG1, GABRB3, GABRG2, GAD1, GAD2, HCN1, HCN2, HCN4, KCNA1, KCNA2, KCNB1, P2RX7, SCN9A, SCN10A, SCN11A, SLC12A2, SLC12A5, TRPA1, TRPV1, or any combination thereof.
  • the disorder-related polypeptide may comprise ADORA2A, CACNA1B, CACNA2D1, CACNA2D3, CACNA1H, CALCA, CALCB, GABBR1, GABBR2, GABRA1, GABRA2, GABRG1, GABRB3, GABRG2, GAD1, GAD2, HCN1, HCN2, H
  • the expression cassette of the viral particle may comprise at least one transgene for encoding at least one disorder-related polypeptide.
  • the expression cassette may comprise at least one transgene for encoding at least one polypeptide, wherein the at least one polypeptide may comprise ADORA2A, CACNA1B, CACNA2D1, CACNA2D3, CACNA1H, CALCA, CALCB, GABBR1, GABBR2, GABRA1, GABRA2, GABRG1, GABRB3, GABRG2, GAD1, GAD2, HCN1, HCN2, HCN4, KCNA1, KCNA2, KCNB1, P2RX7, SCN9A, SCN10A, SCN11A, SLC12A2, SLC12A5, TRPA1, TRPV1, or any combination thereof.
  • the method may comprise delivering a viral particle encoding at least one artificial miRNA targeting ADORA2A, CACNA1B, CACNA2D1, CACNA2D3, CACNA1H, CALCA, CALCB, GABBR1, GABBR2, GABRA1, GABRA2, GABRG1, GABRB3, GABRG2, GAD1, GAD2, HCN1, HCN2, HCN4, KCNA1, KCNA2, KCNB1, P2RX7, SCN9A, SCN10A, SCN11A, SLC12A2, SLC12A5, TRPA1, TRPV1 or any combination thereof.
  • the expression cassette is under control of a promoter.
  • the promoter may comprise a ubiquitous or neuron-specific promoter.
  • the ubiquitous promoter may comprise CBA, CAG, CAGG, CMV, H1, U6 or 7SK.
  • the neuron-specific promoter may comprise SYN1, NSE, CAMKII, or TUBB3.
  • promoter is regulated by an exogenous small molecule.
  • the method may comprise regulating transgene expression by an exogenous small molecule.
  • the expression cassette may also encode artificial miRNA.
  • the artificial miRNA is configured to target endogenous, affected gene, as well as nucleotide sequences to improve RNA stability.
  • the nucleotide sequences to improve RNA stability may comprise WPRE.
  • the expression cassette e.g., the nucleotide sequences
  • the expression cassette is configured to detarget expression from liver and/or DRG.
  • sf-6501845 Attorney Docket No.:15979-20190.40 Methods for treating Charcot-Marie-Tooth Diseases
  • Charcot-Marie-Tooth Diseases is a neurodegenerative disorder that causes abnormalities in both sensory and motor nerves related to feet, legs, hands, and arms.
  • CMT is a heterogenous group of peripheral neuropathies (e.g., affects nerves outside of the brain and spinal cord) affecting dorsal root ganglia and peripheral nerves.
  • CMT Charcot-Marie- Tooth Diseases
  • the method of treating CMT may comprise administering an effective amount of a viral particle of the disclosure.
  • the expression cassette of the viral particle may comprise a transgene.
  • the transgene may comprise PMP22, GJB1, MFN2, TRPV4, NEFH, NEFL, DNM2, or MPZ.
  • the method for treating CMT may comprise delivering a viral particle comprising expression cassette encoding artificial miRNA.
  • the artificial miRNA is configured to target PMP22 or TRPV4.
  • the expression cassette is configured to ‘encode genes comprising GJB1 or MFN1’.
  • the expression cassette is configured to encode both an artificial miRNA targeting MFN2, NEFH, NEFL, DNM2, or MPZ along with targeted genes comprising GJB1 or MFN1.
  • the expression cassette is under control of a ubiquitous promoter or a neuron-specific promoter.
  • the ubiquitous promotor may comprise CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • the neuron- specific promoter may comprise SYN1, NSE, CAMKII, or TUBB3.
  • the promoter may comprise a Schwann cell-specific promoter.
  • the Schwann cell-specific promoter may comprise MBP, MPZ, or PMP22.
  • promoter activity is regulated by an exogeneous small molecule.
  • transgene expression is regulated by an exogenous small molecule.
  • the expression cassette of the viral particle may comprise at least one additional nucleotide sequence.
  • At least one additional nucleotide sf-6501845 Attorney Docket No.:15979-20190.40 sequence is configured to improve RNA stability.
  • at least nucleotide sequence configured to improve RNA stability may comprise WPRE.
  • at least one additional nucleotide sequence may comprise a nucleotide sequence configured to detarget expression from the liver.
  • FD Fabry Disease
  • the method of treating FD may comprise administering an effective amount of a viral particle of the disclosure.
  • the expression cassette may comprise a transgene.
  • the transgene may comprise human GLA.
  • the expression cassette of the viral particle is under control of a ubiquitous promoter or a neuron-specific promoter.
  • the ubiquitous promotor may comprise CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • the Schwann cell-specific promoter may comprise MBP, MPZ, or PMP22.
  • promoter activity is regulated by an exogeneous small molecule.
  • the transgene expression is regulated by an exogeneous small molecule.
  • the human GLA gene is modified to include cell penetrating and/or signal peptides to enhance secretion and uptake.
  • the expression cassette of the viral particle may comprise at least one additional nucleotide sequence.
  • At least one additional nucleotide sequence is configured to improve RNA stability.
  • at least nucleotide sequence configured to improve RNA stability may comprise WPRE.
  • the sf-6501845 Attorney Docket No.:15979-20190.40 at least one additional nucleotide sequence is configured to detarget expression from liver and/or DRG.
  • UBE3A encodes an E3 ubiquitin-protein ligase, which is part of the ubiquitin protein degradation system and is maternally expressed in the brain and spinal cord (central nervous system). AS is caused by materially inherited deletions of UBE3A.
  • AS Angelman Syndrome
  • the method of treating AS may comprise administering an effective amount of a viral particle of the disclosure.
  • the expression cassette may comprise a transgene.
  • the transgene may comprise UBE3A.
  • the viral particle comprises an expression cassette encoding human UBE3A may also comprise an artificial miRNA.
  • the artificial miRNA is configured to target UBE3A-ATS.
  • the expression cassette is under control of a promoter.
  • the expression cassette is under control of a ubiquitous promoter or a neuron-specific promoter.
  • the ubiquitous promotor may comprise CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • the neuron-specific promoter may comprise SYN1, NSE, CAMKII, or TUBB3.
  • promoter activity is regulated by an exogeneous small molecule.
  • the method for treating AS may comprise expressing a transgene regulated by an exogeneous small molecule.
  • the expression cassette may comprise at least one additional nucleotide sequence.
  • the at least one additional nucleotide sequence is configured to improve RNA stability.
  • the at least nucleotide sequence sf-6501845 Attorney Docket No.:15979-20190.40 configured to improve RNA stability may comprise WPRE.
  • the at least one additional nucleotide sequence is configured to detarget expression from liver and/or DRG.
  • the disclosure provides methods of treating Fragile X Syndrome (FXS) by administering an effective amount of a viral particle of the disclosure for expressing/encoding a disorder-related polypeptide of the present disclosure.
  • the disorder-related polypeptide may comprise human FMRP.
  • the expression cassette is under control of a promoter.
  • the expression cassette is under control of a ubiquitous promoter or a neuron- specific promoter.
  • the ubiquitous promotor may comprise CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • the neuron-specific promoter may comprise SYN1, NSE, CAMKII, or TUBB3. In some embodiments, promoter activity is regulated by an exogeneous small molecule.
  • the expression cassette may comprise a transgene. In some embodiments, the transgene may comprise FMR1. In some embodiments, the transgene expression is regulated by an exogenous small molecule.
  • the expression cassette for expressing a disorder-related polypeptide is administered through various routes as provided herein.
  • the expression cassette may comprise at least one additional nucleotide sequence. In some embodiments, the at least one additional nucleotide sequence is configured to improve RNA stability.
  • the at least nucleotide sequence configured to improve RNA stability may comprise WPRE.
  • the at least one additional nucleotide sequence is configured to detarget expression from liver and/or DRG.
  • sf-6501845 Attorney Docket No.:15979-20190.40 Methods for treating Rett Syndrome
  • RS Rett Syndrome
  • the most common causes of RS are mutated variants of the MECP2 gene which affect the cerebral cortex. These gene mutations alter the structure of the MeCP2 protein or reduce the amount produced. The reduction of functional MeCP2 can impair the regulation of gene expression in brain cells and can disrupt alternative splicing of proteins that are important for communications between neurons.
  • the disclosure provides methods of treating Rett Syndrome (RS) by administering an effective amount of a viral particle of the disclosure for expressing/encoding a disorder-related polypeptide of the present disclosure.
  • the disorder-related polypeptide is a wild type disorder-related polypeptide.
  • the disorder-related polypeptide may comprise methyl CpG binding protein 2 (MECP2).
  • the expression cassette is under control of a promoter.
  • the expression cassette is under control of a ubiquitous promoter or a neuron- specific promoter.
  • the ubiquitous promotor may comprise CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • the neuron-specific promoter may comprise SYN1, NSE, CAMKII, or TUBB3. In some embodiments, promoter activity is regulated by an exogeneous small molecule.
  • the expression cassette may comprise a transgene. In some embodiments, the transgene may comprise the transgene MECP2. In some embodiments, the transgene expression is regulated by an exogenous small molecule.
  • the expression cassette for expressing a disorder-related polypeptide is administered through various routes as provided herein.
  • the expression cassette may comprise at least one additional nucleotide sequence. In some embodiments, at least one additional nucleotide sequence is configured to improve RNA stability.
  • the disclosure provides methods of treating Tuberous Sclerosis Complex (TSC) by administering an effective amount of a viral particle of the disclosure for expressing/encoding a disorder-related polypeptide of the present disclosure.
  • TSC Tuberous Sclerosis Complex
  • the disorder-related polypeptide is a wild type disorder-related polypeptide.
  • the disorder-related polypeptide may comprise hamartin, tuberin, or a combination of both.
  • the expression cassette is under control of a promoter.
  • the expression cassette is under control of a ubiquitous promoter or a neuron- specific promoter.
  • the ubiquitous promotor may comprise CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • promoter activity is regulated by an exogeneous small molecule.
  • the expression cassette may comprise at least one transgene.
  • the at least one transgene may comprise the transgene TSC1, TSC2, or a combination of both.
  • the transgenic payload may comprise all or part of human TSC1 and/or TSC2 human cDNA. In some embodiments, the transgenic payload may comprise all or part of human TSC1 and/or TSC2 human cDNA that may include additional flexible linker sequence. In some embodiments, the transgene expression is regulated by an exogenous small molecule. sf-6501845 Attorney Docket No.:15979-20190.40 [0203] In some instances, the expression cassette is modified to include cell penetrating and/or signal peptides to enable secretion and uptake. [0204] In some instances, the genetic payload may comprise at least one additional nucleotide sequence.
  • the at least one additional nucleotide sequence may comprise a nucleotide sequence to facilitate nuclear export. In some embodiments, the at least one additional nucleotide sequence may comprise a nucleotide sequence to improve RNA stability. In some embodiments, the nucleotide sequence to improve RNA stability may comprise a polyA tail. In some embodiments, the nucleotide sequence to improve RNA stability may comprise WPRE. In some embodiments, at least one additional nucleotide sequence is configured to detarget expression from liver and/or DRG. [0205] In some instances, the expression cassette may comprise at least one tagging nucleotide sequence.
  • At least one tagging nucleotide sequence may comprise FLAG, His, Myc or any combination thereof.
  • the viral particle for expressing a disorder-related polypeptide is administered through various routes as provided herein.
  • AAV administration is either intra-CSF (intrathecal, intra-cisterna magna, or intraventricular) or intravenous.
  • Methods for treating Neurofibromatosis type II [0207] Neurofibromatosis type II (NF2) is a disorder characterized by nervous system and skin tumors and ocular abnormalities. Disruption in the function of the protein encoded by the NF2 gene has been implicated in tumorigenesis and metastasis.
  • the disclosure provides methods of treating Neurofibromatosis type II (NF2) by administering an effective amount of a viral particle of the disclosure for expressing a disorder-related polypeptide.
  • the disorder-related polypeptide may comprise a polypeptide expressed by the gene NF2.
  • the expression cassette of the viral particle is under control of a ubiquitous promoter, a neuron-specific promoter, or a Shwann cell-specific promoter.
  • the ubiquitous promotor may comprise CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • the neuron-specific promoter may comprise SYN1, NSE, CAMKII, or TUBB3.
  • the Schwann cell-specific promoter may comprise MBP, MPZ, or PMP22.
  • promoter activity is regulated by an exogeneous small molecule.
  • the expression cassette may comprise at least one transgene. In some instances, transgene expression is regulated by an exogenous small molecule.
  • the transgene may comprise human NF2. In some embodiments, the transgenic payload may comprise all or part of the human NF2 gene, an additional flexible linker sequence, or a combination of both.
  • the expression cassette (e.g., AAV genome, genetic payload, etc.) is modified to include cell penetrating and/or signal peptides to enable secretion and uptake.
  • the expression cassette may comprise at least one additional nucleotide sequence.
  • at least one additional nucleotide sequence may comprise a nucleotide sequence configured to detarget expression from liver and/or DRG.
  • the expression cassette may comprise at least one tagging nucleotide sequence.
  • at least one tagging nucleotide sequence may comprise FLAG, His, Myc or any combination thereof.
  • the viral particle for expressing a disorder-related polypeptide is administered through various routes as provided herein.
  • AAV administration is either intra-CSF (intrathecal, intra-cisterna magna, or intraventricular) or intravenous.
  • Methods for treating Pompe [0215] Pompe is a neuromuscular disease with a caused by autosomal recessive mutations in the acid alpha-glucosidase (GAA) gene. The GAA gene encodes lysosomal alpha-glucosidase, which is essential for the degradation of glycogen to glucose in lysosomes.
  • the disclosure provides methods of treating Pompe by administering an effective amount of a viral particle of the disclosure for expressing/encoding a disorder-related polypeptide of the present disclosure.
  • the disorder-related polypeptide comprises the lysosomal enzyme acid alpha-glucosidase (GAA).
  • the expression cassette is configured for encoding the human GAA polypeptide.
  • the expression cassette is under control of a promoter.
  • the promoter may comprise a ubiquitous promoter, or a muscle-specific promoter.
  • the ubiquitous promotor may comprise CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • the muscle-specific promoter may comprise Spc5-12, Desmin, or ⁇ MyHC.
  • promoter activity is regulated by an exogeneous small molecule.
  • the expression cassette may comprise at least one transgene.
  • at least one transgene may comprise the human GAA gene. In some instances, transgene expression is regulated by an exogenous small molecule.
  • the human GAA gene is modified to comprise cell penetrating and/or signal peptides. In some instances, the human GAA gene may comprise cell penetrating and/or signal peptides to enable secretion and uptake.
  • the expression cassette of the viral particle may comprise at least one additional nucleotide sequence. In some embodiments, at least one additional nucleotide sequence is configured to improve RNA stability. In some embodiments, at least one additional nucleotide sequence configured to improve RNA stability may comprise WPRE. In some embodiments, the at least one additional nucleotide sequence configured to detarget expression from liver and/or DRG.
  • the expression cassette for expressing a disorder-related polypeptide is administered through various routes as provided herein.
  • the method for treating Pompe may comprise delivering the viral particle via IV and/or intra-CSF routes to target both the skeletal muscle and CNS components of the disease.
  • sf-6501845 Attorney Docket No.:15979-20190.40 Methods for treating Globoid cell Leukodystrophy
  • Globoid cell leukodystrophy (GLD; Krabbe disease) an autosomal recessive neurodegenerative disorder caused by a defect in the GALC gene.
  • the GALC gene encodes for the lysosomal enzyme, galactosylceramidase.
  • the disclosure provides methods of treating Globoid cell leukodystrophy (GLD) by administering an effective amount of a viral particle of the disclosure for expressing/encoding a disorder-related polypeptide of the present disclosure.
  • the disorder-related polypeptide comprises the lysosomal enzyme, galactosylceramidase.
  • the method for treating GLD is configured to prevent buildup of cytotoxic galactosylsphingosine, a GALC substrate, which drives death of oligodendrocytes and Schwann cells, leading to widespread myelin loss.
  • the expression cassette of the viral particle is under control of a ubiquitous promoter or a neuron-specific promoter.
  • the ubiquitous promotor may comprise CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • the neuron-specific promoter may comprise SYN1, NSE, CAMKII, TUBB3.
  • the promoter may comprise a Schwann cell-specific promoter.
  • the Schwann cell-specific promoter may comprise MBP, MPZ, or PMP22. In some embodiments, promoter activity is regulated by an exogeneous small molecule.
  • the expression cassette may comprise at least one transgene. In some embodiments, at least one transgene may comprise the human GALC gene. In some instances, transgene expression is regulated by an exogenous small molecule.
  • the human GALC gene is modified to comprise cell penetrating and/or signal peptides to enable secretion and uptake.
  • the expression cassette may comprise at least one additional nucleotide sequence.
  • At least one additional nucleotide sequence is configured to improve RNA stability.
  • at least one additional nucleotide sf-6501845 Attorney Docket No.:15979-20190.40 sequence may comprise WPRE.
  • at least one additional nucleotide sequence is configured to detarget expression from liver and/or DRG.
  • the viral particle is administered through various routes as provided herein.
  • the method for treating GLD may comprise delivering an expression cassette (e.g., AAV genome) via IV and/or intra-CSF routes to target both the PNS and CNS components of the disease.
  • Globoid cell leukodystrophy (GLD; Krabbe disease) an autosomal recessive neurodegenerative disorder caused by a defect in the GALC gene.
  • the GALC gene encodes for the lysosomal enzyme, galactosylceramidase. Without the enzyme, buildup of cytotoxic galactosylsphingosine, a GALC substrate, drives death of oligodendrocytes and Schwann cells, leading to widespread myelin loss.
  • the disclosure provides methods of treating Multiple Sclerosis (MS) by administering an effective amount of a viral particle of the disclosure for expressing/encoding a disorder-related polypeptide.
  • the disorder-related polypeptide comprises a vectorized antibody against CD40L.
  • the expression cassette of the viral particle is under control of a promoter.
  • the promoter comprises a ubiquitous promoter or a neuron- specific promoter.
  • the ubiquitous promotor may comprise CBA, CAG, CAGG, CMV, H1, U6, or 7SK.
  • the neuron-specific promoter may comprise SYN1, NSE, CAMKII, or TUBB3.
  • promoter activity is regulated by an exogeneous small molecule.
  • the expression cassette may comprise at least one transgene.
  • the at least one transgene may encode for a vectorized antibody against CD40L.
  • transgene expression is regulated by an exogenous small molecule.
  • the vectorized antibody is modified to comprise cell penetrating and/or signal peptides to enable secretion and uptake.
  • sf-6501845 Attorney Docket No.:15979-20190.40
  • the expression cassette may comprise at least one additional nucleotide sequence.
  • the expression cassette (e.g., AAV genome) may comprise at least one additional nucleotide sequence configured to improve RNA stability.
  • at least one additional nucleotide sequence configured to improve RNA stability may comprise WPRE.
  • At least one additional nucleotide sequence configured to de-target expression from liver and/or DRG is administered through various routes as provided herein.
  • Methods for treating Gaucher Disease Gaucher Disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in GBA1, the gene encoding glucocerebrosidase (GCase). Reduction or loss of GCase activity leads to the accumulation of toxic lipid substrates, disrupting cellular homeostasis.
  • GD is further GD Type 1 (GD1) patients typically present with splenomegaly, hepatomegaly, and anemia or thrombocytopenia, while GD Type 3 (GD3) patients present with debilitating neurological symptoms and associated systemic manifestations of GD1.
  • GD Type 2 (GD2) is the most severe form of the disease affecting infants before 6 months of age and in most cases causing early death by 2-3 years.
  • GD2 also has CNS symptoms at a much higher severity.
  • Current treatment landscape for GD includes enzyme replacement therapy for GD1 patients that alleviate symptoms but since ERTs fail to enter the brain, they do not help GD2 and GD3 patients that have CNS symptoms.
  • the disclosure provides methods of treating Gaucher Disease (GD) by administering an effective amount of a viral particle of the disclosure for expressing/encoding glucocerebrosidase (GCase).
  • GCase e.g., GCase peptide
  • the method may comprise restoring the loss of GCase activity .
  • intra-CSF dosing for GD2 and GD3; IV dosing for GD1 of the same AAV gene therapy product would be ideal.
  • GBA-PD Parkinson’s disease
  • PD Parkinson’s disease
  • GBA1 is an extremely well-credentialed target in GBA-PD.
  • Current treatment options include Levodopa and/or dopamine agonists for symptom management but most patients notice the effects of these drugs wearing off over time.
  • the disclosure provides methods of treating GBA-PD by administering an effective amount of an expression cassette (e.g., an expression cassette delivered in a rAAV particle) for expressing/encoding GBA1 polypeptide.
  • the method for treating GBA-PD may comprise a one-time administration of AAV-GBA1 delivered via intra-CSF administration to specifically target the CNS.
  • Methods for treating Huntington’s Disease (HD) [0240] HD is caused by a combination of hereditary and somatic expansion of trinucleotide repeats in the gene HTT that drives HD pathology in cerebral cortex, caudate, and putamen.
  • the viral particles of the disclosure can be used for the treatment of HD.
  • treatment of HD involves delivery of an AAV genome encoding artificial microRNA, antisense oligonucleotide, linearized antibody, or nanobody targeting the HTT gene and/or MSH2, MSH3, PMS1, PMS2, FAN1, RRM2B, and MLH3, either alone or in combination, under control of ubiquitous (CBA, CAG, CAGG, CMV, H1, U6, 7SK, or others to be determined) or neuron-specific (SYN1, NSE, CAMKII, TUBB3, or others to be determined) promoters.
  • CBA ubiquitous
  • CAGG CMV
  • H1, U6, 7SK or others to be determined
  • SYN1, NSE, CAMKII, TUBB3, or others to be determined neuron-specific promoters.
  • promoter activity and/or transgene expression is/are regulated by an exogenous small molecule.
  • treatment may encode biological machinery, including DNA binding protein, effector protein, and/or nucleotide guide molecule, required for editing to remove repeat expansions from the HTT gene.
  • sf-6501845 Attorney Docket No.:15979-20190.40 VII. Kits or Articles of Manufacture
  • the expression cassettes e.g., an expression cassette for expressing a disorder-related polypeptide, such as a wild type human disorder-related polypeptide
  • rAAV vectors, particles, and/or pharmaceutical compositions as described herein is/are contained within a kit or article of manufacture, e.g., designed for use in one of the methods of the disclosure as described herein.
  • the system comprises a cannula, one or more syringes (e.g., 1, 2, 3, 4 or more), and one or more fluids (e.g., 1, 2, 3, 4 or more) suitable for use in the methods of the disclosure.
  • the syringe is any suitable syringe, provided it is capable of being connected to the cannula for delivery of a fluid.
  • the system has one syringe.
  • the system has two syringes.
  • the system has three syringes.
  • the system has four or more syringes.
  • the fluids suitable for use in the methods of the disclosure include those described herein, for example, one or more fluids each comprising an effective amount of one or more vectors as described herein, and one or more fluids comprising one or more therapeutic agents.
  • the kit comprises a single fluid (e.g., a pharmaceutically acceptable fluid comprising an effective amount of the vector).
  • the kit comprises 2 fluids.
  • the kit comprises 3 fluids.
  • the kit comprises 4 or more fluids.
  • a fluid may include a diluent, buffer, excipient, or any other liquid described herein or known in the art suitable for delivering, diluting, stabilizing, buffering, or otherwise transporting an expression cassette for expressing a disorder-related polypeptide or rAAV vector composition of the present disclosure.
  • the kit comprises one or more buffers, e.g., an aqueous pH buffered solution. Examples of buffers may include without limitation phosphate, citrate, Tris, HEPES, and other organic acid buffers.
  • the kit comprises a container. Suitable containers may include, e.g., vials, bags, syringes, and bottles.
  • the container is made of one or more of a material such as glass, metal, or plastic. In some embodiments, the container is used to hold a rAAV composition of the present disclosure. In some embodiments, the container may also hold a fluid and/or other therapeutic agent. sf-6501845 Attorney Docket No.:15979-20190.40 [0247]
  • the kit comprises an additional therapeutic agent with a rAAV composition of the present disclosure. In some embodiments, the rAAV composition and the additional therapeutic agent are mixed. In some embodiments, the rAAV composition and the additional therapeutic agent are kept separate. In some embodiments, the rAAV composition and the additional therapeutic agent are in the same container.
  • the rAAV composition and the additional therapeutic agent are in different containers. In some embodiments, the rAAV composition and the additional therapeutic agent are administered simultaneously. In some embodiments, the rAAV composition and the additional therapeutic agent are administered on the same day. In some embodiments, the rAAV composition is administered within one day, two days, three days, four days, five days, six days, seven days, two weeks, three weeks, four weeks, two months, three months, four months, five months, or six months of administration of the additional therapeutic agent. [0248] In some embodiments, the kit comprises a therapeutic agent to transiently suppress the immune system prior to AAV administration.
  • kits further provide cyclosporine, mycophenolate mofetil, and/or methylprednisolone.
  • the rAAV particles and/or compositions of the disclosure may further be packaged into kits including instructions for use.
  • the kits further comprise a device for delivery (e.g., any type of parenteral administration described herein) of compositions of rAAV particles.
  • the instructions for use include instructions according to one of the methods described herein.
  • the instructions are printed on a label provided with (e.g., affixed to) a container.
  • the instructions for use include instructions for administering to an individual (e.g., a human) an effective amount of rAAV particles, e.g., for treating a neurodegenerative disease in an individual.
  • Example 1 Focused ultrasound increases gene delivery to deep brain structure following the administration of a recombinant adeno-associated virus in the cerebrospinal fluid
  • Male Sprague Dawley rats (average 250 g) were ordered from Charles River either pre-cannulated in the cisterna magna or without cannula.
  • the cannula was flushed to keep it open upon arrival with artificial cerebrospinal fluid (Bio-techne/Tocris, cat no 3525) in the volume indicated by the vendor as the void volume of the cannula.
  • the experiments were conducted within 3 days of arrival of the animals to avoid clotting of the cannula prior to use. Animals were kept in a 12/12 light/dark cycle with food and water ad libitum and a temperature of 18° C - 22° C and humidity of 40%-60%. Animal work was performed according to the Canadian Council on Animals Care Policies & Guidelines and approved by the Sunnybrook Research Institute Animal Care Committee.
  • Adeno-associated virus [0252] AAV2-HBKO encoding GFP under a CAG promoter was produced as previously described using polyethyleneimine transfection of HEK293T cells (J. A. Sullivan, et al., Rationally designed AAV2 and AAVrh8R capsids provide improved transduction in the retina and brain. Gene Therapy 25, 205–219 (2016); J. Naidoo, et al., Extensive Transduction and Enhanced Spread of a Modified AAV2 Capsid in the Non-human Primate CNS. Molecular Therapy 26, 1–13 (2018)).
  • Gadolinium Gadodiamide MRI contrast agent, Omniscan, GE Healthcare Canada, Mississauga, ON, Canada
  • ICM injection of gadolinium was injected in the ICM cannula followed by flushing with artificial cerebrospinal fluid corresponding to the dead volume in the cannula.
  • gadolinium was injected in a volume of 45 ⁇ L.
  • MR images were acquired using a 7.0 T MRI instrument (BioSpin 7030; Bruker; Billerica, USA) at 45, 55, 70 and 80 minutes following injection.
  • FUS-MB treatment [0254] For a detailed description of the FUS-MB treatment please see Z. Noroozian, et al., MRI-guided focused ultrasound for targeted delivery of rAAV to the brain. Methods Mol Biol. 1950, 177–197 (2019). Animals were anaesthetized with isoflurane and a tail-vein catheter was inserted followed by hair removal on the animal head to avoid trapping of air bubbles in the ultrasound gel. The animals were placed supine on an MR-compatible sled and T2-weighted MR images were acquired using a 7.0 T MRI instrument (BioSpin 7030; Bruker; Billerica, USA) and used for MR-guided targeting of the FUS.
  • FUS was applied using a 0.58 MHz spherically focused transducer (75 mm outer diameter, 26 mm inner diameter, 60 mm radius of curvature) and an in-house manufactured system (prototype for LP100; FUS Instruments, Toronto, Canada).
  • DEFINITY® microbubbles (0.2 mL/kg) were injected immediately upon FUS application at a fixed pressure of 0.32 MPa.
  • AAV2-HBKO the injection was performed immediately after injection of microbubbles followed by intravenous administration of 0.2 mL/kg gadolinium to visualize BBB permeability on T1-weighted MR images.
  • Tissue collection [0255] Four weeks following AAV delivery, animals were deeply anaesthetized using 75 mg/kg ketamine and 10 mg/kg xylazine followed by transcardial perfusion with 0.9% saline. Brains were collected; one hemisphere was post-fixed for 16 h in 4% paraformaldehyde in 0.1 M PO4, followed by transfer to 30% sucrose, and the other hemisphere was dissected in brain sf-6501845 Attorney Docket No.:15979-20190.40 regions and flash frozen on dry ice. Organs and spines were collected and post-fixed for 16 h in 4% paraformaldehyde in 0.1 M PO4 followed by transfer to PBS.
  • blocking buffer PBS with 0.3% Triton X-100, 3% (w/v) bovine serum albumin, and 10% (v/v) donkey serum.
  • Sections were incubated with primary antibodies in blocking buffer overnight followed by washing in PBS and incubation overnight with secondary antibodies in blocking buffer. Staining with DAPI was performed by incubation for 10 minutes in PBS followed by washing in PBS, mounting on glass slides with polyvinyl alcohol medium and DABCO (Millipore, cat no 10981), and covering with glass coverslips.
  • Primary antibodies include chicken anti-GFP 1:1000 (Abcam, ab13970) and guineapig anti-NeuN 1:500 (Millipore, ABN90). Secondary antibodies were purchased from Jacksom ImmunoResearch and diluted 1:400. [0257] Whole section images were acquired with a 10x objective using a Zeiss Axio Scan.Z1 slide scanner.
  • RNA extraction and qPCR [0258] Brain tissue was homogenized in 1 mL Trizol (Thermo Fisher, cat no 15596018) using a bead homogenizer followed by addition of 200 ⁇ L chloroform and 15 minutes centrifugation at 12,000xg. The supernatant was mixed 1:1 with 70% ethanol and RNA was extracted using PureLink RNA Mini Kit (Thermo Fisher cat no 12183018A) according to manufacturer’s protocol. RNA concentrations were measured using a Nanodrop 2000 (Thermo Fisher).
  • RNA was used for cDNA preparation using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, cat no 4368814) according to manufacturer’s protocol.
  • cDNA was diluted 1:50 and 5 ⁇ L was added to each well of a 385-well plate together with 1 ⁇ L of each sf-6501845 Attorney Docket No.:15979-20190.40 reverse and forward primers (from a 10 ⁇ M dilution), and 7 ⁇ L SYBR Green qPCR master mix (Thermo Fisher, cat no 4472908).
  • GFP forward primer 5'- ACTACAACAGCCACAACGTCTATATCA-3′ SEQ ID NO: 13
  • GFP reverse primer 5’ GGCGGATCTTGAAGTTCACC-3′
  • Hprt1 forward primer 5’-TCCTCAGACCGCTTTTCCCGC-3’ SEQ ID NO: 15
  • Hprt1 reverse primer 5’- TCATCATCACTAATCACGACGCTGG-3’ SEQ ID NO: 16
  • Pgk1 forward primer 5’- ATGCAAAGACTGGCCAAGCTAC-3’ SEQ ID NO: 17
  • Pgk1 reverse primer 5’- AGCCACAGCCTCAGCATATTTC-3’ SEQ ID NO: 18
  • DNA extraction and quantification of GFP genome copies [0259] DNA was extracted from organs using QIAamp DNA FFPE Tissue Kit (Qiagen, cat no 56404) and from blood samples using DNeasy Blood and Tissue Kit (Qiagen, cat no 69504) according to manufacturer’s protocol except for the final elution step being done with a low TE buffer instead of the elution buffer provided in the kit.
  • GFP primers were 50-ACT ACA GCC ACA ACG TCT ATA TCA-30 (SEQ ID NO: 19) and reverse primer 50-GGCGGATCTTGAAGTTCACC-30 (SEQ ID NO: 20) (Invitrogen) and the probe was 50-6- FAM-CCG ACA AGC-ZENAGA AGA ACG GCA TCA-Iowa Black FQ-30 (Integrated DNA Technologies, Coralville, IA, USA).
  • Rpp30 reference gene primers and probe were obtained as a ddPCR Copy Number Assay (Bio-Rad, Part number 10042961, Assay ID dRnoCNS421683336).
  • Spine analysis [0260] Rat spines were excised by disconnecting the ribs and surrounding tissue. Subsequently, the spines were post-fixed overnight in a solution of 4% paraformaldehyde in 0.1 M PO4. The spine underwent a 15-min wash in PBS and was then incubated for approximately sf-6501845 Attorney Docket No.:15979-20190.40 30 days in 10% EDTA in PBS at 37° C, pH 7.5 and on a rotatory shaker.
  • the EDTA solution was changed twice daily, and the bone was regularly inspected until it reached the necessary softness for sectioning. After another 15-min PBS wash, the spine was post-fixed in 4% paraformaldehyde for 60 minutes at room temperature, followed by 15-min PBS wash. Spines were incubated in progressively increasing concentrations of sucrose in 0.1 M PO4, starting from 10% and incrementally reaching 20% and 30% sucrose daily, all at 4° C, shaking.
  • the spine sections were equilibrated to room temperature and washed 3 times 10 minutes in PBS, followed by incubation for 2 hours in blocking solution (PBS with 0.3% Triton X-100, 3% w/v bovine serum albumin, and 10% v/v donkey serum) in a humidify chamber. Sections were incubated overnight with primary antibodies in blocking solution at 4° C followed by three 10-min washes in PBS at room temperature. Subsequently, sections were incubated overnight at 4° C with secondary antibodies in blocking solution, followed by incubation in PBS with DAPI (1:10,000) (Sigma, D9542) for 10 min.
  • blocking solution PBS with 0.3% Triton X-100, 3% w/v bovine serum albumin, and 10% v/v donkey serum
  • Tissues were washed twice for 10 minutes in PBS at room temperature and once for 10 minutes in 0.1 M PO 4 before adding polyvinyl alcohol medium and DABCO (Millipore, 10,981), and covered with a glass coverslip. All solutions were directly applied to the mounted tissue. To prevent solutions from sliding off the slides a hydrophobic pen was utilized to outline the tissue's edges, ensuring the localization of reagents on tissue specimens.
  • Statistics [0261] Normal distribution of qPCR data was determined using Shapiro-Wilk test. Non- transformed data from all brain regions was not normality distributed.
  • MRI magnetic resonance imaging
  • gadolinium gadolinium
  • ICM magnetic resonance imaging
  • MR imaging 45, 55, 70 and 80 minutes post administration was performed.
  • Gadolinium enhancement white was clearly seen in brain areas proximal to the subarachnoid space containing cerebrospinal fluid (e.g., cortical areas, hippocampal formation, cerebellum, and ventral part of midbrain and brainstem) (FIGS.2B-E).
  • Gadolinium enhancement increased with time in brain structures further away from the subarachnoid space, such as the thalamus, when going from 45 minutes to 80 minutes post administration (gadolinium enhancement (white) increased in FIG.2E-e1 compared to FIG.2B-b1). This suggests a gradual diffusion of the ICM-injected gadolinium into the brain parenchyma, perhaps via the perivascular space. The striatum remained without significant gadolinium enhancement at the time points investigated and it was chosen for FUS-targeting as a brain region with poor access to ICM-injected substances in this rat model.
  • gadolinium Because the diffusion of gadolinium into the brain parenchyma continued from 45 minutes to 80 minutes post-injection and AAV is larger ( ⁇ 26 nm and >3,500 kDa) than gadolinium ( ⁇ 0.5 kDa), 120 minutes was chosen as a possible timepoint when ICM- injected AAV could be present in the perivascular space. It remained possible that small amounts of gadolinium in the perivascular space may not have been visible on the MR images. To avoid the potential of losing a significant amount of AAV from the perivascular space into the blood before FUS-MB application, 60 minutes was also chosen as a timepoint for FUS-MB application following AAV injection ICM.
  • Group 4 to determine the relevance of distribution route #2 (FIG.1E) a group of animals was injected with AAV 10 minutes post-FUS-MB application to investigate if AAV is delivered to the FUS-targeted site without potential perivascular pumping (i.e., no AAV in the perivascular space at the time of FUS-MB application) (FIG.2G).
  • Group 5 a group of animals was injected with AAV ICM without FUS-MB application (FIG.2H). FUS was targeted bilaterally in two spots in the striatum (FIGS.2F-G).
  • AAV2-HBKO Modified AAV2
  • AAV2-HBKO modified AAV2
  • FUS-MB FUS-MB targeting to the striatum
  • AAV9 modified AAV9
  • AAV2-HBKO encoded green fluorescent protein (GFP) under control of the ubiquitous promoter CAG.
  • AAV2-HBKO was administered at a dose of 2.16x10 12 GC/kg irrespective of the administration route, which is within the range previously successfully used for FUS-MB-mediated AAV administration (e.g., delivery) to the brain following intravenous injection (1.67x10 12 GC/kg – 1.67x10 13 GC/kg) (see R. H. Kofoed, et al., The engineered AAV2-HBKO promotes non-invasive gene delivery to large brain regions beyond ultrasound targeted sites. Molecular Therapy - Methods & Clinical Development 27, 167–184 (2022)).
  • Four weeks following AAV delivery and bilateral FUS-MB targeting to the striatum the animals were sacrificed.
  • distribution route #2 (FIG. 1E) is likely to be the primary delivery mechanism responsible for GFP protein and mRNA expression seen in animals injected ICM with AAV 120 minutes prior to FUS-MB (FIGS.2J-K).
  • FUS-MB increases delivery of ICM-injected AAV to the striatum [0267]
  • the pilot study demonstrated a tendency towards higher GFP mRNA expression in the striatum when AAV was injected ICM 120 minutes prior to FUS-MB compared to 60 minutes prior to and 10 minutes post FUS-MB.
  • the blood samples suggest a higher level of intravenous AAV 120 minutes compared to 60 minutes following ICM administration.
  • FIG. 4A AAV delivery was therefore compared between animals injected intravenously with AAV during FUS-MB, animals injected ICM with AAV 120 minutes prior to FUS-MB, and animals injected ICM with AAV without FUS-MB application.
  • Animals were sacrificed after 4 weeks and brains analyzed by IHC and qPCR (FIG. 4A).
  • GFP sf-6501845 Attorney Docket No.:15979-20190.40 protein expression (green, white arrows) in the brain were seen in FUS-targeted brain areas in animals injected both intravenous and ICM with AAV, but not in animals injected ICM without FUS-MB application (FIG. 4B).
  • ICM injection of AAV can lead to a high AAV uptake in dorsal root ganglions which can be neurotoxic (see J. Hordeaux, et al., Adeno-Associated Virus- Induced Dorsal Root Ganglion Pathology. Human Gene Therapy 31, 808–818 (2020)).
  • Analysis of GFP mRNA expression in the FUS-targeted striatum showed increased levels of GFP mRNA expression in animals injected intravenously and ICM and treated with FUS-MB compared to animals injected ICM with AAV without FUS-MB application (FIG. 4C).
  • FIG.4C There was no significant difference in GFP mRNA expression in the striatum in animal injected intravenously and ICM with AAV and treated with FUS-MB (FIG.4C).
  • the data in FIG.4C was log10- transformed to obtain normal distribution, and the non-transformed data showed a 5.6-times increase in GFP mRNA expression in the striatum following FUS-MB application in animals injected ICM with AAV compared to animals without FUS-MB treatment.
  • GFP mRNA levels were also measured in brain areas not targeted with FUS-MB, but where gadolinium enhancement in the pilot study suggested a significant delivery of ICM-injected particles (FIGS. 2B-E).
  • ICM injection of AAV, with or without FUS-MB targeting the striatum resulted in significantly increased levels of GFP mRNA expression in the thalamus, midbrain, cerebellum, hippocampus, and brainstem (FIGS.2D-H).
  • the depth of the FUS spots in the z-axis also targets brain structures located dorsally and ventrally relative to the striatum (FIG.4A), (last diagram to the right, turquoise ovals).
  • cortical structures e.g., Cortex 1 (CX1) and 2 (CX2)
  • FIGS. 4A-K demonstrate that FUS-MB can increase delivery to deep brain structures, such as the striatum, even with ICM-injected AAV, which does not otherwise reach these structures efficiently.
  • ICM injection of AAV leads to significantly increased gene delivery to brain areas that are not targeted by FUS-MB compared to intravenous AAV.
  • FUS-MB increases gene delivery to deep brain structures following ICM administration
  • the field of gene therapy is rapidly evolving. Monogenic disorders with a known underlying genetic cause are receiving attention as diseases with a high potential to be treated with gene therapy (see C. G. Limia, et al., Emerging Perspectives on Gene Therapy Delivery for Neurodegenerative and Neuromuscular Disorders. J Pers Med 12, 1979 (2022)).
  • the first monogenic disease that was treated with a one-time gene therapy treatment was spinal muscular atrophy using an intravenous administration of AAV9 in a dose of 1.1x1014 GC/kg (final recommended dose) (see J. R.
  • FUS-MB can be used to increase the transduction volume following ICM administration of AAV to include FUS-targeted deep brain structures, which cannot be reached efficiently by ICM injection alone. Increased delivery to deep brain structures by FUS-MB can enhance the sf-6501845 Attorney Docket No.:15979-20190.40 therapeutic efficacy of gene therapy treatments targeting diseases affecting the entire brain and ensure a better distribution of gene delivery across the brain.
  • This study demonstrates that FUS-MB can expand delivery of ICM-administered AAV vectors to deep brain structures that are poorly reached through ICM injection alone. Further studies are warranted to elucidate the underlying mechanism of action, which is important for the design of future studies and clinical translation.
  • Example 2 Non-human primate (NHP) study evaluating FUS following CSF-administered AAV [0271] It is hypothesized that combining FUS with intra-CSF AAV administration will expand the transduction profile of AAV to targeted deep brain structures enabling translation of this technology for the treatment of neurodegenerative diseases where deep brain structures are affected.
  • NEP Non-human primate
  • Proposed study groups are shown in Table 1.
  • Table 1 Proposed study groups sf-6501845 Attorney Docket No.:15979-20190.40
  • the animals used in this study are 8 cynomolgus macaques (7 on study +1 spare if needed), age ⁇ 2-5 years.
  • Prior to administration prescreen sera from ⁇ 36 animals for levels of neutralizing antibodies to AAV2-HBKO and AAV-SAN006. The study duration will be 3 weeks post-FUS.
  • the test articles are shown in Table 2.
  • Table 2 [0273] Microbubbles are DEFINITY® microbubbles infused IV at 4-16 uL/kg/5min.
  • Gadolinium is used as the tracer. Specifically Gadobutrol at 0.1mM/kg for IV administration and 2.5mL @ 5mg/mL for ICM administration.
  • CT scans are acquired prior to study start to determine skull morphometrics.
  • MRI scans are acquired pre-FUS (T2w and T1w) and pre-necropsy (T2w).
  • Test article dosing will be distributed across 4 groups shown in Table 1.
  • GAD gadobutrol solution
  • T1 MR time course pre-dose, 15min, 30min, 60 min, 90min, 120, 180 min
  • sf-6501845 Attorney Docket No.:15979-20190.40
  • Part b IV GAD with microbubbles (MB) to check the coordinates, parameters, timing for bilateral FUS to caudate and putamen.
  • Group 2 Intravenous dosing of AAV and MB as follows: (1) intravenous AAV at 2.5E 13 total VG/kg (5-8E 13 VG/animal estimating 2-3kg); and (2) intravenous MB co-administered with AAV.
  • Groups 3+4 Direct ICM infusion of AAV, with IV dosing of MB.
  • Direct-CM AAV at 3E 11 total VG/g brain (2.5E 13 VG/animal).
  • Intravenous Microbubbles (MB) delivered via IV administration ⁇ 2 hours post-ICM test article infusion.
  • Focused ultrasound Bilateral striatum (putamen and caudate nucleus).
  • FUS for group 1 will be done during microbubble infusion.
  • FUS for group 2 will be done right after AAV and during microbubble infusion.
  • FUS for group 4 will be done up to 2 hours after AAV dosing, during microbubble infusion.
  • As a viability check animals will be checked twice daily for mortality/moribundity. Detailed clinical observation are carried out weekly.
  • Sample collection comprises collecting blood and cerebral spinal fluid (CSF). Blood sampling is performed predose, immediately after iCM injection of AAV, 15min, 30min, 60min, 6hours, 24 hours, 1 week post dosing, and at necropsy for all groups. CSF sample collection is performed pre-dose and at necropsy for groups 2, 3, 4.
  • Necropsy is performed on 8 animals. Gross necropsy is performed at 3 weeks post- FUS (+/- 2 days). Animals are perfused with nuclease-free phosphate-buffered saline (PBS).
  • PBS nuclease-free phosphate-buffered saline
  • Brain processing comprises cutting into 4 mm coronal slice thickness with brain matrix. Following slicing, the brain is placed on a numbered mat (anterior side face down) in rostrocaudal order, hemisected and digital photographs are taken (photographs are included in the raw data). The slices are transferred to cassettes with the anterior surface of the brain placed face down in the cassettes to maintain left/right orientation. This right hemisphere is frozen for biodistribution and protein analysis. The left hemisphere is saved in 10% neutral buffered formalin (NBF) for histology, then transferred to PBS and stored refrigerated until shipment. The spinal cord processing consists of cutting into 29 segments, with alternate sections frozen for biodistribution and protein analysis.
  • NTF neutral buffered formalin
  • Dorsal Root Ganglia Processing consists of processing the left and right sides. The right side is frozen for biodistribution and protein analysis. The left is saved in 10% NBF for histology, then transferred to PBS and stored refrigerated until shipment. [0283] Representative samples of peripheral tissues are frozen for biodistribution or fixed for histology. [0284] Frozen tissue is analyzed via RT-dPCR and dPCR for transgene expression and biodistribution, respectively.
  • MRIgFUS Magnetic resonance imaging-guided focused-ultrasound
  • AAV adeno-associated viruses
  • NHPs non-human primates
  • Intravenous injection of AAV for gene delivery to the brain requires high doses of vector, results in high peripheral exposure, and poor brain delivery due to inefficient blood-brain barrier crossing.
  • Administration of AAV directly into the CSF is a clinically relevant route of administration and enables transduction of superficial brain regions at relatively low doses, however the distribution of AAV to deep brain structures remains limited.
  • MRI-guided, low-intensity focused ultrasound (MRIgFUS), combined with intravenously injected gas-filled microbubbles (MB), can safely and transiently open the blood brain barrier at specifically targeted locations, and is already in clinical trials for Alzheimer’s Disease, Parkinson’s Disease and ALS. It is also well established that this approach enables AAV vectors to cross from the blood into the brain, but only in the context of intravenous AAV injection, and only to those targeted regions. [0288] It is hypothesized that combining FUS with intra-CSF AAV administration will expand the transduction profile of AAV to targeted deep brain structures enabling translation of this technology for the treatment of neurodegenerative diseases where deep brain structures are affected.
  • MRIgFUS low-intensity focused ultrasound
  • MB gas-filled microbubbles
  • transgene payload DNA (expressing either eGFP or mCherry) was cloned into a plasmid containing AAV2 inverted terminal repeats (ITRs) under control of the CAG promoter (human CMV enhancer, chicken ⁇ -actin promoter, and a chicken ⁇ -actin/rabbit ⁇ -globin hybrid intron).
  • adherent HEK293 cells were transfected using PEI (polyethyleneimine) with a 1:1:1 ratio of three plasmids (containing the ITR, AAV rep/cap and Ad helper).
  • the Ad helper plasmid (pHelper) was obtained from Stratagene/Agilent Technologies (Santa Clara, CA).
  • AAV purification was performed using cesium chloride ultracentrifugation, and virus was titered using ddPCR against the bGH polyA sequence and the concentration of each vector was adjusted accordingly prior to pooling for dose administration. Animal use and care [0290] All experiments were conducted in an AAALAC-accredited institution.
  • ketamine Intramuscular at 0.10 mL/kg
  • isoflurane Standard buprenorphine (0.3mg/mL) was administered intramuscularly as needed and once prior to MR imaging.
  • iCM Intra-cisterna magna
  • FUS Low intensity FUS and MR imaging
  • Insightec ExAblate 4000 low frequency system Insightec, Haifa, Israel
  • 3T MR scanner Seimens SKYRAFIT
  • the head of the animal was coupled to the transducer with degassed water and secured for the duration of the treatment.
  • Baseline T1-weighted images were acquired and used to outline the left caudate and putamen of each NHP for targeting with Insightec ExAblate Neuro software using 1mm grid spacing for sonication spots. Care was taken to avoid placing spots near the lateral ventricle or to adjacent cortical regions.
  • Microbubbles (DEFINITY) were prepared as per manufacturer’s protocol and infused intravenously through a catheter at 5uL/kg/min prior to and for the duration of sonication. Ultrasound was applied at 230 kHz for 180sec at 0.5% duty cycle for each spot. Animals received between 1-3 sonication rounds per target (acoustic doses detailed in the table in FIG.9), depending on the extent of hypointense signal change observed using a modified T2* relaxation map to detect subtle changes in the BBB following each round of sonication. Lack of signal change triggered additional rounds of sonication until signal changes were observed to ensure BBB opening. Animals were removed from the FUS system and repositioned in the MR scanner for follow-up imaging.
  • BBB opening immediately after, and 2 days after sonication was evaluated by axial T1-weighted MR imaging following gadobenate dimeglumine (Bracco MultiHance) injected at 0.2 mmol/kg.
  • T2-weighted scans were run for each animal at 2 days and 20 days post-FUS to monitor for signs of edema following FUS treatment.
  • NHP sample collection [0293] Whole blood was collected into EDTA tubes prior to, 30min, 2-3 hours and 192 hours after AAV infusion for AAV vector genome quantification.
  • sf-6501845 Attorney Docket No.:15979-20190.40 [0294] At necropsy, animals were perfused with chilled RNase/DNase free PBS pH 7.4, and their brains were cut in a brain matrix into 4mm coronal slices and hemisected. The left (FUS treated) and right (FUS untreated) hemispheres from all slabs were frozen on dry ice for biochemical analysis, except for one slab encompassing the anterior caudate nucleus and putamen. This slab was drop fixed in 10% neutral buffered formalin for 36-48 hours at room temperature before embedding in paraffin blocks for subsequent sectioning.
  • Tissue homogenization Tissues were bead homogenized at 4oC in TE buffer (10mM Tris pH7.4, 1mM EDTA) using an Omni Beadruptor set for 20 second cycles 4.7 oscillation/sec., aliquoted, and stored at -80C until further use. Samples used for subsequent RNA analyses were first mixed with QIAzol lysis reagent prior to freezing.
  • Vector genome copies were quantified from extracted gDNA by digital PCR using the QIAcuity Probe PCR Kit (QIAGEN 250102) with probes targeting AAV vector genome sequences specific for each AAV (mCherry for SAN006 and GFP for AAV2HBKO) and endogenous gDNA (TUBB1 intron). Reactions were performed using the QIAcuity 8 digital PCR system (QIAGEN) with manufacturer’s suggested thermocycling conditions. Vector genome and reference gene copies/ ⁇ L were quantified using the QIAcuity Software Suite (QIAGEN), and 2x vector genome copies were divided by reference gene copies to calculate VG/cell.
  • RNA isolation and quantification [0298] RNA was isolated from tissue homogenates using the RNeasy 96 QIAcube HT kit (QIAGEN, 74171) according to manufacturer’s protocol. Briefly, tissue homogenates in QIAzol lysis reagent (QIAGEN, 79306) were thawed and mixed with chloroform (Fisher Scientific, C298-1). The mixture was centrifuged at 4°C and the aqueous phase was transferred to S block (QIAGEN, 19585) for further RNA isolation.
  • QIAGEN QIAcube HT kit
  • RNA isolation was performed by following the steps from QIAcube HT Prep Software with on-column DNase (QIAGEN, 79256) treatment. After RNA isolation, concentration was measured with a NANODROP 8000 (Thermo Fisher Scientific). Reverse transcription was carried out by producing cDNA via QIAcuity OneStep advanced probe kit (QIAGEN, 250132) and using multiplexed primer-probe combinations specific to GFP, mCherry, HPRT and RPP30. Copies/uL of each transgene were normalized to the mean copies of HPRT and RPP30 (Integrated DNA Technology).
  • Tissue sectioning and staining Five ⁇ m-thick sections of brain tissue were cut with a microtome and mounted directly onto charged glass slides. Tissue sections were stained with either hematoxylin and eosin for histopathology assessment, or processed for ISH as follows.
  • Target RNA was detected in tissue sections using automated ISH using a Leica Bond Rx stainer and standard RNAscope detection kits (red- RNAscope 2.5 LSx reagent kit- RED, ACD cat# 322750; brown- RNAscope 2.5 LSx reagent kit- BROWN, ACD cat# 322700) and protocols.
  • ACD 2.5 LS Probes for target detection included: mCherry ACD cat# 431208 and GFP ACD cat# 400288.
  • Probes were used as supplied and all standard RNAcope LSx protocols were used as pre-installed on Leica Bond Rx. Following staining completion, slides were removed from Leica Bond Rx, dehydrated in ethanol and xylene, and coverslip applied by Leica CV5030 coverslipper with Surgipath Micromount mounting medium (Leica, cat# 3801730). Images were collected using Leica Aperio AT2 brightfield slide scanner at 20X magnification.
  • sf-6501845 Attorney Docket No.:15979-20190.40 Results Intra-CSF administration of AAV followed by large volume BBB opening in the NHP caudate and putamen [0300]
  • AAV was administered to four juvenile cynomolgus macaques via direct intra-cisterna magna (iCM) injection, followed by unilateral focused ultrasound targeting the caudate nucleus and putamen in the left hemisphere of each animal. Unilateral targeting was chosen to enable the contralateral hemisphere to be used as a control for iCM AAV without FUS.
  • AAV was administered as a pooled, 1:1 mixture of two capsids, AAV2HBKO and AAV.SAN006, expressing eGFP and mCherry reporter proteins, respectively.
  • Transcranial low intensity FUS was applied to the caudate and putamen using sonication spots distributed across 1 mm grids for each target. Due to the size of the region in some animals, ventral putamen was divided into two separate posterior (A) and anterior (B) sonication grids spread across the same axial plane. Additionally, two animals (1003 and 1004) were treated across two different axial planes (“slices”) per structure to yield more comprehensive coverage (summarized in the table in FIG.9).
  • T2*map A modified T2* mapping (T2*map) sequence was used to detect subtle changes in blood brain barrier (BBB) integrity immediately after each round of sonication, with the detection of signal hypointensity on the T2*map scan indicating sufficient BBB disruption and resulting in the end of sonication (FIG. 5A).
  • BBB opening was confirmed in each animal with administration of intravenous gadolinium (Gd) followed by T1-weighted MR imaging to visualize extravasation of Gd. All animals displayed areas of Gd enhancement precisely overlapping with the targeted regions (FIG.5B). Animals were allowed to recover, and MR scans were repeated two days later to evaluate the extent of BBB-closure (FIG. 5C).
  • FUS had no effect on vector biodistribution to areas outside the caudate or putamen but significantly enhanced intra-CSF AAV2HBKO biodistribution for all animals in the targeted structures, necessarily encompassing the globus pallidus (FIG.6A).
  • FUS greatly enhanced transduction across the rostro-caudal axis of both caudate and putamen (FIG. 6C).
  • AAV.SAN006 exhibited robust transduction across the brain consistent with iCM injection, and FUS treatment did not alter transduction outside the targeted regions (FIG.6B).
  • AAV.SAN006 and AAV2HBKO VG levels were also quantified from DNA extracted from whole blood samples at baseline, 30min after infusion, after sonication was complete, and 7 days after AAV infusion. Both AAVs were present at high levels in the bloodstream throughout the period of time that the BBB was open following FUS. The kinetics of vector efflux from the CSF into the blood were similar, indicating that the differences in striatal transduction following FUS for the two vectors is unlikely to be due to differences in the amount of AAV present in the bloodstream. Vector genome levels were also measured in spinal cord (FIG.6F), dorsal root ganglia (FIG. 6G), liver and spleen (FIG.6H).
  • AAV.SAN006 displayed ubiquitous enhancement of mRNA expression in the FUS-targeted caudate and putamen for all animals exceeding 100-fold improvement in some regions of caudate and putamen (FIGS.7B, 7D). Histological confirmation of MR findings and of enhanced striatal transgene expression in the sonicated hemisphere [0304] While most of the brain was processed and frozen exclusively for molecular analyses, one coronal slab of brain encompassing the anterior caudate and putamen was fixed for histological analyses. Gross examination revealed no notable differences between treated and untreated hemispheres. Upon microscopic evaluation of H&E-stained sections the treated putamen appeared normal for all animals.
  • ISH in situ hybridization

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Nanotechnology (AREA)
  • Dermatology (AREA)
  • Psychology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Optics & Photonics (AREA)
  • Neurosurgery (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Neurology (AREA)
  • Botany (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés pour traiter divers troubles neurodégénératifs comprenant l'administration de particules virales à l'ensemble du cerveau, comprenant à la fois des structures superficielles et profondes. Selon certains aspects, les particules virales sont administrées à de faibles niveaux de dose au CSF, conjointement avec des microbulles suivies par l'application d'ultrasons focalisés (FUS) à une région d'intérêt du cerveau, provoquant ainsi l'entrée des particules virales dans le cerveau.
PCT/US2025/013023 2024-01-25 2025-01-24 Procédés d'administration basée sur fus de particules virales au cerveau Pending WO2025160452A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463625206P 2024-01-25 2024-01-25
US63/625,206 2024-01-25

Publications (1)

Publication Number Publication Date
WO2025160452A1 true WO2025160452A1 (fr) 2025-07-31

Family

ID=94637341

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/013023 Pending WO2025160452A1 (fr) 2024-01-25 2025-01-24 Procédés d'administration basée sur fus de particules virales au cerveau

Country Status (1)

Country Link
WO (1) WO2025160452A1 (fr)

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6566118B1 (en) 1997-09-05 2003-05-20 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US6596535B1 (en) 1999-08-09 2003-07-22 Targeted Genetics Corporation Metabolically activated recombinant viral vectors and methods for the preparation and use
US6723551B2 (en) 2001-11-09 2004-04-20 The United States Of America As Represented By The Department Of Health And Human Services Production of adeno-associated virus in insect cells
US20040224411A1 (en) 2003-02-26 2004-11-11 Clark Kelly Reed Recombinant adeno-associated virus production
US6989264B2 (en) 1997-09-05 2006-01-24 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US7765583B2 (en) 2005-02-28 2010-07-27 France Telecom System and method for managing virtual user domains
US7790154B2 (en) 2000-06-01 2010-09-07 The University Of North Carolina At Chapel Hill Duplexed parvovirus vectors
WO2010148143A1 (fr) 2009-06-16 2010-12-23 Genzyme Corporation Procédés améliorés pour la purification de vecteurs aav recombinants
US8137948B2 (en) 2003-05-21 2012-03-20 Genzyme Corporation Methods for producing preparations of recombinant AAV virions substantially free of empty capsids
WO2015168666A2 (fr) 2014-05-02 2015-11-05 Genzyme Corporation Vecteurs aav pour thérapie génique de la rétine et du snc
WO2023092002A2 (fr) * 2021-11-17 2023-05-25 Voyager Therapeutics, Inc. Compositions et méthodes de traitement de la sclérose latérale amyotrophique et de troubles associés à la moelle épinière
WO2023091949A2 (fr) * 2021-11-17 2023-05-25 Voyager Therapeutics, Inc. Compositions et méthodes de traitement de troubles neurologiques liés à un déficit en bêta glucosylcéramidase

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6989264B2 (en) 1997-09-05 2006-01-24 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US6566118B1 (en) 1997-09-05 2003-05-20 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US7785888B2 (en) 1999-08-09 2010-08-31 Genzyme Corporation Metabolically activated recombinant viral vectors and methods for their preparation and use
US6596535B1 (en) 1999-08-09 2003-07-22 Targeted Genetics Corporation Metabolically activated recombinant viral vectors and methods for the preparation and use
US8093054B2 (en) 1999-08-09 2012-01-10 Genzyme Corporation Metabolically activated recombinant viral vectors and methods for their preparation and use
US7846729B2 (en) 1999-08-09 2010-12-07 Genzyme Corporation Metabolically activated recombinant viral vectors and methods for their preparation and use
US7125717B2 (en) 1999-08-09 2006-10-24 Targeted Genetics Corporation Metabolically activated recombinant viral vectors and methods for their preparation and use
US7790154B2 (en) 2000-06-01 2010-09-07 The University Of North Carolina At Chapel Hill Duplexed parvovirus vectors
US8361457B2 (en) 2000-06-01 2013-01-29 The University Of North Carolina At Chapel Hill Duplexed parvovirus vectors
US6723551B2 (en) 2001-11-09 2004-04-20 The United States Of America As Represented By The Department Of Health And Human Services Production of adeno-associated virus in insect cells
US20040224411A1 (en) 2003-02-26 2004-11-11 Clark Kelly Reed Recombinant adeno-associated virus production
US8137948B2 (en) 2003-05-21 2012-03-20 Genzyme Corporation Methods for producing preparations of recombinant AAV virions substantially free of empty capsids
US7765583B2 (en) 2005-02-28 2010-07-27 France Telecom System and method for managing virtual user domains
WO2010148143A1 (fr) 2009-06-16 2010-12-23 Genzyme Corporation Procédés améliorés pour la purification de vecteurs aav recombinants
WO2015168666A2 (fr) 2014-05-02 2015-11-05 Genzyme Corporation Vecteurs aav pour thérapie génique de la rétine et du snc
WO2023092002A2 (fr) * 2021-11-17 2023-05-25 Voyager Therapeutics, Inc. Compositions et méthodes de traitement de la sclérose latérale amyotrophique et de troubles associés à la moelle épinière
WO2023091949A2 (fr) * 2021-11-17 2023-05-25 Voyager Therapeutics, Inc. Compositions et méthodes de traitement de troubles neurologiques liés à un déficit en bêta glucosylcéramidase

Non-Patent Citations (57)

* Cited by examiner, † Cited by third party
Title
A. PHILIPPIDIS: "Fourth Boy Dies in Clinical Trial of Astellas' AT132", HUMAN GENE THERAPY, vol. 32, 2021, pages 1008 - 1010
BOISON, D., EPILEPSIA, vol. 51, 2010, pages 1659 - 1668
C. G. LIMIA ET AL.: "Emerging Perspectives on Gene Therapy Delivery for Neurodegenerative and Neuromuscular Disorders", 1 PERS MED, vol. 12, 2022, pages 1979
C. G. LIMIA ET AL.: "Emerging Perspectives on Gene Therapy Delivery for Neurodegenerative and Neuromuscular Disorders", J PERS MED, vol. 12, 2022, pages 1979
CLARK ET AL., HUM. GENE THER, vol. 10, 1994, pages 1031 - 1039
CONWAY, JE ET AL., J. VIROLOGY, vol. 71, no. 11, 1997, pages 8780 - 8789
COSTANTINI LC ET AL., GENE THERAPY, vol. 7, 2000, pages 93 - 109
DAILY JENNIFER ET AL: "A Non-Human Primate Biodistribution Study Comparing Multiple Routes of Administration of AAV9", ASGCT 24TH ANNUAL MEETING ABSTRACTS-ABSTRACT 347, 27 April 2021 (2021-04-27), XP093270316, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S1525001621002069> *
DAVIDSON ET AL., PNAS, vol. 97, no. 7, 2000, pages 3428 - 32
E. THÉVENOT ET AL.: "Targeted Delivery of Self-Complementary Adeno-Associated Virus Serotype 9 to the Brain, Using Magnetic Resonance Imaging-Guided Focused Ultrasound", HUMAN GENE THERAPY, vol. 23, 2012, pages 1144 - 1155, XP055556833, DOI: 10.1089/hum.2012.013
EMMANUEL TH�VENOT ET AL: "Targeted Delivery of Self-Complementary Adeno-Associated Virus Serotype 9 to the Brain, Using Magnetic Resonance Imaging-Guided Focused Ultrasound", HUMAN GENE THERAPY, vol. 23, no. 11, 1 November 2012 (2012-11-01), GB, pages 1144 - 1155, XP055556833, ISSN: 1043-0342, DOI: 10.1089/hum.2012.013 *
FERREIRA ET AL., HUM. GENE THER, vol. 25, 2014, pages 180 - 188
FISHER ET AL., J. VIROL, vol. 70, 1996, pages 520 - 532
FUKUDA, A.MBADAUT, J, GENES (BASEL), vol. 4, 2013, pages 435 - 456
GAO ET AL., PNAS, vol. 100, no. 10, 2003, pages 6081 - 6
GAO ET AL., PNAS, vol. 99, no. 18, 2002, pages 11854 - 6
GROTH, A.C. ET AL., PROC. NATL. ACAD. SCI., vol. 97, 2000, pages 5995 - 6000
J. A. SULLIVAN ET AL.: "Rationally designed AAV2 and AAVrh8R capsids provide improved transduction in the retina and brain", GENE THERAPY, vol. 25, 2018, pages 205 - 219, XP036536617, DOI: 10.1038/s41434-018-0017-8
J. HORDEAUX ET AL.: "Adeno-Associated Virus-Induced Dorsal Root Ganglion Pathology", HUMAN GENE THERAPY, vol. 31, 2020, pages 808 - 818, XP055843115, DOI: 10.1089/hum.2020.167
J. NAIDOO ET AL.: "Extensive Transduction and Enhanced Spread of a Modified AAV2 Capsid in the Non-human Primate CNS", MOLECULAR THERAPY, vol. 26, 2018, pages 1 - 13
J. R. MENDELL ET AL.: "Five-Year Extension Results of the Phase 1 START Trial of Onasemnogene Abeparvovec in Spinal Muscular Atrophy", JAMA NEURAL, vol. 78, 2021, pages 834 - 841
J. R. MENDELL ET AL.: "Single-dose gene-replacement therapy for spinal muscular atrophy", NEW ENGLAND JOURNAL OF MEDICINE, vol. 377, 2017, pages 1713 - 1722, XP055555934, DOI: 10.1056/NEJMoa1706198
K. D. FOUST ET AL.: "Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes", NATURE BIOTECHNOLOGY, vol. 27, 2009, pages 59 - 65, XP055023143, DOI: 10.1038/nbt.1515
K. HYNYNENN. MCDANNOLDN. VYKHODTSEVAF. JOLESZ: "Noninvasive MR Imaging-guided Focal Opening of the Blood-Brain Barrier in Rabbits", RADIOLOGY, vol. 220, 2001, pages 640 - 646, XP003004109, DOI: 10.1148/radiol.2202001804
KIM, T.KEBERWINE, J.H, ANAL. BIOANAL. CHEM, vol. 397, 2010, pages 3173 - 3178
KOFOED RIKKE HAHN ET AL: "Efficacy of gene delivery to the brain using AAV and ultrasound depends on serotypes and brain areas", JOURNAL OF CONTROLLED RELEASE, vol. 351, 1 November 2022 (2022-11-01), NL, pages 667 - 680, XP093269999, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2022.09.048 *
KOFOED RIKKE HAHN ET AL: "The engineered AAV2-HBKO promotes non-invasive gene delivery to large brain regions beyond ultrasound targeted sites", MOLECULAR THERAPY- METHODS & CLINICAL DEVELOPMENT, vol. 27, 8 December 2022 (2022-12-08), GB, pages 167 - 184, XP093270017, ISSN: 2329-0501, DOI: 10.1016/j.omtm.2022.09.011 *
KOTIN, R, HUM MOL GENET, vol. 20, no. 1, 2011, pages 2 - 6
KRYSAN, P.J, MOL. CELL BIOL, vol. 9, 1989, pages 1026 - 1033
LEVITT, N ET AL., GENES DEVELOP, vol. 3, 1989, pages 1019 - 1025
M. J. CASTLE ET AL.: "Postmortem analysis in a clinical trial of AAV2-NGF gene therapy for alzheimer's disease identifies a need for improved vector delivery", HUMAN GENE THERAPY, vol. 31, 2020, pages 415 - 422
MARCHI PAOLO M. ET AL: "Delivery of therapeutic AAV9 vectors via cisterna magna to treat neurological disorders", TRENDS IN MOLECULAR MEDICINE, vol. 28, no. 1, 1 January 2022 (2022-01-01), GB, pages 79 - 80, XP093271030, ISSN: 1471-4914, DOI: 10.1016/j.molmed.2021.09.007 *
MARTIN ET AL., HUMAN GENE THERAPY METHODS, vol. 24, 2013, pages 253 - 269
MCLAUGHLIN ET AL., J. VIROL, vol. 62, 1988, pages 1963 - 1973
MIYAZAKI, J ET AL., GENE, vol. 79, no. 2, 1989, pages 269 - 77
N. SHEIKOV ET AL.: "Brain arterioles show more active vesicular transport of blood-borne tracer molecules than capillaries and venules after focused ultrasound-evoked opening of the blood-brain barrier", ULTRASOUND IN MEDICINE AND BIOLOGY, vol. 32, 2006, pages 1399 - 1409, XP025017687, DOI: 10.1016/j.ultrasmedbio.2006.05.015
N. SHEIKOVN. MCDANNOLDS. SHARMAK. HYNYNEN: "Effect of Focused Ultrasound Applied With an Ultrasound Contrast Agent on the Tight Junctional Integrity of the Brain Microvascular Endothelium", ULTRASOUND IN MEDICINE AND BIOLOGY, vol. 34, 2008, pages 1093 - 1104, XP022795620, DOI: 10.1016/j.ultrasmedbio.2007.12.015
NAKAMURA, Y ET AL., NUCLEIC ACIDS RES., vol. 28, 2000, pages 292
O'GORMAN, S ET AL., SCIENCE, vol. 251, 1991, pages 1351 - 1355
P. M. MARCHIL. MARRONEM. AZZOUZ: "Delivery of therapeutic AAV9 vectors via cisterna magna to treat neurological disorders", TRENDS MOL MED, vol. 28, 2022, pages 79 - 80
PASSINI ET AL., J. VIROL, vol. 77, no. 12, 2003, pages 6799 - 810
PECHAN ET AL., GENE THER., vol. 16, 2009, pages 10 - 16
PENZES ET AL., JOURNAL OF VIROLOGY, vol. 95, no. 19, 2021, pages 0084321
R. H. KOFOED ET AL.: "Efficacy of gene delivery to the brain using AAV and ultrasound depends on serotypes and brain areas", JOURNAL OF CONTROLLED RELEASE, vol. 351, 2022, pages 667 - 680
R. H. KOFOED ET AL.: "The engineered AAV2-HBKO promotes non-invasive gene delivery to large brain regions beyond ultrasound targeted sites", MOLECULAR THERAPY - METHODS & CLINICAL DEVELOPMENT, vol. 27, 2022, pages 167 - 184
R. H. KOFOED ET AL.: "Transgene distribution and immune response after ultrasound delivery of rAAV9 and PHP.B to the brain in a mouse model of amyloidosis", MOLECULAR THERAPY - METHODS & CLINICAL DEVELOPMENT, vol. 23, 2021, pages 390 - 405
S. NAKAMURA ET AL.: "Intra-cisterna magna delivery of an AAV vector with the GLUT1 promoter in a pig recapitulates the physiological expression of SLC2A1", GENE THERAPY, vol. 28, 2021, pages 329 - 338, XP037488684, DOI: 10.1038/s41434-020-00203-z
SAUER, BHENDERSON, N, PROC. NATL. ACAD. SCI., vol. 85, 1988, pages 5166 - 5170
SHIMAMURA M ET AL: "Development of efficient plasmid DNA transfer into adult rat central nervous system using microbubble-enhanced ultrasound", GENE THERAPY, NATURE PUBLISHING GROUP, LONDON, GB, vol. 11, no. 20, 22 July 2004 (2004-07-22), pages 1532 - 1539, XP037770755, ISSN: 0969-7128, [retrieved on 20040722], DOI: 10.1038/SJ.GT.3302323 *
URABE, M. ET AL., HUMAN GENE THERAPY, vol. 13, no. 16, 2002, pages 1935 - 1943
VELDWIJK ET AL., MOL. THER, vol. 6, 2002, pages 272 - 278
WANG ET AL., NATURE MEDICINE, vol. 20, 2014, pages 536 - 541
WANG Z ET AL., GENE THER, vol. 10, 2003, pages 2105 - 2111
XIAO ET AL., EXP. NEUROBIOL, vol. 144, 1997, pages 113 - 124
XIAO ET AL., JOURNAL OF VIROLOGY, vol. 72, 1998, pages 2224 - 2232
Y. CHUJ. H. KORDOWER: "Post-Mortem Studies of Neurturin Gene Therapy for Parkinson's Disease: Two Subjects with 10 Years CERE120 Delivery", MOV DISORD, vol. 38, 2023, pages 1728 - 1736
Z. NOROOZIAN ET AL.: "MRI-guided focused ultrasound for targeted delivery of rAAV to the brain", METHODS MOL BIOL, vol. 1950, 2019, pages 177 - 197

Similar Documents

Publication Publication Date Title
US12465657B2 (en) Enhanced delivery of viral particles to the striatum and cortex
AU2021200242B9 (en) AAV vectors for retinal and CNS gene therapy
EP3364970B1 (fr) Thérapie génique pour l&#39;utilisation dans le traitement de la maladie lysosomale
ES2784264T3 (es) Construcciones de ácido nucleico y vectores de terapia génica para su uso en el tratamiento de la enfermedad de Wilson y otras afecciones
JP2015083614A (ja) 神経学的障害を治療するための皮質全体に亘る高レベルの治療薬の分布方法
TW202214864A (zh) 用於藉基因療法治療諸如阿茲海默症之Ｔａｕ蛋白病變的病毒顆粒
WO2025160452A1 (fr) Procédés d&#39;administration basée sur fus de particules virales au cerveau
US20250152744A1 (en) Methods of treating neurodegenerative disorders
HK40097464A (en) Enhanced delivery of viral particles to the striatum and cortex
HK40064049A (en) Aav vectors for retinal and cns gene therapy
HK1246340B (en) Enhanced delivery of viral particles to the striatum and cortex
NZ735290B2 (en) Enhanced delivery of viral particles to the striatum and cortex
HK1234076B (en) Aav vectors for retinal and cns gene therapy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25706141

Country of ref document: EP

Kind code of ref document: A1