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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/48—Nerve growth factor [NGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
• • 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
  • A61K48/005—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 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • 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
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0085—Brain, e.g. brain implants; Spinal cord
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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  • 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
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  • 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
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14171—Demonstrated in vivo effect
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus

Definitions

• the present invention relates generally to methods for treatment of
• neurodegenerative disease such as Parkinson's disease by administering therapeutic nerve growth factors into the mammalian brain.
• Parkinson's disease is a common neurodegenerative disorder characterized clinically by bradykinesia, rigidity, tremor, and gait dysfunction, and pathologically by degeneration of dopamine neurons in the substantia nigra pars compacta (substantia nigra) and by projection to the striatum (including the putamen).
• Present therapies provide satisfactory disease control for most patients, particularly in the early stages. However, no treatments protect against the continued degeneration of these neurons and, over time, all therapies fail.
• Glial-cell-derived neurotrophic factor protects dopamine neurons in in-vitro and animal models of Parkinson's disease. Lin et al.. Science
• Neuxturin is a na turally occurring structural and functional analogue of GDNF (Kotzbauer et al. , Nature 384:467-470 ( 1996)) that has been demonstrated to improve dopaminergic activity in aged monkeys (Herzog et al, Mov Disord 22: 1 124—1 132 (2007)) and protect dopamine neurons in animal models of Parkinson's disease, Horger et al., J Neurosci.
• Adeno-associated type-2 (AAV2)-neuiturin is a vector that has been genetically engineered to express and secrete the human gene for neurturin. Gasmi et al., Mo I Ther 15:62-68 (2007). The AAV2 vector does not induce an inflammatory reaction, has been used safely in clinical trials, and provides long-lasting transgene expression. Bankiewicz et al, Mol Tner 14:564-570 (2006).
• the present invention provides a clinically useful system and protocol for delivery of nerve growth factors into the mammalian brain.
• the invention is particularly useful in treating neurodegenerative conditions in primates, in whom nerve growth factors delivered according to the invention stimulate growth of neurons and recovery of neurological function .
• the invention includes a method for delivery of a therapeutic nerve growth factor to targeted defective, diseased or damaged dopaminergic neurons in the brain of a human subject, such as those impaired in Parkinson's disease.
• the method includes directly delivering a nerve growth factor, a nerve growth factor encoding expression vector, or grafting a donor cell containing such an expression vector into the substantia nigra and, preferably, also into the striatum, for amelioration of the defect, disease or damage in response to the nen'e growth factor.
• striatal delivery is to at least one region of the putamen.
• the total unit dosage of nerve growth factor encoding expression vector delivered to the striatum is greater than the unit dosage of nerve growth factor encoding expression vector delivered into the substantia nigra.
• the unit dosage delivered to the putamen may be up to 3, 4, 5, 6, 7, 8 or 9 times the unit dosage delivered to the substantia nigra.
• the disease is ameliorated by stimulation of repair of or activity in dopaminergic neurons. In related embodiments the disease is ameliorated by reversal of deficits in motor function associated with the Parkinson's disease.
• the nerve growth factor is a GDNF family molecule; for example, GDNF, neurturin, persephin or artemin.
• a recombinant expression vector used to deliver the nerve growth factor to cells for expression which is an AAV vector.
• FIG. 1 provides a graphical illustration of an AAV2-neurturm recombinant expression vector.
• AAV2 ITRs flank the NTN expression cassette, which consists of the CAG promoter, the pre-pro-NGF-NTN hybrid cDNA and the human growth hormone gene polyadenylation signal. The location of the canonical RXXR sequence derived from the NGF pro-domain and the cleavage site are shown.
• the term "about,” when referring to a value can be meant to encompass variations of, in some embodiments, ⁇ 100% in some embodiments ⁇ 50%, in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions,
• the present invention is based on the discover ⁇ ' that that delivery of a nerve growth factor deep in the neurocompromised brain, such as the substantia nigra of the Parkinson's disease brain, can provide enhancement of degenerating neurons.
• the invention provides an effective approach to overcome heretofore unrecognized
• the invention identifies and defines the required parameters of a method for successful regeneration of neurons in the brain with nerve growth factors, especially the neurons whose loss in the substantia nigra is associated with neurodegenerative conditions such as Parkinson's disease.
• the first method parameter defined by the invention is selection of a suitable target tissue.
• a region of the brain is selected for its retained responsiveness to
• CNS neurons which retain responsiveness to
• neurotrophic factors into adulthood include the cholinergic basal forebrain neurons, dopaminergic neurons of the substantia nigra, areas of the striatum, the putamen, the entorhinal cortical neurons, the thalamic neurons, the locus coeruleus neurons, the spinal sensor ⁇ ' neurons and the spinal motor neurons. Loss of functionality in neurons of the substantia nigra is causativeiy associated with the onset of Parkinson's disease.
• the substantia nigra is a relatively small deep brain structure, situated beneath the much larger striatum. Given the surgical risks involved in directly accessing the substantia nigra, delivery to it has been attempted via transport of expressed nerve growth factor from the striatum. However, in the Phase 2 studies described elsewhere above, delivery of an AA V-neurturin construct to the striatum unexpectedly failed to provide sufficient protein to the substantia nigra to achieve therapeutic results in humans comparable to those demonstrated using the same approach in non-human primate models of Parkinson's disease. It is believed that the nigrostriatal pathway may be more degenerated in human Parkinson ' s sufferers than previously understood.
• delivery of a nerve growth factor according to the invention targets the substantia nigra (and preferably also the striatum).
• striatal deliver ⁇ ' different regions of the striatum may be targeted, including for exampl e, the putamen, globus pallidum and caudate nucleus.
• Multiple areas of the brain may be targeted simultaneously, such as to both the putamen and substantia nigra. Additionally, multiple locations of the specific areas may be targeted, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 areas of the putamen and/or substantia nigra each along one or both hemispheres. In human patients, one or more injections per hemisphere or side to the substantia nigra and, preferably, one or more per hemisphere or side to the striatum are believed to be sufficient to achieve a therapeutic amelioration of the disease.
• Dosing of the expression vector delivered to the striatum may also be greater than to the substantia nigra.
• the total dose delivered to the striatum may be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 times the dose deli vered to the substantia nigra in a single course of treatment.
• M ost preferably, the total dose provided to the striatum is from 5 and 10 times the total dose provided to the substantia nigra,
• unit dosage refers generally to the concentration of nerve growth factor/ml of neurotrophic pharmaceutical composition prepared for use in the invention.
• the nerve growth factor concentration is defined by the number of viral particles/ml of neurotrophic composition.
• each unit dosage of nerve growth factor will comprise at least 2.5 ⁇ 1 of a neurotrophic composition, up to 25, 60, 100, 200, 300 ⁇ 1 or more as clinically indicated, wherein the composition includes a viral expression vector in a pharmaceutically acceptable fluid and provides from 10° up to 10 -' ° expressing viral particles per ml of neurotrophic composition.
• a nerve growth factor protein For delivery of nerve growth factor protein, those of ordinary skill in the art will be readily able to convert dosing protocols along these lines to suitable unit doses.
• direct delivery of a nerve growth factor may be achieved by means familiar to those of skill in the art, including microinjection through a surgical incision (see, e.g., Capecchi, Cell, 22:479-488 (1980)), infusion, chemical complexation with a targeting molecule or co-precipitant (e.g., liposome, calcium), electroporation (see, e.g., Andreason and Evans, Biotechniques, 6:650-660 (1988)) and, for delivery of a nerve growth factor encoding expression vector composition, microparticle bombardment of the target tissue (Tang et ai., Nature, 356: 152454 (1992)).
• a surgical incision see, e.g., Capecchi, Cell, 22:479-488 (1980)
• the direct deliver ⁇ ' method employed by the invention obviates a limiting risk factor associated with gene therapy; to wit, the potential for transfection of non-targeted cells with the vector carrying the nerve growth factor encoding transgene.
• delivery is direct and the delivery sites are chosen so diffusion of secreted nerve growth factor takes place over a controlled and predetermined region of the brain to optimize contact with targeted neurons, while minimizing contact with non-targeted cells.
• viral vectors with an operable nerve growth factor encoding transgene have been shown to express human nerve growth factor after delivery to the brain for several years. As such, the invention provides a chronically available source for nerve growth factor in the brain.
• Materials useful in the methods of the invention include in vivo compatible recombinant expression vectors, packaging cell lines, helper cell lines, synthetic in vivo gene therapy vectors, regulatable gene expression systems, encapsulation materials, pharmaceutically acceptable carriers and polynucleotides coding for nervous system growth factors of interest.
• GDNF nerve growth factor
• BDNF brain-derived neurotrophic factor
• NT-3 nerve growth factor-3
• NT-4/5 nerve growth factor-6
• CNTF ciliary neurotrophic factor
• GDNF glial cell line-derived neurotrophic factor
• FGF's 1-15 fibroblast growth factor family
• LIF leukemia inhibitory factor
• IGF-] insulin-like growth factor family
• BMPs bone morphogenic proteins
• BMPs bone morphogenic proteins
• TGF transforming growth factor
• TGF transforming growth factor
• EGF epidermal growth factor
• PDGF platelet-derived growth factor
• the growth factors may be purified, synthesized or produced recombinantly.
• ner growth factor is meant growth factors of any origin which are substantially homologous to and which are biologically equivalent to the nerve growth factors referenced herein.
• Such substantially homologous growth factors may be native to any tissue or species and, similarly, biological activity can be characterized in any of a number of biological assay systems.
• “nerve growth factors” can also include hybrid and modified forms of the molecules, including fusion proteins and fragments and hybrid and modified forms in which certain amino acids have been deleted or replaced and modifications such as where one or more amino acids have been changed to a modified amino acid or unusual amino acid and modifications such as gSycosolations so long as the hybrid or modified form retains the biological activity of the subject nerve growth factor.
• retaining the biological activity it is meant that neuronal repair is achieved or activity promoted, although not necessarily at the same level of potency as that of the nerve growth factor in isolated and purified form or that which has been recombinantly produced.
• pre-pro sequences of nerve growth factors herein is intended to be construed to include pre-pro growth factors containing a pre- or leader or signal sequence region, a pro- sequence region and mature protein.
• the nucleotide sequences of pre- and/or pro- regions can also be used to construct chimeric genes with the coding sequences of other growth factors or proteins and, similarly, chimeric genes can be constructed from the coding sequence of the subject nerve growth factor coupled to sequences encoding pre- and/or pro- regions from genes for other growth factors or proteins (Booth et al., Gene 146:303-8 ( 1994); Ibanez, Gene 146:303-8 (1994); Storici et al., FEBS Letters 337:303-7 (1994): Sha et al, J Cell Biol 114:827-839 (1991), which are incorporated by reference), Such chimeric proteins can exhibit altered production or expression of the active protein species.
• Particularly exemplar ⁇ ' nerve growth factors for use with the invention include the GDNF family of GDNF, neurturin (NRTN), persephin and artemin (e.g., for treatment of Parkinson's disease).
• NRTN neurturin
• persephin e.g., for treatment of Parkinson's disease
• artemin e.g., for treatment of Parkinson's disease.
• the coding sequences for these nerve growth factors are well known to, or readily identifiable by, those of ordinary skil l in the art and need not be repeated here.
• GDNF GDNF
• Gene ID 2668 human GDNF
• GDNF GDNF family
• polypeptide sequences set forth in: www.ncbi, nlm.nih.gov/proteiri/CAG46721.1 (Accession No, CAG46721; human GDNF); wvvw.ncbi.nlm.nih.gov/protein/EAW69140.1 (Accession No. EAW69140; human neurturin— see also, US Patent No, 6,090,778);
• Human nerve growth factors are preferred for use in therapy of human disease according to the invention due to their relatively low immunogenicity as compared to allogenic growth factors. However, other nerve growth factors are known which may also be suitable for use in the invention with adequate testing of the kind described herein. [0044] Recombinant Expression Vectors
• the strategy for transferring genes into target cells in vivo includes the following basic steps: (1) selection of an appropriate transgene or transgenes whose expression is correlated with CNS disease or dysfunction; (2) selection and development of suitable and efficient vectors for gene transfer; (3) demonstration that in vivo transduction of target ceils and transgene expression occurs stably and efficiently; (4) demonstration that the in vivo gene therapy procedure causes no serious deleterious effects; and (5) demonstration of a desired phenotypic effect in the host animal.
• preferred vectors for use in the methods of the present invention are viral and non-viral vectors, such as DNA vectors (e.g, adeno- associated vims (AAV) and adenovirus, especially the former).
• the vector selected should meet the following criteria: 1) the vector must be able to infect targeted cells and thus viral vectors having an appropriate host range must be selected; 2) the transferred gene should be capable of persisting and being expressed in a cell for an extended peri od of time (without causing cell death) for stable maintenance and expression in the cell; and 3) the vector should do little, if any , damage to target cells.
• vectors known to have this capability include DNA viruses such as adenoviruses, adeno- associated virus (AAV), and certain RNA viruses such as HIV-based lentiviruses, feline immunodeficiency vims (FIV) and equine immunodeficiency virus (EIV).
• DNA viruses such as adenoviruses, adeno- associated virus (AAV), and certain RNA viruses such as HIV-based lentiviruses, feline immunodeficiency vims (FIV) and equine immunodeficiency virus (EIV).
• Other vectors with this capability include herpes simplex virus (HSV).
• Construction of vectors for recombinant expression of nervous system growth factors for use in the invention may be accomplished using conventional techniques which do not require detailed explanation to one of ordinary ski ll in the art.
• a specific protocol for construction of an AAV vector useful in the invention is illustrated in the Examples.
• Use of the AAV2 serotype is exemplified; however, other known AAV serotypes might be employed.
• general techniques for vector constmction those of ordinary skill may wish to consult Maniatis et al., in Molecular Cloning: A. Laboratory Manual, Cold Spring Harbor Laboratory, (NY 1982).
• Promoter and enhancer regions of a number of viral and non-viral promoters have also been described (e.g., as to non-viral promoters, Schmidt et al, Nature 314:285 (1985); Rossi and de Crombrugghe, Proc. Natl. Acad. Sci. USA 84:5590-5594 (1987)).
• Methods for maintaining and increasing expression of transgenes in quiescent cells include the use of promoters including collagen type I (1 and 2) (Prockop and Kivirikko, TV " . Eng. J. Med. 311 :376 (1984); Smith and Niles, Biochem. 19: 1820 (1980); de Wet et al, J. Biol. Chem., 258: 14385 (1983)), SV40, chicken ⁇ -actin, and LTR promoters.
• Transgene expression may also be increased for long term stable expression using cytokines to modulate promoter activity.
• cytokines to modulate promoter activity.
• TGF transforming growth factor
• IL interlenkin
• INF interferon
• TGF Tumor necrosis factor
• TGF1 up regulate, and may be used to control, expression of transgenes driven by a promoter.
• Other cytokines that may prove useful include basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).
• Collagen promoter with die collagen enhancer sequence can also be used to increase transgene expression by suppressing further any immune response to the vector which may be generated in a treated brain notwithstanding its immune-protected status.
• anti-inflammatory agents including steroids, for example
• dexamethasone may be administered to the treated host immediately after vector composition delivery and continued, preferably, until any cytokine-mediated inflammatory response subsides.
• An immunosuppression agent such as cyclosporin may also be administered to reduce the production of interferons, which downregulat.es LTR promoter and Coli(E) promoter-enhancer, and reduces transgene expression.
• host cells such as fibroblasts and stem cells (including, without limitation, embryonic stem cells and adult induced pluripotent or totipotent stem cells), may also be utilized for ex vivo delivery of nerve growth factor encoding expression vectors to the brain.
• host cells such as fibroblasts and stem cells (including, without limitation, embryonic stem cells and adult induced pluripotent or totipotent stem cells)
• Preparation of donor cells containing a nerve growth factor transgene encoding expression vector is described in detail in commonly assigned U.S. Pat. No. 5,650,148, the contents of which are incorporated herein.
• the prepara tion is carried out by modifying donor cells by introduction of a vector containing a transgene or transgenes encoding a nerve growth factor protein, which cells are in turn grafted onto the target tissue.
• the preparation of donor cells is known to those of ordinary skill in the art. Briefly, the strategy for transferring genes into donor cells in vitro includes the following basic steps: (1) selection of an appropriate transgene or transgenes whose expression is correlated with CNS disease or dysfunction; (2) selection and development of suitable and efficient vectors for gene transfer; (3) preparation of donor cells (e.g., from primary cultures or from established ceil lines); (4) demonstration that the donor implanted ceils expressing the new function are viable and can express the transgene products(s) stably and efficiently; (5) demonstration that the transplantation causes no serious deleterious effects; and (6) demonstration of a desired phenotypic effect in the host animal.
• nerve growth factor encoding expression vectors may be placed into a pharmaceutically acceptable suspension, solution or emulsion. Similar carriers may be employed for deli very of nerve growth factor protein.
• pharmaceutically acceptable carriers may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
• non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
• Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media
• Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
• Intravenous vehicles include fluid and nutrient replenisbers, electrolyte replenishes (such as those based on Ringer's dextrose), and the like.
• compositions of the present in vention can be administered by any suitable route known in the art including for example intra venous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation.
• administration can be by injection or infusion into the cerebrospinal fluid (CSF).
• CSF cerebrospinal fluid
• administration can be with one or more agents capable of promoting penetration of the molecule across the blood-brain barrier.
• the carrier can also contain other pharmaceuticaily-acceptable excipients for modifying or maintaining the pH, osmolality, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
• the carrier may contain still other pharmaceuticaily-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier.
• excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion into the cerebrospinal fluid by continuous or periodic infusion.
• Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
• composition of nerve growth factor transgenes may be lyophilized using means well known in the art, for subsequent reconstitution and use according to the invention.
• Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formul ation and the route of administration used.
• the specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied.
• the dose wall also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity disclosed herein in assay preparations of target cells. Exact dosages are determined in conjunction with standard dose-response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant
• a colloidal dispersion system may also be used for targeted gene delivery.
• Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
• Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 ⁇ can encapsulate a substantial percentage of an aqueous buffer containing large macro molecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et aL Trends Biochem. Sci., 6:77, 1981).
• LUV large unilamellar vesicles
• a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1 ) encapsulation of the genes encoding the antisense polynucleotides at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells: (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988).
• the composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
• the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
• Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidyiethanolamine, sphingolipids, cerehrosides, and gangliosides.
• diacylphosphatidylglycerols where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
• Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and
• the targeting of liposomes can be classified based on anatomical and mechanistic factors.
• Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific.
• Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries.
• RES reticulo-endothelial system
• Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achie ve targeting to organs and cell types other than the naturally occurring sites of localization.
• a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
• the surface of the targeted gene delivery system may be modified in a variety of ways.
• lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer, V arious linking groups can be used for joining the lipid chains to the targeting ligand.
• non-human primate subjects the process of aging simulates the neurological changes in the brain experienced in aging humans.
• a non-aged animal model that models Parkinson's disease with a high degree of integrity is l -methyl-4-phenyl- 1 ,2,3,6- tetrahydro pyridine (MPTP) treated monkeys (see, e.g., Kordower et al, Exp, Neurology, 160:1-16 (1999)).
• MPTP l -methyl-4-phenyl- 1 ,2,3,6- tetrahydro pyridine
• Such treatment results in extensive degeneration of dopaminergic neurons in the substantia nigra, with concomitant behavioral modification and motor deficits (Example 2).
• Data demonstrating the use and efficacy of the method of the invention in aged non-human primates as well as MPTP treated animals has been previously demonstrated. Further, as discussed in the Examples, data demonstrating the use of the method of the invention in humans has also been demonstrated.
• Clinical evaluation and monitoring of treatment can be performed using the in vivo imaging techniques described above as well as through biopsy and histological analysis of treated tissue.
• neuronal numbers can be quantified in a tissue sample with respect to, for example, TH immunoreactivity.
• Clinical evaluation and monitoring of amelioration of symptoms of disease may be assessed and observed at various time points post-treatment.
• amelioration is observable at or after 30 days, 60 days, 90 days, 180 days, 12 months, 18 months, 24 months, 48 months, 72 months, and longer post-treatment.
• Patients may be assessed with the URPDRS in the practically-defined off state (around 12 h after the last dose of antiparkinsonian drug) and in the best on state (best response to morning dose of antiparkinsonian drug). Fahn et al, Recent Developments in Parkinson 's disease, Macmillan Healthcare Information, Florham Park, NJ 2: 153— -163 (1987).
• the motor subscale (part 3) of the UPDRS may also be done in the practically- defined off state at each visit after 12 months.
• Home diary assessment of motor state, timed motor tests, the dyskinesia rating scale, and the clinical global impression assessment may be done periodically; e.g., at baseline, 6 months, and at each visit thereafter.
• PDQ Parkinson's disease questionnaire
• SF short form
• AAV2-Neurturin Gene Therapy (AAV-neurturin): Differences Between Parkinson's disease and Nonhuman Primate Brains
• a Phase 1 trial in 12 moderately advanced Parkinson's disease patients identified no safety issues, while suggesting possible improvement on several measures of motor function. Marks et ah, Lancet Neurol 7:400-408 (2008). As noted elsewhere above, a subsequent double-blind-controlled Phase 2 trial in 58 subjects further supported the safety of AAV-neurturin, but failed to discern any benefit compared to sham surgery on the primary endpoint (UPDRS-motor-' Off ' at 12- months). However, several secondary endpoints suggested modest clinical benefit, while no measurement favored sham-control.
• AAV-neurturin AAV2-NTRN
• AAV-neurturin was utilized which is an AAV2 vector genetically engineered to express only human NRTN. Gasmi et al., Mol Ther 15:62-68 (2007).
• AAV-neurturin (5.4 x 10 11 vg), distributed via four separate needle tracts per hemisphere and two deposits per tract.
• One subject (#1802) was a 59- year-old man who had been diagnosed 10.2 years earlier and died from a pulmonary embolism on Day 90, post- AAV-neurturin treatment.
• the second subject (#1904) was a 73-year-old man who had been diagnosed 9,5 years earlier and suffered a fatal myocardial infarction on Day 47 post-AAV-neurturin.
• Subject #1904 had baseline UPDRS motor off/on scores of 34/21 and a self-reported diary off time of 1.33 hr/day.
• Subject #1802 had baseline UPDRS motor off/on scores of 51/34 and a self-reported diary off time of 4.7 hr/day.
• Brains were fixed following postmortem intervals of 6 and 13 hours, respectively, with a modified Zamboni's solution and sectioned at 40 ⁇ . Only one hemisphere was available from the second subject. Brains were stained for NRTN and tyrosine hydroxylase (TH), using non-Parkinson's disease aged humans as positive controls. Both brains demonstrated typical pathological features of Parkinson's disease, with marked loss of cells in the substantia nigra, coupled with multiple a-synuciein- stained Lewy bodies.
• TH tyrosine hydroxylase
• TWO young monkeys ( ⁇ 5 years old) were administered 0,5 ⁇ 10' 3 vg of AAV- neurturin per hemisphere in two deposits in a total volume of 25 ⁇ ,, within the striatum and euthanized 1 month later, and an additional young monkey received 1.0 x 10 n vg per hemisphere of AAV-neurturin and was euthanized three months later.
• Three aged monkeys (22-25 years old) were administered 3 x 10 11 vg of AAV- neurturin, unilaterally, via five deposits (30 ⁇ , each) distributed throughout the striatum and euthanized 8 months later.
• Immunoperoxidase labeling was used to visualize NRTN and TH within the human and nonhuman primate striatum and substantia nigra as described in Kordower et al, Ann Neurol,, 60:706-715 (2006).
• a stereologic sampling method, combined computer-assisted imaging software, and the Cavalieri method was used to quantify volume of NRTN expression in the primate striatum.
• two different methods were independently employed to compute volume of NRTN expression in putamen.
• One method employed volumetric analyses based on stereological sampling of 6 to 1 1 sections throughout each putamen. The second method sampled all sections found to contain putamen (19-29 sections per each case).
• the percent of NRTN expressed within each targeted structure was then calculated based on the volumes of the entire target, using values from in-house human and primate histological sections and MRI scans, as well as published values to provide an estimate of nonhuman primate caudate/putamen of ⁇ 1200 mm 3 /hemisphere and human putamen of -4000 mm 3 /hemisphere).
• NRTN Expression For the two Parkinson's disease cases, neurturin expression was quantified in the three available hemispheres 7 weeks or 3+ months post-AAV2-NRTN treatment following death from unrelated causes, NRTN-immunoreactivity was seen in all hemispheres, restricted to the targeted putamen. Two independent, blinded analyses conservatively estimated NRTN protein covered -T5% of the entire putamen by volume. The dosing paradigm employed for AAV -neurturin was intended to distribute the AAV2- NRTN as widely as possible throughout the putamen, while limiting spread to surrounding sites to reduce potential side effects. Kordower et al.
• Tw o young monkeys were administered a particularly low dose of AAV- neurturin (less than 4% of the human Parkinson's disease dose, by relative volume of each targeted stmcture) and euthanized only 1 month later, to provide a conservative estimate of the early-onset bioactivity of AAV-neurturin with low NRTN expression levels.
• the volume of striatal NRTN expression in these two monkeys was estimated to be only 5,6% and 1.8%.
• NRTN retrograde labeling was easily seen within substantia nigra perikarya and anterogradely transported NRTN -1- fibers were seen coursing within the globus pailidus and substantia nigra pars reticulata.
• NRTN was estimated to cover 4, 19, and 25% (mean 16%) of the entire striatum, by volume. Despite the variation in striatal NRTN coverage, NRTN was consistently observed in the substantia nigra, in contradistinction to the Parkinson's disease tissue.
• a mean of -13% of the entire putamen by volume stained for NRTN (8%, 8%, 13%, 15%, and 23%, respectively). Similar to other primate studies, extensive evidence for retrograde!' and anterogradely transported NRTN within the substantia nigra pars compacta and reticulata, respectively, was observed.
• TH-induction following AAV2-NRTN treatment in monkeys was consistently observed in nigrostriatal neurons, was generally robust and typically mirrored the extent and intensity of NRTN expression in the striatum and the nigra. This was evident even at 28 days postdosing, with substantially lower doses, and less NRTN expression, than in the Parkinson's disease cases. Finally, in further contrast to the TH response in Parkinson's disease brain, the area of TH induction in monkey striatum consistently exceeded that of NRTN staining in adjacent sections.
• AAV2-NRTN AAV-neurturin
• Parkinson's disease putamen did not result in labeling of the neuronal cell bodies in the substantia nigra, despite putaminal coverage more than sufficient to produce this response in young, aged, and MPTP-parkinsonian monkeys.
• This distinction suggests a profound difference in the status and function of nigrostriatal neurons in advanced Parkinson's disease versus typical animal models used for Parkinson's disease translational research. As illustrated in the following Examples, the invention has taken this difference into account.
• TH is a major enzyme for dopamine synthesis and a surrogate for functional enhancement of degenerating dopamine neurons.
• the robust TH signal in nonhuman primates in response to AA.V-NRTN is in marked contrast to the limited signal in Parkinson's disease in a number of important ways, including: (1) the intensity of TH signal was far less in Parkinson's disease, (2) it occurred with less frequency and reliability, and (3) it occurred within a much smaller portion of the putamen, well within the region of NRTN
• AAV2 vector was genetically engineered to express only human neurturin (N RTT as discussed in Example I and Gasmi et al., Mol Ther 15:62-68 (2007). It provides targeted and sustained delivery of neurturin (N RTN) to cells of the brain.
• N RTN neurturin
• AAV2-neurturin is being utilized in a Phase 2b multi-center, sham-surgery, double-blinded controlled trial in advanced Parkinson's disease initiated in October 2010. Advanced patients can be expected to have greater degeneration of their nigrastriatal transport pathways than patients in earlier stages of the disease, in which the invention is therefore expected to readily demonstrate efficacy. As of this filing, approximately 20 percent of the 52 subjects have undergone either CERE- 120 administration or sham surgery, with many others enrolled and awaiting surgery, The protocol employs the present invention along the parameters outlined below.
• Stereotactic surgery is done with neuroimaging to plan injection trajectories. Patients are anaesthetized with deep propofol sedation. For patients assigned to active treatment, a gene transfer procedure is done with AAV2 as a vector to deliver DNA- encoding neurturin to the putamen. AAV2-neurturin in a total brain dose of 5.4xlO n vector genomes is administered bilaterally. In these patients, the substantia nigra is directly targeted at two injection sites (reached through burr holes) per side, with a higher dose delivered into the putamen according to the invention, with putaminal delivery being made to three injection sites per side.
• Phase 2b trial was initiated following the successful dosing of six patients in a Phase 1 safety trial that evaluated, for the first time, the feasibility and safety of targeting the substantia nigra with AAA'' -neuxturin, as well as administering a larger dose than had been tested previously.
• the Phase 1 safety database currently reflects follow-up periods ranging from seven to 13 months per patient, and shows no serious adverse events (SAEs) in any of the six subjects dosed, including no effect on weight. Ail patients were discharged from the hospital within 48 hours of surgery, as planned. Consistent with the safety profile observed in the Phase 1 trial, no AAV-neurturin-related serious adverse events have been observed in the ongoing Phase 2 trial.

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