US20060051322A1 - Method for therapy of neurodegenerative disease of the brain - Google Patents

Method for therapy of neurodegenerative disease of the brain Download PDF

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US20060051322A1
US20060051322A1 US10/332,306 US33230605A US2006051322A1 US 20060051322 A1 US20060051322 A1 US 20060051322A1 US 33230605 A US33230605 A US 33230605A US 2006051322 A1 US2006051322 A1 US 2006051322A1
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neurotrophin
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ngf
neurons
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Mark Tuszynski
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Priority to US12/259,950 priority patent/US8486385B2/en
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    • 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
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
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    • 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
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    • 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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    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14171—Demonstrated in vivo effect

Definitions

  • the invention relates to methods for treatment of neurodegenerative disease and methods for delivery of therapeutic neurotrophins into the mammalian brain.
  • Neurotrophins play a physiological role in the development and regulation of neurons in mammals. In adults, basal forebrain cholinergic neurons, motor neurons and sensory neurons of the CNS retain responsiveness to neurotrophic factors and can regenerate after loss or damage in their presence. For this reason, neurotrophins are considered to have great promise as drugs for the treatment of neurodegenerative conditions such as Alzheimer's Disease (AD), Parkinson's Disease (PD), amyotrophic lateral sclerosis (ALS), peripheral sensory neuropathies and spinal cord injuries.
  • AD Alzheimer's Disease
  • PD Parkinson's Disease
  • ALS amyotrophic lateral sclerosis
  • the invention provides a clinically useful protocol for delivery of neurotrophins into the mammalian brain.
  • the invention is particularly useful in treating neurodegenerative conditions in primates, in whom neurotrophins delivered according to the invention stimulate growth of neurons and recovery of neurological function.
  • the invention consists of methods for intraparenchymal delivery of neurotrophins to defective, diseased or damaged cells in the mammalian brain.
  • the invention provides a specific protocol for use in genetically modifying target cholinergic neurons (“target cells”) to produce a therapeutic neurotrophin.
  • target cells target cholinergic neurons
  • the genetic modification of target cells is achieved by in vivo transfection of neurons targeted for treatment, or by transfection of cells neighboring these target neurons (neurons or glia), with a recombinant expression vector for expression of the desired neurotrophin in situ.
  • each delivery site is situated no more than about 500 ⁇ m from a targeted cell and no more than about 10 mm from another delivery site.
  • the total number of sites chosen for delivery of each unit dosage of neurotrophin will vary with the size of the region to be treated.
  • each unit dosage of neurotrophin will comprise 2.5 to 25 ⁇ l of an expression vector composition, wherein the composition includes a viral expression vector in a pharmaceutically acceptable fluid (“neurotrophic composition”) and provides from 10 10 up to 10 15 NGF expressing viral particles per ml of neurotrophic composition.
  • neurotrophic composition is delivered to each delivery site in the brain by injection through a surgical incision, with delivery to be completed within about 5-10 minutes, depending on the volume of neurotrophic composition to be provided.
  • This targeted, regionally specific protocol for nervous system growth factor delivery avoids limitations imposed by diffusion of substances across the blood-brain barrier and through central nervous system (CNS) parenchyma, while avoiding potential adverse effects of neurotrophic factors delivered intact in a non-directed manner to the CNS.
  • CNS central nervous system
  • FIG. 1 is a reprint of the nucleotide sequence coding for human beta nerve growth factor as shown in GENBANK Accession No. X52599.
  • FIG. 2 is a reprint of the nucleotide sequence coding for human NT-3 as shown in GENBANK Accession No. E07844.
  • the invention identifies and defines the required parameters of a method for successful regeneration of neurons in the brain with neurotrophins, especially the neurons whose loss is associated with neurodegenerative conditions with impairment of cognition such as AD.
  • 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 neurotrophic factors.
  • CNS neurons which retain responsiveness to neurotrophic factors into adulthood include the cholinergic basal forebrain neurons, the entorhinal cortical neurons, the thalamic neurons, the locus coeruleus neurons, the spinal sensory neurons and the spinal motor neurons.
  • Abnormalities within the cholinergic compartment of this complex network of neurons have been implicated in a number of neurodegenerative disorders, including AD, Parkinson's disease, and amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease).
  • the cholinergic basal forebrain is a particularly suitable target tissue.
  • magnocellular neurons Ch1-Ch4 provide cholinergic innervation to the cerebral cortex, thalamus and basolateral nucleus of the amygdala.
  • neurons in the Ch4 region which have nerve growth factor (NGF) receptors undergo marked atrophy as compared to normal controls (see, e.g., Kobayashi, et al., Mol. Chem. Neuropathol., 15:193-206 (1991)).
  • NGF nerve growth factor
  • neurotrophins prevent sympathetic and sensory neuronal death during development and prevents cholinergic neuronal degeneration in adult rats and primates (Tuszynski, et al., Gene Therapy, 3:305-314 (1996)).
  • the resulting loss of functioning neurons in this region of the basal forebrain is believed to be causatively linked to the cognitive decline experienced by subjects suffering from neurodegenerative conditions such as AD (Tuszynski, et al., supra and, Lehericy, et al., J. Comp. Neurol., 330:15-31 (1993)).
  • basal forebrain neuronal loss occurs over an intraparenchymal area of approximately 1 cm in diameter.
  • treatment with vector composition at upwards of 10 separate in vivo gene vector delivery sites is desirable.
  • the affected areas of the brain will likely be smaller such that selection of fewer delivery sites (e.g., 5 or fewer) will be sufficient for restoration of a clinically significant number of cholinergic neurons.
  • in vivo gene delivery sites are selected so as to cluster in an area of neuronal loss. Such areas may be identified clinically using a number of known techniques, including magnetic resonance imaging (MRI) and biopsy. In humans, non-invasive, in vivo imaging methods such as MRI will be preferred. Once areas of neuronal loss are identified, delivery sites are selected for stereotaxic distribution so each unit dosage of NGF is delivered into the brain at, or within 500 ⁇ m from, a targeted cell, and no more than about 10 mm from another delivery site.
  • MRI magnetic resonance imaging
  • a further parameter defined by the invention is the dosage of neurotrophin to be delivered into the target tissue.
  • unit dosage refers generally to the concentration of neurotrophin/ml of neurotrophic composition.
  • the neurotrophin concentration is defined by the number of viral particles/ml of neurotrophic composition.
  • each unit dosage of neurotrophin will comprise 2.5 to 25 ⁇ l of a neurotrophic composition, wherein the composition includes a viral expression vector in a pharmaceutically acceptable fluid and provides from 10 10 up to 10 15 NGF expressing viral particles per ml of neurotrophic composition.
  • the neurotrophic composition is delivered to each delivery cell site in the target tissue by microinjection, infusion, scrape loading, electroporation or other means suitable to directly deliver the composition directly into the delivery site tissue through a surgical incision.
  • the delivery is accomplished slowly, such as over a period of about 5-10 minutes (depending on the total volume of neurotrophic composition to be delivered).
  • the direct delivery method employed by the invention obviates a limiting risk factor associated with in vivo gene therapy; to wit, the potential for transfection of non-targeted cells with the vector carrying the NGF encoding transgene.
  • delivery is direct and the delivery sites are chosen so diffusion of secreted NGF takes place over a controlled and pre-determined region of the brain to optimize contact with targeted neurons, while minimizing contact with non-targeted cells.
  • a viral vector with an operable neurotrophin encoding transgene has been shown to express human neurotrophin after delivery to the brain and to the CNS for up to 12 months.
  • the invention provides a chronically available source for neurotrophin 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.
  • Known nervous system growth factors include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5. (NT-4/5), neurotrophin-6 (NT-6), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), the fibroblast growth factor family (FGF's 1-15), leukemia inhibitory factor (LIF), certain members of the insulin-like growth factor family (e.g., IGF-1), the neurturins, persephin, the bone morphogenic proteins (BMPs), the immunophilins, the transforming growth factor (TGF) family of growth factors, the neuregulins, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and others.
  • NGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • NT-4/5 neurotrophin-6
  • CNTF ciliary neurotrophic factor
  • GDNF glial
  • NGF and NT-3 in particular have been tested with promising results in clinical trials and animal studies (see, e.g., Hefti and Weiner, Ann Neurol., 20:275-281 (1986); Tuszynki and Gage, Ann. Neurol., 30:625-636 (1991); Tuszynski, et al., Gene Therapy, 3:305-314 (1996) and Blesch and Tuszynski, Clin. Neurosci., 3:268-274 (1996)).
  • NGF and NT-3 for treatment of the Ch4 region, as in AD are preferred for use in the invention.
  • Human (h) NGF and hNT3 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.
  • other nervous system growth factors are known which may also be suitable for use in the invention with adequate testing of the kind described herein.
  • Coding polynucleotides for hNGF and hNT3 are known, as are coding sequences for neurotrophins of other mammalian species (e.g., mouse, in which the coding sequence for NGF is highly homologous to the human coding sequence).
  • a cDNA including the coding sequence for hNGF is reported in GenBank at E03015 (Kazuo, et al., Japanese Patent Application No.
  • 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 cells 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.
  • 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 period 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 and feline immunodeficiency virus (FIV).
  • AAV adeno-associated virus
  • FMV feline immunodeficiency virus
  • Other vectors with this capability include herpes simplex virus (HSV).
  • a HIV-based lentiviral vector has recently been developed which, like other retroviruses, can insert a transgene into the nucleus of host cells (enhancing the stability of expression) but, unlike other retroviruses, can make the insertion into the nucleus of non-dividing cells.
  • This lentiviral vector has been shown to stably transfect brain cells after direct injection, and stably express a foreign transgene without detectable pathogenesis from viral proteins (see, Naldini, et al., Science, 272:263-267 (1996), the disclosure of which is incorporated by reference).
  • Adenoviruses and AAV have been shown to be quite safe for in vivo use and have been shown to result in long-term gene expression in vivo; they are therefore preferred choices for use in the methods of the invention, where safety and long-term expression of nervous system growth encoding transgenes (persisting for longer than necessary to stimulate regrowth of injured or diseased neurons) is necessary.
  • Those of ordinary skill in the art are familiar with the techniques used to construct adenoviral and AAV vectors and can readily employ them to produce vector compositions useful in the claimed invention (for reference, see, e.g., Straus, The Adenovirus, Plenum Press (NY 1984), pp.
  • Lentiviral-based vectors such as HIV and FIV are currently at earlier stages of development but also are attractive candidates for in vivo gene therapy based upon stability of expression in vivo and safety profiles.
  • Herpes viruses, alpha viruses and pox viruses are also well-characterized virus vectors which may be applied to the methods of the invention.
  • adeno-associated vectors are an especially attractive choice for their lack of pathogenicity and ability to insert a transgene into a host genome.
  • Non-viral delivery methods are also an option for use in the methods of the invention.
  • the plasmid in a “naked” or lipid-complexed form
  • lipoplexes liposome complexed nucleic acids
  • amino acid polymer complexes with nucleic acids and artificial chromosomes are all non-viral gene delivery agents which are demonstrably able to transduce cells and deliver a foreign transgene.
  • Synthetic in vivo gene therapy vectors are also an option for use in the methods of the invention.
  • 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 skill in the art. For review, however, those of ordinary skill may wish to consult Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, (NY 1982).
  • construction of recombinant expression vectors employs standard ligation techniques.
  • the ligation mixtures may be used to transform a host cell and successful transformants selected by antibiotic resistance where appropriate.
  • Vectors from the transformants are prepared, analyzed by restriction and/or sequenced by, for example, the method of Messing, et al., (Nucleic Acids Res., 9:309, 1981), the method of Maxam, et al., (Methods in Enzymology, 65:499, 1980), or other suitable methods which will be known to those skilled in the art. Size separation of cleaved fragments is performed using conventional gel electrophoresis as described, for example, by Maniatis, et al., (Molecular Cloning, pp. 133-134, 1982).
  • Transcription initiation is an early and critical event in gene expression. This depends on the promoter and enhancer sequences and is influenced by specific cellular factors that interact with these sequences.
  • the transcriptional unit of many prokaryotic genes consists of the promoter and in some cases enhancer or regulator elements (Banerji et al., Cell 27:299 (1981); Corden et al., Science 209:1406 (1980); and Breathnach and Chambon, Ann. Rev. Biochem. 50:349 (1981)).
  • LTR long terminal repeat
  • MMV Moloney murine leukemia virus
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • promoter and enhancer regions of a number of non-viral promoters have also been described (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, N. Eng. J. Med. 311:376 (1984); Smith and Niles, Biochem. 19:1820 (1980); de Wet et al., J. Biol. Chem., 258:14385 (1983)), SV40 and LTR promoters.
  • an enhancer sequence may be used to increase the level of transgene expression. Enhancers can increase the transcriptional activity not only of their native gene but also of some foreign genes (Armelor, Proc. Natl. Acad. Sci. USA 70:2702 (1973)).
  • collagen enhancer sequences are used with the collagen promoter 2(I) to increase transgene expression.
  • the enhancer element found in SV40 viruses may be used to increase transgene expression. This enhancer sequence consists of a 72 base pair repeat as described by Gruss et al., Proc. Natl. Acad. Sci.
  • Transgene expression may also be increased for long term stable expression using cytokines to modulate promoter activity.
  • cytokines have been reported to modulate the expression of transgene from collagen 2(I) and LTR promoters (Chua et al., connective Tissue Res., 25:161-170 (1990); Elias et al., Annals N.Y. Acad. Sci., 580:233-244 (1990)); Seliger et al., J. Immunol. 141:2138-2144 (1988) and Seliger et al., J. Virology 62:619-621 (1988)).
  • TGF transforming growth factor
  • IL interleukin-1
  • INF interferon
  • TGF Tumor necrosis factor
  • TGF1 TGF-1
  • TGF1 TGF-1
  • TGF1 TGF-1
  • TGF1 TGF-1
  • TGF1 TGF1
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • Collagen promoter with the 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 downregulates LTR promoter and Coll(E) promoter-enhancer, and reduces transgene expression.
  • neurotrophin encoding expression vectors may be placed into a pharmaceutically acceptable suspension, solution or emulsion.
  • Suitable mediums include saline and liposomal preparations.
  • pharmaceutically acceptable carriers may include sterile aqueous of 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 replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • a composition of neurotrophin transgenes may be lyophilized using means well known in the art, for subsequent reconstitution and use according to the invention.
  • 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 ⁇ m can encapsulate a substantial percentage of an aqueous buffer containing large macro molecules.
  • LUV large unilamellar vesicles
  • 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).
  • liposomes In addition to mammalian cells, liposomes have been used for delivery of operatively encoding transgenes in plant, yeast and bacterial cells.
  • 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.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are 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 distearoylphosphatidylcholine.
  • 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 achieve 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.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand.
  • direct delivery of a neurotrophic composition 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)); electropotation (see, e.g., Andreason and Evans, Biotechniques, 6:650-660 (1988)); infusion, chemical complexation with a targeting molecule or co-precipitant (e.g., liposome, calcium), and microparticle bombardment of the target tissue (Tang, et al., Nature, 356:152-154 (1992)).
  • a targeting molecule or co-precipitant e.g., liposome, calcium
  • Example III In non-human primate subjects (Example III), the process of aging simulates the neurological changes in the brain experienced in aging humans.
  • rats and primates such transections cause retrograde degeneration of cholinergic and non-cholinergic cell bodies in the septal nucleus and nucleus basalis (Ch4 region) of the brain.
  • 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.
  • basal forebrain cholinergic neuronal numbers can be quantified in a tissue sample using, for example, anti-neurotrophin antibody (for immunoassay of secreted neurotrophin) or NGF-receptor (p75) and choline acetyltransferase (ChAT) for labeling of neurons.
  • anti-neurotrophin antibody for immunoassay of secreted neurotrophin
  • p75 NGF-receptor
  • ChAT choline acetyltransferase
  • an expression cassette was cloned containing the following elements: 1) cytomegalovirus promoter (CMV ie ); 2) a multiple cloning site; 3) an internal ribosome entry site followed by the coding sequence for the active, ⁇ -sequence of human nerve growth factor (NGF) or the enhanced form of green fluorescent protein (EGFP); and, 4) a SV40 polyadenylation sequence.
  • CMV ie cytomegalovirus promoter
  • NVF human nerve growth factor
  • EGFP enhanced form of green fluorescent protein
  • the complete cassette was cloned into the vector psub201 (American Type Culture Collection) after XbaI digestion to remove the AAV coding sequences.
  • the coding sequence for human NGF (see, GENBANK Accession No. X52599) was inserted into the multiple cloning site of psub-CXIE resulting in the vector psub-CXIE-NGF.
  • This vector termed psub-CXIE, was used to prepare control GFP expressing virus particles.
  • this vector was used for the production of particles coding for NGF and GFP.
  • Recombinant adeno-associated virus was produced by co-transfection of 18 ⁇ g expression plasmid psub-CXIE or psub-CXIE-NGF, 18 ⁇ g pXX2 and 54 ⁇ g pXX6 per 150 mm plate of subconfluent 293 cells. Transfected cells were harvested 48 h later and adeno-associated virus was purified by Iodixanol density gradient centrifugation and Heparin affinity chromatography. For virus concentration and buffer exchanges BiomaxTM 100K filters were used. Aliquots of virus were stored at ⁇ 80C. The number of viral particles was determined using Southern dot blotting.
  • AAV adeno-associated viral
  • Particles were injected over a time period of 3-5 min. into the right hemisphere at the following coordinates: AP ⁇ 0.3; ML ⁇ 0.5; DV ⁇ 6 from brain surface. The skin was closed and animals were allowed to survive for 2-4 weeks.
  • AAV vector delivery induced increasing zones of transfection with increasing concentration and volume of vector particles. Maximal levels of in vivo gene expression were achieved at the highest concentration of vector and highest volume of injection. Over the two week time period of this experiment, persistent in vivo gene expression was demonstrated. Gene expression was primarily manifested in neurons (>90%) as opposed to glia. No adverse effects of the injections were evident.
  • vector doses of 2.5 to 10 ⁇ l vector stock at a range of 10 10 -10 12 particles per ml were well-tolerated, resulted in optimal vector delivery to the host cholinergic neuronal system, and did not result in adverse events or undesired vector spread beyond the target neuronal nucleus.
  • NGF and enhanced GFP expression were evident for at least two weeks in vivo.
  • aged monkeys received intraparenchymal grafts of autologous fibroblasts genetically modified to produce and secrete human NGF, as previously described. Briefly, autologous fibroblasts obtained from skin biopsies were genetically modified in vitro to produce and secrete the active portion of human NGF. Transduction procedures were carried out using replication-incompetent retroviral vectors derived from Moloney murine leukemia virus (MLV). Transduced cells were selected by growth in the neomycin analog G418.
  • MMV Moloney murine leukemia virus
  • NGF biologically active NGF was verified by induction of neurite outgrowth from PC12 cells as described; production of NGF mRNA was determined by Northern blot; and amounts of NGF produced from cells were assayed by NGF ELISA specific for human NGF and sensitive to 5 pg/ml.
  • Optimal NGF-producing bulk clones were amplified to numbers sufficient for in vivo grafting by serial passaging. Cells were harvested by gentle trypsinization for in vivo grafting.
  • Monkeys underwent pre-operative MRI scans (see, Tuszynski, et al., Gene Therapy, 3:305-314, 1996) to visualize basal forebrain target grafting regions (see, Mesulam et al., J. Comp. Neurol., 214:170-197, 1983). After generating stereotaxic grafting coordinates from MRI scans, each monkey received intraparenchymal grafts of autologous NGF-secreting fibroblasts.
  • Stereotactic coordinates for surgery were generated from magnetic resonance images (MR) of the brain of each subject.
  • MR magnetic resonance images
  • the rostral and caudal boundaries of Ch4 were identified on each subject's MR scan, making reference to primate histological brain sections and to standard primate brain atlases.
  • the total rostral-caudal distance of Ch4 was measured on the MR scan, and five graft injection sites were chosen that were equally distributed over this rostral-caudal distance.
  • VD ventral-dorsal
  • ML medial-lateral
  • mice were placed into a primate stereotaxic apparatus and a midline scalp incision was used to expose the skull.
  • the AP and ML stereotaxic coordinates for the BFC system were used to define the margins of the craniotomy site.
  • a ML zero reference point was obtained by measuring the midpoint of the superior sagittal sinus.
  • the dura was incised and reflected to expose the pial surface.
  • the pial surface at each injection site was used as a VD zero reference point for that injection site.
  • Cells were injected at a concentration of 1.0 ⁇ 10 5 cells/ul (for a total of 10 million grafted cells per animal), a concentration that optimally maintains cells in suspension without clumping but sufficiently concentrated to maximize number of surviving cells in vivo. Monkeys survived for three months before sacrifice.
  • Some control aged subjects received intraparenchymal grafts as noted above. These grafted cells consisted either of autologous fibroblasts transduced to express the reporter gene beta-galactosidase (n 6 monkeys). Beta-gal production was assessed in vitro using a specific anti-beta-gal antibody. Cells were grafted into intraparenchymal sites in numbers identical to those described above for NGF graft recipients.
  • primates were preanesthetized with 25 mg/kg ketamine IM. They were then anesthetized with isoflurane administered by endotracheal intubation. Post-operatively animals were closely monitored, and received supportive care and appropriate analgesics when indicated. Animals were placed in the same primate stereotaxic apparatus (Crist Instruments) that was used to perform MRI scans. A midline scalp incision exposed the skull. A 2.5 ⁇ 5 cm sagittally oriented craniotomy was performed on each side of the hemicranium, and the dura was incised and reflected to expose sites for stereotaxically guided cell injections. Ten ul of cells were injected into each site through a 25 ga.
  • NGF In AD brains, NGF accumulates in regions of basal forebrain cholinergic neurons and is decreased in the basal forebrain, leading to the hypothesis that insufficient retrograde transport of NGF promotes the degeneration of basal forebrain cholinergic neurons observed in AD. In humans, basal forebrain cholinergic neuron dysfunction has been closely linked with age-related cognitive and memory impairment.
  • TrkA the p75 receptor collaborates with the TrkA receptor to form high-affinity binding sites for NGF.
  • activation of TrkA is sufficient for NGF to rescue axotomized cholinergic neurons
  • disruption of NGF binding to p75 reduces NGF binding to TrkA.
  • co-expression of the two receptors can lead to greater responsiveness to NGF.
  • loss of expression may lead to decreased responsiveness to NGF.
  • Expression of both p75 and TrkA is regulated by NGF, so that a loss of NGF signalling further reduces the amount of both p75 and TrkA.
  • p75 expression is a marker for NGF binding, basal forebrain cholinergic neuron dysfunction and cognitive impairment.
  • Coronal sections were cut on a freezing microtome set at 40 um. Every sixth section was processed for p75 immunoreactivity. Briefly, sections were washed thoroughly in Tris-buffered saline (TBS) and endogenous peroxidases were quenched by incubating in a 0.6% hydrogen peroxide solution. Sections were rinsed in TBS and then blocked using 5% donkey serum with 0.5% Triton X-100 in TBS (TBS++). Incubation in primary antibody (monoclonal diluted 1:100 in TBS++) occurred for 24 hours at room temperature.
  • TBS Tris-buffered saline
  • TBS++ Triton X-100
  • Sections were rinsed in TBS++, incubated in secondary antibody (biotinylated donkey-anti-mouse diluted 1:500 in TBS++) for 1 hour, rinsed again in TBS++, and then incubated for 90 minutes using a Vector ABC kit. p75-labeled neurons were then visualized using diaminobenzidine (DAB) as a chromogen. Sections were then mounted and coverslipped.
  • secondary antibody biotinylated donkey-anti-mouse diluted 1:500 in TBS++
  • Ch4i p75-labeled neurons were quantified in Ch4i neurons using stereological procedures. Ch4i was targeted in this study since this region is the principal site of origin of cholinergic projections to cortical regions that modulate memory.
  • Ch4 can be divided topographically into three subdivisions, the anterior (Ch4a), intermediate (Ch4i), and posterior (Ch4p).
  • the anterior subdivision is further divided into medial (Ch4am) and lateral (Ch4al) sectors, which are divided by a vascular structure or rarefication in the density of neurons.
  • Ch4am medial
  • Ch4al lateral
  • Ch4a the characteristic structure of Ch4i, begins to make its appearance.
  • the ansa peduncularis divides Ch4i into ventral (Ch4iv) and dorsal (Ch4id) components.
  • Ch4iv and Ch4id merge into a single nucleus embedded in the intersection of the globus pallidus, putamen, and optic tract.
  • the NeuroZoomTM stereology computer program running on an Apple Macintosh PowerPCTM and connected to a JavelinTM video camera mounted on an Olympus VanoxTM HBT-3 microscope was used to conduct stereology by the well-known West optical dissector method. Briefly, the region of interest (Ch4i) was outlined in NeuroZoom using a 1 ⁇ objective. Specific stereology parameters were then set in NeuroZoom as follows:
  • Ch4i neurons were counted using a 60 ⁇ high numerical aperture (1.40) oil objective. Cells were marked to be included in the count if they met the following criteria: 1) they were p75-labeled; 2) the soma was within the counting frame (or touching the inclusion boundary) but did not touch the exclusion boundary; 3) a clearly visible nucleus was present; and 4) the nucleus was best in focus within the inclusion volume (i.e., the top 12.5% and bottom 12.5% were excluded, and the nucleus was not in focus in either of these exclusion volumes). Multiple group comparisons were made by analysis of variance (ANOVA) with post-hoc analysis using Fisher's least squares difference.
  • ANOVA analysis of variance
  • the number of p75-labeled Ch4i neurons was compared between four groups of rhesus monkeys, two of which were unoperated and two of which received intraparenchymal grafts of genetically-modified fibroblasts.
  • Sections of brain tissue after humane sacrifice of the test animals were cut at 40 um intervals on a freezing microtome. Every sixth section was processed for Nissl stain or hematoxylin and eosin. Immunocytochemical labeling against amyloid was performed using an amyloid-specific monoclonal antibody (anti-A4). Sections lacking primary antibody were processed to verify specificity of labeling.
  • a representative section per subject was quantified from each of the following regions: temporal, frontal, cingulate, insular, parietal and occipital cortices; amygdala and hippocampus; and the intermediate division of the Ch4 region (Nucleus Basalis of Meynert). Sampled sections from each subject were closely matched in region and size. The total number of amyloid plaques per region was quantified and recorded. Observers were blinded to the identity of the tissue being quantified.
  • plaques In aged control animals, plaques typically showed a dense central core and a less dense surrounding halo of immunreactive deposition product, an appearance typical of “mature” plaques observed in AD. This immunolabeling pattern is consistent with previous reports in aged primate brain. However, no increase in amyloid labeling was observed in the aged, NGF-grafted brains, indicating that three months of intraparenchymal NGF delivery does not increase beta-amyloid plaque deposition in the aged primate brain. Thus, the benefits of NGF grafting in the brains of primates exhibiting AD symptoms can be acheived without risk of stimulating amyloid deposition in response to the graft trauma.

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US11446084B2 (en) * 2019-07-12 2022-09-20 Neuralink Corp. Laser drilling of pia mater
US12122844B2 (en) 2020-07-13 2024-10-22 Cornell University Treatment of brain cancers using central nervous system mediated gene transfer of monoclonal antibodies

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US12122844B2 (en) 2020-07-13 2024-10-22 Cornell University Treatment of brain cancers using central nervous system mediated gene transfer of monoclonal antibodies

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