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
• • 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 invention relates generally to the fields of molecular biology and virology, and in particular, to methods for using recombinant adeno-associated virus compositions that express superoxide dismutase or catalase-encoding DNA segments in the treatment of demyelinating disorders of the nervous system and most particularly in the optic nerve.
• the invention concerns the use of rAAV in a variety of investigative, diagnostic and therapeutic regimens. Methods are also provided for preparing rAAVs that express superoxide dismutase or catalase-encoding gene segments for use in such therapies.
• MS multiple sclerosis
• the socioeconomic impact is substantial. In a single year, 75% to 85% of MS patients were unemployed with estimated costs of patient care and lost income of approximately $10 billion.
• Current therapeutic strategies for MS include (Ruuls et al, 1995) nonspecific immunosuppressive drugs (corticosteroids, methotrexate), (Brett and Rumsby, 1993) reduction of lymphocyte trafficking (interferons), and (Guy et al., 1993) inhibition of lymphocyte receptor peptide and induction of anergy (copolymer). Interferons and copolymer must be given one to several times per week. Unfortunately, these agents have many side effects and in many cases are not effective therapy. 1.2.1 EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS
• EAE Experimental allergic encephalomyelitis
• MS human disease multiple sclerosis
• the optic nerve is a frequent site of involvement in EAE and MS (Rao, 1981; Beck et al, 1992; Rizzo and Lessell, 1988).
• myelin-forming oligodendroglia are the primary targets of immune-mediated injury (Raine, 1985; Steinman, 1991; Raine, 1997), although other cell types are affected also.
• Demyelinated axons exhibit hydropic degeneration with dissolution of microtubules and neurofilaments.
• BBB blood-brain barrier
• ROS reactive oxygen species
• NO nitric oxide
• ROS scavengers include catalase and superoxide dismutase.
• Superoxide dismutase dismutes superoxide to hydrogen peroxide (H 2 O ) and catalase detoxifies the H O 2 to H 2 O and O .
• Exogenous catalase has been previously shown to reduce disruption of the BBB and demyelination of the optic nerve in EAE (Guy et al, 1989a; Guy et al, 1989b).
• catalase protein Limitations to the use of catalase protein are several fold. First, catalase must be administered daily, even when conjugated to polyethylene glycol, to prolong the half life of the enzyme (Abuchowski et al, 1977; Guy et al, 1994b). Second, exogenous catalase is effective only during the periods of active BBB disruption when this high molecular weight protein is able to penetrate the central nervous system (CNS). Third, optic neuritis recurs in part due to the inability of the catalase protein to cross the BBB after integrity is restored by the catalase- mediated detoxification of H 2 O . 1.2.2 OXIDANTS
• the least equivocal example of oxidant-mediated lung injury in humans is the acute lung injury resulting from prolonged exposure to elevated levels of O , a frequent and important clinical problem.
• oxygen concentrations exceeding a fraction of inspired oxygen (FiO 2 ) of 0.5 (at 1.0 ami) the likelihood of developing pulmonary oxygen toxicity is accelerated (Warren and Ward, 1997).
• Critical mechanisms of hyperoxia toxicity include the increased production of superoxide anion, hydrogen peroxide, and other activated species that overwhelm antioxidant defenses in both lung endothelial and epithelial cells (Schraufstatter and Cochrane, 1997).
• Free radical-mediated injury is also a major cause of damage occurring in ischemic tissue after reperfusion (Heffher and Fracica, 1996). Ischemia-reperfusion injury has been reported after reexpansion of atelectatic lung, reperfusion of a mechanically occluded pulmonary artery, and lung transplantation (in which context poor tolerance to ischemia-reperfusion remains a major limitation) (Sarris et al, 1994).
• the central mechanism in ischemia-reperfusion lung injury involves neutrophil-endothelium interaction, with highly reactive free radicals generated from various sources in the reperfused tissues, including activated polymorphonuclear leukocytes and the xanthine oxidase system (Heffner and Fracica, 1996).
• a problem described in the report of gene transfer to the optic nerve is that only axons of the optic nerve and their cell bodies in the retina were labeled by the transferred reporter gene 22, whereas in experimental allergic encephalomyelitis and multiple sclerosis, oligodendroglia and endothelia are the targets of mediators of inflammation; thus, for neuroprotection of the optic nerve, the genes that encode for defenses against reactive oxygen species must be transferred to oligodendroglia or endothelia cell types.
• oligonucleotides to a host cell that express a polypeptide useful in the amelioration of such conditions, and in particular, administration of specific adenoviral-based polynucleotide constructs to a mammal are particularly desirable.
• the present invention addresses some of the limitations in the prior art by providing methods and compositions for ameloriation and treatment of effects associated with demyelinating disorders of the central nervous system; for example optic neuritis or allergic encephalomyelitis.
• the invention utilizes recombinant adeno-associated virus to deliver a therapeutic gene to transformed host cells, to protect such cells against the adverse effects of hydrogen peroxide. These effects include the classical demyelination and disruption of the blood brain barrier that are the hallmarks of many demyelating diseases.
• the inventors have described the use of nucleic acid segments that encode polypeptides including, but not limited to, superoxide dismutase (SOD), catalase and the like.
• the eye is known as a readily accessible site for gene transfer (Zolotukhin et al, 1996; Flannery et al, 1997).
• the present invention shows the successful viral-mediated gene transfer of catalase on suppression of EAE in the optic nerve.
• the invention related to suppression of oxidative injury by employing viral-mediated transfer of the human genes that reduce levels of hydrogen peroxide or superoxide in the optic nerves in accepted in vivo models.
• the optic nerve is a frequent site of involvement common to both experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS).
• Catalase activity was increased approximately two-fold each in various cell types of the optic nerve.
• the catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the BBB by 64%, and in vivo levels of H O 2 by 61%.
• rAAV compositions comprise an rAAV that incorporates a polynucleotide sequence that encodes a polypeptide that expresses in a cell and reduces the level of harmful reactive oxygen species, particularly hydrogen peroxide and superoxide.
• Particularly effective antioxidants are human catalase and human superoxide dismutase (SOD) with MnSOD being particularly preferred.
• SOD human superoxide dismutase
• the viral vectors including SOD or Cat genes need not incorporate the entire gene, but may include those segments that express effective amounts of the polypeptide in the cells to effectively scavenge reactive oxygen species.
• the viral vector compositions are used to deliver catalase or SOD to the optic nerve, resulting in prevention of optic nerve demyelation induced by hydrogen peroxide or superoxide. This has been demonstrated in in vivo models for EAE and optic neuritis.
• the rAAV-Cat or rAAV-SOD vectors may transfect endothelial or oligodendroglial cells.
• the presence of catalase has also been demonstrated in astrocytes and microdendroglia as well as in axons.
• a preferred promoter is CMV; however, cell specific promoters for endothelial, oligodendroglial or neuronal cells are also contemplated.
• human myelin basic protein promoter will be useful.
• Other promoters include human platelet dervied growth factor promoter (PDGF) and human vascular endothelial growth factor (VEGF) promoter.
• PDGF platelet dervied growth factor promoter
• VEGF vascular endothelial growth factor
• Such promoters may be employed in a single vector that comprises Cat and/or SOD or in separate vectors.
• a particularly preferred embodiment is the use of AV or rAAV vectors for simultaneous delivery of two different reactive oxygen species scavenger enzymes to the optic nerve. Scavenging of superoxide of superoxide by germ line gene expression increases in the ECSOD, and combined with scavenging of H2O2 by viral-mediated gene tranfer, demyelination of acute experimental allergic encephalomyelitis is decreased by at least 72%.
• compositions comprising the rAAV vectors are envisioned, including rAAV-SOD vector and AV-Cat vector compositions.
• a particularly preferred combination is rAAV-MnSOD and AV-Cat.
• rAAV- MnSOD and rAAV-Cat may be employed.
• the recombinant vector compositions are useful in preventing and/or ameliorating demyelination in conditions such as allergic encephalomyelitis and optic neuritis.
• the experimental allergic encephalomyelitis animal (EAE) model is accepted as a relevant model for multiple sclerosis; thus the long-term expression of reactive oxygen species scavengers, as shown with catalase and SOD, will be effective in treatment regimens for human patients.
• Kits are also contemplated as part of the invention.
• Such kits comprise rAAV compositions comprising a selected polynucleotide sequence encoding a mammalian catalase or superoxide dismutase polypeptide, a device for delivering the rAAV composition and instructions for use.
• the kits may contain AV-Cat vector compositions in combination with the rAAV compositions.
• FIG. 1 is a restriction map of the rAAV-Cat construct
• FIG. 2A is a bar graph showing ⁇ 2-fold increases in the mean number of catalase immunogold particles within astrocytes (astro), oligodendrocytes (oligo), microdendroglia (micro), axons, and endothelia (endo) with inoculations of rAAV-Cat;
• FIG. 2B is a bar graph showing catalase inoculations had the following effect on EAE: reduced demyelination (increased myelin areas);
• FIG. 2 C is a bar graph showing catalase inoculations had the following effect on EAE: reduced optic nerve head edema (smaller areas);
• FIG. 2D is a bar graph showing catalase inoculations had the following effect on EAE: decreased optic nerve cell count;
• FIG. 2E is a bar graph showing catalase inoculations had the following effect on EAE: reduced extravasated immunogold-labeled serum albumin (suppressed disruption of the BBB);
• FIG. 2F is a bar graph showing catalase inoculations had the following effect on EAE: reduced in vivo levels of H 2 O 2 in the optic nerve head (ONH), retrobulbar optic nerve (RON) and the optic nerve sheath (ONS);
• FIG. 3 is a restriction map of the rAAV-MnSOD construct.
• FIG. 4 is a restriction map of the AV-cat construct
• FIG. 5A is a bar graph showing approximately 2-fold increases in the mean number of catalase immunogold particles (AdCat) within astrocytes, ohgodendrocytes, microendroglia, axons and endothelia with inoculations of adenovirus and the catalase gene compared with controls.
• AdCat catalase immunogold particles
• FIG. 5B is a bar graph showing that CAT inoculations had reduced demyelation (increased myelin areas) on experimental allergic encephalomyelitis.
• FIG. 5C is a bar graph showing that CAT inoculations effected reduced nerve head edema (smaller area).
• FIG. 5D is a bar graph showing that CAT inoculations led to decreased optic nerve cell count.
• FIG. 5E is a bar graph showing that CAT inoculations led to reduced extravasated immunogold-labeled serum albumin (suppressed disruption of the blood brain barrier).
• FIG. 5F is a bar graph showing that CAT inoculations reduced in vivo levels of hydrogen peroxide in the nerve head, retrobulbar optic nerve and the optic nerve sheath.
• Viral-mediated gene transfer offers novel and effective treatment regimens of potentially therapeutic proteins to humans.
• the examples described here employ two different approaches; one by injection into the eye and the other into the brain.
• the introduction of transduced proteins into the optic nerve presented a challenge because, unlike glial cells, the cell bodies of axons that comprise the nerve do not reside within the nerve itself, but in the retina. Since axons of the optic nerve do not have the organelles necessary for protein synthesis, transcription and translation of introduced DNA must occur in retinal ganglion cells.
• RT-PCR was used to show that ganglion cells were directly infected by the recombinant virus. While most cells exhibited both the brown mRNA reaction product and blue lacZ protein, thereby indicating transcription of the transgene and translation of the transgene protein, respectively, it was apparent that some cells expressed only the mRNA. This indicated that they were recently transfected, but as yet not translating detectable amounts of the lacZ protein. More important, the brown mRNA reaction product within transduced ganglion cells made it highly unlikely that substantial amounts of the blue ⁇ -galactosidase protein were transferred to ganglion cells from other cell types.
• the blue labeling of the optic nerve head was most likely due to the orthograde axonal transport of ⁇ -galactosidase from retinal ganglion cells, also indicating that most blue cells in the ganglion cell layer were retinal ganglion cells and not displaced amacrine cells.
• CMV promoter Transduction of the cell type(s) affected by the disease process is a common prerequisite for potentially therapeutic gene transfers. For this reason the CMV promoter was selected since it is well known for its potential to drive gene expression in heterogeneous cell types. This promoter supported cellular expression of ⁇ -galactosidase in axons, glia, and endothelia of the optic nerve; however, adenoviral and ⁇ -actin promoters may also be used to drive expression. While a general promoter for transgene expression in different cell types was employed, cell- specific promoters may have an advantage over the CMV promoter by inducing a higher efficiency of transduction in targeted cells.
• cell-specific promoters including the opsin promoter to drive expression exclusively in photoreceptors of the retina with a high rate of efficiency (Flannery et al, 1997).
• CMV promoter to drive expression of hg ⁇
• a much lower rate of transduction efficiency in these same cells has been observed (Ali et al., 1996).
• neuronal promoters neuronal promoters (neuron-specific enolase or platelet-derived growth factor) which drive expression of hg ⁇ in spinal neurons, but not in astroglial cells (Peel et al., 1997).
• the CMV promoter is preferred for transduction of the heterogeneous optic nerve cell population that is affected by the demyelinating inflammation of experimental allergic encephalomyelitis.
• the choice of vector for gene delivery is important, particularly when gene therapy is to be applied to the treatment of human optic neuropathies.
• the vector For transduction of the mammalian optic nerve, the vector must be capable either of incorporating the designated cDNA into the host genome without the need for cell replication, as cell division is limited in the mammalian optic nerve, or of creating a stable episomal state.
• AAV is capable of meeting both these requirements, adenovirus is capable of replication that may be a cause for concern.
• AV and AAV are the only two vectors that have been described for gene delivery to the optic nerve. Any vector associated with treatment regimens for humans must be nonpathogenic.
• the host inflammatory response generated by adenovirus in ocular tissues is particularly well recognized (Hoffman et al, 1997) Unlike adenovirus, AAV is nonpathogenic and does not incite an ocular inflammatory response (Muzyczka, 1992). This is an important consideration in studying the effects of gene therapy on an in vivo inflammatory models of optic nerve demyelination such as experimental allergic encephalomyelitis.
• cellular expression of the transgene protein must persist for the duration of the disease process and perhaps longer to reduce recurrences that occur in optic neuritis and multiple sclerosis. The results described show detection of ⁇ -galactosidase after one year.
• RT-PCR results show the absence of the brown mRNA reaction product in some of the cells that contained only the blue lacZ reaction product suggesting that the transgene gene in these cells was turned off, and is no longer producing mRNA. This finding at the earlier time point helps to explain the reductions in the levels of tissue expression of lacZ seen one year after injection, as ⁇ -galactosidase has a short half-life and would not be expected to persist for long periods in cells that are no longer transcribing lacZ mRNA.
• the cellular structure of the optic nerve consists of myelin-forming oligodendroglia, astrocytes, microglia, endothelia, and axons.
• Oligodendroglia are the cell type most vulnerable to immune-mediated injury in experimental allergic encephalomyelitis, an animal model of multiple sclerosis; however, demyelinated optic nerve fibers are also affected. They exhibit hydropic degeneration with dissolution of microtubules and neurofilaments. Even endothelial cells that appear ultrastructurally intact lose their crucial function of maintenance of the blood- brain barrier.
• candidate gene(s) for transfer may include reactive oxygen species scavengers such as catalase and superoxide dismutase.
• reactive oxygen species scavengers such as catalase and superoxide dismutase.
• superoxide dismutase dismutes superoxide to hydrogen peroxide (H O 2 ) and catalase detoxifies the H 2 O 2 to H O and O 2 .
• ROS Reactive oxygen species
• BBB blood-brain barrier
• Reactive oxygen species include superoxide and nitric oxide, released by infiltrating inflammatory cells, and their metabolites hydrogen peroxide (H 2 O 2 ), peroxynitrite, and hydroxyl radical.
• H 2 O 2 hydrogen peroxide
• hydroxyl radical hydrogen peroxide
• Scavengers of ROS include catalase and superoxide dismutase. The latter dismutes superoxide to H 2 O , and catalase detoxifies the H O 2 to water and molecular oxygen.
• AAV adeno-associated viral
• EAE allergic encephalomyelitis
• MS human disease multiple sclerosis
• the optic nerve is a frequent site of involvement common to both EAE and MS.
• Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE.
• Adeno-associated virus-2 is a human parvovirus which can be propagated both as a lytic virus and as a pro virus (Cukor etal, 1984).
• the viral genome consists of linear single-stranded DNA 4679 bases long (Srivastava et al, 1983), flanked by inverted terminal repeats of 145 bases (Lusby and Berns, 1982).
• AAV requires co-infection with a helper virus. Either adenovirus (Parks et al, 1967) or herpes simplex (Buller et al, 1981) can supply helper function. Without helper, there is no evidence of AAV-specific replication or gene expression (Carter et al, 1983). When no helper is available, AAV can persist as an integrated provirus (Berns et al, 1982).
• AAV Integration apparently involves recombination between AAV termini and host sequences and most of the AAV sequences remain intact in the provirus.
• the ability of AAV to integrate into host DNA is apparently an inherent strategy for insuring the survival of AAV sequences in the absence of the helper virus.
• helper virus When cells carrying an AAV provirus are subsequently superinfected with a helper, the integrated AAV genome is rescued and a productive lytic cycle occurs (Hoggan, 1965).
• AAV sequences cloned into prokaryotic plasmids are infectious (Samulski et al, 1982).
• the wild type AAV/pBR322 plasmid, pSM620 is transfected into human cells in the presence of adenovirus, the AAV sequences are rescued from the plasmid and a normal AAV lytic cycle ensues. This renders it possible to modify the AAV sequences in the recombinant plasmid and, then, to grow a viral stock of the mutant by transfecting the plasmid into human cells (Hermonat et al, 1984).
• AAV contains at least three phenotypically distinct regions (Hermonat et al, 1984).
• the rep region codes for one or more proteins that are required for DNA replication and for rescue from the recombinant plasmid, while the cap and lip regions appear to code for AAV capsid proteins and mutants within these regions are capable of DNA replication (Hermonat et al, 1984). It has been shown that the AAV termini are required for DNA replication. Laughlin et al. (1983) have described the construction of two E. coli hybrid plasmids, each of which contains the entire DNA genome of AAV, and the transfection of the recombinant DNAs into human cell lines in the presence of helper adenovirus to successfully rescue and replicate the AAV genome (See also Tratschin et al, 1984a; 1984b).
• the present invention concerns formulation of one or more of the rAAV compositions disclosed herein in pharmaceutically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
• the nucleic acid segment, RNA, DNA or PNA compositions that express a demyelinating-suppressive polypeptide such as SOD or catalase, as disclosed herein may be administered in combination with other agents as well, such as, e.g., proteins or polypeptides or various pharmaceutically-active agents.
• agents such as, e.g., proteins or polypeptides or various pharmaceutically-active agents.
• the rAAV compositions may thus be delivered along with various other agents as required in the particular instance.
• Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein.
• such compositions may further comprise substituted or derivatized RNA, DNA, or PNA compositions.
• Formulation of pharmaceutically-acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation.
• compositions disclosed herein may be delivered via oral administration to an animal.
• these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
• compositions may include excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
• a binder as gum tragacanth, acacia, cornstarch, or gelatin
• excipients such as dicalcium phosphate
• a disintegrating agent such as corn starch, potato star
• the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both.
• a syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
• any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
• the active compounds may be incorporated into sustained-release preparation and formulations.
• these formulations may contain at least about 0.1 %> of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 60% or 70%> or more of the weight or volume of the total formulation.
• the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
• compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation.
• a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
• the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
• the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth. 4.6.2 INJECTABLE DELIVERY
• compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally as described in U. S. Patent 5,641,515, specifically incorporated herein by reference in its entirety).
• Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
• Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
• Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• a coating such as lecithin
• surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• aqueous solution for parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
• aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
• a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure.
• one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
• Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
• the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
• preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.
• Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• compositions disclosed herein may be formulated in a neutral or salt form.
• Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
• solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
• the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
• carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
• carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
• the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
• pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
• compositions that contains a protein as an active ingredient are well understood in the art.
• such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
• the preparation can also be emulsified.
• the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
• Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U. S. Patent 5,756,353, incorporated herein by reference in its entirety.
• the delivery of drugs using intranasal microparticle resins (Takenaga et al, 1998) and lysophosphatidyl-glycerol compounds (U. S. Patent 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts.
• transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U. S. Patent 5,780,045 (specifically incorporated herein by reference in its entirety).
• compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
• Such formulations may be preferred for the introduction of pharmaceutically-acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein.
• liposomes are generally known to those of skill in the art (see for example, Lasic, 1998; which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy for intracellular bacterial infections and diseases). Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Muller et al, 1990). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, enzymes, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals.
• Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
• MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
• SUVs small unilamellar vesicles
• Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the peptide compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, i.e. in the aqueous spaces and within the bilayer itself, respectively. It is possible that the drug-bearing liposomes may even be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation.
• Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure.
• the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition that markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
• soluble proteins such as cytochrome c
• Cholesterol inhibits this penetration of proteins, apparently by packing the phospholipids more tightly. It is contemplated that the most useful liposome formations for antibiotic and inhibitor delivery will contain cholesterol.
• SUVs have the ability to trap solutes varies between different types of liposomes.
• MLVs are moderately efficient at trapping solutes, but SUVs are extremely inefficient.
• SUVs offer the advantage of homogeneity and reproducibility in size distribution, however, and a compromise between size and trapping efficiency is offered by large unilamellar vesicles (LUVs). These are prepared by ether evaporation and are three to four times more efficient at solute entrapment than MLVs.
• LUVs large unilamellar vesicles
• an important determinant in entrapping compounds is the physicochemical properties of the compound itself. Polar compounds are trapped in the aqueous spaces and nonpolar compounds bind to the lipid bilayer of the vesicle. Polar compounds are released through permeation or when the bilayer is broken, but nonpolar compounds remain affiliated with the bilayer unless it is disrupted by temperature or exposure to lipoproteins. Both types show maximum efflux rates at the phase transition temperature.
• Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
• liposomes The fate and disposition of intravenously injected liposomes depend on their physical properties, such as size, fluidity, and surface charge. They may persist in tissues for h or days, depending on their composition, and half lives in the blood range from min to several h. Larger liposomes, such as MLVs and LUVs, are taken up rapidly by phagocytic cells of the reticuloendothelial system, but physiology of the circulatory system restrains the exit of such large species at most sites. They can exit only in places where large openings or pores exist in the capillary endothelium, such as the sinusoids of the liver or spleen. Thus, these organs are the predominate site of uptake.
• MLVs and LUVs are taken up rapidly by phagocytic cells of the reticuloendothelial system, but physiology of the circulatory system restrains the exit of such large species at most sites. They can exit only in places where large openings or pores exist in the ca
• SUVs show a broader tissue distribution but still are sequestered highly in the liver and spleen. In general, this in vivo behavior limits the potential targeting of liposomes to only those organs and tissues accessible to their large size. These include the blood, liver, spleen, bone marrow, and lymphoid organs.
• Antibodies may be used to bind to the liposome surface and to direct the antibody and its drug contents to specific antigenic receptors located on a particular cell-type surface.
• Carbohydrate determinants may also be used as recognition sites as they have potential in directing liposomes to particular cell types. Usually, it is contemplated that intravenous injection of liposomal preparations would be used, but other routes of administration are also conceivable.
• the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention.
• Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 ⁇ m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention.
• the following techniques are also contemplated as alternative methods of delivering the catalase-expressing or SOD-expressing polynucleotide compositions to a target cell or animal.
• Sonophoresis i.e., ultrasound
• U. S. Patent 5,656,016 specifically incorporated herein by reference in its entirety
• Other drug delivery alternatives contemplated are intraosseous injection, microchip devices, ophthalmic formulations, transdermal matrices and feedback controlled delivery.
• the invention also encompasses one or more compositions together with one or more pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and/or other components, as may be employed in the formulation of particular rAAV-polynucleotide delivery formulations, and in the preparation of anti-demyelinating agents for administration to an animal.
• preferred animals for administration of the pharmaceutical compositions disclosed herein include mammals, and particularly humans. Other preferred animals include murines, bovines, equines, porcines, canines, and felines.
• compositions may include partially or significantly purified compositions, either alone, or in combination with one or more additional active ingredients, which may be obtained from natural or recombinant sources, or which may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing DNA segments encoding such additional active ingredients.
• kits may also be prepared that comprise at least one of the compositions disclosed herein and instructions for using the composition as a therapeutic agent.
• the container means for such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other container means, into which the disclosed rAAV composition(s) may be placed, and preferably suitably aliquoted.
• the kit may also contain a second distinct container means into which this second composition may be placed.
• the plurality of demyelinating compositions may be prepared in a single pharmaceutical composition, and may be packaged in a single container means, such as a vial, flask, syringe, bottle, or other suitable single container means.
• the kits of the present invention will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) are retained.
• RNA, DNA, PNAs and/or substituted polynucleotide compositions disclosed herein will be used to transfect an appropriate host cell.
• Technology for introduction of PNAs, RNAs, and DNAs into cells is well-known to those of skill in the art.
• Non-viral methods for the transfer of expression constructs into cultured mammalian cells include calcium phosphate precipitation, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use.
• viral vectors including retroviruses, baculoviruses, adenoviruses, adenoassociated viruses, vaccinia viruses, Herpes viruses, and the like are well-known in the art, and are described in detail herein.
• a polynucleotide comprising a contiguous nucleic acid sequence that encodes a catalase or a SOD enzyme may be utilized to treat demyelinating disorders in a transformed host cell.
• Such cells are preferably animal cells, including mammalian cells such as those obtained from a human or other primate, murine, canine, bovine, equine, epine, or porcine species.
• the cells may be transformed with one or more rAAV vectors comprising an SOD- or catalase-encoding construct of interest, such that the construct is sufficient to alter, reduce, ameliorate or prevent the effects of demyelination in vitro and/or in vivo.
• assays include, for example, “molecular biological” assays, such as Southern and Northern blotting, RT-PCRTM and PCRTM;
• biochemical assays such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function assay.
• Southern blotting and PCRTM may be used to detect the transgene(s) in question, they do not provide information as to whether the gene is being expressed. Expression may be evaluated by RT-PCRTM for mRNA and/or specifically identifying the protein products of the introduced genes or evaluating the phenotypic changes brought about by their expression.
• Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins.
• Unique physical- chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography.
• the unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by arnino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.
• Assay procedures may also be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed and may include assays for PAT enzymatic activity by following production of radiolabeled acetylated phosphinothricin from phosphinothricin and 14 C-acetyl CoA or for anthranilate synthase activity by following loss of fluorescence of anthranilate, to name two. Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the cells of the animal or human.
• Genomic DNA may be isolated from animal cell lines or any animal parts to determine the presence of the exogenous gene through the use of techniques well known to those skilled in the art. Note that intact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell.
• the presence of DNA elements introduced through the methods of this invention may be determined by polymerase chain reaction (PCRTM). Using this technique, discreet fragments of DNA are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a gene is present in a stable transformant, but does not prove integration of the introduced gene into the host cell genome. It is the experience of the inventors, however, that DNA has been integrated into the genome of all transformants that demonstrate the presence of the gene through PCRTM analysis.
• PCRTM polymerase chain reaction
• Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique, specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition it is possible through Southern hybridization to demonstrate the presence of introduced genes in high molecular weight DNA, i.e., confirm that the introduced gene has been integrated into the host cell genome. The technique of Southern hybridization provides information that is obtained using PCRTM e.g., the presence of a gene, but also demonstrates integration into the genome and characterizes each individual transformant.
• RNA will only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues.
• PCRTM techniques may also be used for detection and quantitation of RNA produced from introduced genes. In this application of PCRTM it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCRTM techniques amplify the DNA. In most instances PCRTM techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridization. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species.
• the delivery of a nucleic acid in a cell, and in particular, an rAAV construct that expresses a SOD or a catalase may be identified in vitro or in vivo by including a marker in the expression construct.
• the marker would result in an identifiable change to the transfected cell permitting ready identification of expression.
• a drug selection marker aids in cloning and in the selection of transformants, for example, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol.
• enzymes such as herpes simplex virus thymidine kinase (tk) (eukaryotic) or chloramphenicol acetyltransferase (CAT) (prokaryotic) may be employed, as well as markers such as green fluorescent protein, luciferase, and the like. Immunologic markers also can be employed.
• the selectable marker employed is not believed to be important, as long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
• the enzyme luciferase is useful as a screenable marker in the context of the present invention.
• cells expressing luciferase emit light which can be detected on photographic or x-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera.
• a photon counting camera is especially useful as it allows one to identify specific cells or groups of cells that are expressing luciferase and manipulate those in real time.
• Green fluorescent protein (g ⁇ ) is also useful as a screenable marker.
• Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent polypeptides, through specific mutagenesis of the underlying polynucleotides that encode them.
• the technique well-known to those of skill in the art, further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
• Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.
• the inventors contemplate the mutagenesis of the contemplated SOD- and/or catalase-encoding polynucleotide sequences to alter the activity or effectiveness of such constructs in inhibiting or altering the demyelinating activity in a transformed host cell.
• the inventors contemplate the mutagenesis of such genes themselves, or of the rAAV delivery vehicle to facilitate improved regulation of the enzyme's activity in vitro and/or in vivo.
• site-specific mutagenesis is well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides.
• site-specific mutagenesis is often used to alter a specific portion of a DNA molecule.
• a primer comprising typically about 14 to about 25 nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.
• site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form.
• Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art.
• Double-stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.
• site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector that includes within its sequence a DNA sequence that encodes the desired peptide.
• An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
• DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
• sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained.
• recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maniatis et al. , 1982.
• oligonucleotide directed mutagenesis procedure refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification.
• oligonucleotide directed mutagenesis procedure is intended to refer to a process that involves the template-dependent extension of a primer molecule.
• template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing.
• vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment.
• PCRTM polymerase chain reaction
• two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence.
• An excess of deoxynucleoside triphosphates is added to a reaction mixture along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides.
• a DNA polymerase e.g., Taq polymerase
• the extended primers By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction product and the process is repeated.
• reverse transcription and PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified.
• Polymerase chain reaction methodologies are well known in the art.
• Another method for amplification is the ligase chain reaction (referred to as LCR), In
• Qbeta Replicase may also be used as still another amplification method in the present invention.
• a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
• the polymerase will copy the replicative sequence that can then be detected.
• An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[ ⁇ -thio]triphosphates in one strand of a restriction site (Walker et al, 1992), may also be useful in the amplification of nucleic acids in the present invention.
• Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e. nick translation.
• a similar method, called Repair Chain Reaction (RCR) is another method of amplification which may be useful in the present invention and is involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection.
• RCR Repair Chain Reaction
• CPR cyclic probe reaction
• a probe having a 3' and 5' sequences of non-target DNA and an internal or “middle" sequence of the target protein specific RNA is hybridized to DNA which is present in a sample.
• the reaction is treated with RNaseH, and the products of the probe are identified as distinctive products by generating a signal that is released after digestion.
• the original template is annealed to another cycling probe and the reaction is repeated.
• CPR involves amplifying a signal generated by hybridization of a probe to a target gene specific expressed nucleic acid.
• modified primers are used in a PCR-like, template and enzyme dependent synthesis.
• the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
• a capture moiety e.g., biotin
• a detector moiety e.g., enzyme
• an excess of labeled probes is added to a sample.
• the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
• nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR.
• TAS transcription-based amplification systems
• NASBA nucleic acid sequence based amplification
• 3SR 3SR
• the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
• amplification techniques involve annealing a primer that has sequences specific to the target sequence.
• DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat-denatured again.
• the single stranded DNA is made fully double stranded by addition of second target-specific primer, followed by polymerization.
• the double stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6.
• a polymerase such as T7 or SP6.
• the RNAs are reverse transcribed into DNA, and transcribed once again with a polymerase such as T7 or SP6.
• the resulting products whether truncated or complete, indicate target-specific sequences.
• ssRNA single-stranded RNA
• dsDNA double-stranded DNA
• the ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
• RNA-dependent DNA polymerase reverse transcriptase
• the RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA).
• RNase H ribonuclease H
• the resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to its template.
• This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
• This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
• a nucleic acid sequence amplification scheme may be used based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic; i.e. new templates are not produced from the resultant RNA transcripts.
• Other amplification methods include "RACE” and “one-sided PCR” which are well-known to those of skill in the art.
• Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide may also be used in the amplification of DNA sequences of the present invention.
• Modification and changes may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a polypeptide with desirable characteristics. As mentioned above, it is often desirable to introduce one or more mutations into a specific polynucleotide sequence. In certain circumstances, the resulting encoded polypeptide sequence is altered by this mutation, or in other cases, the sequence of the polypeptide is unchanged by one or more mutations in the encoding polynucleotide.
• the amino acid changes may be achieved by changing one or more of the codons of the encoding DNA sequence, according to Table 1.
• amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
• the hydropathic index of amino acids may be considered.
• the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
• Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
• hydrophihcity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (- 1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
• an amino acid can be substituted for another having a similar hydrophihcity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
• substitution of amino acids whose hydrophihcity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
• amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophihcity, charge, size, and the like.
• Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
• This example illustrates that viral mediated gene transfer can be successfully accomplished in the optic nerve.
• the duration of foreign gene expression in glial cells, blood vessels, and axons of the optic nerve with a single injection of recombinant adeno- associated virus (rAAV) was investigated in strain- 13 guinea pigs that are susceptible to induction of experimental allergic encephalomyelitis.
• the immediate early cytomegalovirus (CMV) promoter was linked to (1) a lacZ-SV40 polyA reporter gene or (2) the reporter Aquoria victoria green fluorescent protein (g ⁇ ) complementary DNA (cDNA) using standard protocols.
• the final constructs contained either the Escherichia coli -galactosidase gene or a synthetic humanized (hg ⁇ ) gene.
• Each reporter was driven by the CMV immediate early promoter flanked at both sides by wild-type AAV terminal repeats.
• Plasmids pTRCMV-lacZ or pTRCMV-hg ⁇ were packaged into rAAV by transfection into 293 cells (human embryonic kidney cells) that were coinfected with a replication-defective AAV packaging plasmid (pIM45) containing the wild-type AAV genome without the terminal repeats. Cultures were then coinfected with a temperature-sensitive helper virus — adenovirus tsl49 for lacZ or wild-type adenovirus for hgfp at a multiplicity of infection of 10. After 60 hours of incubation, rAAV was harvested by freeze-thawing 3 times.
• Contaminating helper adenovirus was heat inactivated for 30 minutes at 56°C.
• the cellular debris was centrifuged and rAAV was purified through a cesium chloride gradient formed in an SW41 rotor for 48 hours at 200,000g. The gradient was then fractionated and the density was determined by refractometry. Fractions with densities of between 1.38 and 1.4 g/cm 3 were pooled and dialyzed against Dulbecco modified Eagle medium for 4 hours.
• 55 - resultant rAAV-packaged test viruses pTRCMV-facZ and pTRCMV-hgjp were then titered for rAAV by an infectious center assay giving titers of 1 x 10 s and 2 x 10 8 infectious units per milliliter, respectively. They were also tested for contaminating adenovirus by plaque assay for pTRCMV-hg ⁇ and by serial dilution cytopathic effect for pTRCMV-lacZ. Both potentially contaminating viruses were found to be below detection limits; i.e., less than 5 orders of magnitude lower than rAAV.
• Strain- 13 guinea pigs were sedated with 0.2 mL of a 1:1 mixture of ketamine hydrochloride (100 mg/mL) and xylazine (20 mg/mL) by intramuscular injection.
• the pupils were dilated with 2.5% phenylephrine and 0.5% tropicamide followed by a topical anesthetic (proparacaine hydrochloride) administered to the cornea.
• Paracentesis of the anterior chamber with a 25-gauge needle was done to lower the intraocular pressure.
• a 30-gauge needle attached to a syringe was inserted through the pars plana and positioned over the optic nerve head under visualization with a 28-diopter lens and the indirect ophthalmoscope, then approximately 2 x 10 6 to 4 x 10 6 infectious particles of the viral-encapsulated cDNA gene constructs of (1) pTRCMV-lacZ (19 animals), (2) pTRCMV-hg ⁇ (5 animals), or (3) AAV without the promoter and reporter elements (2 animals) were injected into the vitreous of 1 eye of each animal.
• the chest cavity was opened and the animals were perfused by intracardiac injection with 4%> paraformaldehyde and 0.1-mol L phosphate-buffered saline (PBS).
• PBS phosphate-buffered saline
• the globes and optic nerves were immediately dissected out, then the cornea, lens, and vitreous of each eye were removed by incision at the ora serrata.
• the posterior eyecups were immersion fixed in 4% paraformaldehyde and 0.1-mol/L PBS for 15 minutes and washed in 0.1-mol/L PBS (pH 7.4).
• the eye cups were incubated in 5-bromo-4-chloro-3-indolyl ⁇ D-galactoside ( ⁇ -Gal) (1 mg/mL) plus 5-mmol/L potassium ferricyanide, 5-mmol/L potassium ferrocyanide, and 2- mmol/L magnesium chloride in a gently shaking water bath at 35°C overnight. The reaction was terminated by washes in 0.1-mol/L PBS, then the eye cups were cryoprotected in a graded series of 7.5%, 15%, and 30% sucrose buffers in 0.1-mol/L PBS (pH 7.4).
• the optic nerves were trephined from the eye cups, then the specimens were embedded in OCT medium and snap frozen in liquid nitrogen or embedded in LR-white resin (London Resin Co, Ltd, Basingstoke, Hampshire, England) after dehydration in a graded series of ethanol buffers. Cryosections 10- ⁇ m to 30- ⁇ m thick were mounted on gelatin-subbed glass slides for immunostaining and "silanized" glass slides for in situ reverse transcription-polymerase chain reaction (RT-PCR). Semithin (0.5 ⁇ m) and ultrathin (90 nm) LR- white-embedded sections of the optic nerves of the 6-month and 1-year postinjection animals were processed for immunostaining for lacZ at the light and ultrastructural levels.
• LR-white resin London Resin Co, Ltd, Basingstoke, Hampshire, England
• Photographs of specimens were made with bright-field and differential interference contrast optics with a microscope (Axiophot; Carl Zeiss, Inc, Thomwood, NY). Ultrastructural examination of selected tissue was made with a transmission electron microscope (model H 7000; Hitachi, Inc, Tokyo, Japan). Hg ⁇ fluorescent images were collected on a scanning laser confocal microscope (BioRad Lab, Inc, Herculles, CA). The BioRad Al-A cubes were used with argon laser excitation at 514 nm and emission collected at 520 to 560 nm.
• Sections were washed, developed in diaminobenzidine (DAB)-H 2 O 2 substrate (for immunoperoxidase) or 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium substrates (for alkaline phosphatase), and then washed in tap water.
• DAB diaminobenzidine
• 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium substrates for alkaline phosphatase
• Cryostat sections (stored at -70°C) of the eye cups and optic nerves that had previously been reacted with ⁇ -gal, as described above, were washed in 0.1-mol/L PBS. Endogenous peroxidase activity was quenched by incubation in 0.5%) H O 2 for 30 minutes.
• the specimens were digested in 100 ⁇ L of proteinase K (10 ⁇ g/mL) (Sigma- Aldrich) at 37°C for 15 minutes. Heating cycles were performed in a thermal cycler (PTC- 100- 12MS; MJ Research, Woburn, Mass). To prevent evaporation during heating in the thermal cycler, the slides were covered with a 22-mm piece of parafilm. The digestion was stopped by immersing the slides containing the tissue sections in 0.1-mol/L glycine-PBS buffer for 5 minutes, then washed in PBS for 15 minutes.
• RT reverse transcription
• the slides were incubated in 70 ⁇ L of the oligo(dT) mixture (65- ⁇ L deionized water + 5- ⁇ L oligo[dT]) at 70°C for 10 minutes, then they were incubated in 30 ⁇ L of the RT reaction mixture (10 RT buffer [10 ⁇ L] + 10-mmol/L deoxyribonucleoside triphosphates [dNTPs] [5 ⁇ L] + 0.1-mol/L dithiothreitol [10 ⁇ L] + reverse transcriptase [5 ⁇ L]) at 20°C for 10 minutes, then at 42°C for 50 minutes.
• RT buffer [10 ⁇ L] + 10-mmol/L deoxyribonucleoside triphosphates [dNTPs] [5 ⁇ L] + 0.1-mol/L dithiothreitol [10 ⁇ L] + reverse transcriptase [5 ⁇ L] at 20°C for 10 minutes, then at 42°C for 50
• the RT was inactivated by heating to 70°C for 15 minutes and the slides were cooled to 20°C for 10 minutes. All reagents for RT were contained in the Superscript Preamplification System (Life Technologies, Gaithersburg, MD).
• the slides were incubated in 70 ⁇ L of the PCR reaction mixture consisting of 10 PCR buffer (10 ⁇ L), 1.0-mmol/L of each of the 4 dNTPs (deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate) (8 ⁇ L), 10- ⁇ mol/L digoxigenin-deoxyuridine triphosphate (Boehringer Mannheim, Indianapolis, Ind) (1 ⁇ L), 1-mmol/L forward primer 5'-CTG GCG GTA ATA GCG AAG AGG-3' (SEQ ID NO:l) at nucleotide position 105 (1 ⁇ L), 1-mmol/L reverse primer 5'GGT GTA GAT GGG
• a glass coverslip was placed over the slide-PCR mixture and the edges were glued with rubber cement until dry. Then the slides were cycled at 94°C for 15 seconds, 60°C for 15 seconds, 72°C for 60 seconds, for 20 cycles. Reagents for PCR were obtained from the GeneAmp PCR Core Kit (Perkin-Elmer, Norwalk, Conn). After completion of PCR, the coverslips were removed and the slides were washed in 0.1 standard saline citrate at 45°C for 20 minutes, followed by a wash in 0.1%) bovine serum albumin at 20°C for 15 minutes.
• the slides were washed in 0.1- mol/L PBS, then the horseradish peroxidase was developed in DAB-H 2 O substrate (100 ⁇ L) for 10 to 20 minutes. The slides were then washed in tap water. Two controls were included that (1) omitted the RT step to detect nonspecific binding to nuclear or mitochondrial DNA and (2) omitted the primers in the PCR mix to detect nonspecific staining that was unrelated to cDNA amplification.
• ⁇ -Galactosidase tissue staining was absent in eye cups from control animals that received (1) no viral cDNA complexes, (2) AAV without the promoter and reporter gene, or (3) the CMV-hg ⁇ gene constructs that were also processed for lacZ histochemistry.
• lacZ-positive cells immunolabeled dark blue by alkaline phosphatase, were seen in the perivascular space and included glial cells of the interstitial optic nerve.
• LacZ histochemical analysis of unstained retina from eyes that received the lacZ viral constructs demonstrated that most cells of the ganglion cell layer were positive for ⁇ -galactosidase at two weeks. Diffuse ⁇ -Gal labeling of the outer retina was also evident at this time point.
• Retinas injected with the CMV-hg ⁇ viral constructs that served as controls for the histochemical labeling of CMV-lacZ-injected eyes showed some very mild endogenous histochemical staining for ⁇ -galactosidase in the outer retina, supporting detection of endogenous outer nuclear layer activity.
• Confocal fluorescent microscopic examination for hg ⁇ revealed hg ⁇ fluorescence in cells of the ganglion cell layer four weeks after injection of the hg ⁇ viral constructs. No signal in this cell layer was detected in eyes that received no viral cDNA complexes, the AAV without the promoter and reporter gene elements or thelacZ gene construct. However, autofluorescence of the outer retina was seen in both hg ⁇ -
• lacZ histochemical analysis revealed some positively labeled cells in the outer nuclear layer, while ganglion cell labeling was much weaker than earlier.
• the retina was negative for lacZ histochemical staining; however, lacZ immunogold staining was seen in ganglion cells of the retina at one year.
• MnSOD manganese superoxide dismutase
• Cu/Zn SOD copper zinc superoxide dismutase
• Adeno-associated AAV vector pTRUF was used to accept the MxSOD and Cu/Zn SOD cDNAs at the N tl and Sail sites.
• the resulting pTR-MnSOD and pTR-Cu/Zn SOD plasmids were amplified, then purified and packaged into rAAV.
• Five microliters of rAAS MnSOD or rAAV-Cu/Zn SOD were injected over the right optic nerve heads of SJ1/J mice. For controls, the left eyes received no injection.
• the mice were simultaneously sensitized for experimental allergic encephalomyelitis (EAE), the euthanized one month later.
• EAE allergic encephalomyelitis
• levels of MnSOD immunogold in optic nerve cell types were increased by 25% to 97% and by 45% to 150% for Cu/Zn SOD immunogold.
• the eyes of MnSOD transfected mice had a mean myelin area of 28.35 x 10 4 mm 2 that was greater (less demyelination) than a mean of 24.82 x 10 4 mm 2 for the controls.
• the eyes of the Cu/Zn SOD transfected mice had a mean myelin area of 19.32 x 10 4 mm 2 that was less than controls with a mean of 25.27 x 10 4 mm 2 .
• Optic disc edema was reduced 13% by MnSOD (p ⁇ 0.05) while Cu/Zn SOD increased the cell count by 15% (p ⁇ 0.05).
• Blood-brain barrier disruption was reduced by 33% with MnSOD (p ⁇ 0.05), but extravasation of albumin immunogold was increased by 21% with Cu/Zn SOD (p ⁇ 0.05).
• the following example illustrates that adenoviral mediated transfer of the catalase gene suppresses optic neuritis.
• the replication-deficient adenovirus (Ad) containing the human gene for catalase (CAT) and the vector without the catalase gene were provided by Ron Crystal, MD (New York Hospital-Cornell Medical Center, New York), and constructed as previously described (Erzurum, et al., 1993).
• the CAT complementary DNA was under the control of the adenovirus major late promoter. This construct was used to transfect human 293 cells (human embryonic kidney cells), and the resulting Ad-CAT virus was harvested, purified, and concentrated to a titer of lxl 0 10 infectious plaque-forming units per milliliter.
• mice were sedated with methoxyflurane (Metofane; Pitman-Moore Inc, Terre Haute, Ind), by sensitization with homologous spinal cord emulsion in Freund complete adjuvant (Difco Laboratories, Detroit, MI), which was injected subdermally into the nuchal area. While the animals were under sedation, a 32-gauge needle attached to a Hamilton syringe was inserted through the pars plana. The needle tip was visualized in the vitreous with the use of the indirect ophthalmoscope, and it was positioned directly over the optic nerve head.
• methoxyflurane Methodofane; Pitman-Moore Inc, Terre Haute, Ind
• Freund complete adjuvant Difco Laboratories, Detroit, MI
• Ad-CAT Ad-CAT
• mice 5 ⁇ L of Ad-CAT was injected into the vitreous of the right eyes of the mice. This intravitreal injection resulted in transient clouding of the cornea due to the sudden rise in intraocular pressure.
• mice were overdosed with pentobarbital sodium by intraperitoneal injection 1 month after viral and EAE inoculations. They were then perfused by cardiac puncture with fixative consisting of 4% paraformaldehyde in phosphate-buffered sodium buffer (pH 7.4), 0.1 mol/L, or for detection of in vivo H 2 O , with a mixture consisting of cerium chloride, 2 mmol/L; 3-amino- 1,2,4-triazole, 10 mmol/L; the reduced form of nicotinamide adenine dinucleotide, 0.8 mmol/L; phosphate-buffered sodium buffer (pH 7.5), 0.1 mol/L; and 7%sucrose, followed by perfusion with the fixative.
• fixative consisting of 4% paraformaldehyde in phosphate-buffered sodium buffer (pH 7.4), 0.1 mol/L, or for detection of in vivo H 2 O , with a mixture
• the eyes with attached optic nerves were dissected out and further processed by either of the following procedures: For H 2 O 2 localization, tissue specimens were immersion- fixed in 2.5%) gluteraldehyde, then postfixed in 1% osmium tetroxide; sodium cacodylate hydrochloride buffer (pH 7.4), 0.1 mol/L; and 7% sucrose at 0°C, then dehydrated through an ethanol series to propylene oxide, infiltrated, and embedded in epoxy resin that was polymerized at 60°C overnight.
• tissue specimens were immersion- fixed in 2.5%) gluteraldehyde, then postfixed in 1% osmium tetroxide; sodium cacodylate hydrochloride buffer (pH 7.4), 0.1 mol/L; and 7% sucrose at 0°C, then dehydrated through an ethanol series to propylene oxide, infiltrated, and embedded in epoxy resin that was polymerized at 60°C overnight.
• tissue specimens were postfixed in 5% acrolein; sodium cacodylate hydrochloride buffer (pH 7.4), 0.1 mol/L; and 7% sucrose, then dehydrated through an ethanol series and embedded in resin (LR White resin; Ted Pella, Redding, Pa) that was polymerized at 50°C overnight.
• Semithin longitudinal sections 0.5 ⁇ m) of the optic nerve head and retro bulbar nerve were stained with toluidine blue for light-microscopic examination.
• Ultrathin sections (90 nm) were placed on nickel grids for immunohistochemical analysis.
• Nonspecific binding of antibodies was blocked by floating the grids on either 5% normal goat serum in triethanolamine-buffered sodium (pH 7.2), 0.01 mol/L, with polysorbate 20 for 30 minutes for catalase immunostaining, or 2% teleost gelatin and 2% nonfat dry milk in triethanolamine-buffered sodium (pH 7.2), 0.01 mol/L, with polysorbate 20 for 30 minutes for albumin immunostaining. They were then reacted with rabbit anti-CAT antibodies or with rabbit antialbumin antibodies, respectively, in the same buffer for 2 hours at room temperature.
• the grids were reacted with the secondary goat antirabbit IgG antibodies conjugated to 10 nm of gold for 1 hour at room temperature. After washes in buffer, grids were rinsed in deionized water. For examination by low-magnification transmission electron microscopy, the immunogold particles were enlarged by silver enhancement using a kit (Ted Pella), according to the manufacturer's specifications. To check for nonspecific binding of the secondary antibody, control grids were incubated in the buffer, followed by the gold-labeled antibody. Immunolabeled and control specimens were photographed by transmission electron microscopy without poststaining.
• Morphometi ⁇ c analysis was performed in a masked manner, as previously described. Briefly, images of toluidine blue-stained sections of the optic nerve were captured with a video camera mounted on a light microscope, and the digital image was entered into computer memory. After initial calibration with a stage micrometer, the optic nerve head areas were manually traced using the National Institutes of Health (Bethesda, Md) image software and a computer (Macintosh; Apple Computer, Inc, Cupertino, Calif). The number of glial cells and inflammatory cells in the retrobulbar optic nerve were also quantitated by thresholding of the darker staining cell nuclei. Cell-specific catalase activity and extiavasated serum albumin immunogold were similarly quantitated.
• the immunolabeled sections were examined without poststaining using a transmission electron microscope (H-7000; Hitachi Ltd, Tokyo, Japan) operating at 75 kV. Photographs were made at a magnification of 2500. Ten micrographs of each cell type were taken of each optic nerve. The negatives were digitized into computer memory using a scanner (Umax; Umax Data Systems, Fremont, Calif). Silver-enhanced immunogold particles and H2O2 reaction products were enlarged to a final magnification of 7500, thresholded, and counted with the software and computer system.
• Cell-specific catalase activity was quantitated by counting the number of silver-enhanced immunogold particles in endothelial cells, astroglial cells, oligodendroglial cells, axons, and microglial cells. Values were expressed as the mean SEM for each cell type. Mean particle counts for each nerve were obtained by taking the mean value of the 10 micrographs. Each mean value was expressed as the number of particles per unit area. The extent of demyelination was quantitated by threshold measurements of the myelin sheaths that were derived from the axonal micrographs for each optic nerve. Increases in the myelin sheath area (less demyelination) thereby indicated a beneficial treatment effect.
• Grouped t tests were used to assess differences in the myelin areas, optic nerve head areas, optic nerve cell counts, and immunogold and H 2 O 2 particle counts between the CAT- transduced right eyes and the control left eyes and between the left eyes injected with the empty adenovirus and the left eyes that received no ocular injection.
• catalase immunogold was also significantly increased in axons by 1.95-fold (11529 vs 5912; P ⁇ .01) and in oligodendroglia by 1.81-fold (14712 vs 8115; P ⁇ .01) compared with the contialateral control optic nerves.
• catalase immunogold levels were also increased in microglia by 1.45-fold (13916 vs 9614), these differences were not significant (P>.05).
• microglia had the highest endogenous levels of CAT of all cell types in the control optic nerves.
• Representative transmission electron micrographs of the optic nerve inoculated with Ad-CAT showed more catalase immunogold than in the control nerves inoculated with Ad.
• substantially higher levels of catalase activity were achieved with CAT inoculation.
• the left eyes that received the empty adenovirus had a mean myelin area of 26.01.5104/ ⁇ m 2 vs 25.00.6104/ ⁇ m 2 for uninjected left eyes (P>.05).
• CAT-inoculated optic nerves had 30% more myelin (less demyelination), with a mean myelin area of 37.02.0104/ ⁇ m 2 vs 6.01.5104/ ⁇ m 2 (P ⁇ .01) for the control left eyes that received the empty Ad (FIG. 5B).
• Representative transmission electron micrographs of the optic nerve inoculated with Ad-CAT showed less demyelination than the controls. Therefore, gene transfer of catalase achieved therapeutic protection from EAE-induced demyelination.
• Optic disc edema seen in about 40%> of patients with acute optic neuritis, was evident in animals with EAE. Lateral displacement of the peripapillary retina and filling of the optic cup indicated optic disc edema at the light-microscopic level.
• the peripapillary Retinas of SJL/J mice that are highly susceptible to the induction of EAE also showed a genetically induced degeneration of photoreceptors, with the outer nuclear layer reduced to a single cell layer that was symmetric between the right and left eyes. Ultrastructurally, intracellular edema of unmyelinated axons contributed to the optic nerve head swelling.
• Ad-CAT inoculation reduced the optic nerve cell count by 26% to a mean value of 16115 cells 10 5 ⁇ m 2 vs 21816 cells 10 5 ⁇ m 2 for the control left eyes that received the empty adenovirus (FIF. 5D). These differences were significant (P ⁇ .05).
• BBB disruption Disruption of the BBB, a hallmark of both experimental and human optic neuritis, was seen in all animals sensitized for EAE.
• In vivo evaluation of the BBB by contrast-enhanced magnetic resonance imaging reveals enhancement of the optic nerve in most patients with acute optic neuritis and in all animals with acute EAE.
• a standard marker of BBB disruption is the extravasation of serum albumin, which is detected by immunolabeling.
• Transmission election microscopy of the optic nerves revealed albumin immunogold labeling in all animals with EAE.
• Extiavasated albumin immunogold in the perivascular compartment located the foci of BBB disruption in EAE.
• Albumin immunogold confined to the intravascular compartment indicated normal integrity of the BBB.
• adenovirally delivered CAT reduced disruption of the BBB by 61% to a mean value of 25639 extiavasated immunogold particles per 2.6x10 ⁇ m compared with 656121 particles per 2.6x10 6 ⁇ m 2 for the control left nerves that received the empty adenovirus (FIG. 5E). These differences were significant (P ⁇ .05). Representative transmission electron micrographs of the optic nerve inoculated with Ad-CAT show less extiavasated serum albumin than the control left optic nerves, where a marked accumulation of extiavasated albumin immunogold in the perivascular space is evident. Therefore, CAT inoculation markedly improved BBB integrity. 5.3.10 Reduced Levels of H 2 O 2
• Mean particle counts in the optic nerve head were reduced by 81% in CAT-inoculated nerves to a mean of 116 particles per 2.6x10 ⁇ m vs 5921 per 2.6x10 ⁇ m for the control nerves that received the empty adenovirus (P ⁇ .05) (FIG. 5F).
• reaction product counts were reduced by 65% to a value of 8135 with CAT inoculation vs 23170 for control nerves that received the empty adenovirus (P>.05).
• particle counts were reduced 52%) to a mean of 43396 with catalase inoculation vs 900141 in the control nerves that received the empty adenovirus (P ⁇ .05).
• Representative transmission electron micrographs of the optic nerve head inoculated with Ad-CAT exhibited less H 2 O -derived reaction product than the control nerves.
• the restoration of BBB integrity is an important first step in limiting the pathologic effects of EAE.
• the adeno viral-mediated doubling of catalase levels in endothelial cells suppressed the disruption of the BBB by 61%).
• This restoration of BBB integrity might also have a suppressive effect on EAE by restricting not only H 2 O 2 but also other ROS mediators of damage from access to the optic nerve.
• Hydrogen peroxide is a strong oxidant that can diffuse from the sites of generation in the perivascular space and induce peroxidation of myelin and oligodendroglia at remote sites in the interstitial optic nerve. Oligodendroglia are particularly vulnerable to the effects of H 2 O 2 .
• adeno viral titer was 10 3 times higher than that reported in a study using recombinant AAV.
• adenovirus has the theoretical advantage of faster cellular transduction, it has the disadvantage of inciting an inflammatory response that contributes to short-lived cellular transduction, often lasting two weeks.
• Comparisons of the optic nerve cell counts between the control left eyes that received the adenovirus inoculation without CAT and the control left eyes that received no viral inoculation were comparable, and they showed no significant differences, thus suggesting that adenovirus did not substantially increase the inflammatory response in the EAE-induced optic nerve.
• transgene expression with adenoviral vectors incites inflammation in normal tissues, and it is undetectable two months after inoculation.
• Adenovirus vectors will persist longer in animals that do not mount an effective inflammatory response. Persistent adenoviral transduction is impaired by immune mediators such as nitric oxide that are generated by the inflammatory response induced by adenovirus reductions in inflammation induced by ROS scavenging with catalase may prolong the duration of expression of this transgene product in EAE-affected optic nerves one month after adenoviral inoculation.
• AAV Unlike adenovirus, AAV does not incite an inflammatory response; thus, it has provided long-term transgene expression at least as long as 11/2 years. For this reason, AAV is preferred for long-term transgene expression needed for optic nerve protection against future ROS injury by the recurrence of optic neuritis.
• the comparably small size (21 nm) of the AAV particle limits the size of packaged genes for transfer with AAV to about 4.5 kilobases (kb). Although this presented no problem for insertion of the 2-kb CAT, the insertion of larger gene(s), such as the myelin basic protein (MBP), its promoter, or both, is too long for incorporation into AAV.
• MBP myelin basic protein
• Transfer of the MBP gene has the potential to promote remyelination by oligodendroglia that persist in chronically demyelinated nerves, such as those of patients left with poor visual acuity six months or more after an attack of optic neuritis.
• the larger capacity of recombinant adenovirus may accommodate this relatively larger gene, whose transduction in patients blinded by optic neuritis may improve their level of visual function.
• This newly formed myelin should persist in these chronically demyelinated optic nerves because the inflammatory response has long since subsided.
• ROS scavenging by catalase may also promote remyelination by limiting the damage of myelin basic protein in impaired but not destroyed oligodendroglia.
• results show that either viral vector-adenovirus or AAV-may be used to transfer small genes such as CAT to suppress demyelination and perhaps promote remyelination. Because many advances in therapy for MS were first tested in the EAE animal model our findings of the suppression of experimental optic neuritis with CAT gene transfer suggests that this form of therapy may be useful in patients with acute optic neuritis.
• the AAV vector, pTRUF (Zolotukin et al, 1996), was used to accept catalase cDNA at the Notl and SaR sites.
• Catalase cD ⁇ A was obtained from Chiron.
• the resulting pTR-Cat plasmid (FIG. 1, restriction map) was then amplified and purified by using cesium chloride gradient centrifugation.
• the resulting rAAV-Cat construct was regulated by a cytomegalovirus (CMV) immediate early promoter.
• CMV cytomegalovirus
• the resultant rAAV-packaged pTR-Cat was assayed for rAAV by an infectious center assay and gave a titer of 1 x 10 8 infectious units/ml. It also was tested for contaminating Adenovirus by plaque assay and wild-type AAV by infectious center assay. Both potentially contaminating viruses were found to be below detection limits, ⁇ 5 orders of magnitude lower than rAAV.
• mice Experimental allergic encephalomyelitis was induced in 20 SJL/J mice by sensitization with homologous spinal cord emulsion in complete Freund's adjuvant (Difco) that was injected subdermally into the nuchal area.
• Five microliters of rAAV-Cat were injected over the right optic nerve heads of SJL/J mice.
• the left eyes receive rAAV containing the green fluorescent protein (g ⁇ ) gene in place of the catalase gene.
• the mice were simultaneously sensitized to develop EAE (Raine, 1985).
• mice were overdosed with sodium pentobarbital. They were then perfused by cardiac puncture with fixative consisting of 4% paraformaldehyde in 0.1 M PBS buffer (pH 7.4) or, for detection of in vivo H 2 O 2 , with a mixture consisting of 2 raM cerium chloride, 10 mM 3-amino-l,2,4-triazole, 0.8 mM NADH, 0.1 M PBS buffer (pH 7.5), and 7% sucrose followed by perfusion with the fixative (Guy et al, 1994a).
• fixative consisting of 4% paraformaldehyde in 0.1 M PBS buffer (pH 7.4) or, for detection of in vivo H 2 O 2 , with a mixture consisting of 2 raM cerium chloride, 10 mM 3-amino-l,2,4-triazole, 0.8 mM NADH, 0.1 M PBS buffer (pH 7.5), and 7% sucrose followed by perfusion with the fix
• tissue specimens were immersion fixed in 2.5%> gluteraldehyde, postfixed in 1% osmium tetroxide, 0.1 M sodium cacodylate-HCI buffer (pH 7.4), 1% sucrose in the cod, and then dehydrated through an ethanol series to propylene oxide, infiltrated, and embedded in epoxy resin that was polymerized at 60°C overnight; or
• tissue specimens were postfixed in 5.0%> acrolein, 0.1 M sodium cacodylate-HCl buffer (pH 7.4), and 7%> sucrose and then dehydrated through an ethanol series and embedded in LR White (Ted Pella, Redding, PA) that was polymerized at 50°C overnight.
• Morphometiic analysis was performed in masked fashion as described by Qi et al, (1997). Briefly, images of toluidine blue stained sections of the optic nerve were captured with a video camera mounted on a light microscope and then the digital image was entered into computer memory. After initial calibration with a stage micrometer, the optic nerve head areas were manually traced using the NIH IMAGE software and a Macintosh Computer (Apple, Cupertino, CA). The number of glial cells and inflammatory cells in the retrobulbar optic nerve were quantitated also by thresholding of the darker staining cell nuclei. Cell-specific catalase activity and extravasated serum albumin immunogold were similarly quantitated.
• the immunolabeled sections were examined without poststaining by using a Hitachi H-7000 transmission electron microscope (Tokyo, Japan) operating at 75kV. Photographs were made at a magnification of ⁇ 2,500. Ten micrographs were digitized into computer memory by using a UMAX scanner (UMAX Data Systems, Fremont, CA). Silver-enhanced immunogold particles and H 2 O reaction products were enlarged to a final magnification of ⁇ 7,500, thresholded, and counted with the software and computer system.
• Cell-specific catalase activity was quantitated by counting the number of silver-enhanced immunogold particles in endothelial cells, astroglial cells, oligodendroglial cells, axons, and microglial cells. Values were expressed as the mean ⁇ standard error of mean for each cell type. Mean particle counts for each nerve were obtained by taking the mean value of the 10 micrographs. Each mean value was expressed as the number of particles per unit area. The extent of demyelination was quantitated by threshold measurements of the myelin sheaths that were derived from the axonal micrographs for each optic nerve. Increases in myelin sheath area (less demyelination), thereby indicated a beneficial treatment effect. Grouped t tests were used to assess differences in the myelin areas, optic nerve head areas, optic nerve cell counts, immunogold, and H 2 O 2 particle counts between the catalase-transduced right eyes and the control left eyes.
• the levels of catalase were increased by 1.80-fold in astrocytes (135 ⁇ 21 vs. 75 ⁇ 8, P ⁇ 0.05) and by 1.85-fold in endothelial cells (102 ⁇ 11 vs. 55 ⁇ 12, ⁇ 0.05). While the levels of catalase immunogold also were increased in microglia by 1.43-fold (185 ⁇ 20 vs. 129 ⁇ 28), these differences were not statistically significant. Transmission electron micrographs (x2,500) of the optic nerve inoculated with rAAV-Cat showed more catalase immunogold than in the control nerves inoculated with rAAV-g ⁇ . Clearly, substantially higher levels of catalase activity were achieved with rAAV-Cat.
• catalase inoculated nerves exhibited a mean myelin area of 23 ⁇ 3 x 10 4 per ⁇ m, 38%> more myelin (less demyelination) than the contialateral control nerves with a mean of 37 ⁇ 1 x 10 4 per ⁇ m (P ⁇ 0.01) (FIG. 2B).
• Transmission electron micrographs (x2,500) of the optic nerve inoculated with rAAV-Cat had less demyelination than the controls inoculated with rAAV-g ⁇ . Therefore, rAAV gene transfer of catalase achieved therapeutic protection from EAE induced demyelination.
• Optic disc edema seen in s50%) of MS patients with acute optic neuritis (Beck et al, 1992), was evident in EAE animals in which lateral displacement of the peripapillary retina and filling of the optic cup indicated optic disc edema at the light microscopic level.
• Ultrastructural analysis revealed intracellular edema of unmyelinated axons contributing to the optic nerve head swelling. These histopathologic features were seen to some degree in both catalase-transduced nerved and contialateral control nerves.
• Catalase gene delivery by rAAV-Cat reduced optic disc edema, by 20%, with a mean optic nerve head area of 3.00 ⁇ 0.10 x 10 4 ⁇ m vs.
• BBB disruption Disruption of the BBB, a hallmark of MS (Katz et al, 1993), was seen in all animals sensitized for EAE.
• enhanced MRI reveals enhancement of the optic nerve in most patients with acute optic neuritis and in all animals with acute EAE.
• a standard marker of BBB disruption is the extravasation of serum albumin that is detected by immunolabeling.
• Transmission electron microscopy of the optic nerves revealed albumin immunogold labeling in all animals with EAE.
• Extiavasated albumin immunogold labeling in the perivascular compartment located the foci of BBB disruption in EAE.
• Albumin immunogold confined to the intravascular compartment indicated normal integrity of the BBB.
• rAAV-delivered catalase genes reduced disruption of the BBB by 64%, with a mean value of 193 ⁇ 15 extravasated immunogold particles per 2.6 x 10 6 ⁇ m compared with the rAAV-g ⁇ injected nerves with a mean value of 540 ⁇ 93 extravasated particles (FIG. 2E). These differences were statistically significant (P ⁇ 0.05). Transmission electron micrographs (x3.500) of the optic nerve inoculated with rAAV-Cat exhibited less extravasated serum albumin than the controls (x4.000) inoculated with rAAV-g ⁇ in which a marked accumulation of extravasated albumin immunogold in the perivascular space is evident. Thus, catalase gene introduction markedly improved BBB integrity.
• VEGF promoter For anti-ROS expression in endothelial cells, the use of a human VEGF promoter is expected to provide advantages in delivering the anti-ROS genes to endothelial cells. Regulatory elements of the VEGF receptor-2 (Flk-1) gene that mediates endothelial-specific express have been reported (Kappel, et al., 19XX). A second candidate promoter for delivery to endothelial cells is the eNOS 5' sequence.
• reaction product counts were reduced by 66%> to a value of 41 ⁇ 23 with catalase gene inoculation vs. 119 ⁇ 60 for control nerves (P > 0.05).
• particle counts were reduced 75% to a mean of 142 ⁇ 48 with catalase inoculation vs. 421 ⁇ 107 in the control nerves ( ⁇ 0.05).
• Endothelial cells comprising the BBB are the first line of defense against mediators of EAE injury to myelin, axons and oligodendroglia.
• restoration of BBB integrity is an important first step in limiting EAE pathology.
• Viral vector introduction of catalase genes into endothelial cells suppressed disruption of the BBB by 64%>.
• H 2 O is a strong oxidant that can diffuse from the sites of generation in the perivascular space and induce peroxidation of lipids in axonal membranes and myelin at remote sites in the interstitial optic nerve
• restoration of BBB integrity might also have a suppressive effect on EAE by restricting other mediators of damage from access to the optic nerve.
• the marked reductions in perivascular cerium perhydroxide reaction product in catalase transduced nerves suggest that increased intracellular levels of catalase in endothelial cells most likely scavenged H 2 O 2 , thereby contributing to restoration of BBB integrity in EAE animals.
• promoters may be designed for cell-specific expression.
• Cell-specific promoters may have an advantage over the viral promoters by inducing a higher efficiency of transduction in targeted cells.
• protection of endothelial cells, oligodendroglia cells and axons is desirable. This may be accomplished with the use of three viral constructs, one with a cell-specific promoter for endothelial cells, one with a promoter for oligodendroglial cells (Chen et al, 1998), and a third with a neuronal promoter for expression in axons (Peel et al, 1997).
• these promoters may be linked to therapeutic genes in a single construct, although the size constraint of AAV may limit this approach.
• catalase had an overall suppressive effect on EAE, its lack of effectiveness during the initial stages of EAE was in part due to the BBB inhibition of CNS penetration of catalase. Only after extensive BBB disruption by the demyelinating inflammation of EAE, was catalase activity in the optic nerve significantly increased by the intraperitoneal injections. In addition, catalase protein has to be administered daily to maintain this increased activity.
• transgene expression For therapeutic efficacy, cellular expression of transgene product must persist for the duration of the disease process.
• rAAV or adenovirus may be used to drive short term gene expression in the optic nerve for at least one month.
• longer term studies with adenoviral vectors have shown that transgene expression is undetectable two months after inoculation into skeletal muscle (Xiao et al, 1996), but may persist longer in animals with defective immune systems (Hoffman et al, 1997).
• ROS ROS-mediated oxidative stress
• scavenging other ROS mediators including peroxynitrite and nitric oxide, will also suppress EAE.
• passive transfer of inflammatory cells transfected with the immunomodulatory cytokines interleukin 4 or interleukin-10 that effect multiple mediators of tissue injury are also expected to suppress EAE.
• Catalase cDNA (SEQ ID NO:3) Mn SOD (SEQ ID NO:4) Cu/Zn SOD (SEQ ID NO:5)
• compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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