The methodologies of the present invention demonstrate that a critical balance between pro- and anti-amyloidogenic molecules exists that regulates amyloid formation and cell death in Alzheimer's disease and Parkinson's disease. .beta.-Synuclein, the non-amyloidogenic homologue of .alpha.-synuclein, is a negative modulator of .alpha.-synuclein and A.beta. aggregation, having neuroprotective properties against .alpha.-synuclein and A.beta. neurotoxicity and that .beta.-synuclein and therapeutic agents derived therefrom block amyloidogenesis and neurodegeneration in vivo. The method of the present invention establishes that .beta.-synuclein blocks A.beta. aggregation either by direct inhibition of A.beta. amyloidogenesis or indirectly via either .alpha.-synuclein or its 35 a.a. NAC region, inferring neuroprotective characteristics within the effected cells. The generation of a transgenic mouse line and a cell system overexpressing .alpha.-synuclein characterizes the mechanisms by which .beta.-synuclein blocks .alpha.-synuclein and A.beta. aggregation and that this mechanism offers protection to the cell against amyloid formation as seen in the pathologies of Alzheimer's disease and Parkinson's disease.

SUMMARY OF THE INVENTION

It is an object of the present invention to demonstrate the utility of .beta.-synuclein and peptides derived therefrom as anti-amyloidogenic agents in vitro as a method for screening for amyloidogenesis and in vivo as incorporated into novel pharmaceutical compositions and treatments strategies, i.e., gene therapy and peptide infusion, for treatment of neurodegenerative disease.

It is another object of the present invention to provide methods for the use of novel transgenic animals comprising at least one transgene, portion, domain, mutant or derivative thereof that encodes a synuclein protein which is useful in screening for new anti-amyloidogenic agents.

Through the methodologies of the present invention, Applicants seek to demonstrate that a critical balance between pro- and anti-amyloidogenic molecules exists that regulates amyloid formation and cell death in Alzheimer's disease and Parkinson's disease. To this end it is postulated that .beta.-synuclein, the non-amyloidogenic homologue of .alpha.-synuclein, is a negative modulator of .alpha.-synuclein and A.beta. aggregation, having neuroprotective properties against .alpha.-synuclein and A.beta. neurotoxicity and that .beta.-synuclein and agents derived therefrom block amyloidogenesis and neurodegeneration in vivo. The established hypothesis is that .beta.-synuclein may block A.beta. aggregation either by direct inhibition of A.beta. amyloidogenesis or indirectly via either .alpha.-synuclein or its 35 a.a. NAC region, inferring neuroprotective characteristics within the effected cells. Applicants seek to characterize the mechanisms by which .beta.-synuclein blocks .alpha.-synuclein and A.beta. aggregation and whether this mechanism offers protection to the cell against amyloid formation as seen in the pathologies of Alzheimer's disease and Parkinson's disease.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The mechanisms by which altered functioning of .alpha.-synuclein might lead to neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Lewy body disease remain somewhat unclear. It is generally understood that protein aggregation is a common feature in these disorders and that .alpha.-synuclein has shown to be the major constituent of these aggregations. .alpha.-Synuclein is distinct among the various modulators of A.beta. aggregation in that it is by itself an amyloidogenic protein. The amyloidogenic potential of .alpha.-synuclein is related to its 35 amino acid region denominated by the non-A.beta. component of Alzheimer's disease amyloid (NAC).

.alpha.- and .beta.-Synucleins are similar in that they share a highly conserved N-terminal region and a less conserved C-terminal acidic domain. They differ in that .alpha.-, but not .beta.-, synuclein possesses an extremely hydrophobic amyloidogenic NAC domain. The method of the present invention will go to demonstrating that while some molecules stimulate aggregation, .beta.-synuclein inhibits such aggregation and that the NAC domain is necessary for synuclein aggregation and amyloidogenesis both in vitro and in vivo. Additionally, the N-terminal region of .beta.-synuclein appears to be responsible for suppressing the aggregation of .alpha.-synuclein. Using peptides derived from the N-terminal region of .beta.-synuclein Applicants wish to establish that these peptides block .alpha.-synuclein aggregation in vitro and in vivo, thus examining the possibility that specific peptides derived from anti-amyloidogenic proteins have therapeutic potential. Demonstrating that .beta.-synuclein and peptides derived therefrom inhibit the aggregation of A.beta. and NAC in vivo will go to establishing that the anti-amyloidogenic properties of .beta.-synuclein might be used in industrial applications for neurological research and incorporation into novel pharmaceutical compositions and strategies employing gene therapy and peptide infusion. Such utility is of special interest in understanding and combating neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease.

One particularly useful application of the invention is the generation of novel transgenic animals, such as mice, to model different neurodegenerative diseases, in particular, Alzheimer's and Parkinson's Disease. Such transgenic mice will have utility in developing specific and general therapies and screening methods to identify novel anti-amyloidogenic compounds and to otherwise employ the general inventive aspects of the present invention.

Transgenic mice are achieved routinely in the art using the technique of microinjection, as described in U.S. Pat. No. 4,736,866 issued to Leder et al., and as provided by B. Hogan et al. entitled "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., U.S.A. (1986). U.S. Pat. No. 5,574,206 issued to Jolicoeur particularly describes the creation of transgenic mice bearing functional HIV genes and their use in the modeling and study of HIV-mediated diseases. These references are herein incorporated by reference.

The present invention is further directed to a method for the evaluation of the in vivo effects of synuclein on amyloidogenesis and neurodegeneration through the use of such novel transgenic animals. Applicants have generated a number of transgenic mouse lines overexpressing either .alpha.-synuclein (.alpha.-syn tg and PDAPP-J9M tg) or .beta.-synuclein (.beta.-syn tg) to elucidate the in vivo mechanism of amyloidogenesis in Alzheimer's disease. Overexpression of .alpha.-synuclein is defined as any amount of .alpha.-synuclein that is detectable, by RPA, Western blot or like analysis, above normal .alpha.-synuclein levels found in non-transgenic littermates. The .alpha.-synuclein tg mice have been shown to express high levels of APP and A.beta. in addition to exhibiting extracellular amyloidosis which, in an age- and brain region-specific manner, morphologically resembles senile plaques seen in Alzheimer's disease.

The data generated from extensive testing involving such novel transgenic animals and animal models of the present invention, specifically mice, comprising at least one transgene, portion, domain, mutant or derivative thereof, encoding .alpha.-synuclein synuclein or .beta.-synuclein will go toward supporting and confirming their usefulness for in vivo screening for new anti-amyloidogenic agents.

Another useful application of the present invention is the generation of a cell-free system overexpressing .alpha.- or .beta.-synuclein. The analysis of two cell lines (GT1-7 and B103) overexpressing either .alpha.- or .beta.-synuclein suggests that .alpha.-synuclein interacts with A.beta. and modulates A.beta. aggregation. Because A.beta. plays a central role in synaptic pathology and cell death, it is expected that synuclein may effect the neurotoxic effects of A.beta.. Results have shown that .alpha.-synuclein enhances A.beta. toxicity in neuroblastoma cell, whereas, .beta.-synuclein is protective. Such data demonstrating the mechanisms through which synuclein modifies the toxic function of intracellular A.beta. goes to confirming such cell systems usefulness as a method for the in vitro screening for new anti-amyloidogenic agents.

Compositions of the present invention generally comprise a therapeutically effective amount of .beta.-synuclein or derivatives thereof as nucleic acid or gene products in a pharmaceutically acceptable carrier or excipient. Such a carrier can include, but is not limited to, saline, dextrose, water, glycerol, ethanol, or combinations thereof. The formulation of the composition should suit the desired mode of administration. Those of skill in the art are familiar with the principles and procedures discussed in widely known and available sources as Remington's Pharmaceutical Science (17.sup.th Ed., Mack Publishing Co., Easton, Pa., 1985) and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics (8.sup.th Ed., Pergamon Press, Elmsford, N.Y., 1990) both of which are incorporated herein by reference.

In a preferred embodiment of the present invention, the composition comprising .beta.-synuclein or derivatives thereof is formulated in accordance with standard procedure as a pharmaceutical composition adapted for intravenous administration to human beings and other mammals. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

In cases other than intravenous administration, the composition can contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides, such formulations being well established in the art. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate, etc., inert carriers having well established functionality in the manufacture of pharmaceuticals. Various delivery systems are known and can be used to administer a therapeutic of the present invention including encapsulation in liposomes, microparticles, microcapsules and the like.

In yet another preferred embodiment, therapeutics containing .beta.-synuclein or derivatives thereof can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids and the like, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, thriethylamine, 2-ethylamino ethanol, histidine, procaine or similar.

The amount of the therapeutic of the present invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques, well established in the administration of therapeutics. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and the patient's needs. However, suitable dose ranges for intravenous administration are generally about 20 4000 micrograms of active compound per kilogram body weight. Suitable dose ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-responsive curves derived from in vitro or in vivo test systems. Those of skill in the art, applying routine pharmacological techniques, can readily determine a suitable formulation without exercising undue experimentation.

The preferred pharmaceutical composition of the present invention need not be supplied in peptide or protein form, but instead may be administered as a nucleic acid species which can then be conveniently expressed in the afflicted host. This is applicable especially for embodiments that do not lend themselves to extraneous chemical modification at the nucleotide level. For example, those of skill are well aware that certain destabilizing amino acid sequences can be introduced into a peptide by, for example, targeted protease cleavage points, such that the overall peptide is more readily degraded and does not persist to generate unwanted side effects. The converse is also true in that stabilizing amino acids may also be incorporated. U.S. Pat. No. 5,496,721 of Bachmair et al., U.S. Pat. No. 5,652,216 of Kornfelt et al., and U.S. Pat. No. 4,479,898 of Gilvarg et al. site a variety of methods by which amino acids are modified to enhance cell permeability and improve beneficial physico-chemical properties. These references are herein incorporated by reference.

In circumstances where a genetic construct, within the bounds of the invention, is capable of only transient expression, and that to the degree such expression is inadequate to completely fulfill the desired therapeutic function, additional transiently expressing constructs may be administered to supplement the action. Thus, boluses of genetic construct may be delivered, just as may boluses of recombinant and/or purified gene product for example native, modified, or synthetic protein or peptide.

In a preferred embodiment of the present invention, the genetic therapeutic may be administered directly into the individual or ex vivo into removed cells of the individual which are then reimplanted after administration of the therapeutic agent. By either route, the genetic material is introduced into cells that are present in the body of the individual. Routes of administration may include, but are not limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraoccular, and oral, as well as transdermally or by inhalation or suppository. Preferred routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection. Delivery of gene constructs that encode target proteins can confer mucosal immunity in individuals immunized by a mode of administration in which the material is presented in tissues associated with mucosal immunity. Thus, in some examples, the gene construct is delivered by administration in the buccal cavity within the mouth of an individual. Genetic constructs may be administered by means including, but not limited to, traditional syringes, needleless injection devices, or "microprojectile bombardment gene guns". Alternatively, the genetic vaccine may be introduced by various means into cells that are removed from the individual. Such means include, for example, ex vivo transfection, electroporation, microinjection and microprojectile bombardment. After the genetic construct is taken up by the cells, they are reimplanted into the individual. It is contemplated that otherwise non-immunogenic cells that have genetic constructs incorporated therein can be implanted into the individual even if the vaccinated cells were originally taken from another individual.

Claim 1 of 7 Claims

1. A transgenic mouse whose genome comprises a first and second transgenic nucleotide sequence, wherein the first transgenic nucleotide sequence comprises a human .alpha.-synuclein coding sequence or a human non-A.beta. (NAC) region; and, the second transgenic nucleotide sequence comprises a human amyloid precursor protein (APP) coding sequence, each operably linked to a neuronal specific promoter and the human APP coding sequence comprises a Swedish or a Indiana mutation, wherein expression of said first and second transgenic nucleotide sequence results in amyloid deposits in the brain of said mouse.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.