Patent Application
20070157324
This invention provides a system for modeling neurodegenerative and other diseases through somatic gene transfer. In addition, methods of multiple gene transfer, disease analysis and drug testing are provided for.
Numerous methods of gene transfer are known in the art, and are not reviewed in any great detail here. Suffice it to say that in general, methods of gene transfer in vitro are well known and have been practiced for several decades. Methods of in vivo gene transfer are much more recent, but have been successfully applied in such contexts as gene therapy efforts to overcome genetic disorders, and in disease modeling efforts, such as the production of germ-line transgenic animal models, such as gene knockout mice or transgenic mice and other animals expressing heterologous genes. For a global review of Parkinsons and other Neurodegenerative Disorders see Neurodegenerative Dementias: Clinical Features and Pathological Mechanisms, (edited by Christopher Clark and John Trojanowski), McGraw-Hill (2000); and Mitochondrial Inhibitors and Neurodegenerative Disorders, (edited by Paul Sandberg et al.) Humana Press (2000).
In general, the known methods of in vivo gene transfer involve the knockout of single genes present in the genome of an animal model, or the inclusion in the germ-line of a specific transgene in the genome of an animal model. The limitations to such methods include the possibility of inducing terminal illnesses in the animal models, such that either non-viable fetuses are produced, or limited life-span animals are produced. In addition, the effects of multiple gene knockouts or transgenes are extremely difficult to simulate in such systems, due to the complex temporal, gene regulatory and interaction effects in such systems. Furthermore, the germ-line transgenic models currently available tend to provide data on a very slow time scale, and such efforts as drug modeling and disease analysis are delayed by the time-scale of transgenic animal maturation. Accordingly, there remains a need in the art for techniques which address and overcome these limitations. This invention is directed to resolving many, if not all, of these limitations in the art.
For example, Parkinson's disease (PD) is caused by the loss of dopamine-producing neurons in the substantia nigra (SN) and their axons that project to the striatum. Surviving neurons are characterized by two types of Lewy pathology: Lewy bodies and dystrophic Lewy neurites (see Dickson, 1999 and Trojanowski and Lee, 1999 for review). Rare genetic forms of this disease are associated with mutations in α-synuclein (α-syn), a protein that is found in Lewy bodies. Two mutations in α-syn have been linked to familial PD, A53T (Polymeropoulos et al., 1997) and A30P (Krüger et al., 1998). The mechanisms whereby these mutations render individuals susceptible to PD remain unclear and the subject of intensive research efforts, as it is likely to provide insights into the pathophysiology of idiopathic PD as well.
Several models have been developed to study the effects of transgenic α-syn expression in rodents or flies. Interestingly, while the fly model mimics many cardinal features of PD, including age-dependent loss of dopamine neurons, Lewy-like inclusion bodies, and motor deficits (Feany and Bender, 2000), mammalian models have been less successful or consistent. Of five rodent studies demonstrating mutant α-syn transgenic expression in neuronal cytoplasm, for example, three have found Lewy-like neurites (Masliah et al., 2000; van der Putten et al., 2000; Kahle et al., 2000), two have found motor impairments (Masliah et al., 2000; van der Putten et al., 2000), only one loss of dopaminergic striatal nerve terminals (Masliah et al., 2000), and none has observed loss of dopamine perikarya in the SN. Although two of the transgenic lines effectively targeted the SN by using the tyrosine hydroxylase (TH) promoter (Rathke-Hartlieb et al., 2001; Matsuoka et al., in press), others were less successful in producing robust expression of α-syn in the SN (Masliah et al., 2000; van der Putten et al., 2000; Kahle et al., 2000). These differences in the expression and effects of α-syn in transgenic rodent brains have made it difficult to infer to what extent the A30P and A53T variants of α-syn are gain of toxicity mutations in SN dopamine neurons.
This invention provides a system for modeling neurodegenerative and other diseases through somatic gene transfer. In addition, methods of multiple gene transfer, disease analysis and drug testing are provided for. Advantages of the somatic gene transfer (SGT) methodology of this invention include:
Accordingly, objects of this invention include provision of a system which meets any or all of the foregoing criteria. In specific embodiments of this invention, such diseases as Alzheimer's Disease (AD), Parkinson's Disease (PD), and Huntington's Disease (HD) are effectively modeled through somatic gene transfer, as opposed to known methods of germline transgenesis. This patent disclosure demonstrates the present inventors' ability to produce brain aggregates through somatic gene transfer of a mutant form of human tau (P301L), known to be associated with “fronto-temporal dementia with Parkinson's linked to chromosome 17 (FTDP-17)”, or through somatic gene transfer of mutant ∀-synuclein (A30P), known to be associated with PD. This patent disclosure also discloses success in somatic expression of a mutant amyloid precursor protein (APP), and of a mutant presenilin-1 (PS1), mutant forms of each of which are known to be associated with AD.
Specifically, the present disclosure includes critical data on cell loss in an SGT-treated animal model that mimics the cell loss of Parkinson's Disease. This and other data, including behavioral data, indicate the effectiveness of localized SGT according to the present invention to develop appropriate models for neurodegenerative and other diseases having known or suspected relationship to genes deliverable via SGT.
Thus, one object of the invention is to use SGT to induce a specific disease state at a desired stage or age in the life of an organism, such as a laboratory test organism. A specific object is to use SGT to introduce genetic material that results in a laboratory animal model of Parkinson's Disease. Another object is to use SGT according to the present invention to introduce genetic material that results in a laboratory model of other neurodegenerative diseases, including but not limited to Alzheimer's Disease and Huntington's Disease. A related objective is to provide methods to study such neurodegenerative diseases in relation to pharmacologically active substances and or nutrient regimes that are evaluated to determine their ability to slow, reduce, reverse or eliminate the adverse effects of a particular degenerative disease. A related objective is to evaluate the effects of environmental inducers in a model system according to the present invention, alone or in combination with pharmacologically active substances and or nutrient regimes to increase the understanding of the interactions among these classes of inputs.
Thus, a general object of the invention is to evaluate and identify appropriate treatments and/or protocols in such laboratory animal models the slow, reduce, and/or eliminate the disease state so induced by the methods of the invention. Through such studies not only is an appropriate understanding gained of the interaction of life stage and age, physical and nutritional status, and effects of pharmacologically active substances, but more critically to the clinical prognosis of humans afflicted with such diseases, appropriate environmental inducers and/or dietary regimes may are identified or shown irrelevant, and appropriate pharmacologically active substances are identified for appropriate clinical testing in humans.
An additional advantageous aspect of the subject invention pertains to combining genes, or mutant forms thereof, in trans or in cis, in known delivery vehicles (e.g. viral vectors) for simultaneous or proximal delivery to enhance the desired disease effect, or to more broadly analyze the effect of various different gene expressions. For example, a “cocktail” of genetic vectors comprising differing genes could be injected, at the desired site, or could be injected separately by two or more injections. The inventors have discovered that certain cells have been susceptible to expression of certain aberrant genes while other cells are resistant to expression of the aberrant gene product. Delivery of two or more genes via SGT will provide, for the first time, a means to elucidate what molecules, compounds, receptors, or cellular structures are responsible for susceptibility or resistance to certain aberrant proteins and genes.
Furthermore, the subject SGT methods taught herein may be employed in combination with known transgenic model systems (e.g. transgenic mice). For example, SGT could be conducted on transgenic rodents expressing alpha-synuclein, SGT may also introduce alpha synuclein but preferably introduces another aberrant gene. The subject SGT methods make combining different model systems possible. Such combinations provide a more flexible and accurate investigative tool to elucidate cellular mechanisms involved in neurodegenerative diseases.
These and other objects and advantages of the present invention are shown by the following detailed disclosure and examples, which are not to be taken to be limiting of the full scope of the present invention.
This invention provides a system for modeling neurodegenerative and other diseases through somatic gene transfer. In addition, methods of multiple gene transfer, disease analysis and drug testing are provided for. Naturally, variations on this theme, as well as other aspects of the invention and equivalents thereof are to be considered as part of the present patent disclosure.
As opposed to methods of germ-line modification of animals to produce models of various neurodegenerative and other diseases, or methods of transferring genes to achieve therapeutic results, this patent disclosure is directed to methodology wherein a disease state analog is produced in an animal model via somatic gene transfer.
As used herein, the term “somatic gene transfer” or “SGT” is intended to mean the process whereby a gene not normally present in an organism is transferred into that organism in a manner that does not implicate the modification of the germ-line of the recipient organism. In other words, if the recipient organism were to produce progeny, in general, the process of SGT would not result in inheritance of the transferred genes to the progeny. This is not to say that it is impossible for genes transferred to an organism by SGT to be incorporated into the germ line of the recipient organism and thence transferred to progeny. Such events as viral induced gene incorporation, transposon mediated gene integration and the like, could conceivably result in the incorporation of genes transferred by SGT into a recipient organism's germ line. However, it should be understood that this is not a principal purpose of conducting SGT. SGT may, however, be practiced according to the present invention in a recipient which already has a modified germ line. For example, a mouse having a particular gene knockout in its germ plasm may, through SGT, be induced to express one or more other genes. In this manner, it is possible to analyze the effects and interplay of the given gene knockout with the genes transferred by SGT.
SGT is achieved according to the present invention by appropriately cloning genes, known or hereafter discovered, to appropriate gene regulatory signals, such that upon introduction into an organism, the relevant genes introduced by SGT are transcribed and translated appropriately, to exert a biological effect. Those skilled in the art are well familiar with appropriate gene expression promoters, terminators, enhancers, vectors and the like, and this patent disclosure therefore does not review in great detail those methodologies and compositions with which those skilled in the are well familiar.
In one preferred embodiment according to the present invention, SGT is achieved using appropriately constructed viral vectors. Viral vectors that may be used according to this invention include, but are not limited to, lentivirus vectors, herpes virus vectors, adenovirus vectors, retroviral vectors, and equivalents thereof. One preferred viral vector system for this purpose includes the use of recombinant adeno-associated viral (AAV) vectors. AAV's are efficient, their infection is relatively long-lived and is generally non-toxic, unless a toxic transgene is recombined therein. AAV is a small, helper-dependent parvovirus consisting of a single strand 4.7 kb DNA genome surrounded by a simple, non-enveloped icosahedral protein coat. Approximately 85% of the adult human population is seropositive for AAV. However, no pathology as been associated with AAV infection. Adenovirus or herpesvirus is generally required as a helper virus to establish productive infection by AAV. In the absence of helper virus, the AAV genome also amplifies in response to toxic challenge, e.g. UV irradiation, hydroxyurea exposure, and the like. In the absence of either toxic challenge or helper virus, wild-type AAV integrates into human chromosome 19 site-specifically as a function of AAV Rep proteins that mediate the formation of an AAV-chromosome complex at the chromosomal integration site. Up to 96% of the viral genome may be removed, leaving only the two 145 base pair (bp) inverted terminal repeats (ITRs) which are sufficient for packaging and integration of the viral genome. Methods for efficient propagation of recombinant AAV, rAAV, have been developed in the art, including the use of mini-adenoviral genome plasmids, plasmids encoding AAV packaging functions and adenovirus helper functions in single plasmids. Furthermore, methods of rAAV isolation have developed to the point where methods for isolation of highly purified rAAV are a relatively straightforward and rapid undertaking. Likewise for methods of titration of rAAV stocks. Use of green fluorescent protein (GFP) a well-characterized 238 amino acid fluorescent protein is frequently used in a bicistronic arrangement in rAAV to trace rAAV-mediated transgene expression. Promoters for selective and specific expression of rAAV mediated gene transfer have also been identified.
Methods of making and using rAAV and delivery of rAAV to various cells in vivo are disclosed in U.S. Pat. Nos. 5,720,720; 6,027,931; 6,071,889; WO 99/61066; all of which are hereby incorporated by reference for this purpose.
With regard to methods for the successful, localized, long-term and non-toxic transgene expression in the nervous system through SGT using adeno-associated virus (AAV) and selected promoters, reference is made to Klein et al, 1998, Experimental Neurology 150:183-194, “Neuron-Specific Transduction in the Rat Septohippocampal or Nigrostriatal Pathway by Recombinant Adeno-associated Virus Vectors”.
With respect to a method of gene therapy using recombinant AAV with significant persistence through stable expression of the neurotrophic factors NGF or BDNF, and resultant neurochemically quantifiable therapeutic effects, reference is made to Klein et al, Neuroscience 90:815-821, “Long-term Actions of Vector-derived Nerve Growth Factor or Brain-derived Neurotrophic Factor on Choline Acetyltransferase and Trk Receptor Levels in the Adult Rat Basal Forebrain.”
With regard to achievement of quantifiable behavioral effects through somatic transgene expression in the nervous system through AAV vectored expression of BDNF, reference is made to Klein et al, 1999, Brain Research 847:314-320, “Prevention of 6-hydroxydopamine-induced Rotational Behavior by BDNF Somatic Gene Transfer.”
With respect to a review of the state of the art of germline transgenic mouse models for neurodegenerative diseases, including a large number of references in the field of germline transgenic mouse modeling of neurodegenerative diseases, reference is made to Price et al, 1998, Science 282:1079-1083, “Genetic Neurodegenerative Diseases: The Human Illness and Transgenic Models.”
With regard to a review of the state of the art of germline transgenic mouse models for neurodegenerative diseases, including a large number of references in the field of germline transgenic mouse modeling of neurodegenerative diseases, reference is made to PCT Publication WO99/61066, Published 2 Dec., 1999, Avigen, Inc., based on Prior US Applications filed May 27, 1998 and May 18, 1999, “Convection-Enhanced Delivery of AAV Vectors”. However, the review does not substantively address somatic cell transgenesis methodology.
As with the WO99/61066 publication discussed above, reference is made to U.S. Pat. No. 5,720,720, “Convection-Enhanced Drug Delivery”, for its disclosure of methods relating to the delivery of various compounds, including viruses, to the CNS via CED.
With respect to administration of genes to neural precursor cells induced to divide through contact with growth factors to facilitate incorporation of the genetic material into the cell progeny, reference is made to U.S. Pat. No. 6,071,889, “In Vivo Genetic Modification of Growth Factor-Responsive Neural Precursor Cells.” AAV mediated gene delivery is mentioned, although the method appears to be limited to the ex vivo administration of nucleic acids and growth factors to neuronal cells, and the thus treated cells are then administered to the living organism.
With regard to genes known in the art to which reference is made herein, amyloid precursor protein, APP, was described by Hisao et al, 1996, “Correlative memory deficits, AB elevation, and amyloid plaques in transgenic mice,” Science 274:99-102. Presenilin-1, PS-1, was described by Duff et al, “Increased amyloid-beta 42(43) in brains of mice expressing mutant presenilin 1,” Nature 1996, 383(6602):710-713. Tau was described by Nacharaju et al 1999, “Accelerated filament formation from tau protein with specific FTDP-17 missense mutations,” FEBS Letters 447:195-199. Alpha-synuclein was described by Polymeropoulos et al, 1997 “Mutation in the alpha-synuclein gene identified in families with Parkinson's disease,” Science 276:2045-2047; and by Kruger et al., “Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease,” Nat. Genet. 18(1998):106-108. All of these references are hereby incorporated by reference for their teachings of these genes.
Turning now to specific applications of the present invention, wherein SGT is used to induce specific disease states is an appropriate animal model, examples are provided herein of success achieved by the instant inventors in the particularly challenging area of neurodegenerative disease modeling. Those skilled in the art will appreciate that the present invention should not be restricted to the specifics of the examples provided herein, because the operative principles for achieving success in the particularly challenging area of neurodegenerative diseases also apply in less challenging areas of human disease modeling, not requiring delivery of genes to the central nervous system (CNS). In addition, those skilled in the art will appreciate that the present invention is particularly useful in modeling diseases of the CNS, because of the refined control that the present methodology provides in terms of the ability to specifically target selected CNS tissues of interest. In addition, advantages of this invention include:
Accordingly, objects of this invention include provision of a system which meets any or all of the foregoing criteria. In specific embodiments of this invention, such diseases as Alzheimer's Disease (AD), Parkinson's Disease (PD), and Huntington's Disease (HD) are effectively modeled through somatic gene transfer, as opposed to known methods of germline transgenesis. This patent disclosure demonstrates the present inventors' ability to produce brain aggregates through somatic gene transfer of a mutant form of human tau (P301L), known to be associated with “fronto-temporal dementia with Parkinson's linked to chromosome 17 (FTDP-17)”, mutant V-synuclein (A30P), known to be associated with PD. This patent disclosure also discloses success in somatic expression of a mutant amyloid precursor protein (APP), and of a mutant presenilin-1 (PS1), mutant forms of each of which are known to be associated with AD. Other genes of interest with respect to practice of the methods of this invention include, but are not limited to: GAP43, interleukins, especially interleukin-6 (IL-6), gamma-secretase, and combinations thereof. Particularly preferred combinations of genes for transfer to an animal model in accordance with the methodology of this invention include, but are not limited to: APP in combination with presenilin; APP in combination with presenilin plus tau; APP in combination with presenilin plus tau plus IL6; combinations, permutations and variations thereof.
Mutations in the genes for tau and alpha-synuclein can result in abnormal protein deposition, formation of neurofibrillary tangles and Lewy bodies, and death of specific neuron populations. For example, splice site and mis-sense mutations in the tau gene are found in families of neurofibrillary pathology like frontotemporal dementia with Parkinsonism linked to chromosome 17. Transgenic models of neurodegeneration provide functional genomic information about the impact of inherited mutations. Accordingly, somatic cell transgenic models of neurodegeneration are useful for functional genomic studies at particular time points in the lifespan and in particular brain regions. In addition to providing spatio-temporal control of transgene expression, the adeno-associated viral (AAV) vector system enables mixed gene combinations, which are important for complex neurological diseases. Many of these mutant genes are by now well known in the art, having been cloned sequenced and extensively characterized. Accordingly, those skilled in the art, based on the instant disclosure, would be fully enabled to practice the present methods of SGT using such genes known in the art, as well as genes hereafter identified as playing potential roles in development of human neurodegenerative, as well as other human diseases. As a result, the methods disclosed herein provide versatile systems for modeling human diseases, as well as various veterinary diseases, in a rapid, efficient manner, which does not require the delay and complexity of germline disease modeling.
According to the present disclosure, certain specific nucleic acid vector constructs are disclosed by way of exemplary support. Reference is made to
The present inventors have expressed a mutant form of human tau (P301L) using an AAV vector system in the septal nucleus of the basal forebrain and the hippocampus in the adult rat. The vector-derived tau accumulated in cell bodies and dendrites and formed aggregates as observed by co-localization with the reporter gene, green fluorescent protein (GFP), which was bicistronically expressed by the vector (i.e., GFP filled neurons and tau distribution within cell bodies was clustered). The neurofibrillary pathology observed in this model shows abnormal accumulation of tau in neuron cell bodies and dendrites, filaments immunoreactive for hyperphosphyorylated tau, neuritic immunoreactivity for several antibodies that recognized neurofibrillary tangles in Alzheimer's and FTDP-17, and a dramatic induction of reactive astrogliosis. See
Another utility of the present vector system is to apply genes in trans to existing germline mouse and other animal models, for example, by expressing tau in current models of amyloidosis to introduce tangles.
A gene linked to autosomal dominant Parkinson's disease, alpha-synuclein, harboring the A30P mutation, was expressed in the rat substantia nigra. Transduced neurons in this area had aggregates rich in alpha-synuclein and axons with large varicosities (5-10 micrometers in diameter) that were not found in control vector samples. Overexpression of alpha-synuclein in the nigrostriatial pathway also elevated rates of amphetamine-stimulated locomotor behavior, which is apparently consistent with reduced locomotor response in alpha-synuclein knockout mice (Abeliovich et al., 2000). Accordingly, it is concluded that the somatic transgenic models disclosed herein are useful for studying mechanisms of neurodegenerative disease pathogenesis as well as gene structure-function relationships of tau and alpha-synuclein.
Through practice of the SGT method of this invention, we have found in connection with Parkinson's Disease that alpha synuclein overexpression causes a behavioral change in locomotor activity associated with dopamine transmission. In addition, we have found that cellular processes are essentially identical to those seen in standard germline transgenic animal models (see
Through practice of the SGT method of this invention, we have found in connection with Alzheimer's Disease that tau overexpression causes intracellular distribution of the protein that is essentially identical to that seen in the only existing germline transgenic animal model in which neurofibrillary tangles associated with the disease are observed. The neuritic damage observed by the present inventors in using the SGT methodology of this invention is similar to that seen in germline transgenic mice overexpressing the gene.
Reference is made here to
In
Furthermore,
Accordingly, these data demonstrate successful induction of human neurodegenerative disease relevant morphology. Transfer of multiple genes according to this methodology results in variations and combinations of the results seen in these figures.
Adult male rats received intraseptal and intrahippocampal injections of a control AAV vector expressing GFP or a combination of vectors that encode amyloid precursor protein (APP), tau, IL6 and presenilin-1 (see
Evaluation of brain tissues injected with the multiple vectors for the disease-related genes (APP+PS1+tau+IL6) revealed that multiple gene products were formed in the hippocampus. Typical pictures showing this multiple gene expression from the same injected brain, for example, are shown in
The alpha-synuclein gene has been associated with aberrant neuronal pathologies including Parkinson's disease. Using the AAV vector system, this gene was transduced into the substantia nigra of adult male rats, the brain region that degenerates in Parkinson's disease. Eight months later, they were assayed for motor coordination on a rotating rod (
In this example, the direct effects of α-syn transgenic expression in the SN using the adeno-associated virus (AAV) vector system in adult animals were evaluated. AAV vectors have been used to transduce brain neurons efficiently (Kaplitt et al., 1994; McCown et al., 1996; Mandel et al. 1997) without an overt pathological response for at least 1 year (see Peel and Klein, 2000 and Klein et al., 2000 for review). This suggests the suitability using AAV vectors for long-duration laboratory studies of progressive neurological diseases without interference from a secondary adverse effect from the presence of the AAV. While this vector system has been applied for gene therapy historically, it is also well-suited to study gene function at specific ages in targeted brain regions, which proves useful to study neurodegenerative diseases that are age-dependent and associated with specific parts of the brain. Of potentially even greater significance as a useful model system to study neurodegenerative diseases, the present invention can be used in studies that include pre-treatments and/or post-treatments of pharmacologically active substances, nutrient regimes, environmental inducers, or combinations of these.
As used in this invention, the term pharmacologically active substances is defined to be, and includes known or unknown chemical compounds, virus, protein, peptide, amino acid, lipid, carbohydrate, nucleic acid, nucleotide, drug, pro-drug or other substance or any synthetic or natural compounds, and any combinations, mixtures, variants, analogs, or mutants thereof, that demonstrate an affect on or modulate indicators of a neurogenerative disease as set forth herein, including cell loss and behavioral pathologies. Preferably, pharmacologically active substances refer to a substance that slows, reduces or eliminates the adverse effects of a degenerative neurological disease.
As used in this invention, the term nutrient regimes is defined to be any provision of a specific set of nutrient intake, whether oral or provided otherwise, so as to influence the physical and metabolic status of the test organism, and particularly includes antioxidants and compounds or mixtures that provide antioxidant effects, and most particularly includes antioxidants that are known or suspected to facilely cross the blood/brain barrier.
As used in this invention, the term environmental inducers are defined to include compounds and environmental inputs that are known or suspected to aggravate, reduce the effectiveness of, or otherwise deleteriously influence the normal metabolism of a mammalian organism. Environmental inducers include toxins, and particularly include known neurotoxins, and most particularly include neurotoxins shown or believed to promote, induce, or otherwise lead to a neurodegenerative condition. Examples, not meant in any way to be limiting, of the latter subclass of environmental inducers include 6-hydroxydopamine, MPTP (and its MPP+ free radical), and rotenone. Environmental inducers also include known nutrients to which a test organism is exposed in excessive quantity, for instance Manganese (see generally Mechanisms of Degeneration and Protection of the Dopaminergic System, 2001 edited by Juan Segura-Aguila, Programme of Molecular and Clinical Pharmacology, FP Graham Publishing Co., Chapters 3 and 10).
The A30P mutation was selected because rodents normally harbor the A53T form, because it was suggested to oligomerize more readily (Conway et al. 2000), and be more pathogenic than wild-type or the A53T mutation in flies (Feany and Bender, 2000).
The following subsections summarize materials and methods for this example.
DNAs and Transfections
The CBA promoter-containing plasmid, pTR-UF12, was obtained at Vector Core laboratory at the University of Florida. The plasmid contains the AAV terminal repeats (TRs), the only remaining feature (and 4%) of the wild type AAV genome. Flanked by the TRs, the expression cassette of pTR-UF12 includes the following components, in (5′ to 3′) order: 12) a 1.7 kb sequence containing the hybrid CMV immediate early enhancer/chicken β-actin promoter/exon1/intron (Niwa et al, 1991; Daly et al., 1999); 2) the internal ribosome entry site (IRES) from poliovirus, which provides for bicistronic expression (Dirks et al. 1993); 3) green fluorescent protein (GFP) cDNA (Klein et al., 1998); 4) and the polyA tail from bovine growth hormone. The human A30P α-syn cDNA was provided by Dr. M. Farrer and Dr. J. Hardy (Mayo Clinic, Jacksonville, Fla.). The α-syn cDNA was inserted between the CBA promoter and the IRES element of the pTR-UF12 to derive the construct pSyn30. Plasmids were propagated in SURE cells (Stratagene) and CsCl-purified.
The pTR-UF12 and the pSyn30 constructs were tested for in vitro expression by calcium-phosphate transfection of human embryonic kidney 293 cells. GFP fluorescence was observed in cells transfected with either the pTR-UF12 or the pSyn30 DNAs but not in mock-transfected cells. On GFP immunoblots, a monoclonal anti-GFP antibody (Chemicon) labeled a single band at 31 kDa in cell samples that were transfected with either pTR-UF12 or pSyn30, but not in untreated and mock-transfected cell samples. On α-syn immunoblots, an α-syn polyclonal antibody (NACP-98) provided by Dr. Matt Farrer (Mayo Clinic, Jacksonville, Fla.) labeled a single band at 19 kDa in cell samples that were transfected with the pSyn30 DNA, but not in pTR-UF12-transfected, mock-transfected, or untreated 293 cells.
Vector Packaging and Titering
Plasmids were packaged in rAAV by the streamlined method developed by Zolotukhin et al. (1999). Briefly, human embryonic kidney 293 cells at 70% confluence were transfected by the calcium-phosphate method with an AAV terminal repeat-containing plasmid (pTR-UF12 or pSyn30) in an equimolar ratio with the plasmid pDG, which provides the AAV coat protein genes, and adenovirus 5 genes necessary for helper function in packaging (Grimm et al., 1998). Transfections used twenty 15 cm dishes and 80 μg DNA per dish. Three days after, cells and media were harvested and pelleted at 3000×g. The pellet was resuspended in 60 ml of lysis buffer (50 mM Tris pH 8.5, 150 mM NaCl) and freeze-thawed 3 times. The sample was then incubated with 1500 units of endonuclease (Sigma) for 30 min. at 37° C. After, the sample was centrifuged at 3700 RPM and the resulting supernatant was added to a 39 ml Beckman Opti Seal centrifuge tube. Using a peristaltic pump, a discontinuous gradient of iodixinol (OptiPrep, Nycomed) was added to the tube in 4 layers (60, 40, 25, and 15% iodixinol). The 15% iodixinol was dissolved in 1 M NaCl/1×TD. One times TD buffer is 1×PBS, 1 mM MgCl2, 5 mM KCl. The 25% and 40% iodixinols were dissolved in 1×TD. The 60% iodixinol was dissolved in water. Phenol red was included in the 60% and 25% iodixinol to distinguish the layers. The tubes were heat-sealed, placed in a Beckman 70 Ti rotor, and centrifuged at 69000 RPM for 1 hr at 18° C. The AAV was then removed (by pulling the 2nd layer from the bottom of the tube and the interphase between the 1st and 2nd layers), and added to a heparin (Sigma) affinity column. After washing with 1×TD buffer and eluting with 15 ml of 1×TD/1 M NaCl, the sample was concentrated in Millipore Biomax 100 Ultrafree-15 units. Prior to adding the sample, the concentrators were coated with 2% rat serum/lactated Ringer's solution (Baxter) overnight at 4° C. The concentrators were then rinsed twice with lactated Ringer's solution and then spun for 2 min at 2700 RPM. The sample was then added and concentrated to 1 ml, before adding 9 ml of lactated Ringer's solution. This 10 ml volume was again concentrated to 1 ml and then 9 ml of lactated Ringer's solution was added. The 10 ml volume was concentrated to 200 μl, which was removed and placed in a siliconized microcentrifuge tube. The concentrator was then rinsed several times with another 200 μl of lactated Ringer's solution, which was then added to the resulting 400 μl stock of rAAV.
Recombinant AAVs were titered for total particles by a previously described method (Zolotukhin et al., 1999). A 4 μl aliquot of the virus stock was treated with DNAse (Boehringer Mannheim) for 1 hr at 37° C., followed by addition of proteinase K buffer (which denatures the DNAse) and treatment with proteinase K (Boehringer Mannheim) for 1 hr at 37° C., in order to obtain only the encapsidated DNA. Ten times DNAse buffer is 50 mM Tris HCl pH 7.5, 10 mM MgCl and 10× proteinase K buffer is 10 mM Tris HCl pH 8.0, 10 mM EDTA, and 10% SDS. The sample was then phenol: chloroform extracted twice and chloroform extracted once before being precipitated with sodium acetate and ethanol. The pellet was dissolved in 40 μl of water (a 1:10 dilution of the original aliquot). The sample was then quantitated for copies of rAAV DNA using a quantitative-competitive PCR assay. This assay utilizes an internal control of known quantity. The same PCR primers amplify both the internal control and the viral DNA. The small size difference in the PCR product derived from the internal control (GFP DNA with a 100 bp deletion) vs. the full length PCR product from the virus allows for titering the particles of rAAV by titrating increasing amounts of the internal control into this reaction. Titers for the AAVs that were used were 1×1013 particles per ml.
I. Subjects and Stereotaxic Surgery
Male Sprague-Dawley rats (3 months old) were anesthetized with cocktail made up of 3 ml xylazine (20 mg/ml), 3 ml ketamine (100 mg/ml), and 1 ml acepromazine (10 mg/ml) administered intramuscularly at a dose of 0.5-0.7 ml/kg. The injection coordinates for the SN were −5.3 mm bregma, 2.3 mm medial-lateral, 7.8 mm. dorsal-ventral (Paxinos and Watson, 1986). Virus stocks were injected through a 27 ga. cannula connected via 26 ga. I.D. polyethylene tubing to a 10 μl Hamilton syringe mounted to a CMA/100 microinjection pump. The pump delivered 3 μl over 30 min, and the needle remained in place at the injection site for 1 additional min. The cannula was removed slowly (over 2 min), and the skin was sutured and the animal was placed on a heating pad until it began to recover from the surgery, before being returned to their individual cages. All animal care and procedures were in accordance with institutional IACUC and NIH guidelines.
Immunohistochemistry
Anesthetized animals were perfused with 100 ml of cold phosphate-buffered saline (PBS), followed by 400 ml of cold 4% paraformaldehyde in PBS. The brain was removed and equilibrated in a cryoprotectant solution of 30% sucrose/PBS and stored at 4° C. Coronal sections (30 or 50 μm for qualitative or stereological assessments, respectively) were cut on a sliding microtome with freezing stage. Antigen detection was conducted on free-floating sections by incubation in a blocking solution (2% goat serum/0.3% Triton X-100/PBS) for 1 hr at room temperature, followed by primary antibody incubation for 2 hr at room temperature on a shaking platform. Prior to blocking, endogenous peroxidase was quenched by incubation in 0.5% H2O2/PBS for 10 min. Primary antibodies and the dilutions used in this study were: GFP (Molecular Probes polyclonal IgG fraction, 1:1000); α-syn (NACP-98 polyclonal, 1:500); TH (Chemicon polyclonal, 1:500); glial fibrillary acidic protein (GFAP, Chemicon, 1:500). The sections were washed in PBS, and incubated with biotinylated goat anti-rabbit (DAKO, 1:500) secondary antibody for 1 hr at room temperature. The sections were washed with PBS and labeled with horseradish peroxidase (HRP)-conjugated Extravidin (Sigma, 1:1000) for 30 min at room temperature. Development of tissue labeled with HRP was conducted with a solution of 0.67 mg diaminobenzidine (Sigma), 0.13 μl of 30% H2O2 per ml of 80 mM sodium acetate buffer containing 8 mM imidazole and 2% NiSO4. For fluorescent labeling, TRITC-conjugated goat anti-rabbit secondary antibody (Sigma, 1:400) followed the primary antibody. Nuclear counterstaining was performed by incubating the sections for 10 min in DAPI (Sigma, 1 μg/ml) followed by PBS washes. Fluorescent samples were coverslipped using glycerol gelatin mounting media (Sigma).
Stereological Estimates of Dopamine Neurons
The number of SN pars compacta neurons expressing tyrosine hydroxylase immunoreactivity was estimated by an unbiased stereological method, the optical fractionator method of West (1991), using a microscope with a motorized stage. Six to 8 sections regularly spaced throughout the SN pars compacta structure were analyzed. Fifty μm sections were used for stereology. Optical dissectors were 50×50×15 μm cubes spaced in a systematic random manner 100 μm apart and offset 3 μm from the section surface. The fractionator sampling was optimized to yield ca. 150 counted cells per animal, for error coefficients <0.10.
Motor Behaviors
Animals that were injected with either control or α-syn vector unilaterally to substantia nigra (6/group) were challenged with d-amphetamine (free base, 2 mg/kg in saline, i.m.) at 6 and 12 months after gene transfer. The amphetamine was injected 20 min prior to placing the animals in the TruScan activity montoring system (Coulbourn Instruments), which was used to measure rotational behavior, the number of 360° turns in either the clockwise or counter-clockwise direction. Animals that were injected with either control or α-syn vector bilaterally to substantia nigra (8/group) were tested for their motor coordination and agility on a rotating rod (Economex, Colombus Instruments) at intervals of 1, 3, 6, and 12 months after gene transfer. The rod was set at speeds of 12, 24, and 36 RPM and the animals were tested in three trials at each speed for up to 60 sec, unless they fell off the wheel. Rotational behaviors and fall latencies for each vector group were compared by ANOVA.
The Following Subsections Describe the Several Results Parameters Evaluated in this Example.
GFP and α-syn Expression in Substantia Nigra Neurons
The CBA promoter produced robust expression of GFP native fluorescence in SN neurons and their projections with the control vector, pTR-UF12 AAV, throughout the one-year time course of the study (
Immunoreactivity for α-syn accumulated densely in SN pars compacta neurons (
Lewy-Like Axon Morphology
The anterograde labeling of transduced cells with GFP was useful to view the change in neuronal morphology induced by α-syn gene transfer. GFP-expressing nigrostriatal axons could be seen medial to the SN and in the striatum (
Loss of SN Dopamine Neurons
One year after injecting either the control or the α-syn vector into the SN, TH-immunoreactive neuron profiles in the SN were assessed. Expression of GFP in the control group did not appear to alter the density of dopaminergic neurons in this region compared to age-matched, uninjected subjects (
Motor Behavior Tests
Animals expressing α-syn in their SN were compared with controls for two types of motor behaviors. In one paradigm, animals were injected unilaterally with either control or α-syn vector (6/group) and tested for amphetamine-stimulated rotational bias, which is observed towards the lesioned side if there is a large enough loss of dopamine neurons. In another paradigm, animals (8/group) were injected bilaterally and tested for their ability to walk on a rotating rod set at several speeds, which tests locomotor coordination and agility. The animals were tested at intervals between 1 and 12 months after gene transfer with no robust changes observed relative to control with either test. However, there was a trend that approximated statistical significance at several of the intervals for α-syn treated rats to circle towards the side of the α-syn vector injections, and also for α-syn rats to fall off the rotating rod at shorter times compared to controls (p values between 0.05 and 0.10).
The most striking aspect of the results of Example 8 is the fact that the SGT of α-syn resulted in a 53% loss of dopaminergic cells in the SN in the study period. The loss of dopaminergic cells in the SN region is a key characterisitic of PD. These results indicate the suitability of SGT in specific brain regions of test animals for model systems to evaluate the progression of neurological diseases under a variety of controlled conditions.
The somatic gene transfer of Example 8 also was used to evaluate other aspects of PD: cytoplasmic accumulation of α-syn, Lewy-like neurites, and locomotor behavior. The results with the CBA promoter demonstrate the ability of an appropriate promoter to effectively target the SN pars compacta dopaminergic neurons with AAV gene transfer and also to express genes in that region for long intervals using neuron-specific promoters. This is consistent with other studies (Klein et al., 1998; Klein et al., 1999a; Klein et al., 1999b; Patema et al., 2000). Those studies found the NSE promoter vectors to result in neuron-specific transgene expression, which is what was found herein with the CBA promoter. While the present results are consistent with others that vectors based on AAV-2 are limited for infecting glia (Bartlett et al., 1998), expression in astrocytes has been seen using the cell type-specific GFAP promoter in this vector serotype (Peel and Klein, 2000).
The perikaryal immunoreactivity for α-syn densely filled the cytoplasm in clumps including apparent ring formations. Although the presence of classic, singular Lewy inclusion bodies was not detected, this is similar to what was found in transgenic mice expressing the same form of α-syn (Kahle et al., 2000). Without being bound to specific theories or explanations for the lack of the presence of classic, singular Lewy inclusion bodies in the results of this example, some factors that may mitigate against the formation of such classic Lewy inclusion bodies in this example include: the presence of ‘normal’ rat synuclein protein in addition to the SGT-added human α-syn variant; differences in expression or arrangement of the human α-syn variant in rat neurons; and insufficient time of observation.
Despite this apparent lack of Lewy body inclusions in the cell bodies, the striking loss of dopaminergic cells in the SN and the Lewy-like neuronal processes in the SN and the striatum indicate that the present invention does provide a suitable model for the study of neurological diseases.
With regard to the Lewy-like neuronal processes, the α-syn gene transfer resulted in a shift from uniform nigrostriatal axon morphology in controls to heterogeneous morphology characterized by thread-like to sausage- and balloon-like morphologies. These are comparable to samples of Lewy neurite pathology in human PD (Haass and Kahle, 2000; van der Putten et al., 2000) and Lewy-like neurite pathology in transgenic mice (Masliah et al., 2000; van der Putten et al., 2000; Kahle et al., 2000). This structural plasticity occurred at the earliest time point tested, one month after gene transfer, and persisted throughout the one year time-course of the study. These data demonstrate that the induction of Lewy-like neuron pathology in adults by mutant α-syn does not require expression during development.
The apparent lack of Lewy bodies in this model allowed us to ask whether these inclusion bodies are a prerequisite for cell loss. SN pars compacta dopamine neurons degenerated over the course of the study, establishing this approach as an animal model of PD. The lack of cell loss in the transgenic mice could be due to differences in the levels of α-syn expressed within the dopaminergic neurons, or perhaps to compensatory mechanisms during development that were avoided by genetic manipulation in adults. It is also possible that the loss of TH immunoreactivity we observed involved down-regulation of TH levels in intact neurons. This possibility can be addressed by injecting a retrogradelabel such as fluorogold into the striatum prior to gene transfer. Despite the substantial loss of TH immunoreactivity, motor behavior was not significantly impaired. Although 50% losses of dopamine neurons can be sufficient to induce amphetamine-stimulated turning behavior after 6-OHDA lesions in rats (Hefti et al., 1980), it is believed that greater losses are required to induce symptoms in human PD. This difference may be due to the fact that 6-OHDA lesions are typically mapped to the SN sub-region topographically related to the injection site, whereas in PD, the entire SN pars compacta undergoes degeneration. In this model, the spread of the gene transfer affected essentially the entire SN pars compacta. It is therefore possible that for this reason, this genetic model is more similar to human PD than the 6-OHDA lesioning model.
It also is noted that researchers of PD have observed that behavioral effects are not observed until 50 percent or 80 percent of dopaminergic cells in the SN are lost (see chapter 5 of Mechanisms of Degeneration and Protection of Dopaminergic System, pages 105-106, and The absolute number of nerve cells in substantia nigra in normal subjects and in patients with Parkinson's disease estimated with an unbiased stereological method, J. of Neurology, Neurosurgery, and Psychiatry, 1991; 54:30-33). In the rat model, statistically significant behavioral differences may not be consistently observed until greater than the 53 percent loss of dopaminergic cells observed in this example, or without a higher number of subjects observed.
Also, it is noted that some SN dopaminergic neurons expressing α-syn remained at the conclusion of the study, perhaps due to sub-threshold expression levels for toxicity. This hypothesis could be tested by applying higher doses than the one used and potentially by studying dose-response curves of α-syn vector on dopaminergic cell loss to address whether there is more than one population with differential sensitivities to α-syn. In addition to dose-modification, greater expression levels could be accomplished via more efficient vector systems, for example by incorporating the woodchuck hepatitis post-transcriptional regulatory element (Loeb et al., Paterna et al., 2000), or potentially with other AAV serotypes (Davidson et al., 2000).
Alternatively, the survival of some substantia nigra neurons expressing after one year could be due to differences in their processing of the human α-syn. It is -syn may be the toxic form□hypothesized that oligomeric, protofibrillar (Conway et al., 2000; Rochet et al., 2000) and perhaps the surviving cells are able to fibrillize α-syn. There could have been a protective effect in the cells expressing endogenous α-syn and one approach could involve down-regulating rat α-syn with a species-specific ribozyme to potentially block this action. Other differences in the surviving cells could be their ability to ubiquitinate and degrade α-syn, as mutations in the E3 ubiquitin ligase, parkin, are linked to familial PD (Shimura et al., 2001). The model described here should be convenient for combination experiments studying the role of α-syn and other genes in the ubiquitin-proteasome pathway that are linked to PD.
These approaches will hopefully help to uncover why dopamine neurons are selectively vulnerable during PD pathogenesis as well as lead towards novel gene therapy strategies for PD.
Together, these results demonstrate that alpha-synuclein overexpression in a brain region associated with Parkinson's disease causes a behavioral deficit and cellular morphology typical of the disease. This study demonstrates the utility of this somatic transgene approach for modeling because more classic, much slower, mouse transgenic approaches are still attempting to demonstrate these phenomena.
An experiment is envisioned to either enhance or block the toxicity induced by the SGT introduction of α-syn of Example 8. A sufficient number of test organisms are pre-treated with desired environmental stressors at selected concentrations. One example of an environmental stressor is the neurotoxin 6-hydroxydopamine at sub-threshold levels of detection of behavioral deficits. Also, a sufficient number of these organisms are pre-treated with desired nutrient regimes at selected concentrations. One example of a desired nutrient regime is a standard feed supplemented with high level of an antioxidant that targets brain tissue. Also, a sufficient number of these organisms are pre-treated with one or more desired pharmacologically active substances.
The organisms are then treated with the α-syn mutant via SGT according to the method of Example 8. Organisms that were not pre-treated are then provided with treatments (the post-treatment groups), and selected organisms are given treatments before and after the SGT. Some experimental treatments constitute combinations of nutrient regime(s), pharmacologically active substance(s), and/or environmental stressor(s).
At appropriate times selected organisms are selected for behavioral testing and sacrifice for histology. It is expected that the results of this experiment will provide meaningful information regarding which nutrient regimes and pharmacologically active substances are beneficial to improve the condition of the test organisms with regard to the PD symptoms, and which environmental stressors are deleterious and/or their effects are partially or completely counteracted by one or a combination of nutrient regimes and/or pharmacologically active substances.
This experiment is viewed to meet key objectives of the present invention.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.
The teachings of all patents and publications cited throughout this specification are incorporated by reference in their entirety to the extent not inconsistent with the teachings herein.
This application is a continuation of U.S. Ser. No. 10/477,167 filed Jun. 1, 2004, which is a 371 national stage filing of PCT/US02/04019 filed Feb. 11, 2002, which is a continuation-in-part of U.S. Ser. No. 09/780,041, now U.S. Pat. No. 7,098,374, issued Aug. 29, 2006.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10477167 | Jun 2004 | US |
| Child | 11679313 | Feb 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09780041 | Feb 2001 | US |
| Child | 10477167 | Jun 2004 | US |
