Human disease modeling using somatic gene transfer

Abstract

The 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.

Description

[0001] 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.BACKGROUND TO THE INVENTION[0002] 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 ...

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Benefits of technology

[0028] 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.

[0034] 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 remnoved, 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.

[0045] (a) The ability to more precisely control the location to which the genes are transferred (i.e. spatial control of gene expression);

[0046] (b) The ability to more precisely analyze the temporal effects of transferred genes at specific times in the development of otherwise normal organisms (i.e. temporal control of gene expression);

[0053] 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 .alpha.-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.

[0057] 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.EXAMPLE 4Parkinson's Disease Associated CNS Lesions in Animal Models Using SGT of this Invention Induce Similar Behavioral and Morphologic Lesions to those Found in Germline Transgenic Animal Models

Problems solved by technology

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.

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.

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 .alpha.-syn in the SN (Masliah et al., 2000; van der Putten et al., 2000; Kahle et al., 2000).

a. The ability to more precisely control the location to which the genes are transferred (i.e. spatial control of gene expression);

b. The ability to more precisely analyze the temporal effects of transferred genes at specific times in the development of otherwise normal organisms (i.e. temporal control of gene expression);

c. The ability to evaluate the effects of expression of combinations of multiple transgenes, which in a germline transgenic animal would be difficult if not impossible to achieve due to diseases which might prevent the animal model from maturing to the age-appropriate state for modeling onset of a particular, complex human disease, such as Alzheimer's.

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