David M. Yurek, Ph.D. - US grants

The studies proposed in this research grant will examine several techniques for restoring neural function in animals with experimentally- induced neurodegenerative disorders -- in particular, animals with experimental Parkinson's disease. The pharmacological interaction between antiparkinsonian drug therapy [e.g., levodopa] and the development of dopaminergic neurons will also be examined in grafted and intact dopaminergic systems. During the last decade, basic scientific research has provided encouraging evidence that neurons from embryonic donors can be successfully transplanted into the brain of adult recipients. Using embryonic neurons to replace degenerating neurons may provide salutary effects in several diseases associated with the senescence of the nervous system, including Parkinson's and Alzheimer's diseases. Previous neural grafting studies have shown that embryonic dopamine neurons survive transplantation and release dopamine (DA) into the brain parenchyma of animals with experimental parkinsonism, however, fiber outgrowth from those cells is often limited to the immediate vicinity of the graft. More extensive outgrowth is desirable for promoting and maintaining functional recovery of damaged neural systems in animal models as well as human neurodegenerative disorders. Fiber outgrowth of grafted neurons can be extended when embryonic DA neurons are simultaneously co-grafted with their embryonic target cells. The proposed studies are intended to further examine the trophic effect embryonic striatal cells may exert upon grafted or damaged DA neurons using (1) morphological techniques to examine enhanced fiber outgrowth from dopaminergic grafts and (2) neurochemical techniques [in vivo microdialysis] for measuring levels of DA release in co-grafts versus single DAergic grafts. Second, dopamine cells of the nigrostriatal pathway are the most profoundly affected locus of nerve cells in idiopathic Parkinson's disease. The nigrostriatal pathway consists of DAergic cell bodies located within the midbrain that give rise to long axonal projections that course rostrally and terminate at a distal forebrain target site, the striatum. Most of the DA synthesized by these cells is transported and released into striatum, therefore neurodegeneration of these cells results in an extreme loss of the striatal DA. Moreover, there is biochemical, morphological, and neuropharmacological evidence that DA is released in the vicinity of the cell bodies [midbrain] an modulates neural function and affects motor behavior. To date, the vast majority of transplantation studies have focused on restoring DA function in the striatum in order to reverse motor dysfunction; they have not addressed the issue of how DA losses in the midbrain might also affect motor function. The proposed studied intend to examine functional, morphological, and neurochemical changes that occur after neural transplantation of embryonic DA neurons into both the midbrain and forebrain sites of animals with experimentally-induced parkinsonism. Third, recent studies examining the combined treatment of neural grafting while maintaining antiparkinsonian drug therapy suggest that levodopa treatment may interfere with the salutary effects of neural grafts and may even impair the development of grafted DA neurons. These studies were performed in animals of hemiparkinsonism and the effects of levodopa on the function of the intact striatum complicate the interpretation of these data. Therefore, in order to more fully understand this phenomenon and to further characterize it, bilateral grafts of embryonic DAergic tissue will be implanted into the DA-denervated striata of animals with bilateral lesions of the nigrostriatal pathway and graft morphological development, motor function, and neurochemical characteristics of grafted embryonic DA neurons will be assessed. In a complimentary study, the effects of levodopa on the developing nigrostriatal system will also be characterized using immunocytochemical techniques.

Clinical trials have provided encouraging evidence that grafts of fetal dopamine neurons are an effective therapeutic approach toward counteracting the symptoms of Parkinson's disease. Modest therapeutic benefits are observed in grafted patients despite clinical and experimental evidence that survival of grafted cells is low and graft reinnervation is incomplete. The poor survival and limited fiber outgrowth may be a consequence of neural grafts placed ectopically into an environment where the grafted neurons do not receive the proper signals for successful growth and integration into the neural circuitry of the host brain. Gene therapy may be a viable technique to introduce factors [neurotrophic factors] into brain tissue that can potentiate the survival and functional outgrowth of neural grafts, and thus improve the therapeutic value of the graft. In the proposed studies, regulated viral vectors will be injected into the lesioned nigrostriatal pathway of rodents with experimental Parkinson's disease in order to induce transgene expression of several neurotrophic factors that have a history of providing potent neurotrophic support for dopamine neurons. Subsequently, neural grafts will be implanted into lesioned/transduced brain sites and the survival, reinnervation, and function of the grafts will be assessed. Because Parkinson's disease has a higher incidence in the elderly than in the younger population, and recent experimental evidence suggests that the expression of endogenous neurotrophic factors are diminished in the aged striatum following a neurodegenerative lesion, experiments will be performed in young, middle-age, or old rats with experimental Parkinson's disease and the results will be compared within and between each age group. The studies are designed to determine the optimal temporal expression of neurotrophic factors [GDNF, BDNF, FGF-2] that improve graft development and function using regulated viral neurotrophic factors [GDNF, BDNF, FGF-2] that improve graft development and function using regulated viral vectors in young and aged animals with experimental Parkinsonism. These studies will also determine if combinations of viral vectors expressing different neurotrophic factors can be used to improve the therapeutic effects of dopamine grafts.

DESCRIPTION (provided by applicant): The proposed studies will determine the feasibility of compacting plasmid DNA into "nanoparticles" and using these nanoparticles to deliver their payload into cells of the central nervous system (CNS) as a non-viral, gene therapy technique. DNA compacting techniques will be used to form nanoparticles containing condensed DNA plasmids with diameters in the range of 8-12 nanometers. Preliminary data shows that non-targeted DNA nanoparticles (DNPs) can be injected directly into brain and produce long-term transgene expression brain cells, primarily in astrocytes. The principle studies will focus on synthesizing DNPs that can be administered intracerebrally or systemically and increase the transfection efficiency in neurons. Recent studies have demonstrated that bioconjugated quantum rods can be targeted to the transferrin receptor (TfR) and traverse the blood-brain barrier (BBB). These particles have an average size and shape that are similar to our DNPs. As these are key determinants in particle transport, it is reasonable to postulate that DNPs modified to target the TfR may cross the BBB. In preliminary studies we have succeeded in targeting the DNPs to neurons of the hippocampus in brain slices utilizing a ligand to the serpin enzyme complex receptor (sec-R), C105Y, which has been shown to be a novel cell-penetrating peptide;and its receptor, sec-R, has been identified on neurons. Taken together, we hypothesize that unimodal targeting of DNPs to the TfR will enable them to cross the BBB and non-specifically transfect neural cells (neurons and/or glia), while bimodal targeting of DNPs to the TfR and sec-R will enable them to cross the BBB and predominantly transfect neuronal cells. To achieve dopamine neuron specificity, we will use plasmid constructs that contain the tyrosine hydroxylase (TH) promoter;TH is the rate limiting enzyme in the biosynthetic pathway for dopamine. The study design in this two year project will focus on 1) the synthesis of targeted DNPs, 2) transfection efficiency of targeted DNPs in primary neuronal cultures or cell lines, 3) in vivo tracking and transfection efficiency of targeted DNPs using MRI, microPET, and bioluminescent imaging techniques as well as immunohistochemical and protein analyses, 4) toxicity of targeted DNPs in brain, and 5) feasibility of repeated administration of DNPs while maintaining safe and stable transgene expression in brain. Successful results in these studies could then be applied to animal models of neurodegenerative disorders and possibly lead to translational studies for the treatment of neurological disorders, such as Parkinson's disease. PUBLIC HEALTH RELEVANCE: The proposed studies will use a novel nanoparticle technology that allows nucleic acids (DNA) to be compacted near their theoretical limit;this technology almost duplicates the compaction efficiency of viruses. We present preliminary data showing proof-of-concept that these nanoparticles can be used as a non-viral gene therapy for transfecting cells in the brain. In the proposed studies, we will conjugate moieties to the nanoparticles that will target receptors at the blood-brain barrier (BBB) allowing them to cross from the brain vasculature into the brain tissue. Another conjugated moiety will specifically target a novel rapid uptake mechanism that has been identified on neurons in order to increase the neuronal transfection efficient of the nanoparticles. Successful results from our animal studies could then be translated to human studies using these targeted nanoparticles as a form of non-viral, gene therapy to treat various neurological disorders.

DESCRIPTION (provided by applicant): The proposed studies will determine the feasibility of compacting plasmid DNA into nanoparticles and using these nanoparticles to deliver their payload into cells of the central nervous system (CNS) as a non-viral, gene therapy technique. DNA compacting techniques will be used to form nanoparticles containing condensed DNA plasmids with diameters in the range of 8-12 nanometers. In a series of recent publications coming out of our laboratories, we have shown that synthetic nanoparticles containing DNA plasmids can be used to transfect brain cells and establish both short- and long-term transgene activities following a single injection of DNA nanoparticles (DNP) directly into brain tissue. These encouraging results have lead us to propose a series of studies to further characterize and test the potential of using synthetic vectors to deliver therapeutic genes to the brain as a possible treatment for neurodegenerative disorders. My laboratory has considerable experience testing neurotrophic factor therapy as well as cellular replacement therapies in animal models of Parkinson's disease (PD), and we propose to examine the feasibility of delivering a gene encoding for the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) to brain cells as a means to protect the brain against neuronal degeneration that occurs in an animal model of PD. The first set of experiments will expand upon our current knowledge of DNP technology. In Specific Aim 1, we will attempt to further optimize plasmids design and mode of intracerebral delivery of compacted DNA nanoparticles (DNPs), and then assess the immunogenicity of this treatment. In the second specific aim, we will determine if DNP transfection of the lesion brain is greater than in the intact brain, and whether or not the aged brain is more susceptible to DNP transfection than younger brain; this studies will determine if an up-regulation of astrocytes at the site of neurodegeneration actually benefits transfection efficiency of DNPs because our preliminary studies indicate DNPs have a tropism for astrocytes. Finally, our third specific aim will determine whether or not intracerebral infusion of modified hGDNF DNPs prevent neurodegeneration of dopaminergic neurons following a neurotoxic lesion of the nigrostriatal pathway. Successful results in these studies could then be applied to animal models of neurodegenerative disorders and possibly lead to translational studies for the treatment of neurological disorders, such as Parkinson's disease.