William J. Bowers, Ph.D. - US grants

The rapid advances made in the last decade in gene transfer methods have created the opportunity for developing gene therapy for human neurological disease. One goal of therapy is to slow neuron loss, through the expression of neuroprotective genes. The development of gene therapies for chronic diseases critically depends on not only long-term expression of a therapeutic gene product, but also the development of non-toxic gene delivery systems. This project specifically focuses on refining herpes simplex virus (HSV) amplicon vectors, plasmid-based vectors that require a helper virus for packaging. Cytotoxicity associated with amplicon vector stocks appear to be largely a function of helper virus expression of HSV immediate early (IE) gene products. We plan to utilize two approaches to produce amplicon stocks which exhibit reduced cytotoxicity: One includes the use of a three component regulatory packaging system that utilizes tetracycline-dependent expression of IE gene products to increase amplicon to helper titer; the second approach includes the use of Cre recombinase mutants to derive packaging cell lines that cleave loxP-containing helper virus, resulting in reduced helper virus titers and in effect, decreased cytotoxicity in amplicon vector stocks.

The Gene Expression Vector Core (GEVC) will provide Nathan Shock Center-affiliated investigators with state-of-the-art molecular biological resources that facilitate their studies of normal aging and pathophysiology of age-related disease. The GEVC will assist in the development and production of gene transfer reagents based on the herpes simplex virus- based amplicon vectors. These are suitable for in vitro and in vivo gene transfer studies. In addition the GEVC will perform quantitative nucleic acid measurements using "real-time" PCR. While the amplicon vector has been shown to be a very effective vehicle for the delivery of genetic material to numerous tissue and cell types of several animal species, its accessibility to investigators in the aging community will facilitate existing studies and enable new ones. Similarly, the "real-time" quantitative PCR technology has provided to be a reproducible and high throughput method for quantitation of specific nucleic acid sequences (RNA or DNA) from cells and tissues. However, due to the large expense related to initial equipment costs, most investigators are unable to utilize this valuable technology. By taking advantage of the expertise and facilities that already exists at the University of Rochester, the GEVC seeks to provide both amplicon vector-based gene transfer and quantitative PCR technologies to Shock Center investigators locally, regionally and nationally and other researchers pursuing aging-related problems. The GEVC will be housed on the first floor of the newly constructed Arthur Kornberg medical Research Building within the Center for Aging and Developmental Biology. The GEVC will be directed by Dr. William J. Bowers and co-directed by Dr. Howard J. Federoff and, while day-to-day core activities will be performed by a laboratory technical associate. The GEVC scientific oversight committee will consist of the co-directors and Dr. John Olschowka (quantitative PCR consultant).

The existence of multipotent stem cells in the primarily post-mitotic cellular environment of the adult brain has sparked intense research into identification of putative cell type(s) and potential functions. Elucidation of stem cell identity could have an immediate impact regarding its potential use in therapies for neurodegenerative diseases or age-related degeneration. Controversy has arisen regarding the identity of the CNS stem cell and its step-wise lineage progression from stem cell to post- mitotic neuron and/or glia. Recent suggestive studies concerning stem cells located in the subventricular zone (SVZ) have shown that astrocyte- like cells found in the SVZ have the potential to self-renew and/or give rise to cells downstream in the rostral migratory pathway. The development of a novel somatic mosaic approach is described in this application to definitively assess the role of SVZ astrocytes as stem cells, where the overall hypothesis is as follows: Selective removal of GFAP- positive SVZ astrocytes, purported to be multipotent stem cells, will result in the concomitant loss of neuroblastic cells and mature neurons that normally arise from the SVZ. Specifically, a germline-transmitted transgene construct will be used that carries cis recombination elements, loxP, flanking strong transcriptional termination sequences. When activated by the somatic delivery of cre recombinase via a defective herpes amplicon vector a permanent alteration in transgene structure will be catalyzed specifically in the genome of SVZ astrocytes, thereby effecting focal expression of a pro-drug enzyme that sensitizes these cells to a cell death-promoting pro-drug. Cells in which recombination induces transgene expression will also be tagged via co-induction of a histochemical marker, allowing for determination of cell fate in the absence of pro-drug. Experiments described in this proposal constitute the analysis of the properties of the somatic mosaic system that are anticipated to allow for appropriate design, execution, and interpretation of future experiments where the system will be applied to address the function of SVZ astrocytes in the adult rodent.

Gene transfer methodologies have created the opportunity for developing gene therapy for human neurological diseases such as Parkinson's Disease (PD). Adeno-associated virus (AAV) and lentivirus vectors, both of which integrate into the host cell chromosome, have been shown to provide long-term expression of genes and therapeutic efficacy in animal models of PD. These vectors, however, exhibit a number of disadvantages, among them an inability to harbor large transgene segments. Gene therapy vectors based upon the Herpes Simplex virus (HSV) offer numerous advantages for the development of novel therapeutics for PD. These include broad cellular tropism, large DNA packaging capacity that allows for expression of multiple genes, and high transduction efficiency. We propose to modify HSV-derived amplicon vectors to provide stable nigrostriatal cell-specific gene expression. For this pipeline project, we will initially construct novel integrating forms of the amplicon that will direct expression of a reporter gene product specifically within cells of the striatum and substantia nigra. DNA insulator elements will be introduced to facilitate maintenance of the integrated transcription unit in a euchromatic state. These new vectors will be co-administered with an amplicon expressing the Sleeping Beauty transposase into the striata of unlesioned rats, where gene expression duration and cell-type specificity will be experimentally derived. The vectors will then be used to deliver glial cell line-derived neurotrophic factor (GDNF) prior to 6-OHDA-induced lesioning of rats to assess the capacity of the new vector system to confer protection to the nigrostriatal pathway. The proposed studies will yield a novel HSV vector system, provide a detailed understanding of transgene expression in vivo, and evaluate its therapeutic effectiveness in protecting nigrostriatal neurons in a well-established rodent model of PD.

DESCRIPTION (provided by applicant): Alzheimer's Disease (AD) is a neurodegenerative disorder associated with progressive functional decline, dementia and neuronal loss initiated in specific brain regions and progressing by a disease-specific mode. Elucidating the origin(s) of the pathogenic cascade could likely result in the development of novel diagnostic methodologies and potentially stage-specific therapeutics. Inflammatory processes have been proposed as being integral for initiating and/or propagating AD-associated pathology within the brain, as the elaboration of inflammatory cytokine expression and other markers of inflammation is more pronounced in individuals with known AD pathology. Our proposal addresses both the role of inflammation temporally and spatially in pathogenesis as well as examines the interplay between vaccination and inflammation to either slow or exacerbate neurodegeneration. We hypothesize that focal activation of an inflammatory process within the entorhinal cortex of a mouse model of Alzheimer's disease will lead to the exacerbated stepwise propagation of AD-like pathology within the hippocampus and measurable changes in inflammatory mediator transcript levels in the central nervous system. Moreover, peripheral administration of an ABeta-based vaccine delivered via an HSV amplicon vector will attenuate these histological and biochemical and electrophysiological outcomes in a manner dependent upon the form of the delivered immunogen. We propose to create a novel anatomically and temporally controlled inflammation mouse model, that when combined with an established mouse model of Alzheimer's disease, will be utilized to elucidate the role of brain inflammation in propagation of AD-related pathogenesis and how peripheral vaccination modulates this process. Quantitative bionomic technologies will be used in parallel with standard histochemical, biochemical and electrophysiological assays to correlate the molecular mechanisms by which inflammation influences the initiation and propagation of AD-like pathology and degradation of hippocampal-associated synapses.

DESCRIPTION (provided by applicant): Alzheimer's Disease (AD) is a neurodegenerative disorder associated with progressive functional decline, dementia and neuronal loss initiated in specific brain regions and progressing by a disease-specific mode. Elucidating the origin(s) of the pathogenic cascade could likely result in the development of novel diagnostic methodologies and potentially stage-specific therapeutics. Inflammatory processes have been proposed as being integral for initiating and/or propagating AD-associated pathology within the brain, as the elaboration of inflammatory cytokine expression and other markers of inflammation is more pronounced in individuals with known AD pathology. Our proposal addresses both the role of inflammation temporally and spatially in pathogenesis as well as examines the interplay between vaccination and inflammation to either slow or exacerbate neurodegeneration. We hypothesize that focal activation of an inflammatory process within the entorhinal cortex of a mouse model of Alzheimer's disease will lead to the exacerbated stepwise propagation of AD-like pathology within the hippocampus and measurable changes in inflammatory mediator transcript levels in the central nervous system. Moreover, peripheral administration of an ABeta-based vaccine delivered via an HSV amplicon vector will attenuate these histological and biochemical and electrophysiological outcomes in a manner dependent upon the form of the delivered immunogen. We propose to create a novel anatomically and temporally controlled inflammation mouse model, that when combined with an established mouse model of Alzheimer's disease, will be utilized to elucidate the role of brain inflammation in propagation of AD-related pathogenesis and how peripheral vaccination modulates this process. Quantitative bionomic technologies will be used in parallel with standard histochemical, biochemical and electrophysiological assays to correlate the molecular mechanisms by which inflammation influences the initiation and propagation of AD-like pathology and degradation of hippocampal-associated synapses.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is an age-related neurodegenerative disorder associated with progressive functional decline, dementia and neuronal loss affecting approximately 40 percent of persons over the age of 85 years. The demographics make evident that as the median age of the world's population increases, the prevalence and socioeconomic burden of AD will increase substantially. Pathological hallmarks of the disease include amyloid beta (A3) plaques and neurofibrillary tangles, which play a key role in the pathogenic mechanism of Alzheimer's disease, evolving in a temporal and spatial manner. We are interested in understanding the early mechanisms that cause or perpetuate this temporal and spatial progression of AD pathogenesis. Inflammatory processes have been proposed as being integral for initiating and/or propagating AD-associated pathology within the brain, as the elaboration of inflammatory cytokine expression and other markers of inflammation is more pronounced in individuals with known AD pathology. Recently, a 3xTg-AD mouse model of AD that develops both amyloid and tau pathology has been created, which to date is the most disease-relevant model system depicting what occurs in human Alzheimer's disease. We have observed significant up-regulation of the pro-inflammatory cytokine TNF-a in this model prior to the onset of overt amyloid pathology. As TNF-a has been shown to be enhanced in persons with mild cognitive impairment and Alzheimer's disease, the 3xTg-AD mouse provides a novel paradigm in which to investigate the role of this cytokine during early disease stages. We hypothesize that TNF-a mediated inflammation perpetuates disease in an AD model where genetic predisposition to amyloid and tau pathologies exist and that dampening this inflammatory response will diminish the pathological amyloid and tau outcome. Additionally, we hypothesize that the focal induction of an inflammatory event prior to the onset of inflammation will exacerbate pathological outcomes in a regional and temporal manner. We will administer recombinant adeno-associated virus (rAAV) vectors expressing either TNF-a to create a sustained focal inflammatory response or a TNF receptor antagonist to inhibit the endogenous inflammatory response prior to existing pathology. This work will provide major insight into the involvement of inflammation in the temporal and spatial progression of early AD pathogenic events and may potentially elucidate new therapeutic targets.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a progressively dementing disorder characterized by the accumulation in the brain of pathogenic peptides largely comprised of amyloid-beta peptide (Ab), a derivative of the amyloid precursor protein (APP). Ab1-42, the predominant extracellular form, arises from cleavage of APP by two endoproteases, the 2- and 3- secretases, and undergoes assembly into oligomeric forms some of which are postulated to compromise synaptic function. Our laboratory is interested in understanding the early mechanisms that initiate or perpetuate this temporal and spatial progression of AD pathogenesis, and in exploiting an understanding of these mechanisms to derive novel therapeutics. One strategy to treat AD is to deplete the Ab within the parenchymal space and preclude formation of the putative damaging oligomeric forms. Several groups have been successful by introducing antibodies through either active or passive means, although many have incurred untoward immunological events. Among the approaches pursued we seek to deliver to the AD brain recombinant viral vectors that will express a human single chain fragment variable (scFv) antibody directed against Ab oligomeric forms with the goal of facilitating its clearance and preventing synaptic dysfunction and neuronal toxicity. We hypothesize that gene-based passive immunization using single-chain antibodies directed against oligomeric forms of Ab will prevent Ab-engendered synaptotoxicity and will diminish the downstream pathological events that include amyloid plaques, neurofibrillary tangle formation, and associated effects on neuronal viability. Recombinant adeno-associated virus (rAAV) vectors expressing the engineered antibodies will be administered to 3xTg-AD mice, a mouse model of AD that develops both amyloid and tau pathology, prior to the initial appearance of intraneuronal Ab (2 months of age). This work will provide mechanistic insight into the involvement of oligomeric Ab in the temporal and spatial progression of early AD pathogenic events and will potentially lead to the development of new anti-Ab therapeutics designed for early-stage intervention. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is an insidious neurodegenerative disorder that wields significant societal and economic impact. The development of more refined therapeutics directed at disease targets presently believed to be early mediators of the disease and their detailed assessment in state-of-the-art animal models will usher in a new class of therapeutics with the potential to be more than symptom-ameliorating, but truly disease course-modifying.