Xandra Owens Breakefield - US grants

Inherited variations in catecholamine metabolism are thought to underlie individual differences in neurophysiology and susceptibility to disease. These studies are aimed at understanding the genetic basis of variations in the structure and regulation of monoamine oxidases (MAO, E.C.1.4.3.4.), the enzymes primarily responsible for the degradation of amine neurotransmitters. Efforts will be directed first to cloning cDNAs for MAO using mRNA from several species - bovine, rodent and human, and several molecular biologic strategies including expression vector libraries, immunoaffinity purification of mRNA and synthetic oligonucleotides. Molecular genetic and biochemical methods will then be used to determine whether the A and B forms of MAO, which appear to have different structures and discrete functions in the nervous system, are encoded in separate genes and subject to distinct regulatory controls. Genes will be isolated from human genomic libraries and characterized with respect to sequence and intron/exon positions. Their chromosomal location will be determined using somatic cell hybrids and/or in situ hybridization to metaphase chromosomes. Linkage analyses will be carried out in human pedigrees to establish whether inherited variations in platelet MAO-B activity are determined by the structural gene for MAO-B using DNA polymorphisms and genomic blotting. Regulation of gene expression will be monitored by the sizes and amount of mRNA present under culture conditions where levels of MAO-A or MAO-B are changing. These include increased MAO-A activity in rat sympathetic neurons manifesting adrenergic versus cholinergic phenotype and in human skin fibroblasts exposed to glucocorticoids, as well as elevation of MAO-B activity in newborn mouse astrocytes as they proceed through their normal developmental sequence. Monoamine oxidase offers several intriguing phenomena for understanding neural functions - how multiple forms of the same enzyme have evolved and are used to effect different functions, how inherited variations in activity are determined, and how genes are developmentally and hormonally regulated.

Inherited variations in proteins of the nervous system have been hypothesized to underlie differences in mood, behavior, neurophysiology, neuroanatomy and response to drugs in normal individuals, as well as in neurologic and psychiatric patients. These studies will focus on the two neural proteins, catechol-0-methyltransferase (COMT) and Beta-nerve growth factor (Beta-NGF) which are amenable to biochemical and genetic analysis for which inherited variations in function are thought to occur in humans. For COMT, the number, location and structure of human genes coding for multiple forms of this enzyme will be assessed. Further, it will be established whether inherited variations in COMT activity, measured in red blood cell lysates and thought to affect drug metabolism, are caused by allelic variations in structural gene locus for this enzyme. This will elucidate the relationship between multiple forms of COMT, and the molecular basis of normal variations in activity. The role of Beta-NGF in the inherited neurologic disease, familial dysautonomia, will be assessed by establishing whether or not specific alleles for the structural gene are co-inherited with the disease state in affected families. Further, a source of authentic human Beta-NGF will be sought using antibodies prepared against recombinant human "Beta-NGF" and a cloned DNA probe for the human Beta-NGF mRNA. By characterizing the authentic form of this human protein it will be possible to investigate its role in other neurologic diseases. Methods to be employed include protein biochemistry, e.g. gel electrophoresis, peptide mapping, microsequencing; immunological techniques, e.g. immunoblotting, immune precipitation, immunoaffinity adsorption of polysomes, radioimmunoassays; molecular biologic techniques, e.g. in vitro translation of mRNA, cDNA cloning in expression and non-expression plasmids, restriction endonuclease mapping, nucleotide sequencing, Southern and Northern blotting; and cell culture procedures, e.g. transformation of lymphocytes, analysis of somatic cell hybrids, radioreceptor assays.

The continuing theme of this Neurogenetics Center is to elucidate the molecular etiology and to modify the course of hereditary neurologic diseases in humans. The current goals are: to determine the function of genes responsible for the hereditary neuroplasias - neurofibromatosis type 1 (NF1) and 2 (NF2), and tuberous sclerosis type 1 (TSC1) and 2 (TSC2), and to develop improved means of gene delivery to cells in the nervous system. These studies will also provide basic information on development and signaling mechanisms in the nervous system. PI Gusella - Characterization of Merlin Expression. The distribution and function of the NF2 gene product, merlin, will be evaluated in normal cells, including proliferating and differentiated Schwann cells, and in schwannoma and meningioma tumors using genetic, biochemical and immunologic methods. PI Ramesh, Co-investigators Haines and Short - Molecular Genetics of TSC2. Genetic analysis will be undertaken to resolve the roles of the TSC1 and TSC2 genes in tuberous sclerosis. This will involve detailed mutational analysis of the TSC2 gene (and the TSC1 gene when it becomes available), as well as immunochemical and biochemical resolution of the cellular distribution of the TSC2 protein, tuberin, and its interaction with other proteins in normal and tumor tissue. PI Buckler - Cloning and Characterization of the TSC1 Gene. Molecular genetic studies will be undertaken to identify the TSC1 gene. Once it is identified, RNA analyses and immunocytochemical and biochemical studies will be undertaken to elucidate its expression and function. PI Breakefield - Gene Delivery to the Nervous System. New modes of delivery will be developed using virus vectors to achieve more widespread and stable gene delivery to the nervous system. In one scheme, retrovirus vectors will be used in combination with migratory astrocytic lines; and in another scheme, amplicon and recombinant herpes simplex virus-derived vectors will be generated to allow limited replication in glia-derived cells, to promote latency in neurons, and to reduce toxic functions. This project will also generate retrovirus vectors for functional analysis of the NF2 and TSC gene products and will explore the role of the NF1 gene in neurofibroma formation by Schwann cells. These projects will be supported by Cores Tissue Culture, Linkage Analysis and Database Core (PI Haines), and Neuropathology (PI Louis, Co-investigator Short). Collectively these studies provide a continuum of research on hereditary human neurologic diseases from positional cloning of disease genes to functional analysis of the gene products to gene therapy, with an emphasis on disrupted growth regulation and signalling mechanisms in the phakomatoses.

Research will be directed towards developing a herpes simplex virus vector that can be used for gene transfer into neurons. Model genes to be transferred are E. coli lac Z encoding the histochemical marker enzyme - beta-galactosidase, and mouse BNGF encoding the peptide growth factor - beta-nerve growth factor (NGF). The immediate aims are to optimize expression of transferred genes, to evaluate whether these genes will be expressed when the vector is in a latent state in neurons, and to analyze expression of these genes in the rat central nervous system. Vector constructs will be made including various promoter and regulatory elements. Expression will be evaluated in continuous cell lines and primary neural cultures, as well as in sections of rat brain. Beta-galactosidase will be assessed by in situ and solution assays of enzyme activity, as well as by immunoassay; beta-NGF by bioassays, radioimmunoassays and immune precipitation and gel electrophoresis. Virus stocks of vectors will be assessed by restriction mapping and Southern blots; mRNA transcribed from vectors by Northern blots, S1 analysis and in situ hydrization histochemistry. Herpes vectors appear ideally suited for gene transfer into adult neurons in localized regions of the nervous system. This type of gene transfer has potential applications in the treatment of diseases such as diabetes insipidus, the Lesch-Nyhan syndrome, Parkinson disease, Huntington disease and Alzheimer disease.

biomedical equipment purchase; DNA; nucleic acid chemical synthesis;

The monoamine oxidases, MAO-A and MAO-B, are the primary enzymes involved in degradation of biogenic amines in mammals. They control levels of amine neurotransmitters, such as dopamine, norepinephrine and serotonin, in the nervous system and metabolize amines that can act as false transmitters or neurotoxins. Humans inherit wide variations in levels of these enzymes. Normal variations are thought to influence the susceptibility of individuals to pathogenic processes involving amines, such as Parkinson disease (PD). Loss of activity is hypothesized to affect the development of the nervous system and possibly to be responsible for a type of X-linked mental retardation (XLMR). The proposed studies are designed to elucidate structural differences in alleles for the MAOA gene that control levels of enzyme activity. Sequence variations in the MAOA gene will be assessed in genomic DNA from male, cultured skin fibroblast lines which vary over 100- fold in activity. This will be carried out by PCR amplification of exons and 5' flanking sequence, followed by analysis of single strand conformational polymorphisms (SSCPs) and direct sequencing of variant fragments. Correlations will be made between specific sequence differences and levels of MAO-A activity and mRNA; the latter will be determined by northern and slot blot analysis, as well as evaluation of mRNA turnover by 4-thiouridine incorporation and affinity chromatography. Critical polymorphisms in the MAOA gene that control levels of activity will be assessed in control and PD males, where differences in allele frequencies have been noted in preliminary studies. To screen for an MAO deficiency state, genomic DNA from several hundred undiagnosed XLMR patients will be assessed for structural integrity of the MAO genes by multiplex PCR analysis of all exons, and SSCP analysis of conserved exons. In order to understand the effects of MAO deficiency on neuronal development, MAOA and MAOB genes will be disrupted in mice by homologous recombination in embryonic stem cells, formation of chimeric embryos and subsequent evaluation and breeding of mice. These studies will elucidate means by which the MAOA gene itself controls levels of activity, the extent of genetic variation in the MAO genes in the human population, and the effects of this variation on neuronal development and susceptibility to neuronal degeneration.

Variations in monoamine oxidase (MAO) activities in humans have been implicated as having a role in a number of human diseases, including alcoholism, psychiatric illness and Parkinson disease. Levels of activity for both forms of the enzyme, MAO-A and MAO-B, are genetically determined, as measured in skin fibroblasts and platelets, respectively; and both forms are critical to the metabolism of biogenic amines in the central nervous system and throughout the body. Full length cDNA sequences for MAO-A and MAO-B have been described and both genes have been mapped to the p11 region of the human X chromosome. The proposed studies will focus on the development of new molecular genetic techniques to resolve whether allelic variations exist for nucleotide and deduced amino acid sequences of MAO-A and MAO-B. New techniques will involve polymerase chain reaction (PCR) amplification of cDNAs combined with direct sequencing, chemical cleavage and denaturing gel electrophoresis to detect single base pair differences. Synthetic oligonucleotide primers will be used for first-strand cDNA synthesis of overlapping regions of 200-500 bp MAO-A and MAO-B mRNAs from skin fibroblasts and lymphocytes, respectively followed by PCR amplification. GC-rich clamps will be joined to the primers to increase the sensitivity of denaturing gels to detect differences among homo- and heteroduplex cDNAs from different individuals. Chemical cleavage will be carried out on heteroduplex cDNAs from pairs of male samples using a Maxam- Gilbert sequencing ladder to pinpoint the region of variation. Direct sequencing will be carried out by the dideoxy method using synthetic primers. These studies will provide a basis to establish whether allelic variations in the MAO loci exist and contribute to hereditary variations in MAO activity levels in the human population. The identification of sequence differences will also allow assessment of the correlation between specific MAO alleles and human disease states in larger populations, whether these differences occur in coding or regulatory elements of the MAO genes.

Torsion dystonia is a hereditary neurologic disease affecting brain function in over 100,000 individuals in North America. Symptoms include uncontrollable muscle spasms, spastic dysphonia, contortions of the limbs, torticollis, and blepharospasm. There are no neurochemical or neuropathological clues as to the cause of this disease. Dystonia is inherited as an autosomal dominant condition in many ethnic populations, with a high incidence in Ashkenazic Jews. Our group has located a gene responsible for generalized dystonia in Jewish and non-Jewish families on human chromosome 9q32-q34 by linkage analysis. Here we propose a systematic, molecular genetic approach to identification of this dystonia gene. We will define the location of the gene as precisely as possible; construct a genetic and physical map of the 9q32-q34 region; and characterize transcribed sequences from this genomic region expressed in the brain. Additional affected families that show linkage to this region will be ascertained, and information will be summed across families to define the critical region (2-3 cM) containing the gene by tightly linked, flanking markers. The location of the disease gene will be further specified by co-inheritance of specific alleles for surrounding loci. Additional genomic clones will be identified in the critical region using somatic cell hybrids containing portions of human chromosome 9q, YAC clones, and 'walking" procedures. These genomic clones will be physically mapped by somatic cell hybrid panels; pulse field gel electrophoresis; and in-situ hybridization to metaphase chromosomes. New clones will be screened for repeat sequences to determine highly informative polymorphisms, and genetic distances will be established in large reference pedigrees. Genomic fragments will be evaluated for encoded sequences by testing for sequences conserved among species, by hybridization to RNA on Northern blots, and by probing cDNA libraries. Brain cDNAs will be characterized by sequencing and evaluated for a role in dystonia by hybridization to DNA from control and affected individuals to look for gross rearrangements and by comparison of coding sequences generated by PCR amplification. These studies will serve to generate a detailed map of human chromosome 9q32-q34, and to elucidate brain-expressed transcripts coded therein. Eventually this work will lead to identification of the dystonia gene and understanding of its function, which in turn should provide insight into the molecular etiology of this disease state and help in designing new therapies.

The goal of this research is to clone and characterize the DYT1 gene responsible for onset of torsion dystonia in childhood. This is a severely debilitating movement disorder inherited as an autosomal dominant condition with reduced penetrance. Disregulation of movement is believed to originate in the brain without apparent neuronal degeneration. Identification of this disease gene should help elucidate the pathogenesis of this and other forms of dystonia, which afflict 100,000 people in Northern America, and help illuminate molecular mechanisms of movement control in the basal ganglia. At this time all the genomic DNA and a number of cDNAs encoded in the critical region (350-10(X) kb) on chromosome 9q32 have been cloned. Cloned genomic sequences will be assembled into a contiguous stretch and transcribed sequences will be identified within it by "exon amplification". Exons will be used to isolate cDNAs, which will be sequenced and evaluated for mutations. The search for incriminating mutations in the DYT1 gene will be carried out initially by PCR amplification and search for single strand conformational polymorphism (SSCPs) in exons from genomic DNA and in cDNAs generated by reverse transcriptase from patient's cells. Northern blot analysis will be undertaken to evaluate the distribution and size of the corresponding mRNAs in rat tissues and cultured cells from patients and controls. Genomic DNA in the critical region will also be searched for di-, tri- and tetranucleotide repeats which may expand in the disease state. Identification of a mutation will be followed by isolation of full length cDNAs, evaluation of exon structure and sequencing of candidate genes in affected individuals. Once the DYT1 gene identity has been confirmed, antibodies will be generated to deduced amino acid sequences and used in immunoblots and immunocytochemistry to evaluate the size and cellular localization of the encoded protein, dystonin. Our collaborator, Dr. Ann Graybiel (MIT), will evaluate the neuronal localization of this protein in basal ganglia in newborn and adult rat brains by immunocytochemistry an in situ hybridization. These studies should provide us with insights into the possible function of dystonin in the basal ganglia.

Torsion dystonia is a movement disorder originating in the basal ganglia which affects over 200,000 individuals in North America and the USSR. Symptoms include uncontrollable muscle spasms, spastic dysphonia, contortions of the limbs, torticollis and blepharospasm. It is estimated that at least half of the cases are hereditary, with at least two genes being responsible for autosomal dominant forms of the disease and one X- linked recessive disease gene. The disease gene responsible for the most common hereditary form of the disease, which occurs in the Ashkenazic Jewish as well as other populations, has been mapped by Dr. Breakefield's and Dr. Fahn's groups to chromosome 9q34. A common genetic haplotype for the Jewish disease allele has helped to define the chromosomal region to 1 cM with highly informative flanking markers. The purpose of the USA-USSR collaborative study is to expand genetic analysis using highly polymorphic markers in this chromosomal region in order to elucidate how many different hereditary forms of this disease are caused by this disease gene, to trace the origin of the mutation in the Jewish population, and to accumulate different mutations in this disease gene which may serve to incriminate and thus identify it. This study will include clinical and genetic analysis of dystonia patients and family members in the USSR, including video recorded examinations and extensive pedigree collection. In addition, DNA will be extracted from blood samples obtained from patients and their family members and analyzed for highly polymorphic markers in this chromosomal region. Data will be analyzed through the parent NIH grant application as well as collaborative studies undertaken by Dr. Breakefield and Dr. Fahn through funding from the Dystonia Medical Research Foundation. The availability of this rich source of patient material will be invaluable in elucidating the number of different genes and mutations causing dystonia, and in identifying the disease gene on chromosome 9q34.

Torsion dystonia is a movement disorder originating in the basal ganglia which affects over 200,000 individuals in North America and the USSR. Symptoms include uncontrollable muscle spasms, spastic dysphonia, contortions of the limbs, torticollis and blepharospasm. It is estimated that at least half of the cases are hereditary, with at least two genes being responsible for autosomal dominant forms of the disease and one X- linked recessive disease gene. The disease gene responsible for the most common hereditary form of the disease, which occurs in the Ashkenazic Jewish as well as other populations, has been mapped by Dr. Breakefield's and Dr. Fahn's groups to chromosome 9q34. A common genetic haplotype for the Jewish disease allele has helped to define the chromosomal region to 1 cM with highly informative flanking markers. The purpose of the USA-USSR collaborative study is to expand genetic analysis using highly polymorphic markers in this chromosomal region in order to elucidate how many different hereditary forms of this disease are caused by this disease gene, to trace the origin of the mutation in the Jewish population, and to accumulate different mutations in this disease gene which may serve to incriminate and thus identify it. This study will include clinical and genetic analysis of dystonia patients and family members in the USSR, including video recorded examinations and extensive pedigree collection. In addition, DNA will be extracted from blood samples obtained from patients and their family members and analyzed for highly polymorphic markers in this chromosomal region. Data will be analyzed through the parent NIH grant application as well as collaborative studies undertaken by Dr. Breakefield and Dr. Fahn through funding from the Dystonia Medical Research Foundation. The availability of this rich source of patient material will be invaluable in elucidating the number of different genes and mutations causing dystonia, and in identifying the disease gene on chromosome 9q34.

Torsion dystonia is a movement disorder originating in the basal ganglia which affects over 200,000 individuals in North America and the USSR. Symptoms include uncontrollable muscle spasms, spastic dysphonia, contortions of the limbs, torticollis and blepharospasm. It is estimated that at least half of the cases are hereditary, with at least two genes being responsible for autosomal dominant forms of the disease and one X- linked recessive disease gene. The disease gene responsible for the most common hereditary form of the disease, which occurs in the Ashkenazic Jewish as well as other populations, has been mapped by Dr. Breakefield's and Dr. Fahn's groups to chromosome 9q34. A common genetic haplotype for the Jewish disease allele has helped to define the chromosomal region to 1 cM with highly informative flanking markers. The purpose of the USA-USSR collaborative study is to expand genetic analysis using highly polymorphic markers in this chromosomal region in order to elucidate how many different hereditary forms of this disease are caused by this disease gene, to trace the origin of the mutation in the Jewish population, and to accumulate different mutations in this disease gene which may serve to incriminate and thus identify it. This study will include clinical and genetic analysis of dystonia patients and family members in the USSR, including video recorded examinations and extensive pedigree collection. In addition, DNA will be extracted from blood samples obtained from patients and their family members and analyzed for highly polymorphic markers in this chromosomal region. Data will be analyzed through the parent NIH grant application as well as collaborative studies undertaken by Dr. Breakefield and Dr. Fahn through funding from the Dystonia Medical Research Foundation. The availability of this rich source of patient material will be invaluable in elucidating the number of different genes and mutations causing dystonia, and in identifying the disease gene on chromosome 9q34.

The recent finding of a family in the Netherlands with a documented X- linked deficiency of monoamine oxidase A (MAOA)associated with episodic violent behavior (Brunner et al, 1993) has prompted us to evaluate the frequency of this deficiency state in males. We plan to undertake a genetic screen for mutations in the MAOA gene in adult males with a history of episodic impulsive or violent behavior, as compared to control males and males with undiagnosed X-linked mental retardation, Parkinson disease or hypertension DNA from blood samples will be obtained from several hundred affected individuals whose family history is consistent with X-linked mode of transmission of an episodic impulsive behavioral disorder. When possible blood samples will also be analyzed for altered amine metabolites by our collaborator, Dr. Dennis Murphy (NIMH), in which cases only individuals with abnormal levels of amines will be analyzed genetically. The integrity of the entire coding region of the MAOA gene will be determined by amplification of all 15 exons, using the polymerase chain reaction (PCR)and primers in the intronic and untranslated regions. Amplified fragments 150-250 kb in size will be analyzed for single strand conformational "polymorphisms" (SSCP)by resolution on non-denaturing acrylamide gels. Altered fragments will be sequenced directly to resolve sequence variations. This pilot study will provide an estimate of the frequency of the MAOA deficiency state as a basis for future investigations of this syndrome and the phenotypes associated with it.

This project is directed toward gene therapy for brain tumors by developing novel herpes simplex virus type 1 (HSV) derived vectors and evaluating their efficacy and toxicity in rodent models. The focus will be on: achieving gene delivery to tumor cells which have migrated away from the main mass; extending delivery to both dividing and non-dividing tumor cells; increasing the selectivity of therapeutic gene expression for tumor cells; and evaluating toxicity of the vectors. Recombinant virus and amplicon type vectors will be combined to achieve replication- conditional and replication-incompetent vector systems. Recombinant virus vectors will be combined to achieve replication-conditional and replication-incompetent vector systems. Recombinant virus vectors will serve as helper virus for the amplicon vector and will bear mutations in the immediate early gene, ICP27, which is essential for virus replication, as well as other virus gene, e.G. gamma 34.5, to reduce neurovirulence, and ribonucleotide reductase to make virus propagation selective for dividing cells. For replication-conditional vectors, the ICP27 gene will be placed in the amplicon under tumor-selective promoter elements, including the GFAP promoter, and the nestin enhancer. In this 'piggy back' system, propagation of the amplicon and recombinant virus vectors would be mutually dependent and cell-selective. Amplicons will be further modified so as to enhance their retention in dividing tumor cells by incorporation of elements from the adenoassociated virus (AAV)-, the rep gene and the ITRs, needed for integration into the host cell genome. Further, we will generate therapeutic vectors by incorporation of genes for prodrug-activating enzymes, cytochrome P4502B1 and HSV-thymidine kinase under control of tumor-selective regulatory elements. We will attempt to expand the range of the by-stander effect by targeting the prodrug activating enzyme to the cell surface so that toxic metabolites can be generated in the extracellular space. Gene delivery will be assessed in rat and human glioma lines and control lines in culture. Gene delivery to tumor cells and normal cells, as well as neuropathogenicity and systemic toxicity, will also be evaluated in rodents bearing intracerebral tumors following administration of the vectors directly into the tumor mass, through the ventricles or through the blood stream with temporary disruption of the blood-brain-barrier. Models will be developed to explore the effects of reactivation of latent wild type virus, immune response to the virus, and generation of recombinant virus. In combination with other projects in this Program these studies should provide a new generation of vectors to be evaluated in preclinical trials for potential use in humans.

alpha synuclein; disease /disorder etiology; genetic polymorphism; Parkinson's disease; posttranslational modifications; nucleic acid sequence; dystonia; nerve /myelin protein; complementary DNA; nucleic acid probes; animal genetic material tag; laboratory rat; laboratory mouse; human genetic material tag; human tissue;

Torsion dystonia is one of the most common and least well understood of movement disorders in humans. Affected individuals manifest contracted, twisting postures due to abnormal neurotransmission in the basal ganglia. Early onset torsion dystonia, the most common and severe of the hereditary dystonias, is a dominant disorder with reduced penetrance that manifests during a window of childhood development. Most cases of this disease are caused by inframe deletions in the DYT1 (TORIA) gene, which encodes a novel AAA+ chaperone protein, torsinA, with three homologous family members. The goals of this Program are to elucidate the location and function of torsinA in the nervous system during development and adult life, and to determine how mutations in torsinA perturb these functions. Our hypothesis is that defects in torsinA interfer with vesicle trafficking causing abnormal release of dopamine in the striatum, and, in turn, to alterations in the modeling of neuronal circuitry in the basal ganglia with subsequent compromise of motor control and learning. One Project will focus on determining whether torsinA is a component of vesicle trafficking and release mechanisms, and how expression of mutant torsinA affects signaling, receptor density and dopamine homeostasis in the striatum of humans and mouse models. A Second Project will investigate the distribution of the torsins in the developing mouse brain and how expression of mutant torsinA affects neuronal migration, process extension and synaptogenesis in targeted regions using transgenic mice and knock-out. A Third Project will explore the role of torsin in development of the nervous system in Drosophila and in protein trafficking in specific neuronal populations, and identify genes which can modify torsin-related functions. A Fourth Project will generate and characterize transgenic and conditional knock-out mice for torsinA with respect to motor control and learning and in response to pharmacologic manipulations of dopamine receptors. These Projects will be supported by an administrative core, and cores for biochemistry and antibody generation and clinical sample collection, databasing and pilot studies. Findings should provide insight into dysfunction of dopaminergic neurons, also involved in Parkinson's disease, and potential therapeutic interventions.

DESCRIPTION (adapted from the application) This application is directed toward achieving gene replacement in mammalian cells to correct genetic deficiency states using F-plasmid-based herpes simplex virus type 1 (HSV) amplicon (famplicon) vectors bearing elements of adenoassociated virus (AAV). Two strategies will be employed: 1) to increase the frequency of homologous recombination at endogenous mutant loci, large exogenous genomic fragments (50-150 kb) will be delivered by famplicons packaged in HSV virions, using a helper virus-free system. 2) to integrate transgene sequences into the AAVS1 site, the ITR elements and rep gene of AAV will be incorporated into famplicon vectors. Model systems will include cultured fibroblasts from patients with autosomal recessive deficiency states, GM1 gangliosidosis, and ataxia telangiectasia (AT), for which the corresponding genes, BGAL and ATM, are 65 and 150 kb in size, respectively. Recovery of function will be assessed at the single cell level by restoration of acidic betagalactosidase activity for GM1, and by decreased sensitivity to gamma irradiation for AT. Gene replacement by genomic integration will also be evaluated in mutant mice with the hypogonadal (hpg) deficiency state using the 8kb mouse gene LHRH- GAP, as a function of incorporation of the human AAVS1 locus into the mouse genome. Conditions will be developed for efficient delivery of transgenes to the hypothalamus by stereotactic injection of vectors in order to try to restore reproductive functions in hpg mice. This application extends existing technologies developed by Dr. Breakefield and Dr. Fraefel in a novel manner to achieve gene correction in normal human cells and in mouse models of human disease.

This project will focus on developing novel targeted viral vectors and imaging delivery and transgene expression in tumor cells and in mouse tumor models. Based on over ten years experience developing novel vectors and therapeutic transgenes for tumor therapy, it is clear that one of the major limitations to the effectiveness of this new treatment paradigm is insufficient gene delivery to tumor cells in vivo. In order to compare and optimize different delivery strategies, it is imperative to be able to quantitatively assess vector targeting and transgene delivery in animals, and eventually in human patients. Our goals, then, are to help develop technology, which can be widely applied to a variety of vector/tumor paradigms in order to effect on-site visualization of gene therapy in vivo. Our model vector will be the plasmid-based amplicon vector, which incorporates non-coding elements of herpes simplex virus type 1 (HSV), and hence combines ease of construction, highly efficient gene delivery, and minimal inherent toxicity. These vectors will be targeted to tumor cells by incorporation of the peptide/protein ligands, which bind to tumor- enriched receptors, into the envelope of the virion. These tumor receptors are believed to have an important role in the oncogenesis of specific tumor types and hence serve both as diagnostic markers and eventual therapeutic targets. Virions will be imaged in several ways, including: 1) 111/IN-oxine labeling or genetically modifying the viral envelope protein (gC) with metallothionein known to bind 99m/Tc to image genetically modifying the viral envelope protein (gC) with metallothionein known to bind. 99m/Tc to image mass distribution; and 2) use of proteases as reporters to rapidly monitor transgene expression in cells using previously developed near infrared fluorescent imaging probes (Nature Biotech 1999; 17:375-378). We will attempt to amplify the imaging signals conferred by transgene expression by genetic amplification of the transgene within the host cell nucleus. In addition, we will try to enlarge the domain of imaging by expression of proteins which can be translocated from transduced cells through the extracellular space to surrounding cells or which will mediate fusion of transduced cells with neighboring cells. And finally, we will elicit and monitor tumor cell killing through apoptosis, as mediated by expression of ICE which induces cell suicide. In vivo imaging will play a critical part in this project to test the novel approaches. The developed vector systems may ultimately be used to follow transgene delivery and expression in patients.

Description (provided by applicant): Meningiomas are brain tumors arising from the arachnoidal cells of the meninges that may occur sporadically, or in association with the inherited disorder, neurofibromatosis 2 (NF2). They account for about 25% of brain tumors and cause significant morbidity. A little more than half of sporadic meningiomas are caused by loss of merlin, the NF2 tumor suppressor protein, while the remainder is due to unknown causes.. As part of this Program Project grant, in 1993, we successfully isolated the NF2 gene and subsequently, we developed the reagents necessary to analyze merlin function and performed a basic characterization of merlin's structure and expression, finding that virtually all germline and somatic NF2 mutations involve elimination of merlin protein expression. In the past grant period, we focused on an examination of genetic changes in the initiation and progression of meningioma, using both merlin(-) and merlin(+) tumor specimens, and on the effects of these changes on cultured meningioma cells compared with their wild-type arachnoidal cell counterparts. In the coming grant period we plan to employ what we have learned in both genetic and cell biological studies to 1) identify and characterize genes involved in initiating meningioma tumor formation;2) identify and characterize genes involved in progression of these tumors to a more aggressive state;and 3) extend the strategy of whole genome analysis using array comparative genomic hybridization to other tumors from related disorders, including a) comparing pain-associated schwannomas from schwannomatosis to the schwannomas without associated pain seen in NF2 patients;b) comparing sporadic angiomyolipomas with angiomyolipomas found in association with tuberous sclerosis and c) performing an initial assessment of chromosomal changes in the periungual fibromas of tuberous sclerosis. Our findings from the previous grant period suggest that genetic changes can be a useful diagnostic tool in distinguishing different types of meningiomas that have very different prognosis for the patient. Thus, our investigations are likely to have a direct impact on improving diagnostic certainty and classification of meningiomas and result in better management of patients with these tumors. However, the longer-term impact will be the discovery of the specific molecular mechanisms that cause tumor growth and, by consequence, the delineation of targets against which new, specific therapeutic drugs can be developed to prevent the enlargement or recurrence of meningiomas and the morbidity and mortality that they cause.

DESCRIPTION (provided by applicant): The long term goal of this research is to understand the function of the novel protein, torsinA, which is mutated in early onset torsion dysconia. This neurologic disease is inherited as an autosomal dominant condition with reduced penetrance and a developmental window of susceptibility in childhood and adolescence. The torsins are members of the AAA+ superfamily of chaperone proteins involved in protein configurational changes. Studies to date suggest that torsinA may be involved in vectorial membrane movement and/or response of cells to oxidative stress. Only two in-frame mutations resulting in loss of amino acids in the carboxy terminal of this protein have been found in patients with early onset dystonia. In these studies we will screen for additional mutations in patients at the genomic and transcript levels. This analysis will be complemented by generation and expression of targeted mutations in the protein to elucidate structure/function determinants of ATPase activity, oligomerization, and posttranslational modifications. Cellular correlates will include the formation of whorled membrane inclusions in cells overexpressing the GAG-deleted form of torsinA found in most patients. In parallel, a search will be undertaken to identify partner proteins and their binding domains to torsinA, using the yeast two hybrid system, co-immunoprecipitation, affinity binding to purified protein, and protein chip assays. Immunocytochemistry will be used to visualize the cellular location of partner proteins relative to torsins.The predicted function of torsinA as a sensor to oxidative Stress will be examined by characterizing the posttranslational modifications to torsins which result from exposure to hydrogen peroxide and by evaluating whether expression of wild type or mutant forms of torsin act to protect or sensitize cells to this oxidative stress. TorsinA's predicted function in membrane and protein movement will be assessed by monitoring the morphology of the endoplasmic reticulum and vesicles, as well as constitutive and regulated secretion of proteins and vesicle recycling in cultured cells and neurons expressing mutant forms. These studies should help elucidate the function of this novel class of chaperone proteins and reveal how specific mutations in torsinA can disrupt cell function. Dystonia represents a special class of neurologic diseases, which do not manifest apparent neurodegeneration. This class of diseases may be amenable to therapy informed by the molecular etiology of dysfunction at the cellular level.

CORE ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): This research effort will focus on gene delivery of fluorescent proteins to neurons in the retina using HSV vectors in order to elucidate details of synaptic circuitry. In Aim 1, targeted gene delivery to specific neuronal subtypes will be undertaken by selective infection and by placing transgene expression under a cell specific promoter. The major heparin sulfate binding domain of the virion will be replaced with BDNF to increase infection of retinal ganglion cells that express high levels of the TrkB receptor on their cell surface. Transcriptional targeting will be undertaken by using the Thy-1 promoter, specifically expressed in this same neuronal population, either to drive expression of a reporter gene in HSV amplicon vectors, or by blocking expression of a reporter under a strong promoter in the vector through insertion of a IoxP-flanked stop codon between these sequences with subsequent infection of Thy-1-Cre transgenic mice to activate transgene expression. In Aim 2, we will attempt to label specific synaptic pathways by vector-mediated delivery of informative fluorescent fusion proteins. A GFP-tetanus toxin heavy chain fusion protein will be used to selectively label presynaptic contacts of the infected neurons. A BDNF-GFP fusion protein will be used to label synaptic partners of the infected cell which are enriched for TrkB-BDNF receptors. Aim 3 will undertake labeling of synaptically connected neurons by expression of reporter genes carried in a replicating vector system intended to have minimal-to-no toxicity for the host neurons. Vector propagation will be controlled by placing an essential viral gene for ICP4 under a tight tetracycline regulated (tet-on) promoter in an HSV amplicon vector and coupling infection with a mutant helper virus deleted for this gene. Both vector and helper virus will express reporter genes, and co-infection with both will allow drug-dependent regulation of the levels of virions produced and passed onto synaptically connected neurons. The proposed targeting and labeling methods should be adaptable to other types of receptors and synaptic connections throughout the nervous system and should be very useful in dissecting and monitoring synaptic circuitry in living and fixed preparations.

DESCRIPTION (provided by applicant): Effective gene therapy for hereditary neurologic disease will require the development of safe vectors, which can confer stable transgene expression on neurons. As a model disease we have chosen ataxia-telangiectasia (A-T), an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition and radiation sensitivity. These studies will focus on the development of plasmid-based HSV amplicon vectors to achieve site-directed integration of transgenes into the cell genome or to correct mutations in endogenous genes by homologous recombination (HR). Replacement vectors will incorporate elements of AAV, including ITRs and Rep, to target insertion of the ATM cDNA into an AAVS1 site engineered into the genome of Atm knock-out mice. Vectors for HR will include 8 and16 kb regions of the normal mouse gene spanning a 9 bp deletion in the Atm gene in knock-in mice, as well as sequences and expression cassettes for elements that should increase the frequency of HR, such as Rad51 and HSV ICP8. Successful integrations will be evaluated in cultured fibroblasts and neuroprecursor cells from these mice, as well as in primary neuronal cultures and in neurons in the cerebella of these mice by expression of the transgene (immunocytochemistry and RT-PCR) and analysis of recombinant events (PCR, Southern blots and sequencing). Functional recovery will be assessed following injection of vectors in the newborn mouse cerebellum by restoration of the normal apoptotic response of neurons to ionizing radiation, as well as by long-term improvement in motor function, reduction of oxidative damage and expression of ATM. These therapeutic vector designs have applications for a broad range of neurologic diseases allowing delivery and stable expression of large transgene cassettes and the potential for permanent correction of gene defects in neurons.

Description: The administrative component of this PSO (PPG) will have both scientific and organizational responsibilities. This Program Project will involve four interrelated research projects supported by two core facilifies. Dr. Breakefield will serve as the Program Director and liaison between laboratories. She will be assisted in administrative responsibilities by an experienced Grants manager, Ms. Robin Sobolewski. Dr. Breakefield will make all final decisions on the operations of the PSO after extensive discussions with involved individuals. Dr. Breakefield will be advised by an Internal Advisory Committee consisting of three senior members of the PPG: Dr. Pradeep Bhide, Dr. David Standaert and Dr. Laurie Ozelius, as well as two invesfigators at Partners (MGH/BWH) with related expertise - Dr. James Gusella (human genetics and neurologic disease) and Dr. David Kwiatkowski (mouse models for neurologic disease and cell biology). Combination conference calls/onsite meetings will be held at least twice a year with this group as issues arise needing advice. This group will also be present at the External Advisory meeting. The functions of this committee will consist of critical review of key data, strategic guidance in planning future experiments and collaborations, advice on any conflicts that arise within the PPG group or with collaborators, and appropriate adjustments resulting from any changes in Pl status or their locations. Patient issues will be handled by the physician/scientists in the group, Drs. Nutan Sharma and David Standaert, as well as our collaborator. Dr. Susan Bressman, with genefic counseling issues covered by Drs. Sharma and Bressman, with assistance from Ms. Trisha Multhaupt-Buell, a Genetic Counselor in the Movement Disorder Clinic. Advice on research strategy and evaluation of progress will be undertaken in consultation with a standing External Advisory Committee on a yearly basis at a day-long meeting at MGH. This committee will consist of five individuals (two, James Gusella and David Kwiatkowski also serving as members of the Internal Advisory Committee) with combined expertise in human genetics, neuronal development, cell biology, mouse models and clinical aspects of dystonia. (According to NINDS guidelines the additional names of members of the committee and letters from them will be provided when the application is funded). A detailed progress report incorporafing critiques from the external review prepared by the External Advisors and Internal non-PPG Advisors will be distributed to all investigators in the Center and incorporated into the progress report of the non-competitive confinuation applications. The scientific oversight will be carried out primarily by Dr. Breakefield, who will advise investigators on strategic and technical issues, promote collaboration among projects, monitor the functions and effectiveness of core facilities, and assess the scientific quality and progress of the work. This will require active interactions with all groups. Dr. Breakefield will confer with other members of the PSO on a weekly/monthly basis through informal discussions of progress and strategy via direct contact (Drs. Bhide, Sharma and Breakefield are located in adjacent space at MGH), phone calls, e-mail, tri-annual internal PSO meetings spread throughout each year, the annual External Advisor review and other national/international meetings attended by subsets of this group. In the annual progress reports we will include all interactions among Pis and collaborators in this PPG, as well as all attendees at the internal PPG meetings and External Reviews. To make sure there is ongoing exchange among Pis in the Northeast and Dr. Standaert in Georgia, in addition to the common meetings attended by Dr. Standaert and other Pis in this group, as well as biannual conference calls of the Internal Advisory Committee, we now plan to institute a monthly conference call among all Pis with exchange of data images using internet based technology for slide sharing (Glance.net). Through her close contacts with all the participants in this Center, Dr. Breakefield will be able to stimulate collaborative interactions and resolve potential conflicts before they arise. Given the high creativity and productivity of the investigators, their natural tendency to collaborate and their long track record of interaction and commitment to dystonia research, her main job will be to promote communication of research findings and sharing of reagents/resources within and among outside groups, and to guide investigators toward common foci and goals. Organizational aspects will be handled by Dr. Breakefield's administrative co-ordinator, Ms. Robin Sobolewski. These aspects will include setting up meetings, distributing memos and notices, assembling internal and external reports and interacting with administrators working for the other investigators at MGH, UAB and Mt. Sinai. Administrative issues that will be handled by both Dr. Breakefield and Ms. Sobolewski include rebudgeting of funds, changes in personnel, issues of indirect cost rates, and input into institutional space allocations. They will prepare all progress reports and applications related to this Program Project. Dr. Breakefield has had extensive administration experience. She served as Director of the Molecular Neurogenetics Division at the Shriver Center for over four years. She has directed a NINDS Program Project grant on "Molecular Genetics of Inherited Neurological Diseases" s\nce it commenced funding in 1987 and has been actively involved at her institution and nationally in reviewing clinical gene therapy protocols. She serves on the Board of Directors of the American Society of Gene Therapy, on the Scientific Advisory Board of the Bachmann-Strauss Dystonia - Parkinson Research Foundation, on an NINDS Study Secfion (NSD-B) and as an advisor in the Cure Dystonia Inifiative ofthe Dystonia Medical Research Foundafion. Her primary research Interest has been in dystonia since 1981 and during that fime she has interacted and collaborated with many scientists in this field which provides a nexus of information and resources for the current research efforts by this group. Ms. Robin Sobolewski has had over 16 years training and experience in business, and medical and scientific management, including administrative and fiscal management of two NIH POIs and many R01/R21s and foundation grants. Dr. Breakefield and Ms. Sobolewski work in neighboring offices at MGH-East. Ms. Sobolewski's office space is equipped with a Hewlett Packard (with links to MGH investigators and other collaborators, and reference database and search programs), a laser-jet printer, and a copy machine. Dr. Breakefield will devote 10% of her effort and Ms. Sobolewski 33% to this administrative core.

This project will focus on expanding the therapeutic range of HSV-1 vectors for treatment of brain tumors and on creating zones of resistance to tumor growth in the normal brain by developing novel vectors and delivery modalities. The current proposal plans to increase the therapeutic capacity of the oncolytic HSV-1 vector, MGH2 in two ways: by enhancing infection of glioblastoma (GBM) cells through targeting surface antigens by virions;and by combining oncolytic vectors with amplifying/integrating HSV/AAV amplicon vectors to increase and sustain therapeutic transgene levels. We will also attempt to create a zone of resistance to tumor growth in normal brain via AAV vectors and neuroprecursor cells (MFCs) expressing secretable therapeutic proteins. NPCs and chimeric immune receptor (CIR) lymphocytes will be used to target invasive tumor cells. All studies will be carried out in culture and in intracranial human glioma models in nude mice using bioluminescence imaging reporters to track vector and protein delivery, cell fate and tumor growth. An immunocompetent syngeneic rat GBM model will also be used in evaluating CIR lymphocytes. Aim 1 will explore selective infection of glioblastoma cells expressing the mutant EGF receptor, EGFRvIII, by modification of the glycoprotein C envelope protein of HSV-1 virions for presentation of antibodies to this receptor. Aim 2 will evaluate the ability of CIR lymphocytes to target to EGFRvIII and VEGFR receptors in tumor foci. In Aim 3, AAV vectors and genetically modified NPCs will be used to deliver secretable forms of the tumorspecific apoptotic protein, TRAIL, and the anti-angiogenic factor, Flkl to normal brain to create a region of resistance to tumor growth. Aim 4 will employ a dual gene delivery system combining hybrid amplicon vectors incorporating p5 and ITR DNA elements from AAV, and Rep 78/68 proteins as fusions with the HSV-1 virion tegument protein, VP16. Co-infection with oncolytic HSV-1 or a subset of HSV-1 genes will be evaluated for replicative amplification and genomic integration of amplicon-encoded transgenes. This project will use expertise on oncolytic virus (Project 1, Chiocca), in vivo imaging (Project 3, Weissleder), biostatistics (Core A, Finkelstein), HSV vector stocks (Core B, Krisky/Glorioso), and human GBM cells and neuropathology (Core C, Louis). These studies are designed to complement and increase the therapeutic impact of the HSV-1 oncolytic vector strategy for cancer treatment and to explore the possibility of creating regions of the brain resistant to tumor growth by sustained release of therapeutic proteins, using means deemed compatible with human trials.

1H-Thieno(3,4-d)imidazole-4-pentanoic acid, hexahydro-2-oxo-, (3aS-(3aalpha,4beta,6aalpha))-; 2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo(1,2-a)pyrazin-3-one; 21+ years old; 72-kDa Gelatinase; 72-kDa Type IV Collagenase; 72kD type IV Collagenase; AAV vector; APO2 Ligand; APO2L; Address; Adeno-Associated Viruses; Adult; Amino Acid Sequence; Amino Acids; Apo-2 Ligand; Apo-2L; Apoptosis; Apoptosis Pathway; Arts; Astrocytoma, Grade IV; Athymic Nude Mouse; Avidin; Binding; Binding (Molecular Function); Biochemical; Bioluminescence; Biosynthetic Proteins; Biotin; Blood; Brain; Brain Neoplasia; Brain Neoplasms; Brain Tumors; CD31; Capsid; Catalytic Core; Catalytic Domain; Catalytic Region; Catalytic Site; Catalytic Subunit; Cell Communication and Signaling; Cell Death, Programmed; Cell Isolation; Cell Line; Cell Lines, Strains; Cell Segregation; Cell Separation; Cell Separation Technology; Cell Signaling; Cell Surface Proteins; Cell surface; Cell-Extracellular Matrix; CellLine; Cells; Chemistry; Chimera Protein; Chimeric Proteins; Cleaved cell; Collaborations; Condition; Corynebacterium Diphtheriae Toxin; Cultured Cells; Cultured Tumor Cells; DNA Synthesis Factor; Data; Dependovirus; Detection; Development; Diagnosis; Diagnostic; Diphtheria Toxin; Dose; ECGF; ECM; EGFP protein; Effectiveness; Encephalon; Encephalons; Endopeptidases; Endothelial Cell Growth Factor; Endothelial Cells; Endothelium; Engineering; Engineerings; Environment; Esteroproteases; Estrogen Receptors; Evaluation; Extracellular Matrix; FGF; Fibroblast Growth Factor; Fibroblast Growth Regulatory Factor; Firefly Luciferases; Fluorescence; Fusion Protein; GFP; Gelatinase A; Gelatinase Neutrophil; Gene Delivery; Generalized Growth; Generations; Genes; Genetic; Glial Cell Tumors; Glial Neoplasm; Glial Tumor; Glioblastoma; Glioma; Grade IV Astrocytic Neoplasm; Grade IV Astrocytic Tumor; Green Fluorescent Proteins; Growth; HBGF; Human; Human, Adult; Human, General; Hypoxia; Hypoxic; IV drip; Image; Imidazo(1,2-a)pyrazin-3(7H)-one, 6-(4-hydroxyphenyl)-2-((4-hydroxyphenyl)methyl)-8-(phenylmethyl)-; Immunologic, Luciferase; Implant; Improve Access; Infusion; Infusion procedures; Injection of therapeutic agent; Injections; Intracellular Communication and Signaling; Intravenous Drip; Intravenous Infusion; Killings; Label; Lentiviral Vector; Lentivirus Vector; Libraries; Ligase; Link; Location; Luciferases; Luciferases, Firefly; Luciferases, Renilla; Luciferases, Sea Pansy; LysoTracker; MMP-2; MMP2; MMPs; MR Imaging; MR Tomography; MRI; Magnetic Resonance Imaging; Magnetic Resonance Imaging Scan; Mammals, Mice; Man (Taxonomy); Man, Modern; Matrix Metalloproteinase-2; Matrix Metalloproteinases; Mediating; Medical Imaging, Magnetic Resonance / Nuclear Magnetic Resonance; Metallopeptidases; Metalloproteases; Metalloproteinases; Methods; Mice; Mice, Athymic; Mice, Nude; Modality; Modeling; Molecular Biology, Protein Sequencing; Molecular Interaction; Monitor; Murine; Mus; NMR Imaging; NMR Tomography; Neoplasms of Neuroglia; Neoplastic Cells, Cultured; Nervous System Neoplasms; Nervous System, Brain; Neuroglial Neoplasm; Neuroglial Tumor; Normal Tissue; Normal tissue morphology; Nuclear Magnetic Resonance Imaging; Nude Mice; Numbers; Organ; Oxygen Deficiency; PDGFR; PECAM1; PECAM1 gene; PEX; Peptidases; Peptide Hydrolases; Peptide Library; Peptide Peptidohydrolases; Peptide Sequence Determination; Peptides; Phenotype; Photinus luciferin 4 monooxygenase; Pilot Projects; Platelet-Derived Growth Factor Receptor; Population; Procedures; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Property; Property, LOINC Axis 2; Proteases; Protein Sequencing; Protein Structure, Primary; Proteinases; Proteins; Proteins, Cell Surface; Proteolytic Enzyme; Proteolytic Enzymes; RT-PCR; RTPCR; Receptor Protein; Receptors, PDGF; Recombinant Proteins; Recombinants; Relative; Relative (related person); Renilla Luciferases; Reporter; Reporting; Research Design; Reticuloendothelial System, Blood; Reverse Transcriptase Polymerase Chain Reaction; Science of Chemistry; Screening procedure; Sensitivity and Specificity; Sequence Determinations, Amino Acid; Sequence Determinations, Protein; Signal Transduction; Signal Transduction Systems; Signaling; Sorting - Cell Movement; Specificity; Strepavidin; Streptavidin; Study Type; Synthetases; TL2; TNF-Related Apoptosis Inducing Ligand TRAIL; TNF-related apoptosis-inducing ligand; TNFSF10; TNFSF10 Protein; TRAIL Protein; Tail; Testing; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Time; Tissue Growth; Tumor Cell; Tumor Cells, Cultured; Tumor Necrosis Factor Ligand Superfamily Member 10; Tumors of Neuroglia; Tumors of the Nervous System; VEGF Receptors; VEGFR; VPF Receptor; Vascular Endothelial Cell; Vascular Endothelial Cell Growth Factor Receptor; Vascular Endothelial Growth Factor Receptor; Vascular Permeability Factor Receptor; Veins; Ventricular; Viral Vector; Virus; Viruses, General; Vitamin H; Work; Zeugmatography; adeno associated virus group; adeno-associated viral vector; adeno-associated virus vector; adult animal; adult human (21+); aminoacid; angiogenesis; base; biological signal transduction; brain tissue; cell sorting; cell transduction; cell type; cellular transduction; cleaved; coat (nonenveloped virus); coelenterazine; coenzyme R; cultured cell line; design; designing; drip infusion; enhanced green fluorescent protein; extracellular; gene product; genetically modified cells; glioblastoma multiforme; imaging; imaging probe; immunocytochemistry; improved; in vivo; intervention therapy; intravenous administration; macromolecule; mature animal; metalloproteinase (general); neoplastic cell; neuropathology; new therapeutics; next generation therapeutics; novel; novel therapeutics; ontogeny; particle; pilot study; protease; protein expression; protein sequence; proteinase; receptor; response; reverse transcriptase PCR; screening; screenings; sensor; sorting; spongioblastoma multiforme; study design; subcutaneous; therapeutic gene; therapeutic protein; tomography; transduced cells; transduction efficiency; tumor; tumor growth; tumors in the brain; vector; vector genome; vein infusion

0-6 weeks old; AAV vector; Adeno-Associated Viruses; Age; Algorithms; Alleles; Allelomorphs; Ancient Neurilemmoma; Ancient Schwannoma; Anti-Oncogenes; Antioncogenes; Apoptosis; Apoptosis Pathway; Apoptotic; Astrocytes; Astrocytus; Astroglia; Athymic Nude Mouse; Bears; Behavior; Behavioral; Benign; Bioluminescence; Blotting, Northern; Body Tissues; Bourneville Disease; Bourneville syndrome; Bourneville-Brissaud disease; Bourneville-Pringle syndrome; Brain; Brain Ventricle; Breeding; CMV promoter; Cancers; Caspase 1, Apoptosis-Related Cysteine Protease; Caspase 1, Apoptosis-Related Cysteine Protease (Interleukin 1, Beta, Convertase); Caspase-1; Cell Death, Programmed; Cell Line; Cell Lines, Strains; Cell/Tissue, Immunohistochemistry; CellLine; Cells; Cellular Expansion; Cellular Growth; Central Nervous System; Central Neurofibromatosis; Cerebral Ventricles; Cessation of life; Clinical; Comparative Genome Hybridization; Condition; Crush Injury; Data; Death; Debulking; Degenerated Neurilemmoma; Degenerated Schwannoma; Dependovirus; Development; Disease; Disease regression; Disorder; Down-Regulation; Down-Regulation (Physiology); Downregulation; Drugs; EC 3.4.22.36; Elevation; Embryo; Embryonic; Emerogenes; Encephalon; Encephalons; Epiloia; Epiloias; Evaluation; Firefly Luciferases; Forecast of outcome; GFP; Ganglia; Ganglion Cysts; Ganglionic Cysts; Ganglions; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Generalized Growth; Genes; Genes, Cancer Suppressor; Genes, LacZ; Genes, Onco-Suppressor; Genes, p53; Genetic; Genetic Alteration; Genetic Change; Genetic Intervention; Genetic defect; Genomics; Giant Cells; Green Fluorescent Proteins; Growth; HSV; Hamartin; Hereditary; Herpes Simplex Virus; Herpes labialis Virus; Herpesvirus hominis; Histology; Human; Human, General; ICE Protease; IHC; IL-1 beta Convertase; IL-1 beta-Converting Enzyme; IL-1b Converting Enzyme; IL1B-Convertase; IL1BC; Image; Immunohistochemistry; Immunohistochemistry Staining Method; Implant; Infant, Newborn; Inherited; Injection of therapeutic agent; Injections; Injury; Interleukin 1-B Converting Enzyme; Interleukin 1-Beta Convertase; Interleukin-1 Beta Converting Enzyme; Interleukin-1 Converting Enzyme; Interleukin-1beta Converting Enzyme; Intervention, Genetic; KIAA0243; Knock-out; Knockout; Knockout Mice; LacZ; LacZ Genes; Lateral Ventricle of Brain; Lateral Ventricles; Lateral ventricle structure; Length of Life; Lentiviral Vector; Lentivirus Vector; Lesion; Location; Longevity; Loss of Merlin Expression; Luciferases, Firefly; Lymphocytes, Null; Malignant Neoplasms; Malignant Tumor; Mammals, Mice; Man (Taxonomy); Man, Modern; Medication; Merlin; Messenger RNA; Mice; Mice, Athymic; Mice, Knock-out; Mice, Knockout; Mice, Nude; Mice, Transgenic; Micro RNA; MicroRNAs; Modeling; Moesin-Ezrin-Radixin-Like Protein; Molecular Biology, Gene Therapy; Monitor; Multinucleated Giant Cells; Murine; Mus; Mutation; Myxoid cyst; NDUL; NF2 Gene Product; NRVS-SYS; Nerve Cells; Nerve Fibers; Nerve Unit; Nervous System; Nervous System, Brain; Nervous System, CNS; Nervous system structure; Neural Cell; Neural Ganglion; Neuraxis; Neurilemma Cell; Neurilemmal Cell; Neurilemmoma; Neurilemoma; Neurinoma; Neurocyte; Neurofibromatosis 2; Neurofibromatosis 2 Gene Product; Neurofibromatosis II; Neurofibromatosis Type 2 Protein; Neurofibromatosis, Acoustic, Bilateral; Neurofibromatosis, Central, NF 2; Neurofibromatosis, Central, NF2; Neurofibromatosis, Type 2; Neurofibromatosis, Type II; Neurofibromin 2; Neurologic; Neurologic Body System; Neurologic Organ System; Neurological; Neurons; Newborn Infant; Newborns; Nodule; Normal Cell; Normal Tissue; Normal tissue morphology; Northern Blotting; Northern Blottings; Nude Mice; Null Cells; Null Lymphocytes; Null Mouse; Oncogenes, Recessive; Oncogenes-Tumor Suppressors; Outcome; Overexpression; P-30; P-30 Protein; P30; P30 Protein; P45; P53; Patients; Pattern; Peripheral Nerves; Peripheral Nervous System; Phakomatosis, Bourneville; Pharmaceutic Preparations; Pharmaceutical Preparations; Phenotype; Photinus luciferin 4 monooxygenase; Polykaryocytes; Pringle disease; Prognosis; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Protein Analysis; Protein Overexpression; Proteins; RNA blot analysis; RNA blotting; RNA, Messenger; Radial; Rapamune; Rapamycin; Rate; Regression; Relative; Relative (related person); Replacement Therapy; Role; Sampling; Schwann Cells; Schwannoma; Schwannomerlin; Schwannomin; Schwannomin Protein; Sciatic Nerve; Seizures; Simplexvirus; Sirolimus; Site; Source; Staging; Structure; Structure of sciatic nerve; Subependymal; Subependymal Giant Cell Astrocytic Neoplasm; Subependymal Giant Cell Astrocytic Tumor; Subependymal Giant Cell Astrocytoma; Synapsins; Syncytium; TP53; TP53 gene; TRP53; TSC1; TSC1 gene; TSC1 protein; TSC1 protein, human; Therapeutic; Therapeutic Effect; Therapeutic Intervention; Therapy, DNA; Thinking; Thinking, function; Time; Tissue Growth; Tissues; Transgenic Mice; Tsc1 [{C0694894}]; Tuberous Sclerosis; Tumor Cell; Tumor Debulking; Tumor Protein p53 Gene; Tumor Suppressing Genes; Tumor Suppressor Genes; Tumor Volume; Type 2s, Neurofibromatosis; Up-Regulation; Up-Regulation (Physiology); Upregulation; Ursidae; Ursidae Family; Ventricular; Virus; Viruses, General; Volume, Tumor; Weight Gain; Weight Increase; Work; acoustic neurofibromatosis; adeno associated virus group; adeno-associated viral vector; adeno-associated virus vector; adenoma sebaceum; beta Actin; body weight gain; body weight increase; cell growth; cell type; cerebral sclerosis; comparative genomic hybridization; cultured cell line; day; design; designing; disease/disorder; drug/agent; epiploia; gene function; gene product; gene replacement; gene therapy; genetic therapy; genome mutation; hereditary multiple system hamartomatosis; herpesvirus; human TSC1 protein; human herpesvirus 1 group; imaging; implantation; in vivo; insight; intervention therapy; lateral ventricle; life span; lifespan; mRNA; malignancy; meningioma; miRNA; mouse model; neoplasm/cancer; neoplastic cell; nerve injury; nervous system development; nestin; nestin protein; neural injury; neurinomatosis centralis; neuromatosis universalis; neuronal; neuropathology; neurospongioblastosis diffusa; newborn human (0-6 weeks); oncosuppressor gene; ontogeny; outcome forecast; overexpress; phacomatosis; postnatal; ranpirnase; recombinase; sciatic nerve; sclerosis tuberosa; shRNA; short hairpin RNA; size; small hairpin RNA; social role; spongioblastosis circumscripta; tuberose sclerosis; tumor; tumor growth; vector; wt gain

Core A / Administration Description: The administrative component of this Center will have both scientific and organizational responsibilities. The scientific aspects will be carried out primarily by Dr. Breakefield, who will advise investigators on strategic and technical issues, promote collaboration among projects, monitor the functions and effectiveness of core facilities, and assesses the scientific quality and progress of the work. This will require active interactions with all groups. She will visit the participating laboratories and discuss Center-related projects with P.l.'s and laboratory workers about once every other week. Drs. Gusella and Ramesh are in the Simches Building at Mass. General Hospital which is a 15 min shuttle bus ride from Mass. General East where Dr. Breakefield is located; Dr. Kwiatkowski is located at the Harvard Medical School (HMS) Longwood Campus (30 min shuttle), which Dr. Breakefield visits every other week for courses, seminars, conferences and collaborations. Any scientific problems or organizational issues that arise will be discussed by an advisory committee consisting of Drs. Xandra Breakefield, Daniel Haber, Robert Martuza and Pradeep Bhide, which will meet on an ad hoc basis. Decisions as to how to maximize productivity of the Center, including scientific judgment and budgeting adjustments, will be made by Dr. Breakefield after extensive discussion with this advisory committee and key investigators involved in particular decisions.

DESCRIPTION (provided by applicant): This study will evaluate the use of RNA in serum exosomes as biomarkers for cancer. Exploration of this new technology will be carried out in a clinical research study on glioma patients, based on our studies demonstrating that serum/plasma exosomes from glioblastoma patients are derived from tumors and contain mutant RNAs and relative levels of mRNAs and microRNAs distinctive to those tumors. Gliomas are the most common type of primary brain tumor in humans occurring at an incidence of about 3 cases/100,000 individuals per year with a 5 year survival rate of about 3%. Treatment is confounded as diagnosis depends on neurosurgical biopsy and pathologic analysis. Once removal of the main tumor mass has been carried out patients are monitored by MRI and neurologic symptoms for signs of tumor regrowth, both of which can give false-positives due to residual effects of brain surgery and chemical/radiation treatment. Our findings are the first to support RNA serum biomarkers which have the potential to report on the presence, burden and classification of distinct subtypes of glioma tumors, i.e. EGFRvIII. This distinctive mutant/variant for glioblastoma, EGFRvIII mRNA in serum exosomes from these patients can in itself be the basis for a marketable product which can inform the clinical status and prognosis of patients, as well as indicate appropriate drug treatments and track therapeutic responses. The objectives of this study will be: 1) to optimize methods for collection and storage of serum/plasma, and isolation of exosomes in order to obtain high yields and integrity of RNA;2) to determine whether the mutant/variant EGFRvIII mRNA in serum/plasma exosomes can serve as a marker of tumor burden and/or progression in glioma patients;and 3) to initiate a systematic assessment of other known genetic changes i gliomas which can be detected in serum/plasma exosomes of the patients. PUBLIC HEALTH RELEVANCE: is study will evaluate the use of RNA in serum exosomes as biomarkers for cancer. Our previous studies have demonstrated that serum exosomes from glioblastoma patients are derived from tumors and contain mutant RNAs and upregulated levels of mRNAs and microRNAs distinctive to these tumors. We will focus on optimization of this technology and relevance for tumor burden and recurrence in glioma patients. This technology has wide applications as most types of cancer also release exosomes into the serum and have distinguishing RNA features.

DESCRIPTION (provided by applicant): Gliomas account for about 60% of all primary CNS tumors. Glioblastoma multiforme (GBM) or grade IV gliomas which comprise two thirds of all gliomas are the most malignant form. Limitation to brain tumor therapy in general is the difficulty in delivering therapeutic agents across the blood-brain barrier (BBB) sufficient to achieve high concentrations at the tumor site. In this project, we will focus on developing a gene delivery system that can change the surface of a tumor cell so that it presents a molecular "beacon" (biotinylated receptors) to attract diagnostic and therapeutic agents to it. This strategy is designed to make tumor cells a selective, "easy target" for different biotinylated-imaging agents (for diagnostics) or biotinylated-diphtheria toxin (for therapy) complexed to streptavidin which binds with extremely high affinity to biotin (Kd = 10-15) and can be internalized into tumor cells. A critical component of this strategy will be to facilitate delivery of these biotinylated therapeutic/diagnostic agents from the vasculature into tumors in the brain using a complex between streptavidin and transferrin. This universal transport vehicle will take advantage of transferrin receptors on blood vessels in the brain which normally facilitate passage of molecules across the blood-brain barrier. This should enhance the delivery of targeted toxins to the brain and therefore increase their therapeutic efficacy for brain tumors by virtue of high affinity binding between biotin on the tumor cell surface and streptavidintoxin complex. The toxin complex will be safe to normal cells which will not take it up by endocytosis, but will be activated in tumor cells by endocytosis mediated by the biotinylated receptors. The advantage of this biotinylation approach is that it allows the imaging of brain tumors with different modalities including Magnetic Resonance (MR), Positron Emission Tomography (PET) as well as Single Photon Emission Computed Tomography (SPECT). The strategies proposed here are designed to have a major impact on the development of more efficient brain tumor therapeutic and imaging systems and can be combined with traditional therapies. The versatility of this technique can be extended to allow the tracking of any cell transduced to express biotin on its surface, including stem cells, and is fully compatible with translation into humans since all components of this system including the biotin-streptavidin system, the different imaging modalities, diphtheria toxin as well as gene transfer have already been used separately in clinical trials in the USA.

Dystonia is the third most common movement disorder in humans, second only to Parkinson's disease and tremor. The most severe form of dystonia, early onset generalized torsion dystonia, inherited as an autosomal dominant condition, results from a mutation in the coding region of TOR1A. This gene has also been implicated in more prevalent forms of adult onset focal dystonias. TorsinA is an AAA+ protein located primarily in the endoplasmic reticulum(ER) and nuclear envelope(NE). The proposed studies are designed to further characterize torsinA in order to elucidate its functions, as well as dysfunctions caused by the DYT1 mutant form. TorsinA is hypothesized to modulate interactions between NE/ER proteins and the cytoskeleton involved in neurodevelopment, synaptic neurotransmission and response to stress. Aim 1 - Evaluate the effect of torsinA status on migration of neurons in culture. The effect of torsinA in neuronal migration will be studied using striatal and cortical neurons from homozygous torsinA knock-out, heterozygous knock-in, transgenic and control mouse embryos. Migration will be monitored in microfabricated channels using vital fluorescent dyes and proteins to monitor speed of migration and intracellular movement of cell organelles, with correlative immunocytochemistry. Aim 2 - Elucidate the ER function of torsinA in processing proteins through the secretory pathway. The focus will be on the function of torsinA in the ER using DTY1 patient and control fibroblasts and mouse neurons with or without torsinA. We will assess secretion of reporter luciferases to monitor release through the secretory and vesicular pathways, as well as processing of normal glycoproteins, the D2 receptor and epsilon-sarcoglycan (mutated in myoclonic dystonia). Immunocytochemistry, cytoskeletal-disrupting drugs and biochemical methods will be used to evaluate potential interactions between torsinA and ER proteins involved in entry into, exit from or movement of the ER. Aim 3 - Explore involvement of torsinA in the ER stress response. We will evaluate whether mutant torsinA confers hypersensitivity to different forms of ER stress in DYT1 patient as compared to control fibroblasts, and in neurons and neural cells expressing human wild-type or mutant torsinA. ER stress will be monitored using a luciferase reporter to monitor the initial delay in protein synthesis, as well as more traditional methods such as splicing of the XBP-1 message, elevation of BiP levels and cell death. This research will enhance understanding of the molecular etiology of dystonia and provide insights into potential therapeutic intervention.

DESCRIPTION (provided by applicant): Advances in the fight against brain cancer provide new opportunities to personalize and improve drug treatment of patients. These have included genome sequencing of hundreds of glioblastoma (GBM) tumors, pathway analysis of upregulated mRNAs, increasing sophistication of drug design to target driver mutations, and the discovery that tumors release exosomes into the circulation that manifest the genotype of individual tumors. This proposal will expand the informational content of a Phase II drug trial for GBM to include a research arm to monitor the genetic status of the tumors in individual patients over time using specific mutations and levels of mRNA and miRNA in serum exosomes as biomarkers. These biomarkers have the potential to elucidate which genetic changes in tumors make patients more responsive to a specific drug, whether compensatory genetic changes occur in the tumor during treatment, and how these influence the overall response to therapy. This clinical trial will evaluate a novel irreversible inhibitor of a kinase signaling pathway which is activated by alterations in the epidermal growth factor receptor (EGFR) in GBM patients. The cohort will consist of recurrent patients with verified EGFR amplification based on tumor biopsies. A subset of tumor samples will be analyzed for mutations in genomic DNA in 17 cancer driver genes by the Molecular Pathology Service - in parallel we will monitor a number of genetic parameters critical for GBM growth in RNA from longitudinal serum exosome samples from 56 patients in this study, all of whom will concurrently have a full clinical workup. All samples will be biobanked and of sufficient size to allow multiple additional assays in future studies. Statistical analysis will be carried out to evaluate correlations between genetic status of tumors and radiographic assessment of tumor volume, progression-free survival and overall survival. We predict that the constellation of genetic changes in individual GBM tumors can predict their susceptibility to specific drugs and that response to drug treatment can be monitored by analysis of RNA from circulating tumor exosomes.

DESCRIPTION (provided by applicant): This Neuroscience Center Core Facility, directed by Drs. Xandra Breakefield and Bakhos Tannous will be an institutionally unique resource which will bring our neuroscience faculty together and enhance their research on neurologic disorders through powerful, synergistic new technologies. The three new proposed cores are truly state-of-the-art. The Imaging Core (directed by Drs. Brad Hyman and Marian DiFiglia) will include array tomography, transmission electron microscopy and in vivo multiphoton imaging of detailed brain structure. The Microfluidics Core (directed by Drs. Daniel Irimia and Mehmet Toner) will provide specialized microfluidic devices and expertise in analysis for separation of cells and cell organelles, and high precision, real-time imaging of cell migration and axon guidance. The Vector Core (directed by Drs. Bakhos Tannous and Xandra Breakefield) will generate a variety of viral vectors for all projects and continue to expand its repertoire with advances in this field now including 10 serotypes of adeno-associated virus (AAV) vectors and coat-deficient rabies virus. The administration organization overseeing this program will include the P30 Director/Co-Director and Core Directors/Co-Directors to monitor ongoing activities and to interface with users; a Steering Committee to review operating procedures and activity reports on an annual basis, and to advise on prioritization issues and core effectiveness; an Advisory Board to offer advice on technology updates and management issues; and an Administrative Core which will logistically support the program with a dedicated senior grants manager All these services will be offered free-of-charge to the NINDS-funded neuroscience investigators at our institution and will serve to create a very dynamic, interactive environment where ideas become reality. This group of neuroscience investigators includes international leaders in the field, as well as junior and mid-level investigators for whom research on neurologic disorders is the main focus of their work and who have made many major contributions in the field. Our research covers a broad range of neurologic disorders due to stroke, injury, brain tumors and seizures, as well as neurodegenerative diseases including Alzheimer's disease, Parkinson's disease and Huntington's disease, and pediatric disorders, such as early onset dystonia, familial dysautonomia, hereditary sensory and autonomic neuropathy and adrenoleukodystrophy. Our vision for this PSO Core Facility is to achieve a deeper level of understanding of disease pathogenesis and to discover means of alleviation by providing technologies which can achieve high resolution of brain structure.

PROJECT SUMMARY (See instructions): The goal of this project is to maximize genetic information about glioblastoma (GBM) tumors by RNA analysis of tumor-derived microvesicles (MVs) in serum and to develop assays for these genetic parameters that can be applied to clinical samples. Aim 1 will focus on improving isolation methods for tumor MVs from serum. This will involve defining expression of antigenic markers for GBM cells and MVs derived from them, as compared to MVs released from normal cells into the serum. GBM-selective antibodies will be used to enrich for tumor-derived MVs in serum by antibody-mediated microfluidic capture. This enrichment should increase our ability to assay tumor mRNA mutations and levels. Aim 2 will characterize biomarker RNA content of longitudinal serum MVs from mice bearing GBM tumors and from pre-operative serum/tumor sets from 100 GBM patients and serum from 100 controls. Assays will be developed to monitor a set of key mRNAs known to be mutated or have altered levels in subtypes of GBM tumors. Assays will include TaqMan qRT-PCR analysis and BEAMing for detection and quantitation. In Aim 3 we will screen for levels of specific RNAs and mutations in serum MVs obtained longitudinally in GBM mice undergoing different treatment modalities and in clinical phase l/ll trials of human GBM patients. Interactions in this P01 are tightly interwoven among projects and cores. We will provide expertise and assay development for analysis of RNA in MVs from oncolytic virus infected tumors and serum with Project 1, and share parallel mouse and human serum samples, as well as antibodies with Project 2 for designation of antigens enriched on GBM cells and comparison of detection thresholds with DMR analysis. Cores B and C will supply serum and tumor samples from human GBM patients/controls and GBM mouse models/controls, respectively. Core B will provide biostatistical oversight for biomarker assay validation, sensitivity and specificity of biomarkers, power calculations and correlations between biomarkers and tumor status. These studies represent a novel approach to biomarkers which can report on the genetic status of brain tumors using blood samples.

Project Leader: Alain Charest. Extracellular RNA (exRNA) is a newly discovered form of cellular communication whereby information from one cell to another is conveyed by RNA molecules. exRNAs are protected from extracellular RNases by encapsulation within membrane vesicles or as amalgamations of RNA and proteins complexes (ribonucleoproteins RNPs). It appears that the production of these entities is not random and is the result of a highly orchestrated machinery, the details of which remain ill-defined. In this proposal, we aim to uncover the molecular mechanisms by which exRNA is encapsulated in extracellular vesicles and RNPs and how therapeutic interventions affect these mechanisms using glioblastoma multiforme (GBM) as a model system. Using genetically engineered mouse models of GBM that are driven by overexpression and activation of EGFR and PDGFRa, the two most common.genetic events found in GBM, we will determine the vesicle and RNP exRNA profiles of EGFR GBMs and PDGFRa GBMs using deep sequencing methods. Once established, we will then study the effect of therapeutic treatment on the dynamics of exRNA production and perform functional studies of exRNA on target cells in vitro and in vivo. We will initially focus on miRNA as it has been shown that miRNA make up a significant proportion of exRNA. Our project relates to the other projects in this U19 application on multiple levels. We will work in close collaboration with Dr. Anna Krichevsky on deciphering and cataloging the exRNA sequence composition of our genetically-defined glioblastoma tumor cells. We will also collaborate with Dr. Stephen Gould on the molecular mechanisms of exRNA production in glioblastoma as a function of EGFR and PDGFRa signaling pathways and with Dr. Xandra Breakefield on the development of methods and reagents to study exRNA transfer to recipient cells. Finally, we will work closely with Dr. Thorsten Mempel who will provide intravital imaging technology to evaluate and study exRNA fluorescent reporters in vivo.

DESCRIPTION (provided by applicant): The American Society of Gene & Cell Therapy (ASGCT) was founded in 1996. ASGCT's mission is to advance knowledge, awareness, and education leading to the discovery and clinical application of genetic and cellular therapies to alleviate human disease. Our Annual Meeting represents the major educational initiative of the Society. This R13 proposal requests support for travel grants for trainees. Over 1/4 of our members are scientists in training working to develop clinically applicable gene and cell-based technologies. Trainee participation in the Annual Meeting is fostered by a distinctive educational program, numerous trainee travel awards, and recognition of outstanding scientific accomplishments through peer-reviewed trainee Excellence in Research Awards. Over the past decade, the ASGCT has offered 520 travel grants and 69 Research Awards, with 160 travel grants made possible by R13 grant awards from the NIH during that same time. Educational opportunities for travel awardees include exceptional plenary speakers, state-of-the-art scientific symposia, and educational sessions that review current thinking on a variety of topics. In addition to leading scientists and clinicians, the program includes ethicists and representatives from the FDA, OBA, and NIH so young scientists may gain insight into the compliance and ethical issues related to human gene therapy and cell therapy. Trainees are active presenters in oral abstract and poster presentations and the top trainee abstracts are recognized at the Presidential Symposium. The size of the ASGCT meeting (approximately 1,600 participants) is ideally suited to expose young scientists to leaders in the field, yet provide opportunities for trainees to present their work at the premier meeting in the field of gene and cell therapy. NIH support for the ASGCT Annual Meeting will allow continued educational and professional advancement of trainees in the field of cell and gene therapy in addition to the unique opportunity to network with leaders in the field.

Prject Leader: Anna Krichevsky. Glioblastoma (GBM) is the most common and malignant brain tumor in adults and also accounts for approximately 10% of pediatric CNS tumors. Despite very aggressive disease management, that usually includes surgery, chemotherapy and radiotherapy, GBM is a fatal disease with median survival time of only 12-15 months. Novel approaches and molecular targets for GBM are, therefore, urgently needed. The discovery of microRNAs (miRNAs), small regulatory RNA molecules that cause post-transcriptional down-regulation of gene expression, truly revolutionized the field of cancer biology. It suggested an entirely new layer of gene regulation that might be involved in progression and maintenance of human neoplasia. Our work over the past 8 years focused on miRNAs that contribute to gliomagenesis, and today we have mounting evidence indicating that GBM growth and invasiveness are closely regulated by miRNAs. Importantly, microvesicles released by GBM (which may represent a means of communication between the tumor and its intracranial environment), contain large amounts of miRNA regulators, including the key oncogenic miRNAs. We hypothesize that these molecules are taken-up by normal cells surrounding the tumor, and have significant effects on the physiology of these cells. MicroRNA-mediated regulation of gene expression in the recipient cells may lead to transformative events in the brain cells, serving either protective or, ultimately, tumor growth-supportive function. To validate this hypothesis, we will: 1) characterize the repertoire of intracellular versus extracellular/released RNA in human and mouse GBM cells, and intracellular RNA in normal brain cells that constitute GBM microenvironment; 2) investigate miRNA transfer between GBM and normal cells in co-cultures in vitro, and its functional effects on the phenotypes ofthe recipient cells, and 3) investigate whether miRNA transfer exists between the xenograft GBM models and normal brain cells in animals in vivo. The proposed work promises to yield significant new insights into the biology of glioma and more generally- miRNA-mediated crosstalk between different cell populations in the brain.

DESCRIPTION (provided by applicant): The goals of this proposal are to improve treatment for gliomas by enhancement of oncolytic therapy and to develop serum microvesicles as biomarkers for genetic and phenotypic properties of individual GBM tumors and their response to therapy. Project 1 (Chiocca/Kaur) will evaluate whether viral RNA in microvesicles produced by HSV-infected glioma cells increases susceptibility of tumors to oncolysis, and assess viral and cellular mRNAs in tumor-derived serum microvesicles as biomarkers of oncolytic therapy. Project 2 (Weissleder/Hahko) will apply nanotechnology-based diagnostic magnetic resonance (DMR) to characterize micro vesicle number and antigenic profiles in serum based on proteins critical to oncogenesis using serum from mice and patients bearing glioblastoma (GBM) and undergoing different treatment paradigms. Project 3 (Breakefield) will characterize and enrich tumor-derived microvesicles serum using antibody-capture microfluidic chambers and quantities levels and mutations in GBM-related RNAs using serum samples as in Project 2. These projects will be supported by: Core A (Hochberg) to provide oversight for programmatic organization and neuropathology; Core B (Carter) to maintain a biorepository of human GBM tumor and longitudinal serum samples from patients undergoing standard-of-care therapy or clinical Phase l/ll trials, to supply de-identified samples to Project 2 and 3, and to carry out biostatistical correlations with clinical parameters; and Core C (Charest) to provide genetically engineered GBM mice with mutations representing driver mutations in two subtypes of human GBMs to Project 1, and de-identified tumor/serum samples and biostatistical correlations with tumor volume and survival time to Projects 2 and 3. This program will develop and evaluate new accessible serum biomarkers to facilitate evaluation of therapeutic paradigms in mouse models and human patients both in terms of real time response to therapy and mechanisms of resistance to therapy.

DESCRIPTION (provided by applicant): Our broad term objective is two-fold: to understand how extracellular RNA (exRNA) modulates the phenotype of normal cells in the tumor environment and to generate new experimental modalities that can elucidate mechanisms underlying this form of communication among cells. Specifically we will focus on understanding how exRNA released from glioblastoma cells modulates the phenotype of normal cells in the vicinity of the tumor. Our overall specific aims will encompass: Aim 1 - Elucidation of basic molecular and cellular mechanisms of exRNA biogenesis by GBM cells, and uptake and function in normal brain cells, using existing and emerging technologies to manipulate these processes. Aim 2 - Full characterization of the exRNA content and intracellular RNA content of human GBM cells, as well as the intracellular RNA content of normal brain cells, and evaluation of functional transfer of exRNAs from GBM cells to brain cells in culture and in GBM mouse brain models. Aim 3 - Evaluation of transfer and fate of exRNA in brain cells, including visualizing RNA transfer in EVs, monitoring mRNA translation and miRNA functions, determining possible genomic integration of transposable elements/oncogenes, and evaluation of effects of non-coding exRNAs on status of genome methylation. Aim 4 - Description of the dependence of exRNA cargo composition, formation and release dynamics as a function of GBM genotype, including activation of EGFR and PDGFRa signaling pathways, the two most common genetic events in human GBM tumors, as well as changes in GBM exRNA in response to radiation and drug treatment. Aim 5 - Development of regulators and reporters of exRNA release and uptake by tailoring fluorescent and other visual labels, vectors, mouse models and reagents for broad applications in monitoring exRNA release, uptake and function in culture and in vivo. These aims will be supported by a shared imaging core carrying our intravital imaging of extracellular vesicles and their interaction with endogenous cells in the brain.

Core Leader: Thorsten R. Mempel. The Intravital Microscopy Core (Core A) has been formulated based on a stated need of U19 investigators and is designed to draw on the considerable experience of its key personnel in multiphoton intravital microscopy (MP-IVM) techniques to provide expertise and specialized equipment and aid in the design and execution of in vivo imaging studies of exRNA- and EV-mediated communication of glioblastomata with their stromal environment and the surrounding, healthy brain tissue. The Specific Aims ofthe Intravital Microscopy Core are as follows: 1. To provide expertise in the design of specific experiments utilizing multi-photon microscopy-based analysis of EV biogenesis by glioblastomata, their properties in the extracellular space and uptake by nonmalignant brain cells, as well as of functional exRNA- and EV-mediated intercellular communication between tumor cells and tumor stroma. 2. To provide access to the required instrumentation and to aid in the execution of MP-IVM experiments using mouse models of glioblastoma developed by the project Pis with assistance of the core as outlined in aim 1. 3. To provide computational resources forthe storage, processing, in-depth analysis, and interpretation of digital imaging data generated in aim 2.

Project Leader: Stephen Gould The biogenesis and intercellular transfer of functional, extracellular RNA (exRNA) is being detected in an ever-increasing array of biological systems. Intercellular traffic of exRNA plays important roles in human health and disease, and exRNA-mediated systems for disease detection, monitoring, and therapy have a high yet currently untapped potential. However, the molecular mechanisms that cells use to generate exRNAs are only poorly understood, and there is virtually nothing known about the mechanisms of exRNA uptake by human cells. This proposal will advance our mechanistic understanding of exRNA biogenesis and uptake using a glioblastoma multiforme (GBM) model system that focuses on the production of exRNAs by GBM cells, and their uptake by surrounding somatic cell types such as astrocytes, microglia, and brain endothelial cells. The successful completion of this proposal will shed light on the mechanisms of intercellular RNA trafficking, test the scope and limitations of these mechanisms, and generate technologies for the engineered production and uptake of exRNA in human and rodent cell systems.

Administrative Organization. There will be three levels of oversight, including: the PSO Directors and Core Directors/Co-Directors to monitor ongoing activities and to interface with users; a Steering committee to review operating procedures and activity reports on an annual basis, as well as to advise on prioritization issues and core effectiveness; and an Advisory Board to offer advice on technology updates and management issues. This organizational structure will be logistically supported by an Administrative Core with a dedicated senior grants manager to assure compliance at all levels. Collectively, this administrative organization will be responsible for ensuring adherence to NINDS Program guidelines; implementing a fair and equitable Core utilization plan; monitoring Core functions, performance, accessibility and user satisfaction; implementing upgrades; securing institutional support and maintaining effective financial administration.

Core C (Vector) will consult with and advise investigators on vector design and applications in the nervous system, and provide high titer, high quality lentivirus, retrovirus, replication-deficient rabies virus and AAV vectors to monitor gene delivery and neural functions in culture and in rodent models of neurologic disorders. This core will also provide specialized expertise and technologies to our investigators to achieve widespread and targeted gene delivery in the nervous system in utero, neonatal, and adult mice.

Project Summary / Abstract This multidimensional project is directed at identifying common etiologic pathways in three early onset dystonias, DYT1 (torsinA; TOR1A), DYT6 (Thap1; THAP1) and DYT25 (G-alpha-olf; GNAL). A three tier strategy will be employed to: 1) encompass candidates by expression profiling in human neurons derived from induced pluripotent stem cells (iPSCs) from patients (Aim 1, Bragg); 2) test candidate modifying genes in a living organism by RNAi screening of locomotion and dopamine metabolites in dtorsin-null Drosophila (Aim 2, Ito); and 3) evaluate implicated biologic processes in cultured neurons from a mouse model of DYT1 and neuronally differentiated iPSCs from patients (Aim 3, Breakefield). Our unifying hypothesis is that defects in these genes interfere with neurotransmitter receptor signaling in the brain, especially via acetylcholine and dopamine, with consequent compromise of synaptic plasticity known to be altered in DYT1 patients. In the case of DYT1 we hypothesize this results from compromise of the nuclear egress of ribonucleoprotein particles (RNPs) carrying mRNAs locally transcribed at synapses; for DYT6 from altered transcription of genes related to synaptic proteins, and for DYT25 from disruption of G coupled protein receptor signaling through dopamine receptors. Gene candidates will be further informed by molecular genetic analyses in dystonia patients (Project 1 and Core B) and by developmental and physiologic analyses in mouse models of these forms of dystonia (Projects 3). Insights into common pathways across dystonias will inform therapeutic strategies.

Core B (Microfluidics) will provide advice/assistance in design, fabrication and implementation of microfluidic tools to monitor cell motility, separate whole cells and cell organelles, and provide high resolution imaging of neuronal functions, e.g. axon growth and guidance.

Core A (Imaging) will provide consultation/assistance with experimental design, training in use of equipment and data analysis, and access to equipment for electron microscopy, array tomography and intravital multiphoton microscopy systems, with a focus on synaptic structure and function.

? DESCRIPTION (provided by applicant): (Overall Abstract) Dystonia is the third most common movement disorder with most cases having a hereditary predisposition. Our overall goal is to elucidate the molecular, cellular and neuronal circuitry defects in hereditary forms of early onset dystonia and to find common pathways in their etiology and potential targeted drugs for therapeutic intervention. Members of our team discovered three genes underlying early onset dystonia, TOR1A (DYT1), THAP1 (DYT6) and GNAL (DYT25) and have developed and characterized mouse and Drosophila models of DYT1 dystonia, as well as initiating studies on mouse models of DYT6 and DYT25 dystonia. We hypothesize that the core pathophysiology lies in abnormal neurotransmitter signaling, primarily in the striatum, which manifests throughout life as abnormal synaptic plasticity. Studies will analyze neuronal cultures, as well as neurophysiology in these dystonia mouse models, as well as enhancer/suppressor genes in Drosophila mutants. Common themes include cholinergic and dopaminergic neurotransmitter interactions in the striatum, transcriptional and functional control of signaling proteins, and transport of mRNAs in ribonucleoprotein particles (RNPs) for translation at synapses. We will approach these themes with an armamentarium of research tools, including state-of-the-art human molecular genetics, primary neuronal and iPS cell-derived neuronal cultures, and Drosophila and mouse models of dystonia examined using genetic and cell biologic methods and electrophysiologic analysis of slice explants and microdialysis in mouse models. This highly integrated P01 will be led by Dr. Xandra Breakefield, Director and Laurie Ozelius, Co-Director and includes: Project 1 - Genes and susceptibility factors in primary torsion dystonia (PI Dr. Laurie Ozelius, Mt. Sinai Sch. Med.); Project 2 - TorsinA as a key link in receptor-mediated signaling (PI Dr. Xandra Breakefield, Co-Is Drs. D. Cristopher Bragg and Naoto Ito, Mass. Gen. Hosp.); Project 3 - Cholinergic and dopaminergic mechanisms in mouse models of dystonia (Dr. David Standaert, Univ. Ala. Birmingham); Core A - Administration; and Core B - Clinical Core (Dr. Nutan Sharma, Mass. Gen. Hosp.). Clinical information and samples feed directly into Projects 1, 2 and 3. These studies will elucidate common molecular pathways involved in human dystonia to inform therapeutic advances.

Project Summary/Abstract Core A - Administration The objectives of the Administrative Core will be to provide oversight for the Research Projects and Clinical Core, and to promote coordination and collaboration within the program and with investigators and organizations outside the program. This core will oversee organizational and scientific aspects, as well as governance of this P01. Scientific leadership will be provided by the Directors, Xandra Breakefield and Laurie Ozelius aided by administrative expertise provided by Ms. Robin Sobolewski. The directors will be assisted in oversight by an External Advisory Committee consisting of five members chosen for their objectivity and overlapping expertise in the research focus and clinical aspects of this P01. All PIs will present their research to External Advisors once a year at a full one day meeting in Boston and External Advisors will write a critique of their views to be submitted to NINDS with the annual progress report. Drs. Breakefield and Ozelius will be advised in their decision-making policies by Internal Advisors, Dr. Merit Cudkowicz, Chief of Neurology at Massachusetts General Hospital (MGH) and Dr. Susan Bressman, Chief of Neurology at Beth Israel Hospital (BIH). Conflict resolution will be resolved by discussion among Drs. Breakefield, Ozelius, Cudkowicz and Bressman and a decision-making vote; in the case of a tie Dr. Breakefield will make the final decision. Drs. Breakefield and Ozelius will advise investigators on strategic and technical issues, promote collaboration among projects, monitor the functions and effectiveness of core facilities, assess the scientific quality and progress of the work, and initiate collaborative interactions with other dystonia investigators. They will also ensure timely meetings of all investigators and external reviewers, interactions with collaborating institutions, compliance with resource sharing policies and monitoring of progress including preparation of progress reports.

Glioblastomas (GBMs) are one of the most difficult cancers to treat. Their success in defying therapy relies in large part on their dynamic cellular heterogeneity and on their ability to subvert their environment, in particular to create an immune suppressive milieu. This project will address the role of the newly discovered communication vehicles in cancer, extracellular vesicles (EVs) which can carry RNA and protein as informational molecules. Our goal is to evaluate to what extent EVs produced by these tumors contribute to an immune suppressive environment through their interaction with tumor-associated macrophages and microglia (TAMs), and to what extent EVs produced by different genetic subtypes of gliomas, and the stem-like cells within these tumors, underlie to their ability to re-define themselves and escape therapy. In Aim 1 we will assess EV-mediated information transfer between glioma tumors and microglia/macrophages in the tumor microenvironment and its effect on immune suppression. In Aim 2 we will evaluate the role of EVs in modulating GBM stem-like cell (GSC) phenotypes and therapeutic resistance. Studies will use syngeneic mouse glioma models as well as human GBM stem-like neurosphere cultures and patient-derived xenograft in mouse models developed with Core C. We will generate an array of vectors to label cells with multiple luciferases and fluorescent proteins, as well as create capsules to contain tumor cells while allowing release of EVs and/or small molecules within the brain. FACS analysis and RNAseq will be used to define the transcriptome of different cell populations in the brain. Biofluids from mice, as well as from GBM patients (Core B), will be sent to Project 2 for protein analysis. Correlative analysis of mRNA/ protein contents and GBM genotypes will be assessed, as well as how EV contents reflect changes in GBM subtype and resistance to therapy. Our findings will be integrated with those of Project 3 in the context of preclinical evaluation of Gene- Mediated Cytotoxic Immunotherapy (GMIC) combined with immune checkpoint inhibition. Organizational oversight of rigor and reproducibility will be provided by Core A. Studies in this project will serve to inform the development of the projected clinical trial by elucidating how EVs influence and report on GBM status, and how they can be manipulated to improve this type of immunotherapy in the context of the GBM brain tumors.

Glioblastoma multiforme (GBM) has an abysmal prognosis despite advances in genomics, new targeted therapies, and increased understanding of its pathobiology. The goal of our Program Project Grant (PPG) is to improve knowledge about GBM biology and develop effective therapies. Investigators in this PPG have made two notable advances, among many: 1- Tumor extracellular vesicles (tEVs) and their contents transfer functional information among cells in the GBM microenvironment, thereby increasing oncogenic behaviors, and 2- a Phase II clinical trial in patients with newly diagnosed GBM utilizing Gene-mediated Cytotoxic Immunotherapy (GMCI) as an adjuvant to standard-of-care (SOC) has shown encouraging, albeit non- definitive, results. The major hypotheses of this PPG's competitive renewal are that GBM therapy can be improved by combining SOC and GMCI with immune checkpoint inhibition, and that therapeutic responses can be monitored in biofluids by assessing protein and RNA content in tEVs. Three synergistic projects and three supporting cores will test these hypotheses. Project 1 (Xandra Breakefield) will evaluate whether glioma EVs and their contents are fundamental regulators of glioma heterogeneity within the tumor and immune suppression in the tumor microenvironment, thus contributing to tumor progression, immuno-evasion, and therapeutic resistance. Project 2 (Ralph Weissleder) will validate novel technological advances from his laboratory, such as single EV analysis (SEA), to assay tEV contents in biofluids, thus overcoming the limitation of scant materials available from preclinical and clinical trials. Project 3 (E. Antonio Chiocca) will test if SOC/GMCI combined with immune checkpoint inhibition will be an effective therapy in preclinical models of glioma and, ultimately, in a randomized clinical trial. Core A (Breakefield/Chiocca) will provide the necessary administrative structure, scientific oversight, and overall leadership functions for the PPG, including basic science (Breakefield) and preclinical/clinical (Chiocca) efforts. Core B (Carter) will maintain the Clinical Sample Core that biobanks cerebral spinal fluid and serum/plasma samples from patients and clinical trials for tEV analysis, as well as serve as the biostatistics resource. Core C (Charest) will provide genetically engineered mouse models of gliomas and mouse/human glioma ?stem-like? cells from different GBM subtypes. Together these investigators will generate the necessary scientific justification and preclinical data to ultimately support the proposed clinical trial of GMCI with immune checkpoint inhibition in newly diagnosed GBM patients, in which tEVs will be evaluated for their potential as biomarkers and modulators of the therapeutic response.

Administrative Core A of this Program project will provide oversight of organizational aspects, including scientific leadership through the Directors, Dr. Xandra Breakefield and Dr. E. Antonio Chiocca, with administrative expertise provided by Ms. Robin Sobolewski. This core will be responsible for assuring the ethical conduct of all scientific investigations including decision-making regarding thematic and personnel changes, and rigor and reproducibility of scientific and clinical data according to NIH guidelines. Other functions of the core include: 1) organization of scientific meetings among investigators, both within this multi- institutional Program Project (MGH, BWH, UCSD and Tufts), including meetings and oversight by the External Advisory Board and Steering Committee, as well as monthly investigator reviews and annual external reviews and with collaborators; 2) facilitation of EV correlative biomarker collaborative studies in support of clinical trials; 3) oversight of data sharing and pre-publication review; 4) programmatic integration to insure synergies, investigator interactions, and timely completion of studies; and 5) transparent and authorized financial conduct.

Project Summary/Abstract Glioblastomas remain one of the most deadly cancers with no breakthroughs in therapy for the past 20 years. Xandra Breakefield has made critical discoveries demonstrating that these tumor cells release extracellular vesicles containing informative nucleic acids and proteins that convert normal brain cells to tumor supportive cells. Working with a team of seasoned investigators she has continued to make breakthrough advances in the use of these vesicles as biomarkers, in elucidating the means by which they subjugate microglia and other cells in their microenvirons, and in exploring how they may be channeled for therapeutic purposes. This team consisting of Drs. Breakefield, Joseph El Khoury/Suzanne Hickman (microglia experts), Thorsten Mempel (T lymphocyte expert), Marike Broekman (neurosurgeon) and Casey Maguire (vector expert) will advance these insights in three interrelated areas: biomarkers, cell-to-cell communication and therapy. Studies are designed to increase sensitivity and reveal clinical correlates of RNA and protein in extracellular vesicle biomarkers from serum/plasma with goals of early detection, informing therapeutic decisions and longitudinal evaluation. They will explore how tumor extracellular vesicles participate in changing the phenotype of microglia, macrophages and astrocytes in the tumor microenvirons, such that they become a ?life support? system for the tumor in defiance of therapy. These insights will be forged into new therapeutic concepts with a focus on engaging the innate and adaptive immune systems to arm the brain against the tumor. This will include increasing cross presentation of extracellular vesicle-derived tumor antigens via microglia to infiltrating T lymphocytes using co- stimulatory molecules. Microglia associated with the tumor will be endowed with increased capacity to release anti-microbial peptides, which are also anti-tumorigenic, to reawaken their sense of the presence of the tumor and to down-regulate program death-ligands that exhaust cytotoxic T lymphocytes. Tumor-associated cells, including reactive astrocytes, microglia and macrophages will be manipulated using systemically administered adeno-associated virus and other vectors (which are clinically compatible) carrying transgene cassettes under promoters that are strongly up-regulated in cells near the tumor, but not in the same cell types in other parts of the brain. Vesicles produced by these cells will also be used to deliver therapeutic cargo to tumors cells to provide sustained therapeutic impact. These studies acknowledge the cytoplasmic continuum of cancer among all the cell types that make up the tumor mass, and strike at this supportive microenvironment which sustains the tumor. Therapeutic strategies are designed to be combinatorial with standard-of-care without increasing morbidity for this devastating disease that has defied current therapeutic approaches.

Our team will evaluate the potential for gene therapy to reduce life threatening symptoms in tuberous sclerosis complex (TSC), which affects 2 million people world-wide. TSC is an autosomal dominant, tumor suppressor disorder caused by an inherited mutation in either TSC1 (encoding hamartin) or TSC2 (encoding tuberin) with somatic loss of the corresponding normal allele leading to hamartomas (focal cell overgrowths) in different organs. Neurological involvement includes cortical tubers, subependymal nodules and subependymal giant cell astrocytomas with hydrocephalus epilepsy, autism, cognitive impairment and mental health issues. We have created a stochastic central nervous system (CNS) mouse model of TSC2 which manifests subependymal overgrowths, hydrocephalus and early death, reflecting what happens in TSC patients. Current standards-of- care for subependymal lesions in the brain involve neurosurgical craniotomy and/or long term treatment with rapamycin, both having potentially damaging effects on brain development. Our thesis is that a life-threatening complication of TSC ? hydrocephalus can be prevented with a less invasive and longer lasting procedure, i.e. intravenous (IV) delivery of an adeno-associated virus (AAV) vector encoding a replacement protein, without compromising use of other standards-of-care if needed. IV delivery should achieve ?extra copies? of the replacement gene in peripheral tissues reducing the likelihood of other life threatening hamartomas forming in the body. We will use our CNS Tsc2 mouse model to evaluate the ability of an AAV vector encoding a condensed version of tuberin (cTuberin) to reduce the size of ependymal abnormalities and extend lifespan. R61 Aim 1 - Evaluation of cTuberin protein in culture, including use of induced pluripotent stem cells from patients differentiated into neural progenitor cells, with cytoxicity and functional assays, Go-no-go: cTuberin must show at least 50% of tuberin activity in suppression of mTOR activity, and normalization of cell size and rate of cell proliferation. Aim 2 - Optimize AAV serotype and promoter with AAV-cTuberin in CNS Tsc2 mouse model and evaluate therapeutic efficacy. Behavior, survival times, neuropathology and whole body pathology will be monitored, as well as vector distribution and the ability of the vector to target cells in the brain and peripheral tissues. Go-no-go: AAV-cTuberin must at least double the average lifespan in this CNS Tsc2 model and show comparable effectiveness to rapamycin (positive control). In R33 Aim 3 - Evaluate dose escalation and potential toxicity of AAV-cTuberin in Tsc2+/- and CNS Tsc2 mouse models, as well as obtaining quantitative measurements of ventricular volumes in treated and non-treated mice by magnetic resonance imaging. Our team of TSC experts includes Drs. Breakefield (preclinical gene therapy for neurologic diseases), Maguire (AAV vectors), Ramesh (biochemistry and cell biology), Stemmer-Rachamimov (neuroanatomy) and Thiele (clinical care). These activities are aimed to carry out preclinical research in support of clinical trials, to secure intellectual property rights through our institution, and to facilitate licensing to biotechnology companies.