1985 — 1991 |
Kazazian, Haig H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
The Genetic Control of Hemoglobin Synthesis @ Johns Hopkins University
This proposal has three major goals. First, we will characterize further the mutations which result in reduced expression of Beta globin genes. Through sequence analysis of Beta-thalassemia alleles associated with different polymorphism haplotypes in various ethnic groups, we expect to discover about 13 new mutations which lead to reduced Beta gene expression. These data, along with those derived from "normal" Beta genes, will greatly expand our knowledge concerning which mutations are detrimental to gene expression and which are tolerated. By 1985 one should be able to present a map of the Beta gene with 30-40 nucleotide positions outside of exons marked for two types of mutations: 1) those that are found in "normal alleles," and 2) those that lead to recuced gene expression. As a corollary to this first objective, we hope to demonstrate that recurrent mutation is at least partially responsible for the high frequency attained by certain mutant alleles in various human populations. Second, we will test the hypothesis that meiotic recombination preferentially occurs within specific DNA sequences in man. We have previously observed two clusters of norandomly associated sequences in the Beta gene region. Randomization of these sequence clusters appears to take place in the DNA region 5' to the Beta gene. Thus, our studies will focus on this region as a potential site for "recombination sequence(s)." Third, we will study the variability of normal Beta globin genes in various populations and construct a phylogenetic tree of human populations. In these latter studies, we aim to contribute to the "molecularization" of population genetics.
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0.946 |
1985 — 1995 |
Kazazian, Haig H |
M01Activity Code Description: An award made to an institution solely for the support of a General Clinical Research Center where scientists conduct studies on a wide range of human diseases using the full spectrum of the biomedical sciences. Costs underwritten by these grants include those for renovation, for operational expenses such as staff salaries, equipment, and supplies, and for hospitalization. A General Clinical Research Center is a discrete unit of research beds separated from the general care wards. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Genetic Control of Hemoglobin Synthesis @ Johns Hopkins University
hemoprotein biosynthesis; hemoglobin; gene expression; genetic regulation; thalassemia; genetic recombination; sickle cell anemia; human subject;
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1 |
1987 — 1999 |
Kazazian, Haig H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Genetics of Hemophilia A @ University of Pennsylvania
DESCRIPTION: Inversions causing hemophilia A in man will be analyzed by extensive analysis of the three A homologous genes on the X chromosome, one of which lies within the factor VIII gene and gives rise to hemophilia A up to 50% of sporadic cases by intrachromosomal homologous recombination with one of the other two A genes. An animal model of hemophilia A will be produced by homologous recombination in mice. This model will be used to study the potential of somatic cell gene therapy and the development of inhibitory antibodies to factor VIII.
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1 |
1988 — 1990 |
Kazazian, Haig H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Molecular Genetics of Cystic Fibrosis @ Johns Hopkins University
The gene for Cystic Fibrosis, the most common lethal genetic disorder in Caucasians, has recently been localized to the middle of the long arm of chromosome 7 (q2.1-q3.1). The long-term objective of this research plan is to understand the molecular basis of Cystic Fibrosis. We will proceed by pursuit of the following aims: 1). To identify an affected individual with a chromosomal deletion or rearrangement involving the CF gene using macro- restriction mapping using the tightly linked, flanking DNA probes Met and D7S8 (J3.11). Any area of rearranged DNA will be explored for the gene which causes CF. 2). To characterize a Met H/Not I polymorphism (discovered with pulse field gel electrophoresis) by pedigree studies and screening of normal individuals. Families that have a known crossover between Met and CF will be tested for the Not I polymorphism and if present, to determine whether the site has recombined with the CF gene or the Met locus. The Not I polymorphism will also be used to clone DNA fragments that contain the Not I restriction sites identified by the Met probe from a human chromosome 7 mouse hybrid cell line. These will then be employed in the search for a deletion and to create a macro-restriction map of the CF locus. 3). To determine the haplotypes defined by the CF-linked polymorphic sites in affected and normal individuals from populations in which CF is uncommon (i.e., Black, Oriental), then to evaluate the extent of linkage disequilibrium of these haplotypes with the CF locus. These data will be compared to those derived from the European population to assess inter- population variation and/or similarities in the haplotypes associated with CF gene. 4). When the CF gene is isolated, to discover the mutant allele or alleles and develop direct detection strategies to allow population screening for heterozygotes.
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0.946 |
1991 — 1993 |
Kazazian, Haig H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Human Transposable Element @ Johns Hopkins University
Retrotransposition events have been well documented in yeast and Drosophila for many years. In these organisms much is known about the details of this mysterious biological phenomenon. In man, until recently we could only surmise that such events occurred, although the presence of highly repeated human elements with sequence homology to retrotransposons of other species strongly suggested their existence. In 1987 our lab found two instances of de novo retrotransposition of L1 sequences causing de novo hemophilia A in man. In this application we first propose to finish tracking the functional precursor of one of these elements and to describe thoroughly its subfamily and the subfamilies' evolution. We will then isolate the functional precursor of the second L1 element which retrotransposed and compare the structure of these two elements. These studies are critical to our knowledge of the biology and propagation of L1 elements. Second, we will use short oligonucleotide probes specific for each subfamily to search for new insertions in de novo cases of autosomal dominant and X-linked dominant disorders. We should be able to clone the gene for one of these disorders through use of the sequence flanking the inserted L1 element. Third, we will study the expression of the functional element found in the first aim in vivo and in vitro. We will discover whether element found in the first aim in vivo and in vitro. We will discover whether this sequence is transcribed after transfection in a "natural state" without an overexpressing promoter, whether the open-reading frames are translated in cells, and whether the second open-reading frame can code for a natural reverse transcriptase. All of these studies should provide fundamental answers to questions as to how certain L1 elements act as retrotransposons. The second aim should lead us to a novel scientific use of these rare events, the cloning of a hitherto unknown disease gene.
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0.946 |
1992 |
Kazazian, Haig H. |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Medical Genetics @ Johns Hopkins University |
0.946 |
1994 — 2010 |
Kazazian, Haig H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Human Transposable Element @ University of Pennsylvania
[unreadable] DESCRIPTION (provided by applicant): Retrotransposons comprise roughly 1/3 of the human genome. Over evolutionary time, they have played an important role in shaping the genome through a number of mechanisms. We now know that L1 elements are the master retrotransposons of mammalian genomes. They can insert into genes causing disease, lead to deletions and duplications through mispairing and homologous crossing over, transduce flanking sequences during retrotransposition events, and mediate the retrotransposition of non-autonomous retroelements. In this proposal, we study the natural activity of retrotransposons in real time, using a mouse model of retrotransposition from a human L1 transgene. We will answer a number of key biological questions. Do the structural characteristics of retrotranspositions from the transgene match those of endogenous insertions? Is retrotransposition more efficient in male or female germ cells? Does age of the animal affect retrotransposition frequency in male germ cells? Do mechanisms exist that extinguish retrotransposition activity from a transgene after a number of generations? We will then optimize our transgene construct and breed animals containing the optimized constructs to homozygosity in order to increase retrotransposition frequency and produce a practical insertional mutagen in the mouse. We believe that an L1-based mutagenesis system that obviates the treatment of ES cells will be a valuable adjunct to chemical and other mutagenesis systems in determining gene function in mice. In another important aim, we pursue our discovery that SVA elements are L1-mediated, non-autonomous retrotransposons. We determine the important sequences in the SVA element for retrotransposition in the expectation that this knowledge will shed light on the mechanism by which L1s mobilize Alu elements. Thus, this focused proposal will provide answers to key questions of L1 biology and likely lead to a useful mutagenesis system for determination of gene function.
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1 |
1996 — 2002 |
Kazazian, Haig H. |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Core--Dna Sequencing @ University of Pennsylvania
The DNA Sequencing Facility was approved and funded by the NCI Core Grant in 1994. The Facility received "excellent" merit at that time. Dr. Haig Kazazian, Chair of Genetics, has served as Facility Director since the inception of this Shared Resource. Dr. Kazazian is a well-known human molecular geneticist who had directed the Genetic Resources Core at Johns Hopkins for five years prior to coming to Penn. The Technical Director of the Facility is Dr. Tapan Ganguly. Dr. Ganguly has over 10 years of experience in cell and molecular biology, and was associated with the genetic Diagnostic Laboratory in Penn's Department of Genetics for three years before assuming this position. During the current project period, the DNA Sequencing Facility has become fully operationalized and well accepted as a major research service to Cancer Center investigators . Usage has grown consistently, with an increase of 14% from 1997 to 1998 alone. New equipment has been purchased in order to meet increasing demand. Additionally, the core has relocated to larger space in order to accommodate its increasing needs. Lastly, automated plasmid purification has been added to the core's service offerings. 53% of total usage is by Cancer Center members, while 34% is by Cancer Center members with peer-reviewed funding.
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1 |
1997 — 2009 |
Kazazian, Haig H. |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Medical Genetics Research Training Grant @ University of Pennsylvania
DESCRIPTION: (provided by applicant) The major goal of this research training program is to train the future leaders of medical genetics who will emerge from a variety of training pathways. Many will have M.D. degrees, others will be M.D.-Ph.D.s. For this training grant, we concentrate on the former two groups. M.D.s and M.D.-Ph.D.s with clinical training in Pediatrics, Medicine, Ob.-Gyn., Psychiatry, Pathology and other specialty areas will be eligible for support from this training grant. Combined pediatrics-genetics residents will also be eligible during their 4th and 5th years of residency. The overall training program for these individuals consists of one clinical year during which the fellow has a substantial clinical load and fulfills a portion of her/his didactic course work in human genetics and molecular biology. This first year is funded by the University of Pennsylvania School of Medicine and the Children's Hospital of Philadelphia (CHOP), while funds for the research years of the overall training program are sought from this training grant. During the second year, the M.D. fellow completes didactic course work and begins research training. During most of the second year and all of the third and often fourth years, the fellow devotes essentially 100% effort to research in a basic science laboratory. Research opportunities are extremely diverse with training in the laboratories of 47 faculty from 15 different departments at Penn. During the research years, the trainee also takes seminar courses, attends journal clubs, research meetings, and departmental research retreats, and carries out minimal clinical activities, not to exceed 5% effort. Training stipends for these two to three years are requested in this application. The M.D. trainee will likely require further research training (not covered by this training grant), which might be another postdoctoral research experience or a protected faculty appointment with considerable mentoring from a senior faculty member. This grant will fund up to three research years of the M.D. investigator in medical genetics and two years for M.D.-Ph.D. fellows.
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1 |
1997 — 2008 |
Kazazian, Haig H. |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Predoctoral Training Program in Genetics @ University of Pennsylvania
[unreadable] DESCRIPTION (provided by applicant): This is a renewal application for training grant support for the Predoctoral Genetics Training Program at the University of Pennsylvania. The goal of the program is to produce exceptional investigators and teachers in genetics who have a broad background in the field and particular expertise in molecular genetics. We have expanded a graduate education program whose faculty represents the entire University of Pennsylvania genetics community, including various departments of the School of Medicine, Arts and Science, as well as the Wistar Institute and the Fox Chase Cancer Center. The combination of a research-oriented medical school and a strong university base provides excellent opportunities for graduate education in modern genetics. The program obtains substantial financial support from Biomedical Graduate Studies, an institutional organization that coordinates curriculum and student recruitment with other graduate programs. [unreadable] [unreadable] The Program in Genetics is part of the Cell and Molecular Biology Graduate Group, the body that administers the core curriculum for predoctoral trainees in genetics. After course work is completed, the student must pass an oral preliminary exam that consists of a defense of a research proposal and covers general genetic knowledge. The student then carries out a significant research project under the direction of a laboratory mentor and the advice of a thesis committee. [unreadable] [unreadable] New enhancements initiated within the last four years include a semester long Genetics Principles Core Course, a special biannual research seminar for the predoctoral students appointed to the grant, and an annual formal presentation of the predoctoral students at the Department of Genetics Research Talks. [unreadable] [unreadable] Students are selected for two years of training grant support after they have completed one or two years of graduate education. Thus, we support students who are highly motivated to carry out genetic research. [unreadable] [unreadable] [unreadable]
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1 |
2000 — 2004 |
Kazazian, Haig H. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Gene Correction For Hemophilia A @ Children's Hospital of Philadelphia
This proposal sets the stage for clinical trials of gene therapy for hemophilia A, while simultaneously initiating studies on a novel vector system. We initially will determine the most effective and safest gene correction strategy for factor VIII (FVIII) deficiency in our mouse model. We have previously shown that functional VIII can be delivered from the suprabasal epidermis to the circulation in a transgenic mouse and a skin explant. We will improve FVII delivery by use of 1) a FVII resistant to inactivation and 2) a species-specific FVIII. We then use the optimal FVIII construct to deliver the gene in a retroviral vector to human keratinocytes. These cells which contain a high fraction of stem cells will be grafted to FVIII-deficient immunodeficient mice to show correction of FVIII-deficiency from transfected keratinocytes. In addition, we follow- up observations suggesting that intradermal injection of an FVIII transgene can result in long-term circulating FVIII. In a second section of the proposal, we seek to improve upon modest long-term correction achieved by mouse FVIII cDNA delivered in an adenoviral vector by tail vein injection. We test a number of variables, including improved gene construct and short-term immunosuppression. We also use a "gutted" adenoviral vector as a delivery vehicle to improve results. These studies should provide sufficient information concerning optimal modes of therapy to justify attempts to correct a dog model of hemophilia A in the later years of the grant. In a fourth Specific Aim, we initiate studies on a novel vector, the endogenous L1 retro-transposon and determine the efficacy of this promising vector in the delivery of the factor IX gene in cultured cells.
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0.91 |
2000 — 2002 |
Kazazian, Haig H. |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Modeling Gene Therapy of Hemophilia a Via Liver Directed Gene Expression
A major problem in liver-directed gene therapy is the development of an immune response to the therapeutic transgene. Previously, we have found that mouse CMV-driven factor VIII cDNA delivered in a first-generation adenovirus to hemophilia A mice provokes a substantial immune response to both factor VIII and adenoviral proteins. This response can be blunted by suppression of T-cell with anti-CD4. Over the past year, it has become clear that adeno-associated virus (AAV) is a safety and perhaps more effective delivery vehicle than adenovirus. In this project, we aim to carry out long-term correction of hemophilic mice and dogs by delivery of FVIII cDNA to liver in an AAV vector. Our goal is to devise the means to deliver the FVII cDNA to liver in an AAV vector. Our goal is to devise the mans to deliver the FVIII cDNA without encountering an immune response. We have recently cloned a short SQ version of the mouse FVIII cDNA driven by a small liver-specific promoter (human alpha-anti-trypsin promoter) is an AAV vector. This and other vectors will be tested for preliminary for therapeutic effect in vitro and in vivo; then in immunosuppressed, FVIII-deficient mice; and finally in immunocompetent hemophilic mice. The total size of immunosuppressed, FVIII-deficient mice; and finally in immunocompetent hemophilic mice. The total size of the mouse FVIII-SQ cDNA in this year is under 4.4 kb, leaving roughly 380 bp for promoter/enhancer combinations. In immunocompetent mice, we will measure FVIII activity, FVIII antigen, and both cellular and humoral immune responses to FVIII. Using the information gained from mice expectations, we will attempt correction of hemophilia A dogs using canine FVIII-SQ cDNA via liver directed expression. We hope to overcome any immune response to FVIII and provide successful long-term treatment of these animal models. The studies are critical to clinical trials of liver-directed therapy of hemophilia A using AAV vectors.
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0.954 |
2005 — 2008 |
Kazazian, Haig H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Human Variation in Retrotransposon Activity @ University of Pennsylvania
DESCRIPTION (provided by applicant): The overarching hypothesis of this proposal is that human subpopulations are acquiring mutational loads and thereby potentially are evolving at different rates. To test this hypothesis, one needs to study processes that have genome wide effects as opposed to those that only alter the expression of a single gene. Such a general, genomewide agent is the L1 retrotransposon. In this proposal, we determine the variation in L1 retrotransposition capability in the human population as a potential surrogate marker for genome-wide, evolutionary drive. Long Interspersed Nuclear Element-1 (LINE-1 or L1) is an abundant retrotransposon that comprises ~17% of human DNA. The vast majority of L1s are fossil relics and can no longer move (i.e., retrotranspose) because of inactivating mutations. We have determined that an individual human genome as represented by the human genome working draft (HGWD) contains roughly 80-100 active L1 retrotransposons, but that only about 6 account for 84% of the observed retrotransposition activity. On the other hand, we have found that "private" or genome-specific L1s are not uncommon, leading us to hypothesize that there are a very large number, perhaps millions, of highly active L1s in the total human gene pool and that the variation in L1 activity among individual human beings is substantial. This grant represents a logical extension of the work of two experienced groups in the field of human and mammalian retrotransposons in an effort to answer an important question in human biology. The groups bring important areas of technical expertise, namely, 1) a technique to display the human-specific L1s of any individual in a denaturing polyacrylamide gel, 2) a high throughput, rapid assay of the relative retrotransposition frequency of any L1 in tissue culture, and 3) high throughput methods to determine presence/absence polymorphisms of different L1s. Using these methods, we will measure the total variation in L1 retrotransposition capability among individuals by determining both presence/absence of various L1s and allelic variation in their retrotransposition capability. We will also determine the fraction of L1s active in the tissue culture assay that are transcribed in vivo. Using this data, we will determine the extent of variation in L1 retrotransposition capability that exists among different individuals and geographic groups.
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1 |
2005 |
Kazazian, Haig H. |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Faseb Conference: Mamalian Mobile Elements @ Federation of American Societies For Exp
[unreadable] DESCRIPTION (provided by applicant): We request partial support for the Federation of American Societies of Experimental Biology (FASEB) conference on Mobile Elements in the Mammalian Genome, to be held from June 4-9, 2005 in Tucson, Arizona. This meeting will highlight exciting recent advances in mammalian mobile element biology, and will provide a needed forum for critical discussions about how mobile elements have impacted, and continue to influence, the evolution of mammalian genomes. The meeting will be organized around four major themes. The first theme will focus on molecular biological and biochemical studies conducted on mammalian mobile elements as well as related elements from other experimental model systems. Recent work in these areas has fostered a great deal of excitement, but also has indicated a need for multifaceted approaches to tackle emerging questions in the field. The second theme will focus on population genetic and evolutionary analysis of mammalian mobile elements. The invited speakers will discuss how mobile elements have impacted (and continue to impact) mammalian genomes, how to find 'active' mobile elements in modern mammalian genomes, and how differential activity of mobile elements influences gene expression and human variation. The third major theme will focus on the use of bioinformatic methodologies to identify new classes of mobile elements in whole genome sequences. This theme is very timely given the plethora of genome sequences that either are completed or will be completed in the coming years. The last theme of the meeting will focus on transposon engineering. The invited speakers will discuss the use of both DNA transposons and retrotransposons in mouse mutagenesis studies and the use of mobile elements as gene delivery agents. Thus, this conference will allow investigators in these interrelated fields to share information about exciting new findings, experimental challenges, and outstanding questions in the area of mammalian mobile elements. [unreadable] [unreadable]
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0.904 |
2007 — 2010 |
Kazazian, Haig H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Preclinical Gene Correction of Hemophilia A @ University of Pennsylvania
[unreadable] DESCRIPTION (provided by applicant): This proposal sets the stage for clinical trials of gene therapy for hemophilia A. Here we concentrate on the use of adeno-associated viral (AAV) vectors with new capsid serotypes for liver directed gene therapy in a large animal model, namely, the hemophilia A dog. This work builds upon our previous success in "curing" the hemophilia A mouse using FVIII gene delivery in two new AAV serotypes, AAV8 and AAV9. Our studies in hemophilia A dogs, while more modestly successful, have also been encouraging with both new AAV serotypes. In this application, we evaluate further these AAV serotypes, beginning with delivery of FVIII cDNA in heavy chain and light chain vectors. We test responses at two different doses for both AAV8 and AAV9, and compare intraportal to intravenous routes of vector administration, We then go on to develop an effective, smaller single chain vector that can be produced in high titers, and test this single chain vector using the best combination of serotype, dose, and route of administration. We will also carry out therapy with our best conditions in young dogs, in an effort to initiate treatment before the onset of serious morbidity and also to prevent inhibitor formation to rFVIII used for conventional therapy. Lastly, we will determine whether FVIII can be readministered to dogs using a different serotype vector from the vector used in the original virus infusion. In all of these studies significant efforts will be made to test the safety of the new AAV vectors, concentrating on studies of neutralizing antibodies, development of transaminitis, and germline transmission of virus. At the end of these studies, we expect to have discovered treatment conditions that consistently produce long-term therapeutic levels of FVIII (>10% of normal) in a large animal, the hemophilia A dog. These results should make clinical trials with AAV vectors an appropriate next step. [unreadable] [unreadable] [unreadable] [unreadable]
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1 |
2009 — 2010 |
Kazazian, Haig H. |
RC1Activity Code Description: NIH Challenge Grants in Health and Science Research |
Augmenting Gwas With Retrotransposon Polymorphisms @ Johns Hopkins University
DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (08) Genomics and specific Challenge Topic, 08-CA-101: Augmenting Genome-Wide Association Studies. Genome-wide association studies (GWAS) are typically carried out to uncover potential correlations between SNP genotypes and disease phenotypes. Often, cancer or neoplastic conditions have been the subject of these studies, and they have provided useful insights into the genetic determinants of cancer etiology. While SNPs are the most common form of genetic variation, there are other forms such as copy number variation (CNV) and retrotransposon insertion polymorphisms (RIPs). Recently, GWAS incorporating CNV data have been completed with positive results, but to date no GWAS experiments have used RIPs as the genotypic marker despite the fact that active human families of retrotransposon (LINE-1, Alu, and SVA) are collectively responsible for over 60 disease-causing mutations. The reason for this omission of an important source of genomic variation has been largely technical: no large-scale detection method has enabled the cataloguing of RIPs. Recently, we have developed a robust method for the detection of human-specific LINE-1 (L1) insertions on a genome- wide scale using a hemi-specific PCR method to amplify L1 3'flanking regions in a manner amenable to next-generation sequencing on Illumina's Genome Analyzer platform. Subsequent analysis of the sequencing results reproducibly provides the location of human-specific L1 insertion locations. This technique can be readily extended to other forms of retrotransposon-induced variation including the Alu and SVA families of retroposons simply by interchanging the hemi- specific primer sequences. Once a significant amount of RIP-induced variation has been catalogued across several distinct populations, we will use these sites to design a genotyping array using Illumina's iSelect custom Infinium assay system. Using this array of RIP markers, we will then carry out a GWAS for prostate cancer on ~1000 cases and ~1000 controls. We fully expect to identify 4,000 or more RIP markers for this assay that will serve as an augmentation to GWAS studies in which RIPs have not yet been considered. Genome Wide Association Studies (GWAS) have proven their utility in identifying genomic variants associated with risk for many diseases. To date, SNPs and CNVs have been used as the variant markers in these studies, omitting a major contributor to Human variation: retrotransposon insertion polymorphisms (RIPs). We propose to use a technique we have developed which identifies RIPs on a genome-wide scale to collect a large number of RIP markers for a genotyping array and then conduct a GWAS on prostate cancer in a proof-of-concept attempt to identify new markers associated with the disease.
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1 |
2010 |
Kazazian, Haig H. |
RC4Activity Code Description: To support multi-year funded research with high impact ideas that may lay the foundation for new fields of investigation; accelerate breakthroughs; stimulate early and applied research on cutting-edge technologies; foster new approaches to improve the interactions among multi- and interdisciplinary research teams; or, advance the research enterprise in a way that could stimulate future growth and investments and advance public health and health care delivery. This activity code could support either a specific research question or propose the creation of a unique infrastructure/resource designed to accelerate scientific progress in the future. It is the multi-year funded companion activity code to the existing RC2; thus ICs need OER prior approval to use the RC4. |
The Role of Retrotransposons in Autism Spectrum Disorders @ Johns Hopkins University
DESCRIPTION (provided by applicant): This application is in response to RFA number RFA-OD-10-005 and addresses thematic area 1: Applying genomics and other high throughput technologies. Retrotransposons are an often-overlooked source of inter-individual genomic variation in mammals. The primary driver of this variation in the human genome is the LINE-1 (L1) retrotransposition, which exists in ~500,000 copies spread across a succession of subfamilies each of which was active at some point in evolutionary history. The currently active subfamily is known as either L1Hs (for Human-specific) or L1-Ta (for Transcribed group a) and consists of some ~1000 members per haploid genome, a small subset of which are actively mobilized. We have developed a technique to locate retrotransposon insertions with good specificity and sensitivity by high-throughput sequencing, and have been using it to find human-specific L1 and SVA (another human retrotransposon) insertions from various individuals. In parallel, the laboratory of Lynn Jorde at the University of Utah has developed a similar next-generation sequencing-based approach to identify Alu insertions on a genome-wide scale. Additionally, we are in the first stages of developing a bead-array platform for the high-throughput detection of known retrotransposon insertion polymorphisms (RIPs) in order to perform an association study using RIPs as an alternate marker to SNPs and CNVs. In our grant application, we will propose to use these technologies to study whether LINE-1 and Alu retrotransposition plays a role in the genetic susceptibility to autism-spectrum disorders (ASDs). This study will follow 3 aims: 1. The association of all (low and high frequency) L1 and Alu polymorphisms with ASDs using our sequencing technique in a limited number of trios consisting of the parents and affected proband, 2. Studying the rate of somatic de novo L1 and Alu insertion in ASD patient genomes, ascertained through the study of discordant monozygotic twins, and 3. The association of moderate- to high-frequency retrotransposon polymorphisms with ASDs using an array-based approach to screen many individuals (RIP-GWAS). PUBLIC HEALTH RELEVANCE: Autism spectrum disorders (ASD) are major neurological conditions affecting a large proportion of the US population of children (1 in 166 live births). To date, the predicted major genetic component of their etiology has been explored using single nucleotide polymorphisms and copy number variations with success in finding the cause of a small minority of ASD cases. Here we propose to add another form of genome variation, retrotransposon-induced polymorphisms (RIPs), to the search for ASD etiology.
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0.946 |
2012 — 2019 |
Kazazian, Haig H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Retrotransposition in Health and Disease @ Johns Hopkins University
? DESCRIPTION (provided by applicant) Although we used to believe that all retrotransposition occurred in the germ line in humans, over the past 10 years it has become clear that somatic retrotransposition, especially of LINE1s (L1s), is prominent, at least in the brain and in epithelial cancers. A number of groups have shown retrotransposition in neural precursor cells and the adult brain. Likewise, a number of groups including our own have shown significant retrotransposition in gastrointestinal cancers. A key finding of our studies is that most L1 insertions in GI cancers occur very early in cancer development. Indeed, some of the insertions are found in low concentration (detected only by nested PCR) in normal cells, but they become essentially clonal in the cancer (detected by conventional PCR). ln this application, we strive to answer two big questions in the field. First, does significant retrotransposition occur in adult somatic tissues outside of the brain and, if so, at what frequency? In this aim we will study L1 insertions in single normal cells near the tumors to find what fraction of the insertions in the tumor are already present in normal cells. We will determine if retrotransposition rate is greater in normal cells or in tumor cells. We will also stuy the frequency of insertions in single normal cells not associated with cancer and from various organs to determine if some tissue types have a greater propensity for retrotransposition than others. The second big question is: Does L1 somatic retrotransposition have any significant role in the etiology of cancer? We need to determine whether we can alter the cellular phenotype towards normal by deletion of somatic L1 insertions found in cancer-related genes in cancer-derived cell lines.
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0.946 |