1985 — 1988 |
Jaenisch, Rudolf |
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. |
Retroviruses and Mammalian Development @ Whitehead Institute For Biomedical Res
The exogenous Moloney leukemia virus (=M-MuLV) will be used to address three general problems of gene activation and gene function in mammalian development: (1) as a model gene to analyze molecular parameters of gene activation in mouse development; (2) as an insertional mutagen to induce developmental mutations; (3) as a vector to correct genetic defects in animals. (1) A collection of 14 substrains of mice (termed Mov-1 to Mov-14), each carrying a single M-MuLV provirus at a distinct Mendelian locus, will be used for analyzing the tissue specific expression, methylation and chromatin structure of both the proviral genome and the host DNA flanking the provirus. The aim of these experiments is to understand the influence of the chromosomal position on developmental expression of a gene which is experimentally inserted into the germ line. The provirus in Mov-13 mice has integrated into the Alpha1 (I) collagen gene blocking its transcription and resulting in a recessive lethal mutation. To understand the role of collagen in embryogenesis, organ cultures will be established in vitro from embryos prior to developmental arrest. The proviral genomes carried on the X-Chromosome of Mov-14 mice will be used as a non-selective marker to study the process of X-inactivation. (2) Retroviruses will be microinjected into mid-gestation mouse embryos to obtain additional embryonic mutations in an analogous approach as that used to derive the Mov-13 substrain. (3) The Mov-13 substrain will be used for exploring the feasibility of correcting a defined genetic defect by introducing the wild type collagen gene into animals. This will be attempted by microinjecting a retrovirus vector which transduces the collagen gene into Mov-13 embryos prior to developmental arrest.
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0.922 |
1987 — 1993 |
Jaenisch, Rudolf |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Retroviruses, Oncogenes &Mammalian Development @ Whitehead Institute For Biomedical Res
The development of techniques which allow the efficient introduction of foreign genetic material into the germ line of animals has made it possible to analyze controls of mammalian development by a combined molecular and genetic approach. In this research proposal previously established methods will be used to introduce genes or cells into the developing mouse embryo. The main goals of these experiments are (1) identification and characterization of genes which are crucial for early development; (2) understanding the function protooncogenes may have in mammalian embryogenesis and the effect of oncogene expression on the developing mouse embryo; (3) The study of patterns of migration and homing of normal and transformed cells in the developing mouse embryo. 1) Insertional mutations will be induced by inserting retroviruses into the germ line of mice by an experimental approach which was successful previously in derivating a lethal mutation of the a1(1) collagen gene. Furthermore, procedures will be established for genetically rescuing mice carrying lethal mutations. As a first approach we will attempt to rescue the collagen mutation on the germ line level by microinjection of the wild type gene into mouse zygotes and on the somatic level by microinjection of a vector transducing the gene into mutant embryos prior to their developmental arrest. 2) Protooncogenes, oncogenes or temperature sensitive mutants of oncogenes will be introduced into the developing mouse embryo by retrovirus infection or by microinjection in the zygote. The expression of the inserted genes will be controlled by tissue specific or inducible promoters. These experiments are designed to test the normal function of protooncogenes in mammalian development and to determine the oncogenic potential of oncogenes. 3) Previous experiments have shown that neural crest cells can be introduced into midgestation embryos and participate in normal morphogenesis. We will use this approach of introducing cells into the developing embryo in an effort to define the parameters that determine the migration and homing of normal and transformed cells in n embryonic environment.
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0.922 |
1989 |
Jaenisch, Rudolf |
R24Activity Code Description: Undocumented code - click on the grant title for more information. |
Developing and Improving Institutional Animal Resources @ Whitehead Institute For Biomedical Res
An institutional animal resource renovation project is described to increase capacity for cage and bottle sanitation to meet the expanded needs of the Whitehead Institute's 9,500 GSF centralized animal facility. This involves purchase and installation of a Getinge cage and bottle washer (model 550), the necessary facility renovations to accommodate the unit, and ancillary equipment to complete the installation and crease the operating efficiency of the cage washing process.
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0.922 |
1991 |
Jaenisch, Rudolf |
S15Activity Code Description: Undocumented code - click on the grant title for more information. |
Small Instrumentation Grant @ Whitehead Institute For Biomedical Res
biomedical equipment purchase;
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0.922 |
1994 — 2000 |
Jaenisch, Rudolf |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Genetic Disease, Cancer, and Mammalian Development @ Whitehead Institute For Biomedical Res
This research program focuses on genetic controls of mammalian development and disease. The program combines established transgenic methodology and embryo manipulation with the most advanced technology of gene targeting by homologous recombination in embryonic stem cells. This technology is used for creating mice carrying precisely engineered mutations in genes affecting fundamental developmental processes. The same genes are known or suspected to be involved in human genetic diseases and in cancer. We propose to investigate (i) the role of the extracellular matrix in morphogenesis, tumor progression and metastasis; (ii) the effect of DNA methylation on embryonic development, genomic imprinting, X inactivation, cancer incidence and the process of aging; (iii) the role of the Wilms tumor gene in urogenital development and in tumorigenesis; (iv) the molecular and biological basis of neural crest and muscle development. The need for refining and advancing the existing embryonic stem cell technology becomes apparent when analyzing embryonic lethal mutations because their study provides little or no information on the role of the mutated gene in later developmental processes. This may be particularly significant for the study of tumor suppressor genes suspected of acting in postnatal life. To overcome these limitations, we propose to generate mice which express a mutation in a specified lineage only. First, we propose to develop the technology of inserting very large fragments of DNA carried on yeast artificial chromosomes (YACs) into the germ line. This technology would allow for molecular complementation.of mouse mutations and may help in positional cloning of disease genes, one focus of the current genome project.
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0.922 |
1997 — 1998 |
Jaenisch, Rudolf |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Introduction of Yeast Artificial Chromosomes (Yac) Into the Mouse Germline @ Whitehead Institute For Biomedical Res
artificial chromosomes; genetic mapping; embryonic stem cell; phenotype; tissue mosaicism; micelles; mutant; gene mutation; gene expression; transfection; genetic strain; genetically modified animals; laboratory mouse;
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0.922 |
2000 — 2008 |
Jaenisch, Rudolf |
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. |
Genomic Imprinting and the Cloning of Mice @ Whitehead Institute For Biomedical Res
DESCRIPTION (adapted from investigator's abstract): The investigators propose a multifaceted approach to gain new insights into the mechanisms of imprinting, its relation to DNA methylation, and its effect on the cloning of mammals. Their principal hypothesis is that one can interfere experimentally with the establishment and/or maintenance of the "imprinting marks" of imprinted genes in order to generate animals which are devoid of imprinting. For this they will use the sequential inactivation and reactivation of DNA methyltransferase Dnmt-1 as a tool to remove methylation marks specifically from imprinted genes or, alternatively, they will use nuclear cloning of nuclei from nonimprinted ES cells to create "imprint-free" embryos or animals. Inactivation and reactivation of Dnmt-1 will be accomplished by conditional Dnmt-1 alleles controlled by the Cre and Flp recombinases, respectively. The phenotype of "imprint-free" embryos or mice should provide new insights into the role of imprinting in mammalian development. The most spectacular outcome of the experiment would be the generation of imprint-free but otherwise normal mice, a result which would argue against any intrinsic role of imprinting in mammalian development. Finally, they will use imprint-free embryos or mice as a source for isolating novel imprinted genes and they will study the nature of imprinted X chromosome inactivation. These experiments are also relevant for defining some of the parameters that are important for successful cloning of mammals by nuclear transplantation. Presumably, mammalian cloning requires "epigenetic reprogramming" of the somatic genome. An important goal is, therefore, to characterize the methylation changes imposed on the genome of the donor nucleus following transfer into the oocyte. These experiments will test the hypothesis that errors in the maintenance of imprinting marks increase during the life of the donor and that this is a major cause for the low success rate of mammalian cloning by nuclear transplantation. So far it has been not possible to employ gene targeting approaches in species such as rats because ES cells are available only from mice. The cloning of mammals by nuclear transplantation has opened novel and exciting opportunities to employ established gene targeting approaches in species that will permit the establishment of model systems crucial for efforts to study complex diseases such as cancer. This program attempts to define the biological parameters which limit the nuclear cloning approach.
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0.922 |
2001 — 2005 |
Jaenisch, Rudolf |
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. |
Dna Methylation, Gene Regulation, and Cancer @ Whitehead Institute For Biomedical Res
DESCRIPTION (appended verbatim from investigator's abstract): Cancer develops as a consequence of genetic and epigenetic changes and much evidence indicates that tumorspecific gain or loss of genomic DNA methylation may play a prominent role in both. However a clear picture has not yet emerged to explain the role(s) of methylation in neoplasia since both global hypomethylation as well as regional hypermethylation changes are found regularly in the same tumor. It is the aim of this proposal to determine which of these mechanisms operates in neoplasia arising in four different tissues and whether the methylation changes which are regularly seen in cancer are causallyrelated or are merely a consequence of other cellular insults that occur during the transformation process. Our approach involves the precise alteration of genomic DNA methylation levels by either inactivation or overexpression of the major mammalian DNA methyltransferases (MTases) Dnmtl and Dnmt3 in specific tissues of tumorprone mice. We will test the following four hypotheses which link DNA methylation and cancer each of which may be of selective advantage to the incipient tumor cell hypermethylation may cause (i) silencing of tumor suppressor genes and/or (ii) induce point mutations; hypomethylation may (iii) increase expression of oncogenes and/or (iv) cause genomic instability. Because methylation changes of genes leading to altered expression are in contrast to mutations reversible DNA MTases have become attractive drug targets for cancer treatment or prevention. Indeed previous results established that inhibition of Dnmtl either by genetic or pharmacological means can act synergistically in vivo to prevent intestinal tumor formation. However DNA MTase inhibitors are not without potential risks as inhibition of Dnmtl resulted in significantly higher somatic recombination (LOH) in ES cells and an increased lymphoma incidence in otherwise normal mice. Therefore information as to the potential benefits and risks of therapies which target DNA MTases is vital and our aim is to assess the consequences of methylation changes on tumor incidence in different tissues. The role of methylation in transcriptional regulation has not yet been defined although it is known to be essential for normal vertebrate development. To investigate a possible causal relation between methylation and gene control we will compare gene expression in matched cell lines where due to inducible Dnmtl deletion the genome is either normal or hypomethylated.
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0.922 |
2003 — 2007 |
Jaenisch, Rudolf |
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. |
Nuclear Cloning and the Reprogramming of the Genome @ Whitehead Institute For Biomedical Res
DESCRIPTION (provided by applicant): In all mammalian species where cloning has been successful, at best a few percent of nuclear transfer embryos develop to term, and of those many die shortly after birth. This has raised questions of nuclear potency and differentiation that were posed half a century ago with the generation of the first amphibians by transfer of a somatic nucleus into the egg. These issues include whether the potency of a nucleus to direct the formation of an animal is lost with increasing age of the donor cell and whether its state of differentiation influences the type of abnormalities seen in the clone. The main problem of nuclear cloning is thought to be caused by faulty epigenetic reprogramming of the donor nucleus. This proposal seeks to understand molecular mechanisms that are responsible for the low survival rate and abnormal phenotypic characteristics of animals derived by nuclear cloning. We propose the following projects: 1. We will test the potency of nuclei derived from donor embryonic and somatic stem cells by nuclear transfer. 2. We will generate cDNA arrays that will be used to establish molecular criteria for nuclear reprogramming. 3. We will design strategies to improve the epigenetic reprogramming of somatic donor cells. Our goal is to convert the epigenetic state of the somatic cell to one that resembles that of an ES cell. 4. We will clone mice from mature neuronal donor cells. This will test whether alterations occur in the genome of neurons as part of normal brain physiology that would restrict nuclear potency. Embryonic and adult stem cells are thought to offer significant potential for regenerative cell therapy. However, major issues of applying this promising approach are unresolved. For example, the epigenetic state of embryonic and adult stem cell nuclei may be more amenable to reprogramming than that of terminally differentiated cells. It is a central focus of this proposal to establish functional and biological parameters that distinguish the epigenetic state of nuclei from stem cells and differentiated cells. This will serve as a molecular basis for altering the potential of somatic cells so they could be reprogrammed into different cell types that can be used in therapeutic approaches. Indeed, if the molecular mechanisms that are responsible for reprogramming could be understood, it might eventually be possible to manipulate a somatic cell and generate an ES cell-like cell without the need for oocytes and nuclear transfer.
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0.922 |
2006 — 2010 |
Jaenisch, Rudolf |
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. |
Epigenetics, Stem Cells, and Cancer @ Whitehead Institute For Biomedical Res
[unreadable] DESCRIPTION (provided by applicant): This proposal addresses (I) the epigenetic control of cancer and (II) the relation between stem cells and cancer stem cells. I. Genome-wide hypomethylation, leading to genomic instability and increased rates of LOH, and regional hypermethylation, leading to silencing of tumor suppressor genes, are both hallmarks of cancer. The focus of Aim 1 is to establish a causal relation between genomic methylation and tumor formation in multistage cancers of the intestine, pancreas and prostate. Two complementary approaches will be pursued that either cause inhibition or ectopic activation of Dnmtl and Dnmt3b, two methyltransferases (MTases) that are crucial for maintaining and establishing genomic methylation. Because epigenetic changes - in contrast to genetic alterations - are reversible, the DNA methyltransferases represent promising targets for drug therapy. This proposal seeks to define the role these enzymes play in the development of cancers in * different tissues. II. Most cancers comprise a heterogeneous population of cells with marked differences in their proliferative potential. In the cancer stem cell concept, minor populations of tumor cells that possess the stem cell property of self-renewal are the only cells that are immortal and can sustain tumor growth. It is the second focus of this proposal to define epigenetic and genetic differences that distinguish normal stem cells from cancer stem cells. Because cancer stem cells are thought to be resistant to conventional cancer therapy it is vital to understand the differences between the tumor-initiating stem cells and the bulk tumor cells on a molecular level. One of the key issues for our understanding of the relation between adult stem cells and cancer stem cells is defining the molecular circuitry that drives self renewal in the normal stem cell and its malignant counterpart. We have shown that activation of Oct4 in cells of the adult mouse leads to a rapid and fully reversible dysplastic proliferation of the intestinal and skin stem/progenitor cell compartment. Oct4 is a pluripotency gene that in combination with Nanog and Sox2 controls the self-renewal of embryonic stem cells. In Aim 2 we will investigate whether these key genes of ES cell renewal function also in self-renewal of adult stem cells and assess their possible role in cancer. We have used nuclear cloning as a novel and unbiased experimental tool to reprogram the genome of cancer cells and to distinguish between genetic and epigenetic changes that determine the malignant cell phenotype. Aim 3 will use nuclear transplantation as a criterion to define the genetic and epigenetic alterations of the cancer cell genome that interfere with reversion to pluripotency. [unreadable] [unreadable] [unreadable] [unreadable]
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0.922 |
2008 — 2017 |
Jaenisch, Rudolf |
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. |
In Vitro Reprogramming of Somatic Cells Into Pluripotent Es-Like Cells @ Whitehead Institute For Biomedical Res
A major focus of research for the next years will be to understand on a more detailed level the molecular mechanisms driving the reprogramming of somatic cells to pluripotent IPS cells. 1. Single-cell analysis of gene expression during cellular reprogramming An unresolved issue is whether activation of specific genes can predict early in the reprogramming process whether a given cell will develop into an iPS cell. Single RNA molecule detection methods will be Used to: a. assess whether a hierarchical program of gene expression leads to IPS cell formation or whether the process is entirely stochastic as suggested by previous observations. b. define markers that at early stages of reprogramming allow the prospective identification of cells that will generate IPS cells. For this, GFP will be inserted into candidate genes to give a marker for prospective isolation of IPS precursors. 2. Stoichiometry of reprogramming factors and quality of iPS cells mRNA mediated reprogramming will be used to systematically titrate the various factors for investigating the effect of factor stoichiometry on the biological properties of the IPS cells. This will allow the optimization of reprogramming with the goal of generating high quality genetically unmodified iPS cells. 3. Transdifferentiation of somatic cells to cells of different lineages Different somatic donor cells such as liver cells and skin keratinocytes will be used for direct conversion into neural precursors and neurons. Stringent reporters will allow the retrospective confirmation of the endodermal arid ectodermal donor cell type.
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0.922 |
2009 — 2013 |
Jaenisch, Rudolf |
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. |
Programming and Reprogramming Human Cells @ Whitehead Institute For Biomedical Res
DESCRIPTION (provided by applicant): The recent success in reprogramming somatic cells into induced Pluripotent Stem (iPS) cells by defined factors has opened exciting possibilities not only for the investigation of complex human diseases in the Petri dish but also for the ultimate application in transplantation therapy. Direct reprogramming provides for the first time the opportunity to generate in vitro and in vivo models of complex disorders such as familial and sporadic Parkinson's disease using patient-specific cells. However, major technical hurdles need to be resolved to realize the full potential of the iPS system for the study and eventual therapy of human disease. The experiments proposed in this application are a direct extension of our work with mouse cells and are aimed at establishing robust methods for reprogramming human somatic cells and setting up effective protocols for manipulating human ES (hES) cells and iPS cells. This proposal has 4 aims: 1. We will devise robust approaches to target genes in hES and iPS cells. Gene targeting is crucial for the reprogramming of somatic cells into iPS cells and for differentiating ES cells and iPS cells in vitro into functional cells that could be used for eventual therapy of degenerative disorders. 2. We will seek to elucidate the molecular mechanisms of reprogramming human somatic cells. Our goals include the design of approaches that avoid genetic alteration of the donor cells, to assess whether insertional mutagenesis contributes to iPS cell generation, and to define the molecular signature of different reprogramming stages. 3. We will establish high throughput screens to identify small molecules that increase efficiency of reprogramming and substitute for the need to introduce any of the factors by retrovirus-mediated gene transfer. 4. We will establish protocols that eventually will allow the study of complex human diseases using patient-specific iPS cells both in the Petri dish as well as under in vivo conditions. Finally, we will establish procedures to derive iPS cells from peripheral human blood samples. It is our hope that the experiments proposed in this application will contribute to solving some of the crucial obstacles that presently hamper the application of this technology to the study of human disease and to ultimately use it for transplantation therapy of degenerative disorders. PUBLIC HEALTH RELEVANCE: This grant proposal seeks to define the molecular mechanisms that bring about the conversion of human somatic cells to a pluripotent state, to devise strategies for assessing the developmental potential of human iPS cells, and to achieve reprogramming without the need for genetic manipulation. It is our hope that the experiments proposed in this application will contribute to solving some of the crucial obstacles that presently hamper the application of the technology to the study of human disease and to eventually use the technology for transplantation therapy of degenerative disorders. Once the experimental obstacles have been overcome, it is our long-term goal to use patient-specific iPS cell lines to study the basis of complex human diseases.
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0.922 |
2010 |
Jaenisch, Rudolf |
RC1Activity Code Description: NIH Challenge Grants in Health and Science Research |
Patient-Specific Ips Cells to Study Myeloproliferative Disease. @ Whitehead Institute For Biomedical Res
DESCRIPTION (provided by applicant): The recent success in reprogramming somatic cells into induced Pluripotent Stem (iPS) cells by defined factors has opened exciting possibilities not only for the investigation of complex human diseases in the Petri dish but also for the ultimate application in transplantation therapy. Direct reprogramming provides for the first time the opportunity to generate in vitro and in vivo models of complex disorders such as familial and sporadic Parkinson's disease using patient-specific cells. The experiments proposed in this application are aimed at using the iPS technology to set up an experimental system to study a human genetic disease of the hematopoietic system. The proposal has three aims: 1. Derivation of iPS cells from peripheral blood: We will establish protocols to isolate iPS cells from myeloid cells of human peripheral blood using vectors that can be deleted by transient Cre expression. 2. Characterization of iPS cell-derived hematopoietic cells (HSCs): We will establish robust protocols allowing in vitro differentiation of iPS cell-derived HSCs and assess their potential to engraft in NOD/SCID mice. To improve engraftment efficiency we will precondition the cells by transient expression of reprogramming factors or oncogenes. 3. Biology and genetics if myeloproliferative neoplasm (MPN): We will establish an experimental paradigm to study the biology and genetic predisposition of myeloproliferative neoplastic disorder (MPN), a disorder characterized by a specific JAK2 mutation. The methods developed in aims 1 and 2 will be used to investigate the pathology of MPN such as assessing whether the JAK2 mutation provides a selective advantage to the growth of hematopoietic cells and whether hematopoietic stem cells derived from skin biopsies of the same patient acquire the mutation when differentiated in vitro or when transplanted into NOD/SCID mice.
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0.922 |
2014 — 2018 |
Jaenisch, Rudolf Myers, Richard H (co-PI) [⬀] Young, Richard |
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. |
An Ipsc Based Platform For Functionally Assessing Genetic and Environmental Risk @ Whitehead Institute For Biomedical Res
DESCRIPTION (provided by applicant): Parkinson's disease (PD) is the second most common chronic progressive neurodegenerative disease worldwide, with a prevalence of more than 1% in the population over the age of 60, thus constituting a major global health problem of the aging population. The disease is primarily characterized by a major loss of nigrostriatal dopaminergic neurons but the genetic etiology leading to the neuronal cell loss has largely remained unknown. Numerous genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) that point to more than 50 genomic loci containing risk variants for sporadic PD. However the identified risk variants predominantly map to poorly understood non-coding regions of the genome, which has impeded functional and molecular understanding of how these genetic variants contribute to the increased risk for PD. Importantly, it has been established that PD GWAS associated loci are typically non-coding and mediate allele-specific effects on distal gene expression, consistent with a central role for disruption on enhancer element function in PD pathogenesis Human induced pluripotent stem cell (hiPSC) technology offers for the first time the unique opportunity to study sporadic diseases whose genetic components are poorly understood, by allowing for the generation of human patient-derived somatic cells such as midbrain dopaminergic neurons which carry all the genetic alterations that contributed to the development of the disease. The overall goal of this project is to establish a transformational paradigm, which overcomes the substantial technical limitations of the iPS system and creates a genetically defined experimental in vitro system for studying the molecular and biological mechanisms of sporadic PD in the dish. To achieve this, we will establish a novel experimental framework to link descriptive GWAS PD hits to genomic regulatory enhancer elements and establish functional assays for connecting PD risk alleles to the expression of disease relevant effector genes. Our aim is to gain molecular understanding of the complex interactions between multiple genetic risk alleles and to identify key genes of which the expression level affects the PD specific cellular phenotype. To identify functional relevant candidate risk variants (PD-eQLTs) we will link PD associated risk alleles to genomic regulatory (enhancer) elements that are relevant for gene activity in neurons by establishing an enhancer map in cultured homogenous dopaminergic neurons as well as in sorted neuronal nuclei from human brain. Insights into these interactions will facilitate devising rational therapeutic strategies. This experimental in vitro platform will be used to define the interactions of environmental risk factors with the genetic determinants that have been predicted from GWA studies to predispose to PD (GxE).
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0.922 |
2014 — 2018 |
Jaenisch, Rudolf |
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. |
Genetically Engineered Human Pluripotent Stem Cells as a Platform to Define the B @ Whitehead Institute For Biomedical Res
DESCRIPTION (provided by applicant): Rett syndrome (RTT), caused by mutation of the DNA binding protein MECP2, is one of the most common causes for mental retardation in females. Both loss of function mutations of the gene as well as overexpression such as seen in the MECP2 duplication gain of function syndrome, lead to RTT-like syndrome indicating that increased MECP2 levels can be equally detrimental to the nervous system as MECP2 deficiency. While it has been well established that MECP2 deficiency causes RTT in a cell autonomous manner, recent evidence points to an additional cell-non-autonomous mechanism based on therapeutic effects of MECP2 expression in astrocytes or on transplantation with wild type microglia. These observations as well as the recognition that re- expression of MECP2 or treatment with small molecules can halt disease progression and can even revert symptoms in the adult Rett mouse suggest that MECP2 is required for the maintenance of neuronal function. While these results are exciting and suggest a rational treatment in humans, it is crucial to assess therapeutic strategies in well-defined experimental systems using human cells as readout. This project seeks to set up a platform that utilizes human RTT neurons for clarifying the roles of MECP2 in gene expression and that permits the evaluation of candidate treatments in culture as well as under in vivo conditions. Using human iPS cell- derived neuronal cultures, the initial goal is to establish an experimental paradigm that allows defining the molecular role o MECP2 in gene regulation and to provide a robust and quantifiable disease-relevant phenotypic readout in human mutant neurons. We will use molecular approaches such as CHIP-seq to map binding sites of MECP2 to 5mC and 5hmC modified genomic sites and dissect the modes of gene activation and repression. Furthermore, we will identify target genes of MECP2 and MECP2- interacting partners and clarify the deregulation of MECP2 target genes in loss and gain of function RTT. A major focus of the proposal is to establish a platform that allows assessing the efficacy of therapeutic strategies to reverse the RTT phenotype of human neurons under in vitro and in vivo conditions. (i) To overcome limitations of conventional neural 2D culture systems we will use RTT ES or iPS cells as starting point to generate human cerebral organoid cultures. This will enable the analysis of cell-cell interactions and of potentia therapeutic agents in a well-defined 3D test system. (ii) We will transplant GFP-marked neuronal precursors into the developing mouse brain to generate animals that carry human MECP2 mutant neurons incorporated into their brain. By allowing the human neurons to integrate into the intact mouse brain, we seek to establish a clinically relevant platform to perform in vivo validation of growth factors and small molecule compounds that could be beneficial for the treatment of RTT patients.
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0.922 |
2017 — 2021 |
Hyman, Bradley T. Jaenisch, Rudolf Kellis, Manolis [⬀] Tsai, Li-Huei (co-PI) [⬀] |
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. |
Epigenomic, Transcriptional and Cellular Dissection of Alzheimer's Variants
Abstract The search for effective treatments for Alzheimer's disease (AD), the leading cause of late-onset dementia, has proven challenging. While recent successes in identifying more than a dozen new genes contributing to late-onset or sporadic AD (sAD) have generated considerable excitement in AD research, it is clear from large population studies including GWAS and whole-exome sequencing projects that many single nucleotide polymorphisms (SNPs) contributing to elevated sAD risk reside in non-coding intragenic or regulatory regions. The biological significance of these noncoding SNPs with respect to sAD pathogenesis is not clear. In the current application, we propose a scalable discovery platform for discerning which AD risk SNPs are associated with functional enhancers in specific neural cell types derived from human induced stem cells (hIPSCs). These hIPSCs, created from fibroblasts of sAD patients with a wealth of phenotypes that clearly lead to AD heterogeneity, will enable us to obtain a high-resolution map of AD risk SNPs associated with enhancers and their putative target genes in varied cell types. We will utilize CRISPR/Cas9/dCas9 technologies to directly determine the cell biological consequences of these AD risk genomic variants via 2D and 3D cytosystems. Our comprehensive strategy will identify novel genetic elements and unexpected regulatory pathways contributing to AD pathogenesis and progression that will lead to new therapeutic avenues.
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1 |
2019 — 2021 |
Jaenisch, Rudolf Young, Richard |
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. |
Transcriptional Condensates, Epigenetic Editing and Rett Syndrome @ Whitehead Institute For Biomedical Res
Abstract Rett syndrome (RTT) is a postnatal progressive neurodevelopmental disorder associated with severe mental disability and autism-like syndromes that manifests in girls during early childhood, and is caused by mutation of the X-linked DNA binding protein MeCP2 (Methyl CpG-binding Protein 2). Mice carrying null alleles of Mecp2 closely mimic symptoms seen in patients and are faithful models of the disease. Importantly, development of RTT-like symptoms can be slowed or even halted in the adult following correction of a mutant Mecp2 allele by transgene-mediated MeCP2 expression. MeCP2 is one of the most abundant proteins in neurons, and most disease-causing mutations cluster in the DNA binding domain (MBD) and in the transcription repression domain (TRD). However, the function of MeCP2 remains enigmatic, with two major hypotheses having been proposed: (i) MeCP2 acts as repressor of transcription or (ii) as an activator of transcription. Clearly, none of these proposed functions can fully explain the complex phenotype of MeCP2 deficiency or overexpression leading to RTT or MECP2 Duplication Syndrome. Based on our preliminary evidence we postulate that MeCP2?s primary function may be to modulate the 3D chromosome architecture through condensate formation. Components of both euchromatin and heterochromatin can form phase-separated condensates, which provide a mechanism to compartmentalize and concentrate biochemical reactions within cells and are produced by liquid-liquid phase separation driven by intrinsically disordered regions (IDRs) of proteins. MeCP2 protein contains a large IDR and we have obtained preliminary evidence that MeCP2 is involved in phase- separated heterochromatin condensates. Thus, beyond MeCP2?s role as a repressor or activator of gene expression, the protein may have a much wider and more complex role in the cell physiology and disease. In this project we will define the contribution of MeCP2 to heterochromatic and euchromatic condensates in normal and mutant neurons and analyze the effect of RTT causing mutations on LLPS. Our goal is to gain insights into the function of MeCP2 as the basis for designing novel therapeutic approaches. Potential new therapies based on this hypothesis will take time to develop into applications. To explore a more immediate approach we will use epigenetic editing as a therapeutic tool to activate the inactive wt MECP2 allele located on the inactive X chromosome. Most importantly, epigenetic editing will restore MeCP2 expression to exactly wild type levels and thus avoid toxic consequences of MeCP2 overexpression. In contrast, other strategies such as using vector-mediated MeCP2 transduction will invariably produce cells that overexpress MeCP2 and thus will result in serious side effects as seen in patients with MECP2 duplication syndrome.
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0.922 |
2021 |
Esvelt, Kevin Jaenisch, Rudolf Marasco, Wayne A. Markoulaki, Styliani Telford, Sam R |
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. |
Heritable Immunization of the White-Footed Mouse Reservoir of Lyme Disease @ Massachusetts Institute of Technology
Project Summary ? ? RFA-AI-19-037 The rising incidence of Lyme disease demands new strategies for prevention. Existing methods such as acaricides, deer reduction, landscaping, and personal protective clothing, are inherently short-term and must be regularly re-applied, maintained and worn. ?The Mice Against Ticks project seeks to develop a durable one-time intervention to disrupt the ecological cycle of Lyme disease transmission for many decades?. The causative agent of Lyme disease ?B. burgdorferi ?is passed back and forth between ticks and their small animal hosts, which serve as zoonotic reservoirs of disease. The white-footed mouse ?P. leucopus? is widely considered to be the most important reservoir because it is both ubiquitous and extremely efficient at acquiring and transmitting pathogens via ticks. Our overarching goal for this proposed project is to heritably immunize white-footed mice against Lyme by encoding protective ?P?. ?leucopus? antibodies targeting ?B. burgdorferi ?outer surface protein A (OspA) in the mouse germline. According to our calculations, combining at least four such antibodies should prevent evolutionary escape by ?B. burgdorferi ?because too many simultaneous OspA mutations would be needed. Crucially, even if these mice are less important in some areas than currently thought, immunization will reduce the number of infected ticks, which in turn will infect fewer secondary reservoirs, which will infect fewer ticks, reinforcing a negative feedback spiral anticipated to greatly reduce the local burden of Lyme disease. We have already successfully isolated anti-OspA antibodies from OspA-immunized ?P. leucopus?, derived putative ?P. leucopus embryonic stem cells, and shown that the albumin locus appears suitable for antibody secretion from the liver. We now seek to (1) identify antibodies that bind to at least four different OspA epitopes, (2) establish a stable embryonic stem cell line and perform germline editing, and (3) generate heritably resistant mice that express antibodies from a cisgenic cassette linked to a reciprocal chromosomal translocation, a naturally occurring form of high-threshold gene drive that would enable the reversible and tightly localized engineering of wild ?P. leucopus populations. Our open and community-guided approach has met with apparent enthusiasm by residents of Nantucket and Martha?s Vineyard, indicating that local communities suffering from tick-borne disease throughout the Northeast and Upper Midwest may wish to immunize their own wild mouse populations in order to help prevent Lyme disease for many decades.
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