Kevin C. Eggan, PhD - US grants

DESCRIPTION (provided by applicant): The development of the fertilized zygote into a complex organism has traditionally thought to be a unidirectional process, with cells in the developing fetus becoming gradually more committed to a specific tissue type. The recent development of mammalian cloning by nuclear transfer (NT) suggests that the mammalian oocyte has the remarkable ability to relieve the constraints imposed by cellular differentiation and return an adult nucleus to a totipotent, embryonic state. Thus, cloning by NT provides a unique opportunity to elucidate the molecular and cellular mechanisms by which an adult cell can be returned to an undifferentiated state, a process termed developmental reprogramming. Aim 1) To determine whether mice can be cloned directly from terminally differentiated cells. Cloned mice have only been generated from terminally differentiated cells using an embryonic stem (ES) cell intermediate, suggesting that passage of the nucleus through an ES cell might be necessary for complete reprogramming. Experiments will be carried out to determine whether this is true or if instead, the oocyte and embryo alone can successfully reprogram the epigenetic state of a terminally differentiated nucleus. Aim 2) To investigate if the transition from a differentiated state to a pluripotent state can be understood through the genome-wide changes in transcriptional activity taking place after NT. Towards this end, the genome wide changes in transcriptional activity taking place after NT are being assessed by microarray analysis. Experiments to characterize the functional importance of these observed gene expression changes between fertilized and cloned preimplantation embryos will be carried out. The experiments proposed here combine molecular, genetic and developmental approaches that will test the limits of reprogramming, elucidate mechanisms governing reprogramming and determine how inadequacies in reprogramming may lead to the inefficient nature of cloning. These studies may have profound importance for evaluating the medical utility of NT technology, deciphering the molecular basis of pluripotency and expanding our understanding of embryonic development and stem cell biology.

DESCRIPTION (provided by applicant): Although we do not fully know if disease study of cells in Petri dishes can fully emulate the developmental progression that occurs in human adult neurodegenerative disease like ALS, new described technical ability to generate Induced Pluripotent Cells (iPS) from ALS patients provides an exceptional tool by which we can explore these issues. Many recent insights into the pathophysiology of ALS come from the study of familial forms of this disease. The ability to actually have human cell lines- representing the natural disease in the most relevant cell types- motor neurons and astrocytes- will provide unprecedented tools to 1) study cell- cell interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery and genetic pathway analysis. Eventually these ALS cell lines will also be useful to compare common and uncommon pathways between ALS and other neurodegenerative iPS models. But - iPS cell biology is exceptionally new and we do not yet have sufficient information about the reliability of the cells generated, their ability to truly reflect human cell biology, recapitulate the protein, genetic and functional characteristics of native motor neurons and astroglia. Before we can embark on extensive use of these cells for basic/translational research- it would be critical to generate a series of cell lines- all produced under identical conditions, from different fALS mutations, to determine how representative they are for cell type specificity and functional biology. The overall proposal will involve four principal investigators, working in tight collaboration, to generate and evaluate familial ALS (fALS) iPS cell lines. Project 1, led by Dr. Eggan will obtain the skin biopsies from FALS and control patients, generate the fibroblast and ultimately the initial iPS lines. We will employ the aid of iZumi, a biotech company to be a central site for uniform protocol iPS cell generation. iPS cell lines with neural/glial characteristics will be sent to the Project 2 Lab- Motor neuron biology, lead by Chris Henderson and to Project 3 lab, Astrocytes- lead by Jeffrey Rothstein. These two projects/labs will determine which of the fALS iPS cell lines have the appropriate characteristics of motor neurons and astroglia, through a series of sequential analyses. Only those cell lines that meet final criteria (as compared to human ES cell and prior work on human astroglia) will then go on for final genetic analysis in the Project 4 lab, lead by Tom Maniatis. PUBLIC HEALTH RELEVANCE: Understanding the pathophysiology and development of new therapeutics for ALS has been an enormous challenge. The ability to actually have human cell lines- representing the natural disease in the most relevant cell types- motor neurons and astrocytes- will provide unprecedented tools to 1) study cell- cell interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery and genetic pathway analysis. Eventually these ALS cell lines will also be useful to compare common and uncommon pathways between ALS and other neurodegenerative iPS models. )

The mouse inner cell mass (ICM) and the embryonic stem (ES) cells derived from it contain two active X chromosomes. Similarly, nuclear reprogramming resets the state of X chromosome inactivation (XCI) in mouse induced pluripotent stem (IPS) cells, which also transcribe both of their X chromosomes. However, the proper status of dosage compensation in human pluripotent stem cells remains to be clarified. As the transcriptional networks that propagates the pluripotent state in mouse ES cells has been tightly linked to the regulation of X chromosome inactivation, it will be of substantial interest to determine whether these gene regulatory processes are also tightly coupled within human pluripotent stem cells. In addition, many diseases result from mutations in X-linked genes. As there is substantial interest in using reprogrammed cells for modeling these conditions in vitro, it will be critically important to understand the state of dosage compensation in both human IPS cells and their differentiated derivatives. Here we propose to combine reprogramming, stem cell and genomic approaches to understand the behavior of the inactive X chromosome during the generation, maintenance and differentiation of human IPS cells. Our specific aims are to: Aim 1) To determine whether female IPS cells inherit the inactive X chromosome of the somatic cells from which they are derived and to determine how stably they maintain this inactive X in the course of long-term culture. Aim 2) To determine whether the loss of cytological hallmarks of X chromosome inactivation is accompanied by X-chromosome-wide relaxation of DNA methylation, chromatin structure and transcriptional silencing. Aim 3) We will determine the specific culture conditions that contribute to instability of X chromosome inactivation that we have observed and identify culture conditions that allow proper maintenance of X chromosome-wide heterochromatin.

DESCRIPTION (provided by applicant): Induced Pluripotent Cells (IPS) from ALS patients could provide an exceptional tool by which we can disease pathophysiology and druggable targets. Many recent insights into the pathophysiology of ALS come from the study of familial forms of this disease. The ability to actually have human cell lines representing the natural disease in the most relevant cell types- motor neurons and astrocytes- provides unprecedented tools to 1) study cell interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery and genetic pathway analysis. We have generated iPS cell lines, under identical conditions, from various familial ALS (fALS) mutations and sporadic ALS (sALS) patients to determine how representative they are for cell type specificity and functional biology. The first set of cells now generated, are ready for use in disease phenotyping paradigms. The overall proposal will involve four principal investigators, working in tight collaboration, to generate and evaluate fALS and sALS cell lines and to use them to develop cell specific phenotypic assays. Project 1, lead by Dr. Eggan will generate new critical IPS line- isogenic liens from selected fALS mutations as well as non-integrating IPS lines- to complement our retroviral based IPS collection. In addition Project 1 will fully evaluate the pluripotency of the lines using a novel genetic scorecard system. iPS cell lines with neural/glial characteristics will be sent to the Project 2 Lab- Motor neuron biology, lead by Chris Henderson and to Project 3 lab. Astrocytes- lead by Jeffrey Rothstein. These two projects/labs will determine which ofthe fALS IPS cell lines have the appropriate characteristics of motor neurons and astroglia, thru sequential analyses. In addition, both groups will generate Zinc-finger based cell specific reporter cell lines for future use in drug discovery assays. Cell lines that meet final criteria (a compared to human ES cell and prior work on human astroglia) will undergo genetic analysis in the Project 4 lab, lead by Tom Maniatis. Finally, to develop useful tools for therapeutics and pathogenesis, Cores 1 and 2 will generate motor neuron and astroglial disease phenotyping assays. PUBLIC HEALTH RELEVANCE: Understanding the pathophysiology and development of new therapeutics for ALS has been an enormous challenge. The ability to actually have human cell lines- representing the natural disease in the most relevant cell types- motor neurons and astrocytes- will provide unprecedented tools to 1) study interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery.

At the time of submission of the orginal ALS iPS Go Grant, there were no well-validated protocols for generating astroglia from human ES or IPS cell lines, although approaches from rodent cells existed. Fortunately, we were able to develop a reliable protocol in both the Henderson lab and the Rothstein labs (Figure 5A). We now have a protocol that works identically in both labs¿with very similar efficiency at differentiation between labs. We have also been able to detect evidence of true astroglial maturation in a number of lines that have advanced sufficiently and these express appropriate astroglial specific proteins and have functional glutamate transport (Figure 5B-D).

DESCRIPTION (provided by applicant): The combined activity of four transcription factors (Oct4, Sox2, cMyc and Klf4) can reprogram adult cells into induced pluripotent stem (iPS) cells. These reprogrammed cells should provide a limitless supply of genetically tailored cell types for transplantation medicine, drug discovery and the study of human disease. Unfortunately, the methods used to deliver reprogramming factors have either raised concerns regarding the future utility of the resulting stem cells, or may not be compatible with industrial scale production of clinically compatible stem cell lines. Here, we propose to determine the mechanisms of action of small molecules that we have shown can facilitate the reprogramming process by either increasing its efficiency or by allowing the omission of one or more reprogramming factors. Our final goal is to move towards the identification of a chemical formulation that can alone reprogram adult cells to a pluripotent state. These reprogrammed cells would be free of genetic manipulation and would be the optimal pluripotent cells for biomedical applications. Specifically the aims are: aim 1) To determine the mechanisms by which newly identified small reprogramming molecules act to replace Klf4 in the reprogramming process; aim 2) To determine the mechanisms by which newly identified small reprogramming molecules that can replace Oct4 act in the reprogramming process; aim 3) To determine whether the reprogramming molecules that we have identified can act synergistically to replace multiple reprogramming transcription factors, thus moving us closer to a completely chemical method for reprogramming.

DESCRIPTION (provided by applicant): A hexanucleotide repeat expansion at C9ORF72 has recently been found in a significant fraction of patients suffering from Amyotrophic Lateral Sclerosis (ALS). However, it remains to be determined whether this mutation acts through a gain of function or loss of function mechanism. Resolving this issue is essential for long term efforts to design and implement therapies that counteract the effects of this mutation in ALS. As preliminary data, we provide evidence that heterozygous and homozygous mice harboring a loss of function mutation in the ortholog of C9ORF72 are viable, survive to adulthood and initially display motor system functionality similar to their wild type littermates. However, as heterozygous animals aged, we found they displayed a significantly increased rate of mortality. Death in heterozygous animals was associated with declining motor function and paralysis that were accompanied by muscle atrophy, denervation and motor nerve degeneration. Homozygous mutant animals displayed a similar but accelerated and quantitatively more severe phenotype. Our preliminary studies suggest that C9ORF72 serves an important dose dependent function in the long-term maintenance of the mammalian motor system. These findings support the hypothesis that reduced C9ORF72 function and haploinsufficiency resulting from the repeat expansion that many patients harbor contributes directly to the development of ALS. Here we propose three aims to increase understanding of the role that C9ORF72 plays in the biology of motor neuron degeneration. First, we will determine the extent to which features of motor neuron degeneration in mice harboring a loss of function mutation in the C9ORF72 ortholog are consistent with those observed in ALS. These studies will allow us to determine to what extent the mice we have generated might have utility as a mouse model for both mechanistic and drug discovery studies. Second, we will determine whether motor neuron degeneration in C9ORF72 ortholog mutant animals occurs through cell autonomous mechanisms in motor neurons, or is due to the non-autonomous influence of mutant immune cells. Third, we will determine the extent to which transcriptional changes found in patient derived motor neurons by the C9ORF72 repeat expansion can be explained by loss of function of the C9ORF72 gene product. If funded, our studies will provide important insight into the extent to which loss of the C9ORF72 gene product contributes to motor neuron degeneration.

? DESCRIPTION (provided by applicant): The recent increase in GWAS discovery power for psychiatric disorders has led to the recognition of an undisputed genetic basis for schizophrenia (SZ). However, the mechanistic basis of the vast majority of these loci remains uncharacterized, hindering the ability to translate genetic findings into novel drug targets and develop new treatments for SZ patients. In this proposal, we overcome these challenges and seek to identify and characterize novel SZ driver genes and causal variants by combining computational and experimental methods, integrating systems-level information to prioritize individual genes and loci, and validating their gene- regulatory and cellular effects in 10 neuronal and 3 glial cell tyes derived from iPS cells. Aim 1: We infer gene co-expression networks and modules using multiple brain regions and developmental stages, and use them to predict schizophrenia driver genes based on their clustering in common networks/modules, and their linking to schizophrenia-associated loci using activity correlation, chromatin conformation and eQTLs. Aim 2: We search for schizophrenia-enriched modules of enhancer regions, discovered by clustering patterns of H3K27ac activity across brain regions, developmental stages, and individuals, using an iterative probabilistic framework for joint prediction of causal driver genes, variants, and regulators. Aim 3: We experimentally validate the gene- regulatory and neuronal/glial cellular phenotypes of predicted schizophrenia driver genes and variants in neuronal and glial cell lines based on targeted sequencing of heterozygous loci overlapping 800 putative driver genes and 10,000 putative causal variants, and systematic profiling of neuronal and glial phenotypes upon knockdown and knockout of 200 candidate genes and bidirectional CRISPR-Cas9 editing of 50 candidate causal variants. If successful, this ambitious proposal has the potential to reveal dozens of new target genes and variants associated with Schizophrenia, and open up new avenues for therapeutic development that may alleviate the personal and societal burden of schizophrenia in our lifetimes.

Genetic studies have identified many specific loci with significant associations to psychiatric disorders. However, unless we can develop useful approaches for systematically turning genetic information into neurobiological insights about brain disorders, there is a danger that costly and hard-won genetic findings will not be exploitable to understand pathophysiology and generate important therapeutic hypotheses. The goal of our collaborative, interdisciplinary effort is to develop powerful, generalizable approaches for discovering how risk variants for psychiatric disorders shape neurobiological processes at multiple levels of analysis, and to identify the processes whose dysregulation underlies disease. To do this, we propose to develop new experimental and inferential systems to bridge a longstanding gap between human genetics and experimental biology. We aim to identify biological causes and effects that span the genetic, molecular, and cellular levels of the nervous system. Our interdisciplinary team will develop new experimental systems that measure genetic influences across levels of analysis (RNA, proteins, and cellular function including physiology) in precise, scalable, well- controlled ways. We will make use of emerging cellular systems including three-dimensional cortical spheroids and organoids, and radically novel ?population in a dish? experimental systems that collect data on cells from hundreds of donors in a shared environment, inferring donor identity at the time of phenotypic readout. The analysis of such systems in turn requires sophisticated inferential strategies and new ideas from computer science. We propose to develop and widely share experimental and computational resources, including cell lines, methods, datasets, and analytic tools. The successful completion of this work will identify key neurobiological processes for multiple psychiatric disorders, and fortify many other scientists in making such connections in their own work. We hope in so doing to create a new kind of interdisciplinary science that ? by combining the strengths of data-driven, unbiased human genetics with the power of emerging experimental systems ? transforms the rate at which human- genetic leads lead to insights about disease mechanisms.

Herpes simplex virus (HSV) is a major cause of human disease and suffering. HSV is a widespread neurotropic DNA virus that initially causes an acute primary infection in the mucosal epithelium and then spreads to sensory ganglia where it establishes a latent lifelong infection. Approximately 54% of the US adult population is HSV-1 seropositive and 16% are HSV-2 seropositive. Primary infection may be asymptomatic but can result in severe disseminated disease including meningitis/encephalitis and pneumonia in the immunosuppressed, including neonates. Disseminated HSV infections exhibit high morbidity and mortality including neurologic sequelae in survivors, even if treated with acyclovir or other nucleoside analogs. Intermittent recurrences in latent disease can cause HSV keratitis, the most common cause of corneal blindness in the US, and recurrent genital herpes which is associated with significant morbidity. No vaccine exists and currently used antiviral medications like acyclovir target viral replication and are ineffective in eradicating latent virus. Our long-term goal is to develop novel CRISPR/SaCas9 based antiviral therapies for latent and acute HSV-1 and -2 infections and ultimately for infections with other clinically important members of the Herpesviridae family. In an attempt to develop new treatments for HSV, several studies have edited lytic HSV genomes, but no study has reported cleavage of latent/quiescent genomes. We have devised a multi-step in vitro screening approach and identified SaCas9 single guide RNAs (sgRNAs) that efficiently cleave quiescent HSV-1 genomes - the first demonstration that CRISPR-SaCas9 can target non-replicating HSV. We will expand our screen to HSV-2 and test the ability of CRISPR/SaCas9 to cleave the HSV-1 and -2 genome and abrogate latent and acute infections in mouse and human sensory neurons derived from pluripotent stem cells in vitro. We will then use an adeno-associated virus (AAV) based delivery system to test SaCas9/sgRNA combinations emerging from our screen for the treatment of HSV keratitis and neonatal encephalitis in animal models of HSV in vivo. In addition, we aim to understand the viral genomic factors modulating the efficiency of cleavage of individual SaCas9/sgRNA at HSV target sequences in the latent and lytic state. This project leverages a strong existing collaboration between two multi-disciplinary groups at Harvard University, which combines expertise in stem cell biology and CRISPR technology (Eggan Lab) and virology (Knipe Lab). If successful, the proposed study will establish the CRISPR/SaCas9 system as a novel prophylactic and therapeutic anti-viral strategy against HSV -1 and -2. This approach could be extended to other members of the Herpesviridae family as well as to other viruses without current effective treatment including RNA viruses. Our results will also pave the way for the generation of transgenic animals with constitutive expression of specific Cas9/guide RNAs conferring resistance against viral infections. This research is aligned with the NIH mission to develop novel biopharmaceutical approaches to the treatment of infection in humans.