Li Cai - US grants

[unreadable] DESCRIPTION (provided by applicant): The long term goal of this proposal is to develop therapeutic interventions in the generation of neurons from neural stem cells (NSC) for replacement therapies of disease and injury in the central nervous system (CNS). Neural stem cells give rise to all of the different cell types in the CNS. Understanding of the complex transcriptional networks that control the process of cell fate determination in NSC is crucial in applications for therapeutic purpose. The generation of diversity of neurons and glial cells is achieved through cell proliferation, cell fate determination and differentiation of embryonic NSC populations into progressively more specialized cell types that make up the CNS. The uniqueness of individual neurons and glial cells is determined by combinatorial patterns of gene expression; and gene expression is largely controlled at the level of DNA sequence (cis-regulatory element, genetic), as well as by chromatin structure (epigenetic). One of the key components in transcription regulation is the enhancer, a non-coding DNA sequence that is often evolutionarily conserved. Upon binding of trans-acting factors, enhancers determine tissue or cell type-specific expression of particular genes. We will choose genes that are crucial to NSC cell fate determination from genome wide gene expression studies and analyze the non-coding DNA regions for their regulatory functions (e.g. as an enhancer) in NSC gene expression during cell fate determination. We will predict (in silico) and functionally characterize (in vivo) the predicted putative enhancers in chick retinal stem cells. We refer to this kind of enhancer as NSC enhancers. Our focus will be on NSC enhancers that are important for the development of the retina. The two specific aims are: 1) to predict evolutionarily conserved NSC enhancers; and 2) to verify and characterize putative NSC enhancers. The successful completion of the proposed study will help to identify transcriptional control networks that are crucial for cell fate determination of neural stem cells. In addition, novel retina-specific enhancers identified from this study can be used to identify novel protein factors that are previously unknown for their function in controlling neural cell fate determination. Our findings will ultimately provide an integrated transcription network that controls NSC cell fate determination in the retina (can also be applied to other developmental systems in general). Such an understanding of the transcriptional networks is fundamental to the development of potential treatments or therapeutic transplants for diseases ranging from retinal degeneration to spinal cord injury. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: The goal of this proposal is to search for DNA sequence elements that exist in the non-protein coding regions of the genome that regulate the generation of nerve cells from neural stem cells. The successful completion of the proposed studies will help the development of potential treatments or therapeutic transplants for diseases ranging from neural degeneration to spinal cord injury. [unreadable] [unreadable] [unreadable]

CBET-0828244
M. Yarmush, Rutgers University New Brunswick

The overall goal of the proposed research is to develop effective strategies to increase the stability and titer of retroviral vectors for gene therapy applications. Recently, considerable effort has been dedicated toward utilizing these, and other viral, vectors in a number of clinical trials. Although several publications have described various degrees of success, the clinical success of retroviral vectors is limited by the rapid rate of viral inactivation and limited viral titers. Thus, a retrovirus with enhanced intrinsic stability and high titer would alleviate some of the practical obstacles limiting the success of retroviral vectors in the clinic. It is likely that these principles could also be extended to other viral vectors.

The proposed research seeks to accomplish the following: (1) To determine the mechanisms underlying the loss of retroviral bioactivity and the relative stability of the immature / mature viral particles; (2) To optimize culture conditions and methods, in order to allow the production of high-titer viral vectors; and (3) To modify the viral reverse transcriptase to achieve higher efficiency viral replication. To achieve these goals, physical, chemical, and molecular manipulation techniques will be used with the objective of slowing the rate of retroviral decay and increasing retroviral titer.

The proposed work will provide excellent training for the graduate fellows involved in the project, as well as to students exposed to it through various courses and training programs conducted by the investigators. In addition, the PI will provide an excellent educational environment for various high school and university-wide undergraduate research programs. The scientific approach was designed not only to establish an excellent scientific program, but also to expose students to state of the art technology and methodology through both classroom instruction and direct laboratory interaction. The research itself will provide valuable new information regarding the mechanisms of retroviral decay and methods to address them. The experimental results are likely to have a broad impact on the scientific and clinical venues which utilize or are developing viral vector delivery protocols. In summary, the proposed work seeks to provide fundamental research and a human base of personnel equipped to solve problems related to the implementation of techniques to improve suitability of viral vectors for gene therapy applications.

[unreadable] DESCRIPTION (provided by applicant): Breast cancer stem cells have been identified as CD44?/low breast tumor cells that exclusively retain the ability to form new tumors in mouse models. Cancer stem cells can arise from mutations in normal stem cells directly, or from mutations in progenitor cells that lead to the regaining of stem cell property. Although normally down-regulated during mammary epithelial differentiation, CD44 is expressed in up to 85% human breast cancers and the ectopic expression of CD44 has been shown to promote metastasis. It is possible that the deregulation of CD44 in breast stem cell is associated with tumorigenic transformation due to mutations in pathways that regulate CD44 expression. Therefore it is extremely important to dissect the molecular mechanism(s) that regulates CD44 expression in breast stem cells and controls CD44 down-regulation during stem cell differentiation. Gene expression can be controlled by genetic elements (e.g., cis-regulators, i.e., non- coding DNA sequences that control cell type-specific expression/suppression of genes), in addition to epigenetic controls such as DNA methylation. Due to the highly complex nature of the genome, finding cis-regulators, which determine cell type-specific (stem cells versus differentiated cell) gene expression, in vertebrates remains a difficult task. However, the fact that many cis-regulators are often evolutionarily conserved across species provides a basis for the identification of these cis-regulators using comparative genomic method. Our approach employing in silico predication and "wet lab" verification methods to identify crucial cis-regulators and trans-acting factors of the CD44 gene is highly innovative. Evolutionarily conserved non-coding regions for the prediction of cis-regulators greatly reduced false positives, hence, work load for experimental verification. It provides an integrative way to decode the complicated transcriptional regulation of CD44. Understanding regulatory mechanisms of CD44 expression could ultimately lead to the identification of pathways that could be mutated during the tumorigenic transformation of breast stem cells or cells further down the differentiation hierarchy and thus may provide new therapeutic targets for treatment of breast cancer. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to dissect molecular mechanisms that regulate the expression of the breast cancer stem cell marker CD44 .Understanding regulatory mechanisms of CD44 expression could ultimately lead to the identification of pathways that could be mutated during tumorigenic transformation of breast stem cells or cells further down the differentiation hierarchy and thus provide novel therapeutic targets for treatment of breast cancer, aimed directly at the origin of the tumor. [unreadable] [unreadable] [unreadable]