Project Title: Engineering of stem-cell targeted retroviral vectors
Project Description: Delivery of therapeutic genes to cells can provide a promising means to treat inherited as well as acquired diseases. For such gene transfer viral vectors based on retroviruses, including lentiviruses, have been designed and used for clinical trials. While their feature of persistent gene expression is beneficial to the gene therapy purpose, retroviruses, in general, suffer from their structural instability as gene delivery vehicle. Pseudotyping retroviruses with the envelope protein of vesicular stomatitis virus (VSVG) has not only improved the stability of retroviruses but also broadened their tropism to a variety of cells.
In this study we aim to develop retroviral gene delivery vectors having a great specificity to target a certain type of cells while maintaining their stability. We are most interested in targeting hematopoietic stem cells and neural stem cells ex vivo and in vivo. To achieve such goals we will engineer, by genetic techniques, VSVG proteins that are ultimately used to package retroviruses. Due to the lack of a structural model for VSVG and limited knowledge on the surface molecules of those stem cells, however, it is hard to rationally modify VSVG given the goals. To overcome such difficulties we will insert randomized short peptide library into VSVG proteins at different locations, then screen and amplify VSVG-pseudotyped vectors that show a high specificity to target cells over multiple rounds, and finally identify the selected peptide sequences from the amplified vectors. The retroviral vectors packaged with VSVG proteins that incorporate the obtained peptide motifs will allow us to genetically modify a specific type of stem cells, and ultimately facilitate the use of stem cells as regenerative medicines against various diseases.

Project Title: Quantative analysis of cellular signaling in viral latency
Project Description: Latent HIV, a pool of replication-competent virus that “hides” in host cells, is the most significant barrier to complete eradication of HIV from a patient. However, the molecular basis of latency remains unknown. Using a retroviral model of HIV-1, I am exploring the role of IKK–NF-kB gene regulation in HIV latency. The Schaffer group has recently demonstrated that stochastic effects in HIV gene transcription potentially underlie the establishment of latency. We hypothesize that the dynamic NF-kB nuclear environment, primarily regulated by IKK, leads to a fluctuating basal transcription rate that contributes to stochastic gene expression and thus the establishment of latency. This hypothesis will be stated in a mathematical model and refined and tested using an array of molecular and cell biology experimental techniques. By combining our model and experiments, I also plan to investigate how pharmacological activators of IKK differentially regulate HIV latency via NF-kB. We hope that this study will yield insights into HIV gene regulation, as well as serve as a platform for designing new signal transduction-modulating drugs that inhibit latency.

Project Title: Homology-directed gene targeting in human ESCs and iPSCs using Adeno-Associated Virus
Project Description: The capability to target a specific gene in human pluripotent cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hIPSCs) via homologous recombination (HR) has broad implications and applications, including fundamental investigations of developmental processes and disease mechanisms, therapeutic gene correction, and high throughput drug discovery and drug toxicity studies. However, a number of factors limit the efficiency of this process, including barriers to DNA delivery and low frequencies of homologous recombination. Gene delivery vectors based on the human parvovirus adeno-associated virus (AAV) has received much attention recently as AAV has not been associated with any human disease, and AAV vectors can mediate long-term gene expression in a wide array of dividing and nondividing cells in vivo. While natural viral variants offer some desirable properties, they possess several limitations. In particular, AAV is not efficient for delivery to all cells, including several types of stem cells that are attractive targets for gene therapy. Therefore, the proposed research aims to use directed evolution approaches to develop AAV vectors that mediate efficient stem cell transduction and gene targeting in pluripotent stem cells.

Project Title: TSC1 and TSC2 modulation of adult neural progenitor cell function in vitro and in vivo.
In a broad sense, I am interested in studying the cellular signaling pathways that regulate stem cell self-renewal and differentiation. I would like to apply this knowledge with my previous research experience, i.e. designing cellular microenvironment s using biomaterials engineered at the molecular level, to develop novel regenerative therapies. 
Project Description: Tuberous sclerosis complex (TSC) is an autosomal dominant negative genetic disorder characterized by the development of benign tumors (hamartomas) in numerous tissue, including kidneys, lung, skin, eyes, heart, and brain. Patients suffering with TSC can exhibit a broad array of symptoms, including renal tumors, progressive lung disease, growths in the skin, epilepsy, and sever cognitive impairment (i.e. mental retardation). The majority of mortality cases in patients with TSC are due to the development of tumors in the brain and kidney.
Although it is known that TSC is often characterized by numerous neurological conditions, including epilepsy (in ~75% of all patients) and cognitive impairment (in ~50% of all patients), the mechanism underlying these conditions is currently unknown. We hypothesize that TSC could alter adult neurogenesis by modulating adult neural progenitor cell function. We will use biological techniques to analyze TSC1 and TSC2 expression, protein products of TSC1 and TSC2 genes, in adult neural progenitor. We will also use RNAi to study whether inhibition of TSC1 and TSC2 activity modulates adult neural progenitor function in vitro and in vivo. The results of this project will lay the groundwork for future study of whether aberrant regulation of adult neurogenesis and adult stem cell function could be contributing factors to neurological symptoms observed in TSC. Finally, this work could further advance understanding of the molecular control mechanisms of adult neurogenesis.

Project Title: Towards a phase diagram of neural stem cell mechanoregulation
Cells can respond strongly and specifically to mechanical cues in their environment, including the viscoelastic properties of the extracellular matrix. Remarkably, the lineage distributions of adult neural stem cells (NSCs) can be directed in vitro by tuning the elasticity (stiffness) of their environment. We propose to develop a phase diagram of stem cell mechanoregulation in three steps: First, we will identify the viscoelastic parameters of the extracellular matrix substrate cells sense. Second, we will determine which parts of the signal transduction machinery must be present in an NSC for it to sense its mechanical environment. To this end, we will make use of lentiviral vectors to precisely modulate expression of targets in the mechanosening pathway. This will allow us to generate a 'phase diagram' of cell response to stiffness of the material, in dependence of signal transduction machinery. Third, we will use lentiviral expression systems to address the question of whether the expression of cytoskeletal proteins characteristic for one lineage is simply correlated with differentiation, or whether it is a cause of differentiation. This is likely to have particularly high impact on the field, as the expression of these cytoskeletal proteins is frequently used as gold standard marker of cell fate. The proposed project requires expertise in materials science, mechanics, cell biophysics, and stem cell biology. We propose to use the support of the HFSP fellowship to combine my expertise in the former two areas with new training in the latter two to tackle the problem of neural stem cell mechanoregulation.

Project Title: Transcription factor network dynamics in hESCs
Project Description: Human embryonic stem cells (hESCs) have the ability to differentiate into all cell types in the body, and thus hold great promise as tools to study development and as therapeutics for the treatment of disease. Our ability to control self-renewal and differentiation decisions is critical to both laboratory and clinical use of these cells.
Recent work has identified a core circuit of transcription factors that controls pluripotency in both human and mouse ESCs. Oct4/POU5F1 and Nanog have been shown to be essential to ESC pluripotency in both mouse and human. Oct4 can also form heterodimers with the transcription factor Sox2 to activate transcription of key pluripotency genes. Genome-scale location analysis revealed that these three transcription factors co-occupy the promoters of many genes that encode regulatory proteins important for stem cell self-renewal, including Oct4, Sox2 and Nanog themselves. There is also evidence to suggest that the levels of these factors must be maintained in a precise range in order to maintain cells in an undifferentiated state.
We aim to create a computational model of this core circuit in order to understand how the dynamics of this transcription factor network drive self-renewal and differentiation decisions. Predictions and hypotheses generated by the model will be validated and refined with cellular and molecular biology experiments in human embryonic stem cells. We are particularly interested in the behavior of the network that underlies neural differentiation. We believe that better understanding of these dynamics will not only increase our knowledge of cell fate decisions in development but also our ability to safely direct differentiation of hESCs to specific lineages.

Project Description: My current project involves analyzing how human immunodeficiency virus (HIV) responds to selective pressure - specifically a lentiviral vector encoding an antisense gene therapy, which is referred to as VRX496. This project is being conducted in collaboration with VirXsys, a biotech company that focuses on gene therapy and this therapy using the VRX496 vector is currently being tested in clinical trials. This lentiviral therapy encodes a 937 bp antisense sequence that targets the envelope of the HIV protein. I will use a variety of molecular biology techniques including Solexa sequencing and quantitative polymerase chain reaction (qPCR) to analyze how the HIV virus mutates in response to treatment with this antisense gene therapy.

Project Title: Designing biomimetic systems for directed dopaminergic differentiation of human embryonic stem cells
Project Description: Rapid progress has been attained in the development of differentiation paradigms to drive different type of neurons from human embryonic stem cells (hESCs), with the fundamental objective of using these cells for replacement and repair of damaged neuronal circuits in the central nervous system. Of particular interest are midbrain dopaminergic neurons because degeneration or loss of function of these neurons is associated with Parkinson’s disease.
Many protocols used to direct hESCs to develop into dopaminergic neurons are highly inefficient, or use co-culture systems of hESCs with cells of animal origin which prevents any downstream clinical application due to possible transfer of animal cells and pathogens. Also, most of these strategies employ classical two-dimensional culturing conditions for neuronal and dopaminergic induction which have limited relevance to the native three-dimensional conditions. Other major challenges that must be overcome to realize the therapeutic potential of hESC-derived dopaminergic neurons are poor survival and integration upon transplantation.
To overcome these obstacles, we aim to design defined three-dimensional biological systems functionalized with bioactive components including recently identified midbrain patterning molecules and neurotrophic factors to support efficient dopaminergic differentiation of hESCs ex vivo. These cellular scaffolds are also designed to act as a temporary extracellular matrix after transplantation to enhance the survival and functional integration in vivo to meet the requirements for cell-based strategies for brain repair.