1. National Health Institute, National Cancer Institute, R01 Title: Nanotechnology enabled targeting of p53 deficiency in human cancer

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1 1. National Health Institute, National Cancer Institute, R01 Title: Nanotechnology enabled targeting of p53 deficiency in human cancer Duration: Total amount: $1,808,042 Investigators: Xiaoming He (contact PI), Xiongbin Lu (PI) Abstract: There has been little change in mortality rate of human colorectal cancer (CRC) in the past decades and the treatment options available for CRC are still very limited today. In CRC and most other human cancers, the tumor suppressor TP53 gene is frequently inactivated by mutation or deletion. Consequently, tremendous effort has been made to restore the p53 activity for cancer therapy. However, no p53-based therapy has been successfully translated into clinical cancer treatment due to the complexity of p53 signaling. Therefore, instead of restoring the p53 activity, identifying vulnerabilities conferred by TP53 deletion or mutation is a novel strategy for fighting against the p53 deficiency in human cancer. In a recent study published in Nature, we confirmed that genomic deletion of TP53 is frequently accompanied by the partial loss of neighboring essential genes and cancer cells with hemizygous TP53 deletion are remarkably vulnerable to further suppression of such neighboring essential genes. We revealed that POLR2A is such an essential gene that is often partially co-deleted with TP53 in human cancers. Such hemizygous loss of TP53/POLR2A occurs in 53, 60, and 41% of colorectal, breast, and pancreatic cancers, respectively. The POLR2A activity is specifically inhibited by α- Amanitin (Ama), a cyclic peptide of 8 amino acids found in the mushroom Amanita phalloides. We demonstrate that low doses of Ama conjugated with antiepithelial cell adhesion molecule (EpCAM) antibody for targeting CRC can result in much enhanced tumor regression in murine models of human CRC with hemizygous deletion of POLR2A without evident systemic toxicity. However, a small fraction of CRC cells are found to be resistant to Ama in a dose-dependent manner. These drug-resistant cells are often called cancer stem-like cells (CSCs) or tumor initiating

2 cells (TICs). A common feature of the CSCs in many cancers (e.g., CRC together with breast and pancreatic cancers) is that they overexpress the variant CD44. Our preliminary data show that the Ama-resistant CRC cells are indeed enriched with CD44, but not EpCAM. Moreover, our studies show that human breast and prostate CSCs can be effectively destroyed in vitro and in vivo using anticancer agent-laden nanoparticles that target the variant CD44 (but not the normal or non-variant CD44 on normal stem cells) with no evident systemic toxicity. Here, we hypothesize that targeted delivery of Ama and/or clinically used anticancer drugs to the variant CD44+ cancer cells with nanoparticles can overcome their drug resistance. We will test this hypothesis with two specific aims: 1, to determine the mechanisms of resistance to the Ama-based POLR2A-targted therapy of human CRCs with hemizygous loss of TP53 and 2, to determine if the combined therapy of Ama and clinically used anticancer drugs co-delivered with the variant CD44- targeting nanoparticles can overcome the drug resistance of human CRCs. It is expected that this project may result in a novel approach to targeting TP53 and could have a major impact on the treatment of colorectal and other cancers harboring TP53 deficiency. 2. National Health Institute, National Institute of Allergy and Infections Diseases, R01 Title: A novel lactic acid bacteria-based norovirus vaccine Duration: Total amount: $3,013,266 Investigators: Xiaoming He (PI), Jianrong Li (Contact PI), Prosper Boyaka (PI), Xi Jiang (PI), and Steven Krakowka (Co-I) Abstract: Human norovirus (HuNoV) infections are responsible for more than 95% of the non-bacterial acute gastroenteritis worldwide and more than 60% of all food-borne illnesses in the US. Evidence suggests that HuNoVs and porcine norovirus (PoNoV) possess high zoonotic potential and, since PoNoV shares the highest identity to HuNoV GII strains, suggesting that swine may serve as reservoirs HuNoV and for emergence of novel HuNoVs and porcine/human GII recombinants.

3 Despite the major efforts, vaccines or antiviral drugs are not available. This is due in major part to the lack of a cell culture system or a small animal model for HuNoV pathogenesis. The overall goal of this proposal is to develop a Lactococcus lactis or lactic acid bacteria (LAB) as the vector to deliver NoV virus-like particles (VLPs) and protrusion (P) particles, and to develop LAB-based "live" NoV vaccines for clinical trials. We have shown that LAB strains expressing VLP and P particles derived from HuNoV-GII.4 induced strong protective immune responses when orally inoculated into gnotobiotic (GN) piglets, the only nonprimate animal model that accurately replicates HuNoV disease. Using this unique animal model, we will determine the dynamics of LAB colonization and the expression, uptake of the NoV VLP and P particles in gastrointestinal tract, and innate immunity induced by LABbased vaccines. We will determine if LAB- based vaccines provide protection against challenge with homologous (HuNoV-GII) or heterologous (PoNoV-GII) viruses. Subsequently, we will determine the mechanism by which LAB-based vaccine induces NoV-specific mucosal, humoral, and cellular immune responses, and define immune correlates of homologous and heterologous protection against NoV strains. Finally, we will determine if microencapsulation and a dendritic cell (DC) targeting peptide that specifically binds mucosal antigenpresenting cells (APCs) will enhance the immunogenicity of LAB-based NoV vaccines and will protect GN piglets from virulent virus challenge. Successful completion of these studies will result in development of safe, stable and efficacious vaccine(s) for the prevention of HuNoV/PoNoV gastroenteritis in humans and swine. This project will also provide a new avenue for vaccine development for other non-cultivable food- and water-borne viruses of human and domestic animal significance. 3. National Science Foundation, CBET-BBE&DMR-BMAT Grant Title: "SusCHEM: Fundamental studies on lyopreservation of adult stem cells Duration:

4 Total amount: $300,000 Investigators: Xiaoming He (PI) Abstract: Lyopreservation of living cells for banking in a state of suspended animation at ambient temperature makes it possible to achieve low-cost maintenance and convenient distribution of the cells. The ready availability of cells is crucial for the wide application and eventual success of cell-based therapies involving tissue engineering, regenerative medicine, cell transplantation, blood transfusion, and stem cell therapy. The objective of this project is to perform a systematic investigation to understand the protective effect of trehalose, a nontoxic sugar, during freezing and drying of trehalose-laden human adiposederived stem cells (hadscs) and in banking the dried cells at ambient temperature. As trehalose has been used for many lower organisms to survive extreme cold and drought in nature, it is hypothesized that trehalose-laden stem cells will behave similarly. This project will have a significant impact on improving healthcare by facilitating the development of cell lyopreservation as an enabling technology for the success of cell-based medical treatments. The use of a nontoxic sugar (trehalose) to replace toxic chemicals (e.g., dimethyl sulfoxide or DMSO) for cell banking is well aligned with the NSF SusCHEM initiative to replace toxic chemicals with earthabundant, inexpensive, and benign materials. To understand the fundamental science of drying living cells for banking in a state of suspended animation at ambient temperature, three specific research tasks are proposed. These include (i) investigation of the effect of freeze-drying parameters on the kinetics of drying trehalose solutions important for lyopreservation of primary hadscs and on the microscopic morphology of the dried products, using freeze-drying microscopy and a benchtop freeze-dryer for small and large samples, respectively; (ii) simulation and experimental quantification of the biophysical responses of the trehalose-laden hadscs to freezing and drying including cell dehydration and intracellular ice formation important for cell survival; and (iii) analysis of the morphology, survival, long-term proliferation, and stem cell properties and functions

5 of the primary hadscs after freeze-drying and banking at ambient temperature. The stem cell properties and functions will be assessed by the expression of stem cell gene and protein markers and their capability of adipogenic, osteogenic, and chondrogenic differentiation. A high survival and intact function of the primary hadscs are anticipated after freeze-drying and long-term storage at ambient temperature under optimal conditions.