Development of Aptamer Conjugates for Targeted Cancer Therapy

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Development of Aptamer Conjugates for Targeted Cancer Therapy
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english
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Xiong, Xiangling
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University of Florida
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Gainesville, Fla.
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Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
Tan, Weihong
Committee Members:
Horenstein, Nicole A
Cao, Yun Wei
Fanucci, Gail E
Dennis, Donn M

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Subjects / Keywords:
aptamer -- cancer -- dna -- targeted -- therapy
Chemistry -- Dissertations, Academic -- UF
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Chemistry thesis, Ph.D.
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Abstract:
Cancer, defined as a class of diseases that are caused by uncontrolled cell division and invasion, is now the leading cause of death worldwide. Great efforts have been made in cancer treatment from different aspects, and targeted therapy is a promising option. Aptamers are single-stranded oligonucleic acid (or peptide)molecules generated by an iterative screening method called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since their discovery in the 1990s, the development of aptamers as specific, high-affinity probes with diagnostic and therapeutic applications has been widely studied. Aptamers that recognize therapeutically important targets such as cancer-associated antigens are of great interests in cancer research. This dissertation investigated the therapeutic applications of aptamers in cancer. Specifically,two types of aptamer conjugates were designed for targeted cancer therapy: one for improved the targeting specificity and efficiency of cells used for adoptive cell therapy, the other for targeted delivery of therapeutic oligonucleotides into cancer cells. We designed an amphiphilic molecule–a lipid-DNA conjugate­–to modify immune cells with targeting ligands (i.e., DNA aptamers) and demonstrated that aptamers anchored on immune cell membranes facilitated cancer cell targeting and induced immunogenic cancer cell death. For targeted delivery, acancer cell specific aptamer was conjugated to antisense oligonucleotides of an oncogenic microRNA. We studied the specific intracellular delivery of theantisense molecule with the aptamer, and monitored its efficacy in silencing target microRNA. Taken together, the development of these probes explored the potentials of aptamers in cancer treatment and expanded the molecular tool box for fighting cancer.
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Statement of Responsibility:
by Xiangling Xiong.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Tan, Weihong.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31

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1 DEVELOPMENT OF APTAMER CONJUGATES FOR TARGETED CANCER THERAPY By XIANGLING XIONG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DO CTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Xiangling Xiong

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3 To my loving parents

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4 ACKNOWLEDGMENTS My deepest gratitude goes to my exceptional research advisor, Dr. Weihong Tan. I am very for tunate to have an advisor who encouraged me to explore my research interests and provided me with tremendous support and opportunities. His patience and guidance helped me overcome my many difficulties. I wish to express my sincere thanks to Dr. Lung Ji Chang of the Molecular Genetics and Microbiology Department, for helping me to develop my background in immunology, and for constructive guidance in building immunological assays. I am also thankful to Dr. Chen Liu of the Pathology Department, for his gener ous help in mice work. I also appreciate my entire committee, including Dr. Nicole Horenstein, Dr. Gail Fanucci, Dr. Donn Dennis, for their helpful discussions and advice during my research. I would also like to thank Dr. Charles Cao, who was willing to participate in my final defense committee at the last moment. I appreciate continuous support from the Department of Chemistry, University of Florida I especially thank Lori Clark and Dr. Ben Smith for all their efforts to make my Ph.D. study smoother. Special thanks goes to Dr. Kathryn R. Williams for her kindness and help with the manuscript. I thank all the Tan group members who supported my study in one way or another. I greatly thank Dr. Kwame Sefah who patiently trained me in cell SELEX. I also owe m y gratitude to Dr. Haipeng Liu for his guidance at the beginning of my graduate study; I also thank Dr. Yan Chen, Dr. Dalia L opez Coln, Dr. Ling Meng, Dr. Zhi Zhu, Dr. Meghan Altman and Dr. Yanrong Wu for their helpful discussions. I am also grateful to D r. Dimitri Van Simaeys, Weijun Chen, Elizabeth Jimenez, Dr. Hui Wang, Dr.

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5 Suwussa Bamrungsap, Guizhi Zhu, Tao Chen, Sena Cansiz, Mingxu You, Lu Peng, Da Han, Cuichen Wu and Liqin Zhang for their friendship and encouragement. Although my name is the only one on the cover of this dissertation, it is a result of successful collaborations with many great scientists. During my five years study, I have also received a lot of help outside the Chemistry Department. I take this opportunity to thank Dr. Shuhong Han, Yu Ling Yeh, Dr. Erika Adriana Eksioglu, Xiaokui Zhang and Hyun M in Jung for kindly providing experimental materials and help in collaborative projects. Last but not least, I would like to thank my family and friends in both China and the US. Without the ir understanding, encouragement and love, I could not have accomplished my educational goal.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES .......................................................................................................... 10 LIST OF FIGURES ........................................................................................................ 11 LIST OF ABBREVIATIONS ........................................................................................... 13 ABSTRACT ................................................................................................................... 17 CHAPTER 1 INTRODUCTION .................................................................................................... 19 Cancer .................................................................................................................... 19 Causes of Cancer ............................................................................................. 20 External factors .......................................................................................... 20 Internal factors ........................................................................................... 21 Cancer Diagnostics .......................................................................................... 23 Current diagnostic methods ....................................................................... 23 New and developing detection methods .................................................... 24 Cancer Therapy ................................................................................................ 25 Traditional treatments ................................................................................ 25 Emerging treatments .................................................................................. 26 Aptamers ................................................................................................................ 27 Characteristics of Aptamers ............................................................................. 28 Generation of Aptamers ................................................................................... 31 Ration ale of selection ................................................................................. 31 CellSELEX process .................................................................................. 32 Applications of Aptamers .................................................................................. 33 Aptamers for diagnostics ........................................................................... 33 Aptamers in biomarker discovery ............................................................... 34 Aptamers as therapeutics .......................................................................... 34 Overview of Dissertation Researc h ......................................................................... 35 2 FUNCTIONAL CELL MEMBRANES WITH LIPOPHOLIC NUCLEIC ACIDS FOR CONTROLLED CELL CELL ADHESION ................................................................ 39 Introductory Remarks .............................................................................................. 39 Materials and Methods ............................................................................................ 40 General Materials ............................................................................................. 40 General Cell Culture Conditions ....................................................................... 40 LipoDNA Synthesis and Materials ................................................................... 41 Imaging of Lip o DNA on Cell Surface ............................................................... 41

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7 Lipid Rafts Staining .......................................................................................... 42 LipoDNA Insertion Study ................................................................................. 42 Quantification of Cell Membrane Anchored LipoDNA ..................................... 42 Cytosolic Stain .................................................................................................. 42 Homotypic and Heterotypic Cell Assembly ....................................................... 43 DNase Treatment of Cell Aggregates ............................................................... 43 Protease Treatment of Cell Aggregates ........................................................... 43 3 D Microtissue Assembly ................................................................................ 43 Targeting Cells in a Cell Mixture ....................................................................... 44 Results and Discussion ........................................................................................... 44 Design and Synthesis LipidAptamer ............................................................... 44 Modification of Cell Surface with Aptamers ...................................................... 45 Co nfocal microscopy study of lipoDNA insertion ...................................... 45 Partition of lipo DNA into different cell membrane domains ....................... 46 LipoDNA insertio n can be regulated ......................................................... 46 Quantification of membraneanchored lipoDNA ........................................ 47 Retention time of membraneanchored lipoDNA ...................................... 47 Aptamer Directed Cell Assembly ...................................................................... 48 Homotypic cell assembly ........................................................................... 48 Heterotypic c ell assembly .......................................................................... 48 The architectures of assembled cells ......................................................... 49 Cell Assembly Was Controlled by MembraneAnchored Aptamer ................... 49 Cell aggregates can be disrupted with DNase ........................................... 49 Cell aggregates can be disrupted with protease ........................................ 50 The impact of quantity and stability of surface anchored aptamers ........... 50 Potential Biomedical Applications of LipoDNA Modified Cells ......................... 51 Aptamer directed synthesis of microtissue mimics .................................... 51 Selective cell targeting in a cell mixture ..................................................... 52 Con clusion .............................................................................................................. 53 3 DNA APTAMER MEDIATED CELL TARGETING AND CANCER THERAPY ........ 63 Introductory Remarks .............................................................................................. 63 Materials and Methods ............................................................................................ 64 General Materials ............................................................................................. 64 General Cell Culture Conditions ....................................................................... 65 Quantitative Analysis of Cell Aggregates .......................................................... 65 Annexin V/Propidium Iodide DoubleStaining Assay ........................................ 66 CFSE Dilution Assay ........................................................................................ 66 MTS Cell Viability Assay ................................................................................... 66 Natural Killer Cell Isolation ............................................................................... 66 Activation of CTL .............................................................................................. 67 Aptamer assisted Immune Cell Killing Assay ................................................... 67 Results and Discussion ........................................................................................... 67 Quantitative Study of Aptamer Mediated Cell Targeting .................................. 67 Quantification of cell targeting efficiency with flow cytometric based assay ...................................................................................................... 68

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8 Cell targeting at physiological conditions ................................................... 68 Cytotoxicity of Lipo DNA ................................................................................... 69 LipoDNA insertion did not compromise cell membrane integrity ............... 70 LipoDNA insertion did not affect the proliferation pattern of CTL .............. 71 LipoDNA did not affect cell viability ........................................................... 71 Natural Killer Cell Based Assays ...................................................................... 72 NK cell isolation and s urface modification .................................................. 72 NK K562 binding assay ............................................................................. 72 NK K562 killing assay ................................................................................ 73 Cytotoxic T Lymphocyte Based Assays ........................................................... 73 Activation of CTL with small molecules ...................................................... 74 CTL Ramos binding assay ......................................................................... 75 CTL Ramos killing assay ........................................................................... 76 CTL K562 binding and killing assay ........................................................... 77 Conclusion .............................................................................................................. 78 4 ENGINGEERING AN APTAMER BASED DUAL FUNCTIONAL MOLECULAR PROBE FOR CANCER THERAPY ......................................................................... 89 Introductory Remarks .............................................................................................. 89 Materials and Methods ............................................................................................ 91 General Materials ............................................................................................. 91 Cell Lines and Culture ...................................................................................... 91 Oligonucleotide Synthesis ................................................................................ 92 Gel Electrophoresis .......................................................................................... 92 Flow Cytometric Analysis of Probe Binding ...................................................... 92 Confocal Microscopy Study of Probe Internalization ........................................ 93 Transfection of Antagomirs ............................................................................... 93 Quantitative Analysis of Endogenous MiRNA221 Level ................................... 93 Western Blot Analysis ...................................................................................... 94 Ce ll Proliferation Assay .................................................................................... 95 Results and Discussion ........................................................................................... 95 Sequence Design and Synthesis ...................................................................... 95 Binding and Internalization Study ..................................................................... 96 AS1411 Antag221 conjugates can recognize target cells .......................... 96 Internalizati on of Antag221 with AS1411 ................................................... 97 Aptamer Delivered Antagomir Can Inhibit MiRNA Function ............................. 98 Quantitative analysis of miRNA221 level ................................................... 98 Analysis of p27Kip1 expression pattern ....................................................... 99 Effect of AS1411Antag221 on Cancer Cells .................................................... 99 AS1411 Antag221 improved TRAIL sensitivity ........................................ 100 AS1411 Antag221 inhibited cancer cell proliferation ................................ 100 Conclusion ............................................................................................................ 101 5 SUMMARY AND FUTURE WORK ....................................................................... 111 Summary of Dissertation ....................................................................................... 111

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9 Future Direction .................................................................................................... 113 Targeting Murine Liver Cancer Cells with Aptamer Modified Murine T Cells .. 113 Targeting Liver Tumors in an HCC Mouse Model .......................................... 114 T Cell Induced HCC Suppression .................................................................. 114 LIST OF REFERENCES ............................................................................................. 115 BIOGRAPHICAL SKETCH .......................................................................................... 136

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10 LIST OF TABLES Table page 1 1 List of Aptamers in Clinical Trails ........................................................................ 36 1 2 Summary of cancer cell SELEX ......................................................................... 37 2 1 Lipid DNA sequences ......................................................................................... 54 2 2 Quantification of lipoDNA on cell surface .......................................................... 54 4 1 List of oligonucleotide sequences ..................................................................... 102

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11 LIST OF FIGURES Figure page 1 1 Citation report on Aptamer from Web of Knowledge.. ........................................ 38 1 2 Scheme of Cell SELEX. ..................................................................................... 38 2 1 Demonstration of cells modified w ith artificial cell adhesion molecules and the chemical structure of LipoDNA molecule. .......................................................... 55 2 2 Confocal microscopic images of LipoDNA TMR treated cells.. ......................... 55 2 3 Flow cytometric analysis of LipoDNA modification. ........................................... 56 2 4 Aptamer mediated homotypic cell assembly. ..................................................... 57 2 5 Aptamer mediated heterotypic cell assembly .................................................... 57 2 6 Different architectures of assembled cells. ......................................................... 59 2 7 Cell assembly can be disrupted by DNase. ........................................................ 60 2 8 Cell assembly can be disrupted by protease. ..................................................... 60 2 9 Diacyllipid tail is required for a firm in sertion. ...................................................... 61 2 10 Schematic diagram of assembly of multiple types of cells and con focal micrograph of microtissue. ................................................................................. 62 2 11 Schema tic demonstration of selective cell targeting and confocal micrographs of cell aggregates. .......................................................................... 62 3 1 Schematic representation of targeting cancer cells (blue) with aptamer modified immune cells ( red). ............................................................................... 79 3 2 Quantification of cell targeting efficiency. ........................................................... 80 3 3 Cell targeting efficiency in different conditions. .................................................. 81 3 4 Cytotoxicity of the lipo DNA probe. ..................................................................... 82 3 5 Aptamer assisted NK cell targeting and killing of K562 cells. ............................. 83 3 6 Activation of CTL with PMA/ionomycin. .............................................................. 84 3 7 CTL Ramos cell binding assay. .......................................................................... 85 3 8 CT L Ramos cell killing assay. ............................................................................. 86

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12 3 9 CTL K562 cell binding and killing assay. ............................................................ 88 4 1 Schematic presentation of the function of miR NA221 and AS1411Antag221. 103 4 2 Probe synthesis and cell binding test. .............................................................. 104 4 3 Confocal microscopy study of probe internalization. ......................................... 105 4 4 Study of the internalization pathway. ................................................................ 106 4 5 Antag221 delivered by AS1411 can inhibit miRNA221 function. ...................... 107 4 6 Relative A549 cell viability. Cell viability was assessed by MTS assay after different treatments. .......................................................................................... 108 4 7 Internalization of TM R labeled probes in U87MG and MDA MB 231 cells. ..... 109 4 8 Representative micrographs of U87MG and MDA MB 231 cells after incubated with different probes. ........................................................................ 110

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13 LIST OF ABBREVIATION S ACT Adoptive cell therapy AML Acute myeloid leukemia ATCC American Type Culture Collection B ALL Acute B Lymphoblastic Leukemia BB Binding buffer BSA Bovine serum a lbumin CA125 Cancer antigen 125 CAM C ell adhesion molecules CD Clust er of differentiation CFSE C arboxyfluorescein succinimidyl ester CMV C ytomegalovirus CT Computed tomography Ct T hreshold cycle CT B Cholera toxin B subunit CTL Cytotoxic T lymphocytes DAG Diacylglycerol DISC Deathinducing signaling complex DMEM Dulbeccos Modified Eagle Medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DNase Deoxyribonuclease dNTP Deoxyribonucleotide triphosphate DPBS Dulbeccos p hosphate buffered saline EGFR Epidermal growth factor receptor

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14 FBS F etal bovine serum FITC Fluorescein i sothiocyanate HCC Hepatocellular carcinoma HER3 Human epidermal growth factor receptor 3 HPLC High performance liquid chromatography HPV human papillomaviruses IgM Immunoglobulin M IP3 Inositol 1,4,5 trisphosphate ITAM Immunoreceptor tyrosinebased activat ion motifs Kd Dissociation constant kD KiloDalton MAB Monoclonal antibody MCP 1 Monocyte chemotactic protein1 MESF Molecules of e quivalent soluble f luorochrome MFI Mean fluorescent intensities MHC Major histocompatibility complex MHC I Major histocompati bility complex class I miRNA microRNA MRI Magnetic resonance imaging mRNA messenger RNA MS Mass spectrometry MSC M esenchymal stem cells MTS 3 (4,5 dimethylthiazol 2 yl) 5 (3 carboxymethoxyphenyl) 2 (4 sulfophenyl) 2H tetrazolium NCI National Cancer Institu te NIH National Institute of Health

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15 NKC Natural killer cell NSCLC Non small cell lung cancer OCCA Ovarian clear cell adenocarcinoma OSA Ovarian serous adenocarcinoma PBMC Peripheral blood mononuclear cell PBS P hosphate buffered saline PBST PBS buffer conta ining Tween 20 PCR P olymerase chain reaction PDGF Platelet derived growth factor PE R Phycoerythrin PEG Polyethylene glycol PET Positron emission tomography PI P ropidium i odide PIP2 Phosphatidylinositol 4,5bisphosphate PKC Protein kinase C PMA 12myrista te 13 acetate PMS P henazine methosulfate PS Phosphatidylserine PSA Prostatespecifi antigen PTK Protein tyrosine kinase PTK7 Protein tyrosine kinase 7 RBC Red blood cell RIPA R adioimmunoprecipitation buffer RISC RNA induced silencing complex RNA Ribonuclei c acid

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16 RNAI RNA interference rt PCR Real time PCR RT PCR Reverse transcript PCR SA Streptavidin SCLC Small cell lung cancer SDF 1 Stromal cell derived factor 1 SELEX Systematic Evolution of Ligands by EXponential enrichment siRNA small interference RNA SPECT Signal photon emission computed tomography StIP1 Stress induced phosphoprotein 1 T ALL Acute T Lymphoblastic Leukemia/Lymphoma TAA Tumor associated antigen TBE Tris borateEDTA TCR T cell receptor TEAA T riethylamine acetic acid buffer TLR T olllike rece ptors TMR Tetramethylrhodamine TNF Tumor necrosis factor TRAIL TNF related apoptosis inducing ligand tRNA Transfer RNA UV Ultraviolet VEGF V ascular endothelial growth factor VWF von Willebrand factor 2 M ercaptoethanol

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17 Abstract of Dissertation Prese nted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DEVELOPMENT OF APTAMER CONJUGATES FOR TARGETED CANCER THERAPY By Xiangling Xiong December 2012 Cha ir: Weihong Tan Major: Chemistry Cancer, defined as a class of diseases that are caused by uncontrolled cell division and invasion, is now the leading cause of death worldwide. Great efforts have been made in cancer treatment from different aspects, and t argeted therapy is a promising option. Aptamers are singlestranded oligonucleic acid (or peptide) molecules generated by an iterative screening method called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since their discovery in the 1990s, the development of aptamers as specific, high affinity probes with diagnostic and therapeutic applications has been widely studied. Aptamers that recognize therapeutically important targets such as cancer associated antigens are of great interests i n cancer research. This dissertation investigated the therapeutic applications of aptamers in cancer. Specifically, two types of aptamer conjugates were designed for targeted cancer therapy : o ne for improve d the targeting specificity and efficiency of cell s used for adoptive cell therapy the other for targeted delivery of therapeutic oligonucleotides into cancer cells W e designed an amphiphilic m olecule a lipid DNA conjugate to modify

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18 immune cells with targeti ng ligands (i.e., DNA aptamers) and demonstra ted that aptamers anchored on immune cell membranes facilitated cancer cell targeting and induced immunogenic cancer cell death. For targeted delivery, a cancer cell specific aptamer was conjugated to antisense oligonucleotides of an oncogenic microRNA. We studied the specific intracellular delivery of the antisense molecule with the aptamer, and monitored its efficacy in silencing target microRNA. Taken together, the development of these probes explored the potentials of aptamers in cancer treatment and ex panded the molecular tool box for fighting cancer.

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19 CHAPTER 1 INTRODUCTION Cancer Cancer is defi n ed as a class of diseases caused by uncontrolled cell division and invasion. There are two ways to classify cancers: by their organs of origin e.g., lung cancer, or by affected tissue types e.g., carcinoma. Cancer is now the second leading cause of death in United States (1). It is estimated that in the US about 1.6 million new cancer cases are to be diagnosed and about 0.6 million people will die of cancer in 2012 more than 1,500 people per day (1, 2) It is encouraging to know that the overall cancer death rates have been declining about 1.5% annually since 1999. The incidence rates of cancers, however, increased in some types and decreased in others during the 10year period from 1999 to 2008 (3). Causes of cancers with i ncreasing trends are unclear and need further studies. The factors account ing for reduced incidence rates vary for different cancers. For instance, lung cancer incidence is continuing to drop due to declining tobacco consumption (4); r educed hormone replacement therapy greatly decreased breast cancer incidence (5); and t he discovery of prostate specific antigen (PSA) as a biomarker for prostate cancer screening may be associate with a decrease in prostate cancer incidence (6). Nevertheless, studies on cancer causes, diagnosis and therapies to help effectively control the diseases are in great need. The National Institutes of Health (NIH) estimate d that in 2005 the overall costs of cancer were more than $200 billion (7) And grants to inv estigate cancer research from numerous sources are increasing steadily. According to National Cancer Institute (NCI) annual fact book, NCI provided about $5 billion funds on cancer research annually from 20022011 (8). Scientists are trying to

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20 understand the development of canc er, construct analytical methods for prognosis and diagnosis and eventually, find a cure for cancer. Causes of Cancer Cancer is not a modern disease. The earliest written record of cancer was dated back to 2500 BC in a papyrus found in Egypt. Cancers from mummified specimens and fossils preserved for thousands of years have also been found. The word cancer, meaning crab in Greek, was first used to describe the disease by Hippocrates the Greak physician, around 400 BC and he also proposed the first cancer theory humoral theory (9). This theory was not challenged for centuries until the p ractice of anatomy disproved it From 1700, with the development of scientific knowledge in natural sciences, human knowledge of cancers and their causes have advanced. Overall, the causes can be classified as external or internal factors. External factor s Substances that cause cancers are defined as carcinogens which can in general, be classified as chemical, physical and biological effects. Exposure to certain chemicals such as aniline, asbestos, benzene, benzidine, cadmium, nickel and vinyl chloride c an cause various types of cancer. Tobacco and alcohol consumption are associated with lung and liver cancer respectively. Exposure to high energy r adiation is a physical factor. The ultraviolet (UV) radiation in sunlight increases the risk of skin cancer UV radiation from i ndoor tanning devices is also considered carcinogenic Ionizing radiation from x rays, radioactive fallout and radon gas increase the risks of leukemia lung, and other cancers Infectious viruses and bacteria increase the risk of developing certain types of cancer as well. For example, l ong term infection with hepatitis B and C viruses causes

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21 liver cancer; human immunodefici ency virus increases the risk of lymphoma and leukemia; EpsteinBarr virus (EBV) is linked to nonHodgkin lymphoma; and human papillomaviruses (HPV) are detected in 99% of cervical cancers. Other factors such as poor diet, lack of physical activities or obesity, may increase the risks of certain types of cancers. Internal factors How can these factors cause cancer ? The common result shared by all factors is ge netic damage and/or mutations A gene is a collective sequence of deoxyribonucleic acid (DNA) that carries inheritable information for protein expression. Transformed cells differ from healthy cells in their protein repertoire, which define their distinctive features. Reviewed by Hanahan and Weinberg (10) in 2011, there are eight ways that cancer cells differ from normal cells Cancer cells: Sustain proliferative signaling Evade growth suppressors Resist cell death Enable replicative immortality Induce angiogenesis Activat e invasion and metastasis Reprogram energy metabolism Evade immune destruction As the understanding of genetics and molecular biology has grown two types of genes closely related to cancer have been identified : oncogenes which promote cell growth and divi sion and tumor suppressor genes. One of the most common oncogenes is the ras gene (11) The Ras protein family control s genes involved in cell growth, differentiation and survival. In normal cells Ras proteins are off until they are switched on by incoming signals but m utations on ras genes can produce Ras proteins that are constantly on with no controlling signal. And in turn, the genes controlled by Ras

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22 proteins are always active to help cell s grow, differentiate and survive. T umor suppressor genes cease cell growth and division, repair DNA mistakes and initiate cell apoptosis. The protein P53 that is encoded by the TP53 gene is a tumor suppressor. Carcinogens can damage TP53 gene and consequently destroy p53 function (12) In addition, p eople who inherited only one functional copy of the TP53 from their parents are more likely develop tumors. However, not every genetic mutation leads to cancer. When the cellular transformation happens and intrinsic tumor suppression mechanisms fail, the immune system as an extrinsic tumor suppress mechanism is engaged. Because transformed cells often express abnormal proteins (antigen s) (13) as do other pathogens such as bacteria and viruses, the innate and adaptive immunity responds to transformed cells and eliminates them before they become clinically apparent (14) The main immune responses to tumor cells are from cytotoxic T lymphocytes (CTL) and Natur e Killer (NK) cells. Activated CTL and NK cells recognize tumor cells via tumor associated antigens (TAA) presented by maj or histocompatibility complex (MHC) molecules (15, 16) Upon binding to TAA, toxins such as perforins or granzyme B are released, which cause cancer cell death. If immunological molecules and cells successfully r emoved all transformed cells, cancer is not able to develop. However, cancerous cells have a variety of well developed ways to evade the immune system (17 19) For example, MHC downregulation makes cancer cells inv isible to immunosurveillance. In addition, some cancer cells can secret e immune suppressive cytokins such as TGF regulatory T cells to dampen immune responses.

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23 Cancer Diagnostics Cancer diagnosis starts with the observation of symptoms or scr eening results suggesting cancer. A series of tests are then used to confirm the presence of cancer and to identify the primary site of malignancy and the involved cell types. Also, the pathological grade and stage of cancer are classified. In general, tw o types of tests, direct imaging and laboratory test s, are currently in clinical practice. Diagnostic information is critical because it determines treatment options. Also, early detection greatly improves therapeutic response and can lead to a better chance of recovery Current diagnostic methods Imaging technologies help visualize solid tumor s inside the body. X ray, computed tomography (CT) and magnetic resonance imaging (MRI) are common ways to view organs and bones and pinpoint the exact tumor site. C ontrast agents that enhance the differences between normal and diseased tissues are applied in some cases. These methods are straightforward, yet not precise For example, t hey cannot identify subtypes of cancer and a small tumor may not be detected. In a ddition, successful use of these methods depends on the experience of the radiologist who examines the imaging results. Therefore, cancer needs to be confirmed with lab tests which differs according to the cancer type. In most cases, a biopsy is performed when removing a tissue sample from the suspected site is possible. The r emoved sample will be processed, stained and examined under a microscope. Histological and morphological features of the sample cells are compared with normal cells to determine if th e tissue is cancerous. Cancer type, grade and stage are determined after the cancer is diagnosed. Cytogenetics and immunohistochemistry may provide genetic and molecular information of cancer.

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24 Although biopsy is more accurate, one disadvantage is the need for sample acquisition. Some tumor sites are difficult to access, if not impossible. More importantly, the sample may be biased if it does not represent the overall situation. Other than biopsy, blood tests that measure blood cells and examine for tumor markers are frequently performed. One example is PSA, a prostate tumor marker approved by FDA. Usually a high level of PSA in the blood indicates the presence of prostate cancer. Although limited by the types of available cancer markers, efforts to discover new biomarkers are increasing. New and developing detection methods Since the common feature of all types of c ancer cells is genetic mutation, in contrast to t raditional cancer diagnostics which rel y on morphological definition and histological examination, new methods focus on molecular profiles of cancerous and normal cells. These methods probe alterations of genes and proteins to achieve early detection, accurate diagnosis and appropriate treatment. The advances in understanding cancer genetics and the rapid development of gene sequencing technologies contribute to precise classification of cancers, selection of treatment and prediction of clinical outcomes. DNA microarrays quantitatively analyze gene expression patterns, and comparisons of cancer and normal cells can help identify the chromosomal alterations related to cancer development (20) The invention of highthroughput next and third generation sequencing technologies may bring new opportunities in clinical cancer diagnostics (21) Another molecular diagnostic focus is on proteins. The direct outcome of genetic mutation is the altered protein expression, so the protein expression pattern in cancer patients may differ from that in normal people. One example is cancer antigen 125 (CA125), whose serum level is used to prognose and diagnose epithelial ovarian cancer

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25 (22) Strategies for discovering novel cancer biomarkers, especially membrane receptors, are under intensive study. Cancer Therapy Traditional treatments The treatment of cancer involves destruction, removal or control of primary or metastatic cancer, with the hope of eradicating entire tumors, preventing recurrence or spread o f primary cancer, and relieving symptoms Depending on the type and stage of cancer, the treatments may vary. Traditionally, there are three types of treatments available. Cancers with solid tumors are primarily removed by surgery. To prevent tumor reoccur rence, nearby tissue as well as lymph nodes may be removed at the same time. Although surgery can physically remove tumors, it cannot cure cancer in most cases and in some rare cases, it even helps the cancer to spread. Chemotherapy uses chemicals to inhibit cancer cell growth or directly kill cell, and it is usually applied to cancer that cannot be removed by surgery. Ideally, drugs for chemotherapy should selectively target cancer cells. Compared with normal cells, cancer cells proliferate more rapidely; therefore drugs that interfere with cell division are used. For example, a commonly used chemotherapy drug, doxorubicin can intercalate genomic DNA and prevent DNA replication. However, no existing chemotherapeutic drugs can precisely distinguish between normal and cancer cells, and they also introduce undesirable toxicity and damage normal tissue. High energy radiations such as X rays, gamma rays are used in radiation therapy. Radiation directly damages cells or affects cell proliferation. Like chemotherapy,

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26 radiation therapy is not cancer cell specific. Moreover, radiation is a potential carcinogen, and the radiation field must be localized to minimize side effects. Emerging treatments New treatments focusing on targeted therapy, prevention of reoccurrence and metastasis are under development. The discovery of antibodies and the emerging knowledge of the human immune system are leading to the development of more specific therapeutics. Likewise, advances in understanding genetic mutations of cancer cells are paving the way for gene therapies. Monoclonal antibodies (MAB) that recognize tumor associated antigens (TAA) have been developed for cancer treatment. MABs can work by themselves to enhance the anti cancer immune response or inhibit cancer cell growth. F or example, the most widely used MAB, rituximab, recognizing CD20 expressed on B cells and can induce antibody dependent cellular cytotoxicity (23, 24) and complement dependent cytotoxicity (25) Another class of MAB, such as trastuzumab, targets the epidermal growth factor receptor (EGFR) family or related proteins to block the transmi ssion of growth signals in epithelial tumors (26) In addition, MABs can be conjugated to chemotherapeutic or radioactive molecules to home the molecules to cancer c ells and greatly reduce side effects (27) Rituximab has been linked to the chelator tiuxetan with a r adioactive isotope Y 90, and the complex showed reduceddose and toxicity (28, 29) Adoptive cell therapy (ACT) is undertaken with the recognition of interaction between the immune system and cancer (30) This regimen harnesses cancer reactive lymphocytes (normally T lymph ocyte and NK cells) from the patient (autologous) or from a donor (allogeneic), activates and expands the lymphocytes ex vivo and infuses them back into the patient to hopefully overcome immune tolerance and induce tumor

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27 regression (31, 32) Lymphodepletion by chemotherapy or total body irradiation to temporally ablate the immune system is given priory to ACT to precondition the patient (33, 34) In general, three types of l ymphocytes are used for ACT. Tumor infiltrating lymphocytes isolated from tumor tissue are under clinical trails for melanoma (35, 36) Cancers caused by virus infections can be targeted by cytotoxic T cells that r espond to viral antigens (37, 38) Genetically engineered T cells that recognize specific TAAs are also under intensive investigation (39, 40) Current studies emphasize on improving the therapeutic efficacy of ACT. Gene therapy refers to a remedy that introduces genetic materials (DNA or RNA) into cells and utilizes cell machinery for therapeutic benefit. One type is the direct use of DNA sequences that encode a functional or therapeutic gene, to replace a mutated gene or produce therapeutic proteins. Another type involves the RNA interference (RNAi) pathway (41) that post tran scriptionally modulates gene activity. RNAi controls the activity of messenger RNA (mRNA), and can be manipulated by small RNA molecules such as microRNA (miRNA) and small interfering RNA (siRNA). Progress on the design and delivery of small RNA molecules for cancer and other diseases has increased rapidly in recent years (42) but there are challenges that limit the use of RNAi in the clinic and call for more investigations (43) Aptamers Aptamers are usually singlestranded oligonucleic acid molecules generated by the iterative screening method called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Aptamers were first reported by two researc h groups independently in 1990: Ellington and Szostak reported a selection of RNA molecules that specifically binds to organic dyes (44) ; and Tuerk and Gold found RNA ligands to

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28 bacteriophage T4 DNA polymerase (45) These specific RNA molecules were given the name aptamer, from the Latin aptus, meaning to fit by Ellington and Szostak (44) The other group, however, defined the process in finding aptamers as SELEX Systematic Evolution of Ligands by EXponential enrichment (SELEX) (45) The discovery of aptamers and invention of SELEX gave rise to a new field in both academia and industry. Since 1990, aptamers have been extensively explored as specific, high affinity probes for diagnostic and therapeutic applications. According to Web of Knowledge, the research on aptamer is growing exponentially (Fig. 11 ). As of 2012, there are nine aptamers in clinical trails by six companies (T able 11 ). A ptamers against therapeutically important targets such as cancer cell membrane proteins have demonstrated the great potential for aptamers in cancer research (46) Characteristics of Aptamers The most commercially successful aptamer is NX1838 (brand name Macugen) which binds to the predominant isoform of vascular endothelial grow th factor, VEF G165, which inhibit s VEFG bi nding to its cellular receptors and induces an anti angiogenesis effect. Macugen was first generated using RNA bases by two independent laboratories in 19 9 0s (47, 48) and further chem ic al modifications have improve d its half life in human serum. The development of Macugen revealed several advantages of aptamers. First, aptamers are highly specific in recognizing closely related proteins or chemical structures. For instance Macugen can discriminate VEFG165 from the VEFG family. Aptamers that differentiate subtle chemical structural variations, such as a methyl or hydroxyl groups or the D vs L enantiomer, have been reported (49 53) The high speci ficity may be attributed to the repeated selection process in whice the highest

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29 binders (potential aptamers) are retained. Moreover, during target recognition process, nucleic acid aptamers undergo target induced conformational changes to better fit into the binding pocket further improving aptamers selectivity (54 58) The dissociation constant s (Kd) of aptamers reported in the literature are in the low nano molar to high picomolar range, comparable to the Kds of antibodies (59) Aptamer s have the advantage of being smaller than antibodies. Depending on the number of nucleotides, aptamers usually weigh 815 kDa. Antibodies on the other hand, normally have weights of around 150 kDa (60) Smaller size leads to better tissue penetration and faster blood clearance Another advantage is that a ptamers are essentially nonimmunogenic (61) Immunogenicity is one of the major concerns when developing new therapeutics with high molecular weight molecules like anti bodies. Once the active molecule is recognized, the immune system produces neutralizing antibodies or initiates other immune responses. Monoclonal antibodies, for example, are usually highly immunogenic and need to be humanized before administration (62) Even so, minor immune response can be observed. On the contrary, immunogenic antibodies to oligonucleotides h ave never been reported (63) In fact, the possible immune response is from tolllike receptors (TLRs) which belong to the innate immune system. Pathogend erived RNA DNA and synthetic oligonucleotides containing the unmethylated CpG motif can activate TLRs and provoke inflammation (64) This problem can be solved by substituting natural bases with 2 O methylribonucleotides in sequences flanking the CpG motif (65)

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30 The latter capability reflects another advantage of aptamer s easy chemical synthesis and modification. P hosphoramidite chemistry complement ed with solid phase synthesis has made oligonucleotide synthesis rapid and inexpensive. Modifications of nucleotides on both the phosphate/ribose backbone and the nucleobases have been studied to improve resistance to enzym atic degradation and reduce off target events (66) Some of these modified nucleotides are compatible with enzymatic steps such as PCR amplification (67) and thus diversify the selection library. Moreover, with the development of phosphoramidite chemistry most of the modifications can be incorporated with the programming of a DNA synthesizer which makes them easy to perform and readily accessible. In addition, nucleotides can be tagged to various functional molecules or moieties such as fluorescent molecules (68 72) nanoparticles (73 76) and siRNA (77 83) for diagnostic and therapeutic purposes, advancing aptamers applications in bioanalytical and biomedical fields. The chemical nature of aptamers also indi cates another important trait: aptamers are cost effective and easy to produce. Antibodies heavily rely on cell based production system s, require complicated manufacturing processes and often suffer batchto batch var iation (84) Aptamers on the other hand, can be reproduced c hemically with little or no variation. However, aptamers are not surrogates of antibodies. One of the major concerns is aptamers may be too specific and only recognize a specific isoform of a protein target For example, because Macugen only targets the V EFG165 isoform, it is not as effective as anti VEFG antibodies that target all isoforms of VEGF. Macugens market share was over taken by antibodies not long after Macugens initial success (85) N onetheless,

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31 aptamers show great potential as chemical antibodies in biomedical areas including diagnosis, biomarker discovery, and ther apy (86) Generation of Aptamers Rationale of selection The invention of polymerase chain reaction (PCR) by Mullis et al in 1986 tremendously revolutionized biomedical research and SELEX is one example. Since its invention in 1990, SELEX has been applied to a variet y of targets, ranging from small molecules such as inorganic dyes to large moi eties like proteins, cells and an entire tumor (44, 45, 4953 83, 87 113) The process starts by generating a randomized RNA or DNA (and later even peptide) sequence library. By permutation and combination of four basic nucleotides into 2040 nucleotide long sequences, this library normally consists of 10141015 d ifferent candidate aptamers that arguably can recognize v irtually any target molecule (84) Each candidate shares a forward and a backward primer region for PCR amplification. An RNA library also contains binding sequences for reverse transcription. To identify aptamer(s) specific to the target of interest, the li brary is incubated with target for a certain period of time, and the binding sequences are eluted and amplified by PCR. This process is iterated 5 25 times until the pool is enriched with repeated binding sequences. To improve the target specificity, count er selection (or negative selection) with a molecule that shares similar motifs wit h the target molecule, e.g. an isoform of a target protein, is incorporated in some selection process es The aim of counter selection is to remove sequences that bind to bot h target and nontarget molecules, leaving the very specific sequences. Negative selection can be performed before and/or after positive selection, depending on the similarity between

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32 the target and nontarget molecules. Finally, the enriched library is sequenced to identify potential aptamers, which are synthesized and validated with original target. Cell SELEX process The c ell membrane is o ne of the most interesting targets for SELEX, because it consists of thousands of different proteins carbohydrates and lipids. These biomolecules retain specific structures and play crucial roles in cellular activities Compared with pure target, such as an organic dye molecule or a protein, a cell membrane is a very complex system. The first report using a cell membra ne as a selection target was reported by the Gold group (87) In this study, they used red blood cell ghosts as a positive selection tar get and demonstrated that SELEX can be applied not only to pure compounds but also to complex targets such as the cell membrane One advantage of cell SELEX is simultaneous generati on of high affinity probes for multiple targets (87) M oreover, membrane proteins are an important but understudied group because of technical difficulties (114) SELEX may provide an alternative way to dem ystify them by first probing them with high affinity aptamers. After the introduction by Gold et al, selection on whole cells (88, 90) especially tumor cells (89, 101) we re reported, and thus SELEX for cancer research became popular. Cell SELEX is particularly useful in identifying s ubtle differences between normal and cancerous cells or differ ent subtypes of cancerous cells, because of the counter selection process. P osit ive selection on target cells (cancer cells) and negative selection on nontarget cells (normal cells or another type of cancer cells) can reveal molecular differences of membrane proteins and may lead to biomarkers for specific cancer types. The general p rocedures of cell SELEX demonstrated in Fig. 12, and literature reported selections on cancer cells are summarized in Table 12.

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33 Applications of Aptamers Because of the diversity of targets, aptamers have been used alon e or conjugated to different functi onal modalities for a variety of applications. In cancer research, aptamers are being developed for diagnostic, biomarker discovery and therapeutic purposes Aptamers for diagnostics Aptamer based diagnostics detect molecular abnormality in diseases. Aptam ers can be chemically conjugated with various signal generating components for acoustic (115117) electrochemical (118 121) and optical (122 124) sensing. Aptamers have been developed for commonly used diagnostic formats, such as sandwich assays (125, 126) and flow cytometric assays (127, 128) whic h were previously dominated by antibodies. In addition, taking the advantage of adaptive recognition, aptamers can be designed into analyteresponsive biosensors, which improves detection sensitivity by reducing background (129133) or amplifying signals (134137) Aptamers can also be used for molecular imaging. The most popular ex vivo imaging technology is fluorescence based. Fluorescently labeled aptamers, either directl y coupled to fluorescent molecules or conjugated to nanoparticles, are used for histological examination of proteins (138 141) cells (73, 142) and tissue slices (98, 113) More importantly, aptamers are suitable for in vivo imaging because of their relatively small size, resulting in rapid tissue penetration and blood clearance, which are essential for obtaining high signal to noise images (143) In response, development of aptamer based p robes for noninvasive medical imaging techniques, including CT, MRI, positron emission tomography (PET) and signal photon emission computed tomography (SPECT), are in progress (144)

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34 Aptamers in biomarker discovery Identification of tumor specific antigens is critical to the understanding of tumor biology and discovery of pharmacological targets. Conventional methods of biomarker discovery, such as mass spectrometric (MS) approaches enable investigators to analyze the entire cell proteome and identify cell specific proteins. However, the most under represented group in proteome analysis remains membrane proteins which are difficult to tackle (114) Aptamers generated from cell SELEX profile membrane protein di fferences among different cells and simultaneously act as affinity probes for protein extraction, which presents unique advant ages in identifying protein molecules on cell membrane surfaces (145) To date, several membrane proteins have been elucidated with aptamers generated from cell SELEX (89, 146, 147) Aptamers as therapeutics The therapeutic effect of aptamers often resembles that of small molecules or a ntibodies. Aptamers targeting proteins relevant to cancer treatment, such as antibodies (147150) adhesion molecules (151 153) tumor markers (89, 154 157) cytokines (158, 159) chemokines (160, 161) and costimulatory receptors (162, 163) have been developed. In most cases, aptamers act as antagonists that inhibit their target proteins activities. For example, one RNA aptamer, A30, that binds to the oligomeric form of human epidermal growth factor receptor 3 (HER3), can inhibit HER3 associated MCF7 breast c ancer cell proliferation and drug resistance (155) Assembly of monomeric aptamers into bivalent or multivalent form may create agonistic aptamers that activate their receptors by causing dimerization or multimerization (163, 164) Other than altering target protein function, another promising application of aptamers, especially those targeting tumor cell membrane proteins, is as targeting

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35 moieties to deliver chemotherapeutic drugs (165, 166) phototherapeutic agents (167) toxins (168) and nanocarrieres (169171) for targeted cancer therapy. Furthermore, aptamers can be internalized into with their receptors for intracellular delivery of molecules for gene therapy (77, 78) Overview of Dissertation Research This dissertation discusses therapeutic applications of aptamers in cancer treatment Specifically, aptamers as targeting moieties for delivery of therapeutic reagents are explored. Two types of therapeutics, immune effector cells and anti sense oligonucleotides, are used as models. Chapter 2 describes the design, synthesis and verification of an aptamer based probe for facile modification of cell membranes with cell adhesion molecules. Chapter 3 discusses the probes potential in cell based immunotherapy of cancer. Chapter 4 describes the design and development of a piece of a cancer cell specific oligonucleotide for cancer gene therapy. Chapter 5 summarizes the overall significance and suggests future directions for this research.

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36 Table 11. List of Aptamers in Clinical Trails Aptamer Target Therapeutic effect Clinical phase Reference ARC1779 VWF Thrombotic microangiopathies and carotid artery diesease Phase II (102) ARC1905 Factor C5 Age related macular degeneration Phase I (103) AS1411 Nucleolin Acute myeloid leukemia Phase II (104) E10030 PDGF B Age related macular degeneration Phase II (105) NOX A12 SDF 1 Chronic lymphocyt ic leukemia, Multiple myeloma and Glioblastoma multiforme Phase I (106) NOX E36 MCP 1 Diabetic nephropathy, Type 2 diabetes mellitus and Lupus nephritis Phase II (107) NOX H94 Hepcidin Anemia of chronic disease in patients with cancer, renal disease or inflammatio n Phase I (108) NU172 Thrombin Heart disease Phase II (109) NX1838 VEGF Inhibit VEGF binding to VEGFR FDA approved drug (110) REG1 Factor IXa Coronary artery disease Phase II (111)

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37 Table 12. Summary of cancer cell SELEX Target cell line Cancer Type Dissociation Constant (nM)* Aptamer Target** Patient Sample Tested Reference HL60 Acute myeloid leukemia 4.51.6 N.A. Bone marrow (94) CEM Acute T lymphblast ic leukemia 0.80.09 PTK 7 Bone marrow (101, 146, 172, 173) Ramos Burkitt s lymphoma N.D. N.A. Primary human chornic lymphocytic leukemia B cells (174) Ramos Burkitt s lymphoma 49.65.5 N.A. N.A. (175) Ramos Burkitt s lymphoma 0.760.13 IgM heavy chain Bone marrow (92, 147) DLD 1 Colon cancer 32.13.4 N.A. Tissue section ( 98) HCT 116 Colon cancer 3.90.4 N.A. Tissue section (98) U251 Glioblastoma 150 Tenascin C N.A. (89) A172 Glioblastoma 61.826.37 N.A. N.A. (100) MEAR M ouse live r hepatoma 4.51 0.39 N.A. Human hepatocarcinoma cell line (147, 176) A549 Non small cell lung cancer 28.25.5 N.A. Tissue section (96) TOV21G Ovarian clear cell adenocarci noma 0.250.08 StIP1*** N.A. (99) CAO 3 Ovarian serous adenocarci noma 3920 N.A. N.A. (99) NCI H69 Small cell lung cancer 38 N.A. Blood sampe (93) *Best Kd among all aptamers reported in the paper **Target of one reported apt amer ***Unpublished data

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38 A B Figure 1 1 Citation report on Aptamer from Web of Knowledge. A) published items in each year B) citations in each year. Figure 1 2 Scheme of Cell SELEX (177)

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39 CHAPTER 2 FUNCTIONAL CELL MEMBRANES WITH LIPOPHOLIC NUCLEIC ACIDS FOR CONTROLLED CELL CELL ADHESION Introduct ory Remarks Living organisms function properly by establishing well organized hierarchical structures. The smallest structural and fu nctional unit is the cell. Multiple populations of cells form ordered tissue architectures by selectively adhering to each other and the extracellular matrix via cellsurface proteins called cell adhesion molecules (CAMs) (178) Cells regulate life activities through complex cell cell and cell environment interactions One important interaction is cell to cell contact, which controls cells migration (179) proliferation (180, 181) differentiat ion (182, 183) and organization of tissues (184) Thus, t he ability to control the cell cell adhesion of multiple cell types is highly desired for understanding tissue biology, tissue engineering and stem cell research. In response, techniques such as layer by layer printing (185) optical tweezer s (186) and dielectrophoresis methods (187) have been explored to build a variety of microscale 2 D or 3 D assemblies. However, for practical applications these methods are limited because they lack the intrinsic molecular encoding for cell cell recognition. Thus, selective cell assembly from bottom up approaches remains an important challenge. Because of their exquisite molecular recognition property synthetic DNA molecules have been programmed to direct the assemblies of a range of nano/microscale structures DNA encoding of a wide v a ri e ty of materials, including inorganic particles (188 190) soft materials (191195) and live cells (196199) has been successfully applied in diagnostics and tissue engineering. F or example, although Bertozzi and coworker reported the assembly of 3D microtissues via DNA hybridization

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4 0 (198) this technique requires the surface modification of target cells. Ideally, modified cells should recognize their target cells spontaneously. In this chapter, we d escribe a str ategy for controlling cell cell adhesion that mimics the natural process based on the hypothesis that aptamer s will induce cellular adhesion upon spontaneous receptor ligand binding. Our method of using aptamer s to mediate the assembly of cell is illustra ted schematically in Fig. 2 1 A key step in our strategy calls for facile DNA modification of the live cell surface in a manner that enables rapid, efficient and controlled formation of specific 3 D interactions for a range of cell types. We demonstrate t hat aptamers anchored on the cell surface in this fashion, can act as artificial CAMs that specifically recognize their target cells and form the desired cellular architecture. Materials and Methods General Materials Unless otherwise stated, all solvents and chemicals were obtained from SigmaAldrich and used without further purification. DNA synthesis reagents were purchased from Glen Research. PEG phosphoramidite (DMT Hexaethyloxy Glycol phosphoramidite) was purchased from ChemGenes Corporation (Wilming ton, MA). Cholera toxin B subunit (Ct B) conjugated with Alexa488 and CellTrackerTM Green CMFDA was purchased from Invitrogen. Quantum FITC 5 MESF kit was purchased from Bangs Laboratories, Inc. DNase I was purchased from BioLabs. P roteinase K w as purchased from Fisher Biotech. General Cell Culture Conditions Jurkat, K562, CCRF CEM (CCL 119 T cell, human acute lymphoblastic leukemia) and Ramos cells (CRL 1596, B lymphocyte, human Burkitt's lymphoma) were obtained

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41 from ATCC (American Type Culture Collection ) and were cultured in complete RPMI 1640 medium (ATCC) supplemented with 10% fetal bovine serum (FBS) (heat ing buffer contained 4.5 g/L glucose and 5 mM MgCl2 in Dulbecco's PB S (Sigma). Binding buffer used for incubation was prepared by adding yeast tRNA (0.1 mg/mL) (Sigma) and BSA (1 mg/mL) (Fisher) into the washing buffer to reduce background binding. LipoDNA S ynthesis and Materials All DNA sequences were synthesized from 3 to 5 using the ABI 3400 synthesizer on the 1.0 micromolar scale. DMT Hexaethyloxy Glycol (PEG) phosphoramidite was coupled to DNA by extended coupling time (900 seconds) on the DNA synthesizer Each DNA probe was coupled with four PEG phosphoramidite units. Lipid phosphoramidite was synthesized by following a previously published procedure (200) and coupled using the DNA synthesizer by ex tended coupling time (900 seconds). After synthesis, the DNA was cleaved and deprotected from the CPG and purified by reverse phase HPLC using a C4 column (BioBasic 4, 200mm x 4.6mm, Thermo Scientific) with 100 mM triethylamineacetic acid buffer (TEAA, pH 7.5) and acetonitrile (030 min, 10 100%) as an eluent. All purified lipo DNA probes were stored in DNase/RNase free water. Imaging of LipoDNA on Cell Surface Cells (200L, 1106 cells/mL ) were suspended in a 96well plate and incubated with L ipo DNA pro bes ( 1M lipid DNA, with TMR fluorescent dye) in cell culture medium at 37oC Cells were then washed twice with washing buffer to remove free probes and resuspended in binding buffer Images were taken and collected in the perpendicular

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42 lateral (x y) plane by laser scanning confocal microscopy equipped with a 488 nm argon laser and a 543/633 nm helium/neon laser. Lipid Rafts Staining CEM cells were incubated with LipoLib TMR at 37C for 15 min. Cholera toxin B subunit (Ct B) with Alex488 was then incubated with cells for 30 min on ice a concentration. Aliquots were analyzed under las er scanning confocal microscopy using the sequential scan mode to eliminate crosstalk between dyes. LipoDNA Insertion Study Cells were incubated with FITC labeled L ipo Lib probes for different time periods with different probe concentrations. Fluorescent signals from labeled cells were examined by FACS flow cytometry. Data were analyzed using WinMDI flow software, and FITC histograms were prepared. Experimental conditions resulting in the largest fl uorescent intensity were used for later labeling procedures Quantification of Cell Membrane Anchored LipoDNA Cells were incubated with FITC labeled lipoLib FITC probes for 2 hrs with different probe concentrations. Fluorescent signals from labeled cell s were examined by FACS flow cytometry. Data were analyzed using WinMDI flow software, and the mean fluorescent intensities from two independent measurements were obtained and compared with known standards ( Quantum FITC 5 MESF kit) to calculate the number of oligonucleotides per cell Cytosolic Stain CellTracker Green CMFDA (Green, Invitrogen) was first dissolved in d imethyl sulfoxide ( DMSO ) in serum free medium. Fresh cells were washed once in PBS buffer and then incubated

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43 in stain solution for 15 min at 37 C. Labeled cells were washed twice in PBS buffer and resuspended i n complete cell culture medium for another 30 min at 37 C before use. Homotypic and Heterotypic Cell Assembly For homotypic cell assembly, CEM and Ramos cells labeled with L ipo Sgc8 TMR L ipo TD05 TMR or L ipo Lib TMR and shaken at 300 rpm for 20 min at room temperature. For heterotypic cell assembly, various types of cells were modified with L ipo Aptamer TMR or L ipo Lib TMR first, and a proper ratio of aptamer targeting cells was combined in binding buffer and shaken at 300 rpm for 20 min at room temperature Aliquots were analyzed under la ser scanning confocal microscopy DNase Treatment of Cell Aggregates CEM cells labeled with LipoTD05 TMR or Lipo Lib TMR probes were mixed with CellTracker Green labeled Ramos cells and gently shaken for 20 min at room t emperature. Aliquots immediately after mixture and after 20 min incubation were taken and analyzed by confocal microscopy. Cell mixtures were resuspended in DNase I reaction buffer with DNase I (2 units) and incubated at 37C for 10 min. Aliquots were asse ssed by confocal microscopy. Protease Treatment of Cell Aggregates After forming homotypic Ramos cell assemblies cell clusters were incubated with 0.1mg/mL proteinase K in PBS at 37 C for 20 min. To quench the proteinase digestion, the sample was quickly mixed with 200 L of cell culture medium and placed on ice. Then the treated cells were washed with 2 mL of binding buffer and used for imaging 3 D Microtissue Assembly LipoTD05 TMR labeled K562 cells were first incubated for 20 min with Lipo Sgc8 FITC labeled Ramos cells (5 :1 Ramos: K562) with shaking. Without further purification,

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44 untreated CEM cells (5:1 CEM: K562) were added to the above mixture. The entire mix ture was incubated for another 20 min with shaking. Aliquots were analyzed by laser scanning confocal microscopy. Targeting Cells in a Cell Mixture CellTracker Green stained Ramos cells and untreated CEM cells were mixed together in a 1:1 ratio. K562 cells modified with either LipoTD05 TMR or lipo Sgc8 TMR probes were coincubated with 5 equiv alents of the above cell mixtures, respectively, and shaken at 300 rpm for 30 min at 25 C. Aliquots were analyzed using laser scanning confocal microscopy. Results and Discussion Design and Synthesis LipidAptamer The membrane anchored aptamer is designed with three distinct moieties (Fig. 2 1 ) T he first moiety is a celltargeting aptamer sequence selected by cellSELEX (92, 101) Two different aptamers, Sgc8, which specifically targets Protein Tyrosine Kinase 7 (P TK7) on CCRF CEM cells (101, 146) and T D0 5 which targets the immunoglobulin heavy mu chain (IgM) on Ramos cells (92, 147) were used for testing. These aptamers exhibit h igh affinity (KdSgc8: 0.8nM, KdTD05: 74nM) and excellent selectivity toward their target cells, as required for mimicking native cell surface ligandreceptor interactions. In addition, because multiple aptamers are presented on each cell surface, multivalent interactions with target proteins can greatly improve binding (201) T he second moiety is a PEG linker which allows DNA to extend out from the cell surface, thereby minimizing chargecharge, nonspecific and steric interactions betw een the cell surface molecules and the DNA. Thus, the PEG linker facilitates the conformational folding of the aptamer, which is important for aptamer target binding The third moiety a synthetic diacyllipid

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45 with two stearic acids tail s, is conjugated at the 5 end as the membrane anchor. By its hydrophobic nature, the diacyllipid tail can firmly insert into the cell membrane with excellent efficiency. T he diacyllipid is conjugated with a phosphoramidite group so that it can be incorporated into nucleotides in the automated DNA synthesizer. The synthesis and characterization of the molecule were described elsewhere (200) Modification of Cel l Surface with Aptamers To illustrate lipid insertion, fluorescent dye molecule TMR (t etramethylrhodamine) or FITC (Fluorescein isothiocyanate) was conjugated to the 3 end of oligonucleotides for confocal microscopy or flow cytometry, respectively TMR ca n be excited at 543 nm with emission at 569 nm, showing a pseudo red color under confocal microscopy. FITC can be excited with 488 nM laser with emission at 521 nM, which can be detected by flow cytometry in channel 1. Confocal microscopy study of lipoDNA insertion After incubation with a LipoDNA TMR probe confocal microscopy images showed cells with red circle s (Fig 2 2A ), indicating that lipo DNA resided on the cell membrane. Although the entire circle was bright, there were some parts with slight ly h igher fluorescence intensity, possibly due to discrete partition of the probe into different cell membrane microdomains. While still in controversy (202) it is believed that cell membranes have lipid raft and nonraft domain s (203, 204) Lipid rafts are enriched in cholesterols, sphingomyelines and saturated phospholipids, which make them more rigid and tightly packed. The other parts of plasma membranes contain more unsaturated phospholipids and are thus more fluidic. Lipid rafts play an important part in signaling transductions (205208) Di fferent lipid additions have been found in membrane associated proteins and have helped recruitment of proteins into distinct

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46 domains. Several lipophilic oligonucleotides were reported to have higher affinity to different lipid domains (209211) Partition of lipoDNA into different cell membrane domains To explore whether our lipoDNA preferentially partitions into certain lipid domains we spotted lipid rafts with fluorescently labeled Cholera toxin B subunit (CT B). CT B is a toxin that has a high affinity to ganglioside GM1, which is concentrated in lipid rafts (212) We assumed that if L ipo DNA has a higher affinity to a certain type of domain, it will preferably insert into it After short time incubation with TMR labeled lipoDNA, cells were stai ned with FITC labeled C T B and examined for colocalization of TMR and FITC signals As previously observed, lipoDNA was found on cell membrane (red), but inhomogeneous ly distributed (Fig. 2 2B) The brighter parts overlapped well with lipid rafts (green) and yielded a yellow color. However, unlike some lipophilic nucleic acids that exclusiv ely cumulate into lipid rafts (211) this lipo DNA probe shows only a preference for rafts. Lon ger interaction time would diminish the differences by showing a more homogenous distribution on the membrane (Fig 2 2 A). One possible reason that the probe prefers lipid rafts to the nonrafts domain may be the saturated stearate chain, which resembles p almitate a fatty acid that plays a role in lipid rafts targeting (213) A previously reported lipid domain specific lipophilic nucleic acid probe also possessed two palmitic acid chains (211) LipoDNA insertion can be regulated We can control the cell surface aptamer density by both initial probe concentration and incubation time. Cells were incubated with FITC labeled lipoDNA and the fluor escence of each cell was monitored with flow cytometry. G enerally, higher initial concentration resulted in more aptamers anchored on the cell surface, and after

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47 concentration, the increment of probe concentration did not significantly improve insertion, indicating that a plateau had been reached (Fig. 2 3A). L ipid insertion could be observed within 15 min, and reached equilibrium after two hours (Fig 2 3B ). Quantification of membrane anchored lipoDNA To estimate the number of copies o f lipo DNA anchored per cell, we used a commercial ly available microspheres whose fluorescence intensities are in Molecules of Equivalent Soluble Fluorochrome (MESF) units. The microspheres are 79 m in diameter, which is comparable to cell size. Fluorescence intensity of a stained cell sample can be quantified in terms of MESF units by direct comparison of fluorescence intensity with microspheres (198) Cells incubated with different concentrations of L ipo DNA were washed and the mean fluorescent intensities (MFI) were compared with standards. As shown in Table 22, depending on different starting concentrations, there were 104105 copies of DNA aptamers per cell. The number was comparable to that of cell receptors such as IgE (214) and integrin (215) Therefore, it is reasonable to expect that aptamers anchor ed on the cell surface can subsequently act as artificial CAMs for cellcell interaction Retention time of membrane anchored lipoDNA One concern of this modification is the persistence of the a ptamer ligand on the cell surface. To investigate this, streptavidin aptamer that recognizes streptavidin (SA) was used. Cells were modified with Lipo SA aptamer first and fluorescently labeled SA was added at different intervals to quantify the amount of surface Lipo DNA. This indirect labeling eliminated fluorescent signals from internalized probes and possible fluorescent quenching during long term incubation. Figure 23C shows that, although the number of lipo DNA on the cell membrane decreased after 24 hours, there was still

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48 a significant number remaining The lost signals may be attributed to endocytosis of lipo DNA, nuclease degradation of DNA and cell division Only a smal l amount of l ipo DNA was left after 3 days, indicating that cell surface modification is temporary Aptamer D irected Cell Assembly Homotypic cell assembly To test whether cell membraneanchored aptamers retained their specific targeting properties we fir st employed a homotypic cell assembly. Suspensions of CEM cells were modified with L ipo Scg8 TMR during which time the cells were put on an orbital shaker and incubated at 300 rpm for 2 0 min. A fter washing to remove free aptamers, the cells were examined by confocal microscopy The Lipo Sgc8 TMR treated CEM cells spontaneously form ed large sequencespecific aggregates (Fig. 2 4 ). In control experiments where CEM cells were functionalized with a non target aptamer (LipoTD05 TMR) or a random sequence ( L ipo Lib TMR), no aggregates were observed (Fig. 2 4, upper panel ). Similar homotypic assemblies were observed in Ramos cells modified with LipoTD05 TMR (Fig. 2 4, lower panel ). The above experiments provided support for our hypothesis that membraneanchored aptamers can induce cellular adhesion in a defined target specific fashion. Heterotypic cell assembly Similar to the homotypic assembly, the heterotypic assembly was also formed in a target specific manner Ramos cells were first treated with Lipo Sgc8TMR an aptamer sequence for CEM cells or with LipoLib TMR, a control probe with no specificity After removing the free DNA, the modified Ramos cells ( fluorescent ) were mixed with unmodified CEM cells (nonfluorescent) Cell clusters were observed for the S gc8 modified cells but not for the Libmodified cells (Fig. 25A). Likewise, when CEM cells

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49 were modified with Ramos targeting aptamer, TD05, heterotypic cell clusters formed as well (Fig. 2 5A). T he generality of this assembly strategy was demonstrated by crosslinking CEM/Ramos with other cell lines such as Jurkat and K562, using similar procedures (Fig. 2 5B ). The architectures of assembled cells It has been demonstrated that the mixing ratio of cells modified with two complimentary DNA sequences yielded different final architectures of the assembled cells (198) To study whether we can control the architectures of aptamer directed cell assembl ies, Sgc8 modified Ramos were mixed with different ratios of unmodified CEM cells. When mixed at a 1:1 ratio, large cell aggregates were observed, within which a number of Ramos and CEM cells were stuck to each other (Fig. 2 6A ) When mixed at a 1:10 ratio, s mall er cell clusters with a defined architecture were observed (Fig. 25A). In each cell cluster, Sgc8 modified fluorescent Ramos cells were surrounded by nonfluorescent CEM cells forming a flower like shape (Fig. 26B). Cell Assem bly Was Controlled b y Membrane A nchored Aptamer To confirm that the cell assembly was induced by DNA aptamers anchored on cell surfaces, the aggregates were treat ed with the nuclease and protease that can destroy either DNA aptamers or target proteins. If the aggregation was caused by aptamer target protein interaction, nuclease and proteinase should be able to reverse the cell assemblies. Cell aggregates can be disrupted with DNase First, the endonuclease Deoxyribonuclease (DNase) I that nonspecifically cleaves single and d oublestranded DNA was used. CEM cells modified with L ipo Lib TMR or LipoTD05 TMR probes were mixed with 5 equivalents of Ramos cells (CellTracker

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50 Greenlabeled). Images obtained immediately after mixing showed that most CEM and Ramos cells remained apart with only a few small aggregates After 20 min incubation, as expected, almost all the TD05 aptamer modified CEM cells were surrounded by Ramos cells, resulting in a yellow ( overlapping of red and green color ) fluorescent signal, while library labeled CEM cells did not form any aggregates with Ramos cells (Fig. 2 7 ) T hese cell mixtures were then incubated with DNase I As shown in Fig. 27 after treatment, the red fluorescent signal on CEM cells disappeared, because the TMR dye molecule had been cleaved from the cell surface. Moreover, fewer aggregates were observed after treatment, indicating that the aggregation caused by aptamer target recognition had been disrupted by removal of DNA aptamers from the modified cell surfaces. It is noteworthy that some small aggregates remained, and t hat the interface between these two cells remained yellow. One possible reason was that the binding between cells was so tight that DNAse was prevented from interacting with the aptamers due to steric hindrance; consequently the aggregates remained intact. Cell aggregates can be disrupted with protease We also treated cellular aggrega tes with proteinase K, a protease known to digest the TD05 target ing protein IgM on Ramos cell surface (147) P roteinase K was added to Ramos cell clusters formed by membraneanchored TD05. After 20 min, the cell aggregates disassembled and a uniform dispersion of individual cells were observed ( Fig 2 8 ). The fluorescent intensity of each cell remained because the aptamers on the cell membrane were intact. The impact of quantity and stability of surface anchored aptamers Similar to natural cell cell interactions, multivalent binding is crucial for effective cellcell adhesion. W hen cells were treated with aptamers at a low concentr ation ( e.g.,

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51 5 0 nM), fewer lipoDNA were labeled on the cell surface, and cell aggregation was minimal. However, at higher aptamer concentrations (>500 n M), cell s aggrega te d within 10 minutes. Despite the high affinity between aptamers and their target proteins, this observation indicates that multiple receptor ligand interactions are still required for cell cell adhesion. This is different from DNA mediated assembly of nanocrystals, where only one copy of DNA is sufficient for assembly (189) At the same time, however, the minimum density of aptamers required for assembly varies according to many factors, including, f or example, length of DNA (200) cell type and aptamer affinit y Effective DNA modification on the cell surface also requires a firm membr ane anchor. In our experiments, when a monoacyllipid was used, under comparable conditions, low cell fluorescence was observed ( Fig. 2 9 ). This finding is consistent with a previous report, where bivalent cholesterol was shown to improve the anchor strengt h of DNA on lipid membranes (216) Potential Biomedical Applications of LipoDNA Modified Cells Our strategy of using membraneanchored aptamers as affi nity ligands mimics natural process es of cel l cell adhesion, which may be applied in cell based therapies, for example, tissue engineering and adoptive cell transfer. As a proof of principle study, we constructed two simplified models to demonstrate these applications. Aptamer d irected synthesis of m icrotissue mimics Tissue engineering is an emerging multidisciplinary field that employs engineering principles to build up functional 3D tissues (217) It is of great interest not only for building tissue repair material s, but also for constructing realistic in vitro tissue models. C ompa red with polymer scaffold assembly, plasma membrane anchored aptamers may act as artificial cell ligands and induce cellular adhesion upon spontaneous receptor -

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52 ligand binding. This may assist cellular implantation of freshly isolated or cult ured cells into damaged tissue (218) To assess this, microtissue mimics that consisted of multiple types of cell were synthesized. In this approach, Ramos cells served as both aptamer modified and target cells. Specifically, LipoTD05 TMR modified K562 cells were first incubated wi th LipoSgc8 FAM modified Ramos, and then unmodified CEM cells were added. As demonstrated in Fig. 2 10, three different types of cells were sequentially assembled into a 3 D microtissue, with defined connectivity. This result also suggested that the modif ication of the cell membrane with artificial CAMs did not interfere with other membrane bound receptor/ligand interactions. Overall, it implied that our strategy could be extended to engineer 3D aggregates with multiple cell types and form c omplex architectures with functional properties. Selective cell targeting in a cell mixture Taking advantage of aptamer specificity, membraneanchored aptamers may act as targeting ligands in controlling modified cells specificity. To test this, K562 cells modified wit h different aptamers were added to a mixed cell sample containing equal amounts of Ramos and CEM cells. We expected that, depending on the specificity of the anchored aptamers, K562 cells would be able to selectively target Ramos or CEM in the mixture. As shown in Fig. 2 11, K562 cells labeled with lipoTD05TMR (red) bound only to Ramos cells (CellTracker Greenlabeled) while lipo Sgc8 TMR labeled K562 cells recognized only CEM cells (nonfluorescent) N o mismatched cell aggregates were observed. The resul ts further proved that membraneanchored aptamers were highly selective in recognizing cell membrane proteins, even in the presence of nontarget

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53 proteins. This feature paved the way for applications in the more intricate physiological environment. Conclusion In conclusion, we have successfully engineered a new type of artificial cell adhesion molecule based on a membrane anchored aptamer We demonstrated the selective assembl y of multiple types of cells via aptamer protein recognition. This process is rap id and target specific and can be performed under typical cell culture conditions. DNA encoded cell capture and assembly based on DNA hybridization have been reported previously (196198) H owever, these methods r equire tedious procedures for cell surface modification and the mechanism of cellular recognition is limited to DNA hybridization. We emphasize the simplicity of our strategy, which, in turn, offers several advantages over previously reported methods. Fir st, we functionalize the cell surface by a simple and rapid non covalent modification. Second, cell recognition is based on specific aptamer protein interaction, eliminat ing the ne ed for target cell modification Third, the use of aptamers could greatly exp and the scope of DNA encoded cell assembly by taking advantage of the sequence diversity of aptamers. Thus, our method could be easily extended to other types of cells, including, for instance, human T cells, which could be modified by a similar method and utilized for therapeutic applications. We believe this strategy can be applied to the study of cell cell communication, bottom up tissue engineering and adoptive cell transfer

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54 Table 2 1. LipidDNA sequences Probe name Sequence Target cell line Lipo S gc8 5 Diacyllipid (PEG) 4 TTT TTT TAT CTA ACT GCT GCG CCG CCG GGA AAA TAC TGT ACG GTT AGA 3 CEM Lipo TD05 5 Diacyllipid (PEG) 4 AAC ACC GGG AGG ATA GTT CGG TGG CTG TTC AGG GTC TCC TCC CGG TGA 3 Ramos Lipo SA 5 Diacyllipid (PEG) 4 TT TT T TTA TTG AC C GCT GTG TGA CGC AAC ACT CAA T 3 Streptavidin Lipo Lib 5 Diacyllipid (PEG) 4 NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN 3 Control sequence, no specificity Table 2 2. Quantification of lipoDNA on cell surface Initial [DNA] (nM) MFI # Molecules per cell 1000 15899.98 708,547 500 4976.64 217,445 200 100 0 2169.81 739.06 0 93,560 31,418 0

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55 Figure 2 1. Demonstration of cells modified with artificial cell adhesion molecules and the chemical structure of LipoDNA molecule A B Figure 2 2. Confocal microsco pic image s of L ipo DNA TMR treated cells. A) Red fluorescent probes were found mainly on the cell surface after two hours incubation. Scale bar: 10 m. B) Confocal image of lipid raft (gre en), LipoDNA TMR (red) and their colocalization on cell membrane. Scale bar: 10 m.

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56 A B C Figure 2 3. Flow cytometric analysis of LipoDNA modification. A) Modification can be controlled with initial probe concentration. B) Modification can be contr olled with incubation time. C) Retention time of cell surfaceanchored Lipo DNA.

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57 Figure 2 4. Aptamer mediated homotypic cell assembly CEM and Ramos cells were divided into three groups and modified with LipoSgc8 TMR, LipoTD05 TMR and LipoLib TMR p robes. Spontaneous self aggregation of CEM or Ramos cells was observed with LipoSgc8 TMR and LipoTD05 TMR modified groups, respectively. Cells modified with mismatched aptamers or control sequences showed no assembly. Scale bar: 100 m. Figure 2 5. Ap tamer mediated heterotypic cell assembly A) CEM and Ramos cells were modified with LipoAptamer TMR or Lipo Lib TMR probes first and mixed with unmodified Ramos or CEM cells, respectively. Aggregated cells were found only in the aptamer modified groups. S cale bar: 100 m. B) H eterotypic assemblies were observed with various types of cells K562 or Jurkat cells were modified with aptamers or Lib and mixed with Ramos and CEM cells. Sequenc e specific heterotypic assemblies betw een modified cells (K562 and Jur kat) and target cells (Ramos and CEM) were observed. Scale bar: 100 m

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58 A B

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59 A B Figure 2 6 Different architectures of assembled cells. A) 1:1 mixture of aptamer modified cells and target cells yielded bulky aggregates with little control of the final architecture. Scale bar: 10 0 m B) Discrete cell clusters formed when aptamer modified cells and target cells were mixed at 1:10 ratio. Scale bar: 1 0 m

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60 Figure 2 7 Cell assembly can be disrupted by DNase. First, CEM cells were modified with L ipo TD05 TMR or LipoLib TMR, resulting in a red fluorescence. Ramos cells were labeled with a green fluorescent cellTracker dye and mixed with modified CEM (red) cells. Ramos (green) cells formed aggregates with TD05 modified CE M (red) cells which partially dissembled after treatment with DNase I (upper row). Mixture of LipoLib TMR modified CEM (red) cells and Ramos (green) cells cells remained apart before and after treatment (lower row). Most CEM cells lost red fluorescence af ter DNase I treatment. Scale bar: 50 m. Figure 2 8 Cell assembly can be disrupted by protease. H omotypic aggregates of Ramos cells (left) disassembled after treat ment with proteinase K at 37oC Scale bar: 100 m.

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61 A B Figure 2 9 Diacyllipid tail is required for a firm insertion. A ) Fluorescence microscopy image of CEM cells modified with monoacyllipid DNA (monolipidlib TMR). B ) Fluorescence microscopy image of CEM cells modified with diacyllipid DNA (dilipid lib TMR).

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62 Figure 2 10. Schematic diag ram of assembly of multiple types of cells and confocal micrograph of microtissue. Scale bar: 100 m Figure 2 11. Schematic demonstration of selective cell targeting and confocal micrographs of cell aggregates. Ramos cells stained with a green fluoresc ent dye were mixed with unlabeled CEM cells at 1:1 ratio. Then K562 cells modified with LipoTD05 TMR or LipoSgc8 TMR were added to the cell mixture K562 cells selectively targeted Ramos or CEM cells depending on the type of aptamer. Scale bar: 50 m.

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63 C HAPTER 3 DNA APTAMER MEDIATED CELL TARGETING AND CANCER THERAPY Introduct ory Remarks Cellbased therapy has shown considerable potential in treating cancer. Certain types of cells, such as killer lymphocytes that naturally attack cancer cells, are being st udied for direct cancer treatment (16, 219, 220) Other cells, like mesenchymal stem cells (MSC), can be genetically engineered to produce therapeutics in situ after delivery (221223) However, one important issue in cell based therapy is the targeted delivery of cells in vivo (224) Targeted delivery not only improves therapeutic efficacy, but also minimizes side effects. In this regard, several approaches have been studied. A straightforward method involves physical delivery of cells to the site of interest, with the help of proper medical devices (225, 226) Other targeting strategies, including the use of native cell homing machinery (227, 228) and expression or coating of cells with targeting ligands, are under intensive study. Carbohydrates (229, 230) short peptides (231, 232) and extracellular domains of cell membrane receptors (40, 233) have all been used as targeting moieties. In recent years, oligonucleotidebased probes, termed aptamers, have been developed with the specificity and aff inity required for diagnostic (234) and therapeutic applications (60) Similar to antibodies, aptamers can specifically recognize a wide range of targets that vary from small molecules to cancer cells (44 45, 53, 101, 235, 236) However, they have additional properties that make them more attractive than antibodies. For instance, aptamers are usually smaller, resulting in better tissue penetration ability. Furthermore, no immunogenic reactions have thus far been reported for any in vivo experiment with aptamers. Finally, since nucleic acids can be

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64 synthesized chemically, aptamers can be readily adapted for modifications to meet different needs. The C hapter 2 described development of a lipophilic oligonuc leotide probe to modify cell membrane with DNA aptamers. We demonstrated that plasma membraneanchored aptamers retained specificity and acted as targeting ligands to interact with membrane proteins. In this chapter, we explore the potential of this probe in adoptive cell therapy (Fig. 3 1) Immune effector cells modified by the probe showed improved affinity to cancer cells while remaining cytotoxic to target cancer cells. A n ovel cytotoxicity ass ay was assembled to assess the potential of this probe in cellbased immunotherapy of cancer. Materials and Methods General Materials All Lipo DNA molecules were synthesized using DNA synthesizer and purified with HPLC as previously in Chapter 2. CellTrace Far Red DDAO SE CellTrackerTM Green CMFDA, Vybrant Apopto sis Kit #2 and CellTraceTM CFSE were purchased from Invitrogen. CellTiter 96 AQueous NonRadioactive Cell Proliferation Assay was purchased from Promega. Interleukin (IL) 2, 7 and 15 were purchased from PeproTech. RosetteSep Human NK Cell Enrichment Cock tail was purchased from Stemcell Technologies. FicollPaque PREMIUM was purchased from General Electric. Rec blood cell (RBC) lysis buffer was made from deionized H2O containing 0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, with the pH adjusted to 7.3. Ionom ycin and 12 myristate 13 acetate (PMA) were kindly provided by Dr. Lung Ji Chang. Purified Mouse Anti Human Perforin was from BD Pharmingen

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65 General Cell Culture Conditions CCRF CEM and Ramos cells were cultured in complete RPMI 1640 medium ( Sigma ) suppl emented with 10% FBS (heat inactivated, GIBCO) and 100 IU/mL K562 cells were obtained from ATCC and were cultured in complete Iscove's modified Eagle's medium (IMEM) supplemented with 10% FBS and 100 IU/mL ptomycin. Natural Killer cells isolated from concentrated fresh blood sample (Life South) were maintained in complete RPMI with 10% FBS and 20U/mL IL2. Cytomegalovirus CD8+ cytotoxic T lymphocytes (CTL) were generously provided by Prof. Lung Ji Chang and cultured in complete RPMI with 10% FBS and 20U/mL IL 2, 5ng/mL IL 7 and 20ng/mL IL15. Quantitative Analysis of Cell Aggregates First, CEM cells were stained with CellTrace Far Red DDAO SE and Ramos cells were stained with CellTracker Green CMFDA accord ing to the manufacturer's protocol After washing, CEM cells were incubated with various concentrations of LipoLib or Lipo TD05 (without any dye molecules) washed, and then incubated with diff erent quantities of Ramos cells and placed in a orbital shaker for 20 min. T he fluorescent signals from channels 1 (green) and 4 (far red) were determined by flow cytometry (Accuri C6 flow cytometer). The thresholds of channels 1 and 4 were set by comparing the fluorescent signals generated from unstained CEM and Ram os cells. The percentage of aggregation was calculated as 100 times the ratio of the double positive (green and far red) population to the total CEM cell (far red) population (Fig. 3 2A). Each set of samples was analyzed in triplicate.

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66 Annexin V/Propidium Iodide Double Staining Assay CTL from normal culture condition or modified with LipoDNA were was hed in cold PBS twice and resuspended in 1 annexinbinding buffer at 1106 cells/mL. Each100 L cell suspension was double stained with 5 L Alexa Fluor 488 a nnexin V and 1 L of the 100 g/mL PI working solution at room temperature for 15 min. The staining was stopped by adding 400 L of cold 1 annexinbinding buffer and kept on ice. The stained cells were analyzed by flow cytometry, using the fluorescence em ission in channel 1 and 3 for annexin V and PI signals, respectively. C FSE Dilution Assay CTL with or without LipoDNA modification were incubated with 1 M CFSE in PBS buffer at 37C for 15 min. After washing, cells were cultured 3 days and the CTL fluor escence was analyzed. MTS Cell Viability Assay The cytotoxicity of lipid Lib probe was tested by standard MTS ((3(4,5 dimethylthiazol 2 yl) 5 (3 carboxymethoxyphenyl) 2 (4 sulfophenyl) 2H tetrazolium) cell proliferation assay (Promega). K562, CEM Ramos a nd CTL were incubated with Lipo Lib, and seeded into a 96well cell culture plate. After 2 days of incubation, cell culture medium was replaced by MTS containing medium. After 4 hours of incubation, absorbance at 490 nm from each sample was read by a micro plate spectrophotometer. Control samples were cells without any modification. Natural Killer Cell Isolation RosetteSep Human NK Cell Enrichment Cocktail was added to human whole blood at 50 L/mL, mixed and incubated for 20 min at room temperature. The mi xture was diluted with an equal volume of PBS containing 2% FBS, and was gently laid on top

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67 of ficoll. The entire solution was centrifuged for 20 min at 1200 g at room temperature with the brake off. Cells between the plasma and the ficoll were collected and lysed with RBC lysis buffer for 10 min at room temperature. Cells were then washed twice and maintained in RPMI with supplements for future use. Activation of CTL CTL (1.2 06 cells/mL) were incubated in RPMI with io nomycin (1 g/m L ) and PMA (25 ng/m L ) at 37C for a certain period of time, washed and resuspended in PBS. Cells were then stained with T cell markers and a control isotype. Aptamer assisted Immune Cell K illing Assay K562 or Ramos cells were washed with PBS buffer and labeled with 1 M carb oxyfluorescein succinimidyl ester (CFSE) as suggested by the manufacturer, and then aliquotted to a 96well microtiter plate at 1104 cells/well. Immune effector cells were added to each well at the desired effector: target cell ( E:T ) ratio. The final reaction volume was adjusted to 200 L. The plate was kept in a humidified atmosphere of 5% CO2 and 37 C for 26 hours. Before flow cytometry (Accuri C6 flow cytometer) analysis, propidium iodide (PI) was added to each sample and incubated at RT in the dark for 30 min to label dead cells. The target cell death was calculated as the number of CFSE and PI positive cells over the total number of CFSE positive cells. Results and Discussion Quantitative Study of Aptamer Mediated Cell Targeting In C hapter 2 we ex amined cell aggregation using microscopy. Here we describe a method to quantify cell aggregation with flow cytometry. We also discuss different factors that may impact binding efficiency.

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68 Quantification of cell targeting efficiency with flow cytometric bas ed assay Aptamer modified cells (CEM) and target cells (Ramos) were separated into different fluorescent channels by prestaining with green and far red fluorescent dyes, respectively. The binding of CEM to Ramos cells generated a new cell population posses sing both colors (Fig 3 2A) After staining, CEM cells were incubated with various concentrations of lipoLib or lipoTD05 probes, and different molar ratios of Ramos cells were subsequent ly added. As presented in Fig. 3 2B 40 95% CEM cells formed aggrega tes with Ramos cells when modified with TD05, compared with less than 5% aggregate formation in DNA library modified CEM cells. The percentage of aggregation can be controlled by the aptamer concentration and the modifiedto target cell ratio. A n increase significantly increase the aggregation percentage. F the increase is small because 1M modification was sufficient to cause 80% 90% cell aggregation, and the number of lipoDNA probes on the cell surface were close to each other (Fig. 23A). On the other hand, since an increased number of target cells can increase the chance of interaction, more aggregates formed when an excess of either modified cell (CEM ) or targ et cell (Ramos) was used. Cell targ eting at physiological conditions To study how other factors would affect targeting, we assessed targeting under different conditions. In the previous assay, we initiated binding at 4C in binding buffer to favor the aptamer conformation. Also, cells were placed in an orbital shaker to facilitate their encounter. If we intended to use the probe to guide immune cell trafficking in the human body, it should be able to bind at physiological conditions. Previously Shi et al

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69 demonstrated that a Cy5 dye labeled TD05 aptamer recognized a Ramos xenograft in nude mice (237) indicating that TD05 aptamer is stable and specific in vivo We first performed the binding assay at 37C in binding buffer without shaking, and compared the targeting efficiency at 4C in binding buffer with shaking. As shown in Fig. 3 3A, although the efficiency is slightly lower than at 4C, it was still over 70%, with a slightly higher background binding from control groups. This result indicated that membrane anchored aptamers were able to fold properly at 37C and form tight binding to targets. We t hen spiked aptamer modified CEM cells and target Ramos cells into whole blood. In this set of experiments, we intended to test the binding in the presence of an excess number of background proteins and cells. After staining and modifications, CEM and Ramos cells were resuspended in 200 L human whole blood (with heparin) instead of binding buffer, and put into an orbital shaker at 37C for 25 min. Cell targeting efficiency was analyzed as previously described and presented in Fig. 33B. The targeting percentage in whole blood dropped about 15% compared with the percentage in binding buffer, giving about 65% Ramos cells targeted by TD05modified CEM cells. This result suggested that proteins and cells presented in human blood may interfere with aptamer mediat ed cell targeting to some degree, but they did not completely block the aptamer function. Therefore, it is reasonable to assume that this method has clinical potential. Cytotoxicity of Lipo DNA Since delivered cells are inten ded for therapy, modification of the cell surface should not affect cellular functions. The components of the probe, including oligonucleotides, PEG polymers and diacyl lipids, are not known to be cytotoxic. To test

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70 whether the insertion had any toxic effect on cells, we monitored the probes short term and long term effect on cells. The short term effect was evaluated by loss of cell membrane integrity while long term effect was assessed by proliferation rate and cell viability LipoDNA insertion did not compromise cell membrane inte grity Cells normally die via apoptosis or necrosis. Apoptosis is also known as programmed cell death which involves a series of naturally occurring biochemical process es Apoptosis can be detected by Annexin V based assay. Annexin V is a cellular protein that has a high affi nity for phosphatidylserine (PS), a phospholipid that is normally found on the cytosolic membrane. When cells undergo apoptosis, PS is relocated to the extracellular surface, and hence can be probed by Annexin V. Necrosis usually caused by external factors such as pathogen infection and toxins. Although apoptotic and necrosic cells die from different processes, the common feature is loss of membrane integrity. This can be detected using dyes hav ing strong affinity for DNA such as propi dium iodide (PI) which is a fluorescent molecule that can intercalate into DNA. PI cannot pass an intact cell membrane and thus cannot stain viable cells. Dying cells that have permeable membranes can be stained by PI and detected by flow cytometry or mic roscopy. Hence, if lipid insertion compromises cell membrane integrity, cells will be stained by PI. After incubat ion with lipo DNA, CTL were stained with Annexin V and PI and examined by flow cytometry (Fig. 3 4A) Statistical analysis showed that minimal cell death occured ( Fig. 3 4B ), indicating that lipoDNA insertion does not compromise cell membrane integrity.

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71 LipoDNA insertion did not affect the proliferation pattern of CTL We next assessed whether lipoDNA modification would affect CTL proliferat ion. Activated CTL can self proliferate in the presence of cytokines and is one of the key cellular functions. In this study, we first stained cells with carboxyfluorescein succinimidyl ester (CFSE), a fluorescent dye that is retained within cells for a pr olonged time. Since CFSE is not transferable between adjacent cells but can be passed from mother cells to daughter cells, it is used to label lymphocytes and track their migration as well as proliferation, both in vitro and in vivo (238) After 3 days, CFSE signal from CTL was monitored using flow cytomet ry (Fig. 3 4C) CFSE was diluted by each cell division and cell populations with different CFSE intensity were detected. The CFSE dilution followed the same pattern in unmodified and modified CTL, indicating that they share the same proliferative response. LipoDNA did not affect cell viability The number of viable cells in proliferat ion was quantified by standard MTS (3(4,5 dimethylthiazol 2 yl) 5 (3 carboxymethoxyphenyl) 2 (4 sulfophenyl) 2H tetrazolium) assay. In the presence of phenazine methosulfate (PMS), MTS can be reduced in viable cells either enzymatically or through direct reaction with NADH or NADPH to form a water soluble formazan product. Because dead cells rapidly lose the ability to reduce tetrazolium products t he production of the purple formazan product is proportional to the number of viable cells in culture and can be determined colormetrically. No modification after 2 days (Fig 3 4D ). Taken together, t hese results suggested that the probe itself is not cytotoxic to either cancer cells or immune effector cells

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72 Natural Killer Cell Based Assays To investigate the cellular effect and targeting ability of the lipid DNA probe on immune effector cells, we started with natural killer (NK) cells, which are killer lymphocytes occurring in innate immunity NK cells provide rapid response to infected and transformed cells. They recognize cells independent of major histocompatibility complex (MHC), and they kill cells that do not express MHC by releasing granule proteins (239) K562, a type of chronic myelogenous leukemia cell line, is often used as a target for in vitro NK cell assays because they lack MHC class I expression (240) The aptamer used in this study was KK1B10, which specifically recognizes K562 cells (94) NK cell isolation and surface modification NK cells were isolated from human whole blood with RosetteSep Human NK Cell Enrichment Cocktail and maintained in cell culture medium with IL2. We first confirmed that KK1B10 aptamers did not recognize NK cells (Fig. 35A) to rule out possible cross activity between aptamers and NK cells. To verify cell surface modification of immune effector cells, diacyl lipid KK1B10 conjugates with fluorophores were synthesized as previously described. By monitoring the fluorescent intensity change of NK cells, we confirmed that lipophilic DNA has high affinity to NK cells, similar to previous observations with cancer cells ( Fig. 3 5B) NKK562 bindi ng a ssay Next, we tested the targeting efficiency of NK cells to K562 cells using the previously described flow cytometric assay. As presented in Fig. 35C unmodified and library modified NK groups showed a higher background binding (about 40%) compared to previously demonstrated CEM Ramos binding assays. Aptamer modified NK cells

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73 targeted 60% K562 cells, which improved NK cell targeting efficiency by 50%. The high background binding was caused by the spontaneous interaction because NK cells and K562 Moreover, unmodified NK cells targeted the same number of K562 cells as Libmodified NK cells, indicating that membraneanchored DNA sequences, at least at numbers used in this study, do not interfere with natural cell cell adhesion. NKK562 k illing a ssay To determine if NK cells modified with the KK1B10 aptamer would impact the killing of K562 cells we set up a NK cytolytic assay, as previously reported (241) Compared with unmodified NK cel ls, control library modified NK cells showed the same killing effect on K562 cells (around 21%, shown in Fig 3 5D) indicating that lipoDNA would not interfere with the cell mediated cytotoxicity. Moreover, NK cells modified with KK1B10 aptamer killed about 30% of K562 cells, significantly more than the percentage killed by unmodified NK cells It is worth mentioning that the incremental killing efficiency correlated well with targeting efficiency The virtue of adding aptamers as extra targeting ligands was revealed in the presence of excess nontarget cells. In this experiment K562 cells were mixed with 4 equivalent s of nontarget Ramos cells first and then incubated with NK cells. Within the same amount of time, NK cells killed fewer K562 cells, but the decrease was considerably smaller in the aptamer modified group, suggesting that aptamer modification can improve cell targeting without affecting immune effector function (Fig 3 5D ). Cytotoxic T Lymphocyte Based Assay s The tests with NK cells suggested t he feasibility of a novel T cell killing model, in which the specific cytotoxicity is controlled by membraneanchored aptamers. Cytotoxic T lymphocyte (CTL) is a type of CD8+ T cells in the adaptive immune system.

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74 Autologous and allogeneic CTLs are current ly in clinical trails in adoptive T cell therapy of cancer (31 39) In this study, a primary immortalized cytomegalovirus (CMV) specific CD8+ cytotoxic T lymphocyte (CTL) clone (HLA A0201) was used as the immune ef fector cell. The target cells were Ramos cells, a B cell Burkitts lymphoma cell line and K562 cells Activation of CTL with small molecules The activation of CTL requires T cell receptor (TCR) specific binding to antigens that presented by MHC class 1 ( MHC I) on target cell surfaces. The specific interaction between TCR and antigen:MHC I leads to the activation of protein tyrosine kinases (PTK) via an immunoreceptor tyrosinebased activation motif (ITAM). PTK activation triggers downstream effectors, inc luding phospholipase C, which mediates the conversion of phophatidylinositol 4,5phosphate (PIP2) into inositol 1,4,5triphosphate (IP3) and diacylglycerol (DAG), resulting in calcium flux and activation of protein kinase C (PKC). The increase of Ca2+ conc entration leads to activation of calcineurin, which regulates IL2 gene transcription, a key factor in CTL activation. The activation of PKC result s in perforin/granzyme release from CTL, cytotoxins that kill target cells (242) One way to bypass the TCR activation pathway is to use small molecule drugs that directly activate downstream effectors. The activation of PKC can be mimi cked using 12myristate 13acetate (PMA) and ionomycin, which cause intracellular Ca2+ increase (243) Because both target cells, Ramos and K562, do not express CMV specific peptide:MHC I on their cell membranes, we used PMA/ionomycin to activate CTL. First, we m onitored the activation of CTL by the expression of CD69, an early T cell activation marker (244) CTL were incubated with PMA and ionomycin for 0, 2, 4, and 6 hours and stained with anti CD4, antiCD8, antiCD69 antibodies and isotype

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75 control antibodies (Fig. 36). As expected, this CTL clone is CD4and CD8+. However, the gradually declining CD8 signals during activation were unexpected. From the light scatter plot, we notice that the morphology of CTL changed dramatically during activation, which may count for the loss of CD8 signals. Because the forward and side scattering reflects a cells surface, size, shape, nucleus and granularity, and the activation involve exocytosis of proteins, it is reasonable to see these changes. Another possible reason is the death of CTL caused by accumulated cytotoxins during activation. The histogram of CD69 suggests that after 2 hours activation, an elevated amount of CD69 was detected on CTL membranes yet the increment was small and prolonged activation time did not affect its expression. It is worth noting that before activation, CTL was stained CD69+, which explains the small change of its expression after activation, but also indicates that this im mortalized CTL clone is constantly active. In fact, we used both PMA/ionomycin activated and untreated CTL in later cytotoxic assays and found that, although activated CTL were more effective, untreated CTL were able to kill target cells (data not presented). Taking all these observations into consideration, we activated CTL with PMA/ionomycin for 2 hours before use. CTL Ramos binding a ssay Prior to binding assay we determined that TD05 aptamer had no specificity toward CTL membrane proteins, but lipoTD05 was able to modify CTLs surface (Fig. 3 7 A & B). The binding assay (Fig. 3 7C) showed that CTL did not recognize Ramos cells naturally, unless they were modified with the targeting aptamers. Within 2 0 minutes, 80% of Ramos cells were targeted by CTL in the cell culture. To test the targeting ef ficiency under more biologically relevant conditions Ramos cells were spiked into peripheral blood mononuclear cells (PBMC) and then incubated with the CTL. The

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76 targeting efficiency was slightly influenced by the spiked PBMC, resulting in targeting of 70% Ramos cells. A similar effect was observed in CEM Ramos binding assays presented previously. Nevertheless, TD05 modification enables CTL targeting of Ramos, an essential step in cell based therapies. CTL Ramos k illing a ssay The perforin/granzyme cell death pathway requires direct contact between effector and target cells (245) Therefore, without a CMV specific peptide presented by MHC I on Ramos cell surfaces, the CMV specific CTL cannot recognize Ramos cells, resulting in only background cytotoxic effect towards Ramos. Assisted by surfaceanchored TD05 aptamers, however, the modified CTL have increased affinity toward target Ramos cells, thereby facilitating and prolonging interactions between effector and target cells. Thus, enhanced killing of Ramos cells should be observed. We designed the CTLRamos cell killing assay in a manner similar to the previously described NK K562 cell killing assay Fig. 3 8A shows representative dot plots from one set of C TL Ramos killing experiments and statistical analysis of 3 independent experiments is presented in Fig. 3 8B. In general, about 15% and 30% of Ramos cells were dead in the aptamer modified CTL group in 3 hours and 6 hours, respectively, while < 5% dead Ram os cells were found in Libmodified or unmodified CTL groups. To confirm that Ramos cell death was caused by CTL, an anti perforin antibody was added to the aptamer modified group. Anti perforin antibody blocks the perforin/granzyme pathway and hence prev ents cell death by this pathway (246) After adding, t he percentage of dead Ramos cells was greatly reduced from 15% to 5%. This result validated that the death of Ramos was, at least largely, cause by perforin released from CTL.

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77 CTL K562 binding and k illing a ssay With the same principle, we tested the effect of CTL on K562 cells to make sure that the cytotoxicity of C TL is not Ramos cell specific. As mentioned earlier, K562 cells do not express MHC I on their cell surfaces, and thus no CMV specific peptide:MHC I complex presented. The binding assay revealed that over 65% K562 cells were targeted by KK1B10 aptamer modi fied CTLs with less than 5% in control groups (Fig 39A). Compared with CTL Ramos targeting assay, the nonspecific binding from unmodified CTL and Lib modified CTL was slightly higher but not significant; aptamer mediated targeting of K562 cells was less effective by showing a 15% drop. In the previous NK K562 binding assay, KK1B10modified NK cells targeted about 60% K562 cells (Fig. 35C), and this efficiency is comparable with that of CTL based K562 targeting. Since the targeting is controlled by membra ne anchored aptamers, we compared two aptamers, TD05 and KK1B10, which are used in two binding assays. Previous studies reported that the dissociation constant of TD05 ( KdTD05: 74 nM ) was lower than that of KK1B10 ( KdKK1B10: 256 nM ). The binding profiles of these two aptamers (Fig. 39C) indicate that there were more TD05 receptors per Ramos cell, because the mean fluorescent intensity was higher. These two factors, higher binding affinity and receptor density, may contribute to better targeting efficiency. The killing of K562 cells was also less effective than Ramos. Only 10% K562 cells were stained PI positive in three hours, yet the difference from control groups was significant. Longer incubation time (6 hours) induced more dead K562 cells in both control groups and the experimental group (Fig. 39B). The reason K562 cells were less sensitive to CTLmediated killing (compared with NK mediated K562 killing and CTL-

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78 mediated Ramos killing) is not clear, but we suspect that the targeting efficiency may be a mi nor factor. Conclusion In conclusion, we demonstrated that the lipoaptamer probe can modify immune effector cells and improve or redirect their specificity. T he noncovalent modification is simple, yet effective, with no cytotoxic effect on modified cells. Using a CMV specific CD8+ CTL clone as immune effector cells we demonstrated, for the first time, a redirected T cell killing in which the specificity is controlled by aptamers. Immune effector cells modified with aptamers can recognize leukemia cells v ia MHC nonrestricted structures, providing an alternative way to target tumor. Further study will focus on in vivo cell trafficking. We believe that the diversity of aptamer targets and the facile modification make this strategy attractive in cell based delivery and therapy

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79 Figure 31. Schematic representation of targeting cancer cells ( blu e) with aptamer modified immune cells (red). After incubating with lipoaptamer probes (shown in expansion), immune cells recognize cancer cells in the cell mixtu re, and kill cancer cells.

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80 A B Figure 32 Quantification of cell targeting efficiency A) Representative examples of a dot plot analysis from one set of CEM Ramos binding assay s. B) Aggregation percentage of CEM cells in different sample sets. CE M to Ramos cell ratios are represented by blue (1:1), red (1:5) and green (1:10) bars Values are means

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81 A B Figure 33 Cell targeting efficiency in different conditions A) CEM Ramos binding at different temperatures. Values are means with S D (n=3). B) CEM Ramos binding in different buffer systems. Values are means with S D (n=3)

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82 A B C D Figure 34 Cytotoxicity of the lipo DNA probe. A ) Representative examples of a dot plot analysis from Annexin V and PI sta ining of unmodified and lipoDNA modified CTL. The lower left square represen ts a healthy cell population. B) Percentage of viable CTL before and after lipDNA insertion analyzed by Annexin V/PI doublestaining assay. Values are means with S D (n=3). C) CFS E dilution assay. D ) Cell viability assay. Values are means with S D (n=3).

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83 A B C D Figure 35 Aptamer assisted NK cell targeting and killing of K562 cells A) KK1B10 aptamer does not recognize NK cells. B) Modification of NK cell with LipoKK1B10 aptamer. C ) NK K562 cell binding assay. D ) NK K562 cell killing assay. Values are means with S D (n=3). The single asterisk indicates a significant difference between aptamer modified and unmodified or Libmodified groups determined by the onet ailed t test at P<0.01.

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84 A B Figure 36 Activation of CTL with PMA/ionomycin. A) Light scattering dot plots of CTL before and after activation. B) Flow cytometric analysis of CTL stained with monoclonal antibodies against CD4, CD8 and CD69, respect ively.

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85 A B C Figure 37 CTL Ramos cell binding assay. A) TD05 aptamer does not recognize CTL. B) Modification of CTL cell with LipoTD05 aptamer. C) CTLRamos binding assay. Values are means with S D (n=3). The single asterisk indicates a si gnificant difference between aptamer modified and unmodified or Libmodified groups determined by the onetailed t test at P<0.01

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86 Figure 38 CTL Ramos cell killing assay. A) Representative examples of a dot plot analysis from one set of CTL Ramos k illing assay s. CFSE and PI positive cells were dead Ramos cells. A01: Ramos only; B01: unmodified CTL and Ramos; C01: lipoLib modified CTL and Ramos; D01: lipoTD05 modified CTL and Ramos. The percentage of dead Ramos was calculated as the number of CFSE and PI positive cells over the number of CFSE positive cells. B ) CTL Ramos cell killing assay. Values are means with S D (n=3). The single asterisk indicates a significant difference between aptamer modified and unmodified or Libmodified groups determine d by the onetailed t test at P<0.01. The double asterisks indicate a significant difference between aptamer modified and anti Perforin treated groups determined by the onetailed t test at P<0.01. A

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87 B

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88 A B C Figure 39 CTL K562 cell binding and killing assay. A) CTL K562 binding assay. B ) CTL K562 cell killing assay. Values are means with S D (n=3). The single asterisk indicates a significant difference between aptamer modified and unmodified or Libmodified groups determined by the onetailed t test at P<0.01. C) Binding profile of TD05 and KK1B10 aptamers to their target cell lines.

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89 CHAPTER 4 ENGINGEERING AN APTAMER BASED DUAL FUNCTIONAL MOLECULAR PROBE FOR CANCER THERAPY Introduct ory Remarks Mic roRNA (miRNA) is a type of non coding endogenous RNA that post transcriptionally regulates one or several messenger RNA (m RNA) Upon binding to untranslated region of mRNA sequences, miRNA silences the translation of mRNA and thus downregulates encoded protein target (247) Similar to small interfering RNA (siRNA), m iRNAs are small (usually 22 nucleotides long), and they belong to RNA interference ( RNAi) machine ry. Since its discovery in 1990s (248) there is increasing evidence that miRNAs play crucial roles in cell proliferation, differentiation and apoptosis (249, 250) Recently, several miRNAs have been found misregulated in various cancer cells causing prolonged cell survival elevated cell proliferation, metastasis and invasion (251, 252) MiRNA 2 21 for instance, has been reported upregulated in glioblastoma (253) hepatocellular carcinoma (254) breast cancer (255) pancreatic cancer (256) bladder cancer (257) prostate cancer (258) and lung cancer (259) and it negatively regulates cell cycle by inhibiting p27K ip1 protein expression. Studies have showed that antisense strands that are perfectly complementary to target miRNA, can inhibit miRNA function(s) (260) These miRNA antagonists, named antagomirs, are showing great promise in gene therapy of cancer (261) One important issue in gene therapy is targeted and efficient intracellular delivery of therapeutic nucleotides. Oligo nucleotide probes, termed aptamers, are intriguing targeting moieties Fir st, aptamers are small in size with good tissue penetrating properties (60) Second, compared with antibodies and peptides, aptamers are not immunogenic (61) Third, some aptamers can target cancer cells specifically and, more

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90 importantly, be internalized by cancer cells (139) This makes intracellular trafficking simple. Fo u rth, nucleotides can be easily synthesized and modified. For example, 2 O methyl modified nucleotides are resistant to riboand deoxyri bonucleases (66) AS1411 is an aptamer that binds and inhibits various types of cancer cell proliferation (262) The AS1411 target has been identified as nucleolin protein, which shuttle s between the cell membrane, cytosol and nucleolus, and is involved in ribosome biogenesis (263) It has been reported that AS1411 interferes with NF way and destabilizes Bcl mRNAs. (264, 265) Another study showed that AS1411 enters cancer cells but not normal cells via macropinocytosis and initiates cell death (266) Alt hough the exact mechanism is under debate, it is clear that AS1411 can specifically target and enter cancer cells, and can inhibit cell proliferation at micromolar concentrations. AS1411 is being evaluated in phase II clinical trials for treatment of relapsed acute myeloid leukemia (104) Previously, DNA and RNA aptamers have been conjugated with siRNAs and have successfully silence d target proteins (77, 78) Here, we report the conjugation of a therapeutic aptamer AS1411, with an antag om ir that inhibit s oncogenic miRNA221. The aptamer/antagomir conjugate was developed to target different types of cancer cells a nd inhibit their proliferation We first demonstrate d the function of this probe in a nonsmall cell lung cancer cell line (A549 cell line) This cell line is responsive to AS1411 but the IC50 is around 6 M (267) A relatively high expression level of miRNA221 has been reported to cause a specific drug resistance in A549 cells (2 59) By incubating A549 cells with the AS1411Antagmir221 conjugates, we expect that antagomir221 can be codelivered into the cytoplasm of the cell, find its miRNA target

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91 and inhibit its function (Fig. 41). We monitored the intracellular level of miRNA221 and its target protein, p27K ip1, to prove the delivery of the probe, and the drug sensitivity on A549 cells was tested to evaluate the efficacy of the probe. Two other types of cancer cells, glioblastoma (U87 MG cell line ) and breast cancer (MDA MB 21) were also used to test the anti proliferation function of the probe. Materials and Methods Genera l Materials All the DNA synthesis reagents were purchased from ChemGenes. Cell culture medi um were purchased from ATCC fetal bovine serum (FBS) (heat inact i vated) was purchased from GIBCO, and penicillin streptomycin was purchased from Cellgro. Alexa Fluor 633 conjugated transferrin from human serum was purchased from Invitrogen. RNAi Human/Mouse Starter Kit (HiPerFect transfection reagent) and BCA Protein A ssay were purchased from Qiagen. Antibodies for western blot were purchased from Cell Signaling Technology. Nonenzymatic cell dissociation solution was purchased from Cellgro. mirVana miRNA Isolation Kit was purchased from Life Technologies. Western Chemi luminescent HRP Substrate was purchased from EMD Millipore. Laemmli sample buffer, Restore Plus Western Blot Stripping Buffer were purchased from Bio Rad. CellTiter 96 cell proliferation assay was purchased from Promega. Cell Lines and Culture A 549 (nonsm all cell lung cancer cell line) and U87MG (glioblastoma cell line) were cultured in Dulbeccos Modified Eagle Medium (DMEM) supplemented with 10% FBS and 100 IU/mL penicillin streptomycin at 37C with 5% CO2. MD MB 231 (breast cancer cell line) was cultur ed in L15 cell culture medium supplemented with 10% FBS

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92 and 100 IU/mL penicillinstreptomycin at 37C. Cells were split at regular intervals and were not allowed to overgrow. Oligonucleotide Synthesis All oligonucleotides were synthesized using standard p hosphoramidite chemistry on an ABI3400 DNA/RNA synthesizer (Applied Biosystems, F oster City, CA, USA). DNA bases were synthesized on the 1.0 micromolar scale. 2 O methyl modified RNA bases and 5 thiol C6 disulfide modifier CED phosphoramidite were conjug ated to DNA by extended coupling time (900 seconds) on the DNA synthesizer. All oligonucleotides were purified by HPLC using a C18 reverse phase column, except the one with cholesterol modification, which was purified in a C4 column. The absorbance at 260 nm of each sample was measured to quantify the probe concentration. All purified oligonucleotides were stored in DNase/RNase free water. Gel Electrophoresis A 2 L sample was diluted in 8 L DNase/RNase free water, mixed with 2 L 6 DNA loading dye and l oaded in a 3% agarose gel A 25 bp DNA ladder was also loaded. The gel was run for 40 min at 100 mV in TBE (tris borateEDTA) buffer and imaged under UV light. Flow Cytometric Analysis of Probe Binding A549 cells were detached by a non enzymatic cell diss ociation solution and resuspended in the cell culture medium without FBS. FITC labeled oligonucleotides were incubated with 2 105 cells in 200 L of cell culture medium for 25 min at 37C. After incubation, cells were washed twice with 1 mL of washing bu ffer and suspended in 200 L PBS. The fluorescence was monitored with a FACScan cytometer (Becton

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93 Dickinson Immunocytometry systems, San Hose, CA) in channel 1. Cells without any label was used as the baseline control. Confocal Microscopy Study of Probe In ternalization Cells were seeded in a 35 mm glass bottom dish (MatTek) and cultured 12 days to reach 70% confluence. TMR labeled oligonucleotides were incubated with cells in culture medium at 500 nM concentration for 4 hours at 37C. Transferrin conjugated with Alexa Fluor 633 was added 30 min prior to imaging. Dishes were then washed twice with PBS and covered with PBS for imaging using an Olympus FV500IX81 confocal microscope (Olympus America Inc, Melville, NY) Images were taken and collected in the perpendicular lateral (x y) plane with a 543 nm helium laser and/or a 633 nm laser under a 60 oil objective. Transfection of Antagom irs Transfection was carried out with RNAi Human/Mouse Starter Kit (HiPerFect transfection reagent) as suggested by the manuf acturer Briefly, A549 cells were seeded one day before transfection in a 24 well plate at 20,000 cells/well. Cells were transfected with Chol Antag221, and AS1411Antag221 at 50 nM concentration. A cell death control siRNA provided with the kit was used as a positive control for transfection, and cells with HiPerFect transfection reagent only or without any treatment were used as negative control s. Quantitative Analysis of Endogenous MiRNA221 Level Total RNA from cells was extracted with the mirVana miRNA Isolation Kit according to the manufacturers protocol. The concentrations of the eluted mRNA samples were quantified with Nanodrop 8000 (Thermo Scientific, DE).

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94 Relative miRNA221 expression was analyzed by real time PCR using TaqMan microRNA Assays (App lied Biosystems) with RNU6B (P/N 4427975, Applied Biosystems) as endogenous control. The reverse transcription (RT) was performed using TaqMan MicroRNA Reverse Transcirption Kit. First, 25 ng of total RNA was reverse transcripted with TadMan following the manufacturer s instruction. Two L RT PCR product were combined with 0.5 L TaqMan 20 real time primers, 5 L TaqMan 2 Universal PCR Master Mix, No AmpErase UNG and 2.5 L nucleasefree water in a 10 L final volume. Samples were plated at 95C for 10 m in, followed by 40 cycles of 95C for 15 seconds and 60C for 60 seconds. Each set of sample was run in triplicates. The threshold cycle (Ct) values of miRNA221 were normalized to the average Ct values of RNU6B from the same cDNA sample and p The fold change of miRNA221 level between the treated A549 cells and untreated A549 cells equals to 2Ct = CtmiRNA 221 C tRNU6B treated A549 untreated A549. Western Blot Analysis Cells were washed with ice cold PBS twice and lysed in radioimmunoprecipitation buffer (RIPA buffer, 50 nM Tris HCl, 150 nM NaCl, 2mM EDTA, 1 % Triton X 100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS), pH 7.5) with proteinase inhibitors for 30 min at 4C. Lysates were collected and centrifuged at 14,000 rpm for 15 min, and the supernatants were collected. Protein concentration was measured using BCA Protein Assay A 100 g sample of protein were mixed with 2 Laemml i sample buffer (containing 5% heated at 95C for 5 min and loaded into 415% Tris HCl gel. The gel was transferred to a specially prepared transfer membrane with Trans Blot Turbo Transfer System (BioRad, CA) for 10 min. The membrane was bl ocked with 5% nonfat dry milk in PBS buffer containing

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95 0.05% Tween 20 (PBST) for 1 hour at room temperature, and then probed with primary antibodies at 4C overnight. After three washings with PBST for 6 min, the membrane was incubated with horseradish per oxidaseconjugated goat anti rabbit IgG antibody in PBST for 1 hour at room temperature. After washing, the membrane was developed with Immobilon Western Chemiluminescent HRP Subst rate for 1 min and exposed with film. Actin which served as a loading control was probed after washing the membrane in Restore Plus Western Blot Stripping Buffer at room temperature for 15 min. Cell Proliferation Assay A549 cells were seeded at 5,000 cells per well in a 96 cell plate the night before the assay. On the first day, 500 nM probes were added and incubated with cells for 48 hours. TRAIL was added at 200 ng/mL on the third day and incubated for 24 hours. Cell culture medium was then replaced with medium containing CellTiter reag ent (5:1 ratio) and incubated for 30 min. The absorbance of each sample at 490 nm was monitored using a plate reader (Tecan Safire microplate reader, AG, Switzerland). The anti proliferation effect on U87MG and MDA MB 231 cells was assessed on a 24well plate. Cells were seeded at 20,000 cells/well and cultured overnight. Probes (500 nM) were added to the cell culture medium and incubated with cells for 48 hours. Cells were washed in PBS once and examined under a microscope. Results and Discussion Sequence Design and Synthesis The dual functional molecular probe consists of two distinct parts: antagMir 221 ( A ntag221) that blocks miRNA 221 function, and AS1411 aptamer for cell targeting and intracellular delivery (AS1411Antag221). These two moieties were separated by five

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96 deoxythymidines to avoid any spatial interference between the aptamer and antag221. Previous studies showed that AS1411 enters cells with its protein target nucleolin, by receptor mediated endocytosis. However, one recent study reported A S1411 enters cancer cells mainly via macropinocytosis, which avoids endosomal trapping and/or lysosomal degradation (266) Nonetheless, a disulfide bond is introduced between AS1411 and Antag221 to help possible lysosomal escape (165) All the RNA bases were methylated at t he 2 hydroxyl group of the ribose to improve the enzymatic stability. Moreover, it has been reported that 2 O methyl modified nucleotides can evade innate immune responses (65) One advantage of this molecule is one step synthesis. Previously reported siRNA delivery with aptamers involve complicated transcription and conjugation steps Our probe can be synthesized from 3 to 5 using a DNA synthesizer following standard phosphoramidite chemistry without further modification. As a targeting control, AS1411 was replaced by a random sequence and conjugated with A ntag221 (LibAntag221). To compare the delivery efficiency, a cholesterol modified antag221 (Chol Antag221) was also syn thesized as described by others (259) The sequence and abbreviation for each probe are listed in Table 41. After purification, probes were verified by gel electrophoresis (Fig 4 2A). Probes with fluorescent dye molecules attached at 3 were also synthesized for flow cytometr ic analysis and confocal microsco py study Binding and Internalization Study AS1411Antag221 conjugates can recognize target cells After synthesis, the first question to be answered was whether the aptamer sequence in AS1411Antag221 conjugates could recognize target cells. We used FITC l abeled AS1411 aptamer as a positive control and tested the binding of FITC labeled

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97 AS1411 Antag221 and Lib Antag221 to A549 cells. As shown in Fig 4 2B, AS1411Antag221 retained the specific binding to A549 cells, with a small shift back compared with AS1 411 aptamer only. The control sequence, Lib Antag221 had no enhanced affinity to the target cell line. This indicated that Antag221 sequence in this probe had little effect on the spatial structure of AS1411. Thus the binding pattern remained. It also poi nted out that wi thout AS1411, Antag221 alone had no specificity to the cancer cell line. Internalization of Antag221 with AS1411 The next step was to confirm the internalization of Antag221 with the help of AS1411 aptamer. A widely accepted AS1411 intracellular trafficking model is that AS1411 binds to cell surface nucleolin protein and enters cell with the endocytosis of the protein which localizes around nucleus (267) In our design, we utilize this feature to bring antag om ir into cytosol where mature miRNAs exist. To test this probes were conjugated with a TMR fluorescent dye for confocal microscopy study Similar to the binding test, the AS1411 aptamer was used as a positive control. Under the confocal microscope, we clearly observed TMR signals from A549 cells (Fig. 43). The overall fluorescent intensity from AS1411 Antag221 treated cells was slightly lower than intensity of cells treated with AS1411 aptamer alone. The control probe, LibAntag221 showed a minimal background fluorescent signal, indicat ing that without aptamer, Antag221 cannot be effectively transported into cells. Differ ent concentrations of probes were also tested and 500 nM were selected because of the high signal to noise ratio. To determine whether probes delivered with AS1411 would be trapped in endosomes, we incubated cells with a dyeconjugated transferrin protein before imaging. Transferrin is a wellestablished endosome indicator which undergoes endocytosis by a clathrin-

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98 dependent pathway. Overlapping images of aptamers and transferrin proteins revealed partial colocalization, indicating some internalized probes w ere in the endosomes (Fig. 4 4). Taken together, we confirmed that the conjugation of AS1411 with Antag221 does not affect aptamer binding, and the antagomir can be intracellularly delivered with aptamers. Aptamer D elivered Antagomir Can I nhibit M iRNA F u nction Quantitative analysis of miRNA221 level Although the confocal images revealed the internalization of the probe, they did not show the binding of Antag221 to miRNA 221. Previous studies showed that reduced miRNA levels were detected with real time PC R (rt PCR) in cells transfected with Chol Antag221 (257, 259) Therefore, to verify that aptamer delivered antagom irs can reach their miRNA targets, the level of miRNA before and after treatment was quantified and compared. In addition, we transfected cells with the Chol Antag 221 probe as a positive control AS1411 Antag221 probe was also transfected to verify the function of Antag221 in the conjugated molecule. The levels of miRNA221 were assessed by real time quantitative PCR and the relative changes wer e compared with those of untreated cells As shown in Fig. 45A, c ells transfected with Chol Antag221 or AS1411Antag221 showed a reduced amount of miRNA221. Cells incubated with AS1411Antag221, that is Antag221 delivered by aptamers, also showed a lower amount of miRNA221 compared with untreated cells. The rt PCR data clearly demonstrated that Antag221 delivered by AS1411 can reach its miRNA target and have comparable effect with transfection.

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99 Analysis of p27K ip1 e xpression pattern To further confirm the effectiveness of the probe, one of the protein targets regulated by miRNA221 was examined. P27K ip1 protein is a cell cycle regulator that arrest s cel l growth at the G1/S transition Several groups have reported independently with different cancer cell lines that p27 expression is regulated by miRNA221 alone or by miRNA221/222 cluster (255, 257259) Overexpression of miRNA221/222 has been related to diminished p27K ip1 express ion and promotion of cell proliferation. Accordingly, transfection of Antag221 to block miRNA221 function can rescue p27 express ion As a matter of fact, this is one of the proposed mechanisms for the oncogenicity of miRNA221. Based on these studies, we pr obed p27K ip1 expression to evaluate aptamer mediated antagomir delivery. Western blot analysis showed that cells incubated with AS1411 Antag221 retrieved p27K ip1 expression, as in cells transfected with Chol Antag221 or AS1411Antag221 (Fig. 3 5B) This fu rther confirmed that Antag221 delivered by AS1411 functioned well and silenced target miRNA. The rt PCR and western blot results demonstrated that Antag221 function was not compromised in the AS1411Antag221 conjugates. Moreover, Antag221 that travelled to gether with AS1411 can target miRNA targets and inhibit their function following the mechanism described above. E ffect of AS1411Antag221 on Cancer Cells The endogenous expression level of m iRNA221 has been related to the sensitivity of anticancer drugs an d cancer cell proliferation rates. W e selected cell models with elevated expression of miRNA221 that can be targeted by AS1411. We expected that Antag221 delivered by AS1411 could inhibit miRNA221 function and initiate anti cancer effects that previously w ere achieved by transfection (255, 257, 259)

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100 AS1411Antag221 improved TRAIL sensitivity O verexpression of miRNA221 has been interpreted by resistance to a cancer drug named TRAIL (t umor necrosis factor (TNF) rel ated apoptosis inducing ligand). TRAIL belongs to TNF family and is known to induce apoptosi s of a variety of cancers while leaving normal cells intact (268, 269) However, there are phenotypes that are resist ant to TRAIL induced apoptosis. Studies showed that TRAIL resistance is correlated with miRNA221/222 overexpression in nonsmall cell lung cancer (NSCLC) and bladder cancer cells and the sensitivity can be restored with antisense sequences to miRNA221/222 (257, 259) In this study, we used A549, a type of NSCLC cell line that can be targeted by AS1411 but is TRAIL insensitive as a model system. A549 cells were seeded in a 96well plate and incubated with AS1411, LibAntag221 and AS1411Antag221 probes. TRAIL wa s added after 48 hours and incubated with cells for another 24 hours. Cell viability was assessed with MTS based colorimetric assay. As demonstrated in Fig. 46, 70% AS1411Antag221 treated A549 cells were found dead with TRAIL, while only 20% cell died in non treated and LibAntag221 treated groups. The improved sensitivity is very likely caused by intracellularly delivered Antag221 because the AS1411 treated group did not show a significant change in TRAIL response. However, possible synergistic effect fr om AS1411 and Antag221 cannot be excluded, since AS1411 targets one of the downstream factors in a TNF related pathway (264) AS1411Antag221 inhibited cancer cell proliferation Abnormally high expression of miRNA221 has also been found in several glioblastoma and breast cancer cell lines, where it promotes cell proliferation (255) We evaluated the probes anti proliferation function on two representative cell lines: U87-

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101 MG, a glioblastoma cell line and MDA MB 21, a breast cancer cell line. Using TMR labeled probes, we fi rst confirmed that AS1411 recognized both cell lines and can bring Antag221 into cells (Fig. 47). We then incubated cells with unlabeled probes for 2 days and examined them under a microscope. Fig. 48 displays a representative picture from each sample. W e clearly observed fewer cells in AS1411Antag221 treated groups compared with untreated or LibAntag221 treated groups. The AS1411 treated groups also showed a reduced number of cells, but not as low as in AS1411Antag221 treated groups. Therefore, we con firmed that, AS1411Antag221 can be used as an effective anti proliferation agent in some types of cancer. Conclusion In conclusion, we have successfully constructed a DNA aptamer based probe for intracellular delivery of therapeutic oligonucleotides. We designed the probe in a way that can be synthesized in one step using an automated DNA synthesizer. Directed by its aptamer moiety, the probe can target and enter cancer cells. Once inside cells, the antagomir moiety can find and inhibit its target miRNA. We monitored both subcellular (miRNA and protein expression level) and cellular (drug sensitivity and proliferation rate) effects to confirm the probes function. It should be noted that the aptamer we used, AS1411 recognizes a wide range of cancer cells and inhibits their proliferation (in most cases) at a high micromolar concentration. The microRNA target, miRNA221 is an oncogene that has been found upregulated in several cancers and promotes their proliferation. We tested the anti cancer activity of the probe on three different cancer types nonsmall cell lung cancer, glioblastoma and breast cancer with a nanomolar concentration. Given the broad targets of both AS1411 and miRNA221, we expect the conjugated probe could be applied to a variety of cancer ty pes.

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102 Table 41. List of oligonucleotide sequences Name Sequence AS1411 Chol Antag221 5 CGA ACA GGT GGG TGG GTT GGG TGG ATT GTT CG 3 5 GAA ACC CAG CAG ACA ATG TAG CT Cholesterol 3 AS1411 Antag221 5 GAA ACC CAG CAG ACA ATG TAG CT S S TTT TT CG A ACA GGT GGG TGG GTT GGG TGG ATT GTT CG 3 Lib Antag221 5 GAA ACC CAG CAG ACA ATG TAG CT S S TTT TT NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NN 3 The bases in A ntag221 were 2 O Methyl modified bases.

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103 Figure 4 1. Schematic presentation of t he function of miRNA221 and AS1411Antag221. Left pathway: Once matured, miRNA221 is released from the nucleus, is incorporated into a RNA induced silencing complex (RISC, not shown), and targets the 3 untranslated region of target mRNAs. One of the mRNA targets regulates p27Kip1 protein expression. When miRNA221 binds to this mRNA, it stops the translation of p27Kip1 protein and promotes cancer cell proliferation. Right pathway: The antisense of miRNA221, Antag221 is delivered by AS1411 into cells, finds its miRNA targets and hybridizes with them. This prevents miRNA from binding to its mRNA targets, and consequently, the protein expression is rescued. The production of p27Kip1 protein inhibits cancer cell proliferation.

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104 A B Fi gure 4 2. Probe synthesis and cell binding test A) Gel electrophoresis of all probes. B) The binding of AS1411Antag221 was tested with A549 cells by flow cytometry. AS1411 aptamer was used as a positive control.

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105 Figure 4 3 Confocal microscopy study of probe internalization. Lib Antag221, AS1411 and AS1411Antag221 with TMR labels were incubated with A549 cells and imaged.

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106 Figure 4 4. Study of the internalization pathway. Alexa Fluor 633 labeled transferrin was used as an endosome indicator. Colocalization of the probe and transferrin revealed that some probes were in endosomes.

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107 A B Figure 4 5 Antag221 delivered by AS1411 can inhibit miRNA221 function. A) Relative amount of miRNA221 in A549 cells after different treatment. The miRNA lev el in untreated A549 cells were set equal to 1. Values are means with S D (n=3) The single asterisk indicates a significant difference between cells incubated with AS1411Antag221 and LibAntag221 determined by the two tailed t test at P<0.01. B) Western b lot analysis of p27Kip1 in A549 cells. The membrane was stripped and reprobed with action antibody as a loading control.

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108 Figure 4 6 Relative A549 cell viability. Cell viability was assessed by MTS assay after different treatments. The number of live A549 cells in untreated samples was set equal to 1. Values are means with S D (n=3)

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109 Figure 4 7 Internalization of TMR labeled probes in U87MG and MDA MB 231 cells.

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110 Figure 4 8 Representative micrographs of U87MG and MDA MB 231 cells after incubated with different probes. Reduced number of cell were observed in AS1411 Antag221 treated samples.

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111 CHAPTER 5 SUMMARY AND FUTURE WORK S ummary of Dissertation As a leading cause of death worldwide, cancer has attracted tremendous attention in scientific world. Aptamers as high affinity and specificity molecular probes are very attractive tools in cancer diagnostics and therapy. In this dissertation, we developed two aptamer based probes and explored their potential in two types of emerging treatments for cancer. The first probe was designed to improve the targeting specificity and efficiency of therapeutic cells used in adoptive cell therapy. A diacyl lipidconjugated aptamer was developed to interact with the plasma membrane and anchor artificial targeting ligands (aptamers) on the cell surface. The probe initially interacts with lipid raft domains, and is distributed throughout the entire membrane within 2 hours. The initial probe concentration and incubation time control the cell surface density of aptamers, which is comparable to that of natural ligands. H owever, the modification is not permanent, and minimal aptamers are detected on the cell surface after 3 days. Nevertheless, membrane anchored aptamers retained their binding affinities and specificities, resulting in aptamer directed homotypic and heterot ypic cell assembly. Dnase I or the protease that can hydrolyze either aptamers or their binding proteins disassembled cell aggregates, confirming that the aggregation is caused by the aptamer protein recognition. Immune effector cells modified with this probe showed improved cancer cell targeting and killing in cellular models, an important feature in adoptive cell therapy. We demonstrated that neither the lipoaptamer nor its insertion is toxic to cells, a

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112 prerequisite for clinical applications. A flow cy tometric based assay was established to quantify aptamer directed cell targeting. We confirmed that aptamer modified cells recognize 60% 80% of target cells under physiological conditions. In one classic natural killer cell mediated in vitro cytotoxic assa y, we found that aptamer modified NK cells have a higher affinity to target cells, and accordingly, caused more cell death. Finally, a novel aptamer directed cytotoxic T lymphocyte assay was constructed. cytotoxic T lymphocyte modified with aptamers target ed cancer cells and initiated specific cell lysis. It is noteworthy that aptamers endowed CTL with a new targeting specificity, and the recognition is independent of major histocompatibility complex a major limitation of CTL based therapy. The second prob e was designed for targeted and intracellular delivery of therapeutic oligonucleotides. The probe consists of two moieties: a targeting aptamer and an anti miRNA antisense sequence. The nucleolin targeting aptamer AS1411 was used because it can target and be internalized in a wide range of cancer cells. In addition, it inhibits certain types of cancer cell growth at micromolar concentrations. The miRNA target was miRNA221, an oncogene upregulated in several types of cancer cells to improve their drug resist ance and promote growth. It has been shown that intracellularly delivered Antag221, the antisense of miRNA221, can inhibit miRNAs function. We demonstrated that after conjugation with AS1411, Antag221 is able to recognize and translocate into cancer cells inhibit miRNA221 activity, and recover tumor suppressor protein expression. Improved drug sensitivity and reduced cell proliferation were observed in different types of cancer cell lines treated with this probe.

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113 Future Direction Hepatocellular carcinoma (HCC) is a type of cancer that originates within the liver. The primary cause of HCC in the US is alcohol abuse. Aggressive surgery or liver transplantation is effective in treating only early stage HCC. Most HCC patients are diagnosed at later stages when effective therapeutic options are few. Therefore, there is a need to develop early diagnosis probes and alternative therapies for HCC. To this end, we will explore the adoptive T cell therapy for HCC in collaboration with Dr. Chen Liu, Dr. Lung Ji Chang and Dr. Roniel Cabrera at the University of Florida. Specifically, we will investigate the effectiveness of aptamer modified T cells in targeting liver tumors in a mouse model. Targeting Murine Liver Cancer Cells with Aptamer Modified Murine T Cells An e pithelialchemically transformed murine liver tumor cell line, BNL 1ME A.7R.1, will initially be used as a cell model for in vitro studies. BNL 1ME A.7R.1 was chosen, because it can develop a liver tumor model in Balb/cJ mice for later studies, and aptamers specific for this cell line are available. Three aptamers, TLS9a and TLS11a generated from a cell based selection (113) as well as AS1411, that showed a high affinity to this cell line, will be conjugated to the lipid molecule for murine T cell modification. T cells will be isolated from murine splenocytes with the MagCellect Mouse CD3+ T cell isolation Kit (R&D Systems) and activated with T cell Activation 96well Assay plates (BD Biocoat). We will confirm that l ipid aptamers can modify murine T cells and direct them to target the liver cancer cell line with the previously established assays. We will compare the targeting efficiency of the three aptamers, when used individually or in combination, to optimize condi tions for further in vivo trafficking studies.

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114 Targeting Liver Tumors in an HCC Mouse Model Next, we will track the aptamer modified T cells in an HCC mouse model. The HCC mouse model will be developed using Balb/cJ m ice challenged with BNL 1ME A.7R.1 ce lls. Murine T cells isolated from splenocytes will be activated, stained with a cell tracker dye, modified with the lipidaptamer probes, and injected back into tumor bearing mice. The whole mouse will be imaged by an in vivo fluorescence imaging system to monitor T cell trafficking at different time intervals. T C ell I nduced HCC S uppression If aptamer modification helps T cell homing to tumor sites, and the HCC specific T cell clone is available from our collaborators, we will study the destruction of HCC in mice. Briefly, tumor bearing mice will be injected with aptamer modified T cells or unmodified T cells, and the tumor size for each mouse will be monitored. A group of untreated mice will be used as a control for evaluating the efficacy of the adoptive T cell based immunotherapy.

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136 BIOGRAPHICAL SKETCH Xiangling Xiong was born in Changde, China to a radiologist and a mechanic. During high school, she was fascinated by chemistry and biology. In 2003, she was admitted to Hunan University, and majored in Chemistry. She joined Dr. Shouzhuo Yaos lab in 2006, and completed her bachelors degree in 2007. Inspired by her undergraduate study and her mother, she decided to pursue her career in cancer research. She was admitted to the University of Florida graduate program in 2007. After five years study under the tutorage of Dr. Weihong Tan, she received her Ph.D. degree in Chemistry in December 2012.