Lipopolymer Mediated siRNA Therapy for Cancer: Focus on Acute Myeloid Leukemia by Breanne Kathleen Landry A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Materials Engineering Department of Chemical and Materials Engineering University of Alberta © Breanne Kathleen Landry, 2015 Abstract   Protein silencing by small interfering RNA (siRNA) is a promising treatment strategy for cancer as over-expression of proteins is largely responsible for cancer cells’ infinite proliferation, evasion of cell death and multi-drug resistance. However, siRNAs require a carrier as their biological instability, negative charge and large molecular weight prevent cellular delivery. In this thesis, I first provide a review of current non-viral siRNA carrier strategies designed to protect and deliver the siRNA to the cell cytoplasm for RNAi activity and then follow with an over-view of the current state of siRNA development with non-viral carriers specifically in leukemia. One promising cationic polymer for siRNA delivery is high molecular weight polyethylenimine (PEI); however, its toxicity is an obstacle for clinical use. This thesis investigates a library of low- molecular weight (2 kDa) PEI with hydrophobic (lipid) modifications as siRNA carriers. The lipid modification renders this otherwise ineffective low-toxic polymer a safe and effective delivery system for intracellular siRNA delivery and protein silencing. We first explore a lipid modified polymer library in adherent cells lines targeting a model protein target, the house-keeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several relevant cancer targets; P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and survivin. These initial studies in adherent cells demonstrated that although the exact formulations for efficient silencing depended on the cell line and protein target, silencing with two of the lipid-modified polymers (caprylic and linoleic acid substitutions) were consistently effective, suggesting that these carriers can be applied clinically. Fine-tuning of the siRNA/polymer composition was however critical for silencing particular targets. We then focus our efforts specifically on Acute Myeloid i i Leukemia (AML), where siRNA therapy development has lagged behind the cancers that are derived from attachment dependent cells, as evident in the included review of current efforts in AML siRNA therapy. We explored the feasibility of the lipid-modified carriers in AML cell lines. Efficient siRNA delivery and silencing of the model protein target, green fluorescent protein (GFP), was achieved with higher functionality than that of 25 kDa PEI, where again caprylic acid and linoleic acid substitutions stood out as the most desirable polymer substitutions. Further work demonstrated effective silencing of an AML therapeutic target CXCR4, a surface expressed adhesion protein that contributes to leukemic cell survival. The suppression of CXCR4 as well as its ligand, SDF-1 (CXCL12), resulted in a decrease in overall cell survival, which was largely attributed to a decrease in cell proliferation without enhanced effects when silencing the two targets simultaneously. The decrease in cell numbers due to CXCR4/SDF-1 silencing occurred both in the absence and presence of human bone marrow stromal cells (hBMSC), suggesting that the proposed approach would be effective in the presence of the protective bone marrow microenvironment. In more clinically related models, siRNA delivery was achieved in all human AML patient cells tested and CXCR4 silencing was demonstrated in some cases, ex vivo. The effects of silencing CXCR4 in an AML subcutaneous in vivo tumor model were also explored. Overall, we found that caprylic and linoleic lipid-substituted PEI2 can provide effective siRNA delivery to leukemic cells and can be employed in molecular therapy of leukemia targeting suitable proteins, such as CXCR4, with therapeutic outcomes. We conclude with a discussion on the further development of siRNA carriers with focus on AML therapy, describing potential enhancements that could move the field forward. ii i Preface   Previous versions of the literature reviews and research presented in this thesis have been published, as described below. All chapters were conceptualized, researched and written under the involvement of H. Uludağ, the supervisory author. Additional acknowledgements are listed at the end of the respective chapters. The research project, of which this thesis is a part, received research ethics approval from the University of Alberta Research Ethics Board, Project Name “siRNA Based Therapies for Leukemias”, No. 687/04/13/D, 01/05/2012. The other research project, of which this thesis is a part, received research ethics approval from the University of Alberta Research Ethics Board, Project Name “Novel strategies to overcome drug resistance in leukemia”, No. Pro00043783, 21/01/2014 and 26/01/2015. Biosafety approval was obtained under UA file # RES0012356. Chapter 1 contains the literature review consisting of two parts. Chapter 1 - Part I, a review on the delivery of siRNA biomolecules, is included within the manuscript published as H.M. Aliabadi, B. Landry, B, C. Sun, T. Tang, and H. Uludağ, “Supramolecular Assemblies in Functional siRNA Delivery: Where do we Stand?” Biomaterials, vol. 33, issue 8, 2546-69. Inclusion of the described paper was limited to the sections I specifically contributed to (Section 1.1 Background on siRNA Carriers was written by Aliabadi with significant insight and contribution by myself and for Section 1.2 A Mechanistic Look At Cellular Delivery Of SiRNA Complexes, I was the primary author.) Figure 1.1 is courtesy of D. Meneskesedag-Erol. Figure 1.2 and Figure 1.4 are courtesy of H.M. Aliabadi. Chapter 1 - Part II contains a siRNA leukemia review expected to be published as B. Landry, J. Valencia-Serna, H. Gül-Uludağ, X. Jiang, A. iv Janowska-Wieczorek, J. Brandwein, and H. Uludağ, “Progress in RNAi Mediated Molecular Therapy of Acute and Chronic Myeloid Leukemia.” As the primary author, I was responsible for the literature review, analysis and manuscript composition. Gül- Uludağ, Jiang, Janowska-Wieczorek, and Brandwein, through their leukemia expertise, ensured accuracy of several ideas covered in the paper. Valencia-Serna provided insight into writing of the manuscript and contributed specifically to the chronic myeloid leukemia (CML) sections of the paper (the major sections that are specific to CML have been removed from the chapter). Chapter 2 contains portions of three published papers, where the major portion came from a paper published as H.M. Aliabadi, B. Landry, R.K. Bahadur, A. Neamnark, O. Suwantong, and H. Uludağ, “Impact of Lipid Substitution on Assembly and Delivery of siRNA by Cationic Polymers.” Macromolecular Bioscience, vol. 11, issue 5, 662-72. Although I was not the first author, I was fully and directly involved in the design of the studies, collection of the data, analysis of the data and review of manuscript. Sections of an additional two papers have also been included and were published as H.M. Aliabadi, B. Landry, P. Mahdipoor, and H. Uludağ, “Induction of Apoptosis by Survivin Silencing through siRNA Delivery in a Human Breast Cancer Cell Line.” Molecular Pharmaceutics, vol. 8, issue 5, 1821-30. H.M. Aliabadi, B. Landry, P. Mahdipoor, C.Y.M. Hsu, and H. Uludağ, “Effective Down-regulation of Breast Cancer Resistance Protein (BCRP) by siRNA Delivery using Lipid-substituted Aliphatic Polymers.” European Journal of Pharmaceutics and Biopharmaceutics, vol. 81, issue 1, 33-42. Included portions were those that I had direct involvement (design, data collection and data analysis). Other portions of the papers, which I was less involved in, were v minimized, briefly mentioned or removed. Figure 2.5 is courtesy of Aliabadi. Lipid- polymers utilized in these studies were synthesized by Neamnark. Chapter 3, 4 and 5 are research papers focused on siRNA therapy for acute myeloid leukemia. As the lead author, I designed, performed and analyzed the studies and wrote the manuscript. Chapter 3 is published as B. Landry, H.M. Aliabadi, A. Samuel, H. Gül-Uludağ, J. Xiiaoyan, O. Kutsch, and H. Uludağ, “Effective Non-viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-substituted Polyethylenimines.” PLoS ONE, vol. 7 issue 8, e44197. Figure 4.1 and Figure 4.2 are courtesy of O. Suwantong. J. Valencia-Serna and B. Sahin provided the data for creation of Figure 3.S3. Chapter 4 is expected to be published as B. Landry, H. Gül-Uludağ, J. Hongxing, and H. Uludağ, “Targeting CXCR4/SDF-1 Axis by Lipopolymer Complexes of siRNA in Acute Myeloid Leukemia.” Chapter 5 involves AML patient cells and tumor model studies and will be included in a future paper. Samuel was an undergraduate summer student who designed and performed experiments under my guidance. Specific cells used in these studies were provided by A. Janowska-Wieczorek (THP-1, KG-1 and HL-60), O. Kutsch (GFP positive THP-1 cells), H. Jiang (human bone marrow stromal cells), and J. Brandwein and Z. Zak (AML patient cells). Drs. Jiang and Gül-Uludağ afforded leukemia research guidance. Dr. Aliabadi provided assistance in the in vivo cancer model through injection of carriers into the tumor, caliper measurements, helped with tumor extractions and general mouse handling. My role in the same in vivo cancer model study included design of the experiment, cells/carrier preparation for injection, mouse weighing and monitoring, tumor extractions, general mouse handling, and all post tumor extraction v i processing and data analysis. Lipid-polymers utilized in these studies were synthesized by A. Neamnark, R. Bahadur K.C. and J. Fife. We conclude with Chapter 6, Conclusions and Future Directions. This chapter includes portions of the future work sections of the two review papers described above (H.M. Aliabadi, et al. Biomaterials, vol. 33, issue 8, 2546-69. and B. Landry et al. In progress.), as well as new content derived from the knowledge gained from the work presented in the thesis.     vi i Acknowledgements   I have had the opportunity to be supported by scholarship funding over the course of my graduate studies. I would like to acknowledge and thank the following institutions for their support: Canadian Institutes of Health Research (CIHR) for the Frederick Banting and Charles Best Canada Graduate Scholarship, University of Alberta for the President’s Prize of Distinction, Alberta Cancer Foundation (ACF) for the Graduate Studentship, and Women and Children’s Health Research Institute Graduate (WCHRI) for the Graduate Studentship. I have also been supported to present at several conferences and would like to thank the granting programs; Profiling Alberta’s Graduate Students Award (University of Alberta), GMN NSERC CREATE Program On Regenerative Medicine and GSA Professional Development Grant (University of Alberta). Over the course of my program I have had the opportunity to work with many talented researchers within our own lab as well in others across the University of Alberta campus. I would specifically like to thank my lab colleagues (past and present) (in alphabetical order); Dr. Meysam Abbasi, Dr. Rajesh Alphonse, Jeremy Fife, Ross Fitzsimmons, Dr. Hilal Gül-Uludağ, Dr. Charlie Hsu, Dr. Vanessa Incani, Dr. Remant KC, Cezary Kucharski, Xiaoyue Lin, Parvin Mahdipoor, Adam Manfrin, Deniz Meneksedag-Erol, Dr. Hamidreza Montazeri, Nesrine Mostafa, Manoj Parmar, Dr. Laura Rose, Anuja Samuel, Basak Sahin, Juliana Valencia-Serna, Dr. Guilin Wang, and Dr. Sufeng Zhang. The exposure to many researchers from different backgrounds (biological sciences, chemistry, dentistry, engineering, medicine, and pharmacy) where those who have completed their time at University of Alberta have followed many different career directions provided a wide range of experiences. I enjoyed working together with these vi ii talented researchers who made the lab a positive research environment. Specifically I would like to thank Dr. Hamidreza Montazeri whom I enjoyed working on many experimental studies together, Cezary Kucharski for guidance in cell culture studies and Dr. Laura Rose whom I spent most of my PhD years with. I would also like to thank my previous undergraduate supervisors, Dr. Robert Burrell and his PhD student at the time, Dr. Patricia Nadworny as well as my first summer research supervisor, Dr. Carolina Goano-Diaz. Dr. Goano-Diaz provided me with my first start in research in the summer of 2006. Dr. Robert Burrell and Dr. Patricia Nadworny furthered my interest in research through subsequent research summers. I would also like to acknowledge the contribution of the co-authors and those listed in the acknowledgment sections in journal publications for all of their help and greatly appreciated contributions. I would specifically like to thank the main contributors; Dr. Hamidreza Montazeri, Dr. Hilal Gül-Uludağ, Juliana Valencia-Serna as well as my supervisor, Dr. Hasan Uludağ. I would also like to thank Anuja Samuel, a summer student who worked with me on the earlier leukemia studies. I would like to thank my family and friends for their support during my studies; my parents (Lorine Hancock-Landry and Malcolm Landry) as well as my siblings (Nathan (Asami), Adam and Jordan), and my nephews (Naoki and Taiki) and my fiancé (Aaron Lim). Lastly, I have greatly appreciated the support and mentorship I received from my supervisor Dr. Hasan Uludağ. I am thankful for both the strong academic support he provides and the positive laboratory environment he maintains, which is essential for good and enjoyable research. ix Table  of  Contents   i.  SCOPE  ....................................................................................................................................  1   1.  SUPRAMOLECULAR  ASSEMBLIES  IN  FUNCTIONAL  SIRNA  DELIVERY  WITH  A  FOCUS  ON   LEUKEMIA  THERAPYX  ................................................................................................................  5   PART  I.  OVERVIEW  OF  SIRNA  THERAPY  WITH  NON-­‐VIRAL  CARRIERS  ...........................................  6   1.1  BACKGROUND  ON  SIRNA  CARRIERS  FOR  SIRNA  THERAPY  .....................................................  7   1.1.1  Liposomes  ....................................................................................................................  10   1.1.2  Lipoplexes  ....................................................................................................................  11   1.1.3  Stable  Nucleic  Acid  Lipid  Particles  (SNALP)  ..................................................................  11   1.1.4  Cationic  Polymers  ........................................................................................................  12   1.1.4.1  Chitosan  ...............................................................................................................................................  12   1.1.4.2  Other  Natural  Polymers  .......................................................................................................................  13   1.1.4.3  Polyethylenimine  (PEI)  .........................................................................................................................  14   1.1.4.4  Dendrimers  ..........................................................................................................................................  15   1.1.4.5  Other  Synthetic  Polymers  ....................................................................................................................  15   1.1.5  Peptides  .......................................................................................................................  17   1.2  A  MECHANISTIC  LOOK  AT  CELLULAR  DELIVERY  OF  SIRNA  COMPLEXES  ...............................  18   1.2.1  Cell  Surface  Binding  .....................................................................................................  21   1.2.2  Cellular  Internalization  .................................................................................................  26   1.2.3  Crossing  Lipid  Membranes  for  Cytoplasmic  Release  ....................................................  29   1.2.4  Transport  within  the  Cytoplasm  ..................................................................................  31   1.3  CONCLUSIONS  TO  NON-­‐VIRAL  SIRNA  THERAPY  ...................................................................  35   1.4  REFERENCES  ........................................................................................................................  36   PART  II.  PROGRESS  IN  RNAI  MEDIATED  THERAPY  OF  LEUKEMIA  ...............................................  51   1.5  LIMITS  OF  CURRENT  LEUKEMIA  THERAPIES  AND  PROMISE  OF  RNAI  ...................................  52   1.6  NON-­‐VIRAL  SIRNA  DELIVERY  FOR  LEUKEMIA  .......................................................................  55   1.7  FUNCTIONAL  CARRIERS  FOR  RNAI  AGENTS  .........................................................................  56   1.7.1  Cationic  Cell  Penetrating  Peptides  in  Leukemia  siRNA  Therapy  ...................................  61   1.7.2  Lipidic  Carriers  in  Leukemia  siRNA  Therapy  .................................................................  62   1.7.3  Polymer  Carriers  in  Leukemia  siRNA  Therapy  ..............................................................  63   1.7.4  Additional  Functionalization  of  Carriers  in  Leukemia  siRNA  Therapy  ..........................  64   1.8  SELECTIVE  DELIVERY  TO  LEUKEMIC  CELLS  ...........................................................................  66   1.8.1  Employing  Ligands  Specific  for  Leukemic  Cells  .............................................................  67   1.8.2  Relying  on  Targeting  to  Improve  Endocytosis  ..............................................................  70   1.8.3  Antibody-­‐Mediated  Targeting  .....................................................................................  71   1.8.4  Aptamers  for  Targeting  ...............................................................................................  73   1.8.5  Targeting  Adhesion  Receptors  .....................................................................................  74   1.9  SIRNA  DELIVERY  IN  LEUKEMIA  AND  RELATED  MODELS  .......................................................  75   1.9.1  Biodistribution  and  Pharmacokinetics  .........................................................................  80   1.9.2  Silencing  Efficiency  .......................................................................................................  81   1.10  THERAPEUTIC  TARGETS  EXPLORED  FOR  RNAI  IN  LEUKEMIA  .............................................  83   x