Engineering Nanodiscoidal Nucleic Acids as the Next-Generation Platform to Deliver Oligonucleotide Therapeutics Open Access

Abstract

Over the past several decades, nucleic acid therapeutics have made successful strides and almost a dozen therapies have received FDA approval. Typically, a DNA or RNA based drug is a short oligomer comprised on average of <25 base pairs and designed to target a specific mRNA sequence within a cell. As exogenous DNA and RNA sequences, these nucleic acids take advantage of Watson-Crick-Franklin base pairing to bind to and perturb native translational activity and initiate degradation. Despite the simple mechanism, delivery of these drugs has been significantly hindered because of several intracellular and extracellular barriers. To overcome these issues, nanoparticles are often the choice delivery vehicle owing to their large surface areas and small sizes. In this dissertation, we investigate the potential for a high-density lipoprotein (HDL) mimetic known as nanodiscs (NDs) to serve as a biomimetic vector for delivering therapeutic oligonucleotides. In Chapter 1, a comprehensive overview on nucleic acid therapeutics, and the advantages endowed by the ND based on its design, properties, and innate targeting features, compared to other NPs are elucidated. Chapter 2 introduces the conceptualization and construction of the initial ND scaffold: a ND composed of DMPC, thiol phospholipids, and a mimetic Apolipoprotein A1 peptide. We demonstrate that this framework can covalently bind 15 copies of DNA/ND and not alter the ND morphology, its selective recognition pathway using the non-endocytic Scavenger Receptor B1, or overall functional activity as a result. Chapter 3 builds off the framework revealed in Chapter 2 to create the nanodiscoidal nucleic acid (NNA) scaffold. The NNA scaffold is maximally packed with the highest density of nucleic acid reported yet to date, beating even our previous record in Chapter 2. Detailed in vitro and in vivo studies highlight the potency of the NNAs as an enhanced therapeutic. We reveal that the constructed scaffold was highly active in lowering the effective dose of a HIF-1-alpha ASO five-fold, to a level that was previously rendered ineffective by pharmaceutical companies. This work culminates in Chapter 4 by exploring the outlook and future of how NNAs are the next promising step towards advancing oligonucleotide therapeutics.

Table of Contents

List of Figures iii

List of Tables iv

Common Abbreviations v

Chapter 1: Introduction 1

1.1: Brief Overview of Nucleic Acid Therapeutics 2

1.2: Challenges of Nucleic Acid Therapeutics 3

1.3: High-Density Lipoproteins: Cholesterol and Cargo Transport 6

1.4: Advantages of HDL as a Transport Scaffold 9

1.5: Structural Properties of ApoA1 and ApoA1 Mimetic Peptides 11

1.6: Phospholipids and Assembly Techniques for Synthetic HDL Structures 17

1.7: Shortcomings in Previous Renditions of HDL Mimics as a Nucleic Acid DDS 22

1.8: Summary and Scope of this Dissertation 25

Chapter 2: Gene Regulation Using Nanodiscs Modified with HIF-1-alpha ASOs 27

2.1: Overview 28

2.2: Introduction 29

2.3: Results and Discussion 32

2.3.1 Incorporating and Assembling Thiol-Functionalized NDs 32

2.3.2: ND Conjugation to DNA 33

2.3.3: DNA bound to the surface of the ND is functional and nuclease resistant 37

2.3.4: ASO-ND conjugates are internalized in a dose- and time-dependent manner 38

2.3.5: The uptake of thiol NDs and ASO-NDs into cells is partially mediated by SRB1 40

2.3.6: Anti-HIF-1-alpha ASO-ND conjugates are active in vitro 42

2.4: Conclusion 46

2.5: Materials and Methods 47

2.5.1: Synthesis and Characterization of Thiol ND 48

2.5.2: DLS and TEM Characterization of Thiol NDs 49

2.5.3: Addition of Maleimide Group to DNA 49

2.5.4: Covalent Linkage of DNA to NDs 50

2.5.5: FRET Analysis of ASO-ND Samples 51

2.5.6: Quantification of DNA Density 51

2.5.7: Zeta (ζ) Potential Measurement of DNA-ND Samples 52

2.5.8: Gel Electrophoresis 52

2.5.9: DNAzyme Kinetics and Nuclease Degradation Assay 52

2.5.10: Cell Culture 53

2.5.11: Confocal Microscopy of HeLa Cells 53

2.5.12: Dose- and Time-Dependent Uptake Measurements using Flow Cytometry 53

2.5.13: SRB1 Mediated Uptake of ASO-NDs 54

2.5.14: RT-qPCR to Assess HIF-1-alpha Levels after EZN-2968 ASO-ND Treatment in vitro 54

2.5.16: Comparing Dose-Dependent Uptake of Anti-HIF-1-alpha ASO/ Anti-HIF-1-alpha ASO-NDs 54

2.5.17: MTT Assay to Assess Cell Viability 55

2.6: Supplementary Information 56

Chapter 3: Nanodiscoidal Nucleic Acids for Gene Regulation 63

3.1: Overview 64

3.2: Introduction 65

3.3: Results and Discussion 68

3.3.1 Screening of Cysteine-Modified ApoA1 Mimetic Peptides 68

3.3.2: NNAs are Internalized into Cells via Scavenger Receptor B1 75

3.3.3: Internalized ASO-NDs and NNAs Undergo Dissociation with 24 h 78

3.3.4: Quantifying NNA and ASO-ND Activity In Vitro 80

3.3.5: NNAs Penetrate into the Hypoxic Core of Tumor Spheroids and and are active 82

3.3.6: Anti-HIF-1-alpha NNAs are Active In Vivo 85

3.4: Conclusion 88

3.5: Materials and Methods 89

3.5.1: Synthesis and characterization of ND and NNA scaffolds 91

3.5.2: Size and morphology characterization of NDs and NNAs 91

3.5.3: Coupling maleimide-DNA onto the ND and NNA scaffolds 92

3.5.4: Quantifying DNA density 92

3.5.5: Bulk solution FRET measurements of DNA-NDs and NNAs 93

3.5.6: Gel electrophoresis and serum degradation assay 93

3.5.7: DNase I assay 93

3.5.8: Cell and spheroid culture 94

3.5.9: Confocal uptake studies on HeLa cells and spheroids 94

3.5.10: SRB1 mediated uptake of ASO-NDs and NNAs into cells and spheroids 95

3.5.11: Sensitized FRET measurements of ASO-NDs and NNAs in HeLa cells 96

3.5.12: RT-qPCR to assess HIF-1-alpha levels after in vitro treatment with EZN2968 97

3.5.13: MTT assay to assess cell viability 97

3.5.14: Mice acclimatization and tail-vein injection of ND scaffold, NNAs, and ASO 98

3.5.15: Murine in vivo and ex vivo fluorescence imaging 98

3.5.16: RNA isolation and RT-qPCR quantification of HIF-1-alpha from mice organs 99

3.6: Supplementary Information 100

Chapter 4: Recap and Future Directions 107

4.1: Summary and Conclusions 108

4.2.: Future Directions 109

References 111

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Keyword	Nanoparticles Lipid Technology Nanodiscs Drug Delivery HDL/sHDL Antisense Technology
Committee Chair / Thesis Advisor	Khalid Salaita, Emory University Vincent Conticello, Emory University Yonggang Ke, Emory University

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