Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-L-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications

Synthetic Biology-Based Approaches to Enhance Transgene Attributes

<p>Synthetic biology facilitates both the design and fabrication of biological components and systems that do not already exist in the natural world. From an engineering point of view, synthetic biology is akin to building a complex machine by assembling simpler parts. Complex genetic machines can also be built by a modular and rational assembly of simpler biological parts. These biological machines can profoundly affect various cellular processes including the transcriptional machinery. In this thesis I demonstrate the utilization of biological parts according to synthetic biology principles to solve three distinct transcription-level problems: 1) How to efficiently select for transgene excision in induced pluripotent stem cells (iPSCs)? 2) How to eliminate transposase expression following piggyBac-mediated transgenesis? 3) How to reprogram cell lineage specification by the dCas9/gRNA transactivator-induced expression of endogenous transcription factors? </p><p>Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs). For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In the first study, we design an excisable loxP-flanked lentiviral construct that a) includes all the reprogramming elements in a single lentiviral vector expressed by a strong EF-1&#945; promoter; b) enables easy determination of lentiviral titer; c) enables transgene removal and cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex Virus-thymidine kinase/ganciclovir (HSV-tk/gan) negative selection; and d) allows for transgene excision in a colony format. With our design, a reprogramming efficiency comparable to that reported in the literature without boosting molecules can be consistently obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy, we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX) in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase. The robustness of this platform enables the implementation of an efficacious and cost-effective protocol for iPSC generation and their subsequent transgenesis for downstream studies.</p><p>Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed "cut-and-paste" mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In the second study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the transposase element is deactivated after transposition. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells.</p><p>Genome editing by the efficient CRISPR/Cas9 system shows tremendous promise with ease of customization and the capability to multiplex distinguishing it from other such technologies. Endogenous gene activation is another aspect of CRISPR/Cas9 technology particularly attractive for biotechnology and medicine. However, the CRISPR/Cas9 technology for gene activation leaves much room for improvement. In the final study of this thesis we show that the fusion of two transactivation (VP64) domains to Cas9 dramatically enhances gene activation to a level that is sufficient to achieve direct cell reprogramming. Targeted activation of the endogenous Myod1 gene locus with this system leads to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. </p><p>In conclusion, this dissertation demonstrates the power of utilizing biological parts in a rational and systematic way to rectify problems associated with cell fate reprogramming and transposon-based gene delivery. Through design of genetic constructs aided by synthetic biology principles, I aspire to make contributions to the related fields of cellular reprogramming, stem cell differentiation, genomics, epigenetics, cell-based disease models, gene therapy, and regenerative medicine.</p>Dissertatio

A robust strategy for negative selection of Cre-loxP recombination-based excision of transgenes in induced pluripotent stem cells.

Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs). For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In this study, we design an excisable loxP-flanked lentiviral construct that a) includes all the reprogramming elements in a single lentiviral vector expressed by a strong EF-1α promoter; b) enables easy determination of lentiviral titer; c) enables transgene removal and cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex Virus-thymidine kinase/ganciclovir (HSV-tk/gan) negative selection; and d) allows for transgene excision in a colony format. A reprogramming efficiency comparable to that reported in the literature without boosting molecules can be consistently obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy, we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX) in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase. The robustness of this platform enables the implementation of an efficacious and cost-effective protocol for iPSC generation and their subsequent transgenesis for downstream studies

A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification

Gene activation by the CRISPR/Cas9 system has the potential to enable new approaches to science and medicine, but the technology must be enhanced to robustly control cell behavior. We show that the fusion of two transactivation domains to Cas9 dramatically enhances gene activation to a level that is necessary to reprogram cell phenotype. Targeted activation of the endogenous Myod1 gene locus with this system led to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. The levels of myogenic marker expression obtained by the activation of endogenous Myod1 gene were comparable to that achieved by overexpression of lentivirally delivered MYOD1 transcription factor

Engineered tissue patches and optical mapping of action potential propagation.

<p>Microfabricated tissue molds cast in PDMS were used to direct the alignment of iPS cell-derived cardiac progenitor cells in three-dimensional biosynthetic tissue constructs. <b>A</b>. PDMS tissue mold (sputtered with chrome for greater contrast). <b>B–C.</b> Zygometer surface profiles of the PDMS mold showing 1600 µm tall hexagonal features. Individual hexagons are 800 µm in length and 200 µm in width. <b>D.</b> An engineered tissue patch removed from its mold, bordered by a nylon mesh frame to facilitate handling. Elliptical void spaces are a result of tissue compaction away from the hexagonal features. <b>E–F.</b> Live brightfield and RFP images of the engineered tissue construct within its mold, showing tissue compaction forming elliptical pores around the hexagonal features and live cardiomyocytes within the tissue patch. <b>G.</b> Isochrone map showing activation times across the tissue patch during propagation (left to right) of an action potential. Blue pixels represent sites of early activation, and red pixels represent sites of late activation. <b>H.</b> False-color images representing a calcium transient traversing a tissue patch from left to right as a result of action potential firing. Red pixels represent a high intracellular calcium concentration, and blue pixels represent a low concentration.</p

Gene expression analysis performed on differentiating mouse iPS cells.

<p>Temporal gene expression analysis was performed over the length of 14 days (7 timepoints) on cultures of differentiating mouse iPS cells: <i>Pou5f1</i> (undifferentiated iPS cells). <i>T</i>, and <i>Mesp1</i> (precardiac mesoderm). <i>Nkx2-5</i>, <i>Gata4</i>, <i>Tbx5</i>, <i>Mef2c</i>, and <i>Myocd</i> (early cardiac transcription factors). Nppa, Myl2, Myl7, Myh6, Myh7, and Tnnt2 (mature cardiomyocyte markers), <i>Atp2a2</i>, <i>Atp2a3</i>, <i>Cacna1c</i>, <i>Casq2</i>, <i>Hc4</i>, <i>Kcnj2</i>, <i>Kcnj3</i>, <i>Kcnk1</i>, <i>Kcnd3</i>, <i>Pln</i>, <i>Ryr2</i>, and <i>Slc8a1</i> (cardiomyocyte electrophysiology genes). All gene expression levels were normalized against day 0 undifferentiated iPS (except for <i>Pou5f1</i>) using the ΔΔCt method. Error bars represent standard deviation. Significant differences in gene expression were determined using one-way ANOVA. Independent variable: differentiation day, and dependent variable: percentage gene expression. In all cases, the p-value was found to be very small (<<0.01; not shown), indicating highly significant changes in gene expression. Holm-Bonferroni multiple comparison t-tests were then performed to determine whether an individual data point on a particular differentiation day was significantly different from that of the previous. Holm-corrected p-values <0.05 were deemed significantly different and denoted by an *.</p

Intracellular microelectrode characterization of iPS cell-derived cardiomyocytes.

<p>The electrophysiology of RFP-expressing cardiomyocytes matured temporally in culture. <b>A</b>. Representative cardiomyocyte action potential traces at the intermediate differentiation stage (IDS, Day 7+7) or late differentiation stage (LDS, Day 7+14). Between IDS and LDS in culture, <b>B</b>. Action potential amplitude was not significantly different (102.4±1.9 mV versus 100.9±2.2 mV). <b>C</b>. Maximum action potential upstroke velocity increased from 149.3±3.5V/s to 179.5±9.3V/s. <b>D</b>. Action potential duration at 80% repolarization decreased from 77.2±3.2 ms to 60.9±1.7 ms. <b>E</b>. Maximum diastolic potential was not significantly different (−74.1±1.1 mV versus −75.9±1.1 mV).</p

Cardiac progenitor characterization and multipotential differentiation capacity.

<p><b>A</b>. The percentage of total cells expressing cell-surface antigens cKit, Flk1, and Sca-1 or the combinations of cKit/Flk1, and cKit/Sca-1 were determined by fluorescence-activated cell sorting before (gray) and after (black) puromycin addition for enriching Nkx2-5(+) cardiac progenitor cells. No RFP(+) cells were detected before addition of puromycin. The percentage of the five cell subpopulations also expressing RFP following puromycin addition is noted above the black columns. Error bars represent standard deviation. *denotes significant change (p<0.05) as determined by t-test statistical analysis. Isotype control antibodies were used as negative control in order to set the gates for the three cell surface antibodies. <b>B</b>. When cultured in suspension the derived iPS cells readily aggregated to form three-dimensional embryoid bodies which temporally differentiated into various cell types including spontaneously contracting cardiomyocytes detected as early as differentiation day 7. <b>C</b>. Following three days of puromycin antibiotic selection aggregates of spontaneously contracting RFP(+) cardiac progenitors were allowed to attach on gelatin-coated polystyrene. The cells in these aggregates stained positive for cardiac-specific actinin. <b>D</b>. Enzymatically dissociated and puromycin selected iPS-derived cardiac progenitors formed large-scale monolayers of spontaneously contracting cells. <b>E</b>. RFP-expressing (Myh6 promoter) and spontaneously contracting cardiomyocytes stained positive for the cardiac-specific marker Tnnt2. <b>F</b>. Acta2(+)/Tagln(+) smooth muscle cells were detected interspersed within the cultures of Act2(+) cardiomyocytes. <b>G–H</b>. Rare colonies of endothelial cells with varying size were detected within the cell monolayers as determined by immunostaining for Vwf.</p

Confocal microscopy of three-dimensional engineered biosynthetic tissue constructs containing iPS cell-derived differentiated cardiac progenitor cells.

<p>Representative images of the immunostained cells within the tissue constructs. This series of images was taken from an area located between the large and small pores within the construct. <b>A.</b> Cells within the biosynthetic tissue constructs significantly compacted the original hydrogel matrix, allowing for the formation of high-density intertwined string-like fibers. The cardiomyocyte cytoskeleton was elongated and aligned along the long-axis of the biosynthetic tissue constructs. <b>B–C.</b> The cytoskeletal sarcomeric arrangement was highly organized and placed perpendicular to the long axis of the biosynthetic tissues. <b>D–F</b>. The cardiomyocytes formed robust electromechanical connections (gap and adherens junctions) as determined by the level of expression and spatial organization of Gja1 and Cdh2 over long distances. These connections were often detected perpendicular to the long-axis of the tissue constructs.</p

iPS cell characterization.

<p><b>A–B</b>. Undifferentiated iPS cells express and co-localize transcription factors Pou5f1 and Nanog in their nuclei. Expression of the surface antigen Fut4 (SSEA-1) is also detected in Pou5f1-expressing iPS cells. Importantly the cells organize in tight compact colonies similarly to colonies of undifferentiated embryonic stem cells. <b>C</b>. Relative gene expression analysis for <i>Pou5f1</i>, <i>Sox2</i>, <i>Nanog</i>, <i>Rex1</i>, <i>Klf4</i>, and <i>Myc</i> following three serial expansion passages of the derived iPS cells in the absence of doxycycline. All gene expression levels are normalized against mouse embryonic fibroblasts using the ΔΔCt method. Error bars represent standard deviation. *, ** and *** indicate <i>p</i><0.05, 0.01, 0.0001, respectively, and computed using one-tailed Student’s t-test for comparison of the gene expression levels measured for iPS cells with that measured for primary MEFs. ^#indicates significant variance amongst all three groups as determined by one-way ANOVA analysis.</p