Syandan Chakraborty

Electrohydrodynamics: A facile technique to fabricate drug delivery systems

Electrospinning and electrospraying are facile electrohydrodynamic fabrication methods that can generate drug delivery systems (DDS) through a one-step process. The nanostructured fiber and particle morphologies produced by these techniques offer tunable release kinetics applicable to diverse biomedical applications. Coaxial electrospinning/electrospraying, a relatively new technique of fabricating core-shell fibers/particles have added to the versatility of these DDS by affording a near zero-order drug release kinetics, dampening of burst release, and applicability to a wider range of bioactive agents. Controllable electrospinning/spraying of fibers and particles and subsequent drug release from these chiefly polymeric vehicles depends on well-defined solution and process parameters. The additional drug delivery capability from electrospun fibers can further enhance the materials functionality in tissue engineering applications. This review discusses the state-of-the-art of using electrohydrodynamic technique to generate nanofiber/particles as drug delivery devices.

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

Summary 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.

Induced Pluripotent Stem Cell-Derived Cardiac Progenitors Differentiate to Cardiomyocytes and Form Biosynthetic Tissues

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors’ ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.

CRISPR/Cas9-Based Genome Editing for Disease Modeling and Therapy: Challenges and Opportunities for Nonviral Delivery

Genome editing offers promising solutions to genetic disorders by editing DNA sequences or modulating gene expression. The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology can be used to edit single or multiple genes in a wide variety of cell types and organisms in vitro and in vivo. Herein, we review the rapidly developing CRISPR/Cas9-based technologies for disease modeling and gene correction and recent progress toward Cas9/guide RNA (gRNA) delivery based on viral and nonviral vectors. We discuss the relative merits of delivering the genome editing elements in the form of DNA, mRNA, or protein, and the opportunities of combining viral delivery of a transgene encoding Cas9 with nonviral delivery of gRNA. We highlight the lessons learned from nonviral gene delivery in the past three decades and consider their applicability for CRISPR/Cas9 delivery. We also include a discussion of bioinformatics tools for gRNA design and chemical modifications of gRNA. Finally, we consider the extracellular and intracellular barriers to nonviral CRISPR/Cas9 delivery and propose strategies that may overcome these barriers to realize the clinical potential of CRISPR/Cas9-based genome editing.

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.

Transdifferentiation of human endothelial progenitors into smooth muscle cells

Access to smooth muscle cells (SMC) would create opportunities for tissue engineering, drug testing, and disease modeling. Herein we report the direct conversion of human endothelial progenitor cells (EPC) to induced smooth muscle cells (iSMC) by induced expression of MYOCD. The EPC undergo a cytoskeletal rearrangement resembling that of mesenchymal cells within 3 days post initiation of MYOCD expression. By day 7, the reprogrammed cells show upregulation of smooth muscle markers ACTA2, MYH11, and TAGLN by qRT-PCR and ACTA2 and MYH11 expression by immunofluorescence. By two weeks, they resemble umbilical artery SMC in microarray gene expression analysis. The iSMC, in contrast to EPC control, show calcium transients in response to phenylephrine stimulation and a contractility an order of magnitude higher than that of EPC as determined by traction force microscopy. Tissue-engineered blood vessels constructed using iSMC show functionality with respect to flow- and drug-mediated vasodilation and vasoconstriction.

Biostable electrospun microfibrous scaffolds mitigate hypertrophic scar contraction in an immune-competent murine model

UNLABELLED Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). In this study, we evaluate the impact of an elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In vitro, fibroblast-seeded PU scaffolds contracted significantly less and demonstrated fewer αSMA(+) myofibroblasts compared to fibroblast-seeded collagen lattices. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. Our data suggest that electrospun ccPU scaffolds meet the requirements to reduce HSc contraction including reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, our data suggest that a more long-term scaffold may possess merit in reducing HSc. STATEMENT OF SIGNIFICANCE In severe burns treated with skin grafting, between 30% and 90% of patients develop hypertrophic scars (HSc). There are no therapies to prevent HSc, and treatments are marginally effective. This work is the first example we are aware of which studies the impact of a permanent electrospun elastomer on HSc contraction in a murine model that mimics the human condition. Collagen coated polyurethane scaffolds decrease αSMA+ myofibroblast formation in vitro, prevent stiffening of scar tissue, and mitigate HSc contraction. Unlike current standards of care, electrospun, polyurethane scaffolds do not lose architecture over time. We propose that the future bioengineering strategy of mitigating HSc contraction should consider a long-term elastomeric matrix which persists within the wound bed throughout the remodeling phase of repair.

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

Significance Delivery remains a significant challenge for robust implementation of CRISPR/Cas9. We report an efficient CRISPR/Cas9 delivery system comprising PEGylated nanoparticles based on the α-helical polypeptide PPABLG. Assisted by the high membrane-penetrating ability of the polypeptide, P-HNPs achieved efficient cellular internalization and endosomal escape. The CRISPR/Cas9 delivery system could reach 47.3% gene editing in cells, 35% gene deletion in vivo, and HeLa tumor growth suppression >71%, demonstrating an advantage over the existing conventional polycationic transfection reagents. Efficient also in knock-in and gene activation, the reported CRISPR/Cas9 delivery system serves to advance gene editing in vitro and in vivo. 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.

Core Transcription Factors, MicroRNAs, and Small Molecules Drive Transdifferentiation of Human Fibroblasts Towards The Cardiac Cell Lineage

Transdifferentiation has been described as a novel method for converting human fibroblasts into induced cardiomyocyte-like cells. Such an approach can produce differentiated cells to study physiology or pathophysiology, examine drug interactions or toxicities, and engineer cardiac tissues. Here we describe the transdifferentiation of human dermal fibroblasts towards the cardiac cell lineage via the induced expression of transcription factors GATA4, TBX5, MEF2C, MYOCD, NKX2–5, and delivery of microRNAs miR-1 and miR-133a. Cells undergoing transdifferentiation expressed ACTN2 and TNNT2 and partially organized their cytoskeleton in a cross-striated manner. The conversion process was associated with significant upregulation of a cohort of cardiac-specific genes, activation of pathways associated with muscle contraction and physiology, and downregulation of fibroblastic markers. We used a genetically encoded calcium indicator and readily detected active calcium transients although no spontaneous contractions were observed in transdifferentiated cells. Finally, we determined that inhibition of Janus kinase 1, inhibition of Glycogen synthase kinase 3, or addition of NRG1 significantly enhanced the efficiency of transdifferentiation. Overall, we describe a method for achieving transdifferentiation of human dermal fibroblasts into induced cardiomyocyte-like cells via transcription factor overexpression, microRNA delivery, and molecular pathway manipulation.

Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis

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 this 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 separation of the transposase element from the polyA sequence after transposition leads to its deactivation. 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.