Jae-Hyung Jang

Controllable delivery of non-viral DNA from porous scaffolds.

The inductive approach to tissue engineering combines three-dimensional porous scaffolds with drug delivery to direct the action of progenitor cells into a functional tissue. We present an approach to fabricate scaffolds capable of controlled, sustained delivery by the assembly and subsequent fusion of drug-loaded microspheres using a gas foaming/particulate leaching process. DNA-loaded microspheres were fabricated from the copolymers of lactide and glycolide (PLG) using a cryogenic double emulsion process. Microspheres were fabricated in four populations with mean diameters ranging from 12.3 microm to 92.5 microm. Scaffolds fabricated by fusion of these microspheres had an interconnected open pore structure, maintained DNA integrity, and exhibited sustained release for 21 days. Control over the release was obtained through manipulating the properties of the polymer, microspheres, and the foaming process. Decreasing the microsphere diameter or the molecular weight of the polymer used for microsphere fabrication led to increased rates of release from the porous scaffold. Additionally, increasing the pressure of CO(2) increased the DNA release rate. The ability to create porous polymer scaffolds capable of controlled release rates may provide a means to enhance and regulate gene transfer within a developing tissue, which will increase their utility in tissue engineering.

DNA Shuffling of Adeno-associated Virus Yields Functionally Diverse Viral Progeny

Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (10(7)), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4-6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology.

Molecular evolution of adeno-associated virus for enhanced glial gene delivery.

The natural tropism of most viral vectors, including adeno-associated viral (AAV) vectors, leads to predominant transduction of neurons and epithelia within the central nervous system (CNS) and retina. Despite the clinical relevance of glia for homeostasis in neural tissue, and as causal contributors in genetic disorders such as Alzheimers and amyotrophic lateral sclerosis, efforts to develop more efficient gene delivery vectors for glia have met with limited success. Recently, viral vector engineering involving high-throughput random diversification and selection has enabled the rapid creation of AAV vectors with valuable new gene delivery properties. We have engineered novel AAV variants capable of efficient glia transduction by employing directed evolution with a panel of four distinct AAV libraries, including a new semi-random peptide replacement strategy. These variants transduced both human and rat astrocytes in vitro up to 15-fold higher than their parent serotypes, and injection into the rat striatum yielded astrocyte transduction levels up to 16% of the total transduced cell population, despite the human astrocyte selection platform. Furthermore, one variant exhibited a substantial shift in tropism toward Müller glia within the retina, further highlighting the general utility of these variants for efficient glia transduction in multiple species within the CNS and retina.

Gene delivery from polymer scaffolds for tissue engineering

The combination of gene therapy with tissue engineering offers the potential to direct progenitor cell proliferation and differentiation into functional tissue replacements. Many approaches to engineering tissue replacements feature a polymer scaffold to create and maintain a space, support cell adhesion, and organize tissue formation. Polymer scaffolds, either natural, synthetic, or a combination of the two, have also been adapted to serve as delivery vehicles for viral and nonviral vectors, which can induce the expression of tissue inductive factors. Gene delivery is a versatile approach, capable of targeting any cellular process through localized expression of tissue inductive factors. The design and application of tissue engineering scaffolds for localized gene transfer are reviewed. Scaffolds are designed either to release the vector into the local tissue environment or maintain the vector at the polymer surface, which is regulated by the effective affinity of the vector for the polymer. Polymeric delivery can enhance gene transfer locally, promote and extend transgene expression, avoid vector distribution to distant tissues, and reduce the immune response to the vector. Scaffolds capable of controlled DNA delivery can provide a fundamental tool for directing progenitor cell function, which has applications with the engineering of numerous types of tissue. The utility of this approach will increase with the development of design parameters that correlate release and transgene expression, and with continued research into the biology of tissue formation.

Directed Evolution of Adeno-associated Virus for Enhanced Gene Delivery and Gene Targeting in Human Pluripotent Stem Cells

Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However, natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities, and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (~50%) to hPSCs, which are importantly accompanied by a considerable increase in gene-targeting frequencies, up to 0.12%. While this level is likely sufficient for numerous applications, we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (>1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus, this study demonstrates that under appropriate selective pressures, AAV vectors can be created to mediate efficient gene targeting in hPSCs, alone or in the presence of ZFN- mediated double-stranded DNA breaks.

Engineering Biomaterial Systems to Enhance Viral Vector Gene Delivery

Integrating viral gene delivery with engineered biomaterials is a promising strategy to overcome a number of challenges associated with virus-mediated gene delivery, including inefficient delivery to specific cell types, limited tropism, spread of vectors to distant sites, and immune responses. Viral vectors can be combined with biomaterials either through encapsulation within the material or immobilization onto a material surface. Subsequent biomaterial-based delivery can increase the vectors residence time within the target site, thereby potentially providing localized delivery, enhancing transduction, and extending the duration of gene expression. Alternatively, physical or chemical modification of viral vectors with biomaterials can be employed to modulate the tropism of viruses or reduce inflammatory and immune responses, both of which may benefit transduction. This review describes strategies to promote viral gene delivery technologies using biomaterials, potentially providing opportunities for numerous applications of gene therapy to inherited or acquired disorders, infectious disease, and regenerative medicine.

Electrospun nanofibrous scaffolds for controlled release of adeno-associated viral vectors

The integration of viral gene delivery with key features of biomaterial scaffolds that modulate viral delivery in a controlled manner offers a promising strategy for numerous tissue engineering applications. In this study adeno-associated virus (AAV), which is widely utilized in human gene therapy as a gene carrier due to its safety and efficient gene delivery capability, was encapsulated within electrospun nanofibrous scaffolds composed of blended mixtures of elastin-like polypeptides (ELP) and poly (ε-caprolactone) (PCL) and was employed to transduce fibroblasts adherent on the scaffolds. Combinatorial interactions between ELP and PCL chains upon physical blending significantly altered the mechanical properties (i.e. wettability, elastic modulus, strain, etc.) of the ELP/PCL composites, thus providing key tools to mediate controlled release of AAV vectors and robust cellular transduction on the fibrous scaffolds. The ability of ELP/PCL composites to manipulate the controlled release of AAV-mediated gene delivery for subsequent high-efficiency cellular transduction will provide tremendous opportunities for a variety of tissue engineering applications.

Drawing Sticky Adeno‐Associated Viruses on Surfaces for Spatially Patterned Gene Expression

In developing tissues, the spatially controlled secretion of extracellular signals creates biointerfaces that modulate cellular processes of differentiation, proliferation, and migration, and provide molecular cues to organize the structure of tissues. Systems that control spatial distribution of extracellular molecules on substrates have been developed in order to induce patterned expression of intracellular inductive factors. 5,6] Alternatively, spatially patterned gene delivery has been employed to overcome limitations found in protein delivery: short protein half-life and systemic toxicity. The development of in vitro model systems that mimic the spatial control of gene expression in tissues or organs is critical to elucidate a variety of biological mechanisms. However, the majority of gene delivery systems rely on simple additions of gene carriers directly to media, which are inherently limited in their ability to spatially control gene expression. Advances in microand nanofabrication technologies have enabled researchers to control locations of gene vectors on surfaces. 11] Existing methods include microfluidics, surface coating, and self-assembly. In general, the techniques involve multiple laborious procedures, typically chemical activation of substrates, resist depositions, pattern generation using a photomask, and gene vector immobilization. Furthermore, expensive equipment is often necessary. Unlike the aforementioned complex processes, this study describes a simple, versatile approach for spatially patterned gene delivery inspired by adhesion of marine mussels. Catecholamine, the key adhesive moiety found in the specialized adhesive proteins of mytilus edulis, was used to formulate “sticky” viruses. The adhesive catecholamine polymer used in this study is poly(ethylenimine)-catechol (PEI-C), which has been used for material-independent layerby-layer assembly and mechanical reinforcement of carbon nanotube fibers. The adeno-associated virus (AAV), which is a safe and efficient parvovirus, was complexed with the PEIC. Because of the underwater adhesive property of PEI-C, the AAV vector complexed with the PEI-C became a highly sticky virus that can stably adhere onto surfaces. Most importantly, by using the sticky viral vectors, we were able to use a micropipette as a “pen” to create viral patterns on substrates. This “genevector drawing” technique bypasses laborious multiple steps and can thus be a versatile platform to control gene expression for the establishment of complex tissues. Branched PEI was conjugated with 3-(3,4-dihydroxyphenyl) propionic acid (DPA) to generate PEI-C, which provides sticky viral vectors (Figure 1A). A novel virus, AAVr3.45, which was specifically designed for neuronal cell

Heparin-coated superparamagnetic nanoparticle-mediated adeno-associated virus delivery for enhancing cellular transduction

Superparamagnetic iron oxide nanoparticles (SPIONs) have been exploited as an elegant vehicle to enhance gene delivery efficiencies in gene therapy applications. We developed a magnetically guided adeno-associated virus (AAV) delivery system for enhancing gene delivery to HEK293T and PC12 cell lines. Wild-type AAV2 and a novel AAV vector, AAVr3.45, which was directly evolved in a previous study to possess diverse cell tropisms, were used as gene carriers. Additionally, the affinity of each viral vector to heparin was employed as a moiety to immobilize virus onto heparin-coated SPIONs (HpNPs). Magnetically guided AAV delivery resulted fast and efficient cellular transduction. Importantly, a short exposure of virus to target cells under a magnetic field (<180min) yielded comparable transduction produced by the conventional gene-delivery protocol (i.e., 24h-incubation of virus with target cells prior to replacing with fresh medium). Additionally, magnetic guidance of AAV encoding nerve growth factor (NGF) produced sufficient functional NGF, leading to robust neurite elongation by PC12 as compared to direct NGF protein delivery or non-magnetic delivery. The successful establishment of a magnetically guided AAV delivery system, with the ability to efficiently and rapidly infect target cells, will provide a powerful platform for a variety of gene therapy applications.

Development of the digital tongue inspection system with image analysis

In oriental medicine, the color and shape of the tongue gives much physiological information about the human body, and many oriental medical doctors have diagnosed patients diseases using these information. This paper shows a Digital Tongue Inspection System that has been developed that consists of the implemented hardware part for tongue image acquisition, the image processing part that includes color interpolation, an edge detection algorithm for tongue area separation and tongue color detection algorithm and a database and user interface system for archiving and managing the acquired tongue images.