Siqi Guo

Targeted Identification of Metastasis-associated Cell-surface Sialoglycoproteins in Prostate Cancer

Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC4ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

Subnanosecond Electric Pulses Cause Membrane Permeabilization and Cell Death

Subnanosecond electric pulses (200 ps) at electric field intensities on the order of 20 kV/cm cause the death of B16.F10 murine melanoma cells when applied for minutes with a pulse repetition rate of 10 kHz. The lethal effect of the ultrashort pulses is found to be caused by a combination of thermal effects and electrical effects. Studies on the cellular level show increased transport across the membrane at much lower exposure times or number of pulses. Exposed to 2000 pulses, NG108 cells exhibit an increase in membrane conductance, but only allow transmembrane currents to flow, if the medium is positively biased with respect to the cell interior. This means that the cell membrane behaves like a rectifying diode. This increase in membrane conductance is a nonthermal process, since the temperature rise due to the pulsing is negligible.

Electro-gene transfer to skin using a noninvasive multielectrode array.

Because of its large surface area and easy access for both delivery and monitoring, the skin is an attractive target for gene therapy for cutaneous diseases, vaccinations and several metabolic disorders. The critical factors for DNA delivery to the skin by electroporation (EP) are effective expression levels and minimal or no tissue damage. Here, we evaluated the non-invasive multielectrode array (MEA) for gene electrotransfer. For these studies we utilized a guinea pig model, which has been shown to have a similar thickness and structure to human skin. Our results demonstrate significantly increased gene expression 2 to 3 logs above injection of plasmid DNA alone over 15 days. Furthermore, gene expression could be enhanced by increasing the size of the treatment area. Transgene-expressing cells were observed exclusively in the epidermal layer of the skin. In contrast to caliper or plate electrodes, skin EP with the MEA greatly reduced muscle twitching and resulted in minimal and completely recoverable skin damage. These results suggest that EP with MEA can be an efficient and non-invasive skin delivery method with less adverse side effects than other EP delivery systems and promising clinical applications.

Evaluation of Delivery Conditions for Cutaneous Plasmid Electrotransfer Using a Multielectrode Array

Electroporation (EP) is a simple in vivo method to deliver normally impermeable molecules, such as plasmid DNA, to a variety of tissues. Delivery of plasmid DNA by EP to a large surface area is not practical because the distance between the electrode pairs, and therefore the applied voltage, must be increased to effectively permeabilize the cell membrane. The design of the multielectrode array (MEA) incorporates multiple electrode pairs at a fixed distance to allow for delivery of plasmid DNA to the skin, potentially reducing the sensation associated with in vivo EP. In this report, we evaluate the effects of field strength and pulse width on transgene expression and duration using a plasmid encoding the luciferase reporter gene delivered by intradermal injection in a guinea pig model followed by EP with the MEA. As expected, the level of luciferase expression increased with the magnitude and duration of the voltage applied. In addition to adjusting transgene expression levels by altering fielding strength, levels could also be controlled by adjusting the plasmid dose. Our results indicate that the design of the MEA is a viable option for cutaneous plasmid DNA delivery by in vivo EP to a large surface area.

Prevention of distal flap necrosis in a rat random skin flap model by gene electrotransfer delivering VEGF165 plasmid

Therapeutic delivery of angiogenic growth factors is a promising approach for treating ischemia observed in skin flaps and chronic wounds. Several studies have demonstrated that vascular endothelial growth factor (VEGF) helps mitigate skin flap necrosis by facilitating angiogenesis. The present study aimed to demonstrate an electrically‐mediated nonviral gene delivery approach using a non‐invasive multi‐electrode array (MEA) for effective treatment of ischemic skin flaps.

Topical Gene Electrotransfer to the Epidermis of Hairless Guinea Pig by Non-Invasive Multielectrode Array

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after gene electrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest gene electrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient.

Optimization of the transductional efficiency of lentiviral vectors: effect of sera and polycations

Lentiviral vectors are widely used as effective gene-delivery vehicles. Optimization of the conditions for efficient lentiviral transduction is of a high importance for a variety of research applications. Presence of positively charged polycations reduces the electrostatic repulsion forces between a negatively charged cell and an approaching enveloped lentiviral particle resulting in an increase in the transduction efficiency. Although a variety of polycations are commonly used to enhance the transduction with retroviruses, the relative effect of various types of polycations on the efficiency of transduction and on the potential bias in the determination of titer of lentiviral vectors is not fully understood. Here, we present data suggesting that DEAE-dextran provides superior results in enhancing lentiviral transduction of most tested cell lines and primary cell cultures. Specific type and source of serum affects the efficiency of transduction of target cell populations. Non-specific binding of enhanced green fluorescent protein (EGFP)-containing membrane aggregates in the presence of DEAE-dextran does not significantly affect the determination of the titer of EGFP-expressing lentiviral vectors. In conclusion, various polycations and types of sera should be tested when optimizing lentiviral transduction of target cell populations.

Induction of protective cytotoxic T-cell responses by a B-cell-based cellular vaccine requires stable expression of antigen

B-cell-based cellular vaccines represent a promising approach to active immunotherapy of cancer complementing the use of dendritic cells, especially in pediatric patients and patients with low bone marrow reserves. B cells can be easily prepared in large numbers and readily home to secondary lymphoid organs, the primary site of induction of cytotoxic T lymphocyte (CTL) responses. However, most B-cell-based vaccines tested so far failed to induce functional and protective CTLs in in vivo models. Here, we show that B-cells activated through the toll-like receptor-9 (TLR-9) and CD40 up-regulate surface expression of major histocompatibility complex and costimulatory molecules, produce IL-12, and exhibit potent antigen-presenting properties in vitro. Importantly, although administration of peptide-coated or transiently transfected B cells fails to induce immune responses, therapeutic immunization with low numbers of genetically modified B cells stably expressing antigen results in an induction of functional CTLs and protection against the growth of tumor in an animal model. After activation, B cells partially loose their ability to home to organized lymphoid tissue because of the shedding of CD62L; however, this property can be restored by expression of protease-resistant mutant of CD62L. In summary, the data presented in this report suggest that genetically modified activated B cells represent a promising candidate for a cancer vaccine eliciting functional systemic CTLs.

Gene electrotransfer enhanced by nanosecond pulsed electric fields

The impact of nanosecond pulsed electric fields (nsPEFs) on gene electrotransfer has not been clearly demonstrated in previous studies. This study was conducted to evaluate the influence of nsPEFs on the delivery of plasmids encoding luciferase or green fluorescent protein and subsequent expression in HACAT keratinocyte cells. Delivery was performed using millisecond electric pulses (msEPs) with or without nsPEFs. In contrast to reports in the literature, we discovered that gene expression was significantly increased up to 40-fold by applying nsPEFs to cells first followed by one msEP but not in the opposite order. We demonstrated that the effect of nsPEFs on gene transfection was time restricted. The enhancement of gene expression occurred by applying one msEP immediately after nsPEFs and reached the maximum at posttreatment 5 minutes, slightly decreased at 15 minutes and had a residual effect at 1 hour. It appears that nsPEFs play a role as an amplifier without changing the trend of gene expression kinetics due to msEPs. The effect of nsPEFs on cell viability is also dependent on the specific pulse parameters. We also determined that both calcium independent and dependent mechanisms are involved in nsPEF effects on gene electrotransfer.

Electroporation based gene therapy — From the bench to the bedside

A critical aspect of gene transfer is effective delivery of the transgene to the appropriate target. Electrically mediated delivery (electroporation) of plasmid DNA has been accepted as a viable approach to achieve effective delivery. One promising area is delivering plasmid DNA to skin. Gene transfer to the skin with electroporation is currently being evaluated for its potential for inducing angiogenesis for wound healing and for delivering DNA vaccines to the skin. Experiments utilizing a plasmid encoding for vascular endothelial growth factor has demonstrated how wound healing could be accelerated. In another study, delivery of a plasmid encoding Hepatitis B surface antigen have demonstrated that high antibody titers can be induced after two applications (prime/boost). Our laboratory has also examined the use of electroporation to delivery plasmid DNA encoding various cytokines as a potential therapy for melanoma. The plasmid is injected directly into the tumor followed by the administration of electroporation. Extensive preclinical work provided the rationale for a Phase I proof of concept first in human trial in patients with accessible cutaneous melanoma metastases. Biopsies of treated lesions showed significant necrosis of melanoma cells within the tumor as well as IL-12 expression. Lymphocytic infiltrate was observed in biopsies from patients in several cohorts. Clinical evidence of responses in untreated lesions suggested there was a systemic response following therapy was observed. Since this trial several other clinical studies utilizing electroporation to deliver plasmid DNA have been initiated. It is clear that this delivery approach has tremendous potential to facilitate the translation of gene transfer protocols from the bench to the bedside.