IL-12 Gene Electrotransfer Triggers a Change in Immune Response Within Mouse Tumors

Metastatic melanoma is an aggressive skin cancer with a relatively low survival rate. Immune-based therapies have shown promise in the treatment of melanoma, but overall complete response rates are still low. Previous studies have demonstrated the potential of plasmid IL-12 (pIL-12) delivered by gene electrotransfer (GET) to be an effective immunotherapy for melanoma. However, events occurring in the tumor microenvironment following delivery have not been delineated. Therefore, utilizing a B16F10 mouse melanoma model, we evaluated changes in the tumor microenvironment following delivery of pIL-12 using different GET parameters or injection of plasmid alone. The results revealed a unique immune cell composition after intratumoral injection of pIL-12 GET. The number of immune memory cells was markedly increased in pIL-12 GET melanoma groups compared to control group. This was validated using flow cytometry to analyze peripheral blood mononuclear cells as well as delineating immune cell content using immunohistochemistry. Significant differences in multiple cell types were observed, including CD8+ T cells, regulatory T cells and myeloid cells, which were induced to mount a CD8+PD1− T cells immune response. Taken together, these findings suggest a basic understanding of the sequence of immune activity following pIL-12 GET and also illuminates that adjuvant immunotherapy can have a positive influence on the host immune response to cancer

Reduction of Plasmid Vector Backbone Length Enhances Reporter Gene Expression

Gene therapy has a wide range of applications for various types of pathologies. Viral methods of gene delivery provide high levels of gene expression but have various safety concerns. Non-viral methods are largely known to provide lower levels of expression. We aim to address this issue by using plasmid DNA with smaller backbones to increase gene expression levels when delivered using non-viral methods. In this study we compare gene expression levels between two vectors with firefly luciferase encoding gene insert using liposome complexes and gene electrotransfer as delivery methods. A 2-fold reduction in plasmid vector backbone size, disproportionately enhanced gene expression levels more than 10-fold in rat tenocytes in vitro, and rat myocardium in vivo, while improvements in delivery to the skin were more moderate

Secretion of Proteins and Antibody Fragments From Transiently Transfected Endothelial Progenitor Cells

In neurodegenerative diseases such as Alzheimer\u27s disease, Parkinson\u27s disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-beta-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-beta-amyloid Fab protein functions in beta-amyloid aggregate solubilization

Cardioporation Enhances Myocardial Gene Expression in Rat Heart

Damage from myocardial infarction (MI) and subsequent heart failure are serious public health concerns. Current clinical treatments and therapies to treat MI damage largely do not address the regeneration of cardiomyocytes. In a previous study, we established that it is possible to promote regeneration of cardiac muscle with vascular endothelial growth factor B gene delivery directly to the ischemic myocardium. In the current study we aim to optimize cardioporation parameters to increase expression efficiency by varying electrode configuration, applied voltage, pulse length, and plasmid vector size. By using a surface monopolar electrode, optimized pulsing conditions and reducing vector size, we were able to prevent ventricular fibrillation, increase survival, reduce tissue damage, and significantly increase gene expression levels

IL-12 Gene Electrotransfer Triggers a Change in Immune Response within Mouse Tumors

Metastatic melanoma is an aggressive skin cancer with a relatively low survival rate. Immune-based therapies have shown promise in the treatment of melanoma, but overall complete response rates are still low. Previous studies have demonstrated the potential of plasmid IL-12 (pIL-12) delivered by gene electrotransfer (GET) to be an effective immunotherapy for melanoma. However, events occurring in the tumor microenvironment following delivery have not been delineated. Therefore, utilizing a B16F10 mouse melanoma model, we evaluated changes in the tumor microenvironment following delivery of pIL-12 using different GET parameters or injection of plasmid alone. The results revealed a unique immune cell composition after intratumoral injection of pIL-12 GET. The number of immune memory cells was markedly increased in pIL-12 GET melanoma groups compared to control group. This was validated using flow cytometry to analyze peripheral blood mononuclear cells as well as delineating immune cell content using immunohistochemistry. Significant differences in multiple cell types were observed, including CD8+ T cells, regulatory T cells and myeloid cells, which were induced to mount a CD8+PD1− T cells immune response. Taken together, these findings suggest a basic understanding of the sequence of immune activity following pIL-12 GET and also illuminates that adjuvant immunotherapy can have a positive influence on the host immune response to cancer

Secretion of proteins and antibody fragments from transiently transfected endothelial progenitor cells

In neurodegenerative diseases such as Alzheimer\u27s disease, Parkinson\u27s disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-beta-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-beta-amyloid Fab protein functions in beta-amyloid aggregate solubilization

Abnormal junctions and permeability of myelin in PMP22-deficient nerves

The peripheral myelin protein-22 (PMP22) gene is associated with the most common types of inherited neuropathies, including hereditary neuropathy with liability to pressure palsies (HNPP) caused by PMP22 deficiency. However, the function of PMP22 has yet to be defined. Our previous study has shown that PMP22 deficiency causes an impaired propagation of nerve action potentials in the absence of demyelination. In the present study, we tested an alternative mechanism relating to myelin permeability

Tuning PAK Activity to Rescue Abnormal Myelin Permeability in HNPP

<div><p>Schwann cells in the peripheral nervous systems extend their membranes to wrap axons concentrically and form the insulating sheath, called myelin. The spaces between layers of myelin are sealed by myelin junctions. This tight insulation enables rapid conduction of electric impulses (action potentials) through axons. Demyelination (stripping off the insulating sheath) has been widely regarded as one of the most important mechanisms altering the action potential propagation in many neurological diseases. However, the effective nerve conduction is also thought to require a proper myelin seal through myelin junctions such as tight junctions and adherens junctions. In the present study, we have demonstrated the disruption of myelin junctions in a mouse model (<i>Pmp22</i>+/-) of hereditary neuropathy with liability to pressure palsies (HNPP) with heterozygous deletion of <i>Pmp22</i> gene. We observed a robust increase of F-actin in <i>Pmp22</i>+/- nerve regions where myelin junctions were disrupted, leading to increased myelin permeability. These abnormalities were present long before segmental demyelination at the late phase of <i>Pmp22</i>+/- mice. Moreover, the increase of F-actin levels correlated with an enhanced activity of p21-activated kinase (PAK1), a molecule known to regulate actin polymerization. Pharmacological inhibition of PAK normalized levels of F-actin, and completely prevented the progression of the myelin junction disruption and nerve conduction failure in <i>Pmp22</i>+/- mice. Our findings explain how abnormal myelin permeability is caused in HNPP, leading to impaired action potential propagation in the absence of demyelination. We call it “functional demyelination”, a novel mechanism upstream to the actual stripping of myelin that is relevant to many demyelinating diseases. This observation also provides a potential therapeutic approach for HNPP.</p></div

PAK inhibitor PF-3758309 blocks actin polymerization via PAK1.

<p>F-actin, phosphorylated PAK1 (S144, T212) and phosphorylated PAK2 (S20) were analyzed in <i>Pak1+/+</i> and <i>Pak1-/-</i> primary Schwann cell culture after the cells were treated with PF-3758309 (9μM) for 12 hour. PF-3758309 suppressed the levels of F-actin in <i>Pak1+/+</i> Schwann cells but failed to do so in <i>Pak1-/-</i> Schwann cells. The total actin and GAPDH were used as loading controls. Note that the specificity of S20 antibody has been demonstrated by Zhan et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref042" target="_blank">42</a>].</p

Conduction block was detected in naïve <i>Pmp22</i>+/- nerves.

<p><b>(A)</b> A diagram shows the setting for the experiments. A1-5 indicates the sites where the stimulation electrodes were placed on surgically exposed sciatic nerve. <b>(B)</b> In conventional NCS, <u>proximal</u> stimulation electrode is inserted blindly into the sciatic notch (white arrow in B). Variations of distances between the electrode and sciatic nerve (array of white asterisks) are not avoidable. This variation was eliminated by surgically exposing the sciatic nerves. Two black dots indicate the sites where <u>distal</u> stimulation electrodes were placed around ankle. <b>(C)</b> Area nearby ankle was dissected to reveal the tibial nerve (arrow in C). Due to the tiny space of this area, distance between the electrode and tibial nerve was highly consistent (two asterisks represent the sites of black dots in B). Thus, it did not require surgical exposure to place the distal stimulation electrodes. Note that needle electrode at the asterisk sites was inserted just through the dermis to avoid any nerve injury. <b>(D)</b> CMAP amplitudes were similar between A1 to A4. <b>(E)</b> CMAP in a <i>Pmp22</i>+/- mouse at A3-A5 showed a >50% reduction of the A2 amplitude. This finding demonstrated a conduction block that was defined as a ≥50% decrease of proximal CMAP amplitude over the distal CMAP amplitude, a stringent criterion used in human NCS [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref041" target="_blank">41</a>]. Conduction block was found in 12 out of 17 studied <i>Pmp22</i>+/- mice, but not in <i>Pmp22</i>+/+ mice. (<b>F</b>) CMAP was recorded from a different mouse and showed a distal latency (3.3ms) 2 times longer than that (1.2ms) in <i>Pmp22</i>+/+ nerve (A2 in D). The doubled distal latency was found in 2 mice out of the 17 <i>Pmp22</i>+/- mice, while the remaining 15 mice had variable degrees of prolonged distal latency. <b>(G)</b> CMAP in this mouse had a duration of 4ms (temporal dispersion) that was about twice longer than that in <i>Pmp22</i>+/+ nerve (A1 in D). In average, the CMAP duration in 17 <i>Pmp22</i>+/- mice (3.9±1.7ms) was significantly longer than that in 7 <i>Pmp22</i>+/+ mice (2.3±0.4ms; p = 0.001; 3–10 month old).</p