996-11 Direct Gene Transfer and Expression with Arterial Iontophoretic Catheter Delivery

Iontophoresis is a technique of molecular delivery which uses electric current to enhance movement of charged molecules into tissues. A porous balloon catheter was tested with a central silver chloride electrode capable of generating a potential gradient across the arterial wall using an adhesive patch placed on the skin to serve as the anode. We hypothesized that this catheter delivery system might effectively transfer negatively charged plasmid DNA into arterial cells in vivo.MethodsTo localize plasmid DNA arterial delivery, a 7 Fr iontophoretic porous balloon catheterwast,’aced into porcine carotid arteries underflouroscopic guidance. 15μg of 35S-Iabeled plasmid DNA (1.4×106 cpm/μg) expressing the heat stable human alkaline phosphatase (hAP) gene with an RSV promoter was infused through the balloon at 6 atm pressure. A constant current density of 2.5 mA/cm2 was maintained for 10 minutes. The ;35S-labeled plasmid DNA delivery was repeated on the contralateral carotid artery under identical conditions with the absence of electric current. 20 minutes after gene transfer, the arteries were fixed in situ and processed for autoradiography. To analyze gene transfer and expression, 8 porcine carotid arterial segments were subject to iontophoretic gene delivery for 10 minutes at 6 atm with a current density of 2.5 mA/cm2 using the RSV hAP plasmid (n=6) or control plasmid (n=2). Animals were sacrificed 5 days after gene delivery and the transfected arteries analyzed by PCR and heat stable alkaline phosphatase histochemistry.ResultsAutoradiography of the arteries which underwent ;35S-labeled plasmid delivery revealed minimal radiolabel in the luminal cells of the control artery in which current was not delivered. In contrast, significant amounts of radiolabel were present in the media and adventitia of the artery subject to current delivery. PCR analysis of the arterial segments studied 5 days after delivery confirmed gene transfer in all hAP segments and was negative in control arteries. Staining for heat stable recombinant alkaline phosphatase activity demonstrated recombinant protein expression in 5% of medial cells and 10% of adventitial cells in arteries which underwent hAP gene transfer. Control arteries were negative for hAP staining.ConclusionsIontophoretic catheter gone delivery can be used to perform direct plasmid DNA delivery with expression of recombinant protein in medial and adventitial cells

Safety and Short-Term Toxicity of a Novel Cationic Lipid Formulation for Human Gene Therapy

Overview summary Although several viral vectors have been widely applied to the treatment of human disease, the development of nonviral vectors is still in their infancy. In this report, a novel cationic lipid, DMRIE/DOPE, has been incorporated into the DNA–liposome formulation that improves transfection efficiencies and allows up to 1,000-fold higher concentrations of DNA to be administered in vivo. In this paper, the safety and toxicity of this formulation is described in two species, mice and pigs, suggesting that it may prove useful for human gene therapy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63224/1/hum.1993.4.6-781.pd

Human Ovarian Tumor Cells Escape γδ T Cell Recognition Partly by Down Regulating Surface Expression of MICA and Limiting Cell Cycle Related Molecules

Background: Mechanisms of human Vc2Vd2 T cell-mediated tumor immunity have yet to be fully elucidated. Methods and Findings: At least some tumor cell recognition is mediated by NKG2D-MICA interactions. Herein, by using MTT assay and PI-BrdU co-staining and Western-blot, we show that these Vc2Vd2 T cells can limit the proliferation of ovarian tumor cells by down regulation of apoptosis and cell cycle related molecules in tumor cells. Cell-to-cell contact is critical. cd T cell-resistant, but not susceptible ovarian tumor cells escape cd T cell-mediated immune recognition by up-regulating pErk1/2, thereby decreasing surface MICA levels. Erk1/2 inhibitor pretreatment or incubation prevents this MICA decrease, while up-regulating key cell cycle related molecules such as CDK2, CDK4 and Cyclin D1, as well as apoptosis related molecules making resistant tumor cells now vulnerable to cd T cell-mediated lysis. Conclusion: These findings demonstrate novel effects of cdT cells on ovarian tumor cells

Stem Cell Therapy with Overexpressed VEGF and PDGF Genes Improves Cardiac Function in a Rat Infarct Model

Therapeutic potential was evaluated in a rat model of myocardial infarction using nanofiber-expanded human cord blood derived hematopoietic stem cells (CD133+/CD34+) genetically modified with VEGF plus PDGF genes (VIP).Myocardial function was monitored every two weeks up to six weeks after therapy. Echocardiography revealed time dependent improvement of left ventricular function evaluated by M-mode, fractional shortening, anterior wall tissue velocity, wall motion score index, strain and strain rate in animals treated with VEGF plus PDGF overexpressed stem cells (VIP) compared to nanofiber expanded cells (Exp), freshly isolated cells (FCB) or media control (Media). Improvement observed was as follows: VIP>Exp> FCB>media. Similar trend was noticed in the exercise capacity of rats on a treadmill. These findings correlated with significantly increased neovascularization in ischemic tissue and markedly reduced infarct area in animals in the VIP group. Stem cells in addition to their usual homing sites such as lung, spleen, bone marrow and liver, also migrated to sites of myocardial ischemia. The improvement of cardiac function correlated with expression of heart tissue connexin 43, a gap junctional protein, and heart tissue angiogenesis related protein molecules like VEGF, pNOS3, NOS2 and GSK3. There was no evidence of upregulation in the molecules of oncogenic potential in genetically modified or other stem cell therapy groups.Regenerative therapy using nanofiber-expanded hematopoietic stem cells with overexpression of VEGF and PDGF has a favorable impact on the improvement of rat myocardial function accompanied by upregulation of tissue connexin 43 and pro-angiogenic molecules after infarction

Human Umbilical Cord Blood-Derived CD34+ Cells Reverse Osteoporosis in NOD/SCID Mice by Altering Osteoblastic and Osteoclastic Activities

Osteoporosis is a bone disorder associated with loss of bone mineral density and micro architecture. A balance of osteoblasts and osteoclasts activities maintains bone homeostasis. Increased bone loss due to increased osteoclast and decreased osteoblast activities is considered as an underlying cause of osteoporosis.The cures for osteoporosis are limited, consequently the potential of CD34+ cell therapies is currently being considered. We developed a nanofiber-based expansion technology to obtain adequate numbers of CD34(+) cells isolated from human umbilical cord blood, for therapeutic applications. Herein, we show that CD34(+) cells could be differentiated into osteoblastic lineage, in vitro. Systemically delivered CD34(+) cells home to the bone marrow and significantly improve bone deposition, bone mineral density and bone micro-architecture in osteoporotic mice. The elevated levels of osteocalcin, IL-10, GM-CSF, and decreased levels of MCP-1 in serum parallel the improvements in bone micro-architecture. Furthermore, CD34(+) cells improved osteoblast activity and concurrently impaired osteoclast differentiation, maturation and functionality.These findings demonstrate a novel approach utilizing nanofiber-expanded CD34(+) cells as a therapeutic application for the treatment of osteoporosis

Open data from the third observing run of LIGO, Virgo, KAGRA and GEO

The global network of gravitational-wave observatories now includes five detectors, namely LIGO Hanford, LIGO Livingston, Virgo, KAGRA, and GEO 600. These detectors collected data during their third observing run, O3, composed of three phases: O3a starting in April of 2019 and lasting six months, O3b starting in November of 2019 and lasting five months, and O3GK starting in April of 2020 and lasting 2 weeks. In this paper we describe these data and various other science products that can be freely accessed through the Gravitational Wave Open Science Center at https://gwosc.org. The main dataset, consisting of the gravitational-wave strain time series that contains the astrophysical signals, is released together with supporting data useful for their analysis and documentation, tutorials, as well as analysis software packages.Comment: 27 pages, 3 figure

Search for Eccentric Black Hole Coalescences during the Third Observing Run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass $M>70$ $M_\odot$) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities $0 < e \leq 0.3$ at $0.33$ Gpc$^{-3}$ yr$^{-1}$ at 90\% confidence level.Comment: 24 pages, 5 figure