[ZucchiniFlower]

Public release date: 1-Mar-2007


University of Kentucky
DNA nanoparticles hold promise in gene therapy for Parkinson's disease


Michael J. Fox Foundation award allows researcher to explore new avenue in Parkinson's therapy development
Nanoparticle Gene Therapy for Parkinson's Disease -- This electron micrograph shows the minute scale of DNA compacted into nanoparticles. After it is condensed, the DNA is delivered to the brain...

http://www.eurekalert.org/pub_releas...-dnh022807.php

University of Kentucky researcher David Yurek was recently awarded $66,000 by The Michael J. Fox Foundation for Parkinson's Research (MJFF) under the foundation's Rapid Response Innovation Awards program. The goal of this newly launched initiative is to move quickly to support innovative research focused on the cause of and cure for Parkinson's disease (PD). In particular, MJFF seeks to fund high-risk, high-reward projects tackling critical scientific roadblocks that if successful, can open new avenues for PD therapy development.

Yurek's project, titled "Nanoparticle Gene Therapy for Parkinson's Disease," examines a relatively new gene therapy approach for treating neurodegenerative disorders. He is testing the feasibility of using a novel technology to condense DNA plasmids into nanoparticles and deliver them to the brain as a means to halt or prevent the neurodegenerative process.

The technology comes from Copernicus Therapeutics, Inc., a biotechnology company in Cleveland, Ohio. Yurek, whose laboratory is one of the first to apply this technology to central nervous system disorders, said this relatively new gene therapy strategy holds potential to help repair faulty genes. It entails transduction, a technique for expressing a particular gene in a cell by delivering DNA into the cell and making the cell synthesize the protein that corresponds to that DNA.

"We plan to use this technology to transduce brain cells so that they express proteins beneficial to the cell's survival," Yurek said.

The MJFF award will allow Yurek to test the feasibility of delivering condensed DNA nanoparticles that encode for a neurotrophic factor to the brain as a means to halt or prevent the neurodegenerative process in an animal model of PD. Neurotrophic factors are capable of protecting neurons from dying, thereby rescuing essential neurons in the brain. In animal studies, neurotrophic factors have revived dormant brain cells, caused them to produce dopamine, and prompted dramatic improvement of symptoms.

PD is a chronic, progressive disorder of the central nervous system, and is the direct result of the loss of cells in a section of the brain called the substantia nigra. Those cells produce dopamine, a chemical messenger responsible for transmitting signals within the brain. Loss of dopamine causes critical nerve cells in the brain, or neurons, to fire out of control, leaving patients unable to direct or control their movement in a normal manner.

The Michael J. Fox Foundation for Parkinson's Research is dedicated to ensuring the development of a cure for PD within this decade through an aggressively funded research agenda. Enormous progress toward finding a cure has been made on many neurological fronts, and scientists' understanding of the brain and how disease affects it has increased dramatically. The foundation seeks to hasten progress further by awarding grants that help guarantee that new and innovative research avenues are thoroughly funded and explored.

The MJFF Rapid Response Innovation Awards support projects that may have little to no existing preliminary data, but that hold potential to significantly impact understanding or treatment of PD.

"Given the extremely tight budget of federal government research funding, MJFF's work in prioritizing and funding new and innovative projects is extremely valuable," Yurek said.

[ZucchiniFlower]

Published online before print July 28, 2005, 10.1073/pnas.0504926102
PNAS | August 9, 2005 | vol. 102 | no. 32 | 11539-11544

NEUROSCIENCE
Organically modified silica nanoparticles: A nonviral vector for in vivo gene delivery and expression in the brain

Dhruba J. Bharali *, Ilona Klejbor

http://www.pnas.org/cgi/content/full/102/32/11539#top

CONCLUSIONS:

ORMOSIL nanoparticles ({approx}30 nm) are relatively easy to produce on a large scale, and the surfaces of these particles are readily modified during synthesis (10, 25). The addition of cationic groups to the surface of the ORMOSIL enhances binding with negatively charged plasmids for successful carriage inside the cells. This binding provides protection of the sensitive DNA structure from environmental insult during the process involved in in vivo transfer. This process of loading the nanoparticles with DNA for transfection is considerably simpler than the production of transfectable material and its encapsulation in viral particles. The present study demonstrates the ability of this formulation of nanoparticles to effectively traverse a biological barrier and transfect cells in vivo.

The efficiency of ORMOSIL-mediated transfection equaled or exceeded that obtained in previous studies using an herpes simplex viral vector (27). Tissue damage was also observed, caused by associated helper virus during herpes simplex virus 1 vector-mediated intrabrain gene transfer (26, 27). These pathological side effects illustrate problems that are associated with the in vivo gene transfers such as (i) carrier toxicity, (ii) injury due to immunological side effects, and (iii) conversion to a pathogenic form during the transfection process (37–39). In contrast, we observed no such toxic effects in the SN or in brain regions surrounding the LV in mice that received ORMOSIL injections over the time period used in this study. Cells displaying EGFP fluorescence for 10 days after injection indicate that the DNA transfection with ORMOSIL did not cause cellular degeneration. Furthermore, we have injected the same lateral brain ventricle twice 14 days apart (4-week experiment) with ORMOSIL/plasmid DNA and found no evidence of systemic or brain-specific toxicity. This apparent lack of toxicity, together with the exceptionally high efficacy of gene transfection, makes the ORMOSIL nanoparticles a promising experimental and potential therapeutic tool.

In vitro DNA transfection techniques have been used for decades now, and yet no nonviral technique has proven to be as effective as the viral vectors in vivo. Therefore, the transition from in vitro to in vivo systems, as reported here, represents a significant leap forward in the development of experimental techniques to study brain biology and the development of therapeutic approaches to neurological disease. In the present study, we have shown that ORMOSIL nanoparticles can be effectively used to introduce genes into the dopaminergic cells of the SNc. This approach should allow for the modeling of Parkinson's disease, which appears to have a diverse genetic/molecular background, by transfecting with mutant {alpha}-synuclein gene, by blocking FGF and glia-derived growth factor signaling with dominant-negative receptor mutants or by knocking down the parkin gene activity by using antisense or small interfering RNA technology (40–43). The ORMOSIL-mediated transfections of the midbrain dopaminergic neurons also would allow testing diverse gene therapeutic strategies for Parkinson's disease as well as for other disorders involving dopaminergic neurons.

The adult mammalian CNS has a limited potential to generate new neurons, making it vulnerable to injury and disease (23). To be able to therapeutically manipulate the endogenous adult NSPCs in the brain SVZ, it is crucial to identify the extracellular and intracellular signals that regulate division and control the fate of these cells (44, 45). In cultured cells and in the developing rat brain, FGFR1 was associated with the peripheral cytoplasm but also with the cell nuclei (46, 47). The cell-surface FGFR mediates the mitogenic effects of extracellular FGFs, whereas the nonmembrane nuclear FGFR1 signals withdrawal from the cell cycle and postmitotic growth (46). FGFR1(SP-/NLS), which does not associate with cell membranes and is expressed specifically in the nucleus, has been shown to stimulate differentiation of human neural progenitor cells in vitro and to directly influence gene activities without affecting the cell survival (28, 36, 48–55). The NSPCs in the adult brain SVZ include relatively quiescent self-renewing pluripotent neural stem cells and developmentally more restricted neural progenitors (35, 56). The single acute BrdUrd injection used in our study is likely to label predominantly the faster-proliferating progenitor cells. The inhibition of BrdUrd incorporation into the cells in SVZ and in rostral migratory stream by transfected FGFR1(SP-/NLS) observed in the present study demonstrates that the nuclear receptor controls the proliferation of the NSPCs in situ. These findings are consistent with our previous in vitro studies that showed that FGFR1(SP-/NLS) induces the neural progenitor cells' withdrawal from the cell cycle and initiates differentiation (46, 57). Thus, ORMOSIL-mediated in vivo transfection of the SVZ cells provides an effective means for elucidating the biology of stem/progenitor cells, allowing for the modification of these developing cells for therapeutic manipulations. ORMOSIL-based interventions could be developed to stimulate neurogenesis and axonal growth, to neutralize potential growth inhibitory molecules, to guide axons to their targets, and to establish new functional synapses. This ability will open perspectives for unraveling mechanisms that control the neural stem cells biology in vivo, providing a promising future direction for effective targeted brain therapy.

In summary, we have developed a synthetic system for the production of an in vivo nonviral transfection vector consisting of amino-terminated ORMOSIL nanoparticles. Intraventricular injection of ORMOSIL/pEGFP-N2 nanoparticles in the mouse brain resulted in the effective transfection and expression of EGFP in neuronal-like cells in periventricular brain regions and the SVZ. In addition, transfection with ORMOSIL/FGFR1(SP-/NLS) nanoparticles resulted in the modulation of the replication cycle of the stem/progenitor cells in the SVZ. These studies provide the groundwork for the use of ORMOSIL nanoparticle formulations for in vivo gene transfer into the CNS and have the potential to provide a safe and efficient mechanism for in vivo gene therapy applications.

*******************

First author: Bouazzaoui, Abdellatif (poster)

Poster board 446 - Tue 11/07, 16:00 - Hall Y
Session 200 - Parkinson's disease II
Abstract A200.6, published in FENS Forum Abstracts, vol. 3, 2006.
Ref.: FENS Abstr., vol.3, A200.6, 2006

Author(s) Bouazzaoui A. (1), Singh S. (1), Noblejas M. I. (1), Kubasch J. (2), Radunz H. E. (2) & Sabel B. A. (1)

Title Treatment of central nervous system (CNS) diseases using non-viral delivery system.

Text The use of non-viral carrier systems in basic medical research has especially attained great importance in the last few years. In our lab, we have developed a delivery system based on Polybutylcyanoacrylate Nanoparticle (PBCA-NP).

This type of nanoparticle allows permeation through the blood brain barrier and protect the substrate from premature biodegradation after in vivo administration.

We have already found in our previous studies that treatment of animals with the Doxorubicin-PBCA Nanoparticle complex leads to an extended life span of a treated animal compared to a control animal.

Binding of nanoparticle with DNA Oligos, and/or siRNA, is likewise possible. After binding of fluorescein isothiocyanate anti-sense oligonucleotide (FITC AON) to PBCA followed by coating with Tween® 80, i. v. injection of this complex leads to a concentration increase in the brain of the injected animals compared to those who received FITC AON alone.

Our next step is to try to use the PBCA-NP as DNA-delivery system for the treatment of CNS diseases like Morbus-Parkinson. In order to reach this goal, we use at the beginning a system based on the GFP-expression (pcDNA3.1-GFP). 250 µg of Cationic PBCA-NP (like DEAE- or Protasan-PBCA-NP) were added to 6, 25 µg DNA and then mixed in a thermomixer for 4 h. After the loading process is done, the PBCA-NP were centrifuged and coated with Tween® 80/NaCl. The DNA-NP-complex was added to 293T-cells. The GFP Expression kinetic was analysed from day 2 until day 6 by real-time PCR and the fluorescent microscopy. The highest expression was found on day 6 after transfection. The GFP expression achieve 3 x 107 compared to cells transfected with naked DNA or PBCA-NP alone.

We could conclude that the gene therapy using the PBCA-NP as targeting system is also possible.


Theme Neurological and psychiatric conditions
Neurodegenerative disorders / Parkinsons disease: Other