Gene Therapy

Magnetic nanoparticle-mediated gene transfer to oligodendrocyte precursor cell transplant populations is enhanced by magnetofection strategies

by Stuart Jenkins

ACS Nano, 2011.
Stuart I Jenkins, Mark R Pickard, Nicolas Granger, Divya M Chari

This study has tested the feasibility of using physical delivery methods, employing static and oscillating field... more This study has tested the feasibility of using physical delivery methods, employing static and oscillating field "magnetofection" techniques, to enhance magnetic nanoparticle-mediated gene transfer to rat oligodendrocyte precursor cells derived for transplantation therapies. These cells are a major transplant population to mediate repair of damage as occurs in spinal cord injury and neurological diseases such as multiple sclerosis. We show for the first time that magnetic nanoparticles mediate effective transfer of reporter and therapeutic genes to oligodendrocyte precursors; transfection efficacy was significantly enhanced by applied static or oscillating magnetic fields, the latter using an oscillating array employing high-gradient NdFeB magnets. The effects of oscillating fields were frequency-dependent, with 4 Hz yielding optimal results. Transfection efficacies obtained using magnetofection methods were highly competitive with or better than current widely used nonviral transfection methods (e.g., electroporation and lipofection) with the additional critical advantage of high cell viability. No adverse effects were found on the cells' ability to divide or give rise to their daughter cells, the oligodendrocytes-key properties that underpin their regeneration-promoting effects. The transplantation potential of transfected cells was tested in three-dimensional tissue engineering models utilizing brain slices as the host tissue; modified transplanted cells were found to migrate, divide, give rise to daughter cells, and integrate within host tissue, further evidencing the safety of the protocols used. Our findings strongly support the concept that magnetic nanoparticle vectors in conjunction with state-of-the-art magnetofection strategies provide a technically simple and effective alternative to current methods for gene transfer to oligodendrocyte precursor cells.

Analysis of biodistribution and engraftment into the liver of genetically-modified mesenchymal stromal cells derived from adipose tissue.

by Gabriele Toietta

Giuliana Di Rocco, Antonietta Gentile, Annalisa Antonini, Silvia Truffa, Giulia Piaggio, Maurizio C Capogrossi, Gabriele Toietta
Cell Transplant. 2012 Mar 28. [Epub ahead of print]

Lifelong elimination of hyperbilirubinemia in the Gunn rat with a single injection of helper-dependent adenoviral vector

by Gabriele Toietta

Gabriele Toietta, Viraj P Mane, Wilma S Norona, Milton J Finegold, Philip Ng, Antony F McDonagh, Arthur L Beaudet, Brendan Lee
Proc Natl Acad Sci U S A. (2005) 102(11):3930-5.

Various cells retrovirally transduced with N-acetylgalactosoamine-6-sulfate sulfatase correct morquio skin fibroblasts in vitro

by Gabriele Toietta

Gabriele Toietta, Giovanni M Severini, Catia Traversari, Shunji Tomatsu, Kazuko Sukegawa, Seiji Fukuda, Naomi Kondo, Paolo Tortora, Claudio Bordignon
Hum Gene Ther. (2001) 12(16):2007-16.

Somatic Cell therapy: A Genetic Rescue for a Tattered Immune System?

by Bryn Williams-Jones

Williams-Jones, B. 2012. “Somatic Cell therapy: A Genetic Rescue for a Tattered Immune System?” BioéthiqueOnline 1/4

The case of Andrew Gobea, the first child to receive experimental gene therapy for SCID, and a reflection on the... more The case of Andrew Gobea, the first child to receive experimental gene therapy for SCID, and a reflection on the associated ethical implications of gene therapy research.

Optimization of ectopic gene expression in skeletal muscle through DNA transfer by electroporation

by Jordan Taylor

Bind-Predict: An algorithm for identifying zinc finger binding motifs in DNA sequences

by Jayakanthan M

Zinc fingers are the most abundant eukaryotic DNA-binding motifs, which recognize DNA triplets with high efficacy and... more Zinc fingers are the most abundant eukaryotic DNA-binding motifs, which recognize DNA triplets with high efficacy and are used to develop chimeric enzymes for genome modification and site specific gene therapy. Considering the importance of zinc finger motifs and their recognition patterns we have developed a web interface with the incorporation of sequence based search algorithm named as Bind-Predict. This algorithm facilitates the identification of target sites of zinc finger proteins (ZFPs) in the given DNA sequences. This paper describes the concepts of the developed algorithm (scoring system) and its applications in predicting ZFP binding sites. Bind-Predict web interface contains dual applications such as a web server (Bind-Predict tool) and a database (Bind-PredictdB). In order to enhance its utility and to validate the prediction results, Bind-Predicttool is interconnected with Bind-PredictdB. The combination of sequence based pattern recognition followed by computing Bind-Predict scores is used to identify the ZFP binding sites in the direct (5′→3′), complementary (3′→5′), double strand DNA (5′→3′ and 3′→5′) and user-defined binding sites of the input sequence. Currently Bind-PredictdB contains 374 entries of zinc finger motifs comprising 91 engineered constructs, 250 naturally occurring and 33 zinc binuclear clusters. Bind-Predict web interface is freely accessible at http://zfp.bicpu.edu.in.

SiO2 nanoparticles biocompatibility and their potential for gene delivery and silencing

by Giuseppe Vecchio

M.A. Malvindi, V. Brunetti, G. Vecchio, A. Galeone, R. Cingolani, and P.P. Pompa

Nanoscale (2011), DOI: 10.1039/C1NR11269D

Despite the extensive use of silica nanoparticles (SiO2NPs) in many fields, the results about their potential toxicity... more Despite the extensive use of silica nanoparticles (SiO2NPs) in many fields, the results about their potential toxicity are still controversial. In this work, we have performed a systematic in vitro study to assess the biological impact of SiO2NPs, by investigating 3 different sizes (25, 60 and 115 nm) and 2 surface charges (positive and negative) of the nanoparticles in 5 cell lines (3 in adherence and 2 in suspension). We analyzed the cellular uptake and distribution of the NPs along with their possible effects on cell viability, membrane integrity and generation of reactive oxygen species (ROS). Experimental results show that all the investigated SiO2NPs do not induce detectable cytotoxic effects (up to 2.5 nM concentration) in all cell lines, and that cellular uptake is mediated by an endocytic process strongly dependent on the particle size and independent of its original surface charge, due to protein corona effects. Once having assessed the biocompatibility of SiO2NPs, we have evaluated their potential in gene delivery, showing their ability to silence specific protein expression. The results of this work indicate that monodisperse and stable SiO2NPs are not toxic, revealing their promising potential in various biomedical applications.

Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction

by Dr Silvia Camporesi

Co-authored paper with Lorena Zentilin et al from the Molecular Medicine Lab at ICGEB, Trieste.

I worked on this project in 2006 while I was a master student in Medical Biotechnology and doing an internship in the Molecular Medicine Lab at ICGEB, Trieste.

Mounting evidence indicates that the function of members of the vascular endothelial growth factor (VEGF) family... more Mounting evidence indicates that the function of members of the vascular endothelial growth factor (VEGF) family extends beyond blood vessel formation. Here, we show that the prolonged intramyocardial expression of VEGF-A165 and VEGF-B167 on adeno-associated virus-mediated gene delivery determined a marked improvement in cardiac function after myocardial infarction in rats, by promoting cardiac contractility, preserving viable cardiac tissue, and preventing remodeling of the left ventricle (LV) over time. Consistent with this functional outcome, animals treated with both factors showed diminished fibrosis and increased contractile myocardium, which were more pronounced after expression of the selective VEGF receptor-1 (VEGFR-1) ligand VEGF-B, in the absence of significant induction of angiogenesis. We found that cardiomyocytes expressed VEGFR-1, VEGFR-2, and neuropilin-1 and that, in particular, VEGFR-1 was specifically up-regulated in hypoxia and on exposure to oxidative stress. VEGF-B exerted powerful antiapoptotic effect in both cultured cardiomyocytes and after myocardial infarction in vivo. Finally, VEGFR-1 activation by VEGF-B was found to elicit a peculiar gene expression profile proper of the compensatory, hypertrophic response, consisting in activation of αMHC and repression of βMHC and skeletal α-actin, and an increase in SERCA2a, RYR, PGC1α, and cardiac natriuretic peptide transcripts, both in cultured cardiomyocytes and in infarcted hearts. The finding that VEGFR-1 activation by VEGF-B prevents loss of cardiac mass and promotes maintenance of cardiac contractility over time has obvious therapeutic implications.—Zentilin, L., Puligadda, U., Lionetti, V., Zacchigna, S., Collesi, C., Pattarini, L., Ruozi, G., Camporesi, S., Sinagra, G., Pepe, M., Recchia, F. A., Giacca, M. Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction.

Binding properties of water soluble dendrimers.

by Barbara Klajnert

Co-authored with E. Pedziwiatr, D. Shcharbin, L. Chonco, P. Ortega, J. F. de la Mata, R. Gómez, M. Bryszewska published in J. Fluoresc. 2009, 19, 267

Dendrimers have been proposed as new carriers for drug delivery. They have distinctive characteristics, such as... more Dendrimers have been proposed as new carriers for drug delivery. They have distinctive characteristics, such as uniform and controlled size, monodispersity and modifiable surface group functionality, which make them extremely useful for biomedical applications. In this study, the binding capacity of water-soluble carbosilane dendrimers was examined. A double fluorimetric titration method with 1-anilinonaphthalene-8-sulphonic acid (ANS) was used to estimate the binding constant and the number of binding centers per dendrimer molecule. The data obtained suggest that ANS interacts non-covalently with the dendrimers. Second generation dendrimers have an open, asymmetric structure that allows them to encapsulate ANS. The ability of the polymers to interact with DNA was assessed by an ethidium bromide (EB) displacement assay. All the dendrimers studied bound to DNA in competition with EB, though the strength of binding varied. Dendrimer interactions with a protein (BSA) were tested using fluorescence quenchers. The dendrimers caused no conformation change in the protein, indicating that interactions between carbosilane dendrimers and BSA are weak and occur preferentially at the protein surface.

Experimental gene transfer to the corneal endothelium

by Daniel Kampik

Transfer of cDNA to corneal endothelial cells has been demonstrated in cell monolayers in vitro, in endothelium of... more Transfer of cDNA to corneal endothelial cells has been demonstrated in cell monolayers in vitro, in endothelium of whole thickness corneas ex vivo and following intracameral injection. Studies examining the feasibility and optimal methods for gene transfer to the cornea have used viral and non-viral vectors, initially histochemical or fluorescent marker genes, and in endothelium of numerous species ranging from mouse to man. As the feasibility of genetic modification of corneal endothelial cells has been successfully demonstrated in a number of cell culture and animal models, there is significant potential for gene transfer in the treatment of human corneal endothelial disease. The two most widely studied applications of gene transfer to endothelium are (i) transduction of immunomodulatory genes to donor corneal endothelium prior to transplantation as a strategy to delay allogeneic rejection and (ii) modulation of apoptosis or induction of replication in human corneal endothelial cells to increase cell density. Although continued improvements in vectors for gene transfer will improve the efficacy and safety of gene therapy, more detailed understanding of the altered biology of endothelium in disease will be necessary to allow selection of appropriate targets for a gene-based treatment approach.

Affinity Recovery of Lentivirus by Diaminopelargonic Acid Mediated Desthiobiotin Labelling

by Rongjun Chen

Published in Journal of Chromatography B 2010

Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a... more Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a 7,8-diaminopelargonic acid (7-DAPA) dependent manner for envelope independent, single-step affinity purification. 7-DAPA, which has little or no affinity for avidin/streptavidin, was synthesised and veri- fied by NMR spectroscopy and mass spectrometry. By expressing the biotin acceptor, biotin ligase and desthiobiotin synthase bioD, DBL cells converted exogenous 7-DAPA into membrane-bound desthiobi- otin. Desthiobiotin on the DBL cell surface was visualised by confocal microscopy and the desthiobiotin density was quantified by HABA-avidin assay. Desthiobiotin was then spontaneously incorporated onto the surface of lentiviral vectors produced by the DBL cells. It has been demonstrated by flow cytom- etry that the desthiobiotinylated lentiviruses were captured from the crude 7-DAPA-containing viral supernatant by Streptavidin Magnespheres® and eluted by biotin solution efficiently whilst retaining infectivity. The practical, high yielding virus purification using Pierce monomeric avidin coated columns indicates a highly efficient biotin-dependent recovery of infectious lentiviruses at 68%. The recovered lentiviral vectors had a high purity and the majority were eluted within 45 min. This 7-DAPA mediated desthiobiotinylation technology can be applied in scalable production of viral vectors for clinical gene therapy.

Non-Invasive Imaging In Gene Therapy

by Jani Räty

In vivo modulation of gene expression by lentiviral transduction in "human immune system" Rag2-/- gamma c -/- mice

by Nicolas Legrand

A.U. van Lent et al.; Methods in Molecular Biology (2010)

Over the last two decades, several humanized mouse models have been used to experimentally analyze the function and... more Over the last two decades, several humanized mouse models have been used to experimentally analyze the function and development of the human immune system. Recent advances have lead to the establishment of new murine-human chimeric models with improved characteristics, both in terms of human engraftment efficiency and in situ multilineage human hematopoietic development. We describe here the use of newborn BALB/c Rag2(-/-)gamma(c) (-/-) mice as recipients of human hematopoietic progenitor cells to produce "human immune system" (HIS) (BALB-Rag/gamma) mice, using human fetal liver progenitors. The two major subsets of the human dendritic cell lineage, namely, BDCA2(+)CD11c(-) plasmacytoid dendritic cells and BDCA2(-)CD11c(+) conventional dendritic cells, can be found in HIS (BALB-Rag/gamma) mice. In order to manipulate the expression of genes of interest, the human hematopoietic progenitor cells can be genetically engineered ex vivo by lentiviral transduction before performing xenograft transplantation. Using this mouse model, the human immune system can be assessed for both fundamental and pre-clinical purposes.

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

by Nicolas Legrand

M. Centlivre et al.; Gene Therapy (2010)

The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze... more The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze inducible gene expression in a human setting, we generated ‘human immune system’ (HIS) mice by transplanting newborn BALB/c Rag2−/−IL-2Rγc−/− immunodeficient mice with human hematopoietic stem cells transduced with a doxycycline-inducible lentiviral vector. We compared several methods of doxycycline delivery to mice, and could accurately measure doxycycline in vivo using a new sensitive detection assay. Two different lentiviral vector designs with constitutive (TRECMV-V14) or autoregulatory (TREAuto-V14) expression of an optimized reverse tetracycline transactivator were used to transduce human hematopoietic stem cells. After transplantation into immunodeficient mice, we analyzed the expression of the green fluorescent protein (GFP) reporter gene in the human hematopoiesis-derived cells that develop and accumulate in the generated HIS mice. We show efficient inducible GFP expression in adult HIS mice containing TREAuto-V14-transduced human cells, whereas GFP expression is poor with the TRECMV-V14 vector. Multiple cycles of doxycycline exposure in the TREAuto-V14 group result in repeated cycles of GFP expression with no loss of intensity. These findings are of major interest for gene therapy and basic research settings that require inducible gene expression.

A sensitive cell-based assay to measure the doxycycline concentration in biological samples

by Nicolas Legrand

W. Kleibeuker et al.; Human Gene Therapy (2009)

Doxycycline (DOX) is widely used as a pharmacological agent and as an effector molecule in inducible gene expression... more Doxycycline (DOX) is widely used as a pharmacological agent and as an effector molecule in inducible gene expression systems. For most applications, it is important to determine whether the DOX concentration reaches the level required for optimal efficacy. We developed a sensitive bioassay for measuring the DOX concentration in biological samples. We used a modified HeLa cell line with the luciferase reporter gene under the control of the DOX-inducible Tet-On system for regulation of gene expression. These HeLaDOX cells constitutively express a novel variant of the rtTA transcriptional activator protein that is highly DOX-sensitive. Incubation of the cells with a DOX-containing biological sample triggers luciferase expression, which can be quantitated by standard methods. This bioassay is sensitive, with a DOX detection limit of 22 ng/ml in plasma. The assay was used to determine the DOX concentration in plasma derived from DOX-treated rhesus macaques and mice. Furthermore, we found that the DOX concentration in murine cerebrospinal fluid is 31-fold lower than the concurrent plasma DOX level. This bioassay for the quantification of DOX concentration in biological samples has several advantages over high-performance liquid chromatography-based and microbiological assays: (1) multiple samples can be assayed in a single experiment; (2) only small sample volumes are required; (3) the assay has a low detection limit; and (4) the assay can be performed in any cell culture laboratory.

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2-/-γc-/-) mouse model

by Nicolas Legrand

O. ter Brake et al.; Gene Therapy (2009)

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can... more RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2−/−γc−/− mice are engrafted with human CD34+CD38− hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4+ T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.

Functional Human Antigen-Specific T Cells Produced In Vitro Using Retroviral T Cell Receptor Transfer into Hematopoietic Progenitors

by Nicolas Legrand

A.U. van Lent et al.; The Journal of Immunology (2007)

In vitro production of human T cells with known Ag specificity is of major clinical interest for immunotherapy against... more In vitro production of human T cells with known Ag specificity is of major clinical interest for immunotherapy against tumors and infections. We have performed TCR{alpha}beta gene transfer into human hemopoietic progenitors from postnatal thymus or umbilical cord blood, and subsequently cultured these precursors on OP9 stromal cells expressing the Notch human ligand Delta-like1. We report here that fully mature, functional T cells with controlled Ag specificity are obtained from such cultures. Using vectors encoding TCR{alpha}beta-chains directed against melanoma (MART-1), viral (CMV), and minor histocompatibility (HA-2) Ags, we show that the obtained Ag-specific T cells exert cytolytic activity against their cognate Ag and expand in vitro upon specific TCR stimulation. Therapeutic applications may arise from these results because they provide a way to produce large numbers of autologous mature Ag-specific T cells in vitro from undifferentiated hemopoietic progenitors.

Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2-/- {gamma}c-/- mice: functional inactivation of p53 in developing T cells

by Nicolas Legrand

R. Gimeno et al.; Blood (2004)

Tumor suppressor p53 plays an important role in regulating cell cycle progression and apoptosis. Here we applied RNA... more Tumor suppressor p53 plays an important role in regulating cell cycle progression and apoptosis. Here we applied RNA interference to study the role of p53 in human hematopoietic development in vivo. An siRNA construct specifically targeting the human tumor-suppressor gene p53 was introduced into human CD34+ progenitor cells by lentivirus-mediated gene transfer, which resulted in more than 95% knockdown of p53. We adapted the human-SCID mouse model to optimize the development of hematopoietic cells, particularly of T cells. This was achieved by the intraperitoneal injection of CD34+ precursor cells into newborn Rag2-/- {gamma}c-/- mice that lack T, B, and NK cells. Robust development of T cells was observed in these mice, with peripheral T-cell repopulation 8 weeks after injection of the precursor cells. Other lymphocyte and myeloid subsets also developed in these mice. Injecting p53 siRNA-transduced CD34+ cells resulted in stable expression and down-modulation of p53 in the mature T-cell offspring. Inactivating p53 did not affect the development of CD34+ cells into various mature leukocyte subsets, including T cells, but it conferred resistance to {gamma}-irradiation and other p53-dependent apoptotic stimuli to the T cells.