Polymers for Improving the In Vivo Transduction Efficiency of AAV2 Vectors

Background: Adeno-associated virus has attracted great attention as vehicle for body-wide gene delivery. However, for the successful treatment of a disease such as Duchenne muscular dystrophy infusion of very large amounts of vectors is required. This not only raises questions about the technical feasibility of the large scale production but also about the overall safety of the approach. One way to overcome these problems would be to find strategies able to increase the in vivo efficiency. Methodology: Here, we investigated whether polymers can act as adjuvants to increase the in vivo efficiency of AAV2. Our strategy consisted in the pre-injection of polymers before intravenous administration of mice with AAV2 encoding a murine secreted alkaline phosphatase (mSeAP). The transgene expression, vector biodistribution and tissue transduction were studied by quantification of the mSeAP protein and real time PCR. The injection of polyinosinic acid and polylysine resulted in an increase of plasmatic mSeAP of 2- and 12-fold, respectively. Interestingly, polyinosinic acid pre-injection significantly reduced the neutralizing antibody titer raised against AAV2. Conclusions: Our results show that the pre-injection of polymers can improve the overall transduction efficiency of systemically administered AAV2 and reduce the humoral response against the capsid proteins

In Vivo Functional Genomic Studies of Sterol Carrier Protein-2 Gene in the Yellow Fever Mosquito

A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. The conventional method for delivery of DNA into insects is to inject the DNA directly into the embryos. Taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediates DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. Our method was able to introduce inducible gene expression vectors transiently into F0 mosquitoes to perform functional studies in vivo without transgenic lines. The high efficiency of expression knockdown was reproducible with more than 70% of the F0 individuals showed sufficient gene expression suppression (<30% of the controls' levels). At the cohort level, AeSCP-2 expression knockdown in early instar larvae resulted in detectable phenotypes of the expression deficiency such as high mortality, lowered fertility, and distorted sex ratio after induction of AeSCP-2 siRNA expression in vivo. The results further confirmed the important role of AeSCP-2 in the development and reproduction of A. aegypti. In this study, we proved that extrachromosaomal transient expression of an inducible gene from a DNA vector vertically delivered via vitellogenic females can be used to manipulate gene expression in F0 generation. This new method will be a simple and efficient tool for in vivo functional genomic studies in mosquitoes

Development of targeted therapy for ovarian cancer mediated by a plasmid expressing diphtheria toxin under the control of H19 regulatory sequences

<p>Abstract</p> <p>Background</p> <p>Ovarian cancer ascites fluid (OCAF), contains malignant cells, is usually present in women with an advanced stage disease and currently has no effective therapy. Hence, we developed a new therapy strategy to target the expression of diphtheria toxin gene under the control of H19 regulatory sequences in ovarian tumor cells. H19 RNA is present at high levels in human cancer tissues (including ovarian cancer), while existing at a nearly undetectable level in the surrounding normal tissue.</p> <p>Methods</p> <p>H19 gene expression was tested in cells from OCAF by the in-situ hybridization technique (ISH) using an H19 RNA probe. The therapeutic potential of the toxin vector DTA-H19 was tested in ovarian carcinoma cell lines and in a heterotopic animal model for ovarian cancer.</p> <p>Results</p> <p>H19 RNA was detected in 90% of patients with OCAF as determined by ISH. Intratumoral injection of DTA-H19 into ectopically developed tumors caused 40% inhibition of tumor growth.</p> <p>Conclusion</p> <p>These observations may be the first step towards a major breakthrough in the treatment of human OCAF, while the effect in solid tumors required further investigation. It should enable us to identify likely non-responders in advance, and to treat patients who are resistant to all known therapies, thereby avoiding treatment failure.</p

Tumor Lysing Genetically Engineered T Cells Loaded with Multi-Modal Imaging Agents

Genetically-modified T cells expressing chimeric antigen receptors (CAR) exert anti-tumor effect by identifying tumor-associated antigen (TAA), independent of major histocompatibility complex. For maximal efficacy and safety of adoptively transferred cells, imaging their biodistribution is critical. This will determine if cells home to the tumor and assist in moderating cell dose. Here, T cells are modified to express CAR. An efficient, non-toxic process with potential for cGMP compliance is developed for loading high cell number with multi-modal (PET-MRI) contrast agents (Super Paramagnetic Iron Oxide Nanoparticles – Copper-64; SPION-(64)Cu). This can now be potentially used for (64)Cu-based whole-body PET to detect T cell accumulation region with high-sensitivity, followed by SPION-based MRI of these regions for high-resolution anatomically correlated images of T cells. CD19-specific-CAR(+)SPION(pos) T cells effectively target in vitro CD19(+) lymphoma

In vitro evaluation of chitosan-EDTA conjugate polyplexes as a nanoparticulate gene delivery system

It was the purpose of this study to evaluate the potential of different molecular-weight chitosan-EDTA conjugates as a carrier matrix for nanoparticulate gene delivery systems. Covalent binding of EDTA to more than one chitosan chain provides a cross-linked polymer that is anticipated to produce stabilized particles. pDNA/chitosan-EDTA particles, generated via coazervation, were characterized in size and zeta potential by electrophoretic light scattering and electron microscopy. Stability was investigated at different pH values by enzymatic degradation and subsequent gel retardation assay. Lactate dehydrogenase assay was performed to determine toxicity. Furthermore, transfection efficiency into Caco-2 cells was assessed using a beta-galactosidase reporter gene. Chitosan-EDTA produced from low-viscous chitosan with 68% amino groups being modified by the covalent attachment of EDTA showed the highest complexing efficacy resulting in nanoparticles of 43 nm mean size and exhibiting a zeta potential of +6.3 mV. These particles were more stable at pH 8 than chitosan control particles. The cytotoxicity of chitosan-EDTA particles was below 1% over a time period of 4 hours. These new nanoplexes showed 35% improved in vitro transfection efficiency compared with unmodified chitosan nanoparticles. According to these results, the chitosan-EDTA conjugate may be a promising polymer for gene transfer

A practical approach for intracellular protein delivery

Protein delivery represents a powerful tool for experiments in live cells including studies of protein-protein interactions, protein interference with blocking antibodies, intracellular trafficking and protein or peptide biological functions. Most available reagents dedicated to the protein delivery allow efficient crossing of the plasma membrane. Nevertheless, the major disadvantage for these reagents is a weak release of the delivered protein into the cytoplasm. In this publication we demonstrate efficient protein delivery with a non-peptide based reagent, in human epithelial carcinoma HeLa cells and primary human skin fibroblasts. Using a fluorescent protein in combination with fluorescence microscopy and fluorescence-assisted cell sorting analysis, we show that the delivered protein is indeed released effectively in the cytoplasm, as expected for a dedicated carrier. Furthermore, we present a step-by-step method to optimize conditions for successful intracellular protein delivery