Nonmyeloablative allogeneic hematopoietic stem cell (HSC) transplantation can cure malignant and nonmalignant diseases affecting the hematopoietic system, such as severe combined immunodeficiencies, aplastic anemia and hemoglobinopathies. Although nonmyeloablative is favored over myeloablative transplantation for many patients, graft rejection remains problematic. One strategy to decrease rejection is to protect donor activated T cells in the graft from methotrexate (MTX) by genetically modifying the cells to express MTX-resistant dihydrofolate reductase (Tyr22-DHFR), leaving the immunosuppressive effects of MTX to act solely on activated host T lymphocytes, shifting the balance to favor allogeneic engraftment.

To evaluate MTX resistance of Tyr22-DHFR⁺ T lymphocytes in vivo, we transplanted dogs with autologous CD34⁺ cells modified with YFP and DHFR-GFP lentivirus vectors. Dogs were then treated with a standard MTX regimen (days 1, 3, 6, and 11) following immune activation with a foreign antigen as a surrogate assay to mimic early transplantation.

DHFR-GFP⁺ gene marking was maintained in CD3⁺CD25⁺ and CD4⁺ T lymphocytes after MTX treatment while the level of T lymphocytes that expressed only a fluorescent reporter (YFP⁺) decreased. These data show that Tyr22-DHFR expression protects T lymphocytes from MTX toxicity in dogs, highlighting a clinically relevant application for preserving donor T lymphocytes during post transplantation immunosuppression.

These findings have implications for clinical translation of MTX-resistant T cells to facilitate engraftment of allogeneic cells following nonmyeloablative conditioning and minimize the risk of rejection. In summary, Tyr22-DHFR expression in T lymphocytes provides chemoprotection from MTX-mediated elimination in the context of immune activation in vivo. This article is protected by copyright. All rights reserved.

Muscle represents an important tissue target for adeno-associated virus (AAV) vector-mediated gene transfer in muscular, metabolic or blood related genetic disorders. However, several studies demonstrated the appearance of immune responses against the transgene product after intramuscular AAV vector delivery which resulted in a limited efficacy of the treatment. Use of microRNAs (miRNAs) that are specifically expressed in antigen-presenting cells (APCs) is a promising approach to avoid those immune responses. Cellular mir-142-3p, which is APC-specific, is able to repress translation of its target cellular transcripts by binding to a specific target sequences.

In this study we explored the potential of mir-142-3p specific target sequences to reduce or abolish immune responses directed against ovalbumin (OVA), a highly immunogenic protein, expressed as transgene and delivered by AAV1 vector administered intramuscularly.

The occurrence of immune responses against OVA transgene following intramuscular delivery by AAV have been previously described and resulted in loss of OVA protein expression. In our study we demonstrate that OVA protein expression was maintained when mir-142-3pT sequences were incorporated in the expression cassette. The sustained expression of OVA protein over time correlated with a reduced increase in anti-OVA antibody level. Furthermore, no cellular infiltrates were observed in the muscle tissue when AAV1 vectors containing 4 or 8 repeats of mir-142-3p target sequences after OVA sequence were used.

Our data provides the evidence that humoral and cellular immune responses that rise against OVA protein after intramuscular delivery can be efficiently reduced by use of mir-142-3p target sequences. This article is protected by copyright. All rights reserved.

Previous studies suggested that reducing positive charge of melittin could increase endosomal release activity and improve BPEI-mediated transfection. AR-23 is a melittin-related peptide (MRP) from Rana tagoi, which shows 81% sequence identity with melittin, but has less positively charged residues than melittin. The aim of this study is to investigate the mechanistic and functional aspects of the interaction of AR-23 with mammalian cells and improvement of BPEI-mediated gene transfection.

AR23 and two AR-23 analogs (AR-20 without positively charged residues and AR-26 with the same positively charged residues as melittin) were analyzed. Circular Dichroism Spectrometry (CD) was used to analyze the secondary structures of the peptides. Peptide-induced depolarization of cell membrane, the membrane-lytic activity of the peptides and their potency in enhancing cellular uptake of calcein were evaluated. The physicochemical characters of complexes were measured and the effect of the peptides on BPEI mediated transfection was determined.

The CD spectra results indicated that positive charge in AR-23 played a curial role in maintaining α-helical conformation, while extra positive charge could not increase α-helical formation. AR-23 displayed similar depolarization ability to melittin. However, AR-23 showed lower membrane lytic activity at physiological condition and higher lytic activity at endosomal pH than melittin and AR-26 which posses more positive charges. As compared to melittin and AR-26, AR-23 with higher endosomal escaping activity resulted in higher enhancement in BPEI-mediated gene transfection while maintaining lower cytotoxicity.

We suggest that AR-23 may be considered as a potential enhancer for improving the transfection efficiency of cationic polymers. This article is protected by copyright. All rights reserved.

Gene therapy is an important treatment for genetic and acquired diseases. The success of gene therapy is largely dependent on the development of suitable vectors for gene transfer. Vectors are desired to overcome cellular barriers and to achieve efficient DNA delivery with low cytotoxicity. To this aim we synthesized and characterized novel comb-like oligoelectrolyte nanocarrier, called BG-2, as non-viral gene delivery vector.

A novel surface-active oligoelectrolyte of comb-like structure was synthesized via controlled radical copolymerization using oligoperoxide Cu⁺² coordinating complex as multi-site initiator of graft copolymerization. The Critical Micellar Concentration was determined by Nile Red fluorescence. Complex formation of DNA with BG-2 was determined by YOYO-1 fluorescence. The physico-chemical properties of DNA in complex with BG-2 have been investigated by electrophoresis, dynamic light scattering and fluorescence spectroscopy. BG-2/DNA complex was demonstrated by Scanning Electron Microscope (SEM). Interactions between BG-2/DNA complex and model membranes were studied. Sensitivity of DNA molecule, complexed with BG-2, against DNase I and serum nucleases was assessed by agarose gel electrophoresis. BG-2 efficiency in transfection of HeLa cells was determined by measuring the luciferase gene expression by luminometer and cytotoxicity was also evaluated.

BG-2 oligoelectrolyte was successful to overcome cellular barriers by forming stable and small sized complexes with DNA, interacting with model membranes in a desirable manner and protecting DNA from nuclease. Transfection efficiency was quite high and cytotoxicity was rather low.

BG-2 appears to be a promising nonviral vector with low cytotoxicity and efficient transfection properties. This article is protected by copyright. All rights reserved.

Non-viral lipid-based gene delivery vectors have been shown to possess better stability and longer circulation time after surface PEG modification. However, surface PEGylation may decrease the transfection efficiency dramatically. Here, we address the hypothesis that down regulating the lysosomal processing with a clinical available proton pump inhibitor omeprazole might decrease sequestration of PEGylated lipid-mu-DNA (LMD) in intracellular organelles, thereby increasing their transfection efficiencies.

LMD nanoparticles were prepared by the self-assembling of cationic liposomes, peptide mu and plasmid DNA. The characteristics of LMD lipopolyplexes were detected by scanning electron microscopy (TEM), photon correlation spectroscopy (PCS) and DNA gel electrophoresis. They were added into cultured cells with or without omeprazole pretreatment. The detailed cellular uptake and subcellular distribution were followed by flow cytometry and confocal microscopy. The gene transfection efficiencies were evaluated in four cell lines as well as in xenograft tumor models.

Lysosome staining revealed that 0.1 mg/ml (290 μM) omeprazole raised the pH value of intracellular acidic organelles and induced alterations in the lysosomal compartments. Confocal microscopy showed that more gene materials distributed from intracellular organelles to cytoplasms with omeprazole treatment. Luciferase gene transfection assay showed that omeprazole at probably non-toxic concentration increased PEGylated LMD transfection efficiency significantly in human (NCI-H1299, HT-1080 and A375) and mouse (4 T1) cell lines, as well as in tumors of H1299 xenograft tumor models (p < 0.05).

Omeprazole might be used as a helper to improve gene transfection efficiencies of some PEGylated gene delivery vectors in vitro and in vivo. This article is protected by copyright. All rights reserved.

Gene electrotransfer is a non-viral method used for DNA delivery into cells. Several steps are involved. One of them is interaction of DNA with the cell membrane, which is crucial before DNA can enter the cell. We analyzed the level of DNA-membrane interaction in relation to electrotransfer efficiency and importance of electrophoretic accumulation of DNA at the cell membrane. Systematic comparison of long duration, short duration and combinations of electropermeabilizing short HV and electrophoretic long LV pulses were performed. The effect of Mg²⁺ ions concentrations on electrotransfer and their effect on DNase activity were explored.

To visualize DNA-membrane interaction TOTO-1 labeled DNA was used. Transfection efficiency was assessed with plasmid DNA coding for GFP.

Higher relative electrotransfer efficiency was obtained by using longer pulses, while shorter pulses preserved cell viability. Short duration pulses enabled higher (24%) overall transfection yield compared to long duration pulses (12%), although higher DNA-membrane interaction was observed. No significant difference in transfection was obtained between different HV-LV pulsing protocols, although highest DNA-membrane interaction was observed with HV + LV pulses. Formation of DNA-membrane complex depended on Mg²⁺ concentration, while DNase inhibitor did not affect gene expression.

Gene electrotransfer is a complex phenomenon, where many factors are mutually affecting the process and DNA-membrane interaction is only the first step. We showed that longer electric pulses are optimal for higher transfection efficiency but reduce viability, while shorter pulses enable moderate transfection efficiency and preserve viability. Thus each application needs careful choice of pulsing protocol. Copyright © 2013 John Wiley & Sons, Ltd.

Many approaches have been suggested as anti-tumor therapy for targeting insulin-like growth factor 1 receptor (IGF-1R), such as monoclonal antibodies and tyrosine kinase inhibitor. We introduced recombinant adenoviruses expressing antisense, dominant negative or short hairpin RNA to IGF-1R. Moreover, we demonstrated that histone deacetylase inhibitor (vorinostat) can increase the transduction efficiency of adenoviruses by increasing CAR-induced transduction and by enhancing the transcription of the adenoviral transgene. In the present study, we showed that the combination of ad-sh (short hairpin) IGF-1R with vorinostat leads to a synergistic enhancement of IGF-1R blockade.

We measured the change in IGF-1R upon cotreatment with vorinostat and ad-shIGF-1R. Changes in transduction efficiency of ad-shIGF-1R were measured by fluorescent microscopy. Changes in apoptotic proportion and cell survival after the cotreatment were measured by the sub-G1 assay and cell counts. The effect of nuclear factor (NF)-κB activation was also measured by NF-κB p65 activation enzyme-linked immunosorbent assay. Drug interactions were analyzed upon cotreatment with ad-shIGF-1R, vorinostat and cisplatin.

Combined treatment of ad-shIGF-1R and vorinostat synergistically suppressed the IGF-1R expression in lung cancer cell lines and also increased the transduction efficiency of ad-shIGF-1R. Ad-shIGF-1R and vorinostat cotreatment increased apoptotic cell death and synergistically suppressed cell growth compared to ad-shIGF-1R or vorinostat treatment alone. Vorinostat suppressed NF-κB activation, which was activated by ad-shIGF-1R. Moreover, triple combination of ad-shIGF-1R, vorinostat and cisplatin demonstrated synergistic cytotoxicity on lung cancer cells.

Vorinostat enhanced the blocking capability of ad-shIGF-1R. The combined treatment of vorinostat and ad-sh-IGF-1R appears to have promising potential as a new therapeutic approach for lung cancer. Copyright © 2013 John Wiley & Sons, Ltd.

Gene transfer into the inner ear is a promising approach for treating sensorineural hearing loss. The special electrochemical environment of the scala media raises a formidable challenge for effective gene delivery at the same time as keeping normal cochlear function intact. The present study aimed to define a suitable strategy for preserving hearing after viral inoculation directly into the scala media performed at various postnatal developmental stages.

We assessed transgene expression of green fluorescent protein (GFP) mediated by various types of adeno-associated virus (AAV) and lentivirus (LV) in the mouse cochlea. Auditory brainstem responses were measured 30 days after inoculation to assess effects on hearing.

Patterns of GFP expression confirmed extensive exogenous gene expression in various types of cells lining the endolymphatic space. The use of different viral vectors and promoters resulted in specific cellular GFP expression patterns. AAV2/1 with cytomegalovirus promoter apparently gave the best results for GFP expression in the supporting cells. Histological examination showed normal cochlear morphology and no hair cell loss after either AAV or LV injections. We found that hearing thresholds were not significantly changed when the injections were performed in mice younger than postnatal day 5, regardless of the type of virus tested.

Viral inoculation and expression in the inner ear for the restoration of hearing must not damage cochlear function. Using normal hearing mice as a model, we have achieved this necessary step, which is required for the treatment of many types of congenital deafness that require early intervention. Copyright © 2013 John Wiley & Sons, Ltd.

The oncolytic adenovirus Delta24-RGD is currently being tested in phase I trials for the treatment of glioblastoma (GBM). Literature suggests that frequently prescribed anticonvulsants for these patients, phenytoin (PHE), valproic acid (VPA) and levetiracetam (LEV), may interfere with cellular mechanisms of cancer or oncolytic virus activity. We therefore investigated the direct effects of these drugs on Delta24-RGD infection and oncolytic activity.

The anticonvulsants PHE, VPA, and LEV were combined with Delta24-RGD treatment in established glioma cell lines as well as on a panel of patient-derived GBM cultures. Effects on infection efficiency were assessed using luciferase-encoding adenoviral vectors. Oncolytic activity was determined by WST-1 assay and viral progeny production was quantified by dilution titration.

IC50 values of the anti-epileptic drugs on the four glioma cell lines were far above clinically-relevant concentrations. At therapeutic concentrations, the anti-epileptics generally did not alter the infection efficiency of RGD-modified adenovirus, nor affect progeny production or oncolytic activity of Delta24-RGD. The only exception was found in U373 cells, where VPA slightly antagonised the oncolytic effect of Delta24-RGD (from 29% to 55% viability, p<0.01) as well as viral progeny production (60% decrease, p<0.01). Oncolysis by Delta24-RGD was not inhibited by the anti-epileptics in any of the patient-derived glioma cultures (n=6). In fact, in one culture a slight enhancement of viral oncolysis by PHE and LEV was found, from 89.7% viability to 76% and 62.4%, respectively (p<0.01)

Therapeutic levels of valproic acid, phenytoin and levetiracetam do not negatively interfere with the infection efficiency or oncolytic activity of Delta24-RGD in patient-derived GBM cells. Therefore, there is no indication that the choice of anticonvulsant for seizure control in glioma patients should take treatment with Delta24-RGD into account. Copyright © 2013 John Wiley & Sons, Ltd.

DNA delivery systems, which transport exogenous DNA to cells, have applications that include gene therapy, tissue engineering and medical devices. Although the cationic nonviral DNA carrier polyethyleneimine (PEI) has been widely studied, the molecular factors and pathways underlying PEI-mediated DNA transfer remain largely unknown, preventing the design of more efficient delivery systems.

HEK 293 T cells were treated with polyplexes formed with PEI and pEGFPLuc encoding for green fluorescent protein (GFP). Transfected cells expressing GFP were flow-separated from treated, untransfected cells. Gene expression profiles were obtained using Affymetrix HG-U133 2.0 microarrays and differentially expressed genes were identified using R/Bioconductor. Gene network analysis using EGAN (exploratory gene association network) bioinformatics tools was then used to find interaction among genes and enriched gene ontology (GO) terms related to transfection. Genes identified by this method were perturbed using pharmacologic activators or inhibitors to assess their effect on DNA transfer.

Microarray analysis comparing transfected cells to untransfected cells revealed 215 genes to be differentially expressed, with the majority enriched to GO processes including metabolism, response to stimulus, cell cycle, biological regulation and cellular component organization or biogenesis pathways. Gene network analysis revealed a coordinated induction of RAP1A, SCG5, PGAP1, ATF3 and NEB genes implicated in cell stress, cell cycle and cytoskeletal processes. Altering pathways with pharmacologic agents confirmed the potential role of RAP1A, SCG5 and ATF3 in transfection.

Microarray and gene network analyses of the sorted, transfected cell population can identify potential mediators of transfection, providing a basis for the design of improved delivery systems. Copyright © 2013 John Wiley & Sons, Ltd.

Genetic treatments of chronic arthritic conditions are essentially dependent on safe and efficient vector systems. To combine features of the efficient transduction of adenovirus vectors with the advantage of stable integration into the host cell genome of apathogenic prototype foamy virus vectors, hybrid vectors (FAD) have been established. In the present study, we have generated and investigated the use of safe FAD vectors for direct gene delivery to joints.

We generated recombinant FAD encoding enhanced green fluorescent protein (EGFP) or human interleukin 1 receptor antagonist protein (IL1RA) cDNA, and explored their transgene expression profile, as well as the bioactivity of the IL1RA transgene in vitro. The feasibility of IL1RA gene delivery to articular tissues was investigated in a pilot study employing direct FAD injections to the knee joints of Wistar rats.

FAD vectors efficiently transduced human or rat fibroblasts with EGFP or IL1RA transgene in vitro. Levels of IL1RA transgene expression were high, stable and functional in vitro. Transduced synovial fibroblasts and high levels of IL1RA protein (10–35 ng/ml) could be detected in vivo in the synovium of Wistar rats 3–5 days after injection of FAD vectors to the knee joints.

Our results indicate that FAD vectors are capable of efficient in vivo gene transfer to synovium and merit further investigation as a means of providing efficient and long-term intra-articular transgene expression for treatment of the arthritides. Copyright © 2013 John Wiley & Sons, Ltd.