Showing papers in "Journal of Gene Medicine in 2009"

In vivo comparison of 2'-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping.

Abstract: Background Antisense-mediated exon skipping is a putative treatment for Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs), the disrupted DMD reading frame is restored, allowing generation of partially functional dystrophin and conversion of a severe Duchenne into a milder Becker muscular dystrophy phenotype. In vivo studies are mainly performed using 2′-O-methyl phosphorothioate (2OMePS) or morpholino (PMO) AONs. These compounds were never directly compared. Methods mdx and humanized (h)DMD mice were injected intramuscularly and intravenously with short versus long 2OMePS and PMO for mouse exon 23 and human exons 44, 45, 46 and 51. Results Intramuscular injection showed that increasing the length of 2OMePS AONs enhanced skipping efficiencies of human exon 45, but decreased efficiency for mouse exon 23. Although PMO induced more mouse exon 23 skipping, PMO and 2OMePS were more comparable for human exons. After intravenous administration, exon skipping and novel protein was shown in the heart with both chemistries. Furthermore, PMO showed lower intramuscular concentrations with higher exon 23 skipping levels compared to 2OMePS, which may be due to sequestration in the extracellular matrix. Finally, two mismatches rendered 2OMePS but not PMO AONs nearly ineffective. Conclusions The results obtained in the present study indicate that increasing AON length improves skipping efficiency in some but not all cases. It is feasible to induce exon skipping and dystrophin restoration in the heart after injection of 2OMePS and unconjugated PMO. Furthermore, differences in efficiency between PMO and 2OMePS appear to be sequence and not chemistry dependent. Finally, the results indicate that PMOs may be less sequence specific than 2OMePS. Copyright © 2009 John Wiley & Sons, Ltd.

Human beta-defensin-3 promotes wound healing in infected diabetic wounds.

Abstract: Background Infected wounds present a major complication in patients with diabetes. Staphylococcus aureus is the most common single isolate in diabetic wounds. Human beta-defensin (hBD)-3 is antimicrobial active and appears to play a key role in the immune response. The present study aimed to analyse the effect of hBD-3 expression in a model of infected diabetic wounds. Methods Excisional wounds were created on the backs of Yorkshire pigs and Ad5-CMV-hBD-3 vectors were microseeded. Wounds were inoculated with S. aureus, covered with a polyurethane chamber and analysed for transgene expression, bacterial infection, re-epithelialization, wound contraction, wound fluid production and blood vessel formation. Results hBD-3-treated wounds showed a total bacterial load of 2.1 × 108 colony-forming units (CFU)/g tissue, versus 1.3 × 109 CFU/g tissue for controls (p < 0.001) at day 4. At day 12, no statistical difference could be detected. Re-epithelialization showed 75 ± 15% wound closure for hBD-3 expressing wounds and 50 ± 16% for controls (p < 0.01). hBD-3 expression was in the range 15–20 ng/ml of wound fluid during day 1–4. The lower dose of 2 × 109 Ad5-CMV-hBD-3 showed no effect, suggesting a dose dependency for hBD-3. Conclusions In the present study, we show that hBD-3 expression significantly promotes wound closure in S. aureus infected diabetic wounds in a preclinical large-animal model. Furthermore, a ten-fold reduction of bacterial growth on day 4 was detected. These findings indicate that beta-defensin-3 may play a major role in diabetic wound healing and wound infections. Copyright © 2008 John Wiley & Sons, Ltd.

Retroviral vector-producing mesenchymal stem cells for targeted suicide cancer gene therapy.

Abstract: Background Mesenchymal stem cells (MSCs) are a promising vehicle for targeted cancer gene therapy because of their potential of tumor tropism. For efficient therapeutic application, we developed retroviral vector-producing MSCs that enhance tumor transduction via progeny vector production. Methods Rat bone marrow-derived MSCs were nucleofected with the proviral plasmids (vesicular stomatitis virus-G protein-pseudotyped retroviral vector components) (VP-MSCs) or pLTR plasmid alone (non-VP-MSCs). The luciferase-based in vivo imaging system was used to assess gene expression periodically. To evaluate the anticancer effects, we administered MSCs expressing herpes simplex virus-thymidine kinase (HSV-tk) into the left ventricular cavity of nude mice engrafted with 9L glioma cells subcutaneously. Results In vivo imaging revealed that administration of luciferase-expressing non-VP-MSCs enhanced the bioluminescence signal at the inoculation sites of 9L cells, whereas no accumulation was observed in mice at the site of the control Rat-1 fibroblasts. Compared to non-VP-MSCs, the administration of VP-MSCs resulted in significant augmentation of the signal with an increase in transgene copy number. Immunohistochemical analysis showed marked luciferase expression at the tumor periphery in mice injected with VP-MSCs, whereas little expression was detected in those injected with non-VP-MSCs. Under the continuous infusion of ganciclovir, systemic administration of VP-MSCs expressing HSV-tk suppressed tumor growth more effectively than non-VP-MSC administration, whereas no anticancer effect was observed without ganciclovir treatment. Furthermore, VP-MSC administration caused no transgene transduction in the normal tissues and organs. Conclusions VP-MSCs accumulated at the site of tumors after intravascular injection in tumor-bearing mice, followed by in situ gene transfer to tumors without transduction of normal organs. When applied to the HSV-tk/ganciclovir suicide gene therapy, more efficient tumor growth suppression was observed using VP-MSCs compared to non-VP-MSCs. This VP-MSC-based system has great potential for improved cancer gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.

Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures.

Abstract: Background Lentiviral vectors (LV) offer several advantages over other gene delivery vectors. Their potential for the integration and long-term expression of therapeutic genes renders them an interesting tool for gene and cell therapy interventions. However, large-scale LV production remains an important challenge for the translation of LV-based therapeutic strategies to the clinic. The development of robust processes for mass production of LV is needed. Methods A suspension-grown HEK293 cell line was exploited for the production of green fluorescent protein-expressing LV by transient polyethylenimine (PEI)-based transfection with LV-encoding plasmid constructs. Using third-generation packaging plasmids (Gag/Pol, Rev), a vesicular stomatitis virus G envelope and a self-inactivating transfer vector, we employed strategies to increase volumetric and specific productivity. Functional LV titers were determined using a flow cytometry-based gene transfer assay. Results A combination of the most promising conditions (increase in cell density, medium selection, reduction of PEI–DNA complexes per cell, addition of sodium butyrate) resulted in significantly increased LV titers of more than 150-fold compared to non-optimized small-scale conditions, reaching infectious titers of approximately 108 transducing units/ml. These conditions are readily scalable and were validated in 3-liter scale perfusion cultures. Conclusions Our process produces LV in suspension cultures and is consequently easily scalable, industrially viable and generated more than 1011 total functional LV particles in a single bioreactor run. This process will allow the production of LV by transient transfection in sufficiently large quantities for phase I clinical trials at the 10–20-liter bioreactor scale. Copyright © 2009 John Wiley & Sons, Ltd.

Neuroprotection in a 6-hydroxydopamine-lesioned Parkinson model using lactoferrin-modified nanoparticles

Abstract: Background Nonviral gene therapy of chronic degenerative diseases such as Parkinson’s disease (PD) is a great challenge as a result of the low tranfection efficiency of nonviral gene vectors. We previously constructed a lactoferrin (Lf)-modified vector, which was demonstrated to be potential for brain gene delivery both in vitro and in vivo .I n the present study, this type of vector was applied to load human glial cell line-derived neurotrophic factor gene (hGDNF). Methods A rat PD model was constructed by the unilateral lesion of striatum using 6-hydroxydopamine (6-OHDA). Lf-modified nanoparticles (NPs) were prepared and characterized. Neuroprotective effects of Lf-modified NPs were examined in the 6-OHDA-lesioned PD model via a regimen of multiple dosing intravenous administrations. Results The size of Lf-modified NPs was 196 ± 10.1 nm, whereas the zeta potential value was 29.35 ± 3.27 mV. Lf-modified NPs could protect themselves from heparin displacement and DNase digestion. The results of the neuroprotective evaluation show that increasing the number of injections of Lf-modified NPs loading hGDNF improved locomotor activity, reduced dopaminergic neuronal loss and enhanced monoamine neurotransmitter levels in PD rats. Five injections of Lf-modified NPs loading hGDNF exhibited much more powerful neuroprotection than a single injection, indicating the effectiveness and feasibility of multiple dosing administrations. The results of toxicity tests demonstrated that the dosage of NPs used in the present study was safe enough for brain gene delivery. Conclusions The findings obtained in the present study suggest that Lfmodified NPs could be developed for potential nonviral gene therapy of chronic brain disorders. Copyright  2009 John Wiley & Sons, Ltd.

Alkylcarboxylate grafting to polyethylenimine: a simple approach to producing a DNA nanocarrier with low toxicity.

Abstract: Background Various strategies have been examined to improve both transfection efficiency and cytotoxicity of polyethylenimine (PEI), a widely used polycationic nonviral gene vector. In the present study, we sought to improve PEI transfection efficiency by combining the osmotic burst mechanism for lysing endocytotic vesicles with the lipid depletion mechanism, which was accomplished by maintaining buffering capacity at the same time as adding a lipid-absorbing hydrophobic shell. Methods PEI was altered via the substitution of various percentages of its primary amines with carboxylate-terminated short, moderate and long alkyl chains, by reaction with bromoacetic, 6-bromohexanoic, 10-bromodecanoic and 16-bromohexadecanoic acids. Modified polymers were complexed with plasmid and the particle size and zeta potential of the polyplexes were determined. Ethidium bromide dye exclusion was used to show the DNA-binding ability of the polymers and their transfection activity and cytotoxicity was evaluated in Neuro2A mammalian cells. Results Decreased DNA-binding ability resulted from increases in either the degree of substitution or hydrocarbon chain length. Particle size and zeta potential measurements demonstrated that modified PEI polymers were able to form nanoparticles in the size range 60–195 nm, and surface charge decreased with an increasing degree of substitution. Higher degrees of substitution resulted in decreased cytotoxicity of polymers. Alkylcarboxylate substitution of primary amines on PEI enhanced transfection efficiencies by up to approximately five-fold relative to underivatized PEI, with the greatest increases occurring with 6-bromohexanoic acid derivatives at degrees of substitution below 10%. Conclusions The results obtained suggest that an appropriate balance between cationic and hydrophobic regions of alkylated PEI yields the optimal nonviral vector with high transfection efficiency and low toxicity. Copyright © 2009 John Wiley & Sons, Ltd.

Improvement of transfection efficiency by using supercoiled plasmid DNA purified with arginine affinity chromatography

Abstract: Background It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufacturing process. Several challenges encountered in the gene therapy field have emphasized the need for the development of novel platforms that allow the recovery of gene vectors and enable efficient transfection of cells. The use of plasmid DNA-based therapeutics relies on procedures that efficiently purify the supercoiled (sc) plasmid isoform. Plasmid DNA (pDNA) purification strategies that use amino acids as immobilized ligands have recently yielded interesting results. Methods The present study describes a strategy that uses arginine-chromatography to specifically purify sc pDNA from other isoforms and Escherichia coli impurities present in a clarified lysate. Results Control analysis shows that the purity of the sc pDNA is 100% with a homogeneity higher than 97% of sc. Furthermore, no RNA was detectable, the protein content was lower than 10 µg/ml and a 117-fold reduction on genomic DNA contamination and 95% endotoxin removal were accomplished. The chromatographic process demonstrated an impressive performance on sc isoform recovery (79% yield). Furthermore, the sc transfection efficiency of COS-7 cells (62%) was significantly higher compared to the efficiency (25%) achieved with a pDNA control. Conclusions With the simplified sc pDNA purification process, a high yield was achieved, sc pDNA was purified under mild conditions and shown to be extremely efficient with respect to cell transfection. Arginine-chromatography is thus an interesting option for use as a late stage plasmid purification step. Copyright © 2008 John Wiley & Sons, Ltd.

In vivo targeted gene delivery by cationic nanoparticles for treatment of hepatocellular carcinoma.

Abstract: Background Transgene expression in vivo for therapeutic purposes will require methods that allow for efficient gene transfer into cells. Although current vector technologies are being improved, the development of novel vector systems with improved targeting specificity, higher transduction efficiencies and improved safety is necessary. Methods Asialoglycoprotein receptor-targeted cationic nanoparticles for interleukin (IL)-12 encapsulation (NP1) or adsorption (NP2) have been formulated by blending poly(D,L-lactic-co-glycolic) acid (PLGA) (50 : 50) with the cationic lipid 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and the ligand asialofetuin (AF), by using a modified solvent evaporation process. Results We present a novel targeted lipopolymeric vector, which improves significantly the levels of luciferase gene expression in the liver upon i.v. administration. Targeted-NP2 particles showed a five- and 12-fold higher transfection activity in the liver compared to non-targeted (plain) complexes or naked pCMV DNA, respectively. On the other hand, BNL tumor-bearing animals treated with AF-NP1 containing the therapeutic gene IL-12, showed tumor growth inhibition, leading to a complete tumor regression in 75% of the treated mice, without signs of recurrence. High levels of IL-12 and interferon-γ were detected in the sera of treated animals. Mice survival also improved considerably. Tumor treatment with AF-NP2 formulations lead only to a retardation in the tumor growth. Conclusions In the present study, we have developed an efficient targeted non-viral vector for IL-12 gene transfer in hepatocellular carcinoma in vivo, by employing non-toxic cationic PLGA/DOTAP/AF nanoparticles. These results demonstrate for the first time that this cationic system could be used successfully and safely for delivery of therapeutic genes with antitumor activity into liver tumors with targeting specificity. Copyright © 2008 John Wiley & Sons, Ltd.

Single-dose lentiviral gene transfer for lifetime airway gene expression.

Abstract: BACKGROUND: Cystic fibrosis (CF) is caused by a defect in cystic fibrosis transmembrane conductance regulator (CFTR) activity, often resulting in an incurable airway disease. Gene therapy into the conducting airway epithelium is a potential cure for CF; however, most gene vectors do not result in long-lived expression, and require re-dosing. Perversely, intrinsic host immune responses can then block renewed gene transfer. METHODS: To investigate whether persistent gene expression could be achieved after a single dosing event, thus avoiding the issue of blocking host responses, we used a gene transfer protocol that combined an airway pretreatment using lysophosphatidylcholine with a human immunodeficiency virus type-1 (vesicular stomatitis virus G pseudotype) derived lentiviral vector to test whether an integrating vector could produce gene expression able to last for a substantial part of the lifetime of the laboratory mouse. RESULTS: We found that a single dose of LV-LacZ produced immediate as well as lifetime mouse airway expression, confirming our hypothesis that use of an integrating vector extends transgene expression. Importantly, LV-CFTR dosing achieved at least 12 months of CFTR expression, representing partial functional correction of the CFTR defect in CF-null mice. CONCLUSIONS: These findings validate the potential of this methodology for developing a gene transfer treatment for CF airway disease.

Intra-articular gene delivery and expression of interleukin-1Ra mediated by self-complementary adeno-associated virus.

Abstract: Background The adeno-associated virus (AAV) has many safety features that favor its use in the treatment of arthritic conditions; however, the conventional, single-stranded vector is inefficient for gene delivery to fibroblastic cells that primarily populate articular tissues. This has been attributed to the inability of these cells to convert the vector to a double-stranded form. To overcome this, we evaluated double-stranded self-complementary (sc) AAV as a vehicle for intra-articular gene delivery. Methods Conventional and scAAV vectors were used to infect lapine articular fibroblasts in culture to determine transduction efficiency, transgene expression levels, and nuclear trafficking. scAAV containing the cDNA for interleukin (IL)-1 receptor antagonist (Ra) was delivered to the joints of naive rabbits and those with IL-1β-induced arthritis. From lavage of the joint space, levels of transgenic expression and persistence were measured by enzyme-linked immunosorbent assay. Infiltrating leukocytes were quantified using a hemocytometer. Results Transgene expression from scAAV had an earlier onset and was approximately 25-fold greater than conventional AAV despite the presence of similar numbers of viral genomes in the nuclei of infected cells. Fibroblasts transduced with scAAV produced amounts of IL1-Ra comparable to those transduced with adenoviral and lentiviral vectors. IL1-Ra was present in lavage fluid of most animals for 2 weeks in sufficient quantities to inhibit inflammation of the IL-1β-driven model. Once lost, neither subsequent inflammatory events, nor re-administration of the virus could re-establish transgene expression. Conclusions scAAV-mediated intra-articular gene transfer is robust and similarly efficient in both normal and inflamed joints; the resulting transgenic expression is sufficient to achieve biological relevance in joints of human proportion. Copyright © 2009 John Wiley & Sons, Ltd.

Hyaluronic acid complexed to biodegradable poly L-arginine for targeted delivery of siRNAs

Abstract: Background Small interfering RNA (siRNA) has been recognized as a new therapeutic drug to treat various diseases by inhibition of oncogene or viral gene expression. Because hyaluronic acid (HA) has been described as a biocompatible biomaterial, we tested the nanoparticles formed by electrostatic complexation of negatively-charged HA and cationic poly L-arginine (PLR) for siRNA delivery systems. Methods Different electrostatic complexes of HA and PLR (HPs) were formulated: HP101 with 50% (w/w) HA and HP110 with 9% (w/w) HA. Results Gel retardation assays showed that HP101 and HP110 could form complexes with siRNAs. The diameters of these complexes were less than 200 nm. Cellular delivery efficiency of siRNAs by HPs depended on cell surface CD44 density. The HP-mediated delivery of siRNAs was highest in WM266.4 cells followed by B16F10 cells and COS-7 cells, in parallel with CD44 surface densities of these cell lines. TC50 values (i.e. the HP concentrations at which 50% of cells were viable after treatment) were used as indicators of cytotoxicity. HP101 showed TC50 values that were 2-fold and 23-fold higher than those of HP110 and PLR, respectively. After delivery into cells, siRNA exerted target-specific RNA interference effects on mRNA and protein levels. Three days after treatment of red fluorescent protein (RFP)-expressing B16F10 cells with RFP-specific siRNA complexed to HP101, cellular fluorescence signals were reduced. Intratumoral administration of RFP-specific siRNA via HP101 delivery significantly reduced the expression of RFP in tumor tissues. Conclusions HP101 may function as a biocompatible polymeric carrier of siRNAs and have possible application to localized siRNA delivery in vivo. Copyright © 2009 John Wiley & Sons, Ltd.

Effect of the content of unmethylated CpG dinucleotides in plasmid DNA on the sustainability of transgene expression.

Abstract: Background Nonviral gene transfer generally suffers from short-term expression of transgenes. We have previously demonstrated that plasmids with reduced CpG content exhibited a more prolonged expression of murine interferon (IFN)-β or IFN-γ, which was effective in inhibiting metastatic tumor growth. A further extension of the duration of transgene expression could be achieved by controlling the number and location of CpG motifs in plasmid DNA. Methods Luciferase-expressing plasmids with differing CpG content were injected into the tail vein of mice by the hydrodynamic injection method. The effects of CpG content on the duration of transgene expression were examined, focusing on cytosine methylation and pro-inflammatory cytokines. Based on the findings, IFN-γ-expressing plasmids were constructed and their transgene expression and inhibitory effect on pulmonary metastasis were evaluated. Results Plasmids with a few CpG motifs showed a prolonged luciferase activity in the liver. Methylation of CpG motifs in plasmids reduced the expression and the extent of this reduction was greater for plasmids with a high CpG content. Pro-inflammatory cytokines hardly affected the expression. pCpG-Muγ, the IFN-γ-expressing plasmid, which contains 20 CpG motifs only in the cDNA region, exhibited a sustained IFN-γ concentration at therapeutic levels, and had a great inhibitory effect on the pulmonary metastasis of tumor cells. Conclusions The duration of transgene expression of IFN-γ was successfully increased by reducing the CpG content of IFN-expressing plasmid vector, which resulted in an increased anticancer activity of IFN gene transfer. Copyright © 2009 John Wiley & Sons, Ltd.

Osteoinduction by microbubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2.

Abstract: Background Bone morphogenetic protein-2 (BMP-2) is believed to participate in bone healing and regeneration. Previous studies using BMP-2 in clinical applications have encountered difficulties that include the lack of an efficient, safe and simple delivery system, and expensive proteins and matrices. The gene transfer approach is a promising option for utilizing BMP-2. Viral vector-mediated gene transfer is efficient, but safety concerns prevent its clinical application for common diseases. Sonoporation is a simple and inexpensive method that only requires a plasmid and a sonoporation device. Methods We used a plasmid-based human BMP-2 construct (pCAGGS-BMP-2) and examined the induction of bone in the skeletal muscle of mice after plasmid transfer by transcutaneous sonoporation. First, an in vitro study was performed to confirm the expression of BMP-2 after gene transfer by sonoporation using pCAGGS-BMP-2. Next, the BMP-2 gene was transferred into the skeletal muscle of mice by transcutaneous sonoporation using pCAGGS-BMP-2. BMP-2 production was assessed via immunohistochemistry, and osteoinduction was verified by radiography, histology and biochemical assays. Results The presence of human BMP-2, alkaline phosphatase and osteocalcin mRNA and the production of the alkaline phosphatase were observed in vitro. Moreover, mature bone was observed in mice sonoporated with pCAGGS-BMP-2, confirming that transcutaneous sonoporation with pCAGGS-BMP-2 caused osteoinduction in the skeletal muscle of mice. Conclusions These results suggest the possibility of the effective clinical use of human BMP-2 gene therapy using transcutaneous sonoporation, and should facilitate clinical applications of BMP-2 gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.

Nitric oxide synthase gene therapy enhances the toxicity of cisplatin in cancer cells.

Abstract: Background Nitric oxide (NO center dot) derived from donor drugs has been shown to be an effective chemosensitizer in vitro We investigated the combination of inducible nitric oxide synthase (iNOS) gene transfer, driven by a strong constitutive promoter (cytomegalovirus; CMV) with the DNA cross-linking agent cisplatin in mouse and human tumour cell lines Methods Proof of principal experiments were performed in the radiation-induced fibrosarcoma-1 (RIF-1) murine cell line Cells were transfected with constitutively expressed CMV/iNOS plasmid DNA using a cationic lipid vector, before exposure to cisplatin In vivo efficacy was determined in an intradermal RIF-1 turnout model, with intraperitoneal administration of cisplatin Additionally, treatment potential was investigated in various human turnout cell lines including human prostate (DU145 and PC3) and human colon (HT29 and HCT116) cancer cell lines Experimental endpoints were established using western blot, Greiss test, clonogenic assay and turnout growth delay Results Transfection of RIF-1 turnout cells in vitro with the CMV/iNOS significantly enhanced the cytotoxicity of cisplatin (02-10 PM) In vivo transfer of CMV/iNOS by direct injection into established RIF-1 tumours caused a significant (p = 00027) delay in turnout growth CMV/iNOS gene transfer in vitro resulted in the strong expression of iNOS DNA in all cell lines, and significantly increased levels of NO center dot in all cell lines except HCT116 Conclusions Significant chemosensitization of cisplatin cytotoxicity was observed in the presence of NO center dot derived from the overexpression iNOS We conclude that p53 status of the various cell lines was unlikely to be responsible for cisplatin-induced apoptosis Copyright (C) 2008 John Wiley & Sons, Ltd

A versatile targeting system with lentiviral vectors bearing the biotin-adaptor peptide.

Abstract: Background Targeted gene transduction in vivo is the ultimate preferred method for gene delivery. We previously developed targeting lentiviral vectors that specifically recognize cell surface molecules with conjugated antibodies and mediate targeted gene transduction both in vitro and in vivo. Although effective in some experimental settings, the conjugation of virus with antibodies is mediated by the interaction between protein A and the Fc region of antibodies, which is not as stable as covalent conjugation. We have now developed a more stable conjugation strategy utilizing the interaction between avidin and biotin.

Subretinal delivery of adeno-associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice

Abstract: Background Adeno-associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer However, one potential barrier to efficacious long-term therapy is the development of immune responses against the vector or transgene product Methods We evaluated cellular and humoural responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression Results Following subretinal administration of vector, splenocytes and T-cells from draining lymph nodes showed minimal activation following stimulation by co-culture with AAV2 Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes Repeat administration of low-dose AAVhRPE65hRPE65 to both eyes of RPE65−/− mice resulted in transgene expression and functional rescue, but re-administration of high-dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye Conclusions These data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose-dependent Low-dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration Copyright © 2009 John Wiley & Sons, Ltd

An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression.

Abstract: Background The nuclear factor kappa B (NFκB) transcription factor, which shuttles between the cytoplasm and the nucleus under specific conditions, is a suitable intracellular target to increase the nuclear import of plasmid DNA. We report the design of an optimized and extended NFκB DNA binding sequence that promotes an efficient plasmid nuclear import. Methods On the basis of structural studies, the 5′-CTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGGACTTTCCAGG-3′ segment (termed 3NF) comprising three 10-bp κB sites (GGGACTTTCC) separated by a 5-bp optimized spacer (AGCTG) was selected for its capacity to ensure the best structural fit with NFκB and to fix simultaneously three proteins. Plasmids encoding luciferase and bearing this sequence (3NF-plasmids) were constructed and their nuclear import and gene expression efficiencies compared with that of plasmids containing classical κB motifs. Results A high luciferase expression was associated with plasmids containing one (p3NF-luc) or two (p3NF-luc-3NF) 3NF sequences. In situ hybridization experiments and quantitative measurement of the number of plasmid copies demonstrated that the nuclear delivery of 3NF-plasmids was more efficient than that of 3NF-free plasmids. Cross-linked immunoprecipitation showed that 3NF-plasmids were recognized by NFκB inside cells upon transfection. The nuclear delivery was inhibited with BAY 11-7085, an inhibitor of NFκB activation. Finally, p3NF-luc-3NF, the most efficient construct for in vitro transfection, had a long-lived luciferase expression in vivo. Conclusions The results obtained in the present study demonstrate the NFκB-mediated nuclear delivery of 3NF-plasmids. Due to its high affinity for fixing several NFκB, the 3NF sequence is a very promising helper for a nonviral gene delivery system. Copyright © 2009 John Wiley & Sons, Ltd.

Adenovirus type 5 with modified hexons induces robust transgene-specific immune responses in mice with pre-existing immunity against adenovirus type 5.

Abstract: Background Adenovirus type 5 (Ad5) is widely used as a vehicle for vaccine delivery in the treatment of infectious disease and cancer. However, the efficacy of Ad5 vectors has been limited in humans because exposure to Ad5 infections results in most adults having neutralizing antibodies against Ad5. To overcome this limitation, the hexon epitope present in the fifth hypervariable region of Ad5 was modified. Methods To evaluate the ability of Ad5 vectors encoding the HIV env protein to induce Ag-specific immune responses in the face of pre-existing anti-Ad5 immunity, mice were administrated intramuscularly with the Ad-Luc vector, and then vaccinated with parental or hexon-modified Ad5 vectors (Ad-HisHIV, Ad-END/AAAHIV or Ad-HIV) at week 8. HIV-specific cell-mediated immune responses were detected through a combination of tetramer assays and intracellular cytokine staining from weeks 8–23. Results The hexon-modified Ad vector was able to escape from anti-Ad5 neutralizing antibody, and mice with the modified vector generated significantly lower individual neutralizing antibody than those immunized with the parental vector. Furthermore, mice with pre-existing anti-Ad immunity immunized with the modified vector generated significantly stronger cell-mediated anti-env responses than those immunized with the parental vector. Conclusions These data demonstrate that Ad5 vector with hexon modification reduce their sensitivity to pre-existing anti-Ad immunity and improve their clinical utility. Copyright © 2009 John Wiley & Sons, Ltd.

Intranuclear fluorescence resonance energy transfer analysis of plasmid DNA decondensation from nonviral gene carriers.

Abstract: Background There has been considerable interest in researching the regulatory mechanisms that control transgene expression. In particular, there is impetus to investigate the intranuclear mechanisms for gene expression in order to improve the transfection efficiency of nonviral gene carriers. Methods To clarify the direct relationship between DNA decondensation and gene expression, plasmid DNA encoding Keima-Red fluorescent protein was doubly-labeled by a pair of donor–acceptor fluorescent dyes (fluorescein and Cy3), and transfected to HuH-7 cells using nonviral gene carriers: polyethylenimine (polyplex) and LipofectAMINE 2000 (lipoplex). Fluorescence resonance energy transfer analysis between the two dyes represents the condensation state of the pDNA. The intranuclear trafficking and condensation state of the pDNA were explored in transgene expressing and non-expressing cells under confocal laser scanning spectromicroscopy. Results The majority of transgene positively expressing cells transfected with polyplex and lipoplex had decondensed pixels in the nucleus. The majority of non-expressing cells transfected with polyplex had only condensed pixels in the nucleus. The majority of non-expressing cells transfected with lipoplex had no pixels in the nucleus. Conclusions Both polyplex and lipoplex marked a strong correlation between pDNA decondensation and transgene expression, yet the major limiting factor for transgene expression was different. In polyplex, the pDNA decondensation after nuclear entry was the major determining factor for transgene expression, whereas the nuclear entry itself was the chief determining step for transgene expression by lipoplex. This imaging technique allowed in situ observation of pDNA in the nucleus, providing important information about DNA behavior for gene expression. Copyright © 2009 John Wiley & Sons, Ltd.

Combining angiogenic gene and stem cell therapies for myocardial infarction

Abstract: Background Transplantation of stem cells from various sources into infarcted hearts has the potential to promote myocardial regeneration. However, the regenerative capacity is limited partly as a result of the low survival rate of the transplanted cells in the ischemic myocardium. In the present study, we tested the hypothesis that combining cell and angiogenic gene therapies would provide additive therapeutic effects via co-injection of bone marrow-derived mesenchymal stem cells (MSCs) with an adeno-associated viral vector (AAV), MLCVEGF, which expresses vascular endothelial growth factor (VEGF) in a cardiac-specific and hypoxia-inducible manner. Methods MSCs isolated from transgenic mice expressing green fluorescent protein and MLCVEGF packaged in AAV serotype 1 capsid were injected into mouse hearts at the border of ischemic area, immediately after occlusion of the left anterior descending coronary, individually or together. Engrafted cells were detected and quantified by real-time polymerase chain reaction and immunostaining. Angiogenesis and infarct size were analyzed on histological and immunohistochemical stained sections. Cardiac function was analyzed by echocardiography. Results We found that co-injection of AAV1-MLCVEGF with MSCs reduced cell loss. Although injection of MSCs and AAV1-MLCVEGF individually improved cardiac function and reduced infarct size, co-injection of MSC and AAV1-MLCVEGF resulted in the best improvement in cardiac function as well as the smallest infarct among all groups. Moreover, injection of AAV1-MLCVEGF induced neovasculatures. Nonetheless, injection of MSCs attracted endogenous stem cell homing and increased scar thickness. Conclusions Co-injection of MLCVEGF and MSCs in ischemic hearts can result in better cardiac function and MSC survival, compared to their individual injections, as a result of the additive effects of each therapy. Copyright © 2009 John Wiley & Sons, Ltd.

By-passing the nonsense mutation in the 4 CV mouse model of muscular dystrophy by induced exon skipping.

Abstract: BACKGROUND: Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by protein-truncating mutations in the dystrophin gene. Absence of functional dystrophin renders muscle fibres more vulnerable to damage and necrosis. We report antisense oligomer (AO) induced exon skipping in the B6Ros.Cg-Dmd(mdx-4Cv)/J (4(CV)) mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin exon 53. Both exons 52 and 53 must be excised to remove the mutation and maintain the reading frame. METHODS: A series of 2'-O-methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) was evaluated both in vitro and in vivo at the RNA and protein levels. RESULTS: Compared to the original mdx mouse studies, induction of exon skipping from the 4(CV) dystrophin mRNA was far more challenging. PMO cocktails could restore synthesis of near-full length dystrophin protein in cultured 4(CV) myogenic cells and in vivo, after a single intramuscular injection. CONCLUSIONS: By-passing the protein-truncating mutation in the 4(CV) mouse model of muscular dystrophy could not be achieved with single oligomers targeting both exons and was only achieved after the application of AO cocktails to remove exons 52 and 53. As in previous studies, the stability and efficiency of PMOs proved superior to 2OMeAOs for consistent and sustained protein induction in vivo.

Engineering adeno-associated virus serotype 2-based targeting vectors using a new insertion site-position 453-and single point mutations.

Abstract: Background Genetic modification of capsid proteins by peptide insertion has created the possibility of using adeno-associated viral (AAV) vectors for receptor specific gene transfer (AAV targeting). The most common site used for insertion in AAV serotype 2 capsids are amino acid positions 587 and 588 located at the second highest capsid protrusion. Reasoning that peptide insertions at the most exposed position augments target receptor interaction, we explored position 453 as a new insertion site. Methods Position 453 was identified in silico. Capsid mutants carrying the model ligand RGD-4C in position 453 with and without R585A/R588A substitutions were compared with respective mutants carrying the ligand in position 587. The accessibility of the inserted ligand was determined by an enzyme-linked immunosorbent assay, whereas the transduction efficiency and specificity of receptor binding were assayed by gene transfer and competition experiments, respectively. Vector biodistribution was determined in mice by quantitative polymerase chain reaction analysis. Results Initially, RGD-4C, inserted at position 453, failed to efficiently bind its target receptor. R585 and R588, located at the neighboring peak and known to mediate primary receptor binding, were identified as interfering residues. R585A and R588A substitutions rendered position 453 mutants superior to those with the ligand in position 587 in target receptor binding and cell transduction efficiency. The in vivo biodistribution was independent of the insertion site, but directed by the inserted ligand when primary receptor binding was avoided. Conclusions Position 453 emerged as a prominent site for the development of targeting mutants. Furthermore, we show for the first time that linearly distant residues can be critical for the efficiency of inserted peptide ligands. Copyright © 2009 John Wiley & Sons, Ltd.

In vitro and in vivo evaluation of small interference RNA-mediated gynaecophoral canal protein silencing in Schistosoma japonicum.

Abstract: Background Schistosomiasis causes liver and intestinal damage and can be very debilitating The pairing of a male worm with a female worm residing in the gynaecophoral canal of male plays a critical role in the development of female parasite Because the male specific gynaecophoral canal protein of Schistosoma japonicum (SjGCP) is found in significant quantities in the adult female worm after pairing, it could play an important role in parasite pairing Methods In the present study, three small interfering (si)RNA duplexes targeting the SjGCP gene were designed, synthesized and the silencing effects were evaluated in vitro as well as in mice infected with S japonicum in vivo Results In vitro studies using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR revealed the reduction of SjGCP at the transcript level Similarly, western blotting and immunofluorescence studies showed its reduction at the protein level after treatment of parasites with siRNAs At a concentration of 200 nM, two siRNAs totally abolished the parasite pairing To evaluate such a pairing inhibitory effect in vivo, mice infected with S japonicum were treated with siRNA and both parasite pairing and burden were evaluated In vivo tests confirmed the in vitro silencing effect of SjGCP siRNA and revealed that the systemic delivery of siRNA significantly inhibited early parasite pairing and the associated burden Conclusions Our preliminary results demonstrated that the SjGCP plays an important role in pairing and subsequent development in S japonicum, and its silencing might have potential as a therapeutic approach for controlling schistosomiasis Copyright © 2009 John Wiley & Sons, Ltd

Treatment of disseminated ovarian cancer using nonviral interleukin‐12 gene therapy delivered intraperitoneally

Abstract: Background The poor prognosis associated with ovarian cancer is primarily the result of delayed diagnosis and the lack of an effective treatment for advanced disease. Use of novel immunotherapy strategies are being evaluated that work to enhance local and systemic immune responses against cancer cells and can possibly work together with traditional cytotoxic chemotherapy regimens to produce more effective treatment options. Methods In the present study, we describe a gene-based therapy whereby the anticancer cytokine interleukin-12 gene (pmIL-12) is formulated with a synthetic polymeric delivery vehicle (PPC) and administered intraperitoneally into a mouse model of disseminated ovarian cancer. Results The administration of pmIL-12/PPC in tumor-bearing mice was associated with a shift towards a Th1 immune state, including significant increases in murine IL-12 (mIL-12) and interferon (IFN)-γ (mIFN-γ) in ascites fluid, with little change in systemic levels of these proteins. The mIL-12 protein was detectable for several days and could be reintroduced with subsequent injections. We show that treatment delayed the onset of ascites formation and improved survival in a dose-dependent manner. A significant decrease in vascular endothelial growth factor was associated with pmIL-12/PPC delivery and believed to play a predominant role in inhibiting ascites accumulation. Administration of pmIL-12/PPC was associated with minimal toxicity and, when combined with standard chemotherapies, resulted in additive improvement in survival. Conclusions Taken together, these results suggest that pmIL-12/PPC may be an effective strategy for inhibiting progression of disseminated ovarian cancer and may offer a new option for treatment of advanced disease that can be used to complement standard therapies. Copyright © 2009 John Wiley & Sons, Ltd.

Topical delivery of interleukin-13 antisense oligonucleotides with cationic elastic liposome for the treatment of atopic dermatitis.

Abstract: Background Interleukin (IL)-13, overproduced in the skin of atopic dermatitis (AD), has been shown to play an essential role in the pathogenesis of the disease. Thus, inhibition of IL-13 production should provide a key step to alleviate disease conditions of the atopic skin. In the present study, IL-13 antisense oligonucleotide (ASO) was designed and formulated with cationic elastic liposome (cEL) to improve transdermal delivery. Methods ASOs were generated against murine IL-13 mRNA (+4 to + 23) and complexed with cEL. Physicochemical properties of IL-13 ASO/cEL complex were examined by DNA retardation and DNase I protection assay. An in vitro inhibition study was performed in T-helper 2 (Th2) cells and cytotoxicity was tested by the XTT assay. The in vivo effect of IL-13 ASO/cEL complex was tested in a murine model of AD. Results In vitro, the IL-13 ASO/cEL complex showed dose- and ratio-dependent inhibition of IL-13 secretion in Th2 cells. At the IL-13 ASO/cEL ratio of 6, maximum inhibition of IL-13 secretion was observed. When applied to the ovalbumin-sensitized murine model of AD, topically administered IL-13 ASO/cEL complex dramatically suppressed IL-13 production (by up to 70% of the control) in the affected skin region. In addition, the levels of IL-4 and IL-5 were also significantly reduced. Moreover, IL-13 ASO/cEL-treated AD mice showed reduced infiltration of inflammatory cells into the epidermal and dermal areas, with concomitant reduction of skin thickness. Conclusions These data suggests that IL-13 ASO/cEL complex can provide a potential therapeutic tool for the treatment of AD and also be applied to other immune diseases associated with the production of Il-13. Copyright © 2008 John Wiley & Sons, Ltd.

Systemic adenoviral gene delivery to orthotopic murine breast tumors with ablation of coagulation factors, thrombocytes and Kupffer cells

Abstract: Background Rapid clearance of adenoviruses from blood by macrophage lineage cells of the liver and spleen, and binding to platelets, hinder their successful systemic use for cancer gene therapy. Vitamin K dependent coagulation factors are important mediators for the adenovirus liver tropism. Here we aim to determine the effects of coagulation factor, thrombocyte and liver macrophage (Kupffer cell) ablation on biodistribution of serotype 5 adenoviruses in mice with orthotopic breast tumors. Methods Prior to intravenous injection of adenoviruses, mice bearing orthotopic breast tumors were pretreated with warfarin to inhibit vitamin K dependent coagulation factor synthesis, an anti-platelet antibody causing thrombocytopenia or an inhibitor of the Kupffer cell scavenger receptor or their combination. Virus availability in blood after injection was determined from blood samples and transgene expression in tissues analyzed 72 hours afterwards with In Vivo Imaging and luciferase assays. Results Warfarin pretreatment reduced gene delivery to liver, spleen and lung. Kupffer cell ablation increased persistence of adenoviruses in blood but didn't affect biodistribution significantly. Depletion of Kupffer cells combined with thrombocytopenia doubled the systemic gene delivery of 5/3 chimeric adenovirus to tumors (p < 0.05). Triple ablation of platelets, Kupffer cells and coagulation factors increased the tumor to liver ratio of systemic adenovirus gene delivery by 81% (p < 0.05). Conclusions Depletion of coagulation factors can reduce transduction of liver, spleen and lung. Kupffer cell depletion is the most feasible method of increasing amount adenovirus in systemic blood flow and in combination with ablation of thrombocytes can increase the transduction of adenovirus to tumors. Copyright © 2009 John Wiley & Sons, Ltd.

Dexamethasone-conjugated polyethylenimine as an efficient gene carrier with an anti-apoptotic effect to cardiomyocytes.

Abstract: Background Dexamethasone is a potent glucocorticoid with anti-inflammatory effects. Dexamethasone can protect ischemic cardiomyocytes from apoptosis. To apply the anti-apoptotic effect of dexamethasone to ischemic disease gene therapy, dexamethasone-conjugated polyethylenimine (PEI-Dexa) was synthesized and evaluated as an anti-apoptotic gene carrier. Methods PEI-Dexa was synthesized with low molecular weight polyethylenimine (PEI2K, 2 kDa). The transfection efficiency and cytotoxicity of PEI-Dexa were evaluated by luciferase assay and the MTT assay. To evaluate the anti-apoptotic effect, PEI-Dexa/DNA complex was transfected into cells and the cells were treated with H2O2. Cell viability and apoptosis level were measured by the MTT assay and caspase-3 assay, respectively. Results A transfection assay into H9C2 rat cardiomyocytes showed that PEI-Dexa had the highest transfection efficiency at an 8 : 1 weight ratio (PEI-Dexa/DNA). At this ratio, PEI-Dexa had higher transfection efficiency than high molecular polyethylenimine (PEI25K, 25 kDa) and PEI2K. In addition, the cytotoxicity of PEI-Dexa was lower than that of PEI25K. To evaluate the anti-apoptotic effect, PEI-Dexa/pSV-Luc or PEI2K/pSV-Luc was transfected into H9C2 cells and the cells were treated with H2O2. PEI-Dexa was found to reduce caspase-3 activity and increase cell viability compared to PEI2K. Heme oxygenase-1 (HO-1) can protect ischemic cardiomyocytes from apoptosis. Therefore, pSV-HO-1 was cloned and transfected into H9C2 cells using PEI-Dexa. The cells transfected with PEI-Dexa/pSV-HO-1 complex had lower caspase-3 activity and higher viability than the cells transfected with PEI-Dexa/pSV-Luc complex after the H2O2 treatment. Conclusions PEI-Dexa is an efficient gene carrier with an anti-apoptotic effect and may be useful for anti-apoptotic gene therapy in combination with pSV-HO-1. Copyright © 2009 John Wiley & Sons, Ltd.

Use of internally nuclease‐protected single‐strand DNA oligonucleotides and silencing of the mismatch repair protein, MSH2, enhances the replication of corrected cells following gene editing

Abstract: Background Gene editing is potentially a powerful technology for introducing genetic changes by using short single-stranded DNA oligonucleotides (ssODNs). However, their efficiency is reduced by the mismatch repair system, especially MSH2, which may suppress gene editing, although findings vary depending on readout and type of oligonucleotide used. Additionally, successfully edited cells are reported to arrest at the S- or G2-phase. In the present study, we evaluate whether a novel ssODN design and down-regulation of MSH2 expression allows the isolation of replicating gene-edited cells. Methods Cultured Chinese hamster ovary cells expressing mutated enhanced green fluorescent protein were targeted with ssODNs of varying design, all capable of restoring fluorescence, which allows the monitoring of correction events by flow cytometry. Converted cells were isolated by cell sorting and grown to determine colony formation efficiencies. MSH2 expression was suppressed with small interfering RNA and the cell cycle distribution of cells transfected with ssODN was quantified by flow cytometry, following propidium iodide or DRAQ5 staining. Results Although efficiency was higher using ssODN end-protected with phosphorothioate, the potential of edited cells to form colonies was lower than those targeted with unmodified ssODN. We established that ssODN transfection itself perturbs the cell cycle and that MSH2 gene silencing increases correction efficiency. In both cases, however, the effect was dependent on the positioning of the protected nucleotides. Importantly, when internally protected ssODN was used in combination with MSH2 suppression, a higher proportion of G1-phase corrected cells was observed 48–64 h after transfection. Conclusions Use of internally protected ssODN and downregulating cellular MSH2 activity may facilitate isolation of viable, actively replicating gene-edited cells. Copyright © 2009 John Wiley & Sons, Ltd.

Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography

Abstract: Background Adenoviral vectors are used extensively in human gene therapy trials and in vaccine development Large-scale GMP production requires a downstream purification process, and liquid chromatography is emerging as the most powerful mode of purification, enabling the production of vectors at a clinically relevant scale and quality The present study describes the development of a two-step high-performance liquid chromatography (HPLC) process combining anion exchange (AIEX) and gel filtration (GF) in comparison with the caesium chloride density gradient method Methods HEK-293 cells were cultured in ten-layer CellStacks™ and infected with 10 pfu/cell of adenoviral vector expressing green fluorescent protein (Ad5-GFP) Cell-bound virus was harvested and benzonase added to digest DNA, crude lysate was clarified by centrifugation and filtration prior to HPLC Chromatography fractions were added to HEK-293 cells and GFP expression measured using a fluorescent plate reader Results Using AIEX then GF resulted in an adenoviral vector with purity comparable to Ad5-GFP purified by CsCl, whereas the reverse process (GF–AIEX) showed a reduced purity by electrophoresis and required further buffer exchange of the product The optimal process (AIEX–GF) resulted in a vector yield of 23 × 107 pfu/cm2 of cell culture harvested compared to 33 × 107 pfu/cm2 for CsCl The process recovery for the HPLC process was 36% compared to 275% for CsCl and total virion to infectious particle ratios of 18 and 11, respectively, were measured Conclusions We present a simple two-step chromatography process that is capable of producing high-quality adenovirus at a titre suitable for scale-up and clinical translation Copyright © 2009 John Wiley & Sons, Ltd

Neuron‐specific RNA interference using lentiviral vectors

Abstract: Background Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result Of the Strict requirement for precise transcriptional initiation and termination Recently, pol 11 promoters have been used to construct vectors for RNA interference (RNAi) By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding the possible promoter choices and eventually allowing cell type specific down-regulation of target genes Methods In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell Culture and in the brain Results We demonstrate robust kockdown of green fluorescent protein using lentiviral vectors driving RNAi from the ubiquitously-expressing promoter of the cytomegalovirus (CMV) and, in addition, we show for the first time neuron-specific knockdown in the brain using a neuron-specific promoter Furthermore, we show that the expression pattern of the presumed ubiquitously-expressing CMV promoter changes over time from being expressed initially in neurons and glial cells to being expressed almost exclusively in neurons in later stages Conclusions In the present study, we developed vectors for cell-specific RNAi for use in the brain This offers the possibility of specifically targeting RNAi to a subset of cells in a complex tissue and may prove to be of great importance in the design of future gene therapeutic paradigms Copyright (C) 2009 John Wiley & Sons, Ltd (Less)