Combined Gene Delivery Research Articles

One Endothelium-Targeted Combined Nucleic Acid Delivery System for Myocardial Infarction Therapy.

Acute myocardial infarction (MI) and ischemic heart disease are the leading causes of heart failure and mortality. Currently, research on MI treatment is focused on angiogenic and anti-inflammatory therapies. Although endothelial cells (ECs) are critical for triggering inflammation and angiogenesis, no approach has targeted them for the treatment of MI. In this study, we proposed a nonviral combined nucleic acid delivery system consisting of an EC-specific polycation (CRPPR-grafted ethanolamine-modified poly(glycidyl methacrylate), CPC) that can efficiently codeliver siR-ICAM1 and pCXCL12 for the treatment of MI. Animals treated with the combination therapy exhibited better cardiac function than those treated with each nucleic acid alone. In particular, the combination therapy of CPC/siR-ICAM1 and CPC/pCXCL12 significantly improved cardiac systolic function, anti-inflammatory responses, and angiogenesis compared to the control group. In conclusion, CPC-based combined gene delivery systems show impressive performance in the treatment of MI and provide a programmed strategy for the development of codelivery systems for various EC-related diseases.

Controlled pVEGF delivery via a gene-activated matrix comprised of a peptide-modified non-viral vector and a nanofibrous scaffold for skin wound healing

Regulating cell function and tissue formation by combining gene delivery with functional scaffolds to create gene-activated matrices (GAMs) is a promising strategy for tissue engineering. However, fabrication of GAMs with low cytotoxicity, high transfection efficiency, and long-term gene delivery properties remains a challenge. In this study, a non-viral DNA delivery nanocomplex was developed by modifying poly (D, L-lactic-co-glycolic acid)/polyethylenimine (PLGA/PEI) nanoparticles with the cell-penetrating peptide KALA. Subsequently, the nanocomplex carrying plasmid DNA encoding vascular endothelial growth factor (pVEGF) was immobilized onto a polydopamine-coated electrospun alginate nanofibrous scaffold, resulting in a GAM for enhanced skin wound healing. The nanocomplex exhibited much lower cytotoxicity and comparable or even higher transfection efficiency compared with PEI. The GAM enabled sustained gene release and long-tern transgene expression of VEGF in vitro. In an excisional full-thickness skin wound rat model, the GAM could accelerate wound closure, promote complete re-epithelization, reduce inflammatory response, and enhance neovascularization, ultimately enhancing skin wound healing. The current GAM comprising a low-toxic gene delivery nanocomplex and a biocompatible 3D nanofibrous scaffold demonstrates great potential for mediating long-term cell functions and may become a powerful tool for gene delivery in tissue engineering. Statement of significanceGene delivery is a promising strategy in promoting tissue regeneration as an effective alternative to growth factor delivery, but the study on three-dimensional gene-activated scaffolds remains in its infancy. Herein, a biodegradable nanofibrous gene-activated matrix integrating non-viral nanoparticle vector was designed and evaluated both in vitro and in vivo. The results show that the nanoparticle vector provided high transfection efficiency with minimal cytotoxicity. After surface immobilization of the nanocomplexes carrying plasmid DNA encoding vascular endothelial growth factor (pVEGF), the nanofibrous scaffold enabled sustained DNA release and long-term transgene expression in vitro. In a rat full-thickness skin wound model, the scaffold could accelerate wound healing. This innovative gene-activated matrix can be a promising candidate for tissue regeneration.

Rapidly Transducing and Spatially Localized Magnetofection Using Peptide-Mediated Non-Viral Gene Delivery Based on Iron Oxide Nanoparticles.

Non-viral delivery systems are generally of low efficiency, which limits their use in gene therapy and editing applications. We previously developed a technology termed glycosaminoglycan (GAG)-binding enhanced transduction (GET) to efficiently deliver a variety of cargos intracellularly; our system employs GAG-binding peptides, which promote cell targeting, and cell penetrating peptides (CPPs), which enhance endocytotic cell internalization. Herein, we describe a further modification by combining gene delivery and magnetic targeting with the GET technology. We associated GET peptides, plasmid (p)DNA, and iron oxide superparamagnetic nanoparticles (MNPs), allowing rapid and targeted GET-mediated uptake by application of static magnetic fields in NIH3T3 cells. This produced effective transfection levels (significantly higher than the control) with seconds to minutes of exposure and localized gene delivery two orders of magnitude higher in targeted over non-targeted cell monolayers using magnetic fields (in 15 min exposure delivering GFP reporter pDNA). More importantly, high cell membrane targeting by GET-DNA and MNP co-complexes and magnetic fields allowed further enhancement to endocytotic uptake, meaning that the nucleic acid cargo was rapidly internalized beyond that of GET complexes alone (GET-DNA). Magnetofection by MNPs combined with GET-mediated delivery allows magnetic field-guided local transfection in vitro and could facilitate focused gene delivery for future regenerative and disease-targeted therapies in vivo.

Gold Nanoparticles Mediated Drug-Gene Combinational Therapy for Breast Cancer Treatment.

BackgroundCancer is a complex heterogeneous disease to which singular modes of treatment mostly fail to produce a desired therapeutic efficacy. Targeting different cellular pathways using combinational therapies has been gaining popularity in cancer treatment, with the added benefit of reducing dosage and side effects.MethodsA gold nanoparticle-mediated drug delivery nanoplatform was developed for co-delivery of doxorubicin and polo-like kinase 1 (PLK1) siRNA. Gold nanoparticles were coated with polyethyleneimine to facilitate assembly of PLK1 on the surface. Doxorubicin was loaded on nanoparticles through a pH-sensitive linker with a thiol group at one terminal end for controlled release.ResultsThe therapeutic efficiency of this co-delivery system was evaluated in 2D and 3D cultured systems. The reduced IC50 value clearly demonstrated the synergistic effect of combined drug and gene delivery over their individual delivery in a cancer treatment model.ConclusionThis study may provide an adaptable, facile platform to investigate drug-siRNA combinations for cancer inhibition.

Bimodal Targeting Using Sulfonated, Mannosylated PEI for Combined Gene Delivery and Photodynamic Therapy.

Photodynamic therapy (PDT) and gene delivery have both been used to target both cancer cells and tumor-associated macrophages (TAMs). Given the complex nature of tumor tissue, there could be merit in combining these strategies simultaneously. In this study, we developed a bimodal targeting approach to both cancer cells and macrophages, employing materials conducive to both gene delivery and PDT. Polymers libraries were created that consisted of cationic polyethyleneimine (PEI) conjugated to the photosensitizer pyropheophorbide-a, with sulfonation (to target selectin-expressing cells) and mannosylation (to target TAMs). Polyplexes, consisting of these polymers electrostatically bound to DNA, were analyzed for transfection efficacy and cytotoxicity toward epithelial cells and macrophages to assess dual-targeting. This study provides preliminary proof of principle for using modified PEI for targeted gene delivery and PDT.

Regenerative Medicine: Novel Approach in Burn Wound Healing

Context: Burn wounds of the skin require a long period to healing, which very often is incomplete, with functional and esthetic consequences for the patients. Stem cells in the traumatized tissue represent the promoters of the healing process and are a primary focus for regenerative medicine, which aims to find and use the triggers for the activation of stem cells of sin tissue. Evidence Acquisition: At present, tissue engineering, composite epithelial autografts, multipotent stem cells and combined gene delivery with stem cell therapy are the approaches used in regenerative medicine. Alongside, the development of 3D scaffolds or matrices is a promising adjunct, as studies investigate the multiple uses of these supports for wound repair. Results: Application of cells to the burn wound could be performed, either by the bedside, as a non-invasive procedure, or in the operating room, with the use of a matrix, scaffold or dermal substitute. Cell spraying, although under use in clinical setting, is not yet supported by conclusive data. Magnetic resonance imaging, optical imaging and positron emission tomography are currently used to assess the viability and location of stem cells, after transplantation. Conclusions: Stem cell therapies in wound care may lessen the morbidities associated with wound healing. An ideal method for the effective administration of stem cells for burn patients has not yet been elucidated. Further comparison of the local and systemic effects in burn patients, associated with each route of stem cell delivery, needs to be performed.

Multifunctional Poly(L-lactide)-Polyethylene Glycol-Grafted Graphene Quantum Dots for Intracellular MicroRNA Imaging and Combined Specific-Gene-Targeting Agents Delivery for Improved Therapeutics.

Photoluminescent (PL) graphene quantum dots (GQDs) with large surface area and superior mechanical flexibility exhibit fascinating optical and electronic properties and possess great promising applications in biomedical engineering. Here, a multifunctional nanocomposite of poly(l-lactide) (PLA) and polyethylene glycol (PEG)-grafted GQDs (f-GQDs) was proposed for simultaneous intracellular microRNAs (miRNAs) imaging analysis and combined gene delivery for enhanced therapeutic efficiency. The functionalization of GQDs with PEG and PLA imparts the nanocomposite with super physiological stability and stable photoluminescence over a broad pH range, which is vital for cell imaging. Cell experiments demonstrate the f-GQDs excellent biocompatibility, lower cytotoxicity, and protective properties. Using the HeLa cell as a model, we found the f-GQDs effectively delivered a miRNA probe for intracellular miRNA imaging analysis and regulation. Notably, the large surface of GQDs was capable of simultaneous adsorption of agents targeting miRNA-21 and survivin, respectively. The combined conjugation of miRNA-21-targeting and survivin-targeting agents induced better inhibition of cancer cell growth and more apoptosis of cancer cells, compared with conjugation of agents targeting miRNA-21 or survivin alone. These findings highlight the promise of the highly versatile multifunctional nanocomposite in biomedical application of intracellular molecules analysis and clinical gene therapeutics.

Combined Nurr1 and Foxa2 roles in the therapy of Parkinson's disease.

Use of the physiological mechanisms promoting midbrain DA (mDA) neuron survival seems an appropriate option for developing treatments for Parkinson's disease (PD). mDA neurons are specifically marked by expression of the transcription factors Nurr1 and Foxa2. We show herein that Nurr1 and Foxa2 interact to protect mDA neurons against various toxic insults, but their expression is lost during aging and degenerative processes. In addition to their proposed cell-autonomous actions in mDA neurons, forced expression of these factors in neighboring glia synergistically protects degenerating mDA neurons in a paracrine mode. As a consequence of these bimodal actions, adeno-associated virus (AAV)-mediated gene delivery of Nurr1 and Foxa2 in a PD mouse model markedly protected mDA neurons and motor behaviors associated with nigrostriatal DA neurotransmission. The effects of the combined gene delivery were dramatic, highly reproducible, and sustained for at least 1 year, suggesting that expression of these factors is a promising approach in PD therapy.

Multifunctional polymeric nanoplexes for anticancer co-delivery of p53 and mitoxantrone.

Co-delivery of the anticancer drug, mitoxantrone (MTO) and the gene encoding tumor suppressor protein p53 was evaluated towards anticancer combinatorial therapy. The nanoplatforms developed herein are assembled by coupling β-cyclodextrin and the cationic polymer, polyethyleneimine to a hydrophilic polymer, pullulan (PPEICD). The β-cyclodextrin serves as a nanocontainer for the drug MTO, while the cationic moiety can condense pDNA. Acid base titrations provided insight into the buffering capacity of the PPEICD conjugate. Cytotoxicity studies by MTT assay in HepG2 and C6 cell lines and hemocompatibility studies confirmed the conjugate to be nontoxic and hemocompatible. In vitro release studies of MTO in phosphate buffered saline pH 7.4 showed an initial burst effect followed by a slow drug release. The released data fitted with the Korsmeyer-Peppas model and their diffusional exponents suggest that the drug release from the polymeric system followed diffusion and non-Fickian transport. Combined drug and gene loaded nanoplexes have a more apoptotic effect than either the drug or gene individually as confirmed by MTT assay and live dead assay. This indicated the significance of the combined drug and gene delivery system and the ability of the nanoplatform to overcome the multidrug resistance (MDR) of MTO. Moreover, preference of asialoglycoprotein receptor (ASGPR) mediated internalization for nanoparticle cellular uptake in HepG2 cell lines was identified by treating with the inhibitor asialofetuin. Cell studies in both HepG2 and C6 cell lines demonstrated that the nanoplatform PPEICD can efficiently and selectively deliver both p53 and MTO to cancer cells inducing high cell death.

Synergistically Combined Gene Delivery for Enhanced VEGF Secretion and Antiapoptosis

With current pharmacological treatments, preventing the remodeling of the left ventricle and the progression to heart failure is a difficult task. Gene therapy is considered to provide a direct treatment to the long-term complications of ischemic heart diseases. Although current gene therapies that use single molecular targets seem potentially possible, they have not achieved success in the treatment of ischemic diseases. With an efficient polymeric gene carrier, PAM-ABP, we designed a synergistically combined gene-delivery strategy to enhance vascular endothelial growth factor (VEGF) secretion and to prolong its antiapoptotic effects. A hypoxia-inducible plasmid expressing both hypoxia-inducible heme oxygenase-1 (HO-1) and the Src homology domain-2 containing tyrosine phosphatase-1 microRNA (miSHP-1) as well as a hypoxia-responsive VEGF plasmid were combined in this study. The positive feedback circuit between HO-1 and VEGF and the negative regulatory role of SHP-1 in angiogenesis enhance VEGF secretion synergistically. The synergy in VEGF secretion as a consequence of the gene combination and prolonged HO-1 activity was confirmed in hypoxic cardiomyocytes and cardiomyocyte apoptosis under hypoxia and was decreased synergistically. These results suggest that the synergistic combination of VEGF, HO-1, and miSHP-1 may be promising for the clinical treatment of ischemic diseases.

IL-33 precursor and bleomycin synergize in inducing lymphocyte accumulation and fibrosis in the lungs

Event Abstract Back to Event IL-33 precursor and bleomycin synergize in inducing lymphocyte accumulation and fibrosis in the lungs Irina G. Luzina1, 2, Virginia Lockatell1, 2, Pavel Kopach1, Nevins W. Todd1, 2 and Sergei P. Atamas1, 2* 1 University of Maryland School of Medicine, United States 2 Baltimore VA Medical Center, United States We and others have previously found that precursor IL-33 (PRIL33) protein is biologically active without proteolytic activation. We reported that the levels of PRIL33 are substantially elevated in the lungs of patients with interstitial lung disease (ILD), which is associated with idiopathic pulmonary fibrosis and systemic sclerosis. PRIL33 remains intranuclear and promotes inflammation in a non-Th2 fashion by not engaging the mature IL-33 receptor T1/ST2. To further assess the biological role of the elevated expression of PRIL33 in ILD, intratracheal bleomycin injury to the lung, alone or in combination with recombinant adenovirus delivery of PRIL33, was used to model ILD in mice. Combined PRIL33 gene delivery and bleomycin challenge had a potentiating effect on pulmonary lymphocytosis; accumulation of collagen; expression of HSP70; and the levels of TGF-β, CCL6, MCP-1, and MIP-1α, with these combined effects significantly exceeding the sum of the effects of PRIL33 gene delivery or bleomycin challenge alone (p < 0.05). By contrast, the combined effects of PRIL33 expression and bleomycin on bronchoalveolar lavage counts of macrophages and neutrophils in these mice were additive of the effects of each of these factors alone. The Th2 cytokines IL-4, IL-5, and IL-13 were not part of this synergy, and gene deficiency of T1/ST2 did not affect the synergistic nature of the “double-hit” from PRIL33 expression and bleomycin injury. It was concluded that elevated PRIL33 expression in ILD is a likely contributor to inflammatory and fibrotic lung injury, and that this contribution is independent of the T1/ST2 receptor or Th2 cytokines. Keywords: interleukin-33, Inflammation, Fibrosis, Lung, Scleroderma, Systemic, Idiopathic Pulmonary Fibrosis Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Luzina IG, Lockatell V, Kopach P, Todd NW and Atamas SP (2013). IL-33 precursor and bleomycin synergize in inducing lymphocyte accumulation and fibrosis in the lungs. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00859 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Sergei P Atamas, University of Maryland School of Medicine, Baltimore, Maryland, United States, satamas@umaryland.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Irina G Luzina Virginia Lockatell Pavel Kopach Nevins W Todd Sergei P Atamas Google Irina G Luzina Virginia Lockatell Pavel Kopach Nevins W Todd Sergei P Atamas Google Scholar Irina G Luzina Virginia Lockatell Pavel Kopach Nevins W Todd Sergei P Atamas PubMed Irina G Luzina Virginia Lockatell Pavel Kopach Nevins W Todd Sergei P Atamas Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Transcripts for combined synthetic microRNA and gene delivery

MicroRNAs (miRNAs) are a class of short noncoding RNAs which are endogenously expressed in many organisms and regulate gene expression by binding to messenger RNA (mRNA). MicroRNAs are either produced from their independent transcription units in intergenic regions or lie in intragenic regions. Intragenic miRNAs and their host mRNAs are produced from the same transcript by the microprocessor and the spliceosome protein complex respectively. The details and exact timing of the processing events have implications for downstream RNA interference (RNAi) efficiency and mRNA stability. Here we engineer and study in mammalian cells a range of synthetic intragenic miRNAs co-expressed with their host genes. Furthermore, we study transcripts which carry the target of the miRNA, thereby emulating a common regulation mechanism. We perform fluorescence microscopy and flow cytometry to characterize the engineered transcripts and investigate the properties of the underlying biological processes. Our results shed additional light on miRNA and pre-mRNA processing but importantly provide insight into engineering transcripts customized for combined delivery and use in synthetic gene circuits.

Multifunctional, multichannel bridges that deliver neurotrophin encoding lentivirus for regeneration following spinal cord injury

Therapeutic strategies following spinal cord injury must address the multiple barriers that limit regeneration. Multiple channel bridges have been developed that stabilize the injury following implantation and provide physical guidance for regenerating axons. These bridges have now been employed as a vehicle for localized delivery of lentivirus. Implantation of lentivirus loaded multiple channel bridges produced transgene expression that persisted for at least 4 weeks. Expression was maximal at the implant at the earliest time point, and decreased with increasing time of implantation, as well as rostral and caudal to the bridge. Immunohistochemical staining indicated transduction of macrophages, Schwann cells, fibroblasts, and astrocytes within the bridge and adjacent tissue. Subsequently, the delivery of lentivirus encoding the neurotrophic factors NT-3 or BDNF significantly increased the extent of axonal growth into the bridge relative to empty scaffolds. In addition to promoting axon growth, the induced expression of neurotrophic factors led to myelination of axons within the channels of the bridge, where the number of myelinated axons was significantly enhanced relative to control. Combining gene delivery with biomaterials to provide physical guidance and create a permissive environment can provide a platform to enhance axonal growth and promote regeneration.

Stem Cell–Mediated Transfer of a Human Artificial Chromosome Ameliorates Muscular Dystrophy

In contrast to conventional gene therapy vectors, human artificial chromosomes (HACs) are episomal vectors that can carry large regions of the genome containing regulatory elements. So far, HACs have not been used as vectors in gene therapy for treating genetic disorders. Here, we report the amelioration of the dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD) using a combination of HAC-mediated gene replacement and transplantation with blood vessel-associated stem cells (mesoangioblasts). We first genetically corrected mesoangioblasts from dystrophic mdx mice with a HAC vector containing the entire (2.4 Mb) human dystrophin genetic locus. Genetically corrected mesoangioblasts engrafted robustly and gave rise to many dystrophin-positive muscle fibers and muscle satellite cells in dystrophic mice, leading to morphological and functional amelioration of the phenotype that lasted for up to 8 months after transplantation. Thus, HAC-mediated gene transfer shows efficacy in a preclinical model of DMD and offers potential for future clinical translation.

Whirlin Replacement Restores the Formation of the USH2 Protein Complex in Whirlin Knockout Photoreceptors

Whirlin is the causative gene for Usher syndrome type IID (USH2D), a condition manifested as both retinitis pigmentosa and congenital deafness. Mutations in this gene cause disruption of the USH2 protein complex composed of USH2A and VLGR1 at the periciliary membrane complex (PMC) in photoreceptors. In this study, the adeno-associated virus (AAV)-mediated whirlin replacement was evaluated as a treatment option. Murine whirlin cDNA driven by the human rhodopsin kinase promoter (hRK) was packaged as an AAV2/5 vector and delivered into the whirlin knockout retina through subretinal injection. The efficiency, efficacy, and safety of this treatment were examined using immunofluorescent staining, confocal imaging, immunoelectron microscopy, Western blot analysis, histologic analysis, and electroretinogram. The AAV-mediated whirlin expression started at two weeks, reached its maximum level at 10 weeks, and lasted up to six months post injection. The transgenic whirlin product had a molecular size and an expression level comparable to the wild-type. It was distributed at the PMC in both rod and cone photoreceptors from the central to peripheral retina. Importantly, the transgenic whirlin restored the cellular localization and expression level of both USH2A and VLGR1 and did not cause defects in the retinal histology and function in the whirlin knockout mouse. Whirlin transgene recruits USH2A and VLGR1 to the PMC and is sufficient for the formation of the USH2 protein complex in photoreceptors. The combined hRK and AAV gene delivery system could be an effective gene therapy approach to treat retinal degeneration in USH2D patients.

Transplanted articular chondrocytes co-overexpressing IGF-I and FGF-2 stimulate cartilage repair in vivo

The combination of chondrogenic factors might be necessary to adequately stimulate articular cartilage repair. In previous studies, enhanced repair was observed following transplantation of chondrocytes overexpressing human insulin-like growth factor I (IGF-I) or fibroblast growth factor 2 (FGF-2). Here, the hypothesis that co-overexpression of IGF-I and FGF-2 by transplanted articular chondrocytes enhances the early repair of cartilage defects in vivo and protects the neighbouring cartilage from degeneration was tested. Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or co-transfected with the IGF-I and FGF-2 gene, encapsulated in alginate and transplanted into osteochondral defects in the knee joints of rabbits in vivo. After 3 weeks, co-overexpression of IGF-I/FGF-2 improved the macroscopic aspect of defects without affecting the synovial membrane. Immunoreactivity to type-I collagen, an indicator of fibrocartilage, was significantly lower in defects receiving IGF-I/FGF-2 implants. Importantly, combined IGF-I/FGF-2 overexpression significantly improved the histological repair score. Most remarkably, such enhanced cartilage repair was correlated with a 2.1-fold higher proteoglycan content of the repair tissue. Finally, there were less degenerative changes in the cartilage adjacent to the defects treated with IGF-I/FGF-2 implants. The data demonstrate that combined gene delivery of therapeutic growth factors to cartilage defects may have value to promote cartilage repair. The results also suggest a protective effect of IGF-I/FGF-2 co-overexpression on the neighbouring articular cartilage. These findings support the concept of implementing gene transfer strategies for articular cartilage repair in a clinical setting.

Enhancement of bone formation by genetically-engineered bone marrow stromal cells expressing BMP-2, VEGF and angiopoietin-1

To explore the potential of combined delivery of osteogenic and angiogenic factors to bone marrow stromal cells (BMSCs) for repair of critical-size bone defects, we followed the formation of bone and vessels in tissue-engineered constructs in nude mice and rabbit bone defects upon introducing different combinations of BMP-2, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) to BMSCs with adenoviral vectors. Better osteogenesis and angiogenesis were found in co-delivery group of BMP-2, VEGF and angiopoietin-1 than any other combination of these factors in both animal models, indicating combined gene delivery of angiopoietin-1 and VEGF165 into a tissue-engineered construct produces an additive effect on BMP-2-induced osteogenesis.

A combined therapeutic approach for pyruvate dehydrogenase deficiency using self-complementary adeno-associated virus serotype-specific vectors and dichloroacetate

We determined the ability of self-complementary adeno-associated virus (scAAV) vectors to deliver and express the pyruvate dehydrogenase E1α subunit gene (PDHA1) in primary cultures of skin fibroblasts from 3 patients with defined mutations in PHDA1 and 3 healthy subjects. Cells were transduced with scAAV vectors containing the cytomegalovirus promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a vector:cell ratio of 200. Transgene expression was measured 72 h later. The transduction efficiency of scAAV2 and scAAV6 vectors was 3- to 5-fold higher than that of the other serotypes, which were subsequently used to transduce fibroblasts with wild-type PDHA1 cDNA under the control of the chicken beta-action (CBA) promoter at a vector:cell ratio of 1000. Total PDH-specific activity and E1α protein expression were determined 10 days post-transduction. Both vectors increased E1α expression 40–60% in both control and patient cells, and increased PDH activity in two patient cell lines. We also used dichloroacetate (DCA) to maximally activate PDH through dephosphorylation of E1α. Exposure for 24 h to 5 mM DCA increased PDH activity in non-transduced control (mean 37% increase) and PDH deficient (mean 44% increase) cells. Exposure of transduced patient fibroblasts to DCA increased PDH activity up to 90% of the activity measured in untreated control cells. DCA also increased expression of E1α protein and, to variable extents, that of other components of the PDH complex in both non-transduced and transduced cells. These data suggest that a combined gene delivery and pharmacological approach may hold promise for the treatment of PDH deficiency.

β7‐integrin‐independent enhancement of mucosal and systemic anti‐HIV antibody responses following combined mucosal and systemic gene delivery

Vaccination strategies that can block or limit heterosexual human immunodeficiency virus (HIV) transmissions to local and systemic tissues are the goal of much research effort. Herein, in a mouse model, we aimed to determine whether the enhancement of antibody responses through mucosal and systemic immunizations, previously observed with protein-based vaccines, applies to immunizations with DNA- or RNA-based vectors. Intranasal (i.n.) followed by intramuscular (i.m.) immunizations (i.n./i.m.) with polylactide-coglycolide (PLG)-DNA microparticles encoding HIV-gag (PLG-DNA-gag) significantly enhanced serum antibody responses, compared with i.m., i.n. or i.m. followed by i.n. (i.m./i.n.) immunizations. Moreover, while i.n./i.m., i.n. or i.m./i.n. immunizations with PLG-DNA-gag resulted in genital tract antibody responses, i.m. immunizations alone failed to do so. Importantly, beta7-deficient mice developed local and systemic antibody responses following i.n./i.m. immunization, or immunization via any other route, similar to those of wild-type mice. To compare the DNA with an RNA delivery system, immunizations were performed with VEE/SIN-gag replicon particles, composed of Venezuelan equine encephalitis virus (VEE) replicon RNA and Sindbis surface structure (SIN). i.n./i.m., compared with any other immunizations, i.n./i.m. immunization with VEE/SIN-gag resulted in enhanced genital tract but not serum antibody responses. These data show for the first time that mucosal followed by systemic immunizations with gene delivery systems enhance B-cell responses independent of the mucosal homing receptors alpha4beta7 and alphaEbeta7.

Tissue‐Engineered Bone Formation With Gene Transfer and Mesenchymal Stem Cells in a Minimally Invasive Technique

The objective of this study was to use a chitosan-alginate gel to implant bone marrow-derived mesenchymal stem cells subcutaneously in a minimally invasive manner and promote bone formation by the simultaneously transferred osteogenic protein (OP)-1 (bone morphogenic protein-7) gene. The complex of polyethylenimine/luciferase plasmid DNA embedded in the gel was able to transfect HEK 293 cells on a culture dish or co-encapsulated in the gel. When injected into the subcutaneous space of mice, luciferase expression was two to three orders of magnitude increased above the background. To examine the efficacy of gene-, cell-, and combined gene- and cell-encapsulated gels in tissue generation, samples were injected into the subcutaneous space of 6-week-old athymic nude mice, and the OP-1 plasmid was studied. At 8 weeks after the injection, the gels only maintained their volumetric shape when human mesenchymal stem cells (hMSCs) were encapsulated, but otherwise the gels were partially dissolved. Transgene expression of OP-1 was clearly detected in the samples after 4 weeks but not after 8 weeks. Type II collagen was detected in all the gels containing the OP-1 plasmid, with or without hMSCs. The samples with the combination of OP-1 DNA and hMSCs revealed strong type II collagen expression as well as osteoid foci. These results suggest that combined gene and hMSC delivery within a chitosan-alginate gel could be an interesting approach for tissue engineering.